(Advances in Parasitology, Volume 91) David Rollinson, Russell Stothard-Elsevier - Academic Press (2016)

VOLUME NINETY ONE
ADVANCES IN
PARASITOLOGY
SERIES EDITOR
D. ROLLINSON J. R. STOTHARD
Life Sciences Department Department of Parasitology
The Natural History Museum, Liverpool School of Tropical
London, UK Medicine Liverpool, UK
d.rollinson@nhm.ac.uk russell.stothard@lstmed.ac.uk
EDITORIAL BOARD
T. J. C. ANDERSON R. C. OLIVEIRA
Department of Genetics, Texas Centro de Pesquisas Rene Rachou/
Biomedical Research Institute, CPqRR - A FIOCRUZ em Minas
San Antonio, TX, USA Gerais, Rene Rachou Research
Center/CPqRR - The Oswaldo Cruz
M. G. BASEZ Foundation in the State of Minas
Professor of Neglected Tropical Gerais-Brazil, Brazil
Diseases, Department of Infectious
Disease Epidemiology, Faculty of R. E. SINDEN
Medicine (St Marys Campus), Immunology and Infection
Imperial College London, Section, Department of Biological
London, UK Sciences, Sir Alexander Fleming
Building, Imperial College of
Science, Technology and
S. BROOKER Medicine, London, UK
Wellcome Trust Research Fellow
and Professor, London School of D. L. SMITH
Hygiene and Tropical Medicine, Johns Hopkins Malaria Research
Faculty of Infectious and Tropical, Institute & Department of
Diseases, London, UK Epidemiology, Johns Hopkins
Bloomberg School of Public Health,
R. B. GASSER Baltimore, MD, USA
Department of Veterinary Science,
The University of Melbourne, R. C. A. THOMPSON
Parkville, Victoria, Australia Head, WHO Collaborating Centre
for the Molecular Epidemiology
of Parasitic Infections, Principal
N. HALL Investigator, Environmental
School of Biological Sciences, Biotechnology CRC (EBCRC), School
Biosciences Building, University of of Veterinary and Biomedical
Liverpool, Liverpool, UK Sciences, Murdoch University,
Murdoch, WA, Australia
J. KEISER
Head, Helminth Drug X.-N. ZHOU
Development Unit, Department Professor, Director, National
of Medical Parasitology and Institute of Parasitic Diseases,
Infection Biology, Swiss Tropical Chinese Center for Disease Control
and Public Health Institute, Basel, and Prevention, Shanghai, Peoples
Switzerland Republic of China
VOLUME NINETY ONE
ADVANCES IN
PARASITOLOGY
Edited by
D. ROLLINSON
Life Sciences Department
The Natural History Museum
London, UK
J.R. STOTHARD
Department of Parasitology
Liverpool School of Tropical Medicine
Liverpool, UK
AMSTERDAM BOSTON HEIDELBERG LONDON

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CONTRIBUTORS
Amy Abruzzi
Edward J. Bloustein School of Planning and Public Policy, Rutgers University,
New Brunswick, NJ, USA
Sukaina B. Alikhan
U.S. Fund for UNICEF, New York, NY, USA
Jason P. Andras
Zoological Institute, University of Basel, Basel, Switzerland; Department of Biological
Sciences, Mount Holyoke College, South Hadley, MA, USA
Frida Ben-Ami
Department of Zoology, George S. Wise Faculty of Life Sciences, Tel Aviv University,
Tel Aviv, Israel
Brian M. Cooke
Infection and Immunity Program, Monash Biomedicine Discovery Institute and Department
of Microbiology, Monash University, VIC, Australia
Ross L. Coppel
David Duneau
Zoological Institute, University of Basel, Basel, Switzerland; Department Ecologie et
Diversit Biologique, University Paul Sabatier-Toulouse III, Toulouse, France
Louis Du Pasquier
Zoological Institute, University of Basel, Basel, Switzerland
Dieter Ebert
Zoological Institute, University of Basel, Basel, Switzerland
Bernard Fried
Lafayette College, Easton, PA, USA
Robin B. Gasser
Faculty of Veterinary and Agricultural Sciences, The University of Melbourne, Parkville,
VIC, Australia
Geoffrey N. Gobert
Molecular Parasitology Laboratory, Infectious Diseases Division, QIMR Berghofer Medical
Research Institute, Brisbane, QLD, Australia
Catherine A. Gordon
ix j
x Contributors
Darren J. Gray
Research School of Population Health, The Australian National University, Canberra, ACT,
Australia
Matthew D. Hall
Zoological Institute, University of Basel, Basel, Switzerland; Monash University, School of
Biological Sciences, Clayton Campus, Melbourne, VIC, Australia
Anja Joachim
Institute of Parasitology, Department of Pathobiology, University of Veterinary Medicine
Vienna, Vienna, Austria
Malcolm K. Jones
Research Institute, Brisbane, QLD, Australia; School of Veterinary Science, University of
Queensland, Brisbane, QLD, Australia
Pasi K. Korhonen
VIC, Australia
Pepijn Luijckx
Zoological Institute, University of Basel, Basel, Switzerland; Department of Ecology &
Evolutionary Biology, University of Toronto, Toronto, ON, Canada
Donald P. McManus
Narla Mohandas
New York Blood Center, New York, NY, USA
Martina Ondrovics
Institute of Parasitology, Department of Pathobiology, University of Veterinary Medicine
Vienna, Vienna, Austria
Nicholas I. Proellocks
Neil D. Young
VIC, Australia
Xing-Quan Zhu
State Key Laboratory of Veterinary Etiological Biology, Key Laboratory of Veterinary
Parasitology of Gansu Province, Lanzhou Veterinary Research Institute, Chinese Academy
of Agricultural Sciences, Lanzhou, Gansu Province, PR China
CHAPTER ONE
Malaria Parasite Proteins and

Their Role in Alteration of the
Structure and Function of Red
Blood Cells
Nicholas I. Proellocks*, Ross L. Coppel*, Narla Mohandasx,
Brian M. Cooke*, 1
*Infection and Immunity Program, Monash Biomedicine Discovery Institute and Department of
Microbiology, Monash University, VIC, Australia
x
New York Blood Center, New York, NY, USA
1
Corresponding author: E-mail: brian.cooke@monash.edu
Contents
1. Introduction 2
2. Trafcking of Parasite Proteins into the RBC 4
2.1 The PEXEL motif 27
2.1.1 Plasmepsin V-mediated PEXEL function 28
2.1.2 PI(3)P-mediated PEXEL function 29
2.2 PEXEL-negative exported proteins 30
2.3 The role of PTEX 32
2.4 Protein trafcking within iRBCs 33
2.4.1 Vesicle-mediated trafcking 34
2.4.2 Chaperones 34
2.4.3 MCs: an external Golgi? 36
3. Exported Parasite Proteins 37
3.1 MC-associated proteins 38
3.1.1 Skeleton-binding protein 1 38
3.1.2 Membrane-associated histidine-rich protein 1 39
3.1.3 Membrane-associated histidine-rich protein 2 39
3.1.4 Ring-exported protein 1 40
3.1.5 Ring-exported protein 2 42
3.1.6 Other less-well characterized proteins associated with MCs 42
3.1.7 Parasite proteins and the tubovesicular network 43
3.2 Parasite proteins in the RBC cytosol or at the RBC membrane skeleton 44
3.2.1 Knob-associated histidine-rich protein 44
3.2.2 P. falciparum erythrocyte membrane protein 3 45
3.2.3 P. falciparum antigen 332 46
3.2.4 Plasmodium helical interspersed sub-telomeric proteins 47
3.2.5 Ring-infected erythrocyte surface antigen 52
Advances in Parasitology, Volume 91
2016 Elsevier Ltd.
j
ISSN 0065-308X
http://dx.doi.org/10.1016/bs.apar.2015.09.002 All rights reserved. 1
2 Nicholas I. Proellocks et al.
3.2.6 Proteins containing DnaJ domains 53

3.2.7 Mature-parasite-infected erythrocyte surface antigen 55
3.2.8 FIKK kinases 56
3.2.9 P. falciparum proteins involved in trafcking of PfEMP1 58
3.2.10 Other less-well characterized exported proteins 59
3.3 Proteins exposed on the surface of infected RBCs 59
3.3.1 P. falciparum erythrocyte membrane protein 1 60
3.3.2 RIFINs 63
3.3.3 STEVOR 64
3.3.4 SURFINS 64
3.3.5 Glycophorin-binding proteins 65
3.3.6 Cytoadherence-linked asexual gene 66
3.3.7 Other less-well characterized putative iRBC surface proteins 67
3.3.8 Exported proteins of sexual stage parasites 68
4. Alteration of Host RBC Proteins during Malaria Infection 69
5. Conclusion 69
References 71
Abstract
Malaria, caused by Plasmodium spp., continues to be a major threat to human health
and a signicant cause of socioeconomic hardship in many countries. Almost half of
the worlds population live in malaria-endemic regions and many of them suffer one
or more, often life-threatening episodes of malaria every year, the symptoms of which
are attributable to replication of the parasite within red blood cells (RBCs). In the case of
Plasmodium falciparum, the species responsible for most malaria-related deaths, para-
site replication within RBCs is accompanied by striking alterations to the morphological,
biochemical and biophysical properties of the host cell that are essential for the para-
sites survival. To achieve this, the parasite establishes a unique and extensive protein
export network in the infected RBC, dedicating at least 6% of its genome to the process.
Understanding the full gamut of proteins involved in this process and the mechanisms
by which P. falciparum alters the structure and function of RBCs is important both for a
more complete understanding of the pathogenesis of malaria and for development of
new therapeutic strategies to prevent or treat this devastating disease. This review fo-
cuses on what is currently known about exported parasite proteins, their interactions
with the RBC and their likely pathophysiological consequences.
1. INTRODUCTION
Malaria in humans, caused by Plasmodium spp., remains a major cause
of morbidity, mortality and socioeconomic hardship in many areas of the
world, particularly Africa, South America and Asia. Almost half of the
Malaria Parasite Proteins and the RBC 3
worlds population lives in malaria-endemic regions making the develop-

ment and implementation of effective intervention strategies a global health
priority (Noor et al., 2014; Sachs, 2002; Snow et al., 2005). All of the clinical
symptoms of malaria are attributable to replication of the malaria parasite
within red blood cells (RBCs) and vary in severity depending on the parasite
species and the immune status of the infected host. In the case of Plasmodium
falciparum, the malaria species responsible for the vast majority of malaria-
related deaths, serious clinical complications arise because parasite-infected
RBCs (iRBCs) sequester in the microvasculature of various organs (Beare
et al., 2006; Cooke et al., 2000).
Parasite replication within RBCs is accompanied by striking alterations
to the morphological, biochemical and biophysical properties of the
iRBC. These changes have been most extensively studied in the case of
P. falciparum and consequently this review will largely concentrate on this
species. Unlike normal RBCs, iRBCs are rigid and poorly deformable
and adhere to the vascular endothelium or to other infected or uninfected
RBCs (Cooke et al., 2001, 2004a, 2005). For the parasite, which must main-
tain the mechanical integrity of the host RBC under haemodynamic stress,
collect nutrients from the extracellular environment, avoid the host immune
system and escape destruction by the spleen, these structural and functional
modications of RBCs appear to be essential for its survival in vivo. On the
other hand, for the infected human, accumulation of iRBCs in the micro-
vasculature, perturbation of blood ow or partial or complete obstruction of
blood vessels by rheologically abnormal iRBCs results in severe and often
fatal clinical complications. Sequestration of iRBCs within the microvascu-
lature of the brain, for example results in cerebral malaria which is associated
with w10e15% mortality rate in some areas of the world (von Seidlein
et al., 2012). Sequestration of infected and uninfected RBCs by the spleen
results in anaemia which on occasion is life threatening. An understanding of
the mechanisms by which P. falciparum alters the structure and function of
RBCs is essential for the future development of new therapeutic strategies
to prevent or treat this severe and devastating disease.
At the molecular level, RBC modications are mediated by a subset of
proteins that are secreted by the parasite beyond its own plasma membrane,
and selectively trafcked to various locations in the iRBC (Cooke et al.,
2004a; Maier et al., 2009). Bioinformatic analysis of the genome of P. falcip-
arum parasites reveals that about 6% of its genome encodes proteins that are
destined for export into the RBC (Hiller et al., 2004; Marti et al., 2004).
While some of these proteins, as well as others involved in their export
and trafcking, have been identied and characterized, the process remains
poorly understood. This review focuses on the current state of our knowl-
edge of exported parasite proteins, their interactions with RBCs and their
likely role in the pathogenesis of falciparum malaria.
2. TRAFFICKING OF PARASITE PROTEINS

INTO THE RBC
During the blood stage of malaria infection, the parasite invades, ma-
tures and replicates in RBCs over a period of 24e72 h, depending on the
parasite species. RBCs are atypical eukaryotic cells in that they do not contain
any of the organelles necessary for de novo protein synthesis or components
for protein trafcking. To survive in this resource-poor cellular environment,
essentially a membrane-bound corpuscle of aqueous haemoglobin, the
parasite must establish its own network of protein trafcking machinery
and extensively embellish the corpuscular architecture. This complex process
involves the export of a large number of parasite proteins to the RBC
(Tables 1e5). The mechanism by which parasite proteins are exported has
been the subject of considerable research, debate and controversy over the
past few years. All species of malaria parasites export proteins into their
host RBC; however, from the evidence amassed so far, P. falciparum builds
the most elaborate export pathways and exports the greatest number of
parasite-encoded proteins. Construction of this export machinery begins
immediately after the parasite has invaded the RBC. Recent evidence sug-
gests that of the parasites invasion-related organelles, the rhoptry contains
components that build the parasitophorous vacuolar membrane (PVM)
and parasitophorous vacuole (PV) in which the parasite resides for the dura-
tion of its lifetime in RBCs as well as proteins that are directly exported into
the iRBC, such as the RhopH complex of proteins (Riglar et al., 2013;
Zuccala et al., 2012). Shortly after rhoptry contents are released during the
initial stages of parasite invasion, proteins are expelled from another parasite
organelle, the dense granules, that contain components required for later
export of parasite proteins (Bullen et al., 2012; Riglar et al., 2013). These
early stages of establishing a protein trafcking infrastructure are critical for
the survival of the parasite as protein export begins very early in the parasites
life cycle, with the Ring-Exported Surface Antigen (RESA) being exported
to RBC membrane within 12 min after invasion (Riglar et al., 2013). Sub-
sequently, export of numerous, functionally diverse proteins into the RBC
then continues throughout the parasites life cycle until the RBC is nally
Table 1 Exported proteins in P. falciparum blood-stage parasites
Malaria Parasite Proteins and the RBC

Protein name Gene id Localization Comments References
SBP1 PF3D7_0501300 (PFE0065w) Maurers clefts Binds to the RBC membrane Blisnick et al. (2000),
skeleton; involved in Cooke et al. (2006),
PfEMP1 trafcking Maier et al. (2007)
MAHRP1 PF3D7_1370300 (MAL 13P1.413) Maurers clefts Involved in Pf EMP1 Spycher et al. (2003,
trafcking 2008)
MAHRP2 PF3D7_1353200 (PF13_0276) Maurers clefts - Involved in the formation of Pachlatko et al. (2010)
tethers tethers
REX1 PF3D7_0935900 (PFI1735c) Maurers clefts Required for formation of Dixon et al. (2008,
Maurers clefts; involved in 2011), Hanssen et al.
Pf EMP1 trafcking (2008a), Hawthorne
et al. (2004)
REX2 PF3D7_0936000 (PFI1740c) Maurers clefts Integral membrane protein in Haase et al. (2009),
Maurers clefts; unknown Spielmann et al.
function (2006)
Pf PTP1 PF3D7_0202200 (PFB0106c) Maurers clefts and Required for formation of Maier et al. (2008), Rug
iRBC Maurers clefts; involved in et al. (2014)
cytoplasm PfEMP1 and STEVOR
trafcking to Maurers clefts
Pf MC-2TM PF3D7_0114100 (PFA0680c) Maurers clefts Two transmembrane domains; Sam-Yellowe et al.
PF3D7_0101300 (PFA0065w) subfamily of STEVOR; (2004), Tsarukyanova
PF3D7_0222100 (PFB0985c) unknown function et al. (2009)
PF3D7_0221500 (PFB0960c)
PF3D7_0324100 (PFC1080c)
PF3D7_0601200 (PFF0060w)
PF3D7_ 0631400 (MAL6P1.15)
PF3D7_0701600 (MAL7P1.5)
5
(Continued)
Table 1 Exported proteins in P. falciparum blood-stage parasitesdcont'd
6
PF3D7_0713100 (MAL7P1.58)
PF3D7_0700800 (MAL8P1.213)
PF3D7_1039700 (PF10_0390)
PF3D7_1101700 (PF11_0025)
PF3D7_1100800 (PF11_0014)
PCRMPs PF3D7_0911300 (PFI0550w) Maurers clefts; Expressed throughout the Thompson et al. (2007)
PF3D7_0718300 (MAL7P1.92) sporozoite parasites life cycle;
PF3D7_1208200 (PFL0410w) surface unknown function
PF3D7_1475400 (PF14_0722)
Sec31p PF3D7_0214100 (PFB0640c) Maurers clefts Vesicle trafcking Adisa et al. (2001)
Sec23p PF3D7_0822600 (PF08_0036) Maurers clefts Vesicle trafcking Wickert et al. (2003a)
Sar1p PF3D7_0416800 (PFD0810w) Maurers clefts Vesicle trafcking Albano et al. (1999)
Pf NSF PF3D7_0303000 (PFC0140c) iRBC cytosol, Vesicle fusion component Hayashi et al. (2001)
possible Maurers
clefts
Pf J23 PF3D7_1001900 (PF10_0023) Maurers clefts Unknown function Vincensini et al. (2005)
ETRAMP PF3D7_0202500 (PFB0120w) iRBC periphery; Small proteins expressed at Birago et al. (2003),
PF3D7_0423700 (PFD1120c) parasite plasma different times throughout Spielmann et al.
PF3D7_0532100 (PFE1590w) membrane (PPM); the RBC cycle; unknown (2003)
PF3D7_0829600 (MAL8P1.6) parasitophorous function
Nicholas I. Proellocks et al.

PF3D7_0936100 (PFI1745c) vacuolar
PF3D7_1001500 (PF10_0019) membrane (PVM);
PF3D7_1033200 (PF10_0323) Maurers clefts
PF3D7_1016900 (PF10_0164)
PF3D7_1102700 (PF11_0039)
PF3D7_1102800 (PF11_0040)
PF3D7_1240100 (PFL1945c)
PF3D7_1302200 (PF13_0012)

PF3D7_1401400 (PF14_0016)
PF3D7_1476100 (PF14_0729)
PF07_0007 PF3D7_0702400 Maurers clefts; Unknown function Heiber et al. (2013),
iRBC cytosol Oehring et al. (2012)
PF07_0008 PF3D7_0702500 Maurers clefts; Unknown function Heiber et al. (2013)
iRBC cytosol
PF08_0003 PF3D7_0830500 Maurers clefts Unknown function; Heiber et al. (2013)
tryptophan/threonine-rich
antigen
PF11_0505 PF3D7_1148900 Maurers clefts Unknown function Heiber et al. (2013)
PF14_0045 PF3D7_1404800 iRBC cytoplasm Unknown function Heiber et al. (2013)
PFF0090w PF3D7_0601900 Maurers clefts Unknown function Heiber et al. (2013)
PFL0065w PF3D7_1201300 Maurers clefts Unknown function; suggested Frech and Chen (2013),
to be part of the ETRAMP Heiber et al. (2013)
family
PFL2515c PF3D7_1252300 Maurers clefts Unknown function Heiber et al. (2013)
MSRP5 PF3D7_1334300 (PF13_0191) iRBC cytosol; Unknown function; Heiber et al. (2013)
external face of the MSP7-related protein
PVM
MSRP6 PF3D7_1334500 (PF13_0192) Maurers clefts Unknown function; Heiber et al. (2013)
MSP7-related protein
SEMP1 PF3D7_0702400 (PF07_0007) Maurers clefts Unknown function Dietz et al. (2014)
MSRP7 PF3D7_1334700 (PF13_0194) iRBC cytoplasm; Unknown function; Heiber et al. (2013)
external face of the MSP7-related protein
PVM
EVP1 PF3D7_0410000 (PFD0495c) TVN Involved in lipid dynamics Tamez et al. (2008)
7
in the TVN
(Continued)
8
TVN-JP1 PF3D7_0310400 (PFC0435w) TVN Possibly required for van Ooij et al. (2008)
formation of the TVN
PfEMP1 Multigene family (w60 copies) iRBC surface Bind to multiple host cell
expressed receptors; key
mediator of virulence
RIFIN Multigene family (w200 copies) iRBC surface Two sub-groups. RIFIN A Cheng et al. (1998),
(involved in antigenic Fernandez et al.
variation) and RIFIN B (1999), Kyes et al.
(localizes to the PV; (1999), Petter et al.
unknown function) (2007)
STEVOR Multigene family (w30 copies) iRBC surface Clonally expressed on the Cheng et al. (1998),
iRBC surface; involved in Niang et al. (2009),
antigenic variation and Sanyal et al. (2012)
regulation of iRBC
membrane mechanical
properties
SURFIN PF3D7_0113100 (PFA0625w) iRBC and merozoite Unknown function Winter et al. (2005)
PF3D7_0113600 (PFA0650w) surface
PF3D7_0115000 (PFA0725w)
PF3D7_0402200 (PFD0100c)

PF3D7_0424400 (PFD1160w)
PF3D7_0800700 (MAL8P1.162)
PF3D7_0830800 (PF08_0002)
PF3D7_081100 (MAL8P1.1)
PF3D7_1301800 (PF13_0074)
PF3D7_1477600 (PF14_0747)
GBP130 PF3D7_1016300 (PF10_0159) iRBC surface Binds to glycophorin; Maier et al. (2008),
(GBPH) PF3D7_1301200 (PF13_0010) involved in altered RBC Nolte et al. (1991),
PF3D7_1401000 (PF14_0010) membrane rigidity; Perkins (1988)
PF3D7_1462300 (PF14_0593) GBHP-2 is essential
CLAG PF3D7_0220800 (PFB0935w) Rhoptries, Part of the RhopH complex; Gardiner et al. (2004),
PF3D7_0302200 (PFC0110w) transferred to possible alternative Kaneko et al. (2001,
PF3D7_0302500 (PFC0120w) newly invaded functions once in the iRBC; 2005), Ling et al.
PF3D7_0831600 (MAL7P1.229) iRBC membrane roles in cytoadhesion and (2004), Nguitragool
PF3D7_0935800 (PFI1730w) and surface nutrient uptake (new et al. (2011, 2014),
exposed permeability pathway Trenholme et al.
NPP); represent a possible (2000)
novel group of rhoptry
proteins localizing to the
iRBC following invasion
PHISTa See PHIST Table Sargeant et al. (2006)
PHISTb See PHIST Table Sargeant et al. (2006)
PHISTc See PHIST Table Sargeant et al. (2006)
RESA PF3D7_0102200 (PFA0110w) iRBC membrane Involved in altering RBC Maier et al. (2008), Pei
membrane mechanical et al. (2007a),
properties; required for Sargeant et al. (2006),
stabilization of spectrin and Silva et al. (2005)
the iRBC membrane under
heat stress
KAHRP PF3D7_0202000 (PFB0100c) iRBC membrane; Essential for knob formation Magowan et al. (2000),
knobs and anchoring PfEMP1 at Rug et al. (2006),
knobs and iRBC membrane Waller et al. (1999,
skeleton 2002)
(Continued)
9
10
PfEMP3 PF3D7_0201900 (PFB0095c) iRBC membrane Binds to spectrin at the iRBC Waller et al. (2007),
membrane skeleton Waterkeyn et al.
(2000)
Pf332 PF3D7_1149000 (PF11_0507) Maurers clefts; Dual role; decrease of iRBC Glenister et al. (2009),
iRBC membrane membrane rigidity and role Hinterberg et al.
in adhesion; binds to actin at (1994), Hodder et al.
the iRBC membrane (2009), Mattei and
skeleton Scherf (1992), Waller
et al. (2010)
DnaJs/Hsp40 See Table 4 Botha et al. (2007),
Sargeant et al. (2006)
MESA PF3D7_0500800 (PFE0040c) iRBC membrane Binds to protein 4.1R at the Bennett et al. (1997),
(PfEMP2) skeleton RBC membrane skeleton; Black et al. (2008),
unknown function Waller et al. (2003)
FIKK Kinases See Table 5 Schneider and
Mercereau-Puijalon
(2005), Ward et al.
(2004)
hyp1 e hyp17 60e70 members Unknown No known function; some Frech and Chen (2013),
gene families members have been Silvestrini et al. (2010)

detected in gametocytes
PfHsp70x PF3D7_0831700 (MAL7P1.228) J-Dots; iRBC Interacts with the exported Kulzer et al. (2012)
cytoplasm type II DnaJs, possible role
in PfEMP1 trafcking
REX3 PF3D7_0936300 (PFI1755c) iRBC cytoplasm; Unknown function Maier et al. (2008),
detected in Silvestrini et al.
gametocytes (2010), Spielmann
et al. (2006)
REX4 PF3D7_0936400 (PFI1760w) iRBC cytoplasm; Unknown function Silvestrini et al. (2010),

detected in Spielmann et al.
gametocytes (2006)
PF14_0758 PF3D7_1478600 iRBC cytoplasm Involved in PfEMP1 Boddey and Cowman
(PfPTP3) trafcking and decreasing (2013), Maier et al.
membrane rigidity (2008)
MAL7P1.171 PF3D7_0730900 Unknown Involved in PfEMP1 Boddey and Cowman
(PfPTP4) trafcking and increasing (2013), Ikadai et al.
membrane rigidity; (2013), Maier et al.
disruption of the gene arrest (2008)
development of
gametocytes at stage I
PF10_0025 PF3D7_1002100 Unknown Involved in PfEMP1 Boddey and Cowman
(PfPTP5) trafcking and increasing (2013), Maier et al.
membrane rigidity (2008)
PF13_0076 PF3D7_1302000 Unknown Involved in PfEMP1 Boddey and Cowman
(PfPTP6) trafcking (2013), Maier et al.
(2008)
HRP2 PF3D7_0831800 (MAL7P1.231) iRBC cytosol Role in haem detoxication Papalexis et al. (2001),
Sullivan et al. (1996)
HRP3 PF3D7_1372200 (MAL13P1.480) Unknown Unknown function Rock et al. (1987)
GARP PF3D7_0113000 (PFA0620c) Unknown Unknown function Maier et al. (2008),
Triglia et al. (1988)
PIESP1 PF3D7_0310400 (PFC0435w) iRBC surface Essential; unknown function Florens et al. (2004),
Maier et al. (2008)
PIESP2/PfE60 PF3D7_0501200 (PFE0060w) iRBC surface or Unknown function Florens et al. (2004),
Maurers clefts Maier et al. (2008),
Vincensini et al.
(2005)
11
(Continued)
12
LSA3 PF3D7_0220000 (PFB0915w) Hepatocyte surface; Unknown function; expressed Aidoo et al. (2000),
unknown in the in the RBC and liver stage; Guerin-Marchand
iRBC liver stage vaccine candidate et al. (1987), Maier
et al. (2008), Moyano
et al. (2007),
Silvestrini et al. (2010)
PfAARP1 PF3D7_1233600 (PFL1620w) iRBC membrane Unknown function Barale et al. (1997b)
PfAARP2 PF3D7_0106700 (PFA0330w) iRBC cytosol Unknown function Barale et al. (1997a)
PfGCN20 PF3D7_1121700 (PF11_0225) iRBC cytosol Unknown function Bozdech et al. (1998)
PfTKL2 PF3D7_1121300 (PF11_0220) IRBC cytosol in Active kinase, also secreted Abdi et al. (2013)
close proximity to beyond the iRBC
membrane
PF07_0087 PF3D7_0721100 Unknown Expressed in trophozoites; Silvestrini et al. (2010)
unknown function
PF11_0508 PF3D7_1149100.1/ Unknown Expressed in trophozoites; Silvestrini et al. (2010)
PF3D7_1149100.2 unknown function
PF13_0275 PF3D7_1353100 Unknown Expressed in trophozoites; Silvestrini et al. (2010)
unknown function
PFA0210c PF3D7_0104200 Unknown Expressed in trophozoites; Silvestrini et al. (2010)
unknown function

PFC0085c PF3D7_0301700 Unknown Expressed in trophozoites; Silvestrini et al. (2010)
unknown function
PFE0050w PF3D7_0501000 Unknown Expressed in trophozoites; Silvestrini et al. (2010)
unknown function
GEXP03 PF3D7_1429600 (PF14_0275) Unknown Expressed in early gametocytes Silvestrini et al. (2010)
GEXP13 PF3D7_0831300 (MAL8P1.205) Unknown Expressed in early gametocytes Silvestrini et al. (2010)
GEXP18 PF3D7_0402400 (PFD0115c) Unknown Expressed in early gametocytes Silvestrini et al. (2010)
GEXP19 PF3D7_0111400 (PFA0550w) Unknown Expressed in early gametocytes Silvestrini et al. (2010)
GEXP22 PF3D7_0935500 (PFI1715w) Unknown Expressed in early gametocytes Silvestrini et al. (2010)
PFA0225w PF3D7_0104400 Unknown Expressed in mature Silvestrini et al. (2010)
gametocytes
MDR5 PF3D7_1339900 (PF13_0218) Unknown Putative ABC transporter Silvestrini et al. (2010)
(MDR family); expressed in
mature gametocytes
PF13_0317 PF3D7_1360000 Unknown Expressed in mature Silvestrini et al. (2010)
gametocytes
FP1 PF3D7_1458000 (PF14_0553) Unknown cysteine proteinase falcipain 1; Silvestrini et al. (2010)
expressed in mature
gametocytes
PFE1615c PF3D7_0532600 Unknown Expressed in gametocytes; Ikadai et al. (2013)
possible role in
gametocytogenesis
PfPI3K PF3D7_0515300 (PFE0765w) Food vacuole; PM/ Type III phosphatidylinositol Vaid et al. (2010)
PVM; iRBC 3-kinase, catalyses
cytosol formation of PI(3)P, PI(3,4)
P2 and PI(3,4,5)P3; found
in multiple locations
including the periphery of
the iRBC; involved in
haemoglobin uptake
Abbreviations: RBC, Red Blood Cell; PV, Parasitophorous Vacuole; PVM, Parasitophorous Vacuolar Membrane; TVN, Tubovesicular Network.
13
Table 2 Protein interactions at the iRBC membrane skeleton

Binding
constants
Interacting proteins Kd (mM) References
MESA/protein 4.1 0.63e1.25 Bennett et al. (1997), Waller et al. (2003)

KAHRP/ankyrin 1.3e8.3 Magowan et al. (2000), Weng et al. (2014)
KAHRP/PfEMP1 0.01e13.0 Oh et al. (2000), Waller et al. (1999, 2002)
KAHRP/actin NQ Kilejian et al. (1991), Oh et al. (2000)
KAHRP/a spectrin 0.0018 Kilejian et al. (1991), Oh et al. (2000),
Pei et al. (2005)
PfEMP1/actin 0.04 Oh et al. (2000)
PfEMP1/a spectrin NQ Oh et al. (2000)
PfEMP3/actin NQ Waller et al. (2007)
PfEMP3/a spectrin 0.07 Pei et al. (2007b), Waller et al. (2007)
Pf332/Actin 0.4 Waller et al. (2010)
PHIST/PfEMP1 w150 Mayer et al. (2012)
PF10_0378 (MEC)/ NQ Kilili and LaCount (2011)
Protein 4.1
SBP1/a spectrin NQ Blisnick et al. (2000)
RESA/b spectrin 0.00088 Pei et al. (2007a)
Kd is the dissociation constant. NQ is not quantied.
lysed and multiple new parasites are released. In general, exported parasite
proteins are directed into the parasites secretory pathway, usually by the
presence of N-terminal signal sequences (Hiller et al., 2004; Marti et al.,
2004); however, in some cases, the presence of a C-terminal transmembrane
domain is also required (Gruring et al., 2012; Saridaki et al., 2009). Once pro-
teins are directed into the parasites endoplasmic reticulum (ER) and enter
the secretory pathway, they are then committed for export into the RBC
by the recognition and cleavage of a signature sequence at the N-terminus
of the protein, termed the Plasmodium EXport ELement (PEXEL) or the
Vacuolar Transport Signal (VTS) (Hiller et al., 2004; Marti et al., 2004).
The proteins are then trafcked into the PV, by a yet unidentied mecha-
nism, and then directed to a transporter in the PVM, termed Plasmodium
translocon of exported proteins (PTEX) (de Koning-Ward et al., 2009),
where they are rst unfolded before being translocated across the PVM
into the RBC cytosol (Gehde et al., 2009; Gr uring et al., 2012), then onto
their nal cellular destination. Several mechanisms for this nal step of the
trafcking process have been proposed and include vesicle transport, trans-
port in soluble complexes and chaperone-mediated transport (K ulzer et al.,
2010; McMillan et al., 2013; Saridaki et al., 2009) (Figure 1).
Table 3 P. falciparum exported proteins containing a PHIST domain

PHIST Gene id PEXEL Comments References
PHISTa
PF3D7_0402000 (PFD0090c) RNLSE Localizes to the PMV; interacts Maier et al. (2008), Parish
with a sub-population of 4.1R; et al. (2013), Sargeant
unknown function et al. (2006)
PF3D7_1478000 (PF14_0752) RNLTE Up-regulated in highly Mok et al. (2007), Sargeant
cytoadherent clones of 3D7 et al. (2006)
PF3D7_1253300 (PFL2565w) RNLAQ Only other PHISTa with Sargeant et al. (2006)
detectable transcript in asexual
3D7 blood-stage parasites
PF3D7_0102000 (PFA0100c) Negative Sargeant et al. (2006)
PF3D7_0424900 (PFD1185w) RNLVQ Sargeant et al. (2006)
PF3D7_0425300 (PFD1210w) Negative Sargeant et al. (2006)
PF3D7_0425400 (PFD1215w) RNLVQ Sargeant et al. (2006)
PF3D7_0601700 (MAL6P1.21) RNLSE Pseudogene Sargeant et al. (2006)
PF3D7_0819500 (MAL8P1.63) RKNIE Sargeant et al. (2006)
ACBP1 PF3D7_1001100.1/ Negative Two annotated genes in the same Frech and Chen (2013),
PF3D7_1001100.2 (PF10_0014/ location, representing different Sargeant et al. (2006)
PF10_0015) isoforms of acyl-CoA-binding
protein 1 (ACBP1)
PF3D7_1001300 (PF10_0017) RTLSE Sargeant et al. (2006)
PF3D7_1100600 (PF11_0012) Negative Pseudogene Sargeant et al. (2006)
PF3D7_1149700 (PF11_0514) Negative Sargeant et al. (2006)
PF3D7_1253100 (PFL2555w) RNLCE Sargeant et al. (2006)
PF3D7_1253900 (PFL2595w) RNLSE Psuedogene Sargeant et al. (2006)
PF3D7_1301100 (MAL13P1.11) RNLSE Pseudogene Sargeant et al. (2006)
15
(Continued)
Table 3 P. falciparum exported proteins containing a PHIST domaindcont'd
16
PF3D7_1301500 (MAL13P1.59) Negative Sargeant et al. (2006)
PF3D7_1372000 (MAL13P1.470) RNLSE Sargeant et al. (2006)
PF3D7_1400900 (PF14_0009) RNLSE Pseudogene Sargeant et al. (2006)
Pfg14.748 PF3D7_1477700 (PF14_0748) Negative Expressed and localized to the PV Eksi et al. (2005), Sargeant
in gametocytes et al. (2006), Silvestrini
et al. (2010)
PF3D7_1479200 (PF14_0763) RNLVQ Sargeant et al. (2006)
Pfg14.744 PF3D7_1477300 (PF14_0744) RSLSE Localized to the iRBC in Eksi et al. (2005), Frech and
gametocytes Chen (2013), Silvestrini
et al. (2010)
Pfg14.745 PF3D7_1477400 (PF14_0745) RSVND Expressed in gametocytes Eksi et al. (2005), Frech and
Chen (2013), Silvestrini
et al. (2010)
GEXP13 PF3D7_0831300 (MAL8P1.205) RKLSE Expressed in gametocytes Frech and Chen (2013),
PHISTb
PfD80 PF3D7_0401800 (PFD0080c) RNLSE Essential; possibly integral Maier et al. (2008), Pease et

membrane protein; localizes al. (2013), Sargeant et al.
to iRBC membrane; highly (2006), Tarr et al. (2014),
phosphorylated Vincensini et al. (2005)
PF3D7_0402100 (PFD0095c) RKLYHE Expressed in trophozoites; Kilili and LaCount (2011),
essential; contains a MEC Maier et al. (2008),
domain binds to IOVs Sargeant et al. (2006),
PF3D7_0424600 (PFD1170c) RILSE Involved in knob formation; Florens et al. (2004), Maier
membrane associated et al. (2008), Sargeant
et al. (2006), Tarr et al.

(2014)
GEXP02 PF3D7_1102500 (PF11_0037) RNLYE Expressed in gametocytes; Maier et al. (2008),
unknown function Sargeant et al. (2006),
PF3D7_1401600 (PF14_0018) RSLYE Involved in iRBC increasing Kilili and LaCount (2011),
membrane rigidity; contains a Maier et al. (2008),
MEC domain Sargeant et al. (2006)
PF3D7_1201000 (PFL0050c) RILSS Membrane associated; two Florens et al. (2004), Maier
PHISTb domains et al. (2008), Sargeant
et al. (2006)
PfG174 PF3D7_0731300 (MAL7P1.174) RILSE Expressed in gametocytes; Maier et al. (2008),
unknown function Sargeant et al. (2006),
Vincensini et al. (2005)
PF3D7_0936900 (PFI1785w) RILSE Up-regulated in women with Sargeant et al. (2006),
pregnancy-associated malaria; Tuikue Ndam et al.
psuedogene (2008)
PF3D7_1252800 (PFL2540w) RILCT Contains a MEC domain Kilili and LaCount (2011),
PF3D7_0601500 (PFF0075c) Negative Contains two MEC domains Kilili and LaCount (2011),
PF3D7_0631100 (PFF1510w) RNLSE Contains two MEC domains; Kilili and LaCount (2011),
binds IOVs Sargeant et al. (2006)
PF3D7_0902700 (PFI0130c) RNLHE Contains a MEC domain; Kilili and LaCount (2011),
pseudogene Sargeant et al. (2006)
PF3D7_0937000 (PFI1790w) Negative Contains a MEC domain; binds Kilili and LaCount (2011),
IOVs Sargeant et al. (2006)
(Continued)
17
18
PF3D7_0201600 (PFB0080c) RNLSS Localizes to iRBC cytosol; Goel et al. (2014), Sargeant
possibly involved in regulation et al. (2006), Tarr et al.
of VAR2CSA expression (2014)
PF3D7_0424800 (PFD1180w) RILST Sargeant et al. (2006)
PF3D7_0532300 (PFE1600w) RNLCE Sargeant et al. (2006)
LyMP PF3D7_0532400 (PFE1605w) RKLCE Localizes to and associates with Oberli et al. (2014),
iRBC membrane skeleton; role Proellocks et al. (2014),
in cytoadhesion Sargeant et al. (2006)
PF3D7_0702100 (MAL7P1.7) RILIE Pseudogene Sargeant et al. (2006)
GEXP09 PF3D7_0831000 (MAL8P1.2) Negative Expressed in gametocytes Sargeant et al. (2006),
PF3D7_1252700 (PFL2535w) RTLFE Localizes to iRBC membrane Sargeant et al. (2006), Tarr
et al. (2014)
GEXP04 PF3D7_1372100 (MAL13P1.475) RSLLG Expressed in gametocytes Sargeant et al. (2006),
PF3D7_1476200 (PF14_0731) Negative Localizes to iRBC membrane Sargeant et al. (2006), Tarr
et al. (2014)
PF3D7_1476300 (PF14_0732) RKLYE Sargeant et al. (2006)

PHISTb + DnaJ
RESA PF3D7_0102200 (PFA0110w) RNLYGE Localizes to the iRBC Maier et al. (2008), Pei
membrane; involved in et al. (2007a), Sargeant et
membrane mechanic and al. (2006), Silva et al.
required for the stabilization of (2005)
spectrin and the iRBC
membrane under heat stress
RESA-2 PF3D7_1149500 (PF11_0512) RNLYG Up-regulated in Clinical isolates; Daily et al. (2005), Maier et
truncation, does not express al. (2008), Sargeant et al.

DnaJ domain; unknown (2006)
function
RESA-like PF3D7_1149200 (PF11_0509) RNLYCE Essential; expressed in Maier et al. (2008),
trophozoites Sargeant et al. (2006),
RESA-like PF3D7_0201700 (PFB0085c) RQLSE Expressed in trophozoites; role in Maier et al. (2008),
increasing membrane rigidity Sargeant et al. (2006),
RESA-like PF3D7_0220100 (PFB0920w) Negative Involved in increasing iRBC Maier et al. (2008),
membrane rigidity and Sargeant et al. (2006)
cytoadhesion
RESA-like PF3D7_1038800 (PF10_0378) RKVCE Contains a MEC domain; binds Kilili and LaCount (2011),
IOVs and 4.1R Maier et al. (2008),
RESA-like PF3D7_1201100 (PFL0055c) RYLCE Contains a MEC domain Kilili and LaCount (2011),
PHISTc
PF3D7_0424000 (PFD1140w) RNLSE Unknown function Maier et al. (2008),
GEXP05 PF3D7_0936600 (PFI1770w) RKLSE Originally described as PHISTb Frech and Chen (2013),
now reclassied as PHISTc; Sargeant et al. (2006),
expressed in gametocytes Silvestrini et al. (2010)
PF3D7_0936800 (PFI1780w) KSLSD Expressed in trophozoites; Maier et al. (2008), Mayer
essential; role in anchoring of et al. (2012), Sargeant
PfEMP1 et al. (2006), Silvestrini
et al. (2010)
19
(Continued)
20
PfPTP2 PF3D7_0731100 (MAL7P1.172) RNLGE Localized to the Maurers clefts; Maier et al. (2008), Regev-
GEXP11 role in trafcking of PfEMP1 Rudzki et al. (2013),
from the clefts to the iRBC Sargeant et al. (2006),
membrane; expressed in Silvestrini et al. (2010)
gametocytes; involved in cell
ecell communication via
exosomes
PF3D7_0801000 (PF08_0137) Negative Expressed in trophozoites; Akinyi et al. (2012), Florens
membrane associated; et al. (2004), Sargeant et
homologue in Pv localizes to al. (2006), Silvestrini et
Schuffners dots al. (2010)
LSAP2 PF3D7_0202100 (PFB0105c) RKFAE Liver-stage protein localized to Sargeant et al. (2006), Siau
the periphery liver-stage et al. (2008)
parasites; minimal expression in
blood-stage schizonts
GEXP20 PF3D7_0219700 (PFB0900c) Negative Expressed in gametocytes Sargeant et al. (2006),
PF3D7_0219800 (PFB0905c) Negative Sargeant et al. (2006)
PF3D7_0532200 (PFE1595c) Negative Sargeant et al. (2006)
PF3D7_0830600 (MAL8P1.4) RILYE Sargeant et al. (2006)
PF3D7_1001700 (PF10_0021) KILCE Sargeant et al. (2006)

PF3D7_1016600 (PF10_0161a) Negative Sargeant et al. (2006)
PF3D7_1016800 (PF10_0163) RVLTE Sargeant et al. (2006)
GEXP12 PF3D7_1148700 (PF11_0503) RTLAS Expressed in gametocytes Sargeant et al. (2006),
PF3D7_1200900 (PFL0045c) KMLCE Sargeant et al. (2006)
Table 4 P. falciparum exported proteins containing a DnaJ domain

ATP
hydrolysis
DnaJ Gene id motif Comments References
Type II
PFA660 PF3D7_0113700 (PFA0660w) HDP J-Dots; interacts with PfHsp70x; possibly Botha et al. (2007), K ulzer
involved in trafcking of PfEMP1 et al. (2010, 2012), Sargeant
et al. (2006)
PFB90 PF3D7_0201800 (PFB0090c) HDP Possible J-Dots; expressed in trophozoites Botha et al. (2007), Sargeant
et al. (2006), Silvestrini et al.
(2010)
PFE55 PF3D7_0501100.1 (PFE0055c) HDP J-Dots; interacts with PfHsp70x; possibly Botha et al. (2007), K ulzer
involved in trafcking of PfEMP1 et al. (2010, 2012), Sargeant
et al. (2006)
Type III
PF3D7_0220100 (PFB0920w) HDP RESA-like (Table 3) Botha et al. (2007), Sargeant
et al. (2006)
PF3D7_1038800 (PF10_0378) HDP Contains a MEC domain; RESA-like Botha et al. (2007), Kilili and
(Table 3) LaCount (2011), Sargeant
et al. (2006)
PF3D7_1039100 (PF10_0381) HDP Contains a MEC domain; involved in Botha et al. (2007), Kilili and
knob formation LaCount (2011), Maier
et al. (2008), Sargeant et al.
(2006)
(Continued)
21
22
Table 4 P. falciparum exported proteins containing a DnaJ domaindcont'd
ATP
hydrolysis
DnaJ Gene id motif Comments References
PF3D7_1149600 (PF11_0513) HDP Unknown function Botha et al. (2007), Maier et al.
(2008), Sargeant et al.
(2006)
PF3D7_1201100 (PFL0055c) HDP Contains a MEC domain; RESA-like Botha et al. (2007), Kilili and
(Table 3) LaCount (2011), Sargeant
et al. (2006)
Type IV
PF3D7_0102200 (PFA0110w) YPY RESA (Table 3) Botha et al. (2007), Sargeant
et al. (2006)
GEXP06 PF3D7_0114000 (PFA0675w) YPK RESA-like but does not contain a PHIST Botha et al. (2007), Kilili and
domain; contains a MEC domain; LaCount (2011), Sargeant
expressed in gametocytes et al. (2006), Silvestrini et al.
(2010)

PF3D7_0201700 (PFB0085c) NPE RESA-like (Table 3) Botha et al. (2007), Sargeant
et al. (2006)
PF3D7_0220400 (PFB0925w) HPE Contains a MEC domain; unknown Botha et al. (2007), Kilili and
function LaCount (2011), Maier
(2006)
PF3D7_0500800 (PFE0040c) NPH MESA; binds to protein 4.1R Bennett et al. (1997), Black
et al. (2008), Botha et al.
(2007), Sargeant et al.
(2006), Waller et al. (2003)
PF3D7_1102200 (PF11_0034) FLD Contains a MEC domain; essential Botha et al. (2007), Kilili and
LaCount (2011), Maier
(2006)
PF3D7_1149200 (PF11_0509) YPP RESA-like (Table 3) Botha et al. (2007), Sargeant
et al. (2006)
PF3D7_1149500 (PF11_0512) YPI RESA-2 (Table 3) Botha et al. (2007), Sargeant
et al. (2006)
GECO PF3D7_1253000 (PFL2550w) YPK Gametocyte erythrocyte cytosolic protein; Botha et al. (2007), Maier et al.
unknown function (2008), Sargeant et al.
(2006)
PF3D7_1401100 (PF14_0013) YPK Botha et al. (2007), Sargeant
et al. (2006)
23
24
Table 5 FIKK kinases predicted or shown to be exported into iRBCs
FIKK Gene id Comments References
FIKK1 PF3D7_0102600 (PFA0130c) Low level of transcription in asexual stages; Nunes et al. (2007), Schneider
unknown function and Mercereau-Puijalon
(2005), Ward et al. (2004)
FIKK3 PF3D7_0301200 (PFC0060c) Unknown function Schneider and Mercereau-
Puijalon (2005), Ward et al.
(2004)
FIKK4.1 PF3D7_0424500 (PFD1165w) Localized to the Maurers clefts; IP of Nunes et al. (2007), Schneider
FIKK4.1 shown to have kinase activity; and Mercereau-Puijalon
transcribed in rings and schizonts; (2005), Ward et al. (2004)
unknown function
FIKK4.2 PF3D7_0424700 (PFD1175w) Transcribed in rings; localized to distinct Kats et al. (2014), Nunes et al.
(R45) dots in the iRBC; shown to have kinase (2007), Schneider and
activity; functions in regulating knob Mercereau-Puijalon (2005),
structure and in turn adhesion; has a role Ward et al. (2004)
in iRBC deformability
FIKK5 PF3D7_0500900 (PFE0045c) Transcribed in schizonts; unknown Nunes et al. (2007), Schneider
function and Mercereau-Puijalon
(2005), Ward et al. (2004)

FIKK7.1 PF3D7_0726200 (MAL7P1.144) Low level of transcription in asexual stages; Nunes et al. (2007, 2010),
has a role in iRBC deformability and Schneider and Mercereau-
suggested to phosphorylate a 250 kDa Puijalon (2005), Ward et al.
protein in schizonts (2004)
FIKK7.2 PF3D7_0731400 (MAL7P1.175) Internal stop codon within sub-domain Nunes et al. (2007), Schneider
VIII in the kinase domain; the and Mercereau-Puijalon
truncation is transcribed in the schizont (2005), Ward et al. (2004)
stage
FIKK9.1 PF3D7_0902000 (PFI0095c) Transcribed in rings and schizonts; Nunes et al. (2007), Schneider
(2005), Ward et al. (2004)
FIKK9.3 PF3D7_0902200 (PFI0105c) Localized to Maurers clefts; Nunes et al. (2007), Schneider
phosphorylated on serine 575; and Mercereau-Puijalon
transcribed in rings; unknown function (2005), Solyakov et al.
(2011), Ward et al. (2004)
FIKK9.4 PF3D7_0902300 (PFI0110c) Transcribed in rings and schizonts; Nunes et al. (2007), Schneider
(2005), Ward et al. (2004)
FIKK9.5 PF3D7_0902400 (PFI0115c) Low level of transcription in asexual stages; Nunes et al. (2007), Schneider
(2005), Ward et al. (2004)
FIKK9.6 PF3D7_0902500 (PFI0120c) Localized to Maurers clefts; transcribed in Nunes et al. (2007), Schneider
rings and schizonts; unknown function and Mercereau-Puijalon
(2005), Ward et al. (2004)
FIKK9.7 PF3D7_0902600 (PFI0125c) Transcribed in rings; unknown function Nunes et al. (2007), Schneider
and Mercereau-Puijalon
(2005), Ward et al. (2004)
FIKK10.1 PF3D7_1016400 (PF10_0160) High level of transcription in ring stages; Nunes et al. (2007), Schneider
(2005), Ward et al. (2004)
(Continued)
25
Table 5 FIKK kinases predicted or shown to be exported into iRBCsdcont'd
26
FIKK Gene id Comments References
FIKK10.2 PF3D7_1039000 (PF10_0380) Phosphorylated on serine 355; transcribed Nunes et al. (2007), Schneider
in rings; unknown function and Mercereau-Puijalon
(2005), Solyakov et al.
(2011), Ward et al. (2004)
FIKK11 PF3D7_1149300 (PF11_0510) Transcribed in schizonts; unknown Nunes et al. (2007), Schneider
function and Mercereau-Puijalon
(2005), Ward et al. (2004)
FIKK12 PF3D7_1200800 (PFL0040c) Transcribed in rings; localized to Maurers Nunes et al. (2007, 2010),
clefts and iRBC membrane; IP of Schneider and Mercereau-
FIKK12 shown to have kinase activity; Puijalon (2005), Ward et al.
has a role in iRBC deformability; (2004)
suggested to phosphorylate a 80 kDa
protein in trophozoites
FIKK13 PF3D7_1371700 (MAL13P1.109) Unknown function Schneider and Mercereau-
Puijalon (2005)
FIKK14 PF3D7_1476400 (PF14_0733/ Internal stop codon following sub-domain Nunes et al. (2007), Schneider
PF14_0734) III within kinase domain; truncation has and Mercereau-Puijalon
low level of transcription in asexual (2005), Ward et al. (2004)
stages; unknown function

iRBC
Figure 1 Schematic representation of putative protein export pathways in P. falciparum-

infected RBCs. Black dots represent a generic exported protein, blue (light grey in print
versions) dots a chaperone and green (grey in print versions) cylinders are the PTEX
translocon. Maurers clefts (MC) and the parasitophorous vacuole (PV) surrounding the
parasite are shown. There are multiple potential pathways that parasite proteins can
take en route to their nal destination in the infected red blood cell (iRBC). One potential
pathway (A) involves a chaperone complex (such as J-Dots) to transport proteins in the
iRBC cytosol. Some proteins are trafcked in soluble complexes to MCs (B) then onto the
iRBC membrane which could be via a chaperone complex (C) or tethers or actin la-
ments (represented by the wavy lines) (D). Exported proteins can also be transported
in the iRBC cytosol in vesicles (E).
2.1 The PEXEL motif

The exact mechanisms and in turn exact reasons by which the parasite attains
such a high level of protein export remain poorly understood. Recent
studies have revealed that the export of many parasite proteins across the
PVM is mediated by a pentameric sequence (RxLxE/D/Q) known as the
Plasmodium Export Element (PEXEL) or, less commonly, the VTS (Hiller
et al., 2004; Marti et al., 2004). Interestingly, in the case of what is probably
the major virulence-determining exported protein in P. falciparum, P. falcip-
arum erythrocyte protein 1 (PfEMP1), the arginine residue (R) at position 1
of the canonical PEXEL sequence is replaced by lysine (K) and there is
debate on whether or not this lysine PEXEL is in fact functional. While
the amino acids at positions 2 and 4 of the PEXEL are less conserved,
they are in general non-polar residues (Marti et al., 2004). It has recently
been shown that PEXEL cleavage and subsequent acetylation are required
for the trafcking of RESA-like proteins, which contain a relaxed PEXEL
of RxLxxE (Boddey et al., 2013). In general, the trafcking of parasite pro-
teins into the RBC is thought to be a multi-step process. The initial steps
of export, which involve PEXEL recognition and processing, are currently
being investigated but two alternative mechanisms of PEXEL function
seem to be emerging. The rst suggests that the PEXEL is cleaved in
the ER by a resident protease, plasmepsin V (Boddey et al., 2010; Russo
et al., 2010), followed by trafcking of the processed protein to the PV
via a classical vesicle-mediated pathway. The second involves the binding
of phosphatidylinositol-3-phosphate PI(3)P to the PEXEL which mediates
export independent of plasmepsin V cleavage (Bhattacharjee et al., 2012).
In either case, once the proteins are in the PV, the next step involves
their translocation across the PVM and into the RBC (Ansorge et al.,
1996; Boddey et al., 2009; Cesbron-Delauw et al., 2008; Charpian and
Przyborski, 2008).
2.1.1 Plasmepsin V-mediated PEXEL function

Trafcking of PEXELated proteins is blocked by treatment of parasites with
brefeldin A, which blocks protein transport from the ER to the Golgi (Chang
et al., 2008). N-terminal processing of the PEXEL still occurs in the pres-
ence of brefeldin A, indicating that processing occurs rapidly within the
ER (Chang et al., 2008). Cleavage of the PEXEL by plasmepsin V in the
ER occurs immediately after the conserved leucine residue at position 3
of the sequence and is an important step in the export process (Boddey
et al., 2010, 2013; Russo et al., 2010; Sleebs et al., 2014). Following cleav-
age, the N-terminal end of the processed protein is then acetylated, which is
thought to direct the mature protein into the PV (Boddey et al., 2009;
Chang et al., 2008). Some PEXELs have a lysine residue at the start of
the PEXEL instead of an arginine residue, such as those found in PfEMP1.
These lysine PEXELs are not cleaved by plasmepsin V but these uncleaved
proteins are still exported. However, replacing the arginine with lysine in
the PEXEL of the knob-associated histidine-rich protein (KAHRP), a
well-characterized-exported protein, cleavage was blocked and the protein
was no longer exported (Boddey et al., 2013). The export of proteins with
the lysine PEXEL, which are not processed by plasmepsin V, such as
PfEMP1, would be consistent with a view that PEXELs that start with a
lysine are trafcked via a plasmepsin V-independent pathway. Interestingly,
inhibition of plasmepsin V activity also showed a decrease in the export of
PFEMP1 (Sleebs et al., 2014). This suggests that while it may not be
directly involved in PfEMP1 trafcking, plasmesin V is critical for export
in general and that the trafcking pathways in P. falciparum are intrinsically
linked. What is clear from these studies is that proteins containing a classical
PEXEL are processed to produce a mature protein which is acylated, and
this maturation is required for these proteins to be correctly exported
into the RBC (Boddey et al., 2009, 2010, 2013; Chang et al., 2008; Russo
et al., 2010).
2.1.2 PI(3)P-mediated PEXEL function

An interaction between PEXEL and PI(3)P has also been suggested to be
involved in parasite protein export (Bhattacharjee et al., 2012). However,
there is some uncertainty around the exact location of PI(3)P in the parasite
(Bhattacharjee et al., 2012; Tawk et al., 2010). PI(3)P has been localized to
both the food vacuole and the apicoplast in the iRBC (Tawk et al., 2010).
Localization at the food vacuole is also consistent with the presence of a PI3
kinase that is required to produce PI(3)P at the same location (Vaid et al.,
2010). In contrast, another study using a similar method localized PI(3)P
to the ER (Bhattacharjee et al., 2012). The surprising nding of PI(3)P in
the ER raises the question of its source as no PI3 kinase has ever been local-
ized to the ER in P. falciparum. In other systems PI(3)P is present in the ER,
but it is due to the import of a PI3 kinase (PI3K) to the ER (Matsunaga et al.,
2010). PI(3)P in other eukaryotes is involved in trafcking proteins to endo-
somes, in retrograde trafcking and in autophagy, but not in protein traf-
cking from the ER. Thus, the presence of a stable population of PI(3)P
in the ER which is involved in protein trafcking would be unique to
P. falciparum, but additional evidence is required before one can be condent
of this unusual nding. This study also showed that although the PEXEL
was able to bind PI(3)P, it was not required for transport once the PEXEL
motif had been cleaved. Furthermore, the necessity of PI(3)P binding for
export is uncertain as there is evidence indicating that the addition of a
sequence that resembles a cleaved PEXEL to non-exported proteins, to
which PI(3)P would be unable to bind, is sufcient to direct proteins for
export. This is more indicative that the cleavage of the PEXEL to the mature
signal sequence is required for export and not for PI(3)P binding (Gr uring
et al., 2012). Interestingly, PEXELs which contain a lysine at the rst
position (and therefore not cleaved by plasmepsin V were also able to
bind PI(3)P (Boddey et al., 2013), raising the possibility that PI(3)P is
used solely for these lysine PEXELs. While it has been shown that
lysine-rich motifs are able to bind PI(3)P (Six and Dennis, 2003), the role of
this in Plasmodium protein trafcking, while very interesting, has not yet
been demonstrated convincingly.
2.2 PEXEL-negative exported proteins

Not all parasite proteins that are exported into the iRBC contain a PEXEL
motif (Gr uring et al., 2012; Haase et al., 2009; Saridaki et al., 2009;
Spielmann et al., 2006) suggesting that there is more than one export
pathway. Collectively, these proteins have been termed PEXEL-negative
exported proteins (PNEPs) and appear to either associate with parasite-
derived organelles known as Maurers clefts (MCs) (Figure 1) either perma-
nently (such as skeleton-binding protein 1 (SBP1) or ring-exported protein 1
(REX1) (Saridaki et al., 2009; Spielmann et al., 2006)) or transiently (such as
PfEMP1 or SURFIN (McMillan et al., 2013; Zhu et al., 2013)). PNEPs can
be further classied into at least four separate classes based on the protein fea-
tures required for export: (i) transmembrane PNEPs (TM-PNEPs) (Gr uring
et al., 2012; Haase et al., 2009; Saridaki et al., 2009; Spielmann et al., 2006);
(ii) soluble PNEPs (S-PNEPs) (Hanssen et al., 2008a; K ulzer et al., 2012;
Spielmann et al., 2006); (iii) PNEPs containing a classical signal peptide
(SP-PNEPs) (Heiber et al., 2013) and (iv) PNEPs containing a PEXEL-
like (non-functional) motif with a lysine residue at position 1 of the
sequence (K-PNEPs) (Boddey et al., 2013; Boddey and Cowman, 2013).
The TM-PNEPs and S-PNEPs both have a sequence within the N-terminal
domain of the protein that resembles a processed PEXEL, which could indi-
cate a similarity in the export process (Gr uring et al., 2012; Haase et al.,
2009). In fact, swapping the N-terminal sequence of REX2 (a TM-
PNEP) with a processed PEXEL still results in protein export; and traf-
cking of both proteins is blocked by brefeldin A, indicating similar passage
through the ER. Taken together, this suggests some overlap in these two
export pathways (Gr uring et al., 2012); however, there are some clear
differences. In the case of REX2 for example, where processing at the
N-terminus by a yet unidentied protease, results in a mature protein begin-
ning with the residues LAE, classical PEXEL-containing proteins are cleaved
after the leucine residue (Boddey et al., 2013; Haase et al., 2009). In addi-
tion, the N-terminus of TM-PNEPs are not acylated as are mature
PEXEL-containing proteins (Chang et al., 2008; Haase et al., 2009), clearly
indicating differences in the export of these groups. While PNEPs have
some similarities with classical PEXEL proteins, each group has unique fea-
tures that are required for export. TM-PNEPs rely on the presence of a
transmembrane domain for trafcking (Gr uring et al., 2012; Saridaki et al.,
2009; Zhu et al., 2013). In fact, for SBP-1 at least, the TM, and not the
N-terminus, is the critical region that is required for export of this protein
(Saridaki et al., 2009). Even though TM-PNEPs contain a TM, there is ev-
idence that this class of proteins, once exported into the RBC, are trafcked
in soluble complexes to MCs (Gr uring et al., 2012; Saridaki et al., 2009).
S-PNEPs do not contain a TM domain, so their trafcking is clearly quite
different. While some of the S-PNEPs, such as REX1 and MAHRP2, are
located at MCs (Dixon et al., 2008; Pachlatko et al., 2010), not all of the
S-PNEPs are MC-resident proteins. The MC-resident S-PNEPs such as
REX1 are trafcked to the MCs in soluble complexes, as are the TM-
PNEPs, indicating a possible overlap in the later stages of protein trafcking
(Dixon et al., 2008). In contrast, PfHsp70x, an S-PNEP, does not associate
with MCs (K ulzer et al., 2012) and therefore the trafcking of this protein
within the iRBC could be different to that of other PNEPs like REX1.
PfHsp70x may in fact complex with DnaJ type II co-chaperones that are
exported via the PEXEL pathway. While there is some suggestion that
PfHsp70x partially co-localizes with MCs (Grover et al., 2013), this could
be more indicative of the function of this chaperone in the trafcking of
other proteins within the iRBC cytosol, possibly even to the MCs (K ulzer
et al., 2012), rather than PfHsp70x being a resident MC protein. Recently,
an additional 13 new PNEPs have been identied and include some of the
members of the merozoite surface protein 7 (MSP7)-related proteins
(MSRP), that have a classical signal peptide with most localizing at MCs
(Heiber et al., 2013), thus adding to the complexity of this group of
exported proteins. The recent discovery of a new export signal in the rodent
malaria parasite, Plasmodium yoelii (Siau et al., 2014), again adds to the pos-
sibility of more proteins being identied further expanding the PNEP
repertoire.
K-PNEPs, which contain a PEXEL-like motif that have a lysine instead
of arginine residue at position 1 of the sequence were originally considered
to be PEXELated proteins (Bhattacharjee et al., 2012; Hiller et al., 2004;
Marti et al., 2004); however, since a lysine at position 1 in the PEXEL blocks
processing by plasmepsin V (Boddey et al., 2013), and by denition PEXE-
Lated proteins are cleaved and acetylated (Boddey et al., 2010, 2013; Chang
et al., 2008; Hiller et al., 2004; Marti et al., 2004; Russo et al., 2010), they
are in fact PNEPs. It has been suggested that PI(3)P can bind this PEXEL-
like motif in K-PNEPs which could represent an alternative mechanism for
export of these proteins (Bhattacharjee et al., 2012).
2.3 The role of PTEX

Translocation of parasite proteins across the PVM is a critical step in their
export into the iRBC and, to date, only one putative transporter which
may mediate this process has been identied; the Plasmodium translocon of
exported proteins (PTEX) (de Koning-Ward et al., 2009). This protein
complex, localized at the PVM, contains the ATPase Hsp101 that is likely
to be the chaperone responsible for unfolding parasite proteins prior to their
export across the PVM (Gehde et al., 2009; Gr uring et al., 2012). The pres-
ence of an ATPase is also consistent with an earlier study that highlighted the
requirement for ATP in the translocation of proteins across the PVM
(Ansorge et al., 1996). In addition, preventing proteins from becoming
unfolded prior to their translocation across the PVM results in their accumu-
lation in close proximity to the PTEX on the inner (parasite) side of the
PVM (Riglar et al., 2013). The PTEX also contains thioredoxin (Trx2),
exported protein 2 (EXP2) and two hypothetical proteins, PTEX150 and
PTEX88 (de Koning-Ward et al., 2009). At present, there is no information
available on the structure of the PTEX, but one model suggests that EXP2
forms a pore in the PVM and interacts with PTEX150 which in turn binds
to Hsp101 (Bullen et al., 2012; de Koning-Ward et al., 2009). The core
components of the complex, PTEX150, Hsp101 and EXP2, have been
shown to be expressed in the later stages of the parasites RBC life cycle
and are present within the apical end of merozoites, specically in dense
granules (Bullen et al., 2012; de Koning-Ward et al., 2009). This timing
of expression and localization of the core PTEX components could indicate
that the complex is preformed in very late-stage parasites (schizonts), ready
to insert in the newly formed PVM as soon as merozoites (released from
rupturing schizonts) invade a new RBC (Bullen et al., 2012). Protein export
in P. falciparum would be required very soon after invasion, so premade
PTEX is consistent with that requirement. The individual core components
of PTEX associate into homo-oligomers before combining into the nal
complex of 1230 kDa (Bullen et al., 2012), which, when imaged by high-
resolution microscopy, forms discreet foci at the PMV (Riglar et al.,
2013). Based on the size of the PTEX complex and the apparent size of
the foci, it has been suggested that these foci are not a single translocon,
but a concentration of numerous translocons located in distinct export
zones; an arrangement analogous to that of the mammalian mitochondrial
TOM complex (Riglar et al., 2013; Wurm et al., 2011). RESA, a protein
exported very early after parasite invasion, has been shown straddling the
PTEX, with the protein detected simultaneously inside the PV and in the
RBC cytosol, an appearance highly suggestive of the export process (Riglar
et al., 2013).
The core components of PTEX, PTEX150, Hsp101 and EXP2, have
been shown by two separate groups to be essential for the function of
this complex, at least in the mouse malaria Plasmodium berghei (Matthews
et al., 2013; Matz et al., 2013). Two independent knockdown approaches
of the PTEX core components, PTEX150 (Elsworth et al., 2014) and
HSP101 (Beck et al., 2014), have conrmed the role of the PTEX in
export of proteins into the iRBC. In both cases, protein export was
inhibited in the absence of a functional PTEX, and interestingly this reduc-
tion was seen for PEXELated proteins and PNEPs indicating that the
PTEX is critical for the export of a wide range of proteins (Beck et al.,
2014; Elsworth et al., 2014). The presence and the role of the two accessory
proteins, PTEX88 and Trx2, in the function of PTEX is the subject of
continuing debate. All ve components have been identied in immuno-
precipitation experiments implying that they do all interact in one complex
(de Koning-Ward et al., 2009; Matthews et al., 2013); however, at present
only PTEX88 has been localized to the PVM or co-localized with the core
PTEX components (Matthews et al., 2013; Matz et al., 2013). One study
using over-expression of GFP-tagged Trx2 showed a small proportion of
the protein localizing at the PVM (Kehr et al., 2010). Taken together
with recent work that has determined that trx2 can be deleted from the par-
asites genome without loss of parasite viability suggests a minor role for
Trx2 in the PTEX complex (Matthews et al., 2013; Matz et al., 2013).
Trx2 knockout parasites do show growth defects (Matthews et al., 2013;
Matz et al., 2013) but this could be explained by a loss of redox capacity
(Matthews et al., 2013; Matz et al., 2013). Deletion of PTEX88 gives
rise to parasites that show a severe growth defect suggesting that while
PTEX88 is not essential for parasite survival, it plays a critical role in the
function of the PTEX (Matz et al., 2013).
2.4 Protein trafcking within iRBCs

Once parasite proteins are exported beyond the PVM and reach the RBC
cytosol, further alternative trafcking pathways are required to move pro-
teins to their nal destination in the iRBC (Figure 1). These pathways are
similar to methods of protein trafcking seen in other eukaryotes, including
the use of vesicles, chaperones and even a potential Golgi-like organelle
(Cooke et al., 2004a).
2.4.1 Vesicle-mediated trafcking

Observations of movement of parasite proteins in iRBCs via vesicles have
largely come from studies of PfEMP1. To date, two distinct populations
of vesicles in iRBCs have been identied; 25 nm vesicle-like structures
(VLS) (Hanssen et al., 2008b; Kriek et al., 2003; Wickert et al., 2003b)
and larger 80 nm electron-dense vesicles (EDV) (Hanssen et al., 2010a;
McMillan et al., 2013; Taraschi et al., 2001; Trelka et al., 2000). Vesicles
are usually observed in the later stages of parasites development, from tro-
phozoites onwards, and are distinct from the classical coated vesicles that
can be found within the parasite itself at any stage of development (Hanssen
et al., 2008b). Interestingly, both types of vesicle have been shown to fuse
with the iRBC membrane suggesting that they are involved in trafcking
their contents to the RBC surface (Hanssen et al., 2010a,b, 2008b; Kriek
et al., 2003; McMillan et al., 2013; Taraschi et al., 2001; Trelka et al.,
2000; Wickert et al., 2003b). Both vesicle types appear in iRBCs after
MCs have been formed and neither type of vesicle are recognized by anti-
bodies against any currently known resident MC protein, indicating that
they are not involved in either the formation of, or trafcking proteins to,
MCs (Hanssen et al., 2010a, 2008b; McMillan et al., 2013).
EDVs, however, do contain PfEMP1 and have therefore been suggested
to play a role in the transport of PfEMP1 to the iRBC surface (Hanssen
et al., 2010a; McMillan et al., 2013; Taraschi et al., 2001; Trelka et al.,
2000). Furthermore, EDVs fuse to the iRBC membrane and appear to
form a dense cup-like shape which may represent precursors of knobs; the
structure to which PfEMP1 attaches (Hanssen et al., 2010a; Taraschi
et al., 2001; Trelka et al., 2000). In addition, EDVs also contain PfEMP3
(Taraschi et al., 2001; Trelka et al., 2000) suggesting their involvement in
trafcking of other proteins in addition to PfEMP1 from MCs to the
iRBC membrane. In contrast, while it has been suggested that VLS are
also involved in trafcking PfEMP1 in iRBCs (Kriek et al., 2003; Wickert
et al., 2003b), there is currently no convincing evidence to support or
conrm this (McMillan et al., 2013).
2.4.2 Chaperones
Molecular chaperones such as the heat shock proteins (Hsps) are required in
a multitude of biological systems for correct folding of proteins. Members of
the Hsp70 and Hsp90 families facilitate the assembly of proteins into higher
order complexes, the unfolding of protein structures and the translocation
of protein across membranes (Bukau and Horwich, 1998; Cheetham and
Caplan, 1998; Lemmon, 2001). All of these proposed functions are likely to
be required by P. falciparum to export its diverse repertoire of proteins into
the RBC and facilitate their correct association with the iRBC cytoskeleton
or their exposure on the iRBC surface. The function of Hsp70 is facilitated
by the co-chaperone Hsp40, also referred to as a DnaJ protein. A sequence-
based classication system for DnaJ proteins has been suggested (Botha
et al., 2007; Cheetham and Caplan, 1998), with only the DnaJ type I and
type II containing all of the required elements necessary to promote
Hsp70-mediated ATP hydrolysis. DnaJ proteins are dened by the presence
of a conserved J domain that comprises four helices and a conserved cata-
lytic triad (HDP), located between the second and third helices (Botha
et al., 2007; Cheetham and Caplan, 1998; Walsh et al., 2004). The J
domain, and in particular the HDP motif, is required for the interaction be-
tween DnaJ proteins and the Hsp70 ATPase domain. This interaction stim-
ulates the hydrolysis of ATP which in turn promotes the interaction of
Hsp70 with its substrate complex (Bukau and Horwich, 1998; McCarty
et al., 1995). The interaction between a DnaJ and Hsp70 has been shown
to occur at least in those found in the cytosol of parasites (Pesce et al.,
2008) and it has been shown that some proteins, such as those containing
a transmembrane domain, are trafcked within the iRBC to MCs in soluble
complexes (Gr uring et al., 2012; Saridaki et al., 2009). This process is likely
to involve molecular chaperones to help stabilize the proteins during
trafcking.
While there are no type I DnaJ proteins shown or predicted to be
exported into the iRBC, bioinformatic analysis does predict that four
type II, four type III and 11 type IV DnaJs are likely to be exported into
the iRBC (Botha et al., 2007). Although this suggests that these chaperones
are likely to be involved in protein trafcking, the type III and IV DnaJs
lack many of the required motifs to function as chaperones and probably
have other roles within the iRBC (Botha et al., 2007). This leaves the
type II DnaJs as the most likely members of this group to be involved in
trafcking within the iRBC. The presence of an exported Hsp70
(PfHsp70x), which interacts with at least two of the type II exported DnaJs
(Kulzer et al., 2012), is highly suggestive of their involvement in protein
trafcking. Whether their role is only to trafc proteins from the PVM
to MCs or to multiple locations throughout the iRBC remains to be deter-
mined, although there is some evidence to suggest a role in trafcking of
PfEMP1 (K ulzer et al., 2012). These type II DnaJs along with Hsp70x
are found only in P. falciparum which is consistent with the suggestion
that they are present for the sole purpose of trafcking P. falciparum-specic
proteins in iRBCs such as PfEMP1 (Botha et al., 2007; K ulzer et al., 2010,
2012).
2.4.3 MCs: an external Golgi?

MCs are parasite-derived organelles present in the cytosol of RBCs infected
with P. falciparum. A number of proteins transiently associate with MCs en
route to the iRBC membrane skeleton (Glenister et al., 2009; Hodder
et al., 2009; Nilsson et al., 2012; Waterkeyn et al., 2000) or iRBC surface
(Wickert et al., 2003b); a role not dissimilar to that of the Golgi in eukaryotic
cells. Protein trafcking between the ER and the Golgi in eukaryotic cells
involves the coat proteins (COP) I and II. Orthologues of COPII, Sec31p
and Sec23p, have been identied in MCs (Adisa et al., 2001; Wickert
et al., 2003a), consistent with a view that MCs are involved in Golgi-like traf-
cking pathways. The presence of vesicle trafcking to and/or from MCs is
also consistent with the presence of P. falciparum N-ethylmaleimide-sensitive
factor (PfNSF), a protein involved in vesicle fusion, in iRBCs, possibly
associated with MCs (Hayashi et al., 2001).
MCs are formed early in the parasites RBC lifecycle and can be visual-
ized by 2 h after invasion. Initially, MCs appear to be highly mobile struc-
tures in the iRBC cytosol but become stationary in the later stages of the
parasites development, at about 22 h post invasion (Gr uing et al., 2011;
McMillan et al., 2013; Spycher et al., 2006). This loss of mobility of MCs
in mature-stage parasites has been suggested to be either due to the forma-
tion of tethers (membranous structures that join MCs to the iRBC mem-
brane skeleton) (Hanssen et al., 2008b) or by direct protein interactions
between MCs and the iRBC membrane skeleton involving either novel
actin-like laments (Cyrklaff et al., 2011) or resident MC proteins such as
SBP1 (Blisnick et al., 2000; Cooke et al., 2006). The role that these attach-
ments play in the trafcking of proteins from MCs is not yet known; how-
ever, movement of PfEMP1 from MCs to the iRBC membrane is not
blocked by cytochalasin D, suggesting that if actin is involved, it is not the
only method by which trafcking occurs (McMillan et al., 2013). While
the association of MCs with the iRBC membrane was not altered by this
cytochalasin D treatment, a separate study (using 10 times the concentration
of cytochalasin D) revealed an increased movement of about one-third of
the MCs (Kilian et al., 2013) indicating actin laments do have a role in
the anchoring of MCs to the RBC membrane skeleton; however, it is un-
likely that this is the only approach.
It was previously thought that proteins became associated with MCs

immediately after budding off the PVM (Spycher et al., 2006). More recent
evidence, however, suggests that MCs are formed much earlier, long before
the export of many of the MC-resident proteins and further that the number
of MCs does not signicantly increase from the ring to trophozoite stages
(Gruing et al., 2011). MCs are present earlier than many of the cleft-resident
proteins (2e4 h post invasion) and, not surprisingly, it has recently been
shown that resident proteins are in fact continually trafcked to mature
MCs within the iRBC (Gr uing et al., 2011; McMillan et al., 2013). The
mechanism by which this occurs is yet to be elucidated. As with the
Hsp70x/DnaJ type II complex, MCs are also unique to P. falciparum suggest-
ing that their sole purpose is to support the trafcking of P. falciparum
exported proteins. Consistent with this is the observation that disruption
of MC proteins can severely disrupt the trafcking and surface display of
PfEMP1 (Cooke et al., 2006; Hanssen et al., 2008a; Spycher et al., 2008).
While the exact mechanism of protein trafcking to and from MCs remains
unknown, it is clear that these organelles play a major role in protein traf-
cking in iRBCs. Interestingly, it has also been suggested that MCs contain
a protein phosphatase (PP1) that is responsible for dephosphorylating SBP1
(Blisnick et al., 2006). Surprisingly, however, inhibition of PP1 activity in
iRBCs affected merozoite release rather than any phenotype that would
reasonably be associated with protein trafcking. While alteration of the
phosphorylation of SBP1 is intriguing, and may be associated with signalling
events involved in the transit of proteins through MCs, a role for PP1 in this
process seems unlikely.
3. EXPORTED PARASITE PROTEINS

Over more than two decades, numerous exported parasite proteins
have been identied and characterized (Tables 1e5). The nal destination
and function of these proteins in iRBCs vary considerably, and some of these
proteins that have been studied in considerable detail will now be discussed
further. Interestingly, a large number of proteins destined for export, partic-
ularly those involved in processes associated with parasite virulence or those
localized in MCs, tend to be encoded by genes located in the sub-telomeric
regions of the parasites 14 chromosomes (Heiber et al., 2013; Sargeant et al.,
2006). These regions of chromosomes often undergo rearrangement, and
this has allowed P. falciparum to expand its repertoire of genes encoding
many of its exported proteins. While the functions of these exported
proteins are likely to be diverse, and include processes such as nutrient ex-
change and biochemical and metabolic processes (Kirk and Lehane, 2013),
this review will focus only on proteins that have been shown to play impor-
tant roles in the structural and functional modication of RBCs and those
that determine parasite virulence.
3.1 MC-associated proteins

The presence of MCs in iRBCs has been recognized for more than a cen-
tury, yet their biogenesis and function remains poorly understood
(Mundwiler-Pachlatko and Beck, 2013). It is becoming increasingly clear,
however, that both resident proteins of the clefts and proteins that tran-
siently associate with them play a central role in parasite virulence.
3.1.1 Skeleton-binding protein 1

Skeleton-binding protein 1 (SBP1) is a 48 kDa integral membrane protein
of MCs (Blisnick et al., 2000) and was the rst in the group of TM-PNEPs
to be identied (Saridaki et al., 2009). Expression of SBP1 begins within
the rst few hours following parasite invasion and is trafcked to MCs
(McMillan et al., 2013). Interestingly, even though SBP1 contains a TM,
it is rst trafcked as a soluble protein, but once at the cleft it orientates itself
in the membrane of the MC with its C-terminus on the outside of the cleft
and the N-terminus extending into the lumen (Blisnick et al., 2000; Saridaki
et al., 2009). Deletion of SBP1 does not affect the formation of either MCs
or knobs, indicating that SBP1 is not involved in the formation of either of
these structures (Cooke et al., 2006; Maier et al., 2007). However, studies
using two different laboratory-adapted lines of P. falciparum (3D7 and
CS2) have shown that SBP1 is essential for the translocation of PfEMP1
onto the surface of iRBCs and their ability to adhere to either Cluster of
Differentiation 36 (CD36) or chondroitin sulphate A (CSA), a phenotype
that was restored upon gene complementation (Cooke et al., 2006; Maier
et al., 2007). While both studies clearly showed a defect in the trafcking
of PfEMP1 to the iRBC surface, there was a distinct difference in the stage
at which the trafcking process was interrupted; in 3D7 parasites PfEMP1
was stalled at MCs (Cooke et al., 2006) and in CS2, trafcking was disrupted
earlier (Maier et al., 2007). While this clearly demonstrates that there are
distinct differences in protein trafcking between different parasite lines, it
may also indicate that SBP1 plays a more general role in the overall function
of MCs and not specically or directly involved in PfEMP1 trafcking. This
notion is in fact further supported by similar ndings in other parasite lines
where abnormal MCs are associated with abnormal trafcking of PfEMP1

(Dixon et al., 2011; Hanssen et al., 2008a; Spycher et al., 2008).
3.1.2 Membrane-associated histidine-rich protein 1

Membrane-associated histidine-rich protein 1 (MAHRP1) is a small (29 kDa)
TM-PNEP localized in MCs (Spycher et al., 2003, 2006). Like SBP1,
MAHRP1 is expressed in late ring-stage parasites and then trafcked to pre-
formed MCs (Gr uing et al., 2011; McMillan et al., 2013). Studies focussing
on the trafcking of MAHRP1, in conjunction with SBP1, revealed for
the rst time the importance of the transmembrane domain (in addition to
the rst 50 amino acids at the N-terminus of the proteins) for their export
into the iRBC and their localization in MCs (Gr uring et al., 2012; Saridaki
et al., 2009; Spycher et al., 2006). Interestingly, domain swapping experi-
ments have shown that either the rst 50 amino acids of MAHRP can be
replaced with the corresponding region from the N-terminus of SBP1 or
its TM replaced with either from the TM from SBP1 or REX2 without
any effect on the trafcking of MAHRP or its localization in MCs. This is
intriguing and indicates that while these proteins do not share much sequence
similarity, they do share common trafcking mechanisms (Gr uring et al.,
2012; Saridaki et al., 2009; Spycher et al., 2006).
MAHRP1 appears to be compartmentalized within MCs and is localized
to regions that are distinct from PfEMP1 (McMillan et al., 2013; Spycher
et al., 2003, 2006). Interestingly, genetic deletion of MAHRP1 showed
that while morphology of MCs and the trafcking of other MC-resident
proteins were unaffected, trafcking of PfEMP1 to MCs or to the iRBC
surface was ablated (Spycher et al., 2008). While the exact function of
MARHP1 remains unclear, it has been suggested that it may play a role
in redox detoxication in iRBCs since it can interact with and enhance
the ability of ferriprotoporphyrin IX to reduce hydrogen peroxide, at least
in vitro (Spycher et al., 2003). It seems unlikely however that this interaction
plays any important role in vivo since it would only function in very local-
ized areas surrounding MCs.
3.1.3 Membrane-associated histidine-rich protein 2

Membrane-associated histidine-rich protein 2 (MAHRP2) is a small (about
18 kDa) MC-resident protein that is the only protein described so far in this
location that appears to be essential for parasite survival (Pachlatko et al.,
2010). Very interestingly, this may suggest a more general role for MCs in
malaria parasite biology other than the trafcking of virulence-related
proteins. While MAHRP2 is highly expressed in trophozoite-stage parasites

(Pachlatko et al., 2010), it can be detected in small puncta in iRBCs as early
as 4 h post invasion (McMillan et al., 2013). The protein contains a
histidine-rich N-terminal region and a small hydrophobic domain of 15
amino acids, which does not function as a TM domain since MAHRP2 is
a peripheral membrane protein (Pachlatko et al., 2010). The exact localiza-
tion of MAHRP2 is intriguing since it seems to localize in distinct tethers
that appear to emanate from MCs and are proposed to help in their associ-
ation with the RBC membrane skeleton (Hanssen et al., 2008b; McMillan
et al., 2013; Pachlatko et al., 2010). MAHRP2 is the only protein so far to be
localized to these tethers and the presence of these structures as early as 4 h
post invasion is somewhat surprising as they are not required to anchor MCs
until around 20 h post invasion. However, it has been noted that the num-
ber of these structures increase from 16 to 22 h post invasion, which would
be consistent with a role in anchoring (McMillan et al., 2013). Over-
expression of MAHRP2 produces more tethers in the iRBC (Pachlatko
et al., 2010), which indicates that MAHRP2 is important to tether forma-
tion. However, MARHP2 cannot be genetically deleted and as such it is
not known whether or not it is the only protein required for tether
formation.
3.1.4 Ring-exported protein 1

Loss of virulence in P. falciparum has long been associated with the loss of the
end of the parasites chromosome 9 (Barnes et al., 1994; Bourke et al., 1996;
Day et al., 1993). Interestingly, this region of the chromosome contains a
cluster of parasite proteins that are expressed in ring-stage parasites (Spielmann
et al., 2006). At least one of these proteins, ring-exported protein 1 (REX1),
has been shown to be important both for the correct formation of MCs and
for the trafcking of PfEMP1 (Dixon et al., 2011; Hanssen et al., 2008a).
REX1 is a member of the S-PNEP group of proteins that requires the rst
36 amino acids of its N-terminus for export into the iRBC (Dixon et al.,
2008). Interestingly, this region can be swapped with the corresponding
region from other PNEPs, such as SBP1, to allow trafcking to the MCs;
however this domain swapping also requires the addition of a TM, to
correctly trafc it to MCs (Gr uring et al., 2012). While this highlights differ-
ences in the trafcking requirements between the S-PNEPs and TM-PNEPs,
it also shows the relative importance of the N-terminal region; however, this
region alone is not sufcient to trafc REX1 (Dixon et al., 2008). Like other
PNEPs, REX1 is trafcked in a soluble form (Dixon et al., 2008), indicating
there may be some overlap in PNEP trafcking within the iRBC. However,
REX1 also requires the presence of the coiled-coil region at its C-terminal
end for efcient trafcking to occur (Dixon et al., 2008). It has been shown
that this region is important for the association of REX1 with the periphery
of the MCs (Dixon et al., 2008; Hanssen et al., 2008a).
REX1 truncation mutants in which the coiled-coil region has been
deleted, not only results in mis-targeting the protein but also abnormal for-
mation and aberrant distribution of MCs (Dixon et al., 2008; Hanssen et al.,
2008a). The absence of this coiled-coil region resulted in a clustering or
stacking of MCs, a phenotype also seen in other truncation of REX1 that
still contained the coiled-coil region and in the parasite lines where
REX1 was deleted (Hanssen et al., 2008a). The REX1 deletion and the
coiled-coil truncation also interfered with trafcking PfEMP1 to the surface
of the iRBC, while in the REX1 truncation which still contained the
coiled-coil region, PfEMP1 was still trafcked to the surface, indicating
that the coiled-coil region is key to PfEMP1 trafcking (Dixon et al.,
2011). Interestingly, in the coiled-coil truncation or REX1 deletion,
PfEMP1 was still trafcked to the abnormal MCs indicating that a REX1
is not required for this stage of PfEMP1 trafcking (Dixon et al., 2011).
The adhesive properties of the iRBC, and presumably the surface exposure
of PfEMP1, were restored to some extent upon complementation of the
REX1 knockout again conrming the requirement of a functional REX1
in this process (Dixon et al., 2011). However, it is not clear if this block
in trafcking is a direct result of REX1 or more likely due to the aberrant
formation of the REX1-negative MCs.
Somewhat surprisingly, truncations of REX1, including the loss of the
coiled-coil region, or complete deletion of REX1 coincided with the abla-
tion of KAHRP expression and in turn knob formation (Dixon et al., 2011).
This ablation was shown to be due to a break in chromosome 2, which en-
codes a number of proteins including KAHRP and two DnaJ proteins
(Dixon et al., 2011). As this occurred in a number of independent mutant
lines it is highly unlikely that this is a coincidence; however, a different
parasite line (D10), which harbours a chromosome 9 deletion, does have
intact knobs indicating that REX1 can be deleted in a knob-positive parasite
(Dixon et al., 2011). While the association between REX1 deletion and
chromosome 2 breakage is unclear, investigations into the specic effects
on KARHP and knob formation centred around the expression of a
KAHRP-GFP chimera in the REX1 knockout lines (Dixon et al., 2011).
In these parasites it was shown that KAHRP does not correctly localize to
the iRBC membrane; however, no knobs were present on the iRBC surface
(Dixon et al., 2011). It is possible that in the REX1 deletion or the REX1
coiled-coil region, truncation alters MC function and therefore protein traf-
cking is disrupted to such a degree that the accumulation of KAHRP is
detrimental to the parasite and there is a positive selection for parasite con-
taining the chromosome 2 deletion. While it is possible that REX1 or at
least the coiled-coil region of REX1 is involved in the correct localization
of KAHRP to the iRBC membrane and in turn formation of knobs, it
also remains possible that the trafcking of KAHRP is reliant on other pro-
teins that are also lost in the chromosome 2 deletion, such as the DnaJs pre-
sent at the end of this chromosome (Dixon et al., 2011).
3.1.5 Ring-exported protein 2

REX2 is a small (13 kDa) protein in the TM-PNEP family that localizes in
MCs with its C-terminus exposed on the cytoplasmic face of cleft facing into
the RBC cytosol (Spielmann et al., 2006). As with other TM-PNEPs, both
the N-terminus and the TM are required for the trafcking of REX2 to
MCs (Haase et al., 2009). REX2 gave the rst indication that these PNEPs
have an N-terminal sequence required for export that is similar to a mature
cleaved PEXEL (Gr uring et al., 2012; Haase et al., 2009). Interestingly, the
rst 10 to 20 amino acids in the N-terminal region of REX2 appear to
contain the critical signal for MC localization since this region alone was
able to direct non-related transmembrane proteins to MCs (Haase et al.,
2009). While currently there is no assigned function to REX2, due to its
location and the association with the loss of cytoadherence via chromosome
breakage, it is likely that REX2 will have an important role in the trafcking
of virulence factors in P. falciparum.
3.1.6 Other less-well characterized proteins associated with MCs

A number of other proteins have been identied in MCs but have not yet
been characterized in detail. Some components required for vesicle traf-
cking have been shown to associate with MCs such as the small GTP-
binding protein Sar1p and the COPII complex proteins, Sec31p and
Sec24p (Adisa et al., 2001; Albano et al., 1999; Wickert et al., 2003a) sug-
gesting that MCs may play a role in vesicle trafcking in iRBCs; possibly
involving transport of PfEMP1 to the RBC surface. MCs also contain a
number of proteins transcribed from multigene families including some
members of the early transcribed membrane proteins (ETRAMPs), which
all contain a single transmembrane domain (Birago et al., 2003; Frech and
Chen, 2013; Spielmann et al., 2003), 13 small (w25 kDa) proteins which
belong to the two transmembrane family PfMC-2TM (a subfamily of the
STEVORS) (Sam-Yellowe et al., 2004; Tsarukyanova et al., 2009) and
possibly two of the cysteine repeat modular proteins (CRMP) (Thompson
et al., 2007). The ETRAMPs are small proteins with a charged C-terminal
domain that varies in length and are differentially expressed throughout the
parasites RBC lifecycle. Some ETRAMPs have been localized at the PVM,
with their N-terminus projecting into the cytosol of the iRBC (Spielmann
et al., 2003), while others are within the iRBC itself, with at least one mem-
ber shown to trafc through MCs (Birago et al., 2003). In P. berghei, UIS4,
the orthologue of ETRAMP10.2 in P. falciparum, which is localized at the
PVM, is also associated with the tubovesicular network (TVN) in liver-stage
parasites (Grutzke et al., 2014; Spielmann et al., 2003) indicating that the
ETRAMPs may have functions in all parasite stages. There are four CRMPs
in P. falciparum that are expressed throughout the parasites lifecycle with
CRMP1 and CRMP2 co-localizing with PfEMP1 at MCs (Thompson
et al., 2007). While the functions of all of these proteins remain to be deter-
mined, their localization in iRBCs, and for some, an association with MCs,
increases the likelihood that they play important roles in the parasites com-
plex protein trafcking pathways.
A novel set of proteins, involved in trafcking PfEMP1 to the iRBC sur-
face, was also identied during a large functional screen (Maier et al., 2008).
At least two of these proteins were localized in MCs (Maier et al., 2008),
again highlighting the role of MCs in trafcking PfEMP1. One of these
proteins contained a PHISTc domain, while the other, recently termed
PfPTP1, was unique (Rug et al., 2014). Both are suggested to play a direct
role in the transport of PfEMP1 to the iRBC membrane (Maier et al., 2008).
One interesting group of recently identied proteins in MCs are the mero-
zoite surface-related proteins (MSRP) MSRP5, MSRP6 and MSRP7
(Heiber et al., 2013). While these are related in sequence to the merozoite
surface protein 7 (MSP7), their localization in MCs imply a very different
function (Heiber et al., 2013) and highlight the difculty in assigning func-
tion, or even localization, based solely on similarity with other known para-
site proteins.
3.1.7 Parasite proteins and the tubovesicular network

In addition to MCs, a separate, complex array of membranous structures can
be visualized by electron microscopy in the cytosol of iRBCs known as the
TVN (Elmendorf and Haldar, 1994). Very little is known about the protein
composition or the function of the TVN but it is thought to be important

for the import of nutrients, particularly lipids such as cholesterol and sphin-
gomyelin (Elmendorf and Haldar, 1994; Lauer et al., 1997, 2000). While it
has been suggested that there is a parasite-encoded sphingomyelin synthase
present in the TVN, there is currently no direct evidence to support this
(Elmendorf and Haldar, 1994). To date, only two proteins have been iden-
tied in the TNV; a TVN junction protein (TVN-JP1) (van Ooij et al.,
2008) and the erythrocyte vesicle protein 1 (EVP1) (Tamez et al., 2008),
and are thought to be important for the formation of the TVN (van Ooij
et al., 2008) and in the lipid dynamics in the iRBC (Tamez et al., 2008).
Further characterization of these two proteins, and the identication of
others, will provide the rst steps to better understand the function of this
complex network and may highlight new and novel potential drug targets
(Elmendorf and Haldar, 1994; Lauer et al., 1997, 2000; Tamez et al.,
2008; van Ooij et al., 2008).
3.2 Parasite proteins in the RBC cytosol or at the RBC

membrane skeleton
The interaction of numerous parasite proteins with the RBC membrane
skeleton underpins the dramatic changes in both the adhesive and the
membrane mechanical properties of iRBCs (Cooke et al., 2004b, 2014;
Cooke and Lim, 2007) (Table 2). In the following sections, we discuss
the latest ndings on the trafcking of these proteins to the RBC mem-
brane skeleton and their effects on the mechanical and adhesive properties
of RBCs.
3.2.1 Knob-associated histidine-rich protein

The knob-associated histidine-rich protein (KAHRP) was the rst protein
to be shown to be essential for the formation of multiple punctate protru-
sions on the iRBC membrane, termed knobs (Crabb et al., 1997). The
gene encoding KAHRP is located in the sub-telomeric region of chromo-
some 2, and spontaneous loss of the end of this chromosome, which often
occurs during long-term in vitro culture of parasite without the selection
for knobs, results in the loss of these structures from the iRBC membrane
skeleton (Waterkeyn et al., 2001). KAHRP contains a typical PEXEL motif,
and is trafcked into the iRBC, where the N-terminal histidine-rich repeats
are required for its localization at the iRBC membrane skeleton and subse-
quent formation of knobs (Rug et al., 2006; Waller et al., 1999; Wickham
et al., 2001).
In parasites expressing truncated forms of KAHRP that still contain the

histidine-rich repeats, KAHRP and PfEMP1 were still localized at the iRBC
membrane (Rug et al., 2006). However, PfEMP1 trafcking is impaired if
KARHP is deleted from parasites (Crabb et al., 1997). Taken together, this
suggests that the histidine-rich repeats are involved in the trafcking of
PfEMP1 to the iRBC membrane along with KAHRP (Crabb et al.,
1997; Rug et al., 2006; Wickham et al., 2001). While the histidine-rich re-
peats are required for the localization of KAHRP, they are not sufcient for
knob formation, which also requires the C-terminal repeats (Rug et al.,
2006). Interestingly, while PfEMP1 was still trafcked to the iRBC surface
in the KAHRP truncation mutants, the ability of iRBCs to bind to CD36
was signicantly reduced, demonstrating that correct display of PfEMP1 at
knobs is critical for cytoadhesion to occur (Rug et al., 2006).
KAHRP has been shown to interact with the conserved acidic terminal
sequence (ATS) region of PfEMP1 (Waller et al., 1999, 2002) as well as the
integral RBC membrane skeleton proteins, spectrin (Pei et al., 2005) and
ankyrin (Magowan et al., 2000; Weng et al., 2014) (Table 2). It is the com-
bination of these interactions that allows KAHRP to anchor PfEMP1 to the
membrane skeleton in iRBCs (Magowan et al., 2000; Waller et al., 1999,
2002). One region in KAHRP that is required for its interaction with
PfEMP1 is the region containing the histidine-rich repeats (Waller et al.,
1999, 2002). The other signicant interaction with KAHRP involves the
rst block of C-terminal repeats; the region required for knob assembly
(Rug et al., 2006). Considering that this region also interacts with both
the ATS region of PfEMP1 and ankyrin (Magowan et al., 2000; Waller
et al., 1999, 2002), it is apparent that this is the region required for anchoring
PfEMP1. However, a recent study could not nd any interaction between
KAHRP and ATS (Mayer et al., 2012), and while this does not disprove the
role for KAHRP, it does highlight the possibility that other proteins may be
required for anchoring different versions of PfEMP1. More work is clearly
required to determine the entire repertoire of proteins that are able to an-
chor all variants of PfEMP1 at knobs.
3.2.2 P. falciparum erythrocyte membrane protein 3

PfEMP3 is a large (>300 kDa) PEXELated protein that is exported into the
iRBC where it interacts with the membrane skeleton (Waller et al., 2007;
Waterkeyn et al., 2000). While the precise function of this protein remains
unclear, it has been shown to interact specically with both spectrin and
actin (Pei et al., 2007b; Waller et al., 2007). This interaction is mediated
by a single motif within PfEMP3 (Waller et al., 2007), suggesting that it

binds at the junction between spectrin and actin. This is further supported
by the observation that PfEMP3 binds specically to the EF region of
a-spectrin (Pei et al., 2007b); a region that is critical for the interaction be-
tween spectrin and actin in the RBC membrane skeleton (Korsgren and
Lux, 2010). This suggests a role for PfEMP3 in the regulation of the mem-
brane mechanical properties of iRBCs by altering the normal spectrineactin
interaction. It is also possible that the ability to interact with at least two
different membrane skeletal proteins would be consistent with the diffused
localization of PfEMP3 at the iRBC membrane (Waller et al., 2007;
Waterkeyn et al., 2000).
While deletion of PfEMP3 from parasites in vitro did not result in any
detectable abnormal phenotype, truncation of PfEMP3 showed a decrease
in the adhesive properties of iRBCs and in the surface display of PfEMP1
(Waterkeyn et al., 2000). The N-terminus of PfEMP3 is required to direct
it to MCs, through which it transits en route to the RBC membrane
(Knuepfer et al., 2005a; Wickham et al., 2001). The truncated PfEMP3
localized to structures below the iRBC membrane (Waterkeyn et al.,
2000), is likely to be at MCs. This accumulation of PfEMP3 in MCs could
disrupt normal MC function, which in turn would disrupt normal PfEMP1
trafcking in these truncation mutants (Waterkeyn et al., 2000), an effect
often seen with the deletion of resident MCs proteins (Cooke et al.,
2006; Dixon et al., 2011).
3.2.3 P. falciparum antigen 332

P. falciparum antigen 332 (Pf332) is a very large (w750 kDa) protein that is
trafcked to the iRBC membrane skeleton via MCs (Glenister et al., 2009;
Hinterberg et al., 1994; Hodder et al., 2009; Nilsson et al., 2012). Initially, in
the early stages of the iRBC life cycle, Pf332 is a peripheral membrane pro-
tein of MCs. As the parasite matures, Pf332 begins to associate with the
iRBC membrane skeleton and by the schizont stage, most of it is in that
location (Glenister et al., 2009; Hinterberg et al., 1994; Hodder et al.,
2009; Nilsson et al., 2012) where it forms a specic interaction with actin
(Waller et al., 2010).
Pf332 is involved in the alteration of the membrane mechanical properties
of iRBCs. Interestingly, this was the rst parasite protein to be described that
caused a decrease in the rigidity of the RBC membrane, indicating that the
parasite induces multiple changes in the membrane skeleton in order to main-
tain its deformability (Glenister et al., 2009; Hodder et al., 2009). Parasites in
which Pf332 had been deleted showed that MCs clump together, resulting
in a reduced amount of PfEMP1 on the surface of the iRBC (Glenister et al.,
2009). However, reduction of PfEMP1 was not seen in an independent
study (Hodder et al., 2009), which could indicate that there are slight differ-
ences between different parasite lines (in this case, 3D7 (Glenister et al., 2009)
or CS2 (Hodder et al., 2009)). However, in 3D7 parasites, there was a clear
decrease in adhesion (Glenister et al., 2009), which was not tested in the CS2
Pf332 knockout lines. This alteration in the adhesive properties of iRBCs
could be the result of either a reduction in PfEMP1 on the iRBC surface
or to an increase in rigidity of the iRBC membrane skeleton interfering
with binding. Due to the fact that P. falciparum exports a large number of
proteins into the iRBC, with a large number of protein functions revolving
around PfEMP1 trafcking (Table 1), it is feasible that different strains of par-
asites require the function of different exported proteins based on the
different variants of PfEMP1 that are being expressed on the surface of the
iRBC. This would be the case for 3D7, which binds to CD36, whereas
CS2 binds to chondroitin sulphate A (CSA) (Cooke et al., 1998).
3.2.4 Plasmodium helical interspersed sub-telomeric proteins

A comparative proteomic analysis of malaria parasites identied a unique
family of proteins, collectively called PHIST (Plasmodium helical inter-
spersed sub-telomeric) proteins, due to the presence of a conserved 150 res-
idue domain containing four consecutive a-helices that do not appear to be
similar in sequence or predicted structure to any other currently known
protein domains (Sargeant et al., 2006). Partial characterization of some
members of this family has revealed their involvement in various pathoge-
nicity-related functions including RBC modication, RBC adhesion and
parasite protein export (Maier et al., 2008). PHIST proteins are classied
into three subgroups, PHISTa, PHISTb and PHISTc, based on the presence
and position of several conserved tryptophan residues (Sargeant et al., 2006).
Interestingly, different phist genes appear to show peak expression at
different stages of the RBC cycle (from early rings through late schizonts)
suggesting different, non-overlapping functions for different PHIST pro-
teins (Sargeant et al., 2006). Although all Plasmodium spp., contain a varying
number of phist genes, in P. falciparum the family has undergone considerable
expansion in number to 75 related members (Eksi et al., 2005; Frech and
Chen, 2013; Sargeant et al., 2006) (Table 3).
Interestingly, some evidence suggests that at least three phist genes are
up-regulated in their expression in either clinical parasite isolates when
compared to laboratory-adapted lines or between clonal laboratory-adapted

lines selected for increased cytoadherence, further supporting an important
role for this gene family in pathogenesis and parasite survival in vivo (Daily
et al., 2005; Mok et al., 2007; Tuikue Ndam et al., 2008). Although func-
tional redundancy within large protein families, such as PHIST, exists, the
highly divergent nature of the proteins within this family, and even within
each sub-class, combined with the varied functions observed for some mem-
bers (Kilili and LaCount, 2011; Maier et al., 2008; Mayer et al., 2012; Oberli
et al., 2014; Proellocks et al., 2014), suggests that this is unlikely. While most
PHIST proteins contain a PEXEL, and thus predicted to be exported, some
members of this family do not contain any such export signatures (Sargeant
et al., 2006). Since all PHIST proteins characterized to date appear to func-
tion within the iRBC, it is reasonable to believe that all members of this
family will be exported into the iRBC, although the lack of obvious export
signals mean that the nal cellular destination of these proteins will need to
be conrmed experimentally.
3.2.4.1 PHISTa
PHISTa proteins are unique to P. falciparum, and its genome contains 26
different members (Table 3) (Eksi et al., 2005; Frech and Chen, 2013;
Sargeant et al., 2006). PHISTa proteins appear to be very short in amino
acid sequence, comprising a signal sequence, a PEXEL motif and PHISTa
domain, which contains two conserved tryptophan residues. One exception
to this is PF3D7_0402000 which contains an extended C-terminus and
interestingly along with PF3D7_1253300 are the only two PHISTas that
have any detectable transcripts in asexual blood stages of laboratory-adapted
parasite lines (Sargeant et al., 2006). PF3D7_0402000 is the best character-
ized of the PHISTa proteins. It is the only PHISTa to be localized in the
iRBC where it appears to localize at the PVM (Parish et al., 2013). The pro-
tein interacts with the RBC membrane structural protein 4.1R; an interac-
tion that appears to be reliant on the conversed tryptophan residues within
the PHISTa domain. Interestingly, the interaction with 4.1R does not take
place at the iRBC membrane skeleton but at the PVM where there appears
to be a sub-population of 4.1R (Parish et al., 2013). However, it is not
currently known how protein 4.1R relocates from the iRBC membrane
skeleton to the PVM. It has been suggested that the phosphorylation of
4.1R in iRBCs helps to disassociate it from the RBC skeleton where it is
then free to localize at the PVM (Parish et al., 2013). It might also be possible
that a sub-population of 4.1R is recruited to the PVM during parasite
invasion. Clearly, more work is required to determine the precise function

of the PHISTa/4.1R complex at the PVM.
The genes encoding the other PHISTa proteins appear not only to be
transcriptionally silent in laboratory-adapted parasite lines, but at least seven
are pseudogenes. The fact that there seems to be selective expression of a
small number of PHISTa proteins could suggest that these proteins are
involved in antigenic variation; such as that seen with the var genes (Sargeant
et al., 2006). This is highlighted by the up-regulation of PF3D7_1478000 in
a clonal parasite line that was selected for increased cytoadherence (Mok
et al., 2007). The likelihood of a specic mutation required to insert prema-
ture stop codons in these PHISTa genes could suggest that the laboratory-
adapted lines have systematically down-regulated some PHISTs as they
are clearly not required for parasite survival in vitro. However, this remains
to be conrmed by analysing a range of both clinical isolates and laboratory-
adapted parasite lines as there are clearly other mechanisms to regulate
expression of this group since many PHISTa proteins are not expressed in
the laboratory strains. This could also suggest that PHISTa proteins have,
as a group, a diverse set of functions that are not restricted to asexual blood
stages. Two other phista genes, PF3D7_1001100.1 and PF3D7_1001100.2,
appear to encode isoforms of the acyl-CoA-binding protein 1 (ACBP1)
(Frech and Chen, 2013; Sargeant et al., 2006), but as yet, there is no evi-
dence to suggest that these genes are expressed in parasites.
3.2.4.2 PHISTb
The PHISTb subgroup is the largest in the family of PHIST proteins with
a total of 31 distinct members (Table 3). Proteins with a PHISTb domain
share the same structural architecture as PHISTa but possess a C-terminal
domain of varying length, which precedes the PHISTb domain. At least
11 PHISTb proteins have been shown to be differentially phosphorylated
throughout the parasites life cycle (Pease et al., 2013) suggesting that
phosphorylation plays a role in the function of at least some of the PHISTb
proteins. A subgroup of seven PHISTb proteins, including RESA and
RESA-like proteins, also contain a DnaJ domain in their variable
C-terminal region (Sargeant et al., 2006). PHISTb proteins are only pre-
sent in species of malaria parasites that infect primates (Sargeant et al.,
2006) and contain four conserved tryptophan residues in the PHIST
domain. A recent study has suggested that the PHISTb domain could
be required for the localization of this group of proteins at the iRBC
membrane (Tarr et al., 2014). In general, PHISTb proteins seem to play
important pathophysiological roles in P. falciparum. This is highlighted by

the up-regulation of PF3D7_0936900 in parasites isolated from women
with pregnancy-associated malaria (PAM) and its possible role in binding
of iRBCs to CSA in the placenta (Tuikue Ndam et al., 2008). Interestingly,
this gene appears to be a pseudogene in the laboratory-adapted parasite line
3D7, suggesting some polymorphism between clinical and laboratory-
adapted parasite lines which, as seen with PHISTa, may highlight the
down-regulation of non-essential PHIST proteins in vitro. A large gene
knockout study in P. falciparum (Maier et al., 2008) revealed that PHISTb
proteins could be either non-essential or essential for parasite survival, at
least in vitro. PHISTb proteins that were deleted from the parasites
genome revealed a diverse range of functions, most of which are required
for the extreme virulence of P. falciparum (Table 3) (Goel et al., 2014; Kilili
and LaCount, 2011; Maier et al., 2008; Proellocks et al., 2014). However,
four could be deleted without any apparent disruption in virulence (Maier
et al., 2008) (Table 3), suggesting that this family may also function in other
processes.
PfD80 (Table 3) is an essential PHISTb that has been suggested to asso-
ciate with MCs (Maier et al., 2008; Vincensini et al., 2005) and has recently
been localized at the iRBC membrane (Tarr et al., 2014). Together, this
suggests that PfD80 plays a role in trafcking proteins between MCs and
the iRBC membrane skeleton. Another PHISTb, PF3D7_0424600, identi-
ed in iRBCs membrane proteomic fraction (Florens et al., 2004) indicating
its association with the iRBC membrane, was shown to be required for the
formation of knobs and cytoadherence (Maier et al., 2008). Some members
of the PHISTb group have been shown to contain a MESA erythrocyte
cytoskeleton-binding (MEC) domain. Of these, four were conrmed to
bind to the RBC membrane skeleton via an interaction with inside out ves-
icles (IOVs) prepared from human RBCs, specically with protein 4.1R
(Kilili and LaCount, 2011). While another PHISTb that contains a MEC,
PF3D7_1401600, not shown to directly bind to IOVs has been shown to
be required for the altered membrane mechanical properties of iRBCs
(Maier et al., 2008). Recent work on another PHISTb, the lysine-rich
membrane-associated PHISTb (LyMP) (Table 3), has also demonstrated
that this protein is able to bind the RBC membrane skeleton; however,
this interaction is mediated through its C-terminal domain not through a
MEC domain (Proellocks et al., 2014). LyMP has also been shown to be
required for efcient cytoadhesion, possibly through its interaction with
PfEMP1 (Oberli et al., 2014; Proellocks et al., 2014).
The RESA-like PHISTb proteins are an interesting subgroup within this

family. However, some confusion appears to exist in the literature. While all
RESA-like proteins are PHISTb proteins, not all PHISTb proteins are
RESA-like and the designation of RESA-like should be reserved only for
those proteins that contain both the PHISTb domain and DnaJ domain
(the hallmark of RESA). To this end, RESA-2, which is a truncated protein
and does not express the DnaJ domain, more resembles the other PHISTb
proteins. RESA-2 can be deleted in vitro without loss of a detectable func-
tion and has been shown to be up-regulated in clinical isolates (Daily et al.,
2005) suggesting it may play a role in antigenic variation. Only one of the
RESA-like genes, PF3D7_1149200, is essential (Table 3) (Maier et al.,
2008) and at present has no function assigned to it. The remaining four
RESA-like proteins all seem to play a role at the iRBC membrane skeleton.
Two of these, PF3D7_1201100 and PF3D7_1038800, contain MEC do-
mains, with PF3D7_1038800 conrmed to bind to both IOVs and protein
4.1R (Kilili and LaCount, 2011). Although binding of PF3D7_1038800 to
4.1R has been detected, the protein does not have any obvious function in
iRBCs when deleted (Maier et al., 2008) in vitro, which may suggest some
functional redundancy among this group. The other two, PF3D7_0201700
and PF3D7_0220100, appear to be involved in modulating the deformabil-
ity of iRBCs (Maier et al., 2008). As a whole, this group of proteins seems to
play important roles in the extreme virulence of P. falciparum malaria para-
sites and clearly warrants further scrutiny.
3.2.4.3 PHISTc
With 18 members, PHISTc proteins are the smallest subgroup of PHIST
proteins but are the most variable in size (262e1219 residues) and the
PHISTc domain itself contains three conserved tryptophan residues.
PHISTc proteins appear to have evolved very early in the evolutionary his-
tory of Plasmodium, as they are present in several species (Sargeant et al.,
2006). Unlike PHISTa and PHISTb proteins, most PHISTc proteins do
not contain a classical PEXEL motif (Table 3). This could indicate that
this group of proteins was evolving prior to the expansion of the exported
protein repertoire seen in P. falciparum. While not much is known about this
group of proteins, current information suggests their involvement in the
structural and functional modications of iRBCs. At least two PHISTc
proteins (PF3D7_0731100 and PF3D7_0936800) have been shown to
associate with PfEMP1. PF3D7_0731100 is localized in MCs and is
required for the trafcking of PfEMP1 from MCs to the iRBC membrane
(Maier et al., 2008). PF3D7_0936800 appears to be essential for parasite

survival (Maier et al., 2008) and has been implicated in anchoring
PEMP1 at knobs (Mayer et al., 2012); however, its presence at knobs re-
mains to be conrmed. The protein also shows polymorphism between
different isolates of P. falciparum specically at the C-terminal end of the
protein (Chookajorn and Hartl, 2006). PF3D7_0731100 was detected in
a proteomic screen of iRBC membranes (Florens et al., 2004), and its
orthologue in P. vivax has been shown to be exported to Schuffners dots
(structures of unknown function in P. vivax -infected RBCs) (Akinyi
et al., 2012). While it is difcult to predict the localization of PHISTc pro-
teins in the absence of classical export signatures, this is highly suggestive
that this PHISTc, as well as others, will be localized in the iRBC cytosol.
As with PHISTa proteins, not all PHISTc proteins are expressed in asexual
blood-stage parasites. One PHISTc protein (LSAP-2), for example, has
been shown to be up-regulated in sporozoites once they come in contact
with the host hepatocyte (Siau et al., 2008). In liver-stage parasites,
LSAP-2 localizes to the periphery of the parasite; however, it is not
exported into the cytosol of the hepatocyte (Siau et al., 2008).
3.2.5 Ring-infected erythrocyte surface antigen

Ring-infected erythrocyte surface antigen (RESA) is a 155-kDa soluble
protein encoded by a gene located in the sub-telomeric region of chromo-
some 1. It was the rst of a RESA-like family of proteins to be identied and
characterized (Table 3) (Aikawa et al., 1990; Brown et al., 1985; Sargeant
et al., 2006). RESA is rst detected in dense granules in merozoite-stage par-
asites (Aikawa et al., 1990) and is then transferred soon after invasion onto
the iRBC membrane (Brown et al., 1985). RESA contains what is referred
to as a relaxed PEXEL, with six amino acids instead of the usual ve, how-
ever, is still functional and, like a classical PEXEL, is cleaved by plasmepsin V
(Boddey et al., 2013). RESA contains both a PHISTb domain and a DnaJ
domain, and while the presence of a J domain implicates RESA as a chap-
erone (Hsp40), it does not contain the other domains required to act as a
functional Hsp40. In addition, RESA does not contain an HPD motif
within the J domain, which is required for the hydrolysis of ATP; a critical
function for the Hsp40 chaperone (Botha et al., 2007). However, RESA
does seem to act as a heat shock protein. There are a number of studies
that have highlighted the increased destabilization of the iRBC membrane
during heat stress in parasites that do not express RESA, as it is suggested
to protect spectrin from unfolding (Diez-Silva et al., 2012; Pei et al.,
2007a; Silva et al., 2005). RESA has also been shown to interact directly
with the b-spectrin repeat 16 region and increase membrane mechanical sta-
bility (Pei et al., 2007a). Interestingly, this is directly downstream of the
ankyrin-binding site on b-spectrin at repeats 14 and 15 (Kolondra et al.,
2008; Korsgren and Lux, 2010). This proximity of binding sites suggests
that stabilizing the spectrineankyrin interaction is a key function of
RESA during heat stress. RESA has also been shown to contribute to the
increased membrane rigidity of iRBCs; an effect which may be exacerbated
during heat stress (Mills et al., 2007; Silva et al., 2005).
3.2.6 Proteins containing DnaJ domains

A number of proteins in P. falciparum contain DnaJ domains and, by deni-
tion, are extended members of the Hsp40 family. However, not all Hsp40s
are alike and there is now a classication system to distinguish between the
different types (Botha et al., 2007). Type I DnaJs contain the J domain with
the HPD motif that is required for ATP hydrolysis, a glycine-rich region, a
conserved C-terminal domain and a zinc zipper, all domains required for
bestowing Hsp40 function. While P. falciparum has a number of type I DnaJs,
none of them are exported. The type II, III and IV DnaJs are represented by
exported proteins in P. falciparum (Botha et al., 2007). The type II DnaJs
contain most of the domains required for the type I but are missing the
zinc zipper and while they are functional similar to type I DnaJs, they cannot
function in the absence of an interaction with Hsp70. Type III and type IV
proteins contain only the J domain; however, type IV DnaJs have an altered
HPD motif and therefore are not functional (Botha et al., 2007). P. falcipa-
rum exports 18 DnaJs into the iRBC; three type II, ve type III and 10 type
IV (Botha et al., 2007; Sargeant et al., 2006) (Table 4).
Type II DnaJs, PFA660, PFB90 and PFE55, are intriguing as they all
show extremely high identity to one another (Botha et al., 2007). Interest-
ingly, only one (PFA660) appears to be essential for parasite survival (Maier
et al., 2008), suggesting PFA660 may be functionally distinct from the other
two non-essential proteins. Both PFA660 and PFE55 have been localized to
mobile structures distributed throughout the iRBC termed J-Dots (K ulzer
et al., 2010). Due to the similarity of these type II DnaJs it is likely that
PFB90 also localizes to similar structures, although this remains to be
conrmed. While J-Dots do not appear to be membrane bound when visu-
alized by electron microscopy, they have been associated with cholesterol
(Kulzer et al., 2010), indicating that they may be protein complexes
requiring cholesterol to form or function correctly. Recently an exported
Hsp70, termed PfHsp70x, was identied in the iRBC and associates with
J-Dots (K ulzer et al., 2012). The export of PfHsp70x relies on an eight
amino acid motif located downstream of the signal peptide that does not
share any obvious conservation with other known export signatures (K ulzer
et al., 2012). PfHsp70x appears to co-localize with J-Dots that contain either
PFA660 or PFE55 (K ulzer et al., 2012). To date, J-Dots have not been
shown to contain more than one version of the type II DnaJ, suggesting
that there may be distinct populations of J-Dots within the iRBC (K ulzer
et al., 2010). The combination of the DnaJ (Hsp40) and an Hsp70 together
does indicate that these J-Dots form a co-chaperone/chaperone complex
which could be required for the trafcking of a range of proteins to the nal
destination. Additionally J-Dots also show a time-dependent co-localization
with PfEMP1, suggesting a role in trafcking of PfEMP1 within the iRBC
during the early stages of parasite development (K ulzer et al., 2012). While
RBCs infected with parasites in which PFB90 and PFE55 had been deleted
did not show any defect in PfEMP1 trafcking (Maier et al., 2008), this
could highlight some functional redundancy between the different popula-
tions of J-Dots. To further support the role of PfHsp70x as a chaperone, it
has recently been shown that PfHsp70x has ATPase activity and high aggre-
gation suppression activity compared to that of PfHsp70-1 (Blatch et al.,
2014). The potential role of PfHsp70x in combination with J-Dots has
also been strengthened by the Hsp40-stimulated ATPase activity of
PfHsp70x in the presence of a promiscuous DnaJ (Hsj1a). That study also
showed that the activity of PfHsp70x could be modulated by small molecule
inhibitors, demonstrating the potential for this specic Hsp70 as a target for
new antimalaria drugs (Blatch et al., 2014).
Type III and type IV DnaJs are diverse groups of proteins. All type III
DnaJs characterized so far have revealed that this group largely resides at
and interacts with the iRBC membrane skeleton (Table 4) (Kilili and
LaCount, 2011; Maier et al., 2008). One of these, PF3D7_1039100, is
required for knob formation, while another, PF3D7_0220100, is involved
in the regulation of the membrane mechanical properties of iRBCs (Maier
et al., 2008). From the limited characterization of the type IV DnaJs, mem-
brane association is again seen with some members, including some of the
RESA-like proteins, and is involved in regulation of iRBC membrane me-
chanics (Tables 3 and 4) (Kilili and LaCount, 2011; Maier et al., 2008). Type
IV proteins have a mutated HPD motif, with PF3D7_0220400 showing the
closest similarity to a functional HPD motif with a HPE motif. However,
this change to a similar amino acid has been shown to ablate the function
of this motif and is therefore still considered a type IV DnaJ (Botha et al.,
2007). While both type III and IV DnaJs contain a J domain they also
show a high degree of diversity in terms of the other domains they contain,
with some containing a PHISTb domain while others contain the MEC
domain while some contain all three domains (Kilili and LaCount, 2011;
Sargeant et al., 2006). Interestingly, there does not seem to be any correla-
tion between the genes that contain these other domains, suggesting that this
diversity has served to broaden the repertoire of these exported DnaJs and
what they can interact with in the iRBC. This high degree of diversity is
likely due to the location of these genes at the telomeres of chromosomes
and is therefore more likely to undergo rearrangement. Over time, this
has produced a diverse family of proteins that all appear to be required for
alteration of the structural and functional properties of iRBCs (Tables 3
and 4).
3.2.7 Mature-parasite-infected erythrocyte surface antigen

The mature-parasite-infected erythrocyte surface antigen (MESA) is a large
(w300 kDa) repetitive protein that is exported into the iRBC during the
trophozoite-stages of parasite development (Coppel, 1992). MESA also con-
tains a DnaJ domain in the C-terminal region of the protein; however, it is by
far the most distant member in the DnaJ family in P. falciparum (Sargeant
et al., 2006). The presence of the J domain may give insights into the func-
tion of this particular DnaJ; however, like RESA, it lacks the HPD motif and
is unlikely to be functional (Table 4) (Botha et al., 2007; Sargeant et al.,
2006). Despite being studied extensively for many years, the precise function
of MESA remains unknown; however it is clear that it interacts with the
RBC membrane, specically with a 30 kDa domain of protein 4.1R located
in the N-terminal region of the protein (Bennett et al., 1997; Black et al.,
2008; Waller et al., 2003). The sequence of the binding domain of MESA
has been used in homology searches to identify 14 parasite proteins in addi-
tion to MESA that are predicted to bind to protein 4.1R (Kilili and LaCount,
2011). Of these, ve were DnaJ proteins (Table 4) and nine were PHISTs;
two of those RESA-like (Table 3). In total, eight of the 14 proteins were
conrmed to bind to IOVs and three were subsequently conrmed to
bind directly to protein 4.1R (Kilili and LaCount, 2011). This clearly dem-
onstrates that this binding domain is important to localize numerous parasite
proteins to the RBC membrane skeleton.
A function for MESA was rst suggested using a mutant parasite line that
did not express MESA as a result of a deletion in the sub-telomeric region of
chromosome 5, which contained the mesa gene (Magowan et al., 1995).

While RBCs infected with these mesa-negative mutant parasites did not
show any difference in their ability to cytoadhere or form knobs when
compared to normal iRBCs (Magowan et al., 1995), the lack of any obvious
phenotype could have been masked by other genes that were also lost as a
result of the relatively large chromosome break. Interestingly, when
compared to wild-type parasites, the parasites that had lost the sub-telomeric
end of chromosome ve showed increased tness when infecting RBCs
decient in protein 4.1R (Magowan et al., 1995). This difference in relative
growth was attributed to the accumulation of MESA in the cytosol of
protein 4.1R-decient RBCs infected with wild-type parasites leading to
a decrease in tness. However, it is also possible that the absence of other
parasite proteins, due to the chromosome ve breakage, may have contrib-
uted to this phenotype. Determination of the precise function of MESA
awaits generation of a targeted gene deletion parasite, which, surprisingly,
never seem to have been generated.
3.2.8 FIKK kinases

Comparative genomic analysis of apicomplexan parasites revealed a novel
and unique putative family of kinases that are collectively called FIKK,
due to the presence of a conserved Phe (F)- Ile (I)- Lys (K)- Lys (K) amino
acid motif (Schneider and Mercereau-Puijalon, 2005; Ward et al., 2004).
FIKKs are characterized by a C-terminal kinase domain that is distinct
from other established eukaryotic protein kinase groups (Ward et al.,
2004). Each FIKK contains a variable N-terminal region that does not
contain any known functional motifs other than a signal peptide and a
PEXEL motif (Schneider and Mercereau-Puijalon, 2005). The variability
of the N-terminal region of the FIKKs, which in other kinases is responsible
for regulation of their enzymatic activity in response to signalling mole-
cules, substrate recognition and/or specic recruitment sites, indicates
that individual FIKKs most likely target different protein components
and carry out non-redundant functions (Nunes et al., 2007). Strikingly,
most apicomplexan parasites, including most Plasmodium spp., contain
only a single kk gene (orthologous to P. falciparum kk8), but in P. falcip-
arum a process of gene expansion has resulted in 21 kk paralogues
(Schneider and Mercereau-Puijalon, 2005; Ward et al., 2004), 19 of which
are expressed at various times during the erythrocytic asexual cycle (Nunes
et al., 2007). The kk genes are distributed throughout 12 of the 14 chro-
mosomes of P. falciparum and are often located in sub-telomeric regions in
close association with var genes. Of the 21 kk genes, two (FIKK7.2 and
FIKK14) are suggested to be psuedogenes, or at least truncations as they
have an internal stop codon within the kinase domain and if expressed
are unlikely to be functional. Both are predicted to be exported and have
detectable transcripts and FIKK7.2 is one of the more highly transcribed
of the FIKK family, specically in schizonts (Nunes et al., 2007). Most
notably, with the exception of FIKK8 and FIKK9.2, all of the 17 remaining
kk genes that are predicted to encode fully functional kinases that are
exported into the iRBC (Table 5) (Nunes et al., 2007; Schneider and
Mercereau-Puijalon, 2005). This specic gene radiation of exported pro-
teins does suggest that the FIKK kinases are involved in iRBC modications
that are unique to P. falciparum.
FIKK kinases are very distinct from other known kinase families, which
makes them attractive targets for new antimalaria drugs. Most of the FIKKs
that are predicted to be exported also have a similar structure in the gate-
keeper position in the kinase domain, suggesting that design of inhibitors
to this region could target the entire FIKK family (Tewari et al., 2010).
Three FIKKs e FIKK4.1, FIKK4.2 (R45) and FIKK12 e have been
demonstrated to have kinase activity using either puried parasite proteins
(Nunes et al., 2007) or recombinant proteins (Kats et al., 2014). Five of
17 FIKK kinases that have been shown to be exported into the RBC are
localized throughout the iRBC (Kats et al., 2014; Nunes et al., 2007). These
kinases seem to localize to areas within the iRBC, such as MCs and at the
iRBC membrane (Nunes et al., 2007) or novel punctate structures
(K-dots) (Kats et al., 2014), that would be consistent with the involvement
of these proteins in iRBC modication. Interestingly, two exported FIKKs
(FIKK9.3 and FIKK10.2) have been shown to be phosphorylated (Solyakov
et al., 2011) indicating that the activity of these kinases could form part of
novel signalling pathways within iRBCs.
While at the present time, potential redundancy among some exported
FIKKs cannot be ruled out, recent data suggest that this is likely to be
limited to only some members of the family. For example, in RBCs infected
with parasite lines from which FIKK4.2 had been deleted, knobs were
signicantly larger than those on RBCs infected with wild-type parasites,
and showed signicantly reduced adhesion to CD36 (Kats et al., 2014).
In contrast, disruptions of kk7.1 or kk12 had no detectable effect on
the adhesive properties of RBCs but the RBCs were less rigid than those
infected with wild-type parasites (Nunes et al., 2010). Furthermore, analysis
of RBC ghost fractions from RBCs infected with wild-type kk7.1- or
kk12-deleted parasite lines using a phosphoprotein-specic dye revealed

clear differences in the levels of phosphorylation in two distinct proteins
of >250 and 80 kDa, respectively (Nunes et al., 2010). Collectively, evi-
dence so far suggests that different members of the FIKK family play distinct
roles in a number of pathophysiologically signicant RBC alterations and
provide strong support for limited redundancy.
3.2.9 P. falciparum proteins involved in trafcking of PfEMP1

At the present time, six proteins have been identied that are involved in
trafcking PfEMP1 to the iRBC surface that collectively are now referred
to as P. falciparum proteins involved in trafcking of PfEMP1 (PfPTPs)
(Table 1) (Boddey and Cowman, 2013; Maier et al., 2008; Rug et al.,
2014). These proteins do not share any similar motifs and, all but one, a
PHISTc protein termed PfPTP2 (Table 3) does not contain any currently
known functional domains. Both PfPTP1 and PfPTP2 are localized in
MCs and PfPTP3 is present in the iRBC cytosol (Maier et al., 2008). The
precise stage at which these proteins are involved in PfEMP1 trafcking dif-
fers slightly with PfPTP2, PfPTP3, PfPTP5 and PfPTP6 important for traf-
cking PfEMP1 from MCs to the iRBC membrane, while PfPTP1 and
PfPTP4 are required for the trafcking of PfEMP1 from the PVM to
MCs (Maier et al., 2008; Rug et al., 2014). Recent evidence suggests that
PfPTP1 interacts with PfEMP1 and forms a complex with SBP1 in MCs,
which is thought to be important for PfEMP1 trafcking (Rug et al.,
2014). PfPTP1 was also shown to be required for the association of MCs
with the RBC membrane skeleton via actin laments (Rug et al., 2014).
Not much is known about the precise roles of this group of proteins in
the trafcking process; however, deletion of PfPTP4, PfPTP5 or PfPTP6
partially reduced the amount of PfEMP1 on the iRBC surface, in compar-
ison deletion of PfPTP1, PfPTP2 or PfPTP3 give rise to iRBCs that are
completely devoid of PfEMP1 on the surface (Maier et al., 2008). This
may indicate that PfPTP1, PfPTP2 and PfPTP3 are essential for trafcking
while PfPTP4, PfPTP5 and PfPTP6 play accessory roles. Interestingly
PfPTP3, PfPTP4 and PfPTP5 also appear to have a role in regulating the
deformability of iRBCs, either by increasing or decreasing membrane rigid-
ity (Maier et al., 2008). PfPTP2 has recently been shown to be important for
cellecell communication via exosomes (Regev-Rudzki et al., 2013). This
function was identied by the reduced efciency in plasmid transfer in a
PfPTP2-deleted parasite line, which was due to a decrease in the number
of exosomes shed into the culture supernatant. This reduction in exosomes
suggests that the function of PfPTP2 is important for the formation of ves-
icles budding from MCs (Regev-Rudzki et al., 2013).
3.2.10 Other less-well characterized exported proteins

The iRBC has been shown to have an overall increase in the level of phos-
phorylation of both host and parasite proteins (Collins et al., 2014; Pantaleo
et al., 2010; Pease et al., 2013; Solyakov et al., 2011; Wu et al., 2009). Tyro-
sine phosphorylation was of particular interest since there are no known
exported tyrosine kinases in Plasmodium. The host tyrosine kinase, Src, has
previously been implicated in this process (Pantaleo et al., 2010); however,
this does not explain all of the phosphorylation seen. A recent study has shed
further light on this with the identication of a tyrosine-like kinase,
PfTKL2, that is enzymatically active and exported to distinct foci in the
iRBC in close proximity to the iRBC membrane (Abdi et al., 2013). Inter-
estingly, PfTKL2 also appears to be secreted out of the iRBC, but precisely
how and why this occurs remains unknown (Abdi et al., 2013).
Two ring-exported proteins, REX3 and REX4, have been shown to be
exported into the iRBC (Spielmann et al., 2006). REX3 is not essential for
parasite survival (Maier et al., 2008) and REX4 has very low levels of expres-
sion with detection of this protein reliant on an over-expressed GFP fusion
(Spielmann et al., 2006). To date, no function has been assigned to either of
these proteins. P. falciparum also contains at least 17 hypothetical gene fam-
ilies (hypehyp17), that encode proteins predicted to be exported (Frech and
Chen, 2013). However, none of these have yet been shown to be exported
and nothing is known about their function (Frech and Chen, 2013). Other
proteins found in iRBCs that have been characterized in some detail that are
not directly linked to iRBC modications include the histidine-rich protein
2, which is implicated in haem detoxication (Papalexis et al., 2001) and the
asparagine and aspartate-rich proteins (PfAARP), that are localized in the
iRBC cytosol but as yet have no known function (Table 1).
3.3 Proteins exposed on the surface of infected RBCs

To date, almost all parasite proteins that have been identied on the surface
of iRBCs are unique to P. falciparum and are generally encoded by multigene
families. While iRBC proteomic studies have attempted to identify all of the
surface-exposed proteins on iRBCs (Florens et al., 2004; Fontaine et al.,
2012; Wu et al., 2009), they have detected only a small number of proteins,
which did not include PfEMP1; highlighting the difculty in detecting rela-
tively low-abundance parasite proteins within the admix of highly abundant
native RBC proteins. In general, proteins exposed on the surface of the

iRBC are widely believed to be important for antigenic variation (necessary
to avoid the host immune response) or for the altered adhesive properties of
the iRBC which is not only crucial for parasite survival in humans but also
plays a central role in the pathogenesis of severe falciparum malaria. A recent
high throughput screen of small molecules found that the adhesion of
iRBCs, at least CSA and ICAM1, could be disrupted, highlighting the pos-
sibility of anti-adhesion-based drugs as a new form of therapy for severe fal-
ciparum malaria (Gullingsrud et al., 2014). This possibility highlights the
importance of this particular area of research that is still in its infancy but
clearly warrants further work.
3.3.1 P. falciparum erythrocyte membrane protein 1

One of the most important and extensively studied proteins expressed by
P. falciparum is its major virulence determinant P. falciparum erythrocyte
membrane protein 1 (PfEMP1). PfEMP1 is encoded by a large multigene
family (w60 members), and while only one copy is expressed on the sur-
face of any given iRBC at one time, the parasite is able to switch PfEMP1
variants in each RBC cycle allowing iRBCs to bind to a wide range of re-
ceptors expressed on the vasculature of the human host (Cooke et al.,
2004b, 2014). Switching between different var genes involves a range of
mechanisms including chromatin silencing and spatial transcription of var
genes at the nuclear periphery (Figueiredo et al., 2002; Voss et al., 2006,
2007; Witmer et al., 2012). The ability of PfEMP1 to switch between
gene variants and their ability to bind to different host receptors has been
reviewed in detail (see (Cooke et al., 2001, 2005; Guizetti and Scherf,
2013; Kirkman and Deitsch, 2012; Scherf et al., 2008; Sherman et al.,
2003) for reviews).
Trafcking of PfEMP1 and its eventual surface display at knobs on the
iRBC surface has been an important focus of research during the past
decade. It has become increasingly apparent that MCs are critical for the
export of PfEMP1 to the iRBC membrane as disruption of a number of
MC-resident proteins ablate the trafcking of PfEMP1 at various stages of
the pathway (Cooke et al., 2006; Dixon et al., 2011; Hanssen et al.,
2008a; Kriek et al., 2003; Maier et al., 2008; Rug et al., 2014; Spycher
et al., 2008). Considering that both MCs and PfEMP1 are unique to P. fal-
ciparum, it is reasonable to assume that these organelles evolved specically to
control PfMEP1 export. This suggests that targeting proteins in MCs could
offer novel therapeutic approaches for prevention and treatment of human
malaria caused by P. falciparum. However, it remains likely that MCs also

have other functions within iRBCs, highlighted by the essential role of
MARHP2 (Pachlatko et al., 2010) for example. Specic details of PfEMP1
trafcking are only just beginning to become clear. While it was originally
believed that PfEMP1 was a PEXELated protein, with a lysine residue at the
rst position in the PEXEL, recent evidence suggests that this is not in fact a
functional PEXEL and that PfEMP1 should actually be considered a PNEP
(Boddey et al., 2013). Additionally, it seems that trafcking of PfEMP1 is
not solely due to a single PEXEL-like motif but a number of domains
that all play a collective role in the export and surface exposure of PfEMP1
(Knuepfer et al., 2005b; McMillan et al., 2013; Melcher et al., 2010). Firstly,
the transmembrane domain of PfEMP1 is required to direct it into the ER
(Knuepfer et al., 2005b). Secondly, the presence of the NTS domain (the
rst 197 amino acids of the protein) directs it into the PVM. Thirdly, the
transmembrane domain (TM) and the ATS is required for its export to
the iRBC membrane (Melcher et al., 2010). However, for translocation
and localization of PfEMP1 on the iRBC surface, a semi-conserved head
group consisting of the NTS, the rst Duffy-binding-like domain (DBL1)
and the rst cysteine-rich interdomain region (CIDR1), together with the
TM and ATS are required (Knuepfer et al., 2005b; Melcher et al., 2010).
Interestingly, the semi-conserved head group can be replaced with the rst
120 amino acids of KAHRP and still be correctly surface exposed (Knuepfer
et al., 2005b), suggesting the N-terminus (semi-conserved head group) is
required to get the protein to the knob structures, while correct surface
display relies only on the TM and ATS domains. As with other TM-con-
taining P. falciparum proteins that are exported, PfEMP1 appears to be
exported in soluble complexes, possibly containing the Hsp70x/DnaJ chap-
erone/co-chaperone, within the iRBC (Knuepfer et al., 2005b; K ulzer
et al., 2012). Before PfEMP1 reaches the iRBC membrane, it is rst inserted
into the membrane of MCs with the conserved C-terminal domain exposed
to the iRBC cytosol (Kriek et al., 2003). Not much is known of how
PfEMP1 is inserted and then subsequently exposed on the surface and clus-
tered at knobs. While MCs have been shown to be important in PfEMP1
trafcking (Cooke et al., 2006; Dixon et al., 2011; Hanssen et al., 2008a;
Kriek et al., 2003; Maier et al., 2008; Rug et al., 2014; Spycher et al.,
2008), to date only one resident MC protein, PfPTP1, has been shown to
interact with PfEMP1 and is critical in trafcking PfEMP1 to MCs (Rug
et al., 2014). While there is little agreement of how PfEMP1 nally moves
from MCs to the iRBC surface, it seems most likely that it involves vesicle
trafcking, possibly directed to the iRBC membrane via protein interactions

involving either MC-resident proteins or actin laments or possible unique
tether-like structures associated with MCs (Cyrklaff et al., 2011; Hanssen
et al., 2010b; Knuepfer et al., 2005b; McMillan et al., 2013; Melcher
et al., 2010; Rug et al., 2014). It is clear that MCs become stationary in
the later stages of the parasites development in RBCs (Gr uing et al.,
2011; Kilian et al., 2013) indicating some form a tethering, either through
membranous structures or proteineprotein interactions. What also seems
likely is that trafcking of PfEMP1 from MCs to the iRBC membrane in-
volves a preformed knob complex already containing PfEMP1 (Hanssen
et al., 2010b; McMillan et al., 2013); however how these are formed, either
in the MCs, or in vesicles after PfEMP1 has left MCs, is not known. In
RBCs containing abnormal haemoglobins, the parasite produces dramati-
cally altered knobs on the surface of the iRBC and these appear to correlate
with a decreased level of cytoadherence (Fairhurst et al., 2012). Interestingly,
MCs in these iRBCs appear not to be anchored as efciently to the RBC
membrane as in normal RBCs (Kilian et al., 2013) and there is evidence
that this is due to disruption of actin laments (Cyrklaff et al., 2011).
Together these ndings imply an important role for actin laments in
anchoring MCs to the iRBC membrane, which in turn can be used to trafc
PfEMP1 to the membrane via vesicle such as the EDV or preformed knobs.
However, there is some evidence to suggest that PfEMP1 is trafcked to and
exposed on the surface of these iRBCs, albeit in a reduced amount (Fairhurst
et al., 2012) suggesting that if these laments are used, it is not the only
pathway that PfEMP1 can utilize. There is also evidence that disruption
of knob formation can disrupt adhesion without affecting PfEMP1 traf-
cking (Kats et al., 2014; Rug et al., 2006), indicating that trafcking and
surface display of PfEMP1 is not reliant on knob formation. It is likely
that there are multiple pathways for the parasite to get PfEMP1 to the sur-
face of the iRBC; however, it is the correct display on intact knobs that is
critical for its function in cytoadherence.
While the formation of knobs is important for the correct display and
function of PfEMP1, not much is known about the precise protein compo-
sition knobs. What is known is that KAHRP is a major component of
knobs. Several studies have shown that KARHP is required for anchoring
PfEMP1 at the knobs, which in turn is important for the interaction of
PfEMP1 with host receptors (Oh et al., 2000; Rug et al., 2006; Waller
et al., 1999, 2002). While these studies used a recombinant VARC and
VARCD based on the var-2 on chromosome 7 (Oh et al., 2000; Su et al.,
1995; Waller et al., 1999, 2002), a separate group was unable to show this
interaction with the recombinant ATS region based on var variants from
chromosome 2, 3, 6 and 8. They did however show an interaction with a
PHIST domain-containing protein (Mayer et al., 2012). While the recom-
binant proteins of the PfEMP1 C-terminal domain are similar among the
variants used, this difference in binding may highlight slight differences in
the variants of PfEMP1 expressed and the type of anchoring mechanism.
While it remains possible that there are other proteins that are localized to
knobs there is yet no detailed analysis of these knobs, which is a clear gap
in our current knowledge and is likely to reveal novel components that
would be critical for the function of knobs and likely to have effects of
the adhesive properties of the iRBC.
3.3.2 RIFINs
RIFINs are small (36e42 kDa) proteins containing two transmembrane do-
mains and are encoded by the repetitive interspersed family (rif ) of genes of
which there are about 160 copies, each located within the sub-telomeric re-
gion of the chromosomes (Cheng et al., 1998; Sargeant et al., 2006). The
RIFINs can be subdivided into two groups, RIFIN A and RIFIN B, accord-
ing to the presence of a conserved peptide in RIFIN A, which is absent in
the B subgroup, and by the number of conserved cysteine residues in the
protein ( Joannin et al., 2008; Petter et al., 2007). For some time, it was
widely accepted that all parasites expressed only one RIFIN, on the surface
of iRBCs (Cheng et al., 1998; Fernandez et al., 1999; Kyes et al., 1999).
While this remains true for the surface-expressed RIFIN, it has been shown
that both subgroups can be expressed in a single iRBC; however, the two
groups show very different localization patterns; RIFIN As being exported
onto the surface whereas RIFIN Bs localizing to the PV (Petter et al., 2007).
This clearly indicates that the two subgroups have distinct functions. RIFIN
As, once exported, rst localize to MCs before being trafcked to the iRBC
surface in a similar manner to PfEMP1 (Fernandez et al., 1999; Khattab and
Klinkert, 2006; Kyes et al., 1999; Petter et al., 2007). While the function of
both subgroups of RIFINs remains unknown the iRBC surface expression
of clonal variants of the RIFIN A subgroup suggests that this group may play
an important role in antigenic variation (Fernandez et al., 1999; Kyes et al.,
1999). There also appears to be a link between the expression patterns of
RIFIN A and PfEMP1 proteins with the switching of the PfEMP1 in an
iRBC accompanied by a switch in the RIFIN A protein expression
(Wang et al., 2009). However, while PfEMP1 does not seem to be
expressed on the surface of iRBC infected with gametocytes, this is not the
case with RIFINS, with both A and B subgroups being expressed in iRBCs
infected with all stages of gametocytes. This suggests that RIFINs may have
diverse roles throughout all blood stages of the parasites life cycle (Petter
et al., 2008).
3.3.3 STEVOR
STEVOR is another family of small (30 kDa) proteins that are clonally
expressed on the iRBC surface (Cheng et al., 1998; Niang et al., 2009).
They are predicted to contain two TM domains, and like RIFINs have a
highly variable region that is predicted to be exposed on the iRBC surface
(Cheng et al., 1998). There are about 30 different stevor genes (in 3D7 para-
site), which form part of the 2TM superfamily (Lavazec et al., 2006; Sargeant
et al., 2006). STEVORs have a PEXEL motif and are trafcked into the
iRBC where they associate with MCs (Przyborski et al., 2005). This initial
localization to MCs requires the presence of at least one of the TM domains
(Przyborski et al., 2005). The exact function of STEVORs is not known;
however, they have been implicated in antigenic variation (Niang et al.,
2009). This becomes apparent when looking at expression of STEVORs
in clinical parasite isolates which show a marked increase in expression levels,
with over 90% of iRBCs expressing a STEVOR, when compared to
laboratory-adapted parasite lines such as 3D7, where only 30% of iRBCs ex-
press a STEVOR (Blythe et al., 2008). STEVORs have also been shown to
play a role in the altered membrane rigidity of iRBCs, where an increase in
STEVOR expression was linked to an increase in iRBC membrane rigidity
in a number of laboratory parasite clones (Sanyal et al., 2012). This implies
that STEVORs may have a dual role in antigenic variation of the iRBC and
in the alteration of the membrane mechanical properties of iRBCs (Niang
et al., 2009; Tiburcio et al., 2012).
3.3.4 SURFINS
SURFINS are a family of 10 large (w300 kDa) exported proteins contain-
ing a single TM domain that are expressed on the surface of both iRBCs and
merozoite stages of the parasite (Winter et al., 2005). They contain a highly
variable region, which is predicted to be exposed on the iRBC surface and
are encoded by the surface-associated interspersed (surf ) genes. surf, as with
the other genes encoding variable surface antigens, are located within the
sub-telomeric region of chromosomes (Winter et al., 2005). SURFINs
are differentially expressed and localized; for example, SURFIN 4.2 is
present on the iRBC surface and the apical regions of the merozoite while
SURFIN 4.1 is found only on the merozoite surface (Mphande et al., 2008;
Winter et al., 2005). The SURFINs that are localized on the iRBC surface
seem to be co-transported to the iRBC membrane with other surface anti-
gens, such as PfEMP1 and RIFINs via MCs (Winter et al., 2005; Zhu et al.,
2013). Interestingly, SURFINs, PfEMP1 and RIFINs all contain a semi-
conserved tryptophan-rich domain (WRD) in their cytoplasmic tails (Frech
and Chen, 2013; Winter et al., 2005), which could be important for traf-
cking of these proteins. The role of WRD in trafcking has been shown
for SURFIN 4.2 where it is required for the export and localization to
the MCs (Zhu et al., 2013). The correct trafcking of SURFINs also re-
quires the transmembrane domain and the rst 50 amino acids (Zhu et al.,
2013) and while SURFINs contain a sequence that resembles a PEXEL
motif, this PEXEL-like motif does not appear to be required for export
(Alexandre et al., 2011; Zhu et al., 2013). There are two independent se-
quences within the rst 50 amino acids that individually are able to direct
export into the iRBC (Zhu et al., 2013). While nothing is known about
the function of these proteins, the localization at the surface in both iRBCs
and merozoites, in combination with their highly polymorphic region, sug-
gests that they are required for antigenic variation, a function important for
all parasite stages.
3.3.5 Glycophorin-binding proteins

Glycophorin-binding protein 130 (GBP130) was rst identied as a large
exported protein that was able to bind to the cytoplasmic tail of glycophorin
in RBCs (Perkins, 1988). Since then, three paralogues of GBP130 have been
identied in P. falciparum (Nolte et al., 1991; Sargeant et al., 2006). The
function of this group of proteins is not well understood; however, at least
one of the paralogues (GBHP-2) has been shown to be essential for parasite
survival in vitro (Maier et al., 2008). GBP130 is by far the most extensively
characterized member of this group. While is has been shown that GBP130
plays a role in decreasing the membrane rigidity of iRBCs (Maier et al.,
2008) (similar to Pf332), most of the analysis of GBP130 involves the traf-
cking of proteins within the iRBC. The PEXEL of GBP130, along with
that of KAHRP, is often used as a typical PEXEL in multiple trafcking
studies (Boddey et al., 2009a, 2013, 2010; Hiller et al., 2004; Marti et al.,
2004; Riglar et al., 2013). GBP130 was used to prove for the rst time
that protein transport from the parasite to the iRBC was a two-stage process
that requires translocation through the PV (Ansorge et al., 1996).
3.3.6 Cytoadherence-linked asexual gene

The cytoadherence-linked asexual gene (CLAG) is encoded by a ve-
member gene family (Table 1) located on chromosomes 2, 3, 8 and 9.
CLAG 2, CLAG 3.1, CLAG 3.2 and CLAG 8 are all similar at the amino
acid level, but show high degree of polymorphism within a distinct region
towards the C-terminus (amino acids 1000e1200) (Iriko et al., 2008). The
function of this family of proteins remained enigmatic until recently, with
proposed functions of different members of the family differing widely.
CLAG 9, the most distinct member of the group, was the rst to be iden-
tied following the deletion of the sub-telomeric end of chromosome 9,
which was associated with a loss of cytoadhesion; a phenotype later
conrmed by targeted disruption of the clag9 gene (Trenholme et al.,
2000). However, subsequent studies failed to document the export of
CLAG 9 or its association with any protein involved in cytoadherence. Later
studies have in fact shown that all members of this family are localized in the
rhoptries and form part of the RhopH complex of proteins that are impli-
cated in the invasion process (Gardiner et al., 2004; Kaneko et al., 2001,
2005; Ling et al., 2004). Interestingly, immunoprecipitation experiments
revealed that while all CLAGs could partake in the formation of the RhopH
complex, each individual complex contains only a single CLAG (also known
as RhopH1), suggesting that this family could be involved in the alternative
pathways used by P. falciparum to invade RBCs (Kaneko et al., 2001, 2005).
CLAG 9 and the entire RhopH complex has been shown to be transferred
into the RBC following invasion (Ling et al., 2004; Vincensini et al., 2008),
which suggests an alternative role for this protein in the iRBC. A recent
study has revealed that CLAG 3.1 and CLAG 3.2, while initially within
the rhoptries, are also transferred into the newly invaded RBC (Nguitragool
et al., 2011, 2014). This study used a high throughput screen to identify a
role for CLAG 3.1 and CLAG 3.2 in nutrient uptake by the parasite and
more importantly that these CLAGs were not only localized at the iRBC
membrane but exposed on the surface of the iRBC. Protease protection ex-
periments revealed that a small proportion of the CLAG 3 proteins were
cleaved from a full-length protein of 160 kDa to a smaller processed version
of about 35 kDa, implying that the protein is largely exposed on the iRBC
surface but with a small portion of the C-terminus projecting inside the
iRBC (Nguitragool et al., 2011). The role of CLAG 9 in adhesion and
CLAG 3.1/3.2 in nutrient uptake may in time reveal the true functional di-
versity of this gene family. While it was believed that the CLAGs (RhopH1)
were required for alternative parasite invasion pathways, this function is yet
to be demonstrated. However, it is becoming increasingly apparent that

CLAG family is not involved in invasion but form a complex with RhopH2
and RhopH3 as a means to transport into the iRBC during parasite invasion
of RBCs. While the role of the RhopH complex in trafcking CLAG into
the iRBC has not been demonstrated, it highlights the possibility that resi-
dent rhoptry bulb proteins are not only required for the initial stages in PVM
development (Zuccala et al., 2012) but also in important processes within
the iRBC, which is supported by other rhoptry bulb proteins like the
RAP2 also localizing to the iRBC following invasion (Douki et al., 2003).
3.3.7 Other less-well characterized putative iRBC surface proteins

A proteomic approach identied 36 putative proteins on the iRBC surface;
however, only two were further characterized in an attempt to validate this
data set (Florens et al., 2004). These proteins, termed parasite-infected eryth-
rocyte surface proteins (PIESP) 1 and 2, appeared to be exported and by
immunouorescence showed a distinct dot-like pattern within the iRBC
(Florens et al., 2004), PIESP2 was also localized to MCs in a subsequent
study (Vincensini et al., 2005). While this study did not conclusively co-
localize PIESP2 with other previously characterized MC proteins, results
from protease protection experiments were consistent with a MCs-resident
protein (Vincensini et al., 2005). It remains unclear whether PIESP2 transits
though MCs en route to the iRBC surface or if the identication of PIESP2
is merely a result of the detection of MC proteins (possible MC surface) in
the original surface proteome. PIESP2 has recently been suggested to
interact with PfPTP1, an integral MC membrane protein, further suggesting
that PIESP2 is in fact resident in MCs (Rug et al., 2014). While PIESP2 has
been deleted in parasites (Maier et al., 2008), no function has yet been
assigned to either of the PIESPs.
Another protein that has been suggested to be on the surface of the
iRBC is the liver-stage surface antigen 3 (LSA3) (Aidoo et al., 2000;
Guerin-Marchand et al., 1987; Moyano et al., 2007). While there is no
experimental evidence that this protein is expressed in blood-stage parasites,
mRNA transcripts have been identied (Moyano et al., 2007). The fact that
this protein contains a PEXEL and is located on the surface of infected he-
patocytes suggests that if it is expressed, it would likely localize on the iRBC
surface (Aidoo et al., 2000; Guerin-Marchand et al., 1987; Moyano et al.,
2007). The gene encoding LSA-3 has been deleted in blood-stage parasites;
however no abnormal phenotype was observed (Maier et al., 2008). Expres-
sion in both the liver- and blood-stage parasites has also been reported for
the P. berghei protein, IBIS1 (Gr

utzke et al., 2014; Ingmundson et al., 2012).
While there is no orthologue of IBIS1 in P. falciparum, it does highlight the
potential for exported proteins to be functional in multiple parasite develop-
mental stages.
3.3.8 Exported proteins of sexual stage parasites

A number of other putative exported proteins have been detected by pro-
teomic analysis with several validated either by the cleavage and acetylation
of the PEXEL motif or by localization data in gametocytes (Eksi et al., 2005;
Silvestrini et al., 2010). These proteins consist of a diverse group including
both PHIST and DnaJ proteins (Tables 3 and 4) (Silvestrini et al., 2010).
Two PHISTa proteins have been detected in gametocytes; Pfg14.478 and
Pfg14.744 and have been shown to be expressed early in gametocyte-stage
parasites and are exported to the iRBC cytosol (Pfg14.744) or to the PV of
gametocytes (Pfg14.748) (Eksi et al., 2005). The early timing of these
PHISTa proteins in gametocytogenesis also suggests that it may play a role
in sexual stage commitment (Eksi et al., 2005). PfPTP4, along with
PF3D7_0532600, are examples of exported proteins that are involved in
the development of sexual stage parasites (Ikadai et al., 2013). Disruption
of these genes resulted in either the lack of stage I gametocytes
(PF3D7_0532600) or parasites that could not develop past stage I (PfPTP4)
(Ikadai et al., 2013). The gametocyte erythrocyte cytosolic protein (GECO)
is a member of the DnaJ family (Table 4) and while the function of this pro-
tein is not known, it remains possible that GECO may be involved in the
regulation of the membrane mechanical properties of iRBCs infected
with gametocyte-stage parasites. The membrane mechanical properties of
gametocyte-infected RBCs are important for sequestration of early stage ga-
metocytes in the host (Tiburcio et al., 2012) and it remains possible that
GECO could be an important player in this process. The changes in
iRBC membrane deformability become particularly interesting in the sexual
stages of P. falciparum. While PfEMP1 is not involved in the sequestration of
RBCs infected with early stage gametocytes, increased RBC rigidity appears
to be the mediator of sequestration of gametocyte iRBCs in the spleen and
bone marrow (Tiburcio et al., 2012). Subsequently, an increase in RBC
deformability allows iRBCs infected with mature-stage gametocytes to be
released back into the circulation. This release coincides with the reduction
of surface-exposed STEVOR, suggesting that the STEVOR was disassoci-
ating from the membrane skeleton (Tiburcio et al., 2012). These changes in
RBC deformability are critical for the correct development of the sexual
stages, which in turn are essential for the human to mosquito transmission
of malaria parasites. While there is limited functional data for these
gametocyte-exported proteins, they are intriguing and further work on
these is warranted (Ingmundson et al., 2014). Importantly, characterization
of exported proteins in gametocytes could reveal novel targets for potential
transmission-blocking agents.
4. ALTERATION OF HOST RBC PROTEINS DURING

MALARIA INFECTION
The importance of phosphorylation in the alteration of the structure
and function of RBCs during malaria infection is becoming increasingly
recognized (Collins et al., 2014; Pantaleo et al., 2010; Pease et al., 2013;
Solyakov et al., 2011; Treeck et al., 2011; Wu et al., 2009). In addition
to the parasites exported kinases such as the FIKK family, there is also ev-
idence that the parasite commandeers RBC kinases such as Src (Pantaleo
et al., 2010). The parasite induces changes in phosphorylation status of
both parasite-encoded and host RBC proteins, particularly those associated
with the iRBC membrane skeleton (Collins et al., 2014; Pantaleo et al.,
2010; Pease et al., 2013; Solyakov et al., 2011; Wu et al., 2009). These
changes include increased phosphorylation of a number of membrane skel-
etal proteins including spectrin, ankyrin, actin and protein 4.1R (Pantaleo
et al., 2010; Solyakov et al., 2011; Wu et al., 2009), all of which have
been shown to interact with parasite-exported proteins that have roles in
both cytoadherence and alteration of RBC mechanical properties. This
clearly indicates RBC modications could also depend on the phosphoryla-
tion status of both host RBC and parasite proteins. It has also become
apparent that the levels of phosphorylation of many proteins including a
range of exported proteins differ throughout the parasites life cycle (Pease
et al., 2013) highlighting the importance of phosphorylation as a key control
mechanism in many processes. The potential role of phosphorylation in
parasite virulence implies that the use of kinase inhibitors could be of interest
not only inunderstanding the molecular mechanisms of virulence but also as
future new antimalaria drugs.
5. CONCLUSION
One of the many intriguing aspects of malaria is the ability of the para-
site, particularly P. falciparum, to alter the host RBC membrane properties.
An increasing number of proteins have been identied that are involved in

the structural and functional changes that occur in iRBCs, and virtually all
are associated with the unique virulence properties of P. falciparum. The
vast majority of proteins involved in virulence are encoded by genes found
in the sub-telomeric regions of the parasites chromosomes, an area that per-
mits recombination of genes resulting in the diverse variety and often large
protein families. There are also a sub-set of proteins that further increase the
complexity of the exported proteins, as they contain a combination of
domains present in multiple protein families, indicating that this mixing of
domains could be important for diversifying the functions of the exported
proteins. Only a small number of the known exported proteins have been
well characterized to date and yet more proteins are being identied on a
regular basis, highlighting that this area of research has still a long way to
go before the export and in turn virulence of P. falciparum is completely un-
derstood. Interestingly the majority of proteins that have been characterized
have functions that result in the modication of the host RBC such as mem-
brane deformability, membrane stability, cytoadhesive properties or anti-
genic variation. While this seems like a limited number of functions for
such a large number of proteins, it clearly highlights the complexity of these
processes and the level of control required by the parasite. One explanation
for the large number of exported proteins required by the parasite is the pos-
sibility that there are sets of proteins that are required for individual var genes,
as highlighted by the expression of particular RIFINs or PHISTs that are
preferentially expressed with the specic var genes (Daily et al., 2005;
Goel et al., 2014; Tuikue Ndam et al., 2008; Wang et al., 2009). What is
clear, is that this high level of protein export, of a number a highly diverse
proteins, has helped the parasite thrive in the human host, and an under-
standing of the interactions between parasite and host proteins could help
in the effort to control this pathogen.
The complexity of roles of exported proteins continues in the iRBC
when comparing the asexual and sexual stages of the parasite, particularly
in the sequestration of the iRBC in the spleen and bone marrow. Asexual
stage parasites rely on the trafcking and correct display of PfEMP1 to
mediate cytoadherence, and while a number of proteins have been identied
that are required for this in some way, the exact mechanisms remain elusive
(Cooke et al., 2006; Dixon et al., 2011; Kats et al., 2014; Maier et al., 2007,
2008; Mayer et al., 2012; Oberli et al., 2014; Proellocks et al., 2014; Rug
et al., 2006; Spycher et al., 2008; Waller et al., 1999, 2002). Cytoadhesion,
however, does not seem to be required for the sexual stages as it was recently
shown that RBCs infected with P. falciparum gametocytes were unable to

adhere to host vascular endothelium (Silvestrini et al., 2012). The sequestra-
tion of RBCs infected with gametocyte stages, and their subsequent release
into the circulation, relies on alterations to the deformability of the iRBC
membrane (Tiburcio et al., 2012). This stark difference between the asexual
and sexual stages highlights the need for specic approaches in the design for
intervention strategies to not only alleviate the disease caused by this seques-
tration of parasites but also in the possibility in the development of transmis-
sion-blocking agents. Recent work has shown the merit of looking for small
molecules to disrupt cytoadhesion (Gullingsrud et al., 2014), clearly demon-
strating the potential for the development of drugs designed to disrupt
cytoadhesion and prevent the more severe symptoms of P. falciparum malaria.
While this research remains in its infancy and still relatively limited, the po-
tential alone makes this a critically important area of research to pursue in the
future. While this remains important, a block in cytoadhesion would not be
effective against the sexual stages. These stages are unlikely to contribute
signicantly to disease due their relatively low number; however, the trans-
mission of mature gametocytes and therefore spread of the disease is still a
major concern. To this end the development drugs to target the alterations
in membrane deformability could be of interest. The disruption of the con-
trol the parasite has in iRBC membrane deformability could disrupt the
maturation of gametocytes or force immature gametocyte into circulation
where they would be more vulnerable to clearance. The possibility of the
development of transmission-blocking agents designed specically to target
this process could also have an additional effect by also targeting the asexual
stages where membrane deformability is also a key function of many
exported proteins. Further understanding of these processes in all stages of
parasite-infected RBC would provide the critical information on the precise
mechanism(s) involved and in turn the knowledge of how to target and
disrupt this process. Further detailed characterization of exported proteins
in P. falciparum is clearly required for a better understanding of these mech-
anisms and the development of future novel intervention strategies.
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CHAPTER TWO
Harnessing the Toxocara Genome

to Underpin Toxocariasis Research
and New Interventions
Robin B. Gasser*, 1, Pasi K. Korhonen*, Xing-Quan Zhux,
Neil D. Young*
*Faculty of Veterinary and Agricultural Sciences, The University of Melbourne, Parkville, VIC, Australia
x
State Key Laboratory of Veterinary Etiological Biology, Key Laboratory of Veterinary Parasitology of Gansu
Province, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou,
Gansu Province, PR China
1
Corresponding author: E-mail: robinbg@unimelb.edu.au
Contents
1. Introduction 88
2. Signicance of Toxocara and Diagnostic Considerations 89
3. Molecular Discovery in Toxocara Prior to Large-Scale Genomic and Transcriptomic 92
Analyses
4. T. canis Genome and Transcriptomes Give First Global Insights Into This Pathogens 93
Molecular Biology
4.1 Genome and gene set 93
4.2 Molecular groups and their key biological and/or biotechnological relevance 94
4.3 Insights into the pathogens biology 97
5. Prospects for New Intervention Targets in Toxocara and Related Parasites 98
6. Conclusion 100
Acknowledgements 101
References 101
Abstract
Parasitic worms, such as atworms (platyhelminths) and roundworms (nematodes),
cause substantial morbidity and mortality in animals and people globally. The ascari-
doid nematode Toxocara canis is a zoonotic parasite of socioeconomic signicance
worldwide. In humans, this worm causes toxocariasis (disease) mainly in underprivi-
leged communities in both the developed and developing worlds. While reasonably
well studied from clinical and epidemiological perspectives, little is understood about
the molecular biology of T. canis, its relationship with its hosts and the disease that it
causes. However, a recent report of the draft genome and transcriptomes of T. canis
should underpin many fundamental and applied research areas in the future. The pre-
sent article gives a background on Toxocara and toxocariasis, a brief account of
2016 Elsevier Ltd.
j
ISSN 0065-308X
88 Robin B. Gasser et al.
diagnostic approaches for specic identication and genetic analysis, and gives a
perspective on the impact that the genome of T. canis and advanced molecular tech-
nologies could have on our understanding of the parasite and the diseases that it
causes as well as the design of new and improved approaches for the diagnosis, treat-
ment and control of toxocariasis.
1. INTRODUCTION
Parasitic worms have a major, chronic impact on human and animal
health worldwide. For example, it is estimated that about two billion people
are infected with soil-transmitted helminths (STHs), such as the large
roundworm (Ascaris), hookworms (Ancylostoma and Necator) and whipworm
(Trichuris), mainly in underprivileged communities in parts of Asia, Africa
and Latin America (Hotez et al., 2009). The disease burden linked to these
parasites is comparable to that of tuberculosis and malaria (Hotez et al.,
2009). Ascaris, for instance, infects more than one billion people, causing
nutritional deciency, impaired cognitive and physical development, usually
in children, and, in severe cases, death (Crompton, 2001). The socioeco-
nomic importance of Toxocara (a related ascaridoid) might be higher than
presently reported. For instance, in poverty-stricken areas of the USA alone,
it has been predicted that millions of people are exposed to or infected with
Toxocara canis (see Hotez and Wilkins, 2009; Barry et al., 2013; Hotez et al.,
2013; Woodhall et al., 2014). Toxocariasis results from the zoonotic
transmission of Toxocara from carnivores, including canids and felids, to
humans (Gasser, 2013; Jenkins et al., 2013; Macpherson, 2013; Dantas-
Torres and Otranto, 2014). Toxocara canis of canids is recognized as the
main causative agent of this disease; the worm has a complex life cycle,
which can also involve rodents and other animals as paratenic hosts (Strube
et al., 2013). In humans, particularly children, following the ingestion of
infective eggs of T. canis, larvae penetrate the intestinal wall and invade
various tissues and cause visceral larva migrans (VLM), ocular larva migrans
(OLM), covert toxocariasis (CT) and/or neurotoxocariasis (NT) (e.g. eosin-
ophilic meningoencephalitis) (Vidal et al., 2003; Nash, 2005; Holland and
Smith, 2006; Rubinsky-Elefant et al., 2010; Finsterer and Auer, 2013;
Nicoletti, 2013; Bowman, 2014; Moreira et al., 2014). Some reports have
also indicated an association between T. canis infection and allergic disorders,
such as chronic pruritus, urticaria and/or asthma (Pinelli et al., 2006, 2008;
Cooper, 2008; Overgaauw and van Knapen, 2013). Both the T. canis-dog
Harnessing the Toxocara Genome 89
and -mouse models (Akao, 2006; Schnieder et al., 2011) represent useful
tools for exploring toxocariasis and the biology of T. canis, parasiteehost in-
teractions at the immunomolecular level.
Major and rapid developments in various genomic and bioinformatic
technologies (Mardis, 2008, 2013; Koboldt et al., 2013) provide unprece-
dented prospects for studying many fundamental aspects of T. canis and tox-
ocariasis. This progress might also provide an avenue to developing
enhanced intervention methods through the identication and characteriza-
tion of novel drug and vaccine targets, and dening genetic or biological
markers for improved diagnostic applications. The present article (1) reviews
some aspects of Toxocara, particularly T. canis, and its animal and human
health signicance, (2) summarizes recent progress on the sequencing of
the T. canis genome and transcriptomes, and (3) emphasizes the prospects
that knowledge of this genome offers for future investigations of the
genetics, biology and epidemiology of T. canis/toxocariasis from both the
human and animal health perspectives, and for the design of diagnostic
approaches and interventions against human toxocariasis.
2. SIGNIFICANCE OF TOXOCARA AND DIAGNOSTIC

CONSIDERATIONS
Parasitic nematodes of the superfamily Ascaridoidea infect all major
groups of vertebrates (cf. Hartwich, 1974; Sprent, 1956, 1958, 1959,
1983, 1992; Anderson, 2000; Bowman, 2014), and some taxa are of partic-
ular socioeconomic importance. Toxocara is a signicant genus because some
species cause clinical disease and are transmissible from animals to humans
(i.e. are zoonotic) (e.g. Pawlowski, 2001; Despommier, 2003; Magnaval
and Glickman, 2006; Macpherson, 2013; Bowman, 2014). In particular,
larvae of T. canis are capable of invading human tissues and causing various
forms of disease (including VLM, OLM, CT and NT). Larvae of other ascar-
idoids, including Toxocara cati, Toxocara vitulorum and Toxascaris leonina, can
also invade the tissues of laboratory animals, but there has been some uncer-
tainty as to what extent they are implicated in human disease (Holland and
Smith, 2006; Bowman, 2014). Toxocara cati appears to be underestimated as a
zoonotic agent (Fisher, 2003), and there is a possibility that Toxocara malay-
siensis (Gibbons et al., 2001) might also be zoonotic in endemic countries,
but it is unclear to what extent.
Extending early studies (Rohde, 1962; Lee et al., 1993), employing mo-
lecular methods, Zhu et al. (1998a) showed that T. canis from Malaysian
cats was genetically more similar to T. cati than T. canis. In a subsequent

morphological study, Gibbons et al. (2001) named this parasite T. malaysien-
sis n. sp. and found three key morphological features (shape of cervical alae
in cross section; spicule length; lip structure) that allow its differentiation
from T. canis, T. cati and other congeners, such as Toxocara apodemi, Toxocara
mackerrasae (from rodents), Toxocara paradoxura and Toxocara sprenti (viver-
rids), Toxocara pteropodis (bats), Toxocara tanuki (canids) and Toxocara vajras-
thirae (mustelids). The discovery of this new species of Toxocara in cats
raised doubts about the identity of T. canis found in cats in other parts of
the world (cf. Calero et al., 1951; Hitchcock, 1953; Ash, 1962; Sprent
and Barrett, 1964; Parsons, 1987; Baker et al., 1989; Scholz et al., 2003)
and encourages genetic comparisons of Toxocara spp. from different hosts
and geographical origins. Also a study from Switzerland raises questions as
to the epidemiological signicance of detecting T. cati eggs in dog faeces
(Fahrion et al., 2011), warranting future investigations.
Clearly, the accurate identication and differentiation of Toxocara species
are central to the specic diagnosis of toxocariasis in any vertebrate host and
to studying their life cycles, epidemiology, and population biology. Tradi-
tionally, ascaridoids have been identied using morphological characters
of different life cycle stages and their predilection site(s) in their host(s)
(Bowman, 2014). Sprent (1983) assessed the taxonomic relevance of struc-
tures such as the caecum, excretory system, labia, male tail and oesophagus.
Other authors described features for specic identication (Nichols, 1956;
Bowman, 1987; Averbeck et al., 1995). For instance, Nichols (1956)
described the diagnostic characters of T. canis and T. cati larvae, and
Averbeck et al. (1995) reported criteria for the differentiation of T. canis
from T. leonina and Baylisascaris procyonis based on the morphology of the
adult and egg stages. Nonetheless, despite existing descriptions and keys,
there are challenges in specically identifying and differentiating some
developmental stages of ascaridoids, or parts thereof, using microscopic
approaches.
Immunological or serological techniques are commonly used for the
diagnosis of T. canis infection or toxocariasis in humans or paratenic hosts.
Usually, an enzyme-linked immunosorbent assay (ELISA) employing
Toxocara excretory/secretory antigen (TES) produced from T. canis larvae
in vitro (de Savigny, 1975) is used to detect serum antibodies, after which
any test-positive sera can then be tested by Western blotting (Fillaux and
Magnaval, 2013). Although useful to estimate seroprevalence, the major
limitation of these techniques is that they are not able to distinguish among
current infection, past infection and exposure with/to Toxocara (Fillaux and
Magnaval, 2013). Moreover, it is also possible that serum antibodies to
Toxocara spp. other than T. canis, other ascaridoids (e.g. Ascaris) and/or other
pathogens (due to infection or exposure) can cross-react with T. canis TES in
ELISA or on immunoblots.
Biochemical and nucleic acid methods have also been useful for
diagnostic, taxonomic and population genetic applications to ascaridoid
nematodes (Nadler, 1986, 1987, 1990). Since the 1990s, methods based
on the polymerase chain reaction (PCR) (Saiki et al., 1988) took over for
the identication and diagnosis of infections as well as genetic analysis of
different developmental stages of nematodes (Gasser, 2006; Gasser et al.,
2006). These techniques have found broad applicability, mainly because
of their ability to specically amplify selected nucleic acid regions from min-
ute (picogram) amounts of genomic DNA isolated from fresh or xed para-
site material (e.g. single nematode eggs and tiny sections of larvae or adult
stages). Central to the application of such techniques is the selection of suit-
able genetic markers for the particular task at hand. As different genes evolve
at different rates, the DNA target (marker) selected should exhibit sufcient
sequence variation to allow the identication of parasites to the taxonomic
level required. For specic identication, a marker sequence should differ
enough among species, with no or minor within-species variation. In
contrast, for the purpose of identifying genetic or population variants,
sequence variation within a species is desirable.
On the one hand, the rst and second internal transcribed spacers (ITS-1
and ITS-2) of nuclear rDNA sequences have been shown to provide useful
genetic markers for the identication and differentiation of ascaridoid species
and diagnosis of infection (e.g. Jacobs et al., 1997; Zhu et al., 1998a,b, 1999,
2000a,b, 2001a,b, 2002; Li et al., 2006, 2007, 2008a,b; Gasser, 2006, 2013).
On the other hand, mitochondrial (mt) markers are useful for population
genetic and systematic studies (e.g. Anderson et al., 1993, 1998; Blouin
et al., 1995, 1997; Viney, 1998; Blouin, 2002; Hu et al., 2004; Hu and
Gasser, 2006; Hu et al., 2007; Jex et al., 2010a,b, 2015). The mt genomes
characterized for T. canis, T cati, T. malayensis and T. leonina (see Jex et al.,
2008; Li et al., 2008b; Liu et al., 2014) have provided a rich source of
markers for population genetic and epidemiological investigations of key
ascaridoids. To enable such studies, conserved primers anking suitably
variable gene regions in mt genomes can now be identied by sliding
window analysis (cf. Mohandas et al., 2014) and then used for PCR-based
analyses (Gasser et al., 2006).
3. MOLECULAR DISCOVERY IN TOXOCARA PRIOR TO

LARGE-SCALE GENOMIC AND TRANSCRIPTOMIC
ANALYSES
In addition to taxonomic and population genetic investigations, many
studies have been focused on understanding the immunobiology, physi-
ology and/or biochemistry of T. canis using various molecular methods. A
key thrust has been on the discovery of genes encoding dominant secreted
and surface molecules of its arrested larval stage. Such molecules are
believed to play key roles in immune evasion (reviewed by Maizels et al.,
2000; Schnieder et al., 2011). The predominant research effort in this area
has been directed at understanding how the parasite is able to neutralize,
block or evade the host immune response for years, and survive in mamma-
lian tissues for so long without growing and developing. This research has
been possible because larvae can be maintained in vitro in serum-free
medium, allowing the production of TES (de Savigny, 1975) for molecular
investigations. Employing cloning and expressed sequence tag-based
methods (cf. Maizels et al., 2000, 2006), the main genes identied encode
major TES components, including aquaporin, asparagyl endopeptidases
(legumains), cathepsins, C-type lectins, mucins, olfactomedin, phosphatidyl-
ethanolamine-binding proteins (PEBP), prohibitin, SCP/TAPS (venom
allergen-like protein homologues; cf. Cantacessi et al., 2009) and superoxide
dismutases as well as orphan proteins encoded by abundant novel transcripts
(ANTs) (e.g. Gems and Maizels, 1996; Loukas and Maizels, 1998, 2000;
Loukas et al., 1998, 1999a,b, 2000a,b; Tetteh et al., 1999; Falcone et al.
2000; Doedens et al., 2001; Zhou et al., 2011), many of which could be
involved in parasiteehost interactions.
Although all of these studies have provided invaluable insights into
selected molecules, much remains to be investigated on the functional and
structural levels. For instance, the application of advanced glycomic technol-
ogies (Robinson et al., 2012; Cummings and Pierce, 2014) should be able to
provide important information about the nature and extent of glycosylated
proteins in TES (cf. Maizels et al., 2006). In addition, knowledge of the
genome and transcriptome of T. canis should allow many aspects of the
developmental and reproductive biology and physiology of this worm as
well as parasiteehost interactions and the pathogenesis of toxocariasis to
be explored, drawing also on information available in public databases for
organisms such as Caenorhabditis elegans (the free-living nematode; Worm-
Base; www.wormbase.org) and Drosophila melanogaster (the vinegar y;
FlyBase; www.ybase.org) as well as parasitic worms (www.parasite.

wormbase.org). Such global molecular discovery should lead to a substantial
improvement in our understanding of T. canis/toxocariasis and, importantly,
could facilitate the development of new interventions and better diagnostic
tests.
4. T. CANIS GENOME AND TRANSCRIPTOMES GIVE

FIRST GLOBAL INSIGHTS INTO THIS PATHOGENS
MOLECULAR BIOLOGY
High-throughput nucleic acid sequencing technologies (e.g.
Morozova and Marra, 2008; Mardis, 2008, 2013; Wang et al., 2009;
Koboldt et al., 2013) are having a major impact in the study of many organ-
isms, including socioeconomically important parasitic worms, on a massive
scale, providing unprecedented opportunities for studying key aspects of
the biology of these critically important pathogens. In particular, RNA-
sequencing (RNA-seq) technology (Wang et al., 2009) has been used for
the rapid de novo sequencing of the transcriptomes of numerous parasitic
worms of human and veterinary health importance (e.g. Cantacessi et al.,
2010, 2011, 2012; Young et al., 2010, 2012, 2014; Godel et al., 2012; Jex
et al., 2011, 2014; Desjardins et al., 2013; Laing et al., 2013; Schwarz
et al., 2015; Tang et al., 2014), yielding substantial datasets and providing
a major step forward in our understanding of the molecular biology of path-
ogens as well as their interactions with their hosts. Recently, an international
team sequenced the 317 Mb draft genome of Toxocara canis employing
massively parallel sequencing and advanced informatics, and also undertook
comparative genomic analyses (Zhu et al., 2015). This section (1) summa-
rises salient features of this genome and associated transcriptomes and (2) em-
phasizes the prospects that knowledge of this genome offers for future
investigations of the immunobiology, epidemiology and genetics of T. canis
from both human, canid and other animal hosts, the pathogenesis of disease
as well as for the design of improved diagnostic tools and new interventions
for toxocariasis.
4.1 Genome and gene set

The draft genome of T. canis is 317 Mb and has a mean GC-content of
40.0% (Zhu et al., 2015). The repeat content of the genome is estimated
at 13.5% (42.9 Mb of DNA); the overall repeat content is similar to that
of the germ line genome of the related nematode Ascaris suum, but higher
than its diminutive (somatic) genome ( Jex et al., 2011; Wang et al., 2012).
Retrotransposon sequences (n > 72,800) represent nearly 70 families (16
LTR, 32 LINE and 20 SINE), with Gypsy, Pao and Copia predominating
for LTRs and CR1, RTE-RTE and L2 for non-LTRs. DNA transposon
sequences (n > 45,200) represent around 60 families, of which MULE-
MuDR, CMC-EnSpm and Novosib predominate. This richness in families
of transposable elements is comparable to selected nematodes genomes
(Ghedin et al., 2007; Dieterich et al., 2008; Jex et al., 2011).
The T. canis genome is predicted to encode 18,596 genes (Zhu et al.,
2015). The mean lengths of these genes, exons and introns are 8416-,
156- and 1133 bp, respectively, with a mean of 7.4 exons per gene, similar
to the genome of Ascaris (cf. Jex et al., 2011). On average, T. canis genes are
most similar in sequence to those of A. suum and are longer than those of
Brugia malayi, Caenorhabditis elegans and Pristionchus pacicus (see C. elegans
Sequencing Consortium, 1998; Ghedin et al., 2007; Dieterich et al.,
2008; Jex et al., 2011; Wang et al., 2012). Overall, more than two thirds
(67.5%) of all T. canis genes have an homologue (BLASTp cutoff: 105)
in A. suum (n 11,658; 62.7%), B. malayi (8395; 45.1%), C. elegans
(9002; 48.4%) or P. pacicus (7968; 42.8%); 5918 T. canis genes have homo-
logues in the latter nematodes, with 1925 shared exclusively with Ascaris,
and 3557 present in one or more species but absent from C. elegans.
Compared with these other nematodes, 6037 genes (32.5%) appear to be
unique to T. canis. In total, 5406 genes (29.1%) of T. canis have homologues
(108) in known biological (KEGG) pathways (Zhu et al., 2015).
4.2 Molecular groups and their key biological and/or

biotechnological relevance
To date, 14,583 (78.4%) of the 18,596 protein-coding genes of T. canis have
been annotated (Zhu et al., 2015), revealing at least 373 peptidases, 458
kinases, 408 phosphatases, 273 receptors, 503 transporters, 127 GTPases,
268 ion channels and 355 transcription factors, with some proteins likely
having multiple functions.
Excretory/secretory (ES) proteins and their proposed roles in the pathogenehost
interplay. Given the central role(s) that some TES proteins play in interac-
tions between T. canis and host (Maizels et al., 2000, 2006), the secretome
of this parasite was dened (Zhu et al., 2015). It represents at least 870 ES
proteins, likely with many distinct functions. Conspicuous were 14 pepti-
dases, including 7 SC serine proteases (S1, S9 and S28 families), 3 aspartic
proteases (A1 family), 3 CA/CD cysteine proteases (C1 and C13 families)
and 1 MA metalloprotease (M14 family) as well as 23 cell adhesion mole-

cules (immunoglobulins, integrins and cadherins), 17 lectins (C-type) and
6 CAP (or SCP/TAPS) proteins (cf. Gibbs et al., 2008; Cantacessi et al.,
2009), proposed to play important roles in host invasion, immune evasion
and/or feeding by parasitic worms.
Toxocara canis larvae invade various tissues, such as brain, eyes and mus-
cles, and can cause clinical disease. These larvae have an exceptional ability
to block or evade host immune attack and can survive for years in tissues.
This ability is associated with the deployment of molecules that are excreted
or secreted by the parasite or released from its surface (Maizels et al., 2006;
Maizels, 2013). In T. canis, 33 molecules, including 7 mucins with abundant
O-linked glycosylations, are predicted to be involved in host interactions
and/or modulating host immune responses; these molecules are likely heavi-
ly targeted by IgM antibodies and bound by various pattern recognition
receptors linked to host dendritic cells responsible for inducing a Th2
immune response (Hewitson et al., 2009).
Other proteins of T. canis predicted to have immunomodulatory
involvement include homologues of the B. malayi cystatin CPI-2 (B cell in-
hibitor), several TGF-b and macrophage initiation factor mimics, numerous
neutrophil inhibitors, oxidoreductases, known to counteract the neutrophil
oxidative burst, as well as ve close homologues of platelet anti-inamma-
tory factor a (Zhu et al., 2015; cf. Hewitson et al., 2009). Examples of T.
canis ES proteins proposed to be involved in immune evasion include
some hidden antigens (cf. Newton and Munn, 1999), such as numerous
C-type lectins, with close homology to vertebrate macrophage mannose
or CD23 (low afnity IgE) receptors, which mimic host molecules
(Hewitson et al., 2009). Other representatives include aquaporin, aspara-
ginyl endopeptidases (legumains), cathepsins, olfactomedin, phosphatidyl-
ethanolamine-binding proteins (PEBP), prohibitin, SCP/TAPS proteins,
superoxide dismutases and numerous hypothetical (orphan) proteins (e.g.
those encoded by ant genes), identied previously in small-scale molecular
studies of T. canis (see Maizels et al., 2006; Maizels, 2013). Although not
found in the draft genome, ants-3, -5, -30 and -34 are abundantly tran-
scribed in tissues of some T. canis specimens (Zhu et al., 2015). Three-
dimensional modelling (Zhu et al., 2015) indicates that ANT-34 is an
RNA helicase and ANT-5 is an RNA-dependent RNA polymerase, in
accord with previous ndings (Callister et al., 2008). The absence of the
ant genes from the draft genome, the similarity in the structures of ANT-
34 and ANT-5 with viral proteins and some inconsistency of transcription
in various stages, sexes or tissues of T. canis suggest that ant transcripts relate
to one or more double-stranded RNA viruses. This hypothesis warrants
testing to assess whether these ANTs are central to the biology of T. canis
and/or play a role in the regulation of transcription (cf. Callister et al.,
2008). Clearly, T. canis possesses a large arsenal of ES proteins that are likely
intimately involved in blocking, evading or modulating immune responses
in host animals. This represents an exciting area for future research.
Enzymes. In T. canis, ve key classes of proteases (metallo-,
cysteine, serine, threonine and aspartic) have been identied, with serine
(60; 16.1%), metallo- (n 165; 44.2%) and cysteine (107; 28.7%) peptidases
being prominent. The main families in these classes are the M13 neprilysins,
M12 astacins and adamalysins as well as M01 aminopeptidases (19) among
the metallopeptidases; C01 papains (i.e. cathepsins), C02 calpain-like
enzymes and C19 ubiquitin-specic proteases among the cysteine
peptidases; and the S08 subtilisins, S09 prolyl oligopeptidases and S01
chymotrypsins among the serine peptidases. These secreted proteases
(e.g. the M12 metallo-, the C01 and C02 cysteine, as well as the S01,
S08 and S09 serine peptidases) are likely of signicance, given their presence
in ES products from many parasitic worms, and their roles in tissue invasion
and degradation (e.g. during migration and/or feeding) and/or in immune
modulation or evasion (McKerrow et al., 2006; Hewitson et al., 2009). ES
peptidases (e.g. cysteine proteases and/or aminopeptidases) could have a
central involvement in these processes in T. canis and may represent vaccine
or drug targets candidates.
Toxocara canis has at least 458 protein kinases, including serine/threonine
protein (67.2%) and tyrosine (13.3%) as well as a small number of atypical or
unclassied kinases (19.4%). The phosphatome of this nematode contains at
least 408 phosphatases, including mainly serineethreonine (w80%), protein
tyrosine (w13%) and a minority of other phosphatases. In addition, some
127 GTPases are encoded in the T. canis genome, including 29 large
(heterotrimeric), 98 small (monomeric) G-proteins of the families Rab,
Ras, Arf/Sar and Rho, and some unclassied molecules. Homologues of
these GTPases in C. elegans include eft-1 (gene Tcan_11808), fzo-1
(Tcan_14313), glo-1 (Tcan_03008) and rho-1 (e.g. Tcan_13740), which
have important roles in embryonic, larval and/or reproductive development
in this free-living nematode. Therefore, some of these enzymes might be
targets for new nematocides.
Transporters, pores and channels. Channel, pore and/or transporter proteins
might also represent drug targets, as some of them are known to bind
particular anthelmintics (e.g. Kaminsky et al., 2009; Keiser and Utzinger,

2010; Lespine et al., 2012; Krucken et al., 2012). Of 156 GPCRs identied
to be encoded in the T. canis genome, 111 represent class A rhodopsins (e.g.
acetylcholine, adrenaline receptors, dopamine, neuropeptide and serotonin),
21 class B secretin receptors (e.g. latrophilin receptors, nematode chemore-
ception and parathyroid hormone), 10 class C metabotropic glutamate/
pheromone family members (e.g. GABA and glutamate receptors) and 14
other receptors. Of the 268 ion channel proteins predicted, many represent
voltage-gated cation channels (transient receptor potential; 10.8%), voltage-
gated cation (mainly K channels, including SLO-1; 31.0%) and the
Cys-loop superfamily (including aniononic, glycine, GABA-A and nico-
tinic; w40%). Moreover, 530 transporters were predicted, such as major
facilitator superfamily (w3.5%), ABC transporters (w11%) (including
P-glycoproteins) and solute carrier family (w67%) (Zhu et al., 2015).
4.3 Insights into the pathogens biology

Using RNA-seq technology, transcription proles were characterized for
different tissues, developmental stages and sexes of T. canis, and groups of
enriched molecules/pathways dened (see Zhu et al., 2015).
Intestine-enriched transcription. The intestinal tract of T. canis is specically
enriched for gene transcripts associated with molecular uptake and degrada-
tion via lysosomes (e.g. vha-2, -4, -6, -8 and -12), the transportation of amino
acids, sugars, lipids and drugs, the metabolism of xenobiotic metabolism
(ugt-22, -34, -45, -49, -63 and cyp14a5) as well as protein digestion (aspartic,
cysteine and metallopeptidases) and fatty acid elongation (elo-2, -3, -4, -5, -6
and -9) (Zhu et al., 2015). This information suggests extensive digestive,
absorptive and detoxifying functions in the gut of T. canis, similar to ndings
for Ascaris (Wang et al., 2013).
Stage-enriched transcription. Following the liverelung migration of larvae
(L3s), dioecious adults establish and live in the small intestine of the canid
host (Schnieder et al., 2011). Compared with L3s, pathways enriched in
the adult stage were predicted to involve carbon metabolism (aldo-2,
c04c3.3, enol-1, fbp-1, gpd-3 and gpi-1), lysosome activity, molecular transport
(ABC transporters), xenobiotic metabolism and DNA replication/repair,
likely relating to digestive and reproductive processes in the worm (Zhu
et al., 2015). Compared with adult T. canis, L3-enriched pathways were
mainly linked to neuronal signalling (ckr-2, dop-1, gar-2 and unc-49), and
cuticle shedding and/or formation (col-121, dpy-5 and sqt-1) (Zhu et al.,
2015), which might be relevant in blocking or evading host immune attack.
Interestingly, current evidence indicates that homologues of some genes

known to regulate dauer in C. elegans are up-regulated (e.g. daf-9, daf-12,
osm-3 and osm-6) or down-regulated (mek-2) in L3 of T. canis (see Zhu
et al., 2015), suggesting that this stage is in an arrested (hypobiotic) state
of development (Maizels et al., 2006; Crook, 2014).
Gender-enriched transcription. At least 3760 genes display gender-enriched
transcription in adult T. canis, of which w26% have no homologues in any
other organisms for which transcriptomic or genomic data are publicly
accessible (Zhu et al., 2015). Female-enriched transcription links to signal
transduction pathways involving mainly acetylcholine receptors and neuro-
active GABA glycine as well as G proteinecoupled receptors linked to germ
cell proliferation and embryogenesis (Zhu et al., 2015). Such transcription
relates to genes encoding glycosyltransferases of N-acetyl glucose/galactos-
amine moieties linked to egg shell synthesis in oogenesis (e.g. gene codes
C36A4.4, F07A11.2 and F21D5.1), oocyte maturation and embryonic
development (ceh-13, elt-3, gsk-3, kgb-1, nrh-1, pha-4, rab-11.1, unc-60,
-130 and vab-2), egg laying (lin-29, nsy-1, rhgf-1, sem - 2 and sox-2) and vulva
development (arf-1.2, ceh-20, let-23, lin-1 and lin-61) (Zhu et al., 2015). In
contrast, male-enriched transcription associates with energy metabolism,
epinephrine-like hormone regulation (tyramine and octopamine), protein
degradation and spermatogenesis (Zhu et al., 2015). Key genes with male-
enriched proles encode proteasome-related enzymes as well as protein-
serine/threonine and -tyrosine kinases relating to spermatogenesis and
sperm. Conspicuous were those linked to the 26S proteasome and associated
recycling of ubiquitin moieties (e.g. pas-1, rpn-1, rps-26, rtp-3 and rpt-5)
likely involved in germ line development and spermatogenesis (cpb-1, fog-
3 and spe-6).
5. PROSPECTS FOR NEW INTERVENTION TARGETS IN

TOXOCARA AND RELATED PARASITES
There is a need for improved treatments against T. canis and related
worms, as only a small number of drugs are effective against larvae in tissues
of accidental (human) or paratenic hosts (Magnaval and Glickman, 2006;
Othman, 2012). Genomic-guided drug target discovery provides an alterna-
tive or complementary means to conventional screening and re-purposing
(Shanmugam et al., 2012). The goal of such discovery is to predict (essential)
genes or gene products, whose inactivation by one or more drugs selectively
kills the nematode, but does not harm the mammalian host. Essentiality can
be inferred from functional information (e.g. lethality) in C. elegans (see

Zhong and Sternberg, 2006; Lee et al., 2008), and this approach appears
to yield credible targets in parasitic nematodes (Campbell et al., 2011). In
T. canis, 715 essential homologues are predicted; 703 of them relate to lethal
gene knock-down phenotypes in C. elegans, of which 230 have ligands that
meet the Lipinsky rule-of-ve (Lipinski, 2004). Eight channels or
transporters (including GABA, acetylcholine receptors and SLO-1 cal-
cium-activated potassium channels) have been inferred, some of which are
recognized targets for anthelmintics including imidazothiazole derivatives
(levamisole), macrocyclic lactones, cyclic depsipeptides or aminoacetonitrile
derivatives (monepantel) (cf. Campbell et al., 1983; Kaminsky et al., 2009;
Keiser and Utzinger, 2010; Kr ucken et al., 2012). Other potential targets
include 57 kinases, 21 peptidases, 14 phosphatases (including homologues
that bind norcantharidins; see Campbell et al., 2011), 5 GTPases and 4
GPCRs. These prioritized target candidates might now be tested in T. canis
in vitro by gene silencing.
Although some stages of the parasite cannot be produced in culture in
vitro, L3s of T. canis can be maintained in vitro in serum-free medium for
extended periods (18 months) (de Savigny, 1975), indicating that this stage
might be amenable to gene silencing by double-stranded RNA interference
(RNAi) or using specic chemical inhibitors. In total, 43 essential effector
genes have been predicted to relate to the RNAi pathway in T. canis (see
Zhu et al., 2015) as well as the genes pash-1 and rde-4 encoding small
RNA biosynthetic proteins and the nuclear RNAi effector gene ekl-1.
However, neither the effector (g-1) nor the RNAi inhibitor (adr-2) genes
appear to be encoded in T. canis, although a full repertoire of argonaute
genes appears to be present. The genes encoding siRNA amplication effec-
tors as well as dsRNA uptake and spreading factors are consistent with those
found in A. suum (see Dalzell et al., 2011), and sid-1 is present in T. canis.
The apparent absence of the sid-2 gene suggests that the uptake of extracel-
lular dsRNA is somewhat limited (McEwan et al., 2012). Evidence of gene
knock-down in A. suum and T. canis (see Xu et al., 2010; Chen et al., 2011;
McCoy et al., 2015; Ma et al., 2015) suggests some promise for testing the
function of conserved and also orphan (potentially parasite-specic) genes
in T. canis larvae. Following such work, chemicals designed to selected tar-
gets would need to be evaluated extensively in vitro and in vivo. For
instance, a subset of such prioritized chemicals could be selected, depending
on cost, availability, molecular properties, safety and/or prior use as drugs,
and tested for anthelmintic effects in a whole-worm motility screening assay
(cf. Preston et al., 2015a) adapted to T. canis, followed by a hit-to-lead phase,

in which structural analogues of hit compounds could be synthesized and
screened to establish structureeactivity relationships (SARs), and then tested
in established assays to predict intestinal absorption, distribution, meta-
bolism, excretion and toxicity (see Preston et al., 2015b). Subsequently,
compounds with desired parameters that are metabolically stable and are
not cytotoxic to mammalian cells could then progress to initial in vivo
testing of anthelmintic effect against T. canis in experimentally infected an-
imals (e.g. dogs or mice), as a basis for nematocide development.
6. CONCLUSION
The recent genomic and transcriptomic exploration of T. canis (see
Zhu et al., 2015) provides a rst global insight into the molecular biology
of this socioeconomically important pathogen of animals and humans
worldwide. Knowledge of transcription in some developmental stages and
tissues has identied a large spectrum of molecules likely to be involved
in hosteparasite interactions, and immune and pathophysiological responses
(cf. Janecek et al., 2015). Now, the application of advanced proteomic and
glycomic techniques (Robinson et al., 2012; Cummings and Pierce, 2014)
should enable the characterization of, for example, glycosylated proteins
in TES products (Maizels et al., 2006; Maizels, 2013). Immune responses
induced by such molecules might also be explored in animals. Investigating
unique groups of molecules, such as the complex array of peptidases, and
TTLs and CAP proteins, as well as understanding their roles in the path-
ogenehost interplay might support the development of new interventions
(drugs or vaccines) and diagnostic methods.
Knowledge of the gene silencing machinery for T. canis might open up
interesting opportunities for functional genomic studies in the larval stage of
this parasite in vitro, particularly for orphan genes and gene products.
Toxocara canis larvae can be maintained in vitro for months, which suggests
that, for instance, gene silencing might be achievable in this stage. Recent
reviews (Lok, 2012; Hagen et al., 2015) also emphasize the prospects that
nucleic acid transfection and transgenesis offer to achieve gene knock-out
or knock-down in parasitic worms. For example, virus-based transduction
(Hagen et al., 2014) may be useful for delivering microRNA-adapted small
hairpin RNAs (shRNAmirs) into the worm to achieve gene silencing, and
would be worthy of evaluation in T. canis. This area deserves attention and
might provide the prospect of being able to test the functions of genes on a
medium to large scale, which has been a major obstacle for most animal-
parasitic nematodes studied to date (Geldhof et al., 2007; Lok, 2012).
Such an advance could lead to an enhanced understanding of biological
and developmental pathways in Toxocara as well as parasiteehost interactions
at the immuno-molecular level using T. canis-dog and/or -mouse models
(Akao, 2006; Schnieder et al., 2011).
In the future, the genome-wide denition of genetic markers for use in
diagnostic and analytical assays should provide a basis for epidemiological
and population genetic studies, and also to address questions regarding the
complex network of biological and/or ecological factors involved in
the immunological idiosyncrasies of receptive hosts in endemic regions, the
role of chronically infected animals as well as parasiteehosteenvironment
interactions. It would also be informative to explore the susceptibility and
resistance of particular species and genotypes of hosts (e.g. human, mouse
and dog) to T. canis infection. Moreover, investigating the relationship
between host genotype and phenotype (degree of disease expression) in
response to T. canis infection and/or intervention strategy (e.g. treatment)
might assist in understanding toxocariasis and its spread.
In conclusion, the availability of the T. canis genome and transcriptome
and the future, integrated use of glycomic, metabolomic and proteomic
technologies should enable investigations of the systems biology of T. canis
and toxocariasis, which could provide prospects for developing radically
new diagnostic and intervention strategies. The ability to explore Toxocara
in this way should also create opportunities to investigate various other
ascaridoids.
ACKNOWLEDGEMENTS
Funding from the Australian Research Council (ARC), the National Health and Medical
Research Council (NHMRC) of Australia (R.B.G. et al.) and the International Science & Tech-
nology Cooperation Program of China (Grant no. 2013DFA31840; X.Q.Z. and R.B.G.) is
gratefully acknowledged. Other support was from the Victorian Life Sciences Computation
Initiative (VLSCI; grant number VR0007) on its Peak Computing Facility at the University
of Melbourne, an initiative of the Victorian Government, and the Australian Academy of Sci-
ence, Alexander von Humboldt Foundation and Melbourne Water Corporation. N.D.Y. holds
a Career Development Fellowship from NHMRC. R.B.G. thanks all current and past group
members as well as collaborators for their contributions to joint research.
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CHAPTER THREE
Coinfection of Schistosoma
Species with Hepatitis B or
Hepatitis C Viruses
Amy Abruzzi*, 1, Bernard Friedx, Sukaina B. Alikhan{
*Edward J. Bloustein School of Planning and Public Policy, Rutgers University, New Brunswick, NJ, USA
x
Lafayette College, Easton, PA, USA
{
U.S. Fund for UNICEF, New York, NY, USA
1
Corresponding author: E-mail: Amy.Abruzzi@Rutgers.edu
Contents
1. Introduction 114
2. Studies Conducted on General Populations 122
3. Studies Conducted on Special Populations 133
3.1 Subjects with chronic liver disease and related conditions 175
3.2 Subjects with primary liver cancer 178
3.3 Subjects with schistosomiasis 181
3.4 Subjects with acute or chronic hepatitis from HBV 187
3.5 Subjects with HCV 189
4. Studies Comparing Subjects with Schistosomiasis and Subjects with HCV 206
5. Concluding Remarks 209
References 224
Abstract
Although a considerable number of studies have been undertaken to date, it is still
controversial as to whether or not coinfection with schistosomiasis increases the
susceptibility to or progression from Hepatitis B virus (HBV) or Hepatitis C virus (HCV)
infection. This review is a closer examination of the key studies conducted on human
populations on clinical factors that were published in English between 1975 and
January 2015. Our review is mainly based on tables containing the salient information,
which are arranged rst by study population, country of study and publication date. We
provide further explanation, clarication and discussion in the text. As such, it includes
both studies that have been conducted on general populations who are largely asymp-
tomatic for clinical disease (Table 3), as well as those focussing on special populations,
which are usually comprised of clinical patients. These special populations have been
presented as follows: subjects with chronic liver disease or related conditions such as
cirrhosis, Table 4; subjects with primary liver cancer, Table 5; subjects with schistosomi-
asis, Table 6; subjects with acute or chronic hepatitis resulting from HBV, Table 7 and,
2016 Elsevier Ltd.
j
ISSN 0065-308X
112 Amy Abruzzi et al.
subjects with HCV, Table 8. We have presented studies that compared two mono-
infected groups with one that is coinfected separately in Table 9, as these offer us
the best basis from which to evaluate if any synergistic effects accompany coinfection.
A number of factors contributed to the results reported in our tables. These
included, but are not limited to: subject selection (i.e. asymptomatic cases typically
drawn from the general population vs subjects presenting to a hospital or clinic with
clinical disease); study design, which directly impacts our ability to infer causality (i.e.
case series, cross-sectional, case-control, cohort study); use and choice of control pop-
ulation (i.e. apparently healthy subjects vs other hospital patients vs none); sample size,
which directly impacts statistical power and can result in a Type II error; geographic
area, which may reect differences in population genetics, public health history, envi-
ronmental differences or any number of other important factors (i.e. Egypt, Brazil,
China); method of testing for schistosomal infections (i.e. stool vs antibody test);
method of testing to determine if advanced schistosomal disease was present (i.e.
ultrasound, liver biopsy vs none); method of serological testing for HBV (i.e. use of
HBsAg alone or with other markers or DNA testing); method of serological testing
for HCV (i.e. use of anti-HCV alone or with RNA testing) and, year of the study, which
reects among other things, technological improvements between tests as well as
possible changes in the frequency of exposure in the populations under study (i.e.
use of parenteral antischistosomal therapy vs the oral antischistosomal medication).
Despite all these differences, throughout this review we have observed general pat-
terns that seem largely consistent with one another. Studies conducted on general,
largely asymptomatic populations tend to support the view that having one of the dis-
eases in question (i.e. schistosomiasis) does not necessarily predispose one to becoming
coinfected with another (i.e. HBV or HCV). Rather, the probability of becoming coin-
fected seems most closely associated with modes of transmission for either HBV or
HCV in schistosome-endemic areas, such as the past use of parenteral antischistosomal
therapy or frequent blood transfusion. Once coinfected, however, the clinical course of
illness for those with Schistosoma-HBV or Schistosoma-HCV infections are typically much
more severe than for mono-infected subjects. The strongest evidence for this was found
in the half-dozen or so prospective cohort studies that systematically monitored disease
progression in their subjects. With respect to HBV infection, coinfection with Schisto-
soma prolonged the carriage state and more often resulted in chronic hepatitis with
greater cirrhosis as well as higher mortality. Much of the same was also observed
with respect to HCV, where coinfection with Schistosoma was associated with a reduced
ability to spontaneously resolve the viral infection and more often resulted in rapid
brosis as well as higher mortality. Furthermore, two of these studies which were fully
comparative in nature, support the supposition that there is a synergistic association be-
tween Schistosoma-HCV for both liver brosis and mortality. Immunological studies, all
conducted on HCV, also generally seem to support this.
The results of our research argue for greater primary prevention for both HBV and
HCV in Schistosoma-endemic populations. Although no vaccine currently exists for
HCV as it does for HBV, additional steps can still be taken to reduce transmission in
high-risk populations. Greater use of the HBV vaccine is particularly advisable. Finally,
additional observational, longitudinal studies conducted on human populations that
Coinfection of Schistosoma Species 113
are fully comparative in nature could help answer some of the remaining questions on
both Schistosoma-HBV as well as Schistosoma-HCV coinfections. Some of these include
the role of active versus past schistosomal infections, the role of genetic variants, as well
as the effect of coinfection on treatment. Future studies should make a particular effort
to use a sufcient sample size to ensure adequate statistical power, which was not
often properly considered in many of the studies we reviewed for this paper.
Abbreviations
Adj Adjusted
AFP Alpha-fetoprotein
ALT Alanine transaminase
Anti-HCV Hepatitis C antibody
AST Aspartate aminotransferase
AVH Acute viral hepatitis
CAH Chronic active hepatitis
CI Condence interval
CLD Chronic liver disease
DHSS Decompensated hepatosplenic schistosomiasis
GE Greater or equal to
HAV Hepatitis A virus
HBsAg Hepatitis B surface antigen
HBsAb Specic antibody to Hepatitis B surface antigen
HBcAg Hepatitis B core antigen
HBcAb Specic antibody to Hepatitis B core antigen
HBeAg Hepatitis B e antigen
HBeAb Specic antibody to Hepatitis B surface antigen
HBV Hepatitis B virus
HBV-DNA Hepatitis B DNA
HCC Hepatocellular carcinoma
HCV Hepatitis C virus
HCV-RNA Hepatitis C RNA
HDV Hepatitis D virus
HDVAb Antibody to Hepatitis D virus
HIS Hepatointestinal schistosomiasis
HIV Human immunodeciency virus
HGV Hepatitis G virus
HSS Hepatosplenic schistosomiasis
ICC Intrahepatic cholangiocarcinoma
ISS Intestinal schistosomiasis
LC Liver cancer
LD Liver disease
LE Less than or equal to
LSch Liver schistosomiasis
LT Less than
MHF Minimal hepatic periportal brosis
NOS not otherwise specied
OR Odds ratio
PAT Parenteral antischistosomal therapy, potassium antimony tartarate
PIIINP Type III procollagen peptide

PPF Periportal brosis
PPT Periportal thickening
NA Not available, unknown, or not specied in original paper
RR Relative risk
Sch Schistosomiasis, schistosome
SchAb Schistosome antibody
Sh Schistosoma haematobium
SHF Schistosomal hepatic brosis
Sj Schistosoma japonicum
SLD Schistosomal liver disease
Sm Schistosoma mansoni
SPF Schistosomal portal brosis
1. INTRODUCTION
This review examines coinfection of selected species of Schistosoma
with Hepatitis B virus (HBV) or Hepatitis C virus (HCV) in human popu-
lations, with an emphasis on the clinical aspects of disease. The schistosomes
are waterborne digeneans of global concern that infect humans when they
come into contact with a snail-transmitted larval stage (the cercaria) via
contaminated water. Infection with schistosomes, particularly the species
Schistosoma mansoni or Schistosoma japonicum, can result in damage to the liver
and more rarely, specic forms of liver cancer. Schistosomiasis has been most
often studied in terms of single infections but its role in concomitant infec-
tions is of increasing concern, particularly in conjunction with viral infec-
tions. HBV and HCV are two such pathogens, infecting nearly 1 in 12
people globally (WHO, 2014, 2015; see also Mohd Hanaah et al., 2013;
Ott et al., 2012) and of particular interest because of the damage they cause
to the liver. HBV is a double-stranded DNA virus of the hepadnavirus fam-
ily, while HCV is an RNA virus with a molecular structure similar to the
family of aviviruses that cause yellow fever or Dengue fever. HVB is often
spread through vertical transmission, i.e. mother to child, but may also be
spread through horizontal transmission such as through contaminated blood
supply. HCV is most commonly spread through contaminated blood supply,
and documented high-risk groups for HCV include intravenous drug users,
health-care workers exposed to needle sticks, haemodialysis patients and
recipients of blood transfusions; HCV is also often spread through sexual
contact and often, patients fall outside of these high-risk groups. Chronic
infection with either HBV or HCV can result in liver brosis, cirrhosis
and decompensation. In addition, both of these viruses are associated with

primary liver cancer. As with schistosomiasis, the majority of the individuals
infected with HBV or HCV live in non-Western countries and may be
unaware they are infected; Egypt in especially notable for having a high
prevalence of all three infections.
Although a considerable number of studies have been undertaken to
date, it is still controversial as to whether or not coinfection with schistoso-
miasis increases the susceptibility to or progression from HBV or HCV
infection (see Gasim et al., 2015; Bahgat, 2014; Van-Lume et al., 2013).
This review is a closer examination of the key studies to date that are relevant
to clinical presentation. As such, it includes both studies that have been
conducted on general populations who are largely asymptomatic for clinical
disease, as well as those focussing on special populations, which are usually
comprised of clinical patients. The special populations referred to are sub-
jects with chronic liver disease (CLD) or related conditions such as cirrhosis;
subjects with primary liver cancer; subjects with schistosomiasis; subjects
with acute or chronic hepatitis resulting from HBV; and, subjects with
HCV. Mostly, of all the studies conducted on general populations, subjects
with CLD and subjects on liver cancer patients were principally concerned
with estimating the frequency of mono and coinfections in these popula-
tions. Tables 3e5 have provided a special column reporting data on the
prevalence and use a summary column to convey the main ndings on
coinfection. Many of the studies conducted on subjects with schistosomiasis,
subjects with acute or chronic hepatitis from HBV, or subjects with HCV,
examined disease severity and progression by contrasting a mono-infected
group against those with coinfection. For these tables, i.e. Tables 6e8, we
have included any exclusion criteria applied to a study population in order
to rule out other possible causes of liver infection or hepatitis. In these tables,
we report prevalence, when applicable, in the summary column in conjunc-
tion with the other results on coinfection. We have chosen to treat studies
that compared two mono-infected groups (i.e. schistosomiasis and either
HVB or HCV infection) against a group of coinfected subjects separately
from the aforementioned categories. As such, these studies offer us the
best basis from which to evaluate if any synergistic effects accompany
coinfection.
Many of the studies appearing in Tables 3e6 tested for both HBV and
HCV in their study populations. Thus, we also present data, whenever avail-
able, on coinfection between HVB and HCV, and tri-infection with schis-
tosomiasis in our tables. Finally, a few of the studies included in this review
tested for other hepatitis viruses, such as Hepatitis D virus, in their study
populations. Hepatitis D virus is a defective hepatropic RNA virus that
requires the presence of HBV as a helper virus for its pathogenicity and
has been shown to be associated with the most severe forms of acute and
chronic hepatitis in many HBsAg seropositive patients (WHO, 2015,
2002). People who are immune from HBV are immune from HDV, while
carriers of HBV are susceptible to it (WHO, 2015). Rather than universally
omit this data from our tables, we have noted it when relevant and discuss in
conjunction with our ndings in the conclusion.
Our review contains numerous tables as in Abruzzi and Fried (2011), in
which we examined coinfection of schistosomes with protozoa, bacteria and
other helminths, and tabular information is followed by text to clarify and
extend the information presented. In order to be included in a table, the
study in question needed to meet certain inclusion criteria. First, the study
needed to be published in a scientic journal that was indexed by
Helminthological Abstracts, MEDLINE or ISIs Web of Science from
1975 onwards, or the study needed to appear as a footnote in other studies
located through these indexes. Our database search terms were simply hep-
atitis and schistosom*, from which we selected studies relevant to either
HBV or HCV coinfections. We mainly utilized Google Scholar to double
check the results from our database searching and to assist us when following
the footnote trail. In addition, the study in question needed to be published
in the English language before January 2015, which was our practical limi-
tation. All specic entry numbers are arranged rst by the country of the
study population and then in ascending chronological order. No papers
were located for this review on species of Schistosoma other than S. mansoni,
Schistosoma haematobium or S. japonicum.
Given the vastness and complexities of the literature, we have only
included studies conducted on human populations in this review. Animal
studies are sparse on SchistosomaeHBV or SchistosomaeHCV coinfections,
and were excluded. In vitro studies, chiey using soluble egg antigens, are
also beyond the scope of this paper, as are HBV vaccine efcacy studies in
schistosome-endemic populations. In order to be included in a table, the
study in question need to include sufcient information on the methods
used to determine the presence of the coinfection and offer a clear presen-
tation of the results. In each section, we also discuss any relevant data on the
mechanisms of coinfection as studied by the specic papers included in our
tables. While understanding that the mechanisms of coinfection are obvi-
ously important, this was not the primary purpose of our review, as stated
earlier. The reader will nd additional references to several key papers

further discussing the mechanisms of coinfection in our conclusion.
All of the studies included in our tables used observational methods, and
many were conducted on clinical patients. In order to better distinguish
between the studies vis-a-vis their robustness for inferring causality, we
indicated the type of epidemiologic study design used (i.e. case series,
cross-sectional, case-control, cohort) as well as their primary objective (i.e.
prevalence, risk factors, disease progression) in our tables. In some important
ways, however, the designs used to study the association between two dis-
eases represent a departure from the traditional exposureedisease paradigm
that underlies this epidemiologic classication. As such, our designations are
best viewed in this context as points along a continuum of increasing meth-
odological rigor. If the study author(s) did not provide sufcient detail on
methods for us to be condent that a higher-level study had been conduct-
ed, a lower-level designation was applied that adequately described the study
in question. When in doubt, we also considered a papers classication in
MEDLINE, which includes descriptors denoting study design when they
are clearly demonstrated in the paper. We also considered the classication
assigned by Van Lume et al. (2013) for the 10 papers they discuss, which we
largely agreed with. Papers that presented only a brief account of methods
and/or results or were only available by scientic abstract do not appear
in our tables, but are occasionally referred to in the text. Case reports or
studies relying solely on autopsy were routinely excluded from our review.
The following study design designations were used in our tables and are
presented here in order of increasing internal validity or robustness.
Case series: A case series is a type of descriptive observational study
design that closely examines a group of patients with a common set of
characteristics, such as a common diagnosis (e.g. patients with schistoso-
miasis, patients with CLD, patients who are anti-HCV), with the aim
of further describing their clinical presentation. Typically, the number of
cases included in this type of study is small in number and only minor
inclusion or exclusion criteria are utilized. In some cases series studies,
subjects are selected for inclusion from consecutive patients presenting
at a medical facility. Case series patients may also be followed over
time for a change in their disease status, but unlike a cohort study,
with no particular disease endpoint mind. Sometimes a comparison pop-
ulation, such as another small group of patients, is used; occasionally,
external control groups are used. In these cases, case series may appear
to be like caseecontrol studies; however, they lack the same level of
rigor with respect to dening case and/or control status as well as control
of confounding factors. (For additional discussion, see Kempen, 2011).
Cross-sectional: Cross-sectional studies are another type of descriptive,
observational study designs, which are used to estimate the frequency of
disease and its correlation with any exposures of interest in a given study
population at a particular point or period in time. In general, cross-
sectional studies do not provide evidence of causality, since they measure
disease and exposure at the same point in time. However, in clinical
studies where exposure measures are valid proxies for past exposures
or indicate permanent exposure characteristics, cross-sectional studies
and caseecontrol studies are largely equivalent (Kramer, 1988).
Caseecontrol: Caseecontrol studies are a type of analytic, observa-
tional design in which a group of subjects who are known to have the
outcome of interest (i.e. hepatocellular carcinoma (HCC)) are rst iden-
tied as cases. A suitable comparison or control group of subjects without
the outcome of interest are then assembled and used for comparison.
They are particularly well-suited to studying rare diseases or diseases
with long latency periods, and are usually undertaken for the purpose
of evaluating the association of specic risk factors with the health
outcome of interest. Matching is often used to improve statistical
efciency and to make cases and controls comparable with respect to
baseline confounding factors, such as age and sex. Caseecontrol studies
may be population based and are sometimes carried out in conjunction
with other designs, such as a cross-sectional survey. Many of the casee
control studies included in this review were conducted on hospital or
clinic patients. Caseecontrol studies provide some evidence of causality
between exposure and disease, provided recall bias was absent or played
only a minimal role in ascertaining exposure.
Cohort: A cohort study is an analytic, observational study that selects a
group of patients who are initially free of the outcome of interest, records
their various exposure statuses, and then follows them over time for the
development of that outcome. Prospective cohort studies are one of the
best known subtypes of this study design, and are well suited to studying
rare exposures and evaluating disease progression, including mortality.
The cohort studies cited in this review all began with patients who
were diagnosed at an early stage in their infection, then followed over
a period of years to systematically monitor the change in their disease
status. As such, cohort studies offer us better evidence for inferring a
causal relationship between exposure and disease than other observa-

tional designs.
There was considerable variety in how schistosomiasis was determined in
the study populations in these papers, which we have indicated in our tables
and discuss in context. Many studies routinely checked for ova in stool and/
or urine on one or more occasion. When live ova were requisite, we have
denoted this in our tables. A substantial number of studies used one or more
schistosome antibody test to detect the presence of infection. Notably, this
test cannot distinguish past from present infections nor can it always distin-
guish between Schistosoma species. Many studies also used an ultrasound,
computerized tomography (CT) scan, and/or liver biopsy to check for
brosis or other hepatic damage. Most often, studies used a combination
of methods, all of which we note in our tables. Many studies also gathered
data from prior medical records or by questionnaire, especially for a patients
history of schistosomiasis or past exposure to infested water. We did not
include this information routinely in our tables unless this data were used
to establish case status in that population. Similarly, since clinical exams
including routing blood work and liver function tests were used in the
vast majority of these studies, we only noted them in our tables where
they were used to dene case status or report on them in our comments
section when pertinent to major ndings. We provide a brief guide to the
most commonly used serological markers used in the evaluation of liver dis-
ease that are covered in this review in Table 1.
Table 1 Serological markers used in the evaluation of liver disease

Abbreviation Name and description
ALT Alanine transaminase; also called alanine aminotransferase; one of

several liver enzymes routinely examined as indicators of
possible liver damage; in healthy individuals, ALT levels are
low.
AST Aspartate aminotransferase; formerly called serum glutamic
oxaloacetic transaminase; as with ALT, the presence of higher
levels of this enzyme in the blood may be indicative of liver
damage; often tested in conjunction with ALT, and sometimes
presented as a ratio of it.
AFP Alpha-fetoprotein; also written as a-fetoprotein; widely used as a
tumour marker to screen for liver cancer, as well as several
other cancers.
Rutherford 2014a,b, WHO 2014, 2015.
Since most readers of this journal may not be familiar with the serological
markers (seromarkers) used to detect the presence of HBV or HCV infections,
we provide a brief synopsis in Table 2. In our tables, we present results for the
seromarker(s) used in that specic study. If a combination of markers or other
diagnostic measure were used to dene disease status in that population, we
indicated it. Virtually all of the studies in our review tested for HVB by
checking for the Hepatitis B surface antigen (HBsAg), which may indicate
an acute or chronic infection. Many papers also used one or more additional
HBV seromarkers in order to make this distinction, and typically reported
data separately for HBsAg seropositivity versus any HBV marker. Similarly,
Table 2 Serological markers of hepatitis B virus or hepatitis C virus infection

Serological maker Description
Hepatitis B virus
HBsAg Hepatitis B surface antigen; indicates carrier state associated
with acute or chronic infection; often used in the diagnosis
of HBV infection and for the screening of blood; this marker
is the earliest indicator of acute infection, appearing without
HBsAb or HBcAb; persistence of HBsAg for more than
6 months in conjunction with other markers is indicative of
chronic infection.
HBsAb Specic antibody to Hepatitis B surface antigen; also written as
anti-HBs; appearance after 1e4 months after onset of
symptoms is indicative of clinical recovery of and subsequent
immunity to in the absence of HBsAg, the presence of
HBsAb with HBcAb indicates previous HBV infection and
immunity to hepatitis B; in the absence of both HBsAg and
HBcAb, HBsAb indicates vaccine-induced immunity.
HBcAg Hepatitis B core antigen; marker of infectious viral material;
Most accurate index of hepatitis B viral replication.
HBcAb Specic antibody to hepatitis B core antigen; also written as
anti-HBc; HBcAb identies all previously infected persons,
including HBV carriers, but does not differentiate carriers
from noncarriers; in the absence of HBsAg and HBsAb, this
marker indicates a recent HBV infection; class type (IgM,
IgG) used for further distinction.
HBeAg Hepatitis B e antigen; indicates patient is infectious; typically
appears during weeks 3e6 of infection; persistence beyond
week 10 indicates progression of infection to chronic state;
Continuous presence is indicative of chronic active liver
disease.
Table 2 Serological markers of hepatitis B virus or hepatitis C virus infectiond

cont'd
Serological maker Description
HBeAb Specic antibody to Hepatitis B e antigen; also be written as
anti-HBeAg; when present in conjunction with HBcAb and
in the absence of HBsAg, HBsAb and core HBV mutants,
this marker indicates convalescence and low contagiousness.
HBV-DNA Hepatitis B virus DNA; maybe detectable by hybridization
assays or polymerase chain reaction (PCR) as soon as 1 week
after initial infection; HBV DNA polymerase is only
performed for research purposes.
Hepatitis C virus
Anti-HCV Hepatitis C antibody; usually detected by enzyme immune
assay (EIA); current tests have higher sensitivity and
specicity than earlier tests, but additional or conrmatory
testing is usually advisable; individuals will still test positive
for anti-HCV, even if they are no longer infected as in the
case of spontaneously resolved infections; alternatively,
patients with compromised immune systems may not
produce enough antibodies for detection by EIA.
HCV-RNA Hepatitis C virus RNA; usually detected by PCR assay;
presence in serum indicates an active infection; often used to
conrm the diagnosis of hepatitis; detects disease in patients
that may be false negative on anti-HCV, such as
immunocompromised patients.
Rutherford 2014a,b, WHO 2002, 2014, 2015.
the vast majority of studies on HCV infection checked for the presence of
HCV antibodies (anti-HCV), which may indicate present or past infection.
Many studies, especially those conducted in recent years, also conducted tests
for HCV-RNA, which indicates the presence of replicating virus. Here, too,
there was variation as to if this was used to conrm active cases of HCV
infection or was simply gathered as additional information on their study
population. It is important to note that the range of studies included in
this review were conducted using different serological tests, or different gen-
erations of the same test, or conducted in such a way (i.e. repeated tests) that
could easily result in varying degrees of sensitivity and specicity. Incorpo-
rating that level of detail in our tables and analyzing it accordingly is beyond
the scope of this review, which is intended as a broad survey searching for
commonalities with suggestions for further study. Finally, when reporting
results, we have indicated when nonsignicant increases were noted. Lack
of statistical signicance in the context of a single study may be due to lack of

statistical power, which is a function of the frequency of the disease in the
population under study and number of factors examined. As such, we also
indicate the sample size used in each investigation.
2. STUDIES CONDUCTED ON GENERAL POPULATIONS

This section reviews the studies conducted on general populations
where schistosomiasis is endemic for the purposes of measuring the preva-
lence of coinfection with HBV and/or HCV. The 14 studies selected for
inclusion in Table 3 were conducted in Brazil, China, Egypt, Ethiopia,
Kenya, the Philippines, Sudan and Yemen, and were published between
1983 and 2012. Most of the countries in this list are represented by one
or two studies; Egypt is best represented with seven. All studies used a
cross-sectional design and ranged in size from 242 to 2038 subjects, with
about half including less than 700 subjects. With a few exceptions, most
were large, population-based surveys conducted in rural village or commu-
nity settings, typically including males and females from a wide range of
ages. A few of these were notable for using random sampling methods to
select study subjects (entry numbers 7, 10, 11) or undertaking village or
country comparisons (entry numbers 1, 8, 10, 14). In addition, two studies
were included in this table that were conducted in Egypt on younger pop-
ulations: one study was conducted on health-care workers who were at high
risk of workplace exposure to hepatitis viruses through needle-sticks or
other forms of contact with contaminated blood (entry number 9); The
other on male military inductees presenting for physical examination (entry
number 4).
Each study tested for one or more species of Schistosoma and either HBV
(13 studies) or HCV (8 studies), including seven studies that tested for both
HBV and HCV. Most of the studies in this table pertain to S. mansoni, with
two studies pertaining to S. japonicum. Twelve of these studies tested for the
presence of Schistosoma ova in stool using one or more samples; ve studies
also used an ultrasound or other form of sonography to check for advanced
disease in their subjects. In addition, ve of the Egyptian studies also
included a urine test for S. haematobium. The two remaining studies used a
Schistosoma antibody test (entry number 9) and subject recall of past history
in conjunction with an ultrasound (entry number 7). Not surprisingly, the
frequency of schistosomiasis in S. mansoni endemic areas was high, with
Table 3 Studies conducted on general populations
Study design
(objective) and Diagnosis of
Coinfection of Schistosoma Species

No. Reference Location (years) study population disease Prevalence Findings on coinfection
1 Serufo et al. Queixadinha and Cross-sectional (prevalence, HBV: HBsAg, Endemic villagers (n 693): Although the prevalence of
(1998) Cap~ao, Minas village comparison) HBsAb, HBcAb 9% HbsAg, 66% Sm HBV markers was
Gerais, Brazil Subjects: 693 residents of Sch(Sm): stool Coinfected: n.a. considerably higher in the
(1994e1997) Queixadinah (93% of PPF: ultrasound Nonendemic villagers Sm endemic village, within
total population), (n 515): 1% HbsAg, the endemic area there was
endemic area for Sm, <1% Sm no association between the
aged 0e86 years, 49% Coinfected: n.a. HBsAg and Sm, with
male; 515 residents of or without PPF
Cap~ao (96% of total There was a comparable
population), prevalence of HBsAg
nonendemic for Sm, carrier state among Sm
aged 0e83 years, 52% and Sm subjects (9% vs
male 7%)
The distribution for chronic
HBsAg carriage was also
comparable
2 Li et al. (1997) Huie Long, Xia Cross-sectional (prevalence, HBV: HBsAg, Subjects (n 879): 18% On average, no association
Shan, Shang Shan severity) HBcAb, HBeAb HBsAg, 42% any between Sj and HBV
farming/shing Subjects: 879 villagers (22% Sch(Sj): stool, HBV marker, 19% Sj status; however, a higher
villages in of total population, abdominal exam (village range: 13e32%) frequency of HbsAg or
Dongting lake village range 17e34%), Coinfected: 9% any HBV HBV (any marker) was
region, Hunan, aged 5e74 years marker w/Sj found among individuals
China (1992) with advanced Sj (43%,
77%) as well as those
reinfected with Sj (23%,
52%), when compared
with the non-Sj infected
(16%, 35%), those newly
infected with Sj (12%,
40%), or those with past
treated Sj infection (17%,
123
43%)
(Continued)
Table 3 Studies conducted on general populationsdcont'd
124
Study design
3 Hyams et al. Nile delta, Egypt Cross-sectional (prevalence, HBV: HBsAg, Subjects (n 298): 3% No association between past
(1986) (1981e1983) duration) HBsAb, HBcAb, HBsAg, 37% any or present HBV infection
Subjects: 324 villagers HBeAg HBV marker, 50% and Sm; the frequency of
(w16% of total Sch(Sm/Sh): stool, Sm, 2% Sh HBsAg chronic carriage
population of farming urine Coinfected: 1% HBsAg (6 months) was the same
village), 36% male, mean w/Sm, 20% any between Sm and Sm
age 26 years HBVw/Sm groups (3% vs 3%); a
Note: 92% of sample in main nonsignicant difference
analysis was noted between the
frequency of any HBV
marker status among Sm
and Sm groups (40% vs
33%)
Insufcient sample size to
detect statistical signicance
noted
4 Hyams et al. US Naval Medical Cross-sectional (prevalence, HBV: HBsAg, Subjects (n 899 to 1234): Among those with Sm, there
(1987) Research Unit, severity, risk factors) HBcAb 7% HBsAG, 26% Sm, was no difference in
Cairo, Egypt Subjects: 1234 Egyptian Sch(Sm, Sh): stool, 20% Sh, 5% HSS HBsAG status (7%
(1982e1983) males, aged 18e24 years, urine Coinfected: 2% HBsAg HBsAg vs 7% HBsAg)
presenting for induction Enlarged liver/spleen: w/Sm, 2% HBsAg A nonsignicant difference
physical examination physical exam w/Sh noted among those who
Subjects were from lower Note: Not all subjects were Sh (12% HBsAg
and middle social classes, provided both stool and vs 8% HBsAg)
Amy Abruzzi et al.

50% rural/urban urine samples A prior history of PAT
increased risk of HBsAG
(11% HBsAg vs 6%
HBsAg)
Coinfection was not associated
with HSS or enlarged liver/
spleen in this population
5 Kamel et al. Saada, in Kafr El Cross-sectional (prevalence, HBV: HBsAg, Subjects (n 1259): 2% Three was no association
(1994) Sheikh governate, severity) HBcAb HBsAg, 24% any between Sm and HBV
Egypt (1992) Subjects: 1259 villagers HCV: anti-HCV HBV marker, 16% anti- infection (any marker; age

(GE 68% of total Sch(Sm, Sh): stool, HCV, 49% Sm adjusted OR 1.13, 95% CI
population), mean age urine Coinfected (n 1157): 12% 0.87e1.48) or between
20 years, 50% male PPF: ultrasound any HBV marker w/ Sm and anti-HCV
Sm, 7% anti- (OR 1.02, 95% CI: 0.7
HCVw/Sm e1.37)
Note: 5% of villagers were The combined infection of
any HBV marker HBV w/HCV was also not
w/anti-HCV associated with Sm status
A greater proportion of
HBV, HCV or
HBVw/HCV status was
noted among individuals
with Sch brosis than
without, but not enough to
reach statistical signicance
6 El-Sayed et al. Bitter lakes area, Cross-sectional (prevalence, HBV: HBsAg, Subjects (n 506): 3% In univariate model, no
(1997) Sinai Peninsula, severity, risk factors) HBsAb, HBcAb HBaAg, 20% HBV, association between Sm
Egypt (September Subjects: 506 residents GE HCV: anti-HCV 10% anti-HCV, 30% and HBV (OR 0.68, 95%
1993) 1 year in area recently Sch(Sm, Sh): stool, Sm, 1% Sh CI 0.4e1.2) or between
reclaimed from the urine; ultrasound Coinfection: 5% Sm and anti-HCV
desert and endemic for on sample, n.o.s. HBVw/Sm, 3% anti- (OR 0.92, 95% CI 0.5, 1.8)
Sm, mean age of HCVw/Sm No association with Sch
20 years, 52% male Note: HBV HBsAg hepatic PPF with either
and/or HBcAB; also, HBV or HCV, adjusted for
LT 1% of residents age and sex
HBsAg w/anti-HCV; PAT was strongly associated
4% of residents were with anti-HCV (OR
HBV w/anti-HCV 7.86, 95% CI 2.8, 22.0),
but not with HBV (OR
1.51, 95% CI 0.5e4.1)
Of note, adults aged 40 and
over were 4 more likely
125
to be infected with HBV
than children
(Continued)
Study design
126
7 Darwish et al. Kalama, semi-urban, Cross-sectional (prevalence, HBV: HBsAg, Subjects (n 796): 10% Recalled past history of Sch
(2001) Nile Delta village, severity, risk factors) HBcAb HBsAg, 40% anti- (GT 1 year) was associated
19 km north of Subjects: 801 persons (88% of HCV: anti-HCV HCV, 3% History of with anti-HCV status
Cairo, Egypt age-eligible residents), History of Sch: Sch LE 1 year, 17 % (adj OR 1.75, 95% CI 1.14,
(1994e1995) aged 10 years and over questionnaire History of Sch GT 1 year 2.67), and may be related to
PPF: ultrasound Coinfected: 2% anti-HCV prior PAT
Note: History of Sch w/History of Sch LE 1 year, This is consistent with the
infection based 11% anti-HCV w. increased risk of Sch PPF
solely on recall. History of Sch GT 1 year observed among anti-
Note: 4% of villagers HCV villagers (adj OR
HBsAg w/anti-HCV; 1.75, 95% CI: 1.01, 3.05)
Data on coinfection of Of note, HCV infection tends
Sch w/HBV n.a. to reach its peak prevalence
in a population at younger
age (60% by age 30) than
HBV (75% by age 40)
8 Blanton et al. Shamarka, Nile Cross-sectional (prevalence, HBV: HbsAg, Egyptian subjects (n 2038): Among those with
(2002) Delta, Egypt and severity, country HBsAb 20% Sm; serosample hepatocelluar damage, Sm
Katheka, Kenya comparison) HCV: anti-HCV, (n 112): 31% HBsAg, intensity was comparable
(1999e2000) Subject groups: 2038 HCV-RNA 39% anti-HCV, 23% between anti-HCV and
Egyptians and 2120 Sch(Sm): stool, Sm anti-HCV groups in both
Kenyans, aged 11 years ultrasound Coinfected: n.a. Egypt (mean EPG: 3 7 vs
or more, 54% and 42% Kenyan subjects (n 2120): 3 6) and Kenya (mean
male, respectively 64% Sm; serosample EPG: 27 4 vs 27 11),
(n 237): 39% HbsAg, respectively
Amy Abruzzi et al.

11% anti-HCV, 71% Coinfection does not appear
Sm to be associated with the
Coinfected: n.a. geographical variation in
Note: brosis observed between
serosample individuals Egyptians and Kenyans
with Sch brosis and their No interaction was found
rst degree relatives; data between Sm infection or
on coinfection with HBV disease and anti-HCV
n.a. status in multivariate model
Subjects (n 842):, LT 1%

9 Abdelwahab National Liver Cross-sectional (prevalence, HBV: HBsAg, In multivariate model,
et al. (2012) Institute, risk factors, occupation) HBsAb, HBcAb HBsAg, 17% anti- SchAb status was not
Menouya Subjects: 842 health-care HCV: anti-HCV, HCV, 35% SchAb associated with anti-
University, Egypt workers (60% of all HCV-RNA Coinfected: 24% anti-HCV HCV status, after
(2008e2010) National Liver Institute Sch: SchAb; Note: w/ScAb adjusting for age and rural
employees), mean age Almost 2/3 of Note: No data on HBV-Sch status (estimates not
32 years (range 17 subjects had been coinfection; also, 0.2% reported)
e59 years), 45% male vaccinated against HbSAg w/anti HCV
HBV
10 Berhe et al. Worke-Mado, Cross-sectional (prevalence, HBV: HBsAg, Subjects (HBV/HCV: Sm was not associated with
(2007) Cheretee, severity, village HBcAb n 1707; Sm: n 2451): markers of HBV or HCV
Chekorso and comparison) HCV: anti-HCV 5% HBsAg, (village status in endemic villagers
Silo-Elgo villages, Subjects: 2451 persons (50% Sch(Sm): stool range 3e8%), 43% any However, in multivariate
Ethiopia (2000) random sample), in Sm PPF/PPT: HBV marker (village analysis, HVB was
endemic villages, mean ultrasound range 41e55%), 5% associated with greater risk
age 18.8, / HBsAg (village range: 3 of Sch PPF/PPT, measured
15.3 years, 52% male, of e8%), 1% anti- using either any HBV
which 70% available for HCV(village range: 1 marker (adj. OR 2.1, 95%
serology e3%), 66% Sm(village CI 1.4e3.3) or as HBsAg
Controls: 349 students from range 50e85%) (adj. OR 3.5, 95% CI 1.9
non-Sm endemic villages Coinfected: n.a. e6.7)
Controls: Controls lacked Sm Patients with heavier Sm
infection and had lower infections (EPG count
proportions of HBV and >100) with any HBV
HCV markers than Sm marker had the highest risk:
endemic villagers adjusted OR 2.5, 95% CI
1.4e4.5
HBV appears to be associated
with poorer health
outcome in Sm infected
patients
(Continued)
127
128
Study design
11 Domingo et al. Santa Rosa, endemic Cross-sectional (prevalence, HBV: HBsAg, Subjects (n 561): 14% HBV, measured by HBsAg or
(1983) area for Sj, in severity) HBsAb, HBcAg HBsAg, 32% Sj by any other HBV marker,
Barugo, Leyte, Subjects: 561 residents (56% Sch(Sj): stool Coinfected: 5% HBsAb w/ was not associated with Sj
Philippines (n.d.) random sample of total Sj status
pop), aged 1e40 years, The frequency of HBsAg
52% male, plus 22 was largely comparable
additional HSS patients, between Sj and SJ
aged 12e66 years, 86% groups (15% vs 13%)
male Similarly, 15% of HSS patients
were HBsAg
N.S. tendency for HBsAG
status to increase with
severity of Sj parasitism
noted: light 14%, moderate
17%, heavy 21%
12 Eltoum et al. Gezira, Sudan (n.d.) Cross-sectional (prevalence, HBV: HBsAg, Subject serosample (n 207): No association was found
(1991) severity) HBsAb, HBcAb, 9% HBsAg, 54% any between past or present
Subjects: 242 villagers (25% HBeAg HBV marker, 37% HBV infection and current
random sample of total Sch(Sm): stool Sm infection with Sm, with or
pop) mean age 18 years, PPF: sonography Coinfected: 2% HBsAg w/ without PPF
58% male Sm, 18% any HBV Both HBsAg, as well as any
Note: serology conducted marker w/Sm HBV marker, were less
Amy Abruzzi et al.

on 85% of sample common among the Sm
(5%, 48%) than among
those who were Sm
(12%, 58%)
Subjects (n 410): 2% anti-

13 Mudawi et al. Um Zukra Village, Cross-sectional (prevalence, HCV: anti-HCV, There was no difference
(2007) Managil province, risk factors) HCV-RNA HCV, 91% history of among anti-HCV
Gezira state, Subjects: 410 villagers, mean History of Sch(Sm): Sm villagers with respect to past
Central Sudan age of 35 years, 45% past stool sample, Coinfected: n.a. recalled history of Sch (2%
(2000) male questionnaire; yes vs 3% no)
Note: All repeat Similarly, anti-HCV was not
reactive anti- associated with a past
HCV samples history of PAT (3% PAT
were HCV- vs 2% PAT)
RNA.
14 Al-Shamiri 32 schools in 5 Sch Cross-sectional (prevalence, HBV: HBsAg Subjects (n 1484): Among Sm tested children
et al. (2011) endemic districts village comparison) HCV: anti-HCV HBsAg: n.a., anti- (n 187), coinfection of
in Taiz Subjects: 1484 children, ages Sch(Sm/Sh): stool, HCV: n.a., 21% Sm Sm with HBsAg
Governate, 5e16 years urine (district range: 0e29%), occurred less often than
Yemen (2007 7% Sh (district range: 0 HBsAg alone (4% vs
e2009) e20%) 10%)
Coinfected: n.a. Among Sh tested children
Note: HBV and HCV status (n 195), coinfection of
determined in 12% Sh w/HBsAg was
serosample of study much more common
population (17%) than HBsAg alone
(6%), mainly due to Al-
Barh village
Anti-HCV was relatively
rare among children and no
association was found with
either Sm or Sh
n.a., not available; n.o.s., not otherwise specied.
129
more than half of the studies nding 49% or more of their populations
infected (range: 20e71%). In comparison, less than 1% of the population
was infected in a non-endemic village in Brazil, which was included as a
comparison population (entry number 1). With respect to other species,
infection with S. japonicum was detected in up to 32% of the study popula-
tions in China and the Philippines (entry number 2, 11); infection with
S. haematobium was detected in up to 20% of study populations in Egypt
based on ova in urine (entry number 4), with most studies detecting it 2%
or less of their study populations (entry numbers 3, 5, 6).
All of the studies testing for HBV in this section reported their estimates
based on the HBsAg seromarker. A handful of these studies also included
additional estimates based on the presence of any HBV marker, which we
also reported in our tables. The overall prevalence of HBsAg markers in
these studies ranged from less than 1e39%, with the higher frequencies
reported in China, Egypt and Kenya (entry numbers 2, 8); More often,
HBsAg seropositivity was detected 10% less or less of the population (entry
numbers 1, 3e7, 9, 10, 12). Among studies testing for a wider range of HBV
seromarkers, evidence of past or present infection was found in 24e54% of
the study population (entry numbers 2, 3, 5, 10, 12). Infection with HCV, as
indicated by anti-HCV seropositivity, was rarely found in Ethiopia (1e3%,
entry number 10) or Sudan (2%, entry number 13), and more often found in
Egypt where it was detected in 10e40% of study populations (entry
numbers 5, 6, 7, 8, 9). The studies that tested both HBV and HCV usually
found a portion of their populations coinfected. This ranged from less than
1e5% depending in part on if the HBsAg or any HBV marker was used in
conjunction with anti-HCV seropositivity (entry numbers 5e7, 9).
Schistosoma-HBV coinfections were detected in 1e9% of study popula-
tions based on HBsAg seropositivity, and 12e20% based on any HBV
marker (entry numbers 2e6, 11, 12). Schistosoma-HCV coinfections, based
on anti-HCV seropositivity, was detected in 2e11% of the village based
study populations in Egypt (entry numbers 5e7). A greater proportion
(24%) of Schistosoma-anti-HCV coinfection was found among Egyptian
health-care workers, but it should be noted that this study tested for
schistosomiasis using the antibody test whereas the other studies used stool
samples, sometimes with ultrasound. Coinfection with S. haematobium and
HBV was generally not reported. The study with the highest S. haematobium
prevalence (20%) found 2% of their study population coinfected based
on HBsAg seropositivity (entry number 4). Similarly, coinfection with S.
haematobium and anti-HCV was not reported presumably due to no or
few cases (entry number 6). None of the studies in our table that tested for
both HBV and HCV reported the proportion of tri-infected individuals. In
addition to these studies, El-Esnawy and Al-Herrawy (2000) surveyed 233
male wastewater workers in Egypt, ages 20e60 years of age. Coinfection
with HBV or HCV and Schistosoma as indicated by antibody status was com-
mon, and was detected in 16% and 40% of the workers, respectively. In
addition, 9% of these men appear to have been triple infected with HBV,
HCV and schistosome antibody positive.
Overall, studies did not nd an association between HBV and S. mansoni
or S. japonicum across the entirety of their study populations, typically when
comparing the proportions of HBsAg seropositivity in those with schistoso-
miasis against those without coinfection (entry numbers 1e4, 10e12, 14).
An increase was noted for HBsAg seropositivity among children with S. hae-
matobium (entry number 14), however, this appears to be mainly due to one
particular village in a multivillage study; a nonsignicant increase in the pro-
portion of HBsAg among S. haematobium positive recruits was also noted
among the young male military recruits (entry number 4). Typically, studies
also found no statistically signicant difference when any HBV marker was
used (entry numbers 2, 5, 6, 11, 12); Only one study noted a nonsignicant
increase of HBV coinfection among individuals infected with S. mansoni
(40% vs 33%, entry number 3).
In addition to estimating prevalence, a number of the studies in this table
examined if coinfection correlated with the severity of disease (entry
numbers 1, 2, 4e8, 10e12). With respect to HBV, several studies that
analyzed patients with advanced schistosomiasis separately from the general
study population reported an association with coinfection. A higher propor-
tion of HBsAg seropositivity was noted among subjects with advanced S.
japonicum (43%) infection or among those reinfected with S. japonicum
(23%), when compared to those with a cured (17%), recent (12%) or no
infection (16%) (entry number 2). A similar pattern was observed when
any HBV marker was used. Another study found an increase among subjects
with S. mansoni related schistosomal periportal brosis/thickening based on
either HBsAg (OR 3.5, 95% CI 1.9e6.7) or any HBV marker (OR 2.1,
95% CI 1.4e3.3), with a 40% higher risk found among subjects with the
heaviest S. mansoni egg counts (entry number 10). In addition, two other
studies reported nonsignicant increases. In entry number 5, a tendency
was noted for subjects with schistosomal brosis to be coinfected with
HBV and/or HCV, while in entry number 11, the frequency of HBsAg
seropositivity increased with the severity of S. japonicum parasitism. As
with HBV, studies did not tend to nd an association between anti-HCV

seropositivity and schistosomiasis across the entire study populations (entry
numbers 5, 6, 8, 10), and to a lesser extent, did for those with advanced
disease in Egypt. One study conducted found a small increase in risk of
anti-HCV among those with schistosomal periportal brosis (entry num-
ber 7), while another noted a nonsignicant increase among those with the
same condition in another (entry number 5). No association was found,
however, in another study examining subjects with more generally dened
hepatocellular damage (entry number 8). Finally, Tavares-Neto (1998) and
Tavares-Neto et al. (2005) did not nd any associations with either HBV or
HCV and schistosomiasis in the investigations they conducted in Brazil,
which included analyses by type and severity of S. mansoni infection.
A few studies gathered additional data with the aim of better elucidating
the timing and/or mode of transmission of the relevant infections (entry
numbers 4, 6, 7, 9, 10, 12, 13). Some of the studies in this section suggest
that infection with schistosomiasis occurs at a younger age in endemic areas,
prior to HBV or HCV infection, which more often occurs later in life (entry
numbers 6, 7, 10). As one study noted, adults aged 40 and over were infected
four times more often than children with HBV (entry number 6). In
addition, another study observed that HCV infection appeared to reach its
peak prevalence at a younger age (60% by age 30) than HBV (75% by age
40) in their study population (entry number 7). Exceptions to this would
be populations where HBV is more often acquired through birth, in which
case coinfection with schistosomes would occur after (see discussion in entry
number 12). There has been a commercially available vaccine for HBV since
the 1980s, which has likely reduced the prevalence of this virus in some pop-
ulations, and therefore coinfection (see entry number 9), which indicated that
almost two-third of the health-care workers under study had been immu-
nized; see also Ott et al., 2012). Currently, there is no effective vaccine to
prevent HCV infection, which along with reduced control of schistosomiasis,
may have increased the frequency of coinfection in others (see Guerra et al.,
2012; Mohd Hanaah et al., 2013; Sanghvi et al., 2013). Of note, a number
of the studies that were conducted in Egypt reported an increased frequency
of HBV or HCV seromarkers among subjects who received parenteral anti-
schistosomal therapy (PAT; also described as potassium antimony tartarate),
which was an older, injection-based treatment for schistosomiasis in use prior
to the development of oral-based treatment (i.e. praziquantel). In this table,
PAT was associated with increased risk for HBsAg seropositivity (entry num-
ber 4) as well as for anti-HCV seropositivity (entry number 6). Less directly,
another study found that HCV status was associated with a past history of
schistosomiasis, which was in turn associated with PAT (entry number 7).
The association was not found in entry number 13, conducted in the Sudan.
The association with PAT was raised in several other studies in this review,
and will be addressed in our conclusion.
3. STUDIES CONDUCTED ON SPECIAL POPULATIONS

This section concerns studies conducted on special populations, typi-
cally patients with clinical liver disease. They vary with respect to if any of
the pathological agents responsible for the disease were unknown or
known at the time the study was undertaken. Most often, studies used a
cross-sectional design to estimate the frequency of the infections among
study subjects or a caseecontrol design to estimate the risk associated
with the infections for a particular health outcome. Case series designs
were also fairly common, with a handful selectively following patients
over time. Less often, prospective cohort studies were used to carefully
monitor and assess progression of disease in one or more groups of patients
(see Tables 6e8).
The studies presented in Tables 4 and 5 were conducted on subjects with
CLD or related conditions, or on subjects with primary liver cancer. The
studies in both of these tables selected patients who were unknown with
respect to schistosomiasis as well as HBV or HCV status. Since these studies
were undertaken in part to estimate the prevalence of these infections in
their patients, we present this data in a separate column in our tables.
The studies presented in Tables 6 and 8, were conducted on patients
previously diagnosed with schistosomiasis or with HCV, respectively. As
such, chronic patients gure prominently in these studies, and as a general
rule a wider range of diagnostic methods were utilized. Some of the papers
discussed here were seeking noninvasive biomarkers that could be used
instead of liver biopsy for prognosis. A number of papers look at immuno-
logical aspects of coinfection with schistosomiasis, particularly when
combined with HCV. The studies in Table 7 are a mixture of the types
discussed above. In all of these studies, however, the hepatitis that is tested
and reported upon is HBV, often with additional data on disease severity.
As mentioned earlier, a few of the studies in this review also tested for
and reported on HDV. Rather than omit it, we include it in our tables
and discuss it where it is relevant.
Table 4 Studies conducted on subjects with chronic liver disease or related conditions
134
Study design (objective)
Location and
No. Reference (years) study population Diagnosis of disease Prevalence Findings on coinfection
1 Waked et al. Liver Institute Cross-sectional HBV: HBsAg Subjects (n 1023): Coinfection with HCV,
(1995) of (prevalence, severity) HCV: anti-HCV 16% HBsAg, 74% as indicated by anti-
Menoya Subjects: 1023 patients Sch(Sm): rectal snip in anti-HCV, 32% HCV status, was
University, with evidence of patients with history active Sch more common in
Egypt CLD, aged of exposure to Coinfected: n.a. patients with active
(1992) 16e75 years, 63% infested water Note: 4% patients Sm (82%) than in
male CLD: ultrasound/liver coinfected with patients without eggs
biopsy HbsAg w/anti- (68%) or those with
HCV dead eggs (63%) in
rectum
2 Abdel-Kader Ain Shams Case series (prevalence, HBV: HBsAg Cases (n 50): 10% A greater proportion of
et al. (1997) University, severity) HCV: anti-HCV HBsAg, 26% anti- patients with minimal
Cairo, Cases: 50 minimal Sch(Sm/Sh): stool/ HCV, 66% Sch hepatic periportal
Egypt (n.s.) hepatic periportal rectal snip/SchAb Coinfected: 10% brosis were infected
brosis patients, MHF: ultrasound; HBsAg w/Sch, with Sch alone than
n.o.s. liver biopsy on 10% anti-HCVw/ were coinfected with
Note: No patient had a some Sch HBsAg or anti-
history of a fever of Note: Sch based on Note: 4% of subjects HCV
unknown origin or positive result to were coinfected
Amy Abruzzi et al.

exposure to one or more tests with HbsAg
cytotoxic drugs or w/anti-HCV
industrial chemicals Data on coinfection
between Sch and
HBV not reported
3 Angelico et al. Medical Cross-sectional HBV: HBsAg, HBcAb Subjects (n 135): 16% Most patients had
(1997) Research (prevalence, severity, HCV: anti-HCV, HBsAg, 67% anti- evidence of past or
Institute, risk factors) HCV-RNA HCV, 85% ongoing coinfections

Alexandria Subjects: 141 Sch(Sm): stool SchAb; (n 126): Patient coinfected with
University consecutive patients w/SchAb 24% active Sm active Sm and
(1993 with overt or SHF/LD/LC: Coinfected: 2% HBsAg displayed
e1995) suspected CLD, ultrasound, liver w/HBsAg portal brosis and
mean age 43 years, biopsy w/active Sm, 10% chronic hepatitis,
70% male; 60% rural Note: HDVAb present anti-HCV w/ while those with
All patients had in 3% of patients active Sm, 63% active Sm and anti-
persistent elevated anti-HCV w/ HCV displayed
ALT w/evidence of SchAb greater cirrhosis and
splenomegaly Note: 7% of patients hepatic malignancies
were coinfected Detection of HCV-
with HBsAg RNA was associated
w/anti-HCV with a more severe
liver disease and
occurred less
frequently in patients
with a history of Sch;
triple infection of
HBsAg w/anti-
HCV w/SchAb
as well as HBsAg
w/anti-HDV
w/SchAb noted in
33% and 3% of
coinfected patients
PAT associated in
general with anti-
135
HCV
(Continued)
Table 4 Studies conducted on subjects with chronic liver disease or related conditionsdcont'd
136
Location and
4 El-Zayadi Cairo Liver Cross-sectional HCV: anti-HCV Subjects (n 928): 54% Anti-HCV occurred
et al. (1997) Center and (prevalence) Sch: SchAb anti-HCV, 66% more often among
Mansoura Subjects: 928 CLD SchAb SchAb subjects than
University, patients, mean age Coinfected: 41% anti- among those who
Egypt (n.s.) 48 years, 66% male HCV w/SchAb were SchAb: Blood
Controls: 500 blood Controls (n 500): donors (16% vs 9%)
donors, mean age 14% anti-HCV, and CLD patients
39 years, 80% male 64% SchAb (62% vs 39%)
used in some analyses Coinfected: 10% anti- Patients with CLD had a
HCV w/SchAb much higher
frequency of
coinfection with anti-
HCV than blood
donor controls (41%
vs 10%)
There was no cross-
reactivity between
the two antibodies in
the testing conducted
on these populations
5 Halim et al. Al-Azhar Case-control (risk factors, HBV: HBsAg Cases (n 50): 12% Coinfection, based on
Amy Abruzzi et al.

(1999) University, severity) HCV: HVC-RNA HBsAg, 74% either HBsAg w/
Egypt (n.s.) Cases: 50 patients Sch: SchAb HCV-RNA, 84% SchAb or anti-
admitted to hospital CLD: ultrasound/liver SchAb HCV w/SchAb
with CLD, aged biopsy/CT scan Coinfected: 10% occurred more often
23e72 years, 60% HBsAg w/ in patients with CLD
males SchAb, 60% than among either
Controls: 51 patients HCV-RNA w/ control group
with other chronic SchAb Coinfection with 2 or
diseases and 50 Other chronic disease more of these diseases

apparently healthy controls (n 51): 2% may potentiate
subjects mainly HBsAg, 43% pathogenesis of liver
selected from HCV-RNA, 15% disease
workers and staff of SchAb Coinfection with HGV
hospital, matched by Coinfected: 2% and SchAb also
age and sex HBsAg w/ fairly common (20%)
SchAb, 22%
HCV-RNA
w/SchAb
Apparently healthy
controls (n 50):
22% SchAb, 0%
HBsAg, 6% HCV-
RNA, 22%
SchAb, Coinfected:
0% HBsAg w/
SchAb, 2% HCV-
RNA w/SchAb
Note: also tested for
HGV
6 Gad et al. Suez Canal Cross- HBV: HBsAg, Subjects (n 240): 8% Anti-HCV status was
(2001) University sectional (prevalence, HBsAb, HBcAb, chronic HBV, 75% far more common
and Suez severity, risk factors) HBeAb chronic HCV, 37% among patients with
Canal Subjects: 240 HCV: anti-HVC, SLD, Coinfected: SLD (75%) than
Authority consecutive patients HCV-RNA; chronic 25% Chronic HCV among volunteer
Hospitals, with suspected CLD, HCV: elevated w/SLD blood donors (20%)
Egypt mean age 45 years, ALT 6 mo w/ Controls: n.a. Patients with SLD who
137
(1998) 78% male anti-HCV were coinfected with
(Continued)
138
Location and
Controls: 50 volunteer Sch(Sm/Sh): urine, chronic HCV, had
blood donors were stool; ultrasound more severe liver
used in some analyses SLD: ultrasound w/ disease with greater
past history of Sch portal hypertension
or stool/urine and complications
from liver cirrhosis
with considerably
higher mean ALT
levels
History of blood
transfusion and PAT
much more common
among coinfected
7 Hassan et al. Ain Shams Case-control (risk factors, HCV: anti-HCV, Cases (n 46): 24% Nitric oxide (NO) levels
(2002) University complications, HCV-RNA anti-HCV & increased
Hospitals, severity) Sch: SchAb HCV-RNA, 67% proportionately with
Egypt Cases: 46 patients with Cirrhosis: ultrasound, SchAb, Coinfected: severity of liver
(1998e liver cirrhosis, mean liver biopsy 22% anti-HCV & cirrhosis, as assessed
1999) age 47 years, 72% HCV-RNA w/ by Childs
male SchAb classication
Amy Abruzzi et al.

Controls: 30 healthy Controls: 0% anti- Coinfection with anti-
subjects, matched by HCV HCV enhanced
age and sex No other data NO levels in SchAb
reported patients compared to
SchAb- patients

There was a positive
correlation between
HCV-RNA and
SchAb titre; Sch is an
important risk factor
involved in the
enhancement of NO
levels and virus
replication, which
may aggravate liver
cell injury and the
development of
cirrhosis
8 Strickland National Case-control (risk factors) HBV: HBsAg Cases (n 237): 6% There was a greater
et al. (2002) Liver Cases: 237 patients from HCV: anti-HCV, HBsAg, 58% anti- proportion of
Institute, Sm endemic area of HVC RNA HCV, 43% HCV- HBsAg patients
Egypt (n.s.) Nile Delta with Sch(Sm): stool RNA, 68% than controls that
possible CLD, mean History of Sch: history of Sch, 8% reported a prior
age 31 years, 55% questionnaire current Sm, history of Sch
male CLD: ultrasound Coinfected: 46% anti- However, no difference
Controls: 212 subjects HCV w/history of was found in the
without liver disease sch, 4% anti- proportions who
matched by age and HCV w/Sm were anti-HCV w/
sex and Controls (n 212): 3% current Sm infection
neighbourhood HBsAg, 47% anti- Reported history of
HCV, 36% HCV- prior PAT was
RNA, 55% associated with anti-
139
(Continued)
140
Location and
history of Sch, HVC status and
11% current Sm, occurred more often
Coinfected: 29% anti- in CLD patients
HCV w/history of (66%) vs controls
Sch, 6% anti- (50%)
HCV w/Sm
Note: 5% patients and
4% controls were
HBsAg w/anti-
HCV
Data on coinfection
between Sch and
HBV not reported
n.a., not available; n.s., not specied; n.o.s., not otherwise specied.
Amy Abruzzi et al.

Table 5 Studies conducted on subjects with primary liver cancer
No. Reference Location (years) and study population Diagnosis of disease Prevalence Findings on coinfection
1 Zhou et al. (2010) Eastern Case-control (risk factors, HBV: HBsAg Cases (n 317): 49% HBsAg and LSch
Hepatobiliary interaction) HCV: anti-HCV HBsAg, LT 1% independently
Surgery Cases: 317 ICC patients, LSch(Sj): liver biopsy
anti-HCV, 5% associated with ICC in
Hospital, aged 21e73 years, ICC: histologically LSch multivariate model:
China (2003 70% male conrmed prior Coinfected: 2% HBsAg RR 9.7, 95%
e2006) Controls: 634 healthy diagnosis HBsAg w/Lsch; CI 6.3, 14.8, LSch
subjects without anti-HCV w/ RR 11.1, 95% CI 3.4,
hepatopathology, LSch n.a. 36.3, no interaction
matched for sex and Controls (n 634): 7% noted; LSch was
age HBsAg, 0% anti- present in nearly equal
HCV, 1% LSch proportions of ICC
Coinfected: n.a. patients that were
HBsAg (5%) and
HBsAg (6%)
2 Mabrouk (1997) Ain Shams Case series (prevalence) HBV: HBsAG Patients (n 34): 21% Among HBsAb- subjects,
University Cases: 34 HCC patients, HCV: anti-HCV, HBsAg, 94% SchAb occurred
Hospital, aged 48e61 years, HCV-RNA anti-HCV, 35% more often in anti-
Cairo, Egypt 77% male Sch: SchAb HCV-RNA, HCV HCC patients
(1995e1996) Controls: 27 non-HCC HCC: liver biopsy/ SchAb: n.a., than in anti-HCV
subjects, n.o.s. used in CT scan, AFP Coinfected n.a. controls (92% vs 61%);
some analyses Note: SchAb status Controls: n.a. anti-HCV and
Note: patients had determined only in Note: 16% patients SchAb appear to be
underlying cirrhosis, anti-HCV w/ coinfected with associated in HCC
but no reported HBsAg subjects HBsAg w/anti- cases
history of alcohol HCV HCC may develop
abuse, hormone use or through a cascade of
toxin exposure Sch followed by HCV
(Continued)
Table 5 Studies conducted on subjects with primary liver cancerdcont'd
infection > cirrhosis >
HCC
3 Badawi and National Cancer Case-control (risk factors, HBV: HBsAg, Cases (n 102): 11% The frequency of
Michael (1999) Institute, severity) HBsAb, HBcAg HBsAg, 91% any HBsAg was higher
Cairo, Egypt Cases: 102 HCC patients Sch(Sm,Sh): stool, HBV marker, 59% among Sch patients
(n.d.) from Nile Delta, mean urine Sch than among those
age 53 years, 78% male HCC: histologically Coinfected: 9% without the parasitic
Controls: 96 subjects conrmed prior HBsAg w/Sch infection (15% vs 5%)
without diagnosis, AFP Controls (n 96): 7% In general, Sch patients
hepatopathology, of any HBV marker, had a higher frequency
comparable age and 12% Sch of HBV markers than
sex Coinfected: n.a. those with no signs of
previous or current
infection
The RR, adjusted for age
and other factors, was
highest for Sch (RR
5.2, 95% CI 2.9e9.3)
and HBsAg (RR
12.5, 95% CI 6.1
e25.6); Sch
increased the severity
of HBV infection and
elevated the risk for
HCC over that
associated with HBV
alone
No interaction in
multivariate model
reported; coinfection
w/Sch appears to
prolong HBsAg
carriage
4 Hassan et al. National Cancer Case-control (risk factors, HBV: HBsAg, Cases (n 33): 15% Among HBsAg
(2001) Institute and interaction) HbcAB HBsAg, 76% subjects, an interaction
University of Cases: 33 HCC patients, HCV: anti-HCV anti-HCV, 21% was noted between
Cairo, Cairo, mean age 55 years, Sch: SchAb SchAB anti-HCV with
Egypt (1995 70% males HCC: histologically Coinfected: n.a. SchAb (OR 10.2,
e1996) Controls: 25 HCC-free conrmed prior Controls (n 35): 3% 95% CI 1.3e79.8) that
subjects comprised of diagnosis HBsAg, 43% anti- was much higher than
nonrelative visitors, HCV, 14% for anti-HCV alone
40% male, mean age SchAb (OR 6.5, 95% CI 1.6
51 years Coinfected: n.a. e26.6) or SchAb
Note: some patients alone (OR 0.2, 95% CI
HBV (n.s.) 0.1e6.2), adj for age,
w/anti-HCV: sex
number not No interactions noted
reported between anti-HCV
and HBV (n.s.), or
between HBV (n.s.)
and SchAb that
affected HCC
development
The presence of past or
current Sch infection
appears to increase the
(Continued)
Table 5 Studies conducted on subjects with primary liver cancerdcont'd
risk of HCC, but only
in the presence of anti-
HCV
Alcohol use, oral
contraceptive use and
smoking all n.s. in
multivariate model
5 Inaba et al. (1984) 7 hospitals in Case-control (risk factors, HBV: HBsAg, Cases: 36% HBsAg Coinfected individuals
Yamanashi interaction) HBsAb (n 62), 57% (HBsAg w/SchST)
prefecture, Cases: 62 liver cancer Sch: SchST(skin test) SchST (n 56) with a daily
Japan (1977 (HCC/hepatoma) HCC/hepatoma: liver Coinfected: n.a. consumption of GE 1
e1979) patients from area biopsy, AFP Controls (n 56): 3% cup of Japanese alcohol
endemic for SJ, 79% HBsAg, 18% were at highest risk of
male, aged 45 HbsAb, 58% disease
e74 years SchST The adjusted RR based
Controls: 62 other Coinfected: n.a. on the matched pair
hospital subjects, analysis was HbsAg
matched for age and (RR 10.0), SchST
sex (RR 9.5), daily
Note: 56 (used in consumption of
matched pair analysis) alcohol (RR 3.2); 95%
CIs were not
presented; trifecta of
factors suggests possible
interaction for liver
cancer
6 Nouh et al. King Adbul Aziz Case series (prevalence) HBV: HBsAg Cases (n 50): 58% The frequency of
(1990) University Cases: 50 HCC patients, Sch (prob. Sm): SchAb HBsAg, 36% HBsAg was higher
Hospital, aged 21e90 years, HCC: liver biopsy/ SchAb among SchAb HCC
Riyadh, Saudi 86% males CT scan, AFP Coinfected: HBsAg patients (66%) than
Arabia (1985 No control group used w/SchAb among SchAb HCC
e1987) Note: The prevalence patients (53%)
of Sch in the Of note: approximately
general population twice the number of
was estimated to be patients tested positive
up to 14% at this for the coinfection than
time reported a known
history of both
hepatitis/jaundice and
Sch
Coinfection appears to be
related to the risk of
HCC
Table 6 Studies conducted on subjects with schistosomiasis
146
Study design
Location (objective) and Exclusion Diagnosis
No Reference (years) study population criteria of disease Findings on coinfection
1 Lyra et al. University of Bahia, Case-Control (severity, No evidence of HBV: HBsAg Patients with HSS were
(1976) Hospital complications) cirrhosis or other HSS (Sm): stool, liver more likely to have a
Professor Edgard Cases: 103 HSS patients causes of biopsy/clinical exam higher frequency of
Santos, Bahia, with viable Sm ova in hepatosplenomegaly DHSS: low serum HBsAg than either
Brazil (1973 stool, mean age albumin/ascites/ of the other patient
e1975) 29 years, 67% male other sign of liver control groups (8% vs
Controls: 134 patients insufciency 1%)
with other illnesses All HSS or DHSS had A greater proportion of
not related to HBV, viable Sm ova in HBsAg was noted
including 66 patients stool among patients with
with HIS, mean age decompensated
34 years, 63% male disease (12%) when
In addition, 600 blood compared with other
donors were used to HSS patients (6%), but
estimate the this difference was not
prevalence of HbsAg statistically signicant
in the local The incidence of
population HBsAg in the
patient control groups
Amy Abruzzi et al.

did not differ from
that observed among
blood donors (both
1%)
Coinfected patients had
more clinical signs of
LD, with greater

inammation of portal
spaces on liver biopsy
2 Pereria et al. Sao Paulo Liver Cross-sectional No alcohol exceeding HBV: HBsAg, HBcAb, Chronic Sch patients
(1994) Unit and (prevalence, severity) 80 g/day, anti- HBsAb, HBV-DNA were more likely to
University of Subjects: 189 consecutive HCV or other HIS (Sm): stool, clinical have at least one HBV
Pernambuco chronic Sch (46 ISS, chronic LD exam marker (44%) and be
Liver Unit, Brazil 143 HSS) patients, HSS: ultrasound, liver HBsAg (10%) than
(1990e1992) ages 8e68 years, 11% biopsy in some controls (20%, 0%),
male Note: All HSS patients respectively
Controls: 50 other had evidence of Overall, there was no
patients undergoing portal hypertension, signicant difference
surgery with negative splenomegaly or PPF in the frequency of
stools and no signs of HBV markers
LD used in some between HIS or HSS
analyses groups, although the
12 patients with
DHSS all had markers
of HBV, with 83%
HBsAg
These patients also had a
greater proportion of
replicating virus (50%)
than any other group,
suggesting that HBV
is a major pathogenic
factor in progression
to the more severe
147
forms of HSS
(Continued)
Table 6 Studies conducted on subjects with schistosomiasisdcont'd
Study design
148
3 Pereira et al. Sao Paulo Liver Cross-sectional All patients were HCV: anti-HCV, Evidence of HCV
(1995) Unit and (complications, HBsAg HCV-RNA infection (anti-
University of severity) No pregnant women, Sch(Sm): stool/rectal HCV and/or HCV
Pernambuco Subjects: 215 chronic Sm or history of alcohol biopsy, case history RNA) was present
Liver Unit, Brazil patients, with various intake exceeding LD: ultrasound/biopsy in 24% patients with
(1990e1993) forms including HIS, 80 g/day or chronic chronic Sch as
HSS, some with liver diseases from compared with 2% of
decompensated other known causes controls
disease, ages 12 Among chronic Sch
e75 years, 53% male patients, there was a
Controls: 50 other greater proportion of
patients admitted to anti-HCV in those
hospital for elective with decompensated
surgical procedures, LD (81%) than those
without Sm or CLD, with less severe
all from same infection (12% or less)
endemic area, aged Overall, 62% of chronic
20e76 years, 42% Sm patients who were
male anti-HCV were
Note: 162 chronic Sm found to be HCV-
subjects included RNA
Amy Abruzzi et al.

from previous study; Concomitant HCV
see Pereria et al. appears to be a major
(1994) (entry factor contributing to
number 2) severity of LD in
patients which
chronic Sch in Brazil
4 Conceicao Fraga Filho Cross-sectional n.a. HBV: HBsAg, anti- HBsAg was more
et al. University (prevalence, severity) HBsAg, anti-HBc common among

(1998) Hospital, Subjects: 398 outpatients Sch(Sm): stool/rectal patients with Sm than
Universidade with clinical Sm, aged biopsy among controls (8% vs
Federal, Rio de 10e62 years, 47.5% HSS: liver biopsy 2%)
Janeiro, Brazil male; clinical forms There was n.s. difference
(1983en.a.) included 6% in HBsAg between
toxaemic, 57% Sch clinical forms Sch,
infection, 25% though HSS form had
hepatointestinal, 12% higher predominance
HSS of HBV markers and
Controls: 50 other presented with more
patients without Sm severe clinical disease,
and normal liver higher frequency of
functions used in cirrhosis and worse
some analyses prognosis than other
Both patients and groups
controls were treated There was no association
by infectious and between HBsAg status
parasitic disease and prior history of
service PAT
5 Aquino et al. Gastroenterological Case series (retrospective; No other liver disease, HBV: HBsAg, HBcAb The frequency of HBV
(2000) Clinic of Santa severity) such as Wilsons HCV: Anti-HCV, and HCV serologic
Casa de Sao Cases: 101 HSS patients, disease, HCV-RNA markers was higher
Paulo, Brazil aged 19e74 years, autoimmune HSS (Sm): stool/rectal among patients with
(1994e1997) 43% male, all from hepatitis or HCC biopsy w/SchAb, HSS (i.e. HBsAg 3%,
northeastern states ultrasound anti-HCV 13%) than
and from Minas All patients were among controls
Gerais SchAb (HBsAg 1%,
149
(Continued)
150
Study design
Controls: Mean values anti-HCV 1%), and
29,406 registered was associated with a
blood donors at same greater proportion of
hospital used in some hepatic cell
analyses decompensation
Among the coinfected,
decomposition was
highest among HSS
patients who were
HBsAg (100%) or
anti-HCV w/
HCV-RNA
(81.8%)
These patients also had
notably higher AST
levels
Presence of viral
coinfection could be
an important factor in
Amy Abruzzi et al.

the decompensation
of patients with HSS
6 Silva et al. Clinics Hospital, Cross-sectional Patients presenting HBV: HBsAg, HBcAb, 34% of HSS patients had
(2011) Federal (prevalence, risk with symptoms of HBsAb viral markers for HBV
University of factors) other diseases and HCV: anti-HCV infection (30%
Pernambuco, Subjects: 230 HSS those with other HSS(Sm): patient HBcAb including
Recife, Brazil patients, mean age clinical forms of Sch history, ultrasound/ 3% HBsAg, with an

(2008) 55 years, 41% male, splenectomy additional 4%
attending Note: only HBcAb HBsAb alone), and
gastroenterology were tested for 7% were anti-HCV
outpatient clinic HBsAg There was a higher
proportion of females
who were coinfected
with HSS and HBV,
aged 50 years and over
Anti-HCV was most
common among
individuals who had
received 6 or more
blood transfusions
7 Li et al. Dongting lake area, Case series (retrospective, Incomplete medical HBV: HBsAg Overall, 43% of patients
(1993) Hunan, China mortality) records without Advanced Sj: stool, were HBsAg, with
(1985e1990) Subjects: 245 patients conrmation of Sj SchAb, autopsy w/ increased trend
who died of advanced and evidence or not liver biopsy towards advanced Sch
Sj infection, ages 9 of HBV Among those with the
e77 years, 93.9% poorest grade of liver
male, 75% farmers; function, 64% were
patients had 50% HBsAg
ascites, 23% Among those cases
splenomegaly, 26% complicated by liver
hepatosplenomegaly, carcinoma, 62% were
19% HCC HBsAG
Note: 70% of cases died
below age 50 years
151
(Continued)
152
Study design
8 Ye et al. A village in Cross-sectional None of the subjects HBV: HBsAg, HbsAb, 57% of subjects had long
(1998) Dongting lake (prevalence) sought medical care HBcAb lasting Sj infection
region, China, Subjects: 205 subjects due because of Sch(Sj): stool HBV was not associated
(n.a.) aged 0e40; All illness with Sj infection in
subjects identied this study population
house to house The frequency of
HBsAg was
comparable between
Sj and Sj subjects
(13% vs 13%), as was
the distribution of
HBcAb (60% Sj vs
53% Sj)
9 Li et al. Dongting lake area, Cross-sectional No patients were HBV: HbsAg, HBsAb Active HBV infection
(2011) Hunan, China (prevalence, risk alcoholics, though HCV: anti-HCV based on HBsAg
factors, severity) 21% had a history of Advanced Sj: patient liver sample was
Subjects: 102 patients alcohol use history, stool, SchAb, present in 44% of the
who underwent ultrasound, liver advanced Sj patients
splenectomy for biopsy In addition, 55% of these
Amy Abruzzi et al.

advanced Sj, aged 17 Note: all Sj patients patients were
e77 years, 64% male, were symptomatic HBsAb and 6% anti-
all longtime residents w/splenomegaly; HCV
of highly endemic 35% of patients had 1 patient was
area, 89% had history fresh eggs in feces; seropositive for both
repeated contact with HDV also tested HBV and HCV
Sj infested water antibodies
Patients who were
coinfected with HBV

had higher brosis and
inammation scores
than patients with
advanced Sj alone
Coinfection with HBV
appears to have
accelerated the
development of liver
brosis
10 Bassily et al. U.S. Naval Medical Cohort (comparative, n.a. HBV: HBsAg, HBsAB Coinfected patients with
(1979) Research Unit disease progression, DHSS (Sm,Sh): stool, HBsAg carried the
and Cairo mortality) urine, liver biopsy antigen for up to
University, Patient groups: 14 DHSS CAH/LD/LC: liver 3 years and had higher
Cairo, Egypt w/HBsAg, HBsAB biopsy, other clinical serum glutamin
(1970e1978) , 9 DHSS w/high exams transaminases, with
HBsAb and Note: All patients had more destructive liver
HBsAg, 12 DHSS Sch lesions on liver cell lesions in the form
w/HBsAg, HBsAb biopsy of CAHor liver
subjects cirrhosis
All male, mean age These patients were
33 years, all farmers refractory to diuretic
Note: patients were re- treatment and had
evaluated at 6 higher mortality rate
e12 months intervals (64% in 3 years)
for up to 36 months compared to 22% and
33% in the other two
groups
153
(Continued)
154
Study design
Chronic active hepatitis,
especially when
related to HBV in
patients with severe
HSS carries a grave
prognosis even when
Sch infection is cured
by specic
chemotherapy
11 Zakaria et al. Endemic Medical Cross-sectional n.a. HBV: HBsAg, HBsAb 90% of cases had a
(1979) Department, (prevalence, severity) Sch(Sm,Sh): stool, urine, present or past Sch
Cairo University, Subjects: 1013 cases rectosigmoidoscopy, infection
Cairo, Egypt presenting to the SchAb A greater portion of
medical department; LD: liver biopsy in Sch patients with present
includes 916 subjects group or past Sch infection
with present/past Sch had evidence of HBV
infection, including infection than controls
432 HSS and 119 Among patients with
cases with ascites, and Sch, 7% and 15% were
Amy Abruzzi et al.

97 subjects without HBsAg or HBsAg,
present/past Sch compared with 2 and
infection 4% of subjects without
Sch
A much greater
proportion of HVB

exposure (either
marker) was found
among Schis patients
with HSS (26%) or
Sch w/ascites (30%),
than among those
with simple Sch
infection (10%)
HBV infection in Sch
patients appears to
correlate with severity
of disease
12 Bassily et al. U.S. Naval Medical Cohort (comparative, Village women who HBV: HBsAg Coinfected villagers who
(1983) Research Unit disease progression, tested positive for Sch (Sm): stool, isotope had chronic HBsAg
and Cairo mortality) HBV were excluded scans showed a substantial
University, Patient groups: 42 male due to social CAH/LD/LC: liver incidence of CAH and
Cairo, Egypt villagers with Sm, 19 constraints biopsy, other clinical liver cirrhosis
(1976e1980) w/chronic HBV, 23 exams compared with other
w/transient HBV, Note: All subjects had study groups
aged 8e68 years, positive stools w/ Over 4 the years of the
mean age 23 mean 452 EPG; also, study, mortality was
Subjects represent 89% subjects were treated 11% in those who
of all males who were in 1978 for Sm and were Sm w/chronic
HBsAg in 1976 reinfected by 1980 HBsAg, while no
village survey; status deaths were observed
(Continued)
155
156
Study design
based on repeated among those with
HBV test in 1978 Sm w/transient
Controls: 10 chronic HBsAg (resolved
HBsAg, Sm hepatitis), or among
subjects, n.o.s. those who were Sm
Note: Many patients and w/chronic HBsAg
all of the controls Sm infected individuals
were followed for an with chronic HBV
additional 2 years, may be at especially
until 1980 high risk for
development of severe
LD, with morbid
outcome
13 Larouze et al. Three villages in Case-control (risk factors) No subject sought HBV: HBsAg, HBsAb, The pattern of HBV
(1987) Nile delta (Abou Cases: 67 subjects with medical care because HBcAb markers was similar in
Goma, Aghour heavy Sm infection of Sch; also: mass Sch(Sm,Sh): stool, urine subjects with heavy
and Sanar), (GE 50 EPG in 2 treatment (i.e. PAT) Sm infections and low
about 20 subsequent survey for Sch had not been grade or Sm free
e30 miles north years) administered in controls: any HBV
Amy Abruzzi et al.

of Cairo, Egypt Controls: 67 subjects these villages marker (58% vs 62%),
(1976e1981) with no or low grade HBcAb (5% vs 8%)
Sm, matched for age, and HBsAb (54% vs
sex and village of 55%)
origin; subjects aged No one in the study
10e29 years, 66% tested HBsAg

male HBV status, based on
any marker, was not
associated with either
Sm status or level of
infection
Sm does not appear to be
a risk factor for HBV
14 Hassanein Theodor Bilharz Cross-sectional (prevalence, No evidence of HBV: HBsAg, HBsAb, 60% of patients were
et al. Research severity) rheumatic disease, HBeAb, HBcAb coinfected; HBV
(1989) Institute, Giza, Subjects: 55 patients from no abnormal kidney HSS (Sm,Sh): stool, markers were found
Egypt (n.s.) endemic areas for Sch functions urine, recto- more frequently in
with hepatosplenic sigmoidoscopic patients with SLF (w/
affection, n.o.s. exam, liver biopsy wo chronic hepatitis),
Controls: 44 healthy Note: No eggs detected than in controls
subjects comprised of in urine 95% of patients with SLF
medical staff of and chronic hepatitis
institute; all subjects had at least one
21 years of age or positive HBV marker,
older used in some compared to 67% of
analyses patients with SLF
alone
The presence of HBV
had no effect on the
level of PIINP in
patients with SLF
(Continued)
157
158
Study design
15 Madwar et al. Tropical Medical Cross-sectional No treatment for Sch HBV: HbsAg, anti- Most patients (80%)
(1989) Institute, Cairo (complications, risk infection for HbsAb, anti-HBcAb, were positive for one
(n.s.) factors): 6 months prior to HBeAg, HBeAB or more HBV
Subjects: 105 outpatients study Sch (Sm, Sh): stool, markers, with 32%
with uncomplicated urine, rectal snip HBsAg
Sm, Sh or Sm w/Sh, Note: Live ova of Sm or Coinfected patients who
aged 9e56 years, 98% Sh detected in all were HBsAg had
male patients greater complaints of
Controls: 40 adult nausea and vomiting
medical staff, n.o.s. and higher mean
used in some analyses serum bilirubin and
aspartate
aminotransferase
levels, and fewer loose
stools
Coinfected patients with
any HBV marker,
were older and more
likely to have received
prior PAT than those
Amy Abruzzi et al.

without coinfection,
less likely to complain
of blood in stools, and
more likely to have
higher serum total
proteins, albumin,
globulin and ALT
16 Khalil et al. Ain Shams Case-control n.a. HBV: HBsAg Overall, patients with
(1994) University, (comparative; Sch (Sm), stool, urine, Sch had a greater
Cairo, Egypt complications) rectal snip, SchAb proportion of
(n.s.) Case groups: 20 ISS; 20 Note: Sm ova detected HBsAg compared
with HSS wo/ascites; in stool of ISS and with the controls
30 HSS w/ascites HSS wo/ascites; Sm (37% vs 3%)
patients ova detected by There was n.s.
Controls: 30 non-Sch, all rectal snip in HSS w/ difference, however,
from Cairo and no ascites, not in stool in the frequency of
past history of HBsAg between
exposure different Sch groups
Note: No other subject representing different
data presented phases of the disease
The higher frequency of
HBsAg across Sch
patients may be
explained by a greater
exposure to iatrogenic
exposure such as prior
PAT treatment
17 Omran et al. Theodor Bilharz Case-Control No alcohol or drug HBV: HBsAg There was no difference
(1994) Research (complications, intake which may Sch (Sm, Sh): stool, between patients with
Institute, Giza, comparative) have interfered with urine, HSS alone or
Egypt (n.s.) Case groups: 17 patients blood coagulation; rectosigmoidoscopy, coinfected with HBV
with HSS, aged 12 all patients had rectal biopsy, liver with respect to
e60 and 13 patients adequate dietary biopsy vitamin K dependent
with HSS and Sch, intake coagulation proteins
159
(Continued)
160
Study design
aged 12e55 years, Prothrobin time and
100% male partial thromboplastin
Controls: 14 healthy time were reduced in
subjects with no all Sch patients
history of Sch, compared with
thrombosis or controls
haematemesis, aged Sch coagulopathy is not
28e36 years necessarily aggravated
Note: All patients were by chronic hep B virus
admitted to hospital infection
18 Mabrouk Ain Shams Case-control n.a. HCV: anti-HCV, A greater proportion of
et al. University (complications) HCV-RNA Sch patients were
(1996) Hospital, Cairo, Cases: 20 Sch patients, Sch: SchAb, liver biopsy HCV-RNA (35%)
Egypt (1993 aged 24e60, 80% when available than in the
e1996) male haemodialysis (30%)
Controls: 27 subjects on or routine check-up
haemodialysis controls (20%)
awaiting kidney Coinfected patients had
transplantation, aged normal liver enzymes
Amy Abruzzi et al.

25e56, 78% male and represent a carrier
and 105 subjects from group who may
the general transmit the disease
population, coming silently to others
for routine check-up,
aged 20e50, 70%
male
Note: All subjects

referred to oncology
diagnostic unit
19 Fahim et al. Ain Shams Case-control No HBV, as measured HCV: anti-HCV Coinfected patients had
(2000) University (comparative, by HBsAg or Chronic Sch(Sm,Sh): the highest elevated
Hospital, Cairo, complications) HBcAb status stool, urine, SchAb, serum levels of ALT
Egypt (n.s.) Case groups: 30 chronic ultrasound and AST activities;
Sch divided by stage; coinfection with
30 chronic Sch w/ HCV appears to
chronic HCV; all aggravate liver
patients aged 35 dysfunction more
e50 years, 100% than infection with
male chronic Sch alone
Controls: 10 healthy
subjects from same
population, n.o.s.
20 Makhlouf Suez Canal Case-control No other hepatitis, HCV: anti-HCV Coinfected had higher
et al. University, (comparative, alcoholism w/ INS (Sm) stool mean IL-5 and IgE
(2006) Ismailia, Egypt complications, chronic renal failure, HSS (Sm, Sh): SchAb, serum levels than all
(n.a.) immunology) diabetes mellitus, stool/rectal snip, other groups
Case groups: 25 ISS; 15 autoimmune, chest urine, ultrasound including those with
HSS; 40 HSS w/ or cardiac diseases; HSS alone
HCV patients, aged 6 no Serum IFN-gamma was
e80 years immunosuppressive also elevated among
Controls: 15 healthy drugs coinfected patients,
individuals from same but less than in
population, n.o.s. patients with INS
alone
161
(Continued)
Study design
162
The Th-0 and Th-2
cytokine pattern and
associated depression
of Th-1 response
observed in coinfected
patients appears to
favour the chronic
form of both S and
HCV, and may play a
role in their
persistence and
severity
21 El-Moamly Suez Canal Case-control (genetics, No HBV, HIV, liver HCV: anti-HCV w/ There was no association
et al. University and severity) transplantation, HCV-RNA between the
(2013) Al-Azhar Cases: 190 chronic Sm autoimmune LD, Chronic Sm: stool, CCR5D32 mutation
University, Egypt w/HCV patients thyroid disease, SchAb, ultrasound and HCV disease
(n.a.) Controls: 220 chronic diabetes mellitus, Note: Only 30% of susceptibility in
Sm patients malaria or other patients had eggs in patients with Sm
wo/HCV; known causes of stool; all were The presence of the
All aged 18e65, 71% LD; no history of SchAb mutation, had a
male, 90% rural drug abuse, alcohol favourable effect on
Amy Abruzzi et al.

residence consumption, or hepatic brosis, with
No other controls used IFN-alpha or less severe disease
immunosuppressive observed in patients
therapy with the mutation
than with no mutant
allele
Most of the HCV
patients were
genotype Type 4a
22 Uemura et al. Kofu City Hospital, Cross-sectional No LD caused by HBV: HBsAg Sch patients with
(1992) Japan (1989 (immunology, autoimmune HCV: anti-HCV elevated ALT levels
e1990) complications) disorders, alcohol, Chronic Sch: SchAb, were more likely to be
Subjects: 96 chronic Sch drug or metabolic ultrasound/CT, liver HBsAg (14%) or
patients, in two disorders, no biopsy, rectal biopsy anti-HCV (53%)
groups based on congestive heart Note: Normal than Sch patients with
serum ALT level and failure ALT ALT normal ALT levels
137 conrmed CLD consistently LT 30 (2%, 0%, respectively)
patients (chronic IU for GE 6 mo; A high proportion of
hepatitis, cirrhosis or elevated ALT ALT CLD patients wo/Sch
HCC) without GE 30 IU at least were also HBsAg
chronic Sj; all patients once during (15%) and anti-
were admitted to 6 months HCV (49%)
hospital Less than 1% of blood
Controls: 649 voluntary donors were HBsAg
blood donors used in or anti-HCV
some analyses Among patients
coinfected with Sj and
HCV, greater liver
cirrhosis and HCC
was found than
brosis; these patients
also had consistently
high ALT levels
163
(Continued)
164
Study design
Data suggest that
coinfection with
HCV may accelerate
the derangement of
liver function
23 Hayashi et al. Tokyo Case series (progression No HBV (HBsAg) HCV: anti-HCV Case review of 9 chronic
(2000) Metropolitan of disease) Chronic Sj: liver biopsy, Sj patients, followed
Komagome Cases: 9 chronic Sj ultrasound, stool, for various lengths of
Hospital, Japan patients, aged 52 SchAb time
(n.a.) e68 years, 4 of Note: No eggs in any Among these, 44% were
whom were heavy stool; not all patients anti-HCV
drinkers were SchAb coinfected
Note: Follow-up varied; Only patients who were
patients were coinfected with HCV
followed for up to developed HCC
4 months to over (n 2), suggesting
12 years that hepatic viral
infection is more
important than Sj in
Amy Abruzzi et al.

promoting the
development of HCC
24 Koshy et al. Kuwait University, Case series (severity) No patients were HCV: anti-HCV The majority of patients
(1993) Kuwait (1990 Cases: 12 consecutive HBsAg Sm: SchAb (83%) in this case
e1991) male Sm patients with Cirrhosis/LD: Liver report were anti-
cirrhosis biopsy, ultrasound HCV on repeated
All with mild to Note: All patients had tests, suggesting that
moderate portal and origins in Egypt w/ HCV may be an

lobular activity and past history of ova in important cause of
mild to severe brosis stools and current cirrhosis in patients
high titres of with Sm
SchAb The coinfected tended
to have greatest
severity of liver disease
as indicated by
ChildePugh score
25 Koshy et al. Al Amiri Hospital, Case-control No clinical signs of HBV: HBsAg Patients with active
(1995) Kuwait (immunology, liver disease, normal HCV: anti-HCV urinary Sch (i.e. ova in
University, complications) blood counts Sch(Sh): bladder biopsy bladder) were more
Kuwait (1993) Cases: 13 male, Egyptian w/SchAb likely to be anti-
urinary Sch patients, Note: No patient had HCV than non-Sch
aged 24e56 years ova found in urine or controls (70% vs 0%)
Controls: 13 males stool; all had high An equal number of
without urinary Sch, titres of SchAb subjects were
aged 24e80 years, HBsAg in both the
reporting to same patient group and the
hospital; controls controls (10% vs 10%)
were from various Elevated serum ALT
countries including levels were noted in
Egypt 22% of coinfected
Note: All subjects, subjects, while all
including controls, subjects who were
were patients referred anti-HCV had
for cystoscopy normal liver function
tests
165
(Continued)
Study design
166
26 Al-Freihi King Fahd Hospital Case-control (risk factors, Exposure to any HBV: HBsAg HBsAg was more
(1993) of King Faisal complications) known risk factors Sch(Sm): ova in stool/ common among
University, Cases: 70 consecutive for HBV/other liver rectal snip or patients with Sm
Dammam, Saudi eligible patients with disease granuloma on liver than among non Sm
Arabia (n.a.) conrmed diagnosis biopsy controls (26% vs 4%)
of Sm, patients, mean Note: All patients had Neither sex nor
age 37, 81% male hepatomegaly and/ nationality was
Controls: 70 apparently or splenomegaly associated with
healthy subjects, HBsAb in Sch
matched for age, sex, patients
nationality and place Coinfected patients had
of residence (Saudi greater derangement
patients only) of hepatic enzymes as
Notes: In order to be indicated by abnormal
eligible, patients had liver function tests
to have known than mono Sm
HBsAg status; all patients (78% vs 42%,
subjects with OR 4.77, 95% CI
HBsAg had test 1.22e20.11)
repeated after 4 Serum albumin levels
Amy Abruzzi et al.

e6 months; subjects were also lower
were mainly Saudi, among coinfected
Yemeni and Egyptian patients than among
mono Sm patients
(61% vs 43%)
27 Mohamed Armed Forces Case-Control No HBV viral HCV: anti-HCV A greater proportion of
et al. Hospital, Riyadh, (comparative, markers, no history HSS (Sm): SchAb, coinfected patients

(1998) Saudi Arabia complications) of alcohol; all stool, ultrasound, had elevated ALT
(1990e1995) Cases: 30 HSS w/anti- patients had gastroscopy; rectal levels compared with
HCV patients negative biopsy on some those with mono HSS
Controls: 30 HSS autoimmune screen Note: Ova in stool LT (83% vs 23%), which
patients wo/HCV and normal ferritin 50% of the time; all ranged between two
All patents aged 25 levels HSS patients had to ve times the upper
e78 years, 77% male evidence of portal limit of normal
No other control groups hypertension Coinfected patients also
used in analysis had greater cirrhosis
(58%) and HCC
(10%) compared to
mono infected
subjects (19% and 0%,
respectively)
The mean age of anti-
HCV patients was
less than that for HCV
patients, which may
indicate that HCV
leads to
decompensated liver
functions earlier in
coinfected HSS
patients
28 Khano et al. Viral Diagnostic and Cross-sectional n.a. HCV: anti-HCV, Only a small proportion
(2004) Parasitology (prevalence) RNA-HCV of patients with a
Department in Subjects: 405 patients Sch(Sm): stool, SchAb clinical suspicion of
167
Dammam, Saudi with clinical suspicion Sch were SchAb
(Continued)
168
Study design
Arabia (1999 of Sch, aged 15 based on the IHA test
e2000) e55 years, 77% male, Of those who were
88% Saudi nationals SchAb (n 39),
Controls: 300 healthy 18% were found to be
blood donors used in coinfected with HCV
some analyses Infection with SchAb
alone as well as
coinfection with
HCV was more
common among non-
Saudis than among
Saudis
Among the coinfected
(n 7), 27% were
Egyptian vs 12% Saudi
all blood donors were
negative for both
SchAb as well as HCV
29 Daneshmend Khartoum Civil Case Series No cirrhosis or chronic HVB: HBsAg, HBcAb, Evidence of past and
Amy Abruzzi et al.

et al. Hospital and (complications, active hepatitis HBsAB present HBV
(1984) University prevalence) Sch (Sm): liver biopsy infection based on
Hospital, Soba, Cases: 20 HSS patients Note: Liver biopsy seromarker prole,
Sudan (n.a.) w/PPF performed as part of indicates that HBV
Controls: 41 normal routine management was found twice as
subjects comprised of on patients; liver often in Sch patients as

medical staff from biopsy not in non-Sch controls
same geographic area performed on HBsAg was 30% in
as patients controls Sch patients vs 15% in
None had controls
schistosomiasis or The largest proportion
jaundice of Sch patients (40%)
were HBsAg,
HBcAb, HBsAB,
compared 10% of
controls; whereas
15% Sch patients
were HBsAg,
HBcAb, HBsAb,
compared with 58%
of controls
HBV may be unusually
common in Sudanese
patients with Sch
30 Itoshima et al. Ibn Sina Hospital, Case Series (comparative, n.a. HBV: HBsAg/anti- No sig. Difference
(1989) Khartoum, Sudan prevalence) HBc between incidence of
(1987) Case groups: 23 Sm Sch(Sm): prior diagnosis; HBV markers
patients, 13 liver some w/advanced between hospital
cirrhosis patients, 6 disease) LD: liver controls (4%
HCC patients biopsy, HBsAg, 60%
Controls: 25 other peritoneoscopy HBcAb), blood
(Continued)
169
170
Study design
hospitalized patients Note: no testing/ donors (24%
in reporting of Sch in HBsAg, 57%
otorhinolaryngology liver cirrhosis and HBcAB), and the
or urology; 21 blood HCC patients Sch patients (22%
donors; all subjects HBsAg, 65%
aged 15 years and HBcAg), HBV
over, 83% male markers occurred
most often between
those with liver
cirrhosis (31%
HBsAg, 77%
HBcAb%) or HCC
(67% HBsAg, 83%
HBcAg)
Amy Abruzzi et al.

Table 7 Studies conducted on subjects with acute or chronic hepatitis from hepatitis B virus
Study design

(objective) and
No. Reference Location (years) study population Exclusion criteria Diagnosis of disease Findings on coinfection
1 Andrade et al. Federal University, Cross-sectional No HCV, no other HBV: HBsAg, HBeAg, Coinfection with Sch was
(2014) Minas Gerais, (prevalence, liver diseases HBeAb, HBV-DNA detected in 31% of
Brazil (1998 severity, risk Note: HDV not Chronic HBV: HBsAg patients with chronic
e2012) factors) tested as >6 mo HBV, of which 61% had
Subjects: 406 adults population came Replicative HBV: HBV replicative CHB and 39%
with chronic from DNA 2000 IU/m were inactive HBV
HBV (HBsAG nonendemic Sch (sm): Patient history, carriers; among the
for >6 months), Brazilian area stool/rectal mucosa coinfected, 70% had SPF;
median age SPF: Ultrasound, liver after controlling for
45 years, 64% biopsy alcohol consumption and
male HBV load, coinfected
patients had signicantly
more severe liver brosis
than HBV mono infected
patients (44% vs 26%);
patients with replicative
CHB and SPF had more
advanced brosis and
severe inammation
compared with patients
wo/SPF (80% vs 44%)
2 Nooman et al. Assiut University Cohort n.a. HBV: HBsAg HbsAg occurred slightly
(1977) Hospital and (comparative, risk Sch(Sm,Sh): Stool, urine, more often among Sch
Assisut University factors, disease rectal snip AVH patients than it did
Fever Hospital, progression, SHF: Liver biopsy among those who were
171
Egypt (n.d.) severity) Sch (47% vs 56%); over
(Continued)
Table 7 Studies conducted on subjects with acute or chronic hepatitis from hepatitis B virusdcont'd
172
Study design
(objective) and
Patient groups: 111 the follow-up period,
patients with coinfected patients had a
acute viral greater duration of
hepatitis and 93 antigenaemia (mean
patients with 95 days 143 days) than
acute viral those with HBsAg
hepatitis w/Sch, alone (mean
aged 2e66 years, 36 days 61 days), and
64% male was not affected by
Note: Patients were specic Sch treatment;
all admitted to Sch infection may
hospital for AVH; prolong the retention of
after release, HBsAg after an acute
followed from attack
6 months to up to
2 years
3 Gaffar et al. (1991) Shebin El Kom Cohort (severity, No Wilsons disease, HBV: HBsAg, HBcAg, Overall, 64% had acute
Fever Hospital, disease haemolysis or HBsAg, HBcAg HBV and 62% were
Egypt (1983 progression, risk cholestasis Acute HBV: HBcAb wo/ Sch
Amy Abruzzi et al.

e1985) factors) No patients had HBsAb Coinfection of Sch w/acute
Patients: 144 acute HAV Sch(Sm,Sh): Stool, urine, HBV was detected in
viral hepatitis rectal snip 37% of patients, of which
patients, Note: Also tested for 25% were tri-infected
predominately antibodies to HDV with HDV
rural, n.o.s. 1 year after admission to
Note: Patients were hospital, the HbsAg
seen on at 3 of 4 carrier rate was nearly

scheduled visits fourfold higher in those
over 1 year with Sch, with greater
splenomegaly, more
persistent and greater
liver function
abnormalities, more sever
histological ndings on
liver biopsies and higher
mortality than those with
acute HBV infection
alone
Splenomegaly increased
from 40% to 69% in
coinfected over
12 months, compared
with 11e20% in those
with HBV alone
Both male sex and
coinfection were
associated with the
prolonged HBsAg carrier
state
4 El-Hawy et al. Al-Azhar University Case series/case- n.a. HBV: HBsAg, HBcAb, Serological markers among
(1993) Hospital, Egypt control (severity, HBeAg, HBcAg the 54 patients with CAH
(1991e1992) risk factors) Sch(Sm,Sh): Stool, urine, indicated that 67% were
Cases: 54 patients sigmoidoscopy HBsAg and 28%
with chronic CAH; liver biopsy HBsAg w/
173
active hepatitis Note: All patients had HDVCoinfection with
(Continued)
Table 7 Studies conducted on subjects with acute or chronic hepatitis from hepatitis B virusdcont'd
174
Study design
(objective) and
(CAH), aged HBsAg in Sch was also fairly
18e43 years, 69% hepatocytes; this study common (39%), with
male also tested for 32% Sch w/HBsAg
Controls: 20 healthy antibodies to HDV; Similar proportions of Sch
subjects matched 28% of patients were were found among those
by age and sex tri-infected with with HBsAg or
(used in some HBV, HDV and HBsAg w/HDV
analyses only) schistosomiasis serological markers (48%
Note: All patients vs 47%), which was
followed for greater than that observed
6 months among controls (22%)
Tri-infected patients (n 7)
showed the greatest
altered liver prole, with
greater advanced liver
disease, and had the
highest mortality
n.a., not available; n.o.s., not otherwise specied.
Amy Abruzzi et al.

3.1 Subjects with chronic liver disease and related

conditions
The eight studies in Table 4 were undertaken in Egypt, where CLD is usu-
ally an attributed infection with S. mansoni. Typical feature of hepatic schis-
tosomiasis, which may also be called schistosomal liver disease, include the
development of hepatic granuloma and periportal brosis, with bleeding
from gastroesophageal varices. For reasons that are not always clear, many
patients with CLD have preserved liver functions, while others have a
more progressive course and die from hepatic failure and or/complications
including HCC. These studies, published between 1995 and 2002, were un-
dertaken to investigate if coinfection with HBV and/or HCV infection may
explain some of these differences.
Of the eight studies in this table, one was a case series (entry number 2);
the other seven were either cross-sectional (entry numbers 1, 3, 4, 6) or casee
control studies (entry numbers 5, 7, 8). With respect to the cross-sectional
studies, two used external controls drawn from blood donors in some analyses
(entry numbers 4 and 6); one cross-sectional study also gathered additional
data on subjects including history of PAT or blood transfusion. All of the
caseecontrol studies used healthy controls matched for age and sex (entry
numbers 5, 7 and 8); one caseecontrol study included a second control group
comprised of chronic disease patients (entry number 5), while another study
matched cases and controls by neighbourhood (entry number 8).
Overall, the studies in this table ranged in size from 46 to 1023 subjects,
with the majority of studies conducted on 250 or fewer subjects. In addition
to being Egyptian, study subjects tended to be male (60e80%), with mean
ages that ranged from approximately 30e48 years. Most subjects were drawn
from patients who presented at a hospital or clinic with symptoms that
included recurrent jaundice, chronic hepatitis, ascites, and a history of gastro-
intestinal bleeding. Sometimes, but not always, persistently elevated serum
alanine transaminase (ALT) levels were noted (i.e. entry numbers 3, 6).
Two of the studies restricted their patients to those with either minimal he-
patic periportal brosis (entry number 2) or liver cirrhosis (entry number 7).
Most studies checked for ova from S. mansoni in stool samples and/or
rectal snip, with a few using a schistosome antibody test as an alternative; in
addition, one study also specically checked for S. haematobium infection
(entry number 6). The proportions infected with schistosomiasis in these pop-
ulations depended on whether it was detected by tests based only on stool
and/or rectal snip (8e32%, entry numbers 1,3 and 8), or relied in whole or
in part on the presence of antibodies (66e84%, entry numbers 2e5, 7). Entry
number 3 was notable for reporting results based on both methods. Two
studies focussed on active schistosomal infections (entry number 1, 3). Among
controls, the proportion infected with schistosomiasis was not always re-
ported; when it was, it varied widely, ranging from 15% to 64% in two studies
both testing for schistosomal antibodies (entry numbers 4, 5). Most subjects
were then examined by ultrasound and/or liver biopsy, from which it was
determined if they had one or more of the following: periportal brosis,
splenomegaly, hepatic decompensation or cirrhosis, or HCC.
All eight studies conducted serum tests for the HCV antibodies and/or
HCV-RNA, six of which also tested for the presence of HBV usually using
the HBsAg marker. HCV was particularly common among patients with
more generally dened CLD, which ranged from 54% to 75% based on
anti-HCV seropositivity (entry numbers 1,3,4,6, 8). A comparable propor-
tion was found for one of the studies using HCV-RNA as their serological
indicator (74%, entry number 5), while a somewhat lower proportion was
reported by another study based on their methods (43%, entry number 8).
HCV infection was detected least often in the two studies conducted on
minimal hepatic periportal or liver cirrhosis patients, which reported 26%
(entry number 2) and 24% (entry number 7) of their patients were anti-
HCV seropositive, respectively. The frequency of HCV infection was not
always reported for control populations depending on the nature of the
study. When it was, it varied widely depending on control population,
with 0, 14 and 47% of controls testing anti-HCV seropositive (entry
numbers 7, 4, 8, respectively), and 6e43% based on HCV-RNA seroposi-
tivity (entry numbers 5, 8). HBV infection was as found far less often, with
6e16% of study populations testing HBsAg seropositive (entry numbers 1e
3, 5, 8). HBsAg seropositivity was even more rare among controls, infecting
approximately 2% or fewer subjects (entry numbers 5, 8); nally, several
studies reported the proportions of their patients who were coinfected
with HVBeHCV, which ranged from 3% to 7% (entry numbers 1e3, 5).
Only one study also noted that HBVeHCV coinfection occurred among
their controls (4%, entry number 8).
Six of the studies in this table reported the frequency of coinfection with
HCV in their study population, which appeared to vary depending on
several factors including patient population and methods of testing for schis-
tosomiasis. Among CLD patients, studies identifying stool-based, active
schistosomiasis infections detected coinfection with HCV in 6e10% of their
populations (entry numbers 8, 3, respectively), whereas studies that utilized a
schistosome antibody test to identify past or present infections found 41e

63% of their populations coinfected (entry numbers 3, 4, 5). The propor-
tions did not appear to vary based on study design or whether anti-HCV
and/or HCV-RNA testing was used. Among controls in these studies, the
proportion coinfected with HCV was 6% for stool-based, active infection
(entry number 8, case-control) and 10e22% when based on schistosomal
antibody test (entry numbers 4, 5, cross-sectional and case-control, respec-
tively). With respect to related conditions, SchistosomaeHCV coinfection
was detected in 22% of liver cirrhosis patients (entry number 7, case-control)
and 10% of patients with minimal hepatic periportal brosis (entry number
2, case series). Both of these studies relied on a schistosome antibody test,
with one study using it as an alternative to stool- and/or rectal snip-based
samples (entry number 2).
Overall, all of the studies that analyzed disease severity among patient
groups, found it associated with SchistosomaeHCV coinfection. Coinfected
CLD patients displayed more severe liver disease than non-coinfected
patients, with greater portal hypertension and/or cirrhosis (entry numbers
1, 3, 5, 6). Several cross-sectional studies connected coinfection to the pres-
ence of live Schistosoma ova in either the stool or rectum. One study found
that coinfected patients were more likely to have active S. mansoni infection
(82%) than patients without eggs (68%) or, with dead eggs in their rectum
(63%) (entry number 1). In another, coinfected patients with active S. mansoni
infection had greater cirrhosis and hepatic malignancies (entry number 3).
Similarly, coinfected patients with active HCV, as detected by the presence
of HCV-RNA, found it associated was a greater severity of liver disease (entry
number 3, 6; See Figure 1). Among cirrhosis patients studied using a casee
control design, coinfection was associated with enhanced nitric oxide levels,
which increased proportionately with the severity of disease (entry number 7).
Fewer studies reported data on SchistosomaeHBV coinfections, which
were considerably less common than with HCV. Among CLD patients, co-
infection was detected in 2% of patients with active S. mansoni infections and
10% when the antibody test was used (entry numbers 3 and 5, respectively);
coinfection was not found among apparently healthy controls in the one
caseecontrol study which reported data on it (entry number 5). Only one
of the crossesectional studies analyzed patients by disease severity, nding
coinfected patients had greater portal brosis and cirrhosis (entry number
3). Notably, these coinfected patients all had active Schistosoma disease. Con-
trary to this, past history of schistosomiasis rather than active disease was asso-
ciated with coinfection with HBV; no other data were provided on these
Figure 1 Liver biopsy from an anti-HCV positive patient excreting schistosomal eggs in
stools. The microphotograph shows parenchymal nodules surrounded by brous septa.
Inammation is observed within portal tracts, septa and at the stromaeparenchyma
interface (cirrhosis with features of chronic aggressive hepatitis). HCV, Hepatitis C virus.
Angelico et al. (1997).
patients (entry number 8, case-control). Finally, neither coinfection with

schistosomiasis and either HBV or HCV was associated with minimal hepatic
periportal brosis (MPF) in the case series reported in entry number 2.
3.2 Subjects with primary liver cancer

This section examines studies conducted on subjects with primary liver can-
cer, with most studies focussing on HCC. HCC occurs more often in males,
typically aged 50 years or older, and has a higher incidence in Africa and Asia
with half of all global cases occurring in China (Jemal et al., 2011). HCC is
strongly associated with scarring of the liver (cirrhosis), which may be caused
by a number of factors including alcohol abuse and autoimmune disorders of
the liver. In developing/non-Western countries in particular, HCC is most
often associated with HBV or HCV infections (Jemal et al., 2011). One of
the studies included in this section examines coinfection among patients
with intrahepatic cholangiocarcinoma (ICC), which is a rare subtype of pri-
mary liver cancer that is also known as bile duct cancer. Bile duct cancer of
the intrahepatic variety frequently emerges in the setting of CLD where it
requires differential diagnosis with respect to HCC (Bragazzi et al., 2012).
Difcult to diagnose, it frequently presents at a late stage when no effective
therapeutic intervention is possible (Bragazzi et al., 2012). The presence of
gallstones in biliary ducts of the liver (i.e. hepatolithiasis) and HCV infection
are established risk factors for ICC, while at present hepatic schistosomiasis,
liver cirrhosis and HBV infection are regarded as probable causes (Bragazzi
et al., 2012).
The six studies in Table 5, dated 1984e2010, were conducted in China,
Egypt, Japan and Saudi Arabia. Most of the studies in this table were con-
ducted on middle aged, male HCC patients (entry numbers 2e6) and
ranged in size from 33 to 102 patients. Four of the studies presented in
this table used a case-control design, with controls comprised of disease-
free subjects of comparable age and sex (entry numbers 1, 3e5); two studies
used matching to better balance these possible confounders (entry numbers 1
and 6). Three of the four caseecontrol studies used multivariate methods to
estimate risk and checked for the presence of statistical interaction between
key factors (entry numbers 1, 4 and 5). The remaining two studies used a
case series design to evaluate the frequency of coinfection, one of which
included a minimally described control group (entry numbers 2 and 6).
In all of the studies, liver cancer was histologically conrmed, either
through a biopsy done at the time of the study or previously as determined
by a review of the patients medical records. Two of the six studies pertained
to infections with S. japonicum; the other four studies all pertain to S. mansoni
and/or S. haematobium infections. The majority of these studies relied on a
schistosome antibody test to determine infection; only one of the casee
control studies checked stool and urine for evidence of current Schistosoma
infections (entry number 3). Among HCC patients in S. mansoni and
S. haematobium areas, the prevalence of Schistosoma infection was 59% based
on ova in stool/urine (entry number 3) and 21e36% in studies testing for
schistosome antibodies (entry numbers 4, 6). The prevalence of schistosomi-
asis among the controls in the caseecontrol studies ranged from 12% based
on stool/urine (entry 3) to 14% based on an antibody test in the one study
that tested for it (entry number 4). In S. japonicum areas, the prevalence was
57% among HCC patients based on a schistosome antibody test, which was
comparable to the frequency observed among their controls (58%, entry
number 5). Evidence of liver schistosomiasis due to S. japonicum was slightly
higher, however, among ICC patients than their controls (5% vs 1%, entry
number 1).
All six of the studies in this table tested for the presence of the HBsAg
marker. Three of these studies (entry numbers 1, 2, 4) also tested for anti-
HCV seropositivity, with one also testing for the presence of HCV-RNA
(entry number 2). The frequency of HBsAg among HCC patients in these
studies ranged from 11% to 58% (entry numbers 2e6), compared with
approximately 3% in any reported control population (entry numbers 4, 5).
Among ICC patients, HBsAg was found in 49% of patients and 7% of

controls (entry number 1). The frequency of HCV infection was higher
than observed for HBV among HCC patients, which were found to be
76% (entry number 4) and 94% (entry number 2) anti-HCV seropositive.
Notably, both of these study populations were in Egypt, where coinfection
between HVB and HCV was also found in 16% of the study population
(entry number 2). Among controls, 43% were found to be anti-HCV sero-
positive in the only caseecontrol study report such data (entry number 4).
Compared with HCC patients, the frequency of HCV among ICC patients
was considerably lower, occurring in less than 1% among ICC cases and
absent in controls (entry number 1).
Few studies reported the frequency of coinfection in their study popu-
lations. Of the two caseecontrol studies that did, one study found 9% of
HCC patients and 2% of ICC patients were coinfected with HBV and schis-
tosomiasis (entry numbers 3, 1, respectively). All studies conducted analyses
of their data for coinfection, however, and found an association between
HBV and schistosomiasis (entry numbers 1, 3, 5, 6) or between HCV and
schistosomiasis (entry numbers 2, 4). Among HCC patients, the frequency
of HBsAg was higher among Schistosoma patients with ova in stool and/or
urine than among those without the parasitic infection (15% vs 5%, entry
number 3); similarly, the frequency of HBsAg seropositivity among HCC
patients was higher among those tested positive for schistosome antibodies
than among those who tested negative in a case series study (66% vs 53%,
entry number 6). As mentioned earlier, several caseecontrol studies used
multivariate methods to estimate the risk associated with specic factors.
One study found that schistosomiasis (OR 5.2, 95% CI 2.9e9.3) in
conjunction with HBV (OR 12.5, 95% CI 6.1e25.6) elevated the risk of
HCC over that observed for HVB alone (entry number 3). The absence
of a reported interaction suggests this effect may be additive. Elsewhere, a
multiplicative interaction was noted for the risk of HCC, this time associated
with coinfection with S. japonicum and HBsAg infection in conjunction with
the daily consumption of one cup or more of Japanese alcohol (RR 10.0,
95% CI not reported, entry 5). Autopsy studies of HCC patients have also
suggested an association between S. japonicum and HBV (Nakashima
et al., 1975; Kojior et al., 1986). Both HBsAg seropositivity (RR 9.7,
95% CI 6.3e14.8) and liver schistosomiasis (RR 11.1, 95% CI 3.4, 36.3)
were also found to be independent risk factors for ICC, again without
interaction, suggesting an additive rather than a multiplicative effect (entry
number 1).
Finally, two studies in this section (entry numbers 2, 4) evaluated the

effects of SchistosomaeHCV coinfection among HBsAg negative subjects.
The rst of these was a case series, and found that Schistosoma antibodies
occurred more often in anti-HCV positive patients than in controls who
were also anti-HCV seropositive, which could not be attributed to other
likely factors such as alcohol abuse, hormone use or greater toxin exposure
(92% vs 61%, entry 2). The other study followed a caseecontrol design
(entry number 4) and estimated an interaction between anti-HCV and
Schistosome antibody seropositivity (OR 10.2, 95% CI 1.3, 79.8) that was
greater than the sum of anti-HCV (OR 6.5, 95% CI 1.6,26.6) and Schis-
tosome antibody seropositivity (OR 0.2, 95% CI 0.1e6.2) alone, using a
multivariate model. In addition, a higher proportion was also reported by
El-Tonsy et al. (2014), who found that 61% of the anti-HCV HCC patients
they examined were coinfected with schistosomiasis based on an antibody
test. In this study, he also found that coinfected patients had a younger
mean age and more often had tumours that were multifocal and larger in
size than in subjects with HCV alone. This, in conjunction with the other
results reported above, suggests a more aggressive course of disease for coin-
fected subjects.
3.3 Subjects with schistosomiasis

The 30 studies in Table 6 were all conducted on patients with schistosomi-
asis, many with an advanced form of the disease, and ranged in publication
date from 1976 to 2013. Egypt and Brazil are best represented, with 12 and 6
studies each, respectively. The remaining studies were conducted in China,
Japan, Kuwait, Saudi Arabia and the Sudan, and are each represented by two
or three studies. Accordingly, most these studies in this table pertain to the
S. mansoni species. Ten of the studies also tested for S. haematobium ova in the
urine. Nine of these were conducted in Egypt, in populations where
S. mansoni infections were more common; the remaining study was
conducted on Egyptians in Kuwait and focussed exclusively on urinary
schistosomiasis (entry number 25). In addition, ve of the studies in this table
pertained to S. japonicum infection (entry numbers 7e9, 22 and 23). The vast
majority of these studies used multiple tests to determine the presence and
extent of schistosomal infection. These methods routinely included check-
ing for the presence of ova in stool and/or in the rectum through rectal snip,
the use of the schistosomal antibody test, and the use of an ultrasound and/or
a liver biopsy to check for the presence of granuloma and evaluate the extent
of damage to the liver. Only a few studies relied on stool and/or urine
checks (entry number 8 and 13) or the schistosomal antibody test (entry
number 18) as the sole or main method. Several studies in this table reported
that all of their study subjects had viable ova in their stools (entry numbers 1,
12 and 15). Just as often, studies indicated that only a portion of their subjects
had viable ova, even after multiple samples were checked (entry numbers 9,
21, 23, 27). Usually these subjects were at an advanced stage of schistosomi-
asis, when the inammatory reaction and scarring of the intestinal wall is
such that it can prevent deposited eggs from moving into the intestinal
lumen and exiting through the stool (Li et al., 2011).
The studies in this table ranged in size from 9 to over 900 study subjects,
about two-thirds of which were conducted on patient groups of around 100
or less; most compared disease severity between coinfected and mono-
infected schistosomal subjects and were careful to exclude subjects with
other possible causes of CLD, including alcohol abuse and other hepatitis
viruses other than those of interest. Eleven of the studies used a case-control
design (entry numbers 1, 13, 16e21, 25e27), two of which matched con-
trols by sex and age. There were also 11 studies using a cross-sectional design
(entry numbers 2e4, 6, 8, 9, 11, 14, 15, 22, 28) as well as 6 studies best
described as case series (entry numbers 5, 7, 23, 24, 29 and 30). Many of
the cross-sectional (entry numbers 2e4, 22 and 28) and case series (entry
numbers 5, 29 and 30) studies used a control group in one or more analysis,
which were typically comprised of blood donors, medical staff or occasion-
ally a selection of other patients. Only two of the studies in this section fol-
lowed a prospective cohort design (entry numbers 10 and 12), which was
used to evaluate the progression of disease. Several of the caseecontrol
studies included patients at various stages of schistosomal disease, and so
were able to make additional comparisons pertaining to coinfection (i.e.
entry numbers 16, 19 and 20). A few of the studies, chiey caseecontrol,
compared mono and coinfected subjects for immunological or genetic
differences (entry numbers 20e22 and 25).
More than half of the studies in this table tested for the presence of HBV
(21 studies) in their schistosomiasis patients; 14 of the studies tested for HCV,
including 5 that tested for both HBV and HCV. In the studies concerned
with HBV infection, the HBsAg seromarker was used most often to
determine infection with data on any additional HBV markers reported
separately. In the studies concerned with HCV, infection was always
determined by the presence of the anti-HCV seromarker, sometimes with
additional testing for HCV-RNA. Overall, many of the studies using a non-
schistosomal control group found a higher proportion of both HBsAg in
their schistosomal patients. This seemed to vary less by study design than it
did by country, severity of schistosomiasis in the patient population and
composition of the control population. In two cross-sectional studies
conducted in Brazil, HBsAg seropositivity was found in 8e10% of schisto-
somiasis patients spanning various stages of the disease, compared with 0e
2% of other patients who were used as controls (entry numbers 2 and 4).
The proportion of HBsAg seropositivity in schistosomal patients versus
non-schistosomal controls was usually higher in most other countries such
as Japan (14% vs less than 1%, cross-sectional, entry 22); Saudi Arabia
(26% vs 4%, caseecontrol, entry number 26); Sudan (30% vs 15%, case
series, entry number 29) and Egypt (37% vs 3%, case-control, entry number
16), respectively. Occasionally, differences in the frequency of this marker
were not statistically signicant, particularly in some of the smaller case series
studies (i.e. entry number 30, 22% schistosomal patients vs 4% hospital
controls). In at least one other small study, this time a case-control, the pro-
portion of patients and controls infected with HBsAg was identical (10% vs
10%, entry number 25).
The studies that tested for HCV were even more consistent in their
ndings, all reporting greater anti-HCV seropositivity among schistosomi-
asis patients than in their control populations and spanning a range of study
designs (entry number 3, 5, 18, 22, 25, 28). This generally ranged from 13%
to 35% in most studies. It was appreciably higher among schistosomal
patients with elevated ALT levels (53%, cross-sectional, entry number 22)
and among active urinary schistosomiasis cases (70%, case-control, entry
number 25). Studies conducted in countries other than Egypt usually found
that less than 2% of controls were anti-HCV seropositive; in these studies,
which included caseecontrol, cross-sectional and case series designs, the
controls were comprised of volunteer blood donors or they were other
non-schistosomal patients (entry numbers 3, 5, 22, 25, 28). A much higher
proportion of HCV infection was found among controls in a caseecontrol
Egyptian study, specically replicative virus, which reported 30% of haemo-
dialysis patients and 20% of the general population attending the hospital for
routine checkups were infected (entry number 18). Many countries were
represented by at least one study that tested for the presence of both viruses
in their study populations. Studies conducted in Brazil, Kuwait and Japan all
found greater anti-HCV seropositivity than HBsAg seropositivity in their
study subjects (entry numbers 5, 6, 22, 25). The main exception to this
was China, where a cross-sectional study conducted on advanced S. japoni-
cum cases who underwent a splenectomy found 44% HBsAg seropositive
compared with 6% anti-HCV seropositive (entry number 9). Coinfection

with both HBV and HCV was generally not reported in these studies; again,
the only exception was entry number 9, which reported one coinfected case.
Unfortunately, none of the studies conducted in Egypt that were located for
this table (entry numbers 10e21) tested for both HBV and HCV in the same
study population.
The most interesting ndings of the studies reviewed in this section came
from comparing mono and coinfected schistosomal groups. We begin by
reviewing the ndings for coinfection with HBV, which typically found
that HBsAg seropositive schistosomal patients had more severe disease, as
indicated by greater brosis, greater cirrhosis, chronic hepatitis or liver
cancer, than mono-infected schistosomal subjects across every type of study
design (entry numbers 1, 2, 5, 7, 9e12, 14). The association of HBV
coinfection with greater liver brosis and inammation in schistosomal
patients was particularly well-illustrated in entry number 9, which was con-
ducted on patients with advanced S. japonicum infection (see Figure 2).
Studies were particularly consistent in nding an association between
HBV and decompensated liver disease (entry numbers 1, 2 and 5). In one
of the cross-sectional studies, 83% of subjects with decompensated hepatos-
plenic schistosomiasis were HBsAg seropositive; a greater proportion of
these patients also had replicating virus than the other groups, further sup-
porting the supposition that HBV infection plays an important role in disease
Figure 2 HBV infection in liver section shown by immunohistochemical staining of

HBsAg. A., Patient with both advanced schistosomiasis and HBV infection. The brown
granules present in the cytoplasm denote an active HBV infection (400 magnica-
tion). B., Patient with advanced schistosomiasis only. No brown granules are evident
in the cytoplasm (400 magnication). HBV, Hepatitis B virus; HBsAg, Hepatitis
B surface antigen. Li et al. (2011).
progression in schistosomal patients (entry number 2). Notably, two of the

studies in this table were prospective cohorts with follow-up periods that
lasted for up to 2 or 4 years, and both found greater progression of disease
among the patients who were coinfected (entry numbers 10 and 12). Mor-
tality was also higher among the coinfected in these two prospective studies
(11% of the coinfected in entry number 12, 64% of the coinfected in entry
number 10), compared with no deaths among mono-infected schistosomi-
asis subjects. Similarly, a case series found a high proportion of patients (43%)
who died of advanced S. japonicum infection in China were HBsAg seropos-
itive; in this study, coinfection was also often found in patients with the
poorest liver functions (64%) or in patients with HCC (62%) (entry number
7). More generally, coinfection with HBV was associated with greater
derangement of liver functions in a number of cross-sectional and casee
control studies, in particular higher serum ALT, aspartate aminotransferase
(AST) and bilirubin levels (entry numbers 15, 22, 26). Greater amounts of
vomiting and nausea were also sometimes noted among coinfected patients
(entry number 15).
In contrast with the results reported above, two caseecontrol and two
cross-sectional studies did not nd an association between disease severity
and coinfection with HBV (entry number 4, 8, 13, 16). Of interest, two
of these studies examined only subjects stool or urine to determine the level
of schistosomiasis infection, and both reported that none of their study
subjects had sought medical care for their disease (entry numbers 8 and
13). This seems to imply that none of the subjects in these studies were expe-
riencing advanced, clinical disease. These studies, one cross-sectional and the
other caseecontrol, were also both village-based whereas most of the other
studies in this table were conducted on hospital patients, making them an
interesting point of comparison with the studies conducted on general pop-
ulations that we presented earlier in Table 3. Entry numbers 4 and 16 each
compared clinical forms of schistosomiasis, usually intestinal schistosomiasis
with hepatosplenic schistosomiasis, and neither found a signicant difference
in the frequency of HBsAg seropositivity between their schistosomal groups.
Entry 4, which used a cross-sectional design with a comparison group,
reported that patients with the hepatosplenic form had a higher predomi-
nance of HBV markers and presented with more severe clinical disease,
including greater cirrhosis and worse prognosis than the other groups. Entry
16, which used a comparative case-control design, failed to provide addi-
tional data on study subjects that would have assisted us in making a better
evaluation of its ndings.
Most studies that examined HCV found that coinfected schistosomal

patients had more severe liver disease than mono-infected schistosomal sub-
jects, with greater cirrhosis, decompensated disease or HCC (entry number
3, 5, 22, 23, 24, 27). The proportions coinfected were often dramatically
high and indicative of active infection with HCV. For example in a matched
caseecontrol study conducted on chronic schistosomiasis patients in Brazil,
81% of subjects with decompensated disease were anti-HCV compared
with 12% of those with less severe infection; this study also tested for
HCV-RNA, and found that overall 62% of chronic schistosomiasis subjects
also had active HCV infection (entry number 3). A Brazilian case series
reported that an even higher proportion of their decompensated hepatos-
plenic schistosomiasis patients had active, RNA-conrmed, HCV infection
(82%, entry number 5). Similarly, 83% of male schistosomiasis patients with
cirrhosis in Kuwait tested anti-HCV seropositive on repeated tests; in this
case series study, coinfected patients also had the greatest severity of disease
as indicated by ChildePugh score (entry number 24). In a caseecontrol
study conducted in Saudi Arabia, hepatosplenic schistosomiasis patients
who were anti-HCV positive had greater cirrhosis (58%) and HCC (10%)
than those who were anti-HCV negative (entry number 27). The authors
of this study also noted that coinfected subjects also had a lower mean age
than mono-infected subjects, suggesting that HCV may result in faster
progression to severe disease. This was also supported by a case series that
followed nine chronic schistosomiasis patients in Japan for up to 12 years
and found that HCC only developed in coinfected cases (entry number
23). Of related interest, Iida et al., 1999 documented that a slightly greater
proportion of patients developed haematoma who were triple infected with
SchistosomaeHCVeHBV compared than did SchistosomaeHCV patients
without the concomitant HBV infection (33% vs 26%); these proportions
were based on a comparison of two small groups, with 9 and 31 patients
respectively, and were not statistically signicant.
Consistent with these ndings, several studies also reported that coin-
fected subjects had abnormally elevated ALT and AST liver enzyme blood
levels, which are usually indicative of greater liver damage (entry numbers
19, 22, 25, 27). This includes the only study in our review conducted exclu-
sively on patients with active urinary schistosomiasis (entry number 25). In
this caseecontrol study, 22% of patients coinfected with anti-HCV had
elevated ALT levels, while levels among all mono-infected urinary schisto-
somiasis patients were normal. The proportion of patients displaying liver
enzyme derangement was greater when the subject was coinfected with
S. mansoni or S. japonicum and HCV. In a caseecontrol study conducted in

Saudi Arabia, for example, 83% of patients with hepatosplenic schistosomi-
asis had elevated ALT levels compared with 23% of mono-infected subjects,
with values that were two to ve times the upper limit of what is generally
considered normal (entry number 27). An exception to this was the casee
control study conducted in Egypt, which reported normal liver enzymes
in coinfected patients with HCV-RNA (entry number 18). In addition,
two of the more recent caseecontrol studies in this table examined immune
responses or looked for genetic variants that might be associated with the
disease severity among the coinfected (entry numbers 20 and 21, respec-
tively). As discussed in more detail in Section 3.5, patients with Schisto-
somaeHCV coinfection displayed greater Th0-Th2 and lesser Th1
responses compared with mono-schistosomal patients, which appears to
favour the chronic form of both diseases and may play a role in their persis-
tence and severity (entry number 20). The second of these studies, also con-
ducted in Egypt, checked for the presence of a genetic mutation that may
increase HCV susceptibility in schistosomal patients, but failed to nd an
association; as in most other Egyptian studies, the anti-HCV seropositive
patients were mainly genotype 4a (entry number 21). In comparison,
genotype 1a and 3a are usually found in Brazil, and are frequently found
coinfected subjects (Alvarado-Mora et al., 2012).
3.4 Subjects with acute or chronic hepatitis from HBV

The four studies in Table 7 all pertain to coinfection between schistosomiasis
and Hepatitis B, and were conducted on patients with acute or chronic hep-
atitis. Acute hepatitis is generally dened in these studies and elsewhere as an
inammation of the liver that lasts less than 6 months, while chronic hepatitis
lasts for longer than this period. Most studies began by testing for the pres-
ence of both HBV and schistosomiasis in their hepatitis patient population
and compared frequencies between groups. As noted earlier in this review,
there are causes other than HBV or HCV that can produce inammation of
the liver. For example, these include other infections such as mononucleosis,
chicken pox, as well as drug or alcohol abuse, toxins, fatty liver disease,
trauma, and autoimmune hepatitis (WHO, 2014). It is not always clear
why some individuals with viral hepatitis recover during the acute stage,
while others progress to the chronic form of the disease.
The four studies in this table ranged in date from 1977 to 2014, and
include one of the oldest studies in our review, as well as one of the most
recent. The studies ranged in size from 54 to 406 subjects, which tended
to be male and represented a range of ages. The three older studies in this
section were all conducted in Egypt; the most recent study is from Brazil
(entry number 1). Two of the studies were prospective cohorts undertaken,
at least in part, to see if coinfection with schistosomiasis plays a role in the
progression and severity of Hepatitis B (entry numbers 2, 3). In these studies,
subjects were selected with acute viral hepatitis and followed over time to
see if they developed chronic hepatitis and evaluated for other complica-
tions. The other two studies used a cross-sectional or a caseecontrol design,
the latter in conjunction with a case series analysis with a modest amount of
follow-up, to evaluate if the frequency of coinfection in their subjects was
associated with disease severity (entry numbers 1, 4).
All of the studies used HBsAg as the main seromarker of interest. Three
of the papers also used additional markers or methods that reect some of the
advancements made in detecting HBV infection during this time period (en-
try numbers 1, 3, 4). All of the subjects who were diagnosed with schisto-
somiasis in these studies had histological conrmation of S. mansoni ova
based on stool or rectal snip, with most studies also obtaining a liver biopsy.
The three studies based in Egypt also checked for the presence of S. haema-
tobium, which was generally absent or rare in these study populations.
Despite a span of more than 20 years in publication dates and a number
of other differences, most studies found about one-third of their study pop-
ulations were coinfected with S. mansoni and HBV, with frequencies
ranging from 31% to 37% (entry numbers 1, 3, 4). Entry 2, one of the oldest
studies in this review, reported that 22% of their cohort was coinfected. The
four studies were also largely in agreement with respect to ndings on dis-
ease progression and severity. In one of the cohort studies following acute
hepatitis patients over time (entry number 2), coinfected subjects had a
greater duration of antigenemia (mean 95 days 143 days) than those
testing HBsAg seropositive alone (mean 36 days 61 days). Interestingly,
this study noted that a greater proportion of schistosomiasis was not always
found among those who were HBsAg seropositive (entry number 2). This
was interpreted by the authors as indicating that subjects already suffering
from schistosomiasis are not by nature more susceptible to HBV. Once
infected with HBV, however, patients with an underlying schistosomal
infection appear to have a tendency to remain chronically infected and
experience greater disease progression than mono-infected schistosomal
subjects. Of particular concern, treatment for the underlying schistosomiasis
in this study, did not shorten the carriage rate of HBV observed in these
patients.
In the other prospective cohort (entry number 3), the HBsAg carrier rate
was nearly fourfold higher among the coinfected when compared to mono-
infected HBV acute hepatitis patients after 1 year of follow-up. In addition,
coinfected patients were found to have greater splenomegaly, more persis-
tent and greater liver function abnormalities with accompanying histological
changes and higher mortality than mono-HBV subjects. This nding is
echoed by entry number 1, which also found that coinfected patients had
more severe liver brosis than mono-infected HBV patients (44% vs
26%); this cross-sectional study also reported that patients with replicative
HBV and schistosomal portal brosis had more advanced brosis and severe
inammation than any other cases. Finally, three of the four studies in this
table tested for the presence of Hepatitis D in their populations and two
reported its frequency in coinfected subjects (entry numbers 3, 4). The pro-
portions that were triple infected were of note in both of these studies,
approximately 9% of all patients with acute viral hepatitis in entry number
3 and 13% of all chronic active hepatitis patients in entry number 4. Entry
number 4, which used a caseecontrol design for its main analysis, also noted
that patients who were triple infected showed the greatest alterations in liver
prole, displayed the most advanced liver disease, and had the highest
mortality.
3.5 Subjects with HCV

The 16 studies selected for inclusion in Table 8 were all conducted on sub-
jects with HCV infection, and all but one was conducted on Egyptians. The
studies ranged in publication date from 1998 to 2014, and included a greater
share of more recent publications than some of our other tables. More than
half of these studies used caseecontrol (entry numbers 2, 3, 5, 6, 8, 10 and
12) or cohort (entry number 1 and 7) designs, which typically compared
carefully selected groups of HCV mono-infected subjects with subjects
coinfected with schistosomiasis versus other controls. The remaining studies
were all cross-sectional (entry numbers 4, 9, 13e16), with the exception of
one case series (entry 11). Most of the studies in this table were undertaken
for purposes of evaluating immunological or other physiological differences
associated with coinfection. Severity of disease and other complications were
often examined in the studies using case-control and cross-sectional designs,
while disease progression was evaluated by the two prospective cohorts (i.e.
entry numbers 1, 7). A few studies, two of which used a caseecontrol
design, were undertaken for the purpose of evaluating a noninvasive
Table 8 Studies conducted on subjects with hepatitis C virus
190
Study design
(objective) and
No Reference Location (years) study population Exclusion criteria Diagnosis of disease Findings on coinfection
1 Kamal et al. Ain Shams Cohort (comparative, No HBV, HAV, Acute HCV: ALT Viral loads were higher in
(2001b) University, disease progression, HEV, (20 normal) w/ coinfected at baseline,
Cairo, Egypt severity, immunology) autoimmune, or anti-HCV w/ otherwise comparable to
(1992e1994) Patient groups: 15 acute alcoholic or drug- HCV-RNA mono HCV patients
HCV and 17 acute related causes Sch: History, stool/ with respect to age,
HCV w/Sch patients, rectal biopsy gender, peak ALT at
mean age 28 years, SchAb, ultrasound entry, source of HCV
66% males (genotype 4) infection
Patients were and absence of brosis; at
consecutive, follow-up, 33% of mono
symptomatic and acute HCV patients had
followed for a mean of recovered vs 0% of
72 4.6 months coinfected; based on
paired liver biopsies
taken at entry and again
after 6 years, coinfected
had dramatically higher
brosis progression rates
compared to mono
HCV subjects (0.53 vs
Amy Abruzzi et al.

0.1 units per year);
coinfected subjects had
either absent or transient
weak HCV-specic
CD4 T cell responses
with Th0/Th2 cytokine
production, which at
week 12 was inversely
correlated with brosis
progression
2 El-Refaei et al. Al Azhar University Case-control No HCC, HAV, Chronic HCV: anti- Coinfected subjects had
(2003) Hospital, Cairo, (Comparative, HBV, HDV, HIV, HCV w/elevated fewer late differentiated
Egypt (n.a.) immunology) EpsteineBarr virus, ALT GE HCV-specic CD8 T
Case groups: 14 chronic pregnancy, history 6 months; cells compared to HCV
HCV and 13 chronic of alcoholic liver detectable HCV- mono infected subjects,
HCV w/Sm patients, disease or RNA but were comparable
ages 18 years and over autoimmune Sch(Sm): Patient with respect to early
Controls: 6 local Subjects hepatitis; none of history w/stool, differentiated cells
without evidence of the subjects had SchAb Net CD8 T cell responses
Sm or HCV, matched received IFN-alpha were comparable
by age therapy between groups as were
IL-15 levels
Ccoinfection appears to
target a specic subset of
memory CD8 T cells
in HCV infection
3 El-Refaei et al. Al Azhar University Case-control (comparative, No HCC, HAV, Chronic HCV: anti- Coinfected subjects had
(2004) Hospital, Cairo, immunology, HBV, HDV, HIV, HCV w/elevated altered cytokine proles,
Egypt (n.a.) genetics) EpsteineBarr virus, ALT GE with lower IFN-gamma
Case groups: 15 chronic pregnancy, history 6 months; and higher IL-10 levels
HVC and 23 chronic of alcoholic liver detectable HCV- than mono-infected
HCV w/Sm patients, disease or RNA subjects; however, the
191
(Continued)
Table 8 Studies conducted on subjects with hepatitis C virusdcont'd
192
Study design
(objective) and
aged 18 years and over autoimmune Sch(Sm): Patient decrease in IFN-gamma
Controls: 10 local hepatitis, previous history, w/stool,
levels observed in the
individuals, matched IFN-alpha therapy SchAb coinfected did not
by age without w/ribavirin appear to be associated
evidence of Sm or with a decrease in the
HCV number of HCV-
specic T cells that
produced IFN-gamma;
Egyptians infected with
HCV genotype 4 can
mount HCV-specic T
cell responses (both
CD4 and CD8)
despite the prevalence of
concomitant Sch
4 El-Shorbagy Zagazig University, Cross-sectional (severity, No signs or symptoms Sch: stool, SchAb, Nearly half of all patients
et al. (2004) Egypt (2000e risk factors) of advanced liver ultrasound, liver tested positive for SchAb
2003) Subjects: 109 HCV- disease, or other biopsy Coinfected patients had
RNA patients, aged signicant medical HCV: anti-HCV w/ greater hepatic brosis
Amy Abruzzi et al.

5e78 years, 71% male diseases other than HCV-RNA than those with mono
those under study HCV (OR 7.6, 95% CI
1.9e35.5) coinfection,
along with age GE
45 years and a positive

history of blood
transfusion was
associated with severe
hepatic pathology, and
may warrant special
attention with more
intensive follow-up
5 ElSammak Alexandra Case-control (comparative, No HBsAg, HCV: anti-HCV, Coinfected patients
et al. (2006) University, complications) nonorgan specic HCV-RNA displayed higher serum
Alexandria, Case groups: 30 HCV, 30 autoantibodies, SHF: Ultrasound, activin A levels
Egypt (n.a.) HCV w/Sch hepatic hereditary defects, SchAb compared to those with
brosis and 30 HCV history of alcohol HCC/other LD: HCV alone, along with a
w/Sch associated consumption, use ultrasound, concomitant reduction
HCC patients of certain elevated AFP, liver in serum IGF-1; Activin
Mean ages 44e50 years, medications biopsy A levels were highest
73e93% male including among those with HCC
Controls: 30 healthy corticosteroids and and lowest among
subjects, mean age heparin controls; activin A may
43 years, 80% male represent a potential
prognostic tool to
determine the severity of
liver cirrhosis as it
correlated with
ChildePugh score and
appears to be a predictor
for the development of
HCC
193
(Continued)
194
Study design
(objective) and
6 Emam et al. Zagazig University Case-control (comparative, No HIV, HBV, liver Chronic HCV: anti- While HCV-RNA viral
(2006) Hospitals, immunology, cirrhosis, HCC, or HCV w/HCV- load was comparable
Zagazig, Egypt complications) alcoholic liver RNA w/elevated between mono and
(n.a.) Case groups: 18 chronic disease; no patients ALT for GT coinfected HCV groups,
HCV and 17 chronic had IFN-gamma 6 months coinfected subjects had
HCV w/Sch patients, and/or ribavirin Sch: Patient history, lower IFN-gamma and
mean age 45 years, treatment within w/SchAb, stool/ higher IL-4 and IL-10
57% male 12 months prior to rectal snip levels in comparison
Controls: 15 healthy sample collection with monochronic
subjects with no HCV patients or healthy
evidence or history of control
HBV, HCV, or Sm, It was also noted that IL-4
matched for age and and IL10 levels did not
sex correlate with one
another, or with
histological activity
index or HCV viral load
in any group
7 Kamal et al. Ain Shams Cohort (comparative, Patients had no Acute HCV: anti- Patients were followed for
(2006) University, disease progression, alcohol HCV for 6 months progression of disease,
Amy Abruzzi et al.

Cairo, Egypt severity) consumption, no Chronic HCV: anti- with paired biopsy at
(1992e Patient groups: 22 acute HIV, or HBV; No HCV w/elevated start and end of study
2001) HCV and 20 acute patient had active ALT (10) and At entry, coinfected had
HCV w/Sm patients, Sch HCV-RNA higher HCV-RNA titres
mean age 29 years, Sch(Sm): Ova in and TNF-alpha levels
62% male stool/rectal biopsy than mono infected
Note: An additional 45 w/SchAb groups, but otherwise
HCV patients, 58% SHF: Paired liver were similar with respect
coinfected w/Sm, biopsy to age, sex, peak ALT,

were used as a Note: no patient had source of infection,
validation cohort for clinically active HCV genotype and level
the YKL-40 Sch of liver brosis
biomarker Within 2 years, coinfected
Patients were followed subjects exhibited
for 96 4.6 months greater increases in
TFG-B levels and YKL-
40, suggesting that the
brotic process was
progressing with changes
in the extracellular
matrix
By the end of the follow-
up period, coinfected
had more rapid
progression to brosis
than mono HCV
subjects (0.61 vs 0.1 units
per year)
Coinfected also developed
evidence of portal
hypertension with
splenomegaly and
oesophageal varices,
independent of liver
brosis
195
(Continued)
196
Study design
(objective) and
8 Raslan et al. National Research Case-control (comparative, No extrahepatic Chronic HCV: anti- Both IGF-1 and IGFBP-3
(2007) Centre, Cairo, severity) failure, metabolic HCV w/elevated were lower in subjects
Egypt (n.a.) Case groups: 17 chronic disease, recent ALT for GT with coinfection than in
HCV and 13 chronic systemic infection, 6 months HCV alone and
HCV w/Sch patients, or active variceal Sch: SchAb indicative of more severe
aged 27e67 years, bleeding, recent Cirrhosis: Ultrasound liver disease
60% male, 47% liver alcohol intake, Among the coinfected,
cirrhosis corticosteroid mean serum IGFBP-3
Controls: 16 healthy therapy were negatively
subjects, matched for correlated with age and
age and sex AST levels, and
positively correlated
with serum albumin and
prothrombin
Data suggests that
coinfection with Sch
may have additional
harmful effect on hepatic
function beyond that
observed on HCV alone
Amy Abruzzi et al.

9 Abbas et al. National Liver Cross-sectional No B-cell Chronic HCV: anti- Coinfection with Sch was
(2009a) Institute, (prevalence, risk malignancy, HCV w/HCV- detected in 62% of the
Menofeya factors) immune liver RNA chronic HCV related
University, Subjects: 119 consecutive disease, chronic Sch (Sm): history, chronic liver disease
patients with chronic rheumatic disorders abdominal patients
Menofeya, Egypt HVC related chronic or chronic ultrasound w/ The prevalence of Sch
(2006e2007) liver disease; 82% male infections from SchAb, liver coinfection was

hepatropic agents biopsy signicantly higher in
CG: cryocrit level HCV infected patients
>1% without
cryoglobulinemia (CG)
compared with patients
with it; the risk of mixed
CG in HCV infected
patients might be
suppressed by the
presence of Sm
coinfection and
accompanying Th2
response
10 Abbas et al. National Liver Case-control (comparative, No cirrhosis or other HCV: anti-HCV w/ Grade of inammation and
(2009b) Institute, genetics, forms of chronic HCV-RNA stage of brous showed
Menofeya complications) liver disease before no association with IL-
University, Case groups: 54 HCV and combination 10 polymorphisms
Menofeya, Egypt 55 HCV w/Sch therapy Frequency of SM
(2007e2008) patients, aged 19 Sch (Sm): History, coinfection and IL-10
e67 years, 74% male stool w/SchAb, genotypes/haplotypes
Controls: 62 healthy rectal biopsy when were not sig different
subjects, aged 18 possible between nonresponders
e56 years, 65% male, Note: 70% patients and responders to
without evidence of treated with combination therapy
Sm, HCV or HBV, w/ PegIFN-alpha and
normal LFT ribavirin for
197
(Continued)
198
Study design
(objective) and
Note: all subjects were 12 weeks, 30%
born in same untreated
hyperendemic rural
area where prevalence
of HCV and SM is
highest (>15%)
11 Abdel-Aziz National Liver Case series (biomarker) No cirrhosis, HBV, Chronic HCV: anti- 50% of chronic HCV cases
et al. (2012) Institute, Cases: 100 chronic HCV autoimmune HCV w/HCV- included in this study
Menouya patients, aged 21 hepatitis RNA for 6 months were SchAb
University, e60 years, 65% male Sch: SchAb There was no difference in
Menouay, Patients were followed Fibrosis: liver biopsy the ability to use serum
Egypt (2010 before, during and hyaluronic acid (HA) as
e2012) after therapy and for marker of liver brosis
6 months later between mono and
coinfected HCV groups
HA appears to be a sensitive
marker of liver brosis,
regardless as to the
etiologic agents involved
Amy Abruzzi et al.

12 Ramadan et al. Ain Shams Case-control (comparative, No HBsAg, HIV, Chronic HCV: anti- Coinfected patients had
(2012) University, pathogenesis, liver cirrhosis or HCV w/HCV- higher mean TNF-alpha
Cairo, Egypt inammatory renal disease, or RNA w/elevated levels than healthy
(n.a.) response) other causes of AAT for GE subjects or subjects with
Case groups: 30 chronic hepatocellular 6 months mono HCV
HCV and 30 chronic injury such as Sch(Sm): Ova in stool, Super oxide dismutase
HCV w/Sch patients, alcohol and drug SchAb, liver (SOD) levels were lower

mean group aged related injuries biopsy among all HCV
46e48 years subjects, with slightly
Controls: 20 healthy lower levels among the
subjects, mean age coinfected
45 years There is a cause and effect
relationship between
increased levels of TNF-
alpha and decreased
levels of SOD, relative to
the progression of
chronic HCV, especially
with bilharzial
coinfection
13 Esmat et al. Ain Shams Cross-sectional No other liver Chronic HCV: anti- Coinfection with Sch was
(2013) University, (prevalence, severity, diseases, HCV w/HCV- detected in 25% of the
Cairo, Egypt diagnostics) decompensated RNA chronic HCV patients
(n.a.) Subjects: 231 chronic liver cirrhosis, Sch: SchAb There was an association
HCV patients, aged 18 HCC, liver biopsy Fibrosis: ultrasound, between SchAb status
e60 years contraindication, liver biopsy and the presence of liver
or unt for IFN brosis
and ribavirin As compared with biopsy,
treatment, or the sensitivity of
BMI 30 kg/m2; broscan was impaired
no prior antiviral in coinfected patients,
therapy particularly in subjects
with portal brosis with
rare septa or in subjects
199
with numerous septa
without cirrhosis
(Continued)
200
Study design
(objective) and
14 Allam et al. National Liver Cross-sectional Patients were Spontaneously resolved 48% of the study subjects
(2014) Institute, (prevalence, severity, unaware of HCV HCV: anti-HCV tested positive for SchAb
Menouya virology) status at time of the wo/HCV-RNA A nonsignicant difference
University, Subjects: 141 health-care initial investigation Current HCV: anti- was noted in the
Menouya, workers and had not yet HCV w/HCV- frequency of
Egypt (n.a.) Mean age 41 years, 65% received standard RNA spontaneously resolved
male, 70% rural of care at the time Sch (Sm): SchAb, HCV cases between the
residents this study was ultrasound coinfected and mono
Note: This is a follow-up undertaken Note: Most had HCV infected HCV groups
of Abdelwahab et al. genotype 4 (24% vs 33%)
(2012), reported in Periportal brosis found in
Table 3 coinfected subjects
No data on the time (25%), whereas
elapsed between echogenic liver was
studies is presented found in 25% of mono-
infected subjects
Overall, coinfected had
comparable viral
clearance, RNA levels
Amy Abruzzi et al.

and indicators of liver
inammation to those
with mono HCV
infection
Note: It is unclear what span
of time took place
between tests conducted
on study subjects
15 Helal et al. Al-Jimi and Tawam Cross-sectional No HBsAg, history HCV: anti-HCV 52% of anti-HCV
(1998) Hospitals, United (prevalence, severity) of alcohol or drug Sch: SchAb patients were coinfected
Arab Emirate Subjects: 44 patients, aged abuse, autoimmune LD: liver biopsy with Sch
(1991e1994) 20e55 years, 80% liver disease Portal and septal
male Note: Serum samples inammation was
Note: All patients were taken prior to present in varying
Egyptians and all therapy degrees in all subjects
patients with Sch were Coinfected subjects had
male slightly more cirrhosis
and mild CAH, while
than mono-infected
subjects had slightly
more moderate CAH
Overall, no sign differences
between coinfected and
mono infected HCV
groups; anti-Sch
positivity did not
enhance the severity of
HCV hepatic pathology
In addition, SchAb status
did not give a false
(Continued)
201
202
Study design
(objective) and
positive reading for anti-
HCV
16 Tanaka et al. Kofu in Yamanashi, Cross-sectional (virology) n.a. HCV: anti-HCV Coinfection with Sj was
(2005) Katayama in Subjects: 113 HCV-1b w/HCV-RNA present in 57% of HCV-
Hiroshima and patients from endemic Sch (Sj): SchAb 1b subjects
Chikugo in Saga/ Sj areas, mean group w/ultrasound/CT HCC occurred more often
Fukuoka ages 67e70, 51% male HCC: patient among the coinfected
Prefectures, Japan Controls: 18 individuals history, conrmed than HCV alone (45% vs
(2001) with HCV-1b from by ultrasound/ 23%)
nonendemic Sj, mean CT/liver biopsy The molecular
age 67 years, 50% male evolutionary analysis
indicates that the
estimated spread of
HCV in previously Sj
endemic areas in Japan
coincides with injection
treatment for Sj
conducted in 1921
n.a., not available.
Amy Abruzzi et al.

alternative to liver biopsy that could be used to monitor disease severity (i.e.
entry numbers 5 and 12).
Compared with others in this review, the studies in this table also tended
to be conducted on a small number of patients, with carefully selected study
populations and exclusion criteria. The largest among them was a cross-
sectional study conducted on 231 subjects (entry number 13); the vast
majority of the other studies involved less than 100 patients. Most were
particularly careful to exclude patients with HBV, HDV or other liver con-
ditions, and several noted if their data were gathered prior to subjects
receiving standard treatment. In terms of the case-controls, controls were
typically comprised of similarly aged individuals; four of these studies used
matching to balance age and sex confounders, and occasionally other factors
(entry numbers 2, 3, 6 and 8). As elsewhere, study subjects in this table
tended to be male, and mean ages between 41 and 48 years were common.
The two prospective cohorts were notable for having study populations
with particularly younger mean ages (28 years, entry number 1; 29 years,
entry number 7), as was appropriate since substantial follow-up time was
involved.
All of the studies in this section began with subjects with conrmed
HCV infections. Typically, studies used more than one test to check for
anti-HCV seropositivity and conrmed active status with HCV-RNA.
Most studies were conducted on chronic HCV patients typically infected
with the virus for at least 6 months (entry numbers 2,3,8,11e13), or patients
with active disease that had been present for an unknown or unspecied
amount of time (entry numbers 4, 6, 9, 10, 14, 16). The two cohort studies
both followed patients diagnosed with acute hepatitis subjects over time
(entry numbers 1 and 7). None of the other studies in this table were lon-
gitudinal, though the one case series (entry number 11) included 6 months
of follow-up on HCV patients, and two of the cross-sectional studies made
use of data collected during other time periods in their write-ups (entry
number 14 and 16).
With respect to Schistosoma species, the 14 studies that were conducted in
Egypt as well as the one from the United Arab Emirates were principally
concerned with S. mansoni coinfections; the one study conducted in Japan
utilized a population-based cross-sectional design and pertained to S. japoni-
cum (entry number 16). All of the studies used a schistosome antibody test on
their subjects, always in conjunction with other diagnostics such as stool,
rectal snip, ultrasound and/or liver biopsy, depending on purpose of the
investigation. Studies also varied as to whether schistosomiasis was a current
or past infection, in part reecting the difference in design between a cohort

and caseecontrol study (i.e. entry number 7 vs entry number 12, respec-
tively). Only a few of the cross-sectional studies estimated the frequency
of coinfection in their patients, as most studies began by selecting study
groups for comparison. Among those reporting prevalence, estimates varied,
with SchistosomaeHCV coinfection detected in 25% of chronic HCV
patients (entry number 13, Egypt) and 62% and 57% of active HCV disease
patients (entry numbers 9, 16, from Egypt and Japan, respectively).
We begin by discussing the two prospective cohort studies, both by
Kamal et al. that were conducted to evaluate disease progression (entry
numbers 1 and 7). Both studies selected acute HCV patients who were
then followed over time for a period ranging from about 6 to 8 years, and
used a paired liver biopsy at the beginning and end in conjunction with
other measures. At the start of each study, coinfected patients had higher
viral titres than the mono-HCV groups, but were otherwise comparable
with respect to age, sex, extent of brosis and other important indicators.
In entry number 1, coinfected patients had active schistosomiasis whereas
in entry number 7, active disease was not present. Nevertheless, their results
with respect to disease progression were remarkably similar. At follow-up,
entry number 1 found that 33% of mono-infected HCV patients had
resolved their infection compared with 0% of the coinfected; when severity
of disease was compared, the coinfected had dramatically higher rates of
brosis progression compared to mono-infected HCV subjects (0.53 vs
0.1 units per year, respectively). Similarly, in entry number 7, coinfected
subjects also had more rapid brosis than mono-infected HCV subjects
(0.61 vs 0.1 units per year, respectively). The coinfected also had developed
evidence of portal hypertension with splenomegaly and oesophageal varices,
independent of liver brosis. These results appear to be in keeping with
many of the cross-sectional and caseecontrol studies in this table that
observed greater pathology among the coinfected when compared to their
mono-infected HCV subjects (entry numbers 4, 5, 8, 12, 13, 16). The
highest risk observed was noted in a cross-sectional study conducted on
HCV-RNA patients (entry number 4), which estimated a 660% increase
in the risk of hepatic brosis was associated with an active Schistosoma-active
HCV concomitant infection (Odds Ratio 7.6, 95% CI 1.9e35.5). Higher
levels of brosis among coinfected subjects were also described in Hano
et al. (2011).
As mentioned earlier, many of the studies in this section were under-
taken to examine immunological differences and many compared the
cytokine proles of study subjects. Most studies were in agreement,

observing that SchistosomaeHCV coinfected subjects had dominant Th2
responses while mono-infected HCV subjects had dominant Th1 responses.
Two caseecontrol studies found that coinfected subjects had lower IFN-
gamma and higher IL-10 levels than mono-infected HCV subjects (entry
numbers 3, 6). One of these studies also noted higher IL-4 levels among
coinfected subjects, but noted that these were not correlated with IL-10
levels or with viral load (entry number 6). In another caseecontrol study,
IL-10 polymorphisms were not associated with grade of inammation, stage
of brosis or responsiveness to combination therapy for HCV infection
(entry number 10). Interestingly, in a cross-sectional study coinfected sub-
jects were also found to have a lower prevalence of cryoglobulinemia
than mono-infected anti-HCV seropositive subjects, which was attributed
to the tipped Th2 immune response associated with schistosomiasis coinfec-
tion (entry number 9).
In keeping with the immune responses described above, coinfected sub-
jects were also found to have fewer early CD4 T cells than mono-infected
patients, which was associated with greater disease progression in one of the
cohorts we described earlier (entry number 1); a caseecontrol study found
fewer late differentiated HCV-specic CD8 T cells than mono-infected
subjects, which was associated with greater pathogenesis (entry number 2).
At least two studies reported higher mean TNF-alpha levels in coinfected
subjects, indicative of an increased inammatory response (entry numbers
7 and 12); one of these was a prospective cohort, which found it to be
associated with the increased rate of brosis observed in coinfected patients
(entry number 7). A caseecontrol study reported that coinfected subjects
had higher levels of serum actin A in conjunction with a reduction in
IGF-1, which was associated with more severe liver disease and a higher
risk of HCC (entry number 5). Lower IGF-1 as well as IGFBP-3 were found
among coinfected subjects in another caseecontrol study (entry number 8),
where they served as early predictors of hepatic dysfunction and were asso-
ciated with other indicators of more severe liver disease. Finally, an associ-
ation with HCC was also noted in entry number 16, a population based
cross-sectional study, which found that coinfected subjects developed
HCC nearly twice as often as mono-infected HCV subjects (45% vs 23%).
Two studies appear to be in direct disagreement with the results discussed
above (entry numbers 14, 15), and both were cross-sectional in design. One
study was an update to Abdel-wahab et al. (2012) (Table 3, entry number 9),
which retested asymptomatic anti-HCV seropositive health-care workers
with known schistosomal antibody status (entry number 14). Overall,

mono- and coinfected subjects were found to have comparable levels of viral
clearance, HCV-RNA titres, and indicators of liver inammation, though
several nonstatistical differences such as higher periportal brosis among
the coinfected were noted. It was not clear, however, how much time
had elapsed between the anti-HCV tests reported on in this study, only
that the patients had not yet received treatment prior to being retested.
The other study, conducted on Egyptian patients in the United Arab
Emirates, reported no signicant differences between coinfected and
mono-infected patients with respect to the severity of their hepatic pathol-
ogies (entry number 15). Neither of these studies used any external compar-
ison populations, nor was noteworthy for utilizing any additional methods
that would have strengthened the level of inference that could be drawn
from them.
4. STUDIES COMPARING SUBJECTS WITH

SCHISTOSOMIASIS AND SUBJECTS WITH HCV
The 11 studies selected for inclusion in Table 9 were published be-
tween 2000 and 2011. All but two of the studies were conducted in Egypt.
The other two were conducted in Brazil. All of the studies were small, con-
ducted on 100 or fewer subjects, and all focussed on schistosomiasis and
HCV infections. Ten out of the 11 studies used a caseecontrol or cohort
design to compare two mono-infected groups with one that is coinfected,
as well as a nondiseased control group for comparison. The remaining study
was a case series that also compared a small series of patients, but lacked suf-
cient detail on inclusion or exclusion criteria or other methods to justify a
caseecontrol designation. We did not identify any studies for inclusion in
this table on schistosomiasis and HBV that followed this type of fully
comparative design and met our other inclusion criteria.
As in Tables 7 and 8, studies were often undertaken to compare disease
severity, immunological and/or genetic differences, and most studies were
careful to eliminate subjects with other viral infections and liver diseases
from their study population. All of the studies in this section used stool-
based exams to check for ova from S. mansoni, often in conjunction with
ultrasound to evaluate advanced disease. Three of the eight caseecontrol
studies used matching to control differences by age and sex (entry numbers
4, 6 and 8); matching was also used in one of the cohort studies, to make
patient groups more comparable with respect to age, sex and duration of
infection (entry number 5). A few studies conducted in Egypt also checked
for ova in the urine from S. haematobium, which was generally absent (entry
numbers 3, 10, 11). Two caseecontrol studies reported that all of their pa-
tients had ova in stool samples (entry numbers 8 and 9); this is in contrast
with two other caseecontrol studies that reported ova in less than half of
their schistosomiasis patients (entry numbers 10 and 11). With respect to
testing for HCV, eight of the studies used HCV-RNA to conrm disease
status. Based on this testing, three caseecontrol studies specically studied
chronic HCV patients (entry numbers 4, 6 and 7). The other, a prospective
cohort, specically followed acute anti-HCV patients for disease progres-
sion (entry number 5).
The two prospective cohorts in this section each followed patients for
more than 6 years (entry number 3 and 5). The rst of these studies (entry
number 3), found that over the observation period, coinfected subjects had
greater progression of disease, resulting in higher liver-related mortality
(48%) compared with mono-HCV (12%) or mono-schistosomal (3%)
subjects. HCC only developed in coinfected subjects (11%), and not in
either mono-infected group. Of interest, most coinfected subjects were
HCV Genotype 4 (92%) compared with 62% of mono-HCV subjects.
Coinfected subjects also had higher HCV titres and long duration of
HCV than mono-HCV subjects in this study. Unfortunately, not all subjects
were at the same stage of disease at the start of this study, which makes
additional comparisons difcult. The other prospective cohort in this section
(entry number 5), also found greater disease progression in coinfected
subjects. In particular, liver brosis was greatly accelerated in coinfected
subjects with 0.58 units per year compared with 0.1 units per year in
mono-infected HCV patients. Few mono-schistosomal patients had any
progression of brosis, suggesting that the effects are multiplicative in coin-
fected subjects, rather than additive. Compared with mono-HCV subjects,
coinfected subjects also had higher degrees of interface hepatitis, periportal
necrosis and a lower magnitude and breadth of intrahepatic HCV-specic
CD4 T cells responses. The authors of this study suggested that the
enhancement of progression of liver brosis is associated with the failure
to develop HCV-specic Th1 responses, particularly during the early phase
of chronic infection.
As noted in a number of other studies in this review, distinctive cytokine
proles, particularly tipped towards the Th2 response, were identied for
coinfection subjects. Most of the results in this section were studies using
caseecontrol designs. In entry number 4, coinfected subjects had IL4 and
IL10 levels that were comparable to or higher than those observed in mono-
schistosomal subjects, and IFN-gamma and IL-18 levels that were consider-
ably lower than mono-HCV subjects. This study suggested that infection
with Schistosoma preceded HCV infection in coinfected subjects, which
inhibited the ability of coinfected subjects to mount HCV-specic Th1
responses. This same cytokine pattern, with IL4 and IL10 levels meeting
or exceeding those observed in mono-schistosomal subjects and IFN-
gamma levels below those observed for mono-HCV subjects, was reported
in both entry numbers 6 and 8. In addition, entry number 6 also reported
that coinfected patients had higher titres of HCV-RNA with reduced
CD4 T cell response, which were also noted in the prospective cohorts
discussed above (entry numbers 3 and 5, respectively). Higher IL4 levels
were also found for coinfected subjects in entry number 10, a caseecontrol
study which found them correlated with greater portal vein diameter, more
pronounced brosis, and greater portal hypertension.
Taken together, these studies suggest that the dominance of the Th2
response observed in coinfected patients may result in increased viral
replication, and is likely to be related to the greater brosis observed in these
patients than in either HCV or schistosomiasis alone. Regardless of study
design, all of these studies were in agreement that coinfected subjects do
not respond to HCV infection the way that mono-HCV subjects do, the
latter typically demonstrating a strong Th1 response. In addition, at least
one study (entry number 8) indicated that coinfected subjects displayed
lower levels of Th1 cytokines than observed among healthy controls as
well as mono-schistosomiasis subjects, suggesting that coinfected suffer
from additional immunologic suppression or impairment. In addition,
most of the caseecontrol studies, but not all, reported that the coinfected
subjects had higher mean ALT and mean AST levels than either mono-
infected groups, which was often correlated with degree of brosis (entry
number 4, 6, 7, 10, 11). These ndings appear to be consistent with those
reported by Wahib et al. (1998), Kamal et al. (2001a) and El-Shazly et al.
(2001), which were not included in our table. Finally, one caseecontrol
study found that coinfected subjects had a higher frequency of a heterozy-
gote mutant of the lymphotoxin-alpha genotype; more generally, lympho-
toxin-alpha is a member of the TNF superfamily which may be associated
with increased susceptibility to HCV (entry number 11).
In contrast with the ndings reported above as well as those described in
Section 3.5, one study in this section reported that coinfected patients had
higher TNF-alpha levels than either mono-schistosomal or mono
anti-HCV patients (entry number 1). Notably, this study used case series
design and provided little data on patients, making it difcult to access their
comparability. The results of this study still suggested, however, that immu-
noregulation of coinfection differs from each disease in isolation. One of the
caseecontrol studies found no difference in the degree of brosis between
coinfected subjects and either mono anti-HCV seropositive or mono-hep-
atosplenic schistosomiasis patients based on evaluation by liver biopsy or ul-
trasound, respectively (entry number 2); it should be noted that 91% of the
schistosomal subjects in this study presented with severe brosis. Coinfected
subjects did, however, display higher brosis markers such as alkaline phos-
phatase, bilirubin and gamma globulin than other mono-infected groups. It
should also be noted that this particular study did not use matching between
cases and controls, and lacked additional detail on possible confounders be-
tween the comparison populations.
5. CONCLUDING REMARKS
In this review, we have been concerned with identifying the clinical
effects of coinfection between Schistosoma and HBV or HCV. A number
of factors contributed to the results reported in our tables. These included,
but are not limited to subject selection (i.e. asymptomatic cases typically
drawn from the general population vs subjects presenting to a hospital or
clinic with clinical disease); study design, which directly impacts our ability
to infer causality (i.e. cross-sectional vs prospective cohort study); use and
choice of control population (i.e. apparently healthy subjects vs other hos-
pital patients vs none); sample size, which directly impacts statistical power
and can result in a Type II error; geographic area, which may reect differ-
ences in population genetics, public health history, environmental differ-
ences or any number of other important factors (i.e. Egypt, Brazil, China);
method of testing for schistosomal infections (i.e. stool vs antibody test);
method of testing to determine if advanced schistosomal disease was present
(i.e. ultrasound, liver biopsy vs none); method of serological testing for HBV
(i.e. use of HBsAg alone or with other markers or DNA testing); method of
serological testing for HCV (i.e. use of anti-HCV alone or with RNA
testing); and, year of the study, which reects among other things, techno-
logical improvements between tests as well as possible changes in the fre-
quency of exposure in the populations under study (i.e. use of parenteral
anti-schistosomal therapy vs the oral anti-schistosomal medication).
Despite all these differences, throughout our tables we have observed

general patterns that seem largely consistent with one another. As has
been noted elsewhere (i.e. Gasim et al., 2015; Bahgat, 2014; Van-Lume
et al., 2013), studies conducted on general, largely asymptomatic popula-
tions tend to support the view that having one of the diseases in question
(i.e. schistosomiasis) does not necessarily predispose one to becoming coin-
fected with another (i.e. HBV or HCV). Rather, the probability of
becoming coinfected seems most closely associated with mode of transmis-
sion for either HBV or HCV in schistosome-endemic areas. In Table 3,
several cross-sectional studies reported an increased risk for both HCV as
well as HBV from the use of parenteral anti-schistosomal therapy (see entry
number 4, Hyams et al., 1987; entry number 6, El-Sayed et al., 1997; entry
number 7, Darwish et al., 2001). These ndings were echoed in a number of
the other studies presented in various tables (see Gad et al., 2001; Strickland
et al., 2002) and have been much discussed elsewhere (Frank et al., 2000; El
Sabah et al., 2011; Sanghvi et al., 2013). Overall, there seems to be general
agreement that the insufcient sterilization of the syringes used to administer
PAT helped spread these viruses in many schistosome-endemic areas, partic-
ularly in Egypt. In addition, frequent blood transfusions, which are associ-
ated with hepatosplenic schistosomiasis, appear to have increased the
probability of becoming coinfected with HCV in Brazil and perhaps in other
geographic areas (see Silva et al., 2011).
Once coinfected, however, the clinical course of illness for those with
SchistosomaeHBV or SchistosomaeHCV infections are typically much
more severe than for mono-infected subjects. The strongest evidence for
this may be inferred from eight prospective cohort studies we reported on
in our tables that systematically monitored disease progression in their sub-
jects. Namely, in Table 6: entry number 10, Bassily et al., 1979; entry num-
ber 12, Bassily et al., 1983; In Table 7: entry number 2, Nooman et al., 1977;
entry number 3, Ghaffar et al., 1991; In Table 8: entry number 1, Kamal
et al., 2001a,b; entry number 7, Kamal et al., 2006; and in Table 9: entry
number 3, Kamal et al., 2000; entry number 5, Kamal et al., 2004. The re-
sults of these studies are very consistent with one another. With respect to
HBV infection, coinfection with Schistosoma prolonged the carriage state
and more often resulted in chronic hepatitis with greater cirrhosis as well
as higher mortality (Bassily et al., 1979, 1983; Noonman et al., 1977).
Much of the same was also observed with respect to HCV, where coinfec-
tion with Schistosoma was associated with a reduced ability to spontaneously
Table 9 Studies comparing subjects with schistosomiasis and subjects with hepatitis C virus
Study design

(objective) and
1 Morais et al. Cidade University, Case series (comparative, HIV, HBV HCV: anti-HCV, Coinfected patients had
(2006) Recife, Brazil immunology, HCV-RNA higher TNF-alpha levels
(n.a.) severity) HSS(Sm): stool, than either mono-
Case groups: 3 HSS, 23 ultrasound, liver infected groups, while
HCV and 11 HSS w biopsy mono-HSS patients
HCV patients displayed higher TNF-
Controls: presented in Beta levels
graphs, but nowhere IL-13 levels were similar
described between all patient
Note: further details on groups
patients n.a. Results suggest that
immunoregulation of
coinfection differs from
each disease in isolation
2 Morais et al. Hospital dad Case-control Other causes of liver HCV: anti-HCV There was no difference in
(2010) Clinicas da (comparative, disease, liver w/HCV-RNA brosis degree between
Universidade severity, biomarkers) transplantation, prior HSS (Sm): stool, coinfected subjects and
Federal de Case groups: 22 HSS, 39 interferon therapy, ultrasound mono HCV patients
Pernambuco, HCV and 19 HSS w/ immunosuppressive who were both anti-
Recife, HCV, aged 18 therapy, HBV, HIV HCV w/HCV-
Pernambuco, e65 years RNA, based on
Brazil (n.a.) Controls: 13 non HSS, histology evaluation, or
non-HCV subjects, between coinfected
ages 21e57 years subjects and mono HSS
from nonendemic based on ultrasound
211
areas of Pernambuco However, coinfected
(Continued)
Table 9 Studies comparing subjects with schistosomiasis and subjects with hepatitis C virusdcont'd
212
Study design
(objective) and
patients did display
higher brosis markers
such as AP, bilirubin and
gamma-globulin,
compare to either
mono-infected patient
group
Best indicators for
distinguishing between
mind and severe brosis
varied by group, with
TNF-alpha and alkaline
phosphatase best for
mono HCV subjects,
while total bilirubin was
the best indicator for
coinfected patients; no
biomarker was
identied for mono
Amy Abruzzi et al.

HSS subjects, as 91%
presented with severe
brosis according to
ultrasonography
3 Kamal et al. Liver Unit, Ain Cohort (comparative, Serological evidence of HCV: anti-HCV Compared with mono
(2000) Shams University risk factors, disease active HAV, HBV, w/HCV-RNA HCV patients,
Hospital, Cairo, progression) HDV infection, Sch (Sm, Sh): history coinfected patients had
Egypt (1992e Patient groups: 30 Sm, 33 autoimmune hepatitis, or current higher HCV titres and
1999) HCV, and 63 Sm cytomegalovirus, infection, stool, longer duration of HCV
w/HCV subjects; Epstein Barr virus, or urine, rectal infection (9 vs 13 years),
mean group ages other hepatic parasites biopsy, with greater clinical
38e44 years, 67% ultrasound signs of liver disease,
male Note: all patients including cirrhosis, at
Note: all Egyptians from had active HCV the start of study; over
rural and urban areas infection the observation period,
in Cairo, Nile delta coinfected patients had
and Upper Egypt greater progression of
areas disease, resulting in
Patients followed for higher liver-related
72e76 months mortality (48%)
compared with mono
HCV (12%) and mono
Sm (3%); the
development of HCC
was only observed in
coinfected patients
(11%), not in either
mono infected group
HCV Genotype 4
observed in 62% mono
infected HCV subjects
213
(Continued)
214
Study design
(objective) and
vs 92% in the coinfected
subjects
On average, coinfected
patients had acquired
HCV infection at a
younger age than those
infected with HCV
alone (aged 19 vs age
30)
PAT was associated with
HCV in coinfected
patients, whereas blood
transfusion was
associated with mono
HCV patients
4 El-Kady et al. National Liver Case-Control n.a. Chronic HCV: anti- Coinfected subjects had
(2004) Institute, (comparative, HCV w/HCV- IL-4 and IL-10 levels
Minuya immunology, RNA that were comparable to
University, El- complications, Sch (Sm): stool/ or higher than mono
Minuya, Egypt severity) rectal snip w/ Sm subjects, and IFN-
Amy Abruzzi et al.

(n.a.) Case groups: 15 Sm, 20 SchAb gamma and IL-18 levels
chronic HCV and 20 Note: All Sm patients that were considerably
Sm w/chronic HCV had ova in stool/ lower than mono HCV
patients, group mean rectum subjects
ages 40e46 years, 66 This dominate Th2
e75% male cytokine prole suggests
Controls: 5 healthy infection with Sm
subjects, matched for preceded HCV in
age and sex with no coinfected subjects, and
evidence of liver inhibits their ability to
disease mount an HCV-specic
Th1 response
Coinfected patients had
high brosis scores, with
high ALT and AST than
other groups
5 Kamal et al. Ain Shams Cohort (comparative, No subject had received Acute HCV: anti- Coinfected subjects had
(2004) University, Cairo, immunology, disease antiviral or immune- HCV w/HCV- accelerated liver brosis
Egypt (n.a.) progression) modulatory treatment RNA, w/ALT compared with mono-
Patient groups: 23 acute before entry or during (GT 10 normal) HCV subjects (0.58 vs
HCV for 6 follow-up; other Sch(Sm): stools/ 0.1 units per year),
e10 months, 20 HSS causes of hepatitis rectal biopsy, despite similar baseline
and 25 acute HCV ruled out SchAb, liver necroinammatory
w/HSS subjects biopsy scores and the absence
HCV groups matched of brosis
by age, sex and Few mono-schistosomal
duration of HCV subjects had progression
infection (all of brosis
genotype 4) Coinfected subjects also
HSS groups matched for had higher degrees of
duration of Sch interface hepatitis,
infection periportal necrosis and
(Continued)
215
Study design
216
(objective) and
Note: Patients were lower magnitude and
followed for breadth of intrahepatic
96 8.7 months HCV-specic CD4T
cell responses compared
with subjects with
mono HCV subjects
The enhancement of
progression of liver
brosis is associated with
the failure to develop
HCV-specic CD4
Th1 response during the
early phase of chronic
infection favours the
development of liver
damage and progression
of disease
6 El-Kady et al. National Liver Case-Control n.a. Chronic HCV: anti- Coinfected subjects had
(2005) Institute, (comparative, HCV w/HCV- cytokine proles that
Minuya immunology, RNA were similar to mono-
Amy Abruzzi et al.

University, El- complications) Sch (Sm): stool/ Sm subjects, with higher
Minuya, Egypt Case groups: 15 Sm, 20 rectal snip w/ IL-4 and IL-10 and
(n.a.) chronic HCV and 20 SchAb lower IFN-gamma and
Sm w/chronic HCV Note: All Sm patients IL-18 levels
patients, group mean had ova in stool/ Coinfected patients had
ages 40e46 years, rectum signicantly higher
66e75% male HCV-RNA titres, with
Controls: 5 healthy an inverse relationship
subjects, matched for between virus load and
age and sex with no C4 T-cell responses
evidence of liver Suggests dominance of the
disease Th2 response may result
in increased viral
replication, resulting in
more aggressive
progression to brosis
ALT and AST levels were
much higher in
coinfected than other
groups
7 El-Masry et al. National Liver Case-control Seromarkers for HAV, Chronic HCV: anti- Coinfected patients had
(2006) Institute, (comparative, HBV, HDV HCV w/HCV- higher serum laminin
Minuya severity) infections, alcohol RNA, liver concentrations than
University, El- Case groups: 34 Sm, 58 consumption, biopsy mono infected or
Minuya, Egypt chronic HCV and 68 smoking Sch (Sm): stool/ control groups
(n.a.) Sm w/chronic HCV rectal snip w/ This was positively
patients, mean group SchAb correlated with brosis
ages 25e40 years, Note: All Sm patients grading scores and
65% males had ova in stool/ highest in mono Sm
Controls: healthy rectum patients, followed by
controls, matched for coinfected patients
age and sex Coinfected patients also
had higher ALT and
AST levels than all other
217
groups
(Continued)
Study design
218
(objective) and
8 Fahmy et al. Zagazig University Case-control Hepatitic infections Active HCV: anti- Th2 cytokines (IL-4 and
(2006) Hospitals, Egypt (comparative, other than HCV, HCV w/HCV- IL-10), were highest in
(2005e2006) immunology) parasitic infections RNA w/elevated all groups compared
Case groups: 9 active Sm, other than Sm ALT with controls, with
13 active HCV and Active Sch (Sm): coinfected patients
12 active Sm w/ stool/rectal snip displaying the highest
active HCV patients, w/SchAb levels of IL-4 levels
group age range 27 Note: All Sm patients Both coinfected and mono
e61 years, 53% male had ova in stool/ Sm infected patients had
Controls: 10 apparently rectal snip high levels of IL-10
healthy non-Sm, non Mono HCV-infected
HCV subjects, aged patients displayed the
32e57 years, 50% highest levels of Th1
male, drawn from response cytokines (IL-2
University and IFN-gamma), while
coinfected subjects
displayed levels lower
than that observed in
apparently healthy
controls or mono Sm
subjects
Amy Abruzzi et al.

Coinfection of Sm with
HCV results in a strong
Th2 response that leads
to suppression of the
Th1 response needed to
control HCV infection
9 Ahmed et al. Al Azhar University, Case-control n.a. HCV: HCV-RNA All groups had higher
(2008) Cairo, Egypt (comparative, liver Sch (Sm): stool, mean ALT, AST and
(n.a.) functions, severity) SchAb alpha-glutathione-S-
Case groups: 16 Sm, 20 Note: All Sm transferase compared
Sm w/HCV and 19 Patients had ova with controls; relative to
HCV patients, aged in stool one another
28e60 years, 64% Mono Sch patients had the
male highest mean values,
Controls: 20 healthy mono HCV patients
subjects, aged 21e55, had the lowest, with
80% male coinfected patients in
between
ALT was more strongly
correlated with brosis
in subjects than other
biomarkers
10 ElSammak et al. Medical Research Case-control HBV infection, HCV: anti-HCV All patients had enlarged
(2008a) Institute (comparative, autoimmune liver w/HCV-RNA liver/spleen
Teaching immunology, disease, alcohol Sch (Sm, Sh): stool, Coinfected subjects had
Hospital, severity) consumption, use of urine, SchAb IL-4 levels that were
Alexandria Case groups: 22 SHF, 22 certain medications SHF/LD: higher than mono-Sm
University, Egypt HCV and 22 SHF w/ including ultrasound subjects, high IL-4 levels
(n.a.) HCV patients, mean contraceptives, Note: Sm ova found were correlated with
group ages 48 malignancy, hypo- in 10 patients, no greater portal vein
e54 years, 60e64% thyroid disease, Sh ova detected diameter, more
males pregnancy and other All patients had pronounced brosis and
Controls: 22 non-Sch, portal hypertension
219
(Continued)
220
Study design
(objective) and
non-HCV, mean age concomitant acute active HCV Increased IL-4 secretion
49 years, 41% male infection infection may downregulate Th1
cell-mediated immune
effecter mechanisms
important in the host
defence against HCV
infection
Coinfected patients also
had higher AST and
ALT levels compared
with other groups
11 ElSammak et al. Medical Research Case-control HBV, autoimmune HCV: anti-HCV, All patients had enlarged
(2008b) Institute (comparative, hepatitis, metabolic HCV-RNA liver/spleen; only 10
Teaching pathology, genetics) liver disease, Wilsons Sch(Sm): stool, subjects found to be
Hospital, Case groups: 22 SHF, 22 disease, history of SchAb, excreting Sm eggs, all
Alexandria HCV, 22 SHF w/ alcohol consumption ultrasound light infections
University, Egypt HCV, mean group and malignancy Note: Sm ova found Patients with mono HCV
(n.a.) ages 50e54 years, in 10 patients, no had a higher frequency
53% male Sh ova detected of homozygote
Amy Abruzzi et al.

Controls: 22 apparently lymphotoxin-alpha
healthy subjects, (LT-alpha) mutant,
mean age 51 years, while coinfected
64% male patients had a higher
frequency of LT-alpha
heterozygote mutants
compared
Lymphotoxin-alpha is a
member of the TNF
superfamily which may
be associated with
susceptibility; Patients
with mono SHF had a
higher frequency of
wild-type LT-apha
genotype, as did
controls
Overall, LT-alpha
polymorphisms may
play a role in the
susceptibility to HCV,
but do appear to affect
susceptibility to Sch
infection
Additional data is needed
on coinfection
n.a., not available.
221
resolve the viral infection and more often resulted in rapid brosis as well as
higher mortality (Kamal et al., 2000, 2001a,b, 2004, 2006).
The key question is if the effect of coinfection with Schistosoma and HBV
or HCV is synergistic, i.e. if the combined effect is greater than the sum of
each disease in isolation. The best evidence for this may be inferred from the
two prospective cohort studies we presented in Table 9 (i.e. Kamal et al.,
2000 and Kamal et al., 2004), which each compared two mono-infected
groups with one coinfected group which were similar with respect to base-
line confounding factors. Both of these studies pertain to coinfection with
Schistosoma and HCV. The earlier of the two studies documented differences
in mortality between mono and coinfected groups, while the later study
documented differences in the rate of brosis. In the rst study, mortality
among the coinfected (48%) was considerably more than the sum of that
observed among mono-infected HCV (12%) or mono-infected S. mansoni
(3%) subjects during the 72e76 month follow-up period of the study
(Kamal et al., 2000). More generally, the coinfected patients in this study
were characterized by having more advanced liver disease with higher
histologic activity and a higher incidence of cirrhosis and HCC than either
subjects from either mono-infected group. In addition, coinfected subjects
also had higher HCV-RNA titres with a predominance of HCV genotypes
4 when compared with the mono-infected HCV group.
Similarly, in the later cohort study, the rates of liver brosis among the
coinfected (0.58 units per year) were again much higher than the sum of
those observed for mono-infected HCV (0.1 units per year) or mono-
infected S. mansoni (less than 0.1 units per year) subjects during the 96 or
so months of follow-up. In both studies, the effect of coinfection appears
to be multiplicative rather than additive, supporting the supposition that a
synergistic relationship exists between HCV and schistosomiasis. This study
also compared HCV-specic intrahepatic and peripheral CD4 T cell
proliferative responses and cytokine production between coinfected and
mono-infected HCV patients. At the start of the study, subjects in the
HCV infection mono-group had stronger multispecic intrahepatic
HCV-specic CD4 Th1 responses than did coinfected subjects. The
coinfected group was characterized as having no T cell responses or weak,
narrowly focussed responses that over time were maintained only in the
liver. In sum, the rate of progression of brosis observed in these subjects,
as well as HCV virus load, was found to be inversely correlated with
intrahepatic HCV-specic CD4 T cell response. This suggests that the
more rapid progression of liver brosis is associated with a failure to develop
early, multispecic HCV-specic CD4 Th1 responses in coinfected sub-

jects, and is most likely due to an earlier infection with schistosomiasis
that triggered a prior Th2 cytokine response. Numerous studies, all conduct-
ed on HCV, generally seem to support the idea of a reduced Th1 host
response in coinfected subjects (see Fahmy et al., 2006; and more recently
Loffredo-Verde et al., 2015). Unfortunately, we lack recent comparative
observational studies that would allow us to draw the same level of inference
about the effect of coinfection with HBV and schistosomiasis, though the
results of the prospective cohort study undertaken by Gaffar et al., 1991
(entry number 3) suggests a certain similarity. For a more detailed discussion
on the mechanisms of coinfection between schistosomiasis, hepatitis C and
B, we suggest the reader consult the recent reviews by Gasim et al. (2015)
and Baghat (2014), which summarized the immunological research from a
wider range of studies beyond the scope of our analysis; in conjunction
with this, these authors also provided a more comprehensive discussion of
advances in treatment including antiviral therapy.
In conclusion, the results of our research argue for greater primary
prevention for both HBV and HCV in Schistosoma-endemic populations.
Although no vaccine currently exists for HCV as it does for HBV, additional
steps can still be taken to reduce transmission in high-risk populations (see
Anwar et al., 2008; Lemoine et al., 2013, 2014; Vineas and Wild. 2014).
Furthermore, vaccination against HBV would also prevent subjects from
becoming triple infected, either with SchistosomaeHBVeHCV or
SchistosomaeHBVeHDV, and thus possibly worsening their clinical course
as was sometimes documented in a few of our studies (see Zakaria et al.,
1993, 1994).
Additional observational, longitudinal studies conducted on human popu-
lations that are fully comparative in nature could help answer some of the
remaining questions on both SchistosomaeHBV as well as SchistosomaeHCV
coinfections. Some of these include the role of active versus past schistosomal
infections, the role of genetic variants (see Dessein et al., 2013), as well as the
effect of coinfection on treatment. While a thorough discussion of treatment
is outside the scope of this review, it has been documented that Schistosomae
HCV subjects are less responsive to antiviral therapy than mono-HCV
infected subjects (see Abdel-Rahman et al., 2013; El-Zayadi, 2009); and
while SchistosomaeHBV coinfected patients may now fare better, additional
work on larger populations is still needed (see Huang et al., 2013). Finally,
in designing these studies, researchers must also take care to use a sufcient
sample size to ensure adequate statistical power, particularly in longitudinal
studies where loss-to follow-up is a well-known problem. Surprisingly, only a

few studies examined in this review calculated the statistical power needed
either in the design of their study or presented it when evaluating their nd-
ings (i.e. Hyams et al., 1986; Kamel et al., 1994; Al-Freihi, 1993).
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CHAPTER FOUR
Recent Advances in Elucidating

Nematode Moulting e Prospects
of Using Oesophagostomum
dentatum as a Model
Martina Ondrovics*, Robin B. Gasserx, 1, Anja Joachim*, 1
*Institute of Parasitology, Department of Pathobiology, University of Veterinary Medicine Vienna,
Vienna, Austria
x
Faculty of Veterinary and Agricultural Sciences, The University of Melbourne, Parkville, VIC, Australia
1
Corresponding authors: E-mail: Anja.Joachim@vetmeduni.ac.at; robinbg@unimelb.edu.au
Contents
1. Introduction 234
2. Nematode Cuticle 235
3. Towards Understanding the Moulting Process 236
4. Oesophagostomum dentatum as a Model for Studying Fundamental 239
Developmental Processes
5. Elucidating Aspects of Moulting in Oesophagostomum dentatum and Its 241
Implications
6. Future Prospects and Concluding Remarks 253
References 255
Abstract
There are major gaps in our knowledge of many molecular biological processes that
take place during the development of parasitic nematodes, in spite of the fact that un-
derstanding such processes could lead to new ways of treating and controlling parasitic
diseases via the disruption of one or more biological pathways in the parasites. Progress
in genomics, transcriptomics, proteomics and bioinformatics now provides unique
opportunities to investigate the molecular basis of key developmental processes in
parasitic nematodes. The porcine nodule worm, Oesophagostomum dentatum,
represents a large order (Strongylida) of socioeconomically important nematodes,
and provides a useful platform for exploring molecular developmental processes,
particularly given that this nematode can be grown and maintained in culture in vitro
for periods longer than most other nematodes of this order. In this article, we focus on
the moulting process (ecdysis) in nematodes; review recent advances in our
understanding of molecular aspects of moulting in O. dentatum achieved by using
2016 Elsevier Ltd.
j
ISSN 0065-308X
234 Martina Ondrovics et al.
integrated proteomic-bioinformatic tools and discuss key implications and future

prospects for research in this area, also with respect to developing new anti-nematode
interventions and biotechnological outcomes.
1. INTRODUCTION
Nematodes are one of the most abundant and diverse groups of organ-
isms on our planet and include free-living as well as parasitic species (Platt,
1994). The latter group imposes a substantial public health and economic
burden worldwide, as members represent signicant pathogens of humans,
plants and animals.
Soil-transmitted helminthiases caused by Ancylostoma duodenale, Necator
americanus, Trichuris trichiura and/or Ascaris lumbricoides represent some of
the most prevalent, chronic infectious diseases in humans, with more than
1 billion infected people worldwide (Bethony et al., 2006a; Brooker
et al., 2006; Colley et al., 2001; Harhay et al., 2010; Hotez et al., 2009).
Most cases of infection occur in underprivileged countries, and infected chil-
dren in these areas suffer from impairments in physical, intellectual and/or
cognitive development (Bethony et al., 2006a). Plant-parasitic nematodes,
including Meloidogyne, Heterodera, Globodera, Pratylenchus and Radopholus
spp., represent a major threat to the worlds agriculture by reducing food
production, with estimated economic losses of 80 billion US dollars per
year (Jones et al., 2013; Nicol et al., 2011). Parasitic nematodes of livestock,
including Haemonchus, Ostertagia, Teladorsagia, Trichostrongylus and Oesopha-
gostomum species, cause substantial economic losses due to poor productivity,
failure to thrive, repeated anthelmintic treatments to control infections and
deaths (Coles, 2001; McLeod, 1995; Newton and Meeusen, 2003; Smith,
1997). The extensive use of anthelmintics to control nematode infections
of livestock has led to the emergence and spread of resistance against
multiple classes of nematocidal compounds (Kaplan, 2004; Sutherland and
Leathwick, 2011; Wolstenholme et al., 2004). As a rational consequence,
much effort has been spent on identifying new targets to develop new inter-
vention and control strategies (Bethony et al., 2006b; Knox, 2000; Newton
and Meeusen, 2003; Newton and Munn, 1999). The identication of nem-
atode-specic gene products involved in crucial fundamental biological pro-
cesses essential for the parasites development represents a promising
approach to the discovery of novel and selective intervention targets.
Recent Advances in Elucidating Nematode Moulting 235
Current advances in omic technologies and recent progress in the develop-

ment of improved bioinformatic tools have proven highly valuable, as the inte-
gration of these high-performance techniques in hypothesis-driven research
allows improved investigations of fundamental processes and pathways gov-
erning nematode development (Bennuru et al., 2009; Cantacessi et al.,
2010, 2012a, 2012b; Huang et al., 2013; Mulvenna et al., 2009). To date,
the transcriptomes and/or genomes of several nematode species of socioeco-
nomic importance have been sequenced and annotated, providing extensive
information to complement functional research of nematodes, such as Ascaris
suum (see Jex et al., 2011), Brugia malayi (see Ghedin et al., 2007), Haemonchus
contortus (see Laing et al., 2013; Schwarz et al., 2013), N. americanus (see Tang
et al., 2014), Oesophagostomum dentatum (see Cantacessi et al., 2010; Tyagi
et al., 2015), and T. trichiura (see Foth et al., 2014; Jex et al., 2014). An inte-
grative approach implementing omic technologies and bioinformatics for
hypothesis-driven studies allows for optimized explorations of pathways and
processes governing nematode biology and enables in-depth investigations
of key developmental phenomena, such as moulting. Ultimately, an improved
understanding of fundamental biological processes in parasitic nematodes is
vital for the discovery of potential new intervention targets and developing
novel control strategies against these pathogens.
2. NEMATODE CUTICLE
Nematodes, together with arthropods, nematomorphs (horsehair
worms), onychophorans (velvet worms), tardigrades (waterbears), kino-
rhynchs and priapulids belong to the clade of ecdysozoans (Aguinaldo
et al., 1997), as they share the common feature of shedding the exoskeleton
or cuticle to be able to grow and reach maturity.
The composition of the cuticle of the members of the ecdysozoan clade
varies signicantly. While the cuticle of arthropods consists mainly of chitin
and matrix proteins (Merzendorfer, 2013), the main component of nema-
tode cuticle is collagen (Cox et al., 1981; Page and Johnstone, 2007), sug-
gesting that the enzymatic cascades mediating the release of the
exoskeleton in nematodes is distinct from those of arthropods. Cuticle col-
lagens are encoded by a large gene family (>170 members) and they are
expressed in a sophisticated temporal series (Johnstone and Barry, 1996).
Collagen biosynthesis is a complex, multistep process that requires several
enzymes (including prolyl 4 hydroxylase, protein disulphide isomerase and
peptidyl prolyl cis-trans isomerase) and is accompanied by several co- and
posttranslational modications (Bachinger, 1987; Page and Johnstone, 2007;

Steinmann et al., 1991; Winter and Page, 2000). Collagens are indispensible
for the formation of the nematode cuticle. Therefore, changes in expression
of specic collagen genes or key enzymes in the collagen biosynthetic
pathway in the free-living nematode Caenorhabditis elegans results in signi-
cant defects in body shape, larval viability and/or moulting (e.g. Edens et al.,
2001; Frand et al., 2005; Rual et al., 2004; Simmer et al., 2003; Page and
Johnstone, 2007).
In addition to cross-linked collagens, cuticlin proteins and glycoproteins
as well as lipids build the complex, highly structured extracellular matrix of
the exoskeleton (Lee, 2002; Page and Johnstone, 2007). The nematode-
specic cuticlins represent the insoluble material of the external and, to a
minor extent, the internal cortical layer (Cox et al., 1981). The outermost
layer of the cuticle is lipid-rich and covered by a loosely associated,
negatively charged surface coat, comprised predominantly of glycoproteins.
The nematodes cuticle is vital for the worms viability. It supports body
shape, enables motility via attachments to body wall muscles, and provides
an interaction site with and a barrier against the environment, as it provides
protection from unfavourable environmental conditions and the hosts
immune response.
3. TOWARDS UNDERSTANDING THE MOULTING

PROCESS
Ecdysis (kdyo, ekduo, to take or strip off ), the moulting of the
cuticle, occurs four times during nematode development from the rst-stage
larvae (L1s) to the mature adults that are able to sexually reproduce. Prior to
moulting, ribosomes, endoplasmic reticulum and Golgi bodies become
prominent in the epidermis, particularly in the seam cells of the lateral cords,
as a sign of intense biosynthetic activity (Lee, 2002). The moulting process is
preceded by a period of decreased general activity and feeding, known as
lethargus, when the old cuticle begins to disconnect from the underlying
hypodermis (Page and Johnstone, 2007). During apolysis, the old cuticle
is separated, allowing the newly synthesized one to be secreted in the space
between the two layers. The moulting cycle is completed with ecdysis,
when the old cuticle is completely shed and the worm emerges to the
next stage with a new cuticle (Lee, 2002; Singh and Sulston, 1978).
In animal-parasitic nematodes, the cuticle of the second-stage larvae
(L2s) is retained and envelops the third-stage larvae (L3s) as a sheath
(Rogers and Sommerville, 1957). This sheath (or uvea) protects the larvae
from unfavourable environmental conditions and enables free-living stages
of parasitic nematodes to survive in the environment for weeks to months
(Lee, 2002). The sheath is released in the gastrointestinal tract of the
mammalian host, stimulated by a change in pH, and an increase in temper-
ature and carbon dioxide concentration (Rogers and Sommerville, 1968).
The stimuli of the host cause the secretion of an exsheathment uid contain-
ing several enzymes (such as collagenases, lipases and proteinases), which is
excreted into the space between the sheath and the L3 cuticle (Rogers
and Sommerville, 1968). This uid facilitates the digestion of the ring
regions in the cuticular sheath surrounding the L3 and allows the larva to
escape via a prespecied aperture, which, in several nematode species, is
located at the anterior end of the sheath (Silverman and Podger, 1964).
Ecdysis of the second-stage cuticle in the ensheathed larvae of animal
parasitic nematodes is called exsheathment (Lee, 2002; Rogers and
Sommerville, 1957), and takes place in the region of the gastrointestinal tract
anterior to the preferred location of the adult nematode (Hertzberg et al.,
2002). The exsheathment process is of particular interest for a better under-
standing of the developmental biology of nematode parasites in general and,
more specically, for studying the moulting process, as it marks the transition
from a free-living to a parasitic lifestyle (Hertzberg et al., 2002) and, further-
more, marks the nal step of the moulting process.
While there is considerable knowledge regarding the morphological
changes that take place during the development of several nematode species,
the biochemical and molecular changes underlying the moulting process, to
reach the next developmental stage, are not fully understood. In the last two
decades, several studies, primarily using the free-living model nematode
C. elegans, have elucidated the molecular mechanisms and key molecules
involved in moulting and signicantly improved our understanding of
this process. For instance, a genome-wide RNA-mediated interference
(RNAi)-based screen revealed more than 150 genes which are involved
in the moulting of C. elegans, including matrix components, sterol sensing
proteins, nucleic acid binding/interacting proteins, signalling proteins, tran-
scription factors, secreted peptides, peroxidases, transmembrane proteins and
other novel genes (Frand et al., 2005). In their screen, Frand et al. (2005)
reported the signicance of two nuclear hormone receptors (NHRs),
NHR-23 and NHR-25, in the moulting cycle of C. elegans. The two tran-
scription factors regulate changes in gene expression required for the cutic-
ular moults, and their role in C. elegans moulting was implicated previously
in independent studies (Gissendanner and Sluder, 2000; Kostrouchova et al.,

1998). NHR-23 and NHR-25 are orthologues, respectively, of the hor-
mone ecdysone responsive gene products DHR3 and Ftz-F1 of Drosophila
melanogaster (see Asahina et al., 2000; Gissendanner and Sluder, 2000;
Kostrouchova et al., 1998, 2001), indicating the involvement of neuroendo-
crine pathways in mediating nematode moulting. Furthermore, moulting in
C. elegans requires cholesterol, the biosynthetic precursor of all steroid
hormones, as well as the low-density lipoprotein receptor-like protein
LRP-1, which is a receptor of sterols and mediates the endocytosis of these
organic molecules (Yochem et al., 1999).
Besides the identication of several genes linked to moulting in C. elegans,
the importance of hydrolases/peptidases during this process has become
evident, as they are crucial for controlling the synthesis of new cuticle,
releasing the old sheath and also mediating signalling events during the moult-
ing process itself (Craig et al., 2007; Frand et al., 2005; Novelli et al., 2004;
Suzuki et al., 2004). Furthermore, proteases play crucial roles during moult-
ing, as they are needed for the digestion of cuticle-anchoring proteins during
apolysis and for the processing of proenzymes. Different hydrolases (EC class
3) have been the focus of several studies of the development of parasitic nem-
atodes. Pyrophosphatases have been identied to be involved in development
and ecdysis of larval stages in the roundworm A. suum (see Islam et al., 2003,
2005), and cysteine proteases are required for the moult from L3s to the
fourth-stage larvae (L4s) in Ascaris (Rhoads et al., 1998, 2001), Onchocerca
volvulus (see Lustigman et al., 1996) and B. malayi (see Guiliano et al.,
2004). Similarly, metalloproteases play essential roles in cuticle collagen
processing and moulting in free-living (Davis et al., 2004; Novelli et al.,
2004) as well as in parasitic nematodes (Gamble et al., 1996; Hotez et al.,
1990; Stepek et al., 2011). Interestingly, the activity of the above-mentioned
hydrolases can be blocked by specic inhibitors (e.g. o-phenanthroline for
metalloproteases; iodoacetamide or peptidylmonouoromethylketone for
cysteine peptidases; sodium uoride for pyrophosphatases), resulting in a
severe impairment of larval development by impeding ecdysis (Feng et al.,
2007; Guiliano et al., 2004; Hotez et al., 1990; Islam et al., 2003, 2005;
Lustigman et al., 1996; Ondrovics et al., 2013).
Although some advances have been made to understand the moulting
process in different nematode species, neither the molecular and biochem-
ical mechanisms nor pathways that initiate, regulate and complete this multi-
faceted process are well understood. Profound insights into the mechanisms
of moulting are fundamentally important, as this step represents a hallmark
of nematode development and may provide the basis for the identication of
novel intervention strategies against parasites of this clade. The parasite is
particularly vulnerable during its moult; the newly developed cuticle must
be protected, while the old cuticle is cast off and degrades, and connections
between muscles and exoskeleton disintegrate and regenerate.
Investigating the moulting process in parasitic nematodes is of particular
interest for several reasons: (1) the moulting cycle is imperative for a parasite
to develop, reach maturity and reproduce; (2) moulting is conserved within
the nematode phylum, and studies in one species may allow extrapolation to
closely related taxa (Aguinaldo et al., 1997); (3) this process involves a com-
plex series of events which entail metabolic, physiological and behavioural
changes (Craig et al., 2007; Ewer, 2005); (4) key molecules governing
moulting in parasitic nematodes appear to lack sequence homology to those
of their host. It appears that molecules involved in nematode moulting
constitute possible targets for new interventions, as the disruption of this
developmental pathway would prevent the completion of the life cycle.
One of the major challenges would undoubtedly be the development of
one or more intervention strategies that target the parasite but have no or
minor adverse effects on the host.
4. OESOPHAGOSTOMUM DENTATUM AS A MODEL FOR

STUDYING FUNDAMENTAL DEVELOPMENTAL
PROCESSES
The free-living nematode C. elegans represents the most extensively
studied model invertebrate, as it offers major advantages for scientic
research due to its simple, rapid life cycle and its dened number of cells.
Furthermore, the gene expression of C. elegans can be readily manipulated,
resulting in the generation of useful information on gene/protein function
(cf. www.wormbase.org). This model nematode has also been used to study
fundamental processes that might be comparable to parasitic nematodes.
Nonetheless, although some aspects of structure, development and repro-
duction are similar between parasitic and free-living nematodes, some char-
acteristic traits are likely to be associated with parasitism to an extent that we
do not yet fully understand.
The porcine nodule worm, O. dentatum (order Strongylida), has proven
useful as a unique parasitic nematode model to study fundamental aspects of
molecular biology and developmental processes (e.g. Daugschies, 1995;
Gasser et al., 2007; Joachim and Ruttkowski, 2008; Joachim et al., 1998,
2001, 2005; Ondrovics et al., 2012, 2013, 2014). A number of phylogenetic

studies of nuclear ribosomal DNAs have shown that O. dentatum is relatively
closely related to a number of socioeconomically important nematodes of
the order Strongylida, including A. duodenale, H. contortus and N. americanus
(Blaxter, 1998; Chilton et al., 2006; Jex et al., 2010), rendering it a valuable
model for parasitic nematodes of this order. Caenorhabditis elegans, whose
genome was published in 1998 (C. elegans Sequencing Consortium,
1998) is another member of clade V (Blaxter, 1998), which supports its
use for comparative functional analyses of homologous/orthologous genes.
Similarly, both the genome and the transcriptome of O. dentatum have
recently become available (Cantacessi et al., 2010; Tyagi et al., 2015; see
www.nematode.net). Both transcriptomic and genomic data sets will repre-
sent a powerful tool for exploring pathways governing the molecular
biology and enabling detailed investigations of developmental processes in
this model parasitic nematode.
The relatively short and direct life cycle of O. dentatum is advantageous for
studying the parasites development and cuticular moulting (Kotlan, 1948).
With the release of unembryonated eggs in the hosts faeces, the free-living
phase of the parasites life cycle begins. Under favourable environmental con-
ditions, the eggs develop to L1s, followed by L2s (Kotlan, 1948); feeding on
nutrients and microbes in the faecal matter, they develop through to infective
L3s. The infective stage of O. dentatum is an L3 ensheathed by the second-
stage cuticle (enL3) and does not feed, but is dependent on nutrient reserves
within the larva to live and survive. The L3s migrate into the surrounding
environment and are ingested by the porcine host. With the exsheathment
of enL3s to the exsheathed L3s (xL3s) in the small intestine, the parasitic phase
of the nematodes life cycle begins (Rogers and Sommerville, 1957). The
xL3s migrate to the large intestine, penetrate the mucosal layer, produce le-
sions and undergo histotropic development (Stockdale, 1970). Within nod-
ules in the submucosa, composed of aggregations of neutrophils and
eosinophils (Stockdale, 1970), the L3s moult to L4s (McCracken and Ross,
1970). After 6e17 days, L4s emerge to establish in the intestinal lumen and
then, after a nal cuticular moult, develop to mature adults that reproduce.
The prepatent period of O. dentatum is w17e20 days (Talvik et al., 1997),
although longer periods have been observed (Kotlan, 1948; McCracken
and Ross, 1970; Stewart and Gasbarre, 1989).
Oesophagostomum dentatum represents an excellent model to study the
moulting cycle. The exsheathment process can be induced experimentally
in O. dentatum L3s. By exposing L3s to hypochlorite solution (10e12%
v/v), L3s are stimulated to exsheath within a short time frame (Talvik et al.,
1997). In vitro exsheathment with hypochlorite does not have adverse ef-
fects on larval viability compared to the in vivo process, as the cuticular
sheath is not lysed by the action of hypochlorite (Joachim et al., 2005).
The larvae actively emerge from the cuticle through a prespecied aperture
near the apical cap (Joachim et al., 2005), similar to the physiological process
(Silverman and Podger, 1964). Thus, the induction of exsheathment in vitro
(Ondrovics et al., 2014) is considered to mimic the natural process in the
host animal, and thus offers the opportunity of elucidating this process in
O. dentatum. In addition, an in vitro cultivation system of the parasitic stages
of O. dentatum (see Daugschies and Watzel, 1999; Joachim et al., 2001;
Joachim and Ruttkowski, 2008) offers numerous possibilities to study the
nematodes biology, development and moulting. Under optimized culture
conditions, xL3s of O. dentatum perform two cuticular moults and develop
through to the sexually differentiated adult stages (Daugschies and Watzel,
1999; Joachim and Ruttkowski, 2008). Although in vitro-cultured larval
stages may be morphologically and biochemically slightly different from
larvae recovered from the porcine intestine, the basic biological and devel-
opmental functions appear to be retained, as larvae produced in vitro are
capable of developing into adults following rectal transplantation into the
pig host (Joachim et al., 2001). Thus, in vitro assays for the exsheathment
and cultivation of O. dentatum provide useful systems for studying some as-
pects of nematode development and moulting.
5. ELUCIDATING ASPECTS OF MOULTING IN

OESOPHAGOSTOMUM DENTATUM AND ITS
IMPLICATIONS
The elucidation of moulting in O. dentatum commenced more than
10 years ago with studies on the involvement of eicosanoids in and their
impact on the exsheathment of L3s (Joachim et al., 2005). Eicosanoids are
bioactive lipids that can be detected in the homogenates and the excre-
tory/secretory products (ESPs) of larvae of O. dentatum (see Daugschies
and Joachim, 2000). The addition of inhibitors of eicosanoid synthesis,
such as the inhibition of prostaglandin H-synthase (cyclooxygenase) by ace-
tylsalicylic acid or inhibition of lipoxygenase by diethylcarbamacine, resulted
in the reversible suppression of sodium hypochlorite-induced exsheathment
of O. dentatum L3s (Joachim et al., 2005). Moreover, the action of the gluta-
thione S-transferase (GST) inhibitors sulphobromophthalein, indomethacin
and ethacrynic acid reversely impeded the in vitro development of

O. dentatum L3s to L4s, as they did not moult to reach the next larval stage
(Joachim and Ruttkowski, 2008; Joachim et al., 2011). As GSTs are enzymes
that are involved in the biosynthesis of eicosanoids by transforming the
instable prostaglandin H2 to the stable and bioactive metabolite prosta-
glandin D2 in O. dentatum (see Joachim and Ruttkowski, 2011), these results
indicate the crucial roles of metabolites of the eicosanoid pathway in
exsheathment and the completion of the moulting cycle.
In recent studies, we used a proteomic approach combined with in vitro
assays and extensive bioinformatic analyses to explore the moulting and
exsheathment process in O. dentatum (see Ondrovics et al., 2013, 2014).
Since the moulting cycle of nematodes is a complex sequence of events
that involves changes in behaviour, physiology and metabolism as well as
protein expression pattern of larvae, these studies aimed at identifying and
characterizing molecules that are involved in this hallmark of nematode
development using a comparative proteomics approach. In a rst step, the
in vitro moult from L3 to L4 of O. dentatum (which, in vivo, takes place
in the submucosa of the porcine host during the parasites histotropic phase)
was targeted by the addition of specic hydrolase inhibitors (1,2-epoxy-3-
(pnitrophenoxy)-propane, iodoacetamide, o-phenanthroline or sodium
uoride) to larval cultures. Each of these inhibitors impeded >90% of
moulting from L3s to L4s; subsequent comparison of the proteomic prole
of moulting-inhibited L3s with that of uninhibited controls by two-
dimensional gel electrophoresis identied 21 differentially expressed proteins
in moulting-inhibited L3s (Ondrovics et al., 2013). Similarly, the in vitro
exsheathment of enL3s to xL3s of O. dentatum was investigated employing
a lter-based technique to generate L3s during the process of exsheathment.
L3s before, during and after exsheathment were compared by two-
dimensional difference gel electrophoresis (2D-DIGE), and 11 proteins
were overexpressed exclusively during exsheathment (Ondrovics et al., 2014).
Analysis of these differentially expressed protein spots by MALDI-TOF
mass spectrometry identied 28 proteins involved in different biological
processes, including energy metabolism [fructose-bisphosphate aldolase
(FBA), glucose-6-phosphate 1-dehydrogenase (GPD), UTP-glucose-1-
phosphate uridylyltransferase (GPU), 4-hydroxybutyrate-CoA transferase
(HCT), malate dehydrogenase (MDH), propionyl-CoA carboxylase
(PCC), phosphoenolpyruvate carboxykinase [GTP] (PEPCK) and pyruvate
dehydrogenase (PDH)], stress response and hostepathogen interactions
(aspartyl protease inhibitor (API), calreticulin (CRT), heat shock 12.6 kDa
protein (HSP-12.6), heat shock 60 kDa protein (HSP-60), heat shock

70 kDa protein (HSP-70) and peroxiredoxin (PRDX)), structure and
motility [actin (ACT), cuticlin-1 (CUT-1), disorganised muscle protein 1
(DIM-1), intermediate lament protein B (IFB), peptidyl-prolyl cis-trans
isomerase (cyclophilin, CYN), tropomyosin (TPM) and troponin T
(TNT)], signalling and interaction [14-3-3 protein, GDP dissociation inhib-
itor (GDI), LIM domain protein (LIM), phosphatidylethanol-amine binding
protein homologue (PEBP), receptor for activated protein kinase C 1
(RACK-1), transthyretin-like protein 5 (TTL-5) and probable voltage-
dependent anion-selective channel (VDAC)] and development and growth
(all proteins mentioned above). Interestingly, nine proteins (API, CUT-1,
CYN, FBA, IFB, PCC, PEPCK, TPM and TTL) were inferred to have
signicant roles in the nematode moulting process (Figure 1 and Table 1).
As nematode moulting requires a series of metabolic, structural,
biochemical and physiological changes, we assumed that key molecules
involved in this process are less abundantly expressed in moulting-inhibited
L3s compared with their controls, and/or overexpressed in L3s during
exsheathment, but not before or after this transition. This expression pattern
was characteristic for all molecules identied, except for three proteins
(DIM-1, LIM and PEBP), which were upregulated following the inhibition
of moulting. Their upregulation might be a consequence of this inhibition,
or they could be part of a regulatory system that requires detailed study.
The downregulation of enzymes involved in energy metabolic pathways
(i.e. FBA, GPD, GPU, HCT, MDH, PCC, PDH and PEPCK), mainly in
carbohydrate metabolism in the moulting-inhibited larvae, and/or the
overexpression in larvae during exsheathment were conspicuous. These
enzymes appear to provide the nematode larvae with the energy required
to enable moulting and the sloughing of the old cuticle during ecdysis
following the lethargus phase. Notable were proteins (API, CRT, HSP-
12.6, HSP-60, HSP-70 and PRDX) involved in the stress response and
modulation of the host immune attack. These molecules are likely to ensure
parasite survival by modulating and suppressing the hosts immune response(s)
immediately after the exsheathment of L3s. Furthermore, heat shock proteins
might be involved in stress resistance when xL3s moult to L4s in the nodules
during the histotropic phase of development, while peroxidases might play a
protective role, limiting the damage caused by host-derived reactive oxygen
species (Henkle-D uhrsen and Kampk otter, 2001). Clearly, all molecules
involved in the stress response and/or hostepathogen interactions are likely
to be critical for the survival of the parasite in the vertebrate host, particularly
244
Signalling &
interaction
Mediating
Me and regulating pathways
Host- on
Protein interaction
pathogen
interaction Structure and
motility
Immuno-
Stress
St
modulation
response Active movement for cuticle shedding
Cuticle formation and remodelling of
Protection and survival attachments
1st & 2nd Adaption to new host environment Collagen biosynthesis 4th
moult Establishment of infection moult
Exsheathment 3rd moult
Egg L1/L2 enL3 xL3 L4 Adult
Provision of energy
Synthesis of key components of cuticle
Energy Eicosanoid
Martina Ondrovics et al.

metabolism metabolism
Figure 1 Major biological pathways involved in the exsheathment and/or third moult of Oesophagostomum dentatum larvae. The pro-
teins downregulated in the in vitro moulting-inhibited and/or overexpressed in L3s during exsheathment were assigned to various pathways
related to the moulting process in O. dentatum. enL3, ensheathed third-stage larvae; L1/L2, rst-/second-stage larvae; L4, fourth-stage larvae;
xL3, exsheathed third-stage larvae. Adapted from Joachim and Ruttkowski (2008), Joachim et al. (2011), Ondrovics et al. (2013) and Ondrovics
et al. (2014).
Table 1 Characterization of Oesophagostomum dentatum proteins inferred to be involved in in vitro moulting and/or exsheathment
Recent Advances in Elucidating Nematode Moulting

Interaction with
DAF-2 homologue
Biological Expression Key role in moulting of Caenorhabditis Secretion
process Protein pattern process elegans prediction
Energy Fructose-bisphosphate Y in miL3s Provision of energy / /

metabolism aldolase
Glucose-6-phosphate [ in L3dxs Provision of energy Interaction via /
1-dehydrogenase GCS-1 and
DAF-16
UTP-glucose-1- [ in L3dxs Provision of energy / /
phosphate
uridylyltransferase
4-hydroxybutyrate-CoA Y in miL3s Provision of energy / /
transferase
Malate dehydrogenase Y in miL3s Provision of energy Direct interaction /
Propionyl-CoA [ in L3dxs Provision of energy, Interaction via /
carboxylase Y in miL3s synthesis of fatty CIF-1and
acids as cuticle DAF-16
components,
modulator of key
regulator of
moulting
Phosphoenolpyruvate [ in L3dxs Provision of energy Overexpression in /
carboxykinase Y in miL3s dauer
Pyruvate dehydrogenase Y in miL3s Provision of energy / /
(Continued)
245
Table 1 Characterization of Oesophagostomum dentatum proteins inferred to be involved in in vitro moulting and/or exsheathmentd
246
cont'd
Interaction with
DAF-2 homologue
Stress response Aspartyl protease Y in miL3s Modulation of hosts / Nonclassical
and hoste inhibitor immune reaction,
pathogen inhibition of hosts
interactions immune effectors
depending on
proteolysis,
regulation of
endogenous aspartyl
proteases to control
moulting
Calreticulin Y in miL3s Modulation of hosts / /
immune reaction,
protection of key
proteins
Heat shock 12.6 kDa [ in L3dxs Modulation of hosts Direct interaction Nonclassical
protein immune reaction,

stress resistance
Heat shock 60 kDa Y in miL3s Modulation of hosts / /
protein immune reaction,
stress resistance
Heat shock 70 kDa [ in L3dxs Modulation of hosts Direct interaction /
protein Y in miL3s immune reaction,
stress resistance
Recent Advances in Elucidating Nematode Moulting
Peroxiredoxin Y in miL3s Modulation of hosts Interaction via /
immune reaction, GCS-1 and
stress resistance DAF-16
Structure and Actin [ in L3dxs Active movement to / /
motility shed old cuticle,
remodelling of
cuticle attachments
Cuticlin-1 [ in L3dxs Cuticle component / Nonclassical
Disorganized muscle [ in miL3s / / /
protein 1
Intermediate lament Y in miL3s Active movement to / /
protein B shed old cuticle,
remodelling of
cuticle attachments
Peptidyl-prolyl cis-trans Y in miL3s Collagen biosynthesis / /
isomerase
Tropomyosin Y in miL3s Active movement to / /
shed old cuticle,
remodelling of
cuticle attachments
Troponin T [ in L3dxs Active movement to / /
Y in miL3s shed old cuticle,
remodelling of
cuticle attachments
Signalling and 14-3-3 protein Y in miL3s Regulation of protein Direct interaction /
interaction interactions and key
pathways
247
(Continued)
Table 1 Characterization of Oesophagostomum dentatum proteins inferred to be involved in in vitro moulting and/or exsheathmentd
cont'd
Interaction with
248
DAF-2 homologue
GDP dissociation [ in L3dxs Regulation of Direct interaction /
inhibitor signalling pathways
LIM domain protein [ in miL3s / / /
Phosphatidylethanol- [ in miL3s / / /
amine binding
protein homologue
Receptor for activated Y in miL3s Regulation of /
protein kinase C 1 transcription and
translation,
membrane
trafcking and signal
transduction
Transthyretin-like [ in L3dxs Role in hormonal Classical
protein 5 transport, regulation
of nervous system
pathways, mediation
and regulation of
moulting pathways

Probable voltage- Y in miL3s Calcium signalling and /
dependent anion- ion transport
selective channel
The expression pattern of the proteins identied by Ondrovics et al. (2013, 2014) are listed. Proteins were assigned to main biological processes and their role in the
moulting cycle was inferred. The different expression patterns of the molecules are listed. Interactions with DAF-2 homologues of Caenorhabditis elegans were identied
with Gene Orienteer (www.geneorienteer.org/; Interaction with DAF-2 homologue of C. elegans). Classical or nonclassical protein secretion was predicted by SignalP
(http://www.cbs.dtu.dk/services/SignalP/) or SecretomeP (http://www.cbs.dtu.dk/services/SecretomeP/), respectively. CIF-1, COP9/Signalosome and
eIF3 complex shared subunit; GCS-1, gamma-glutamine cysteine synthetase; L3dxs, third-stage larvae during exsheathment; miL3s, moulting-inhibited third-stage
larvae; /, not known.
during and after moulting and/or exsheathment. The downregulation of

the structural proteins ACT, IFB, TNT and TPM in moulting-inhibited
O. dentatum L3s and/or their overexpression in L3 during exsheathment indi-
cate their role(s) in movement to shed their old cuticle during ecdysis, as they
are known to be linked to myobril assembly and muscle contraction
(Hapiak et al., 2003; Krause et al., 1989; Myers et al., 1996; Ohtsuki et al.,
1986). Several of the identied proteins, including 14-3-3 protein, GDI,
RACK-1, TTL-5 and VDAC, have diverse roles in various protein interac-
tions and/or in fullling pathway regulatory functions. While 14-3-3 pro-
teins bind phosphoserine and phosphothreonine residues (Durocher et al.,
2000), thereby regulating key biological processes through interaction with
their partners (Aitken, 2006; Tzivion et al., 2001), GDI might regulate signal-
ling pathways, as deduced from its involvement in vesicular trafcking in
mammals (e.g. Bianchi et al., 2009; Huang et al., 2004). RACK-1 is known
to regulate transcriptional and translational processes, membrane trafcking
and signal transduction (Sklan et al., 2006), and TTL-5 proteins are suggested
to be involved in hormonal transport (Parkinson et al., 2004) and regulation
of nervous system pathways in nematodes (Jacob et al., 2007). Furthermore,
VDACs function as anion channels and are involved in calcium signalling
pathways and ion transport (De Pinto et al., 2010). These molecules are likely
to regulate the progress of moulting by mediating protein interactions and/or
regulating various signalling pathways required during this sophisticated
developmental process. However, as they can have a diverse set of functions,
their precise roles during moulting and exsheathment need to be examined in
further detail.
In-depth analyses further inferred nine proteins, namely API, CUT-1,
CYN, FBA, IFB, PCC, PEPCK, TPM and TTL, in the moulting process
of O. dentatum. In accord with their proposed function during moulting/
exsheathment, all the above-mentioned molecules were downregulated in
in vitro moulting-inhibited L3s and/or overexpressed during the exsheath-
ment of L3s of O. dentatum.
The differential expression of the energy metabolic enzymes FBA, PCC
and PEPCK (GTP) was of particular interest. PEPCK has been identied
previously in several parasitic nematode species (Davila et al., 2006; Geary
et al., 1993; Hewitson et al., 2011; Iglesias et al., 2005; Klein et al., 1992;
Mulvenna et al., 2009; Rios and Nowak, 2002; Rohrer et al., 1986;
Simcock et al., 2012) and mainly functions in anaerobic respiration by
the carboxylation of phosphoenolpyruvate to oxaloacetate, thereby
introducing the products of glycolysis into mitochondrial metabolism
(Saz and Lescure, 1969). PEPCK is essential for the anaerobic metabolism in
gastrointestinal nematodes, and the energy obtained is required during the
moulting process. Accordingly, this enzyme exhibits its highest activity dur-
ing the moult from L3 to L4 in anisakid nematodes (Davila et al., 2006;
Iglesias et al., 2005). In contrast to its function in nematodes, vertebrate
PEPCK acts primarily in gluconeogenesis by converting oxaloacetate to
phosphoenolpyruvate, which is processed to glucose (Saz, 1981). As the
biochemical pathway and the kinetic properties of nematode and vertebrate
PEPCKs differ signicantly, PEPCK has been proposed as a potential target
for selective inhibition (Geary et al., 1993), a hypothesis that needs profound
testing in the future. PCC is also crucial for energy metabolism of parasitic
nematodes as it catalyses the rst step in fatty acid synthesis, converting
acetyl-CoA, Mg2 ATP and bicarbonate to malonyl-CoA (Wakil et al.,
1983). Furthermore, this enzyme might be important during moulting/
exsheathment in O. dentatum, since fatty acids, as key components of the
newly formed cuticle, need to be synthesized. Interestingly, specic knock-
out of homologous proteins of PCC results in a phenotype with severe moult
defects in C. elegans (see Li and Paik, 2011). In addition to its function as an
enzyme in fatty acid metabolism, PCC might interact with selected gene
products, similar to its homologue in C. elegans for which modulating roles
of mlt-10, a key regulator of moulting, have been proposed (Frand et al.,
2005). FBA, another enzyme of carbohydrate metabolism involved in
glycolysis by catalyzing the conversion of fructose-1,6-bisphosphate to
glyceraldehyde-3-phosphate and dihydroxyacetone phosphate (Inoue et al.,
1997), was inferred to function during moulting in O. dentatum. This enzyme
has been reported in various nematode species (Craig et al., 2006; Inoue
et al., 1997; McCarthy et al., 2002; Moreno and Geary, 2008; Yan et al.,
2013) and might serve in providing energy during moulting. The involve-
ment of FBA in moulting in nematodes was supported by evidence for
O. volvulus that FBA was highly expressed in L3s, where the cuticle separates
during moulting, thus assisting in the transformation by providing energy
(cf. McCarthy et al., 2002).
The proteins CUT-1, CYN, IFB and TPM may be involved in the
moulting process of O. dentatum by processing cuticle formation and remod-
elling structural attachments. Cuticlins, including CUT-1, represent the
insoluble component of nematode cuticle and have been described in
various nematode species (Favre et al., 1998; Lewis et al., 1999; Sebastiano
et al., 1991; Timinouni and Bazzicalupo, 1997). CUT-1 is vital for the nem-
atodes body structure, and during moulting it constructs the cuticle and
provides body shape and structure (Favre et al., 1998; Lewis et al., 1999;
Page and Johnstone, 2007). Consistent with its function as a cuticular pro-
tein, cut-1 mRNA was most abundant preceding the moult in Brugia (Lewis
et al., 1999). During nematode moulting, the peptidyl-prolyl cis-trans isom-
erase, CYN, assists in the trimerization of imino-rich collagen by the slow
cis-trans proline isomerization during the collagen biosynthetic pathway
(Page and Johnstone, 2007). This molecule is important for the correct
implementation of moulting by assisting in collagen processing, required
for proper cuticle synthesis. Furthermore, during moulting the outer struc-
ture of the nematode needs to be remodelled, and the cuticle needs to be
detached from hypodermis and muscle to enable the separation of the old
cuticle, and then reattached when the new cuticle is being synthesized.
The structural proteins IFB and TPM might assist in disrupting and recon-
necting specic muscle attachments during moulting in O. dentatum, as indi-
cated by homologues in C. elegans that are known to connect body wall
muscles to the external cuticle and are strongly associated with moulting
in this free-living nematode (Francis and Waterston, 1991; Frand et al.,
2005; Labouesse, 2006). Additionally, in B. malayi, intermediate lament
proteins were found to be abundantly represented in ESPs of larvae during
their transition from L3s to L4s, further conrming their likely function as
structural and remodelling components needed during the moulting in para-
sitic nematodes (Bennuru et al., 2009).
Based on the ndings of our studies (Ondrovics et al., 2013, 2014),
TTL-5 and API are proposed to have key functions in the moulting process
of O. dentatum larvae. TTL-5, a putatively secreted molecule, belongs to one
of the largest groups of nematode-specic proteins (Parkinson et al., 2004).
As TTL proteins play key roles in hostepathogen interactions they may
enable O. dentatum to cope with the host environment during the moult
from L3 to L4 in their tissue granuloma, as well as aid L3s to adapt to the
change from the free-living to the parasitic lifestyle during exsheathment.
Moreover, TTL-5 is likely to mediate and regulate several pathways
required for proper moulting, as this protein is involved in several signalling
pathways and hormone transport ( Jacob et al., 2007; Parkinson et al., 2004).
Bennuru et al. (2009) showed TTLs in ESPs of B. malayi to be abundantly
present during the moult of L3 to L4, further conrming their role during
moulting/exsheathment in parasitic nematodes. Furthermore, aspartyl pro-
tease inhibitors are unique to nematodes (Shaw et al., 2003) and, in addition
to their immunomodulatory function (Delaney et al., 2005; Shaw et al.,
2003), they might be involved in developmental processes such as moulting.
These inhibitors might regulate endogenous nematode aspartyl proteases to

control nematode moulting and exsheathment, in which proteases play
essential roles (e.g. Iglesias et al., 2001).
Remarkably, several O. dentatum proteins have homologues in C. elegans
that are interaction partners of the receptor tyrosine kinase DAF-2, a key
regulator of dauer formation in C. elegans (see Hu, 2007; Kimura et al.,
1997). According to the dauer hypothesis, similar molecular mechanisms
are involved in the entry into and exit from dauer in C. elegans and the tran-
sition from the free-living to the parasitic lifestyle in parasitic nematodes
(Crook, 2014). Six protein homologues (namely GDI, GPD, HSP-12.6,
HSP-70, PCC and PEPCK; Ondrovics et al., 2014) linked to dauer forma-
tion in C. elegans were overexpressed in O. dentatum L3s during exsheath-
ment. Additionally, three C. elegans homologues, namely MDH
(Samuelson et al., 2007), 14-3-3 protein (Li et al., 2007) and PRDX (Hu,
2007; Olahova et al., 2008; Wang et al., 2010), which were downregulated
in the in vitro moulting-inhibited L3s of O. dentatum appear to interact with
DAF-2 (Table 1). These observations support the theory of conserved
mechanisms functioning in dauer formation in C. elegans and in the in vitro
transition from the free-living to the parasitic phase of O. dentatum L3s, and
indicate that the respective conserved mechanisms and pathways might also
apply to moulting in nematodes in more general terms.
Interestingly, the proteins HSP-70, PCC, PEPCK and TNT were
detected in both studies using independent methodological approaches
(Ondrovics et al., 2013, 2014). These molecules were downregulated in
the in vitro moulting-inhibited L3s of O. dentatum and overexpressed in
L3 during exsheathment. Caenorhabditis elegans homologues of three of the
four proteins, namely HSP-70, PCC and PEPCK, are also interaction part-
ners of DAF-2 (Hu, 2007; Kimura et al., 1997), suggesting the presence of
conserved mechanisms of moulting in a wide range of nematodes. The pu-
tative involvement of these proteins in both the moulting from L3 to L4 and
the exsheathment from enL3s to xL3s indicate that these proteins might act
on all four moults in O. dentatum. This hypothesis is further supported by a
study by Frand et al. (2005), who reported that inactivation of the majority
of genes identied to be involved in moulting in C. elegans prevented the
ecdysis of several larval stages.
Molecules that are involved in the fundamental moulting process and
crucial for the parasites development and reproduction are of particular in-
terest, as new intervention strategies against parasitic nematodes could block
or disrupt essential biological pathways.
In this regard, immunogenic proteins (e.g. API, CUT-1, HSP-12.6,

HSP-60, HSP-70 and TTL-5) might serve as potential vaccine candidates,
as nematodes exploit heat shock proteins (including HSP-12.6, HSP-60
and HSP-70) to modulate or suppress the hosts immune responses and
support parasite survival (cf. Dakshinamoorthy et al., 2012; Shiny et al.,
2011; Wang et al., 2009). Moreover, the antigenic nematode-specic pro-
teins API, CUT-1 and TTL-5 lack homology to mammalian host proteins
and are unlikely to have adverse effects on mammals, and thus might repre-
sent promising immunogens. Aspartyl protease inhibitors have been
described previously as potential immunogens, as specic IgE responses
to APIs of Trichostrongylus colubriformis have been shown to correlate with
reduced faecal egg counts in sheep (Shaw et al., 2003). To achieve
adequate protection against parasitic nematode infections, target antigens
need to be accessible to the hosts immune system. In this regard, immu-
nization with somatic antigens of parasitic nematodes, such as CUT-1,
might be able to induce immunity in the host. In addition, four of the sug-
gested vaccine candidates may be secreted through classical (TTL-5) or
nonclassical (API, CUT-1 and HSP-12.6) secretory pathways, as predicted
by SignalP (Petersen et al., 2011) and SecretomeP (Bendtsen et al., 2004),
respectively (Ondrovics, 2014). In hookworms, for example, vaccination
of mice with recombinant Ancylostoma-secretory proteins, designated
ASP-1 and ASP-2, provided signicant protection against subsequent chal-
lenge infection (reviewed in Hotez et al., 1996), and vaccination with a
43 kDa immunodominant glycoprotein of Trichinella spiralis induced high
levels of protection against larval infection in mice (Robinson et al.,
1995), indicating that ESPs from parasitic nematode larvae might contain
vaccine targets. Nonetheless, some (hidden) antigens were also effective
against blood-feeding nematodes (cf. Newton and Meeusen, 2003), as
high levels of specic antibodies are ingested when feeding on blood,
but might be less effective against nematodes, such as O. dentatum, which
do not feed on blood.
6. FUTURE PROSPECTS AND CONCLUDING REMARKS

While a range of molecules have been predicted to be involved in the
moulting/exsheathment in O. dentatum larvae (Joachim and Ruttkowski,
2008; Joachim et al., 2005, 2011; Ondrovics et al., 2013, 2014), their active
role during these processes needs to be assessed in functional studies. Gene
silencing by RNAi represents an approach for revealing specic functions
of selected gene products. In C. elegans, RNAi has been extensively used to

examine gene functions (Frand et al., 2005; Maine, 2008; Yanos et al.,
2012), but, despite the similarities between parasitic and free-living nema-
todes, appropriate validation requires studies in the parasite itself. Studies
on RNAi in parasitic nematodes have had divergent results (Geldhof
et al., 2006; Kotze and Bagnall, 2006; Visser et al., 2006). Gene silencing
often differs in reproducibility in and susceptibility of different parasitic
nematode species and their developmental stages, as the site of gene expres-
sion and the efciency of double-stranded RNA (dsRNA) uptake inuence
the outcome of gene knock-down of a specic target transcript (Geldhof
et al., 2007). Nonetheless, successful specic gene knock-down following
RNAi for some genes in parasitic nematodes is encouraging (Samarasinghe
et al., 2011; Selkirk et al., 2012). Moreover, a sequence similarity survey for
orthologues of C. elegans RNAi pathway proteins in various parasitic nem-
atode species, including O. dentatum, revealed that most facets of the RNAi
pathway are well represented across a number of parasitic nematode species,
indicating the presence of the machinery required to facilitate an RNAi
response (Dalzell et al., 2011). With the optimization of in vitro culture con-
ditions, dsRNA preparation and its delivery route, it should be possible to
develop this technique to examine the functions of parasite gene, which
would signicantly contribute to the assessment of the precise roles of
genes/proteins and, subsequently, to the evaluation of their potential as
candidates for intervention.
The extensive use of anthelmintic drugs to treat parasitic nematode
infections has led to the worldwide emergence and increase of anthelmintic
resistance (Kaplan, 2004; Sutherland and Leathwick, 2011; Wolstenholme
et al., 2004). As a consequence, much effort has been spent on the identi-
cation of candidate target antigens for use in vaccination or as novel drug
targets for nematode control (Bethony et al., 2006b; Claerebout et al.,
2003; Knox, 2000; Newton and Meeusen, 2003; Newton and Munn,
1999). Undoubtedly, a better understanding of the parasites biology and
their key developmental processes paves the way for designing new inter-
vention strategies.
In conclusion, the integrative approach, combining in vitro assays with
transcriptomic, proteomic and bioinformatic techniques, to investigate
moulting in O. dentatum offers a unique opportunity to elucidate this key
fundamental process. For particular proteins (e.g. CYN, CUT-1, IFB and
TPM), reported roles in the moulting process in related nematode species
were conrmed in O. dentatum; previous ndings of proteins with predicted
functions during the nematode moult were afrmed (e.g. FBA, PCC and
PEPCK), and new molecules (e.g. API and TTL-5) were suggested to
play important roles during moulting/exsheathment. The molecules
predicted as key players in moulting in O. dentatum could be part of one
or more conserved developmental pathways linked to moulting and/or
developmental processes in nematodes. Taken together, the porcine nodule
worm O. dentatum represents a useful model organism to study such funda-
mental molecular processes. The information available for research on
O. dentatum has grown tremendously in recent years, and there are
numerous tools and opportunities for scientic research of this and related
nematodes, providing a foundation for tackling a range of fundamental
questions and for developing new methods of intervention.
ACKNOWLEDGEMENTS
MO is recipient of a DOC-fFORTE-fellowship of the Austrian Academy of Sciences.
Currently, RBGs research is supported by the Australian Research Council (ARC) and the
National Health and Medical Research Council (NHMRC) of Australia; it is also supported
by a Victorian Life Sciences Computation Initiative (VLSCI), grant number VR0007, on its
Peak Computing Facility at the University of Melbourne, an initiative of the Victorian Gov-
ernment. Other support from the Australian Academy of Science, Alexander von Humboldt
Foundation and Melbourne Water Corporation is gratefully acknowledged.
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CHAPTER FIVE
A Population Biology
Perspective on the Stepwise
Infection Process of the Bacterial
Pathogen Pasteuria ramosa
in Daphnia
Dieter Ebert*, 1, David Duneau*, x, Matthew D. Hall*, {,
Pepijn Luijckx*, jj, Jason P. Andras*, #, Louis Du Pasquier*,
Frida Ben-Ami**
*Zoological Institute, University of Basel, Basel, Switzerland
x
Department Ecologie et Diversit Biologique, University Paul Sabatier-Toulouse III, Toulouse, France
{
Monash University, School of Biological Sciences, Clayton Campus, Melbourne, VIC, Australia
jj
Department of Ecology & Evolutionary Biology, University of Toronto, Toronto, ON, Canada
#
Department of Biological Sciences, Mount Holyoke College, South Hadley, MA, USA
**Department of Zoology, George S. Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv, Israel
1
Corresponding author: E-mail: dieter.ebert@unibas.ch
Contents
1. Introduction 266
2. The DaphniaePasteuria System 268
3. Steps of the Infection Process in the DaphniaePasteuria System 269
3.1 Step 1. Host encounter with parasite transmission stages 270
3.2 Step 2. Activation of dormant parasite spores 274
3.3 Step 3. Attachment of activated spores 275
3.4 Step 4. Host penetration 280
3.5 Step 5. Early within-host phase 281
3.6 Step 6. Late within-host phase 286
3.7 Step 7. Host death and spore competence 287
4. Using the Stepwise Model to Address Evolutionary Questions 289
4.1 How much host variation can be explained by each step? 289
4.2 Genetic basis of disease expression 291
4.3 Evolution of resistance and its costs 293
4.4 Expression and evolution of virulence 294
4.5 Hosteparasite coevolution 298
4.6 The evolution of host range 300
5. Conclusions 300
References 302

2016 Elsevier Ltd.
j
ISSN 0065-308X
266 Dieter Ebert et al.
Abstract
The infection process of many diseases can be divided into series of steps, each one
required to successfully complete the parasites life and transmission cycle. This
approach often reveals that the complex phenomenon of infection is composed of
a series of more simple mechanisms. Here we demonstrate that a population biology
approach, which takes into consideration the natural genetic and environmental vari-
ation at each step, can greatly aid our understanding of the evolutionary processes
shaping disease traits. We focus in this review on the biology of the bacterial parasite
Pasteuria ramosa and its aquatic crustacean host Daphnia, a model system for the
evolutionary ecology of infectious disease. Our analysis reveals tremendous differences
in the degree to which the environment, host genetics, parasite genetics and their in-
teractions contribute to the expression of disease traits at each of seven different steps.
This allows us to predict which steps may respond most readily to selection and which
steps are evolutionarily constrained by an absence of variation. We show that the ability
of Pasteuria to attach to the hosts cuticle (attachment step) stands out as being
strongly inuenced by the interaction of host and parasite genotypes, but not by envi-
ronmental factors, making it the prime candidate for coevolutionary interactions.
Furthermore, the stepwise approach helps us understanding the evolution of resis-
tance, virulence and host ranges. The population biological approach introduced
here is a versatile tool that can be easily transferred to other systems of infectious
disease.
1. INTRODUCTION
In parasiteehost interactions, the parasite must pass through a series of
steps (or stages) to successfully complete its life and transmission cycle
(Combes, 2001; Schmid-Hempel, 2011). It must encounter the host, enter
it, survive the hosts immune response, reproduce and release transmission
stages. The stepwise nature of this infection process is well understood for
many human, animal and plant infections and in some cases we even
know the interacting genes for some of the steps (e.g. Dodds and Rathjen,
2010; Ferrandon, 2013; Gutjahr and Parniske, 2013; Lemaitre and
Hoffmann, 2007; Nakajima and Akutsu, 2014; Sarker and Paredes-Sabja,
2012; Schulenburg et al., 2007; van Schie and Takken, 2014). Indeed, life
and transmission cycles depicting the steps of the infection process have
long been part of parasitology and infectious disease textbooks (Burnet
and White, 1972; Cox, 1993). However, while we have claried details
about the infection processes for many diseases, we rarely look at these steps
from a population biology perspective, which considers natural variation
among host and parasite genotypes and how they are modied by the
Evolution and the stepwise infection process 267
environment (Dybdahl et al., 2014; Schmid-Hempel and Ebert, 2003). Es-

timates of variation in trait expression at given steps of the infection process
are usually not included in pictures of life and transmission cycles. In most
cases, such estimates do not even exist. Understanding natural variation is,
however, essential to understanding how evolution and the environment
shape infection processes.
Mechanistic and population biological approaches can yield very
different conclusions about the expression and evolution of disease-related
traits. A gene that plays a key role in the infection process, for example, is
largely irrelevant for the evolution of the disease if all individuals share the
same variant of the gene (Hueckelhoven et al., 2013). In contrast, genes
with allelic variation segregating in a population may be exposed to selection
and lead to an adaptive response (Schmid-Hempel and Ebert, 2003) even if
their overall contribution to disease expression is small. Thus, knowing
where genetic variation exists in the infection process would enable us to
better understand the evolution of disease traits. Furthermore, nongenetic
factors that cause variation in the expression of disease traits must also be
considered, as they can inuence the rate of adaptive evolution. Some steps
of an infection process may stand out in being more prone to environmental
variation such as climate, environmental stressors and competition among
parasites. Other steps may be inuenced by genetic interactions among
host and parasite genotypes or even complex combinations of host, parasite
and environmental factors.
Although we know little about the sources and consequences of differen-
tial variation in the individual steps of the infection process for most diseases,
some studies have noted that such differences exist and are important.
For example, the experimental manipulation of early steps in the infection
process can reveal very different disease outcomes (Behrens et al., 2014;
Dhondt et al., 2007; Martins et al., 2013). In part this can be attributed to
variation in the contribution of genetic and environmental factors inu-
encing trait expression during different steps (Martins et al., 2013; Wargo
et al., 2012). Furthermore, steps in the infection process without genetic vari-
ation are less likely to evolve in response to our measures to control diseases
and might therefore be good targets for therapy (He et al., 2014). This relates
to the idea that parasite control strategies could be made evolution proof by
targeting genetically constrained infection steps, thereby preventing or delay-
ing evolution of parasite resistance (Koella et al., 2009; Read et al., 2009).
One system for which we have a thorough understanding of the
infection process is Daphnia and its bacterial parasite, Pasteuria ramosa
(Duneau et al., 2011; Hall and Ebert, 2012). Over the last two decades,
studies of the ecology and evolution of this system have produced a
detailed picture of the steps of the infection process within an environ-
mental and evolutionary context. The system has become a model for
the study of the ecology, evolution and coevolution of infectious diseases
(Decaestecker et al., 2007; Ebert, 2008). In this review, we apply a popu-
lation biology approach to this system, explicitly considering the sources of
natural variation that inuence the different steps of the infection process
and how this variation affects disease expression. We examine, in turn,
the effects of host genetics, parasite genetics, the environment and their
interactions on each of the seven steps in the infection process. We
highlight the developmental and phylogenetic constraints on these
disease-related traits. Finally, we apply the insights of this analysis to issues
regarding host and parasite evolution and coevolution, the genetics of
disease expression and resistance, the evolution of host ranges and the
evolution of virulence.
2. THE DAPHNIAePASTEURIA SYSTEM

Pasteuria ramosa is a common bacterial endoparasite of Daphnia and
related Cladocera, reported in Eurasia and North America (Andras and
Ebert, 2013; Auld et al., 2012a; Goren and Ben-Ami, 2013; Green,
1974). In natural populations, it can reach a prevalence of 100% in adult
hosts (Duncan and Little, 2007) and has strong tness consequences,
sterilizing hosts and reducing life expectancy (Ebert et al., 1996). It is there-
fore believed to play a major role in the ecology and evolution of its hosts
(Auld et al., 2012a; Ebert, 2005). Research on this system is facilitated by
the cyclic parthenogenetic reproduction of the hosts (Box 1), which allows
clonal replication of host genotypes but also enables genetic crosses among
clones (Luijckx et al., 2012). Because of its predominantly asexual mode of
reproduction, research on Daphnia is carried out mostly with females.
Therefore, unless mentioned otherwise, we report here results for females.
Pasteuria can be cloned as well, and transmission stages (spores) can be kept
frozen (Luijckx et al., 2011). The genetic characteristics of the parasite
are most clearly seen in clones, as isolates (propagation of spores from
eld-collected infected hosts) often harbour multiple genotypes (Luijckx
et al., 2011; Mouton et al., 2007) and therefore diminish the genetic
resolution.
Box 1 The Daphnia model

Daphnia is a genus of planktonic freshwater crustaceans with a worldwide
distribution. Adults are 1e5 mm in size and reach maturity in 6e12 days (at
20 C) (Ebert, 1992). They grow throughout their life with a lifespan, under
laboratory conditions, of 50e150 days. Daphnia reproduce primarily by means
of cyclic parthenogenesis, i.e. produce mostly genetically identical daughters
and sons, but can also reproduce sexually by producing haploid eggs that
require fertilization by males. Sexual eggs require an obligate resting phase.
The combination of sexual and asexual reproduction provides powerful
means for genetic crossing designs, allowing the estimation of genetic
and nongenetic variance components (Ebert et al., 1993). Under natural
conditions, Daphnia undergoes sexual reproduction about once a year
(Lampert, 2011).
In the past few years D. pulex and D. magna have become model systems in
ecological genomics (Colbourne et al., 2011; Ebert, 2011; Smirnov, 2014), open-
ing up new possibilities for combining functional and evolutionary genetics with
ecology and epidemiology, in particular in the elds of ecotoxicology and envi-
ronmental health. Daphnia is one out of 13 ofcial model organisms for biomed-
ical research in the National Institutes of Health, USA (http://www.nih.gov/
science/models/).
3. STEPS OF THE INFECTION PROCESS IN THE

DAPHNIAePASTEURIA SYSTEM
The infection process of the DaphniaeP. ramosa system is well docu-
mented, and two decades of research allow us to present its major steps in
detail. Although certain processes may happen in parallel, e.g. the parasite
might have different routes to enter the host, or different components of
the immune system may act in parallel to thwart the parasite, the major
steps can be presented as a chain-like sequence. Processes occurring in
parallel could affect the evolution of the system; however, we currently
know too little to address this topic for the PasteuriaeDaphnia system.
Here we use a seven-step sequence to map the infection process in this
system, but the distinctions between steps are not always clear. Indeed
some steps could be subdivided, or two steps may be clustered into one.
Nevertheless, the model presented here has proven convenient to examine
the underlying mechanisms. The number of steps for other systems may be
different.
3.1 Step 1. Host encounter with parasite transmission stages

Infection processes begin with an encounter of host and parasite (Figure 1,
Table 1). Hosts often reduce the chance of encounter by avoiding contact
with infected hosts or avoiding locations where there is a greater likelihood
of encountering parasite transmission stages. Pasteuria infects Daphnia hosts via
environmental transmission stages (in the following called spores) encoun-
tered either in the water (free oating spores) or deposited in the sediment
of ponds and lakes (Ebert, 2005; Ebert et al., 1996). Vertical and mixed
mode transmission was never reported for any Pasteuria species (Ebert,
2013; Ebert et al., 1996). For the water-to-host route of transmission,
Daphnia ingest Pasteuria spores along with food particles oating in the water
while lter-feeding (Ebert, 2005; Smirnov, 2014), thus producing a conict
between the benets of food uptake and the risk of infection (Hall et al.,
2007). The higher the density of spores in the water, the higher the likeli-
hood of infection, with the form of this density dependence being well
approximated by mass action (Ben-Ami et al., 2008b; Regoes et al., 2003).
Figure 1 Schematic representation of the seven infection steps in the host (clockwise
from the encounter step at the left). Encounter happens when spores ltered from the
free water or the sediment come in contact with the host (step 1). Spores will then be
activated by the host (step 2) and may attach to the gut wall (step 3). Attached parasites
penetrate the gut wall (step 4) and enter the body cavity, where they multiply (steps 5
and 6). Eventually the host is killed by the parasite (step 7) and spores are released from
the decaying cadaver. Both male and female Daphnia may become infected.
Even a single spore can cause an infection, although with very low likelihood
(Luijckx et al., 2011). While experimental studies have primarily used well-
mixed suspensions of spores in water to achieve controlled infections, in na-
ture it is not clear how common the water-to-host route for transmission is.
However, sloppy feeding by predators and water turbulence may indeed lead
to spores being suspended in the water (Auld et al., 2014; Hall et al., 2010;
Goren and Ben-Ami, 2015).
Under natural conditions, it is believed that infection most likely occurs
via a sediment-to-host route when animals browse on and in the sediment
surface and stir up particles that they lter from the water (Ebert, 2005;
Horton et al., 1979). Spores are released from decaying host cadavers on
the pond or lake oor, resulting in a clustered distribution of spores (Ebert,
2005). Experimental exposure of Daphnia to pond sediments frequently
leads to infection, even when sediment from cores are used, which can be
several decades old (Andras and Ebert, 2013; Decaestecker et al., 2002,
2004; Jansen et al., 2010). This sediment-to-host route has been linked to
differences in host behaviour, which varies strongly among Daphnia geno-
types (Decaestecker et al., 2002). Negatively phototactic Daphnia genotypes
stay lower in the water column and tend to be found more in habitats with
sh than in shless habitats. They even move downward when sh kairo-
mones are added to the water (De Meester, 1993, 1996; De Meester
et al., 1995), a behavioural change that reduces their likelihood of encoun-
tering predatory sh. However, being closer to the pond sediments has costs
in that it increases the likelihood of exposure to sediments and, thus, parasite
spores. In contrast to the water-to-host route, transmission via the sediment-
to-host route is not density dependent, because the spore bank in the pond
sediments, which accumulates over months and even years, decouples the
current production of transmission stages from infection of new hosts and
thus dampens epidemiological and evolutionary dynamics (Auld et al.,
2014; Ebert et al., 1997). The combination of direct (from water) and indi-
rect (via spore bank) transmission is expected to increase the long-term
persistence of the parasite in a host population, as it expands the range of
environmental conditions under which transmission is possible. This is anal-
ogous to the epidemiological dynamics of mixed-mode transmitted parasites
(Ebert, 2013).
Negatively phototactic Daphnia magna clones have a higher infection rate
than Daphnia that remain higher in the water, and addition of sh kairo-
mones cause not only a downward movement of the Daphnia, but also an
increase in infection rates (Decaestecker et al., 2002). These differences
Table 1 Description of steps in the infection process of Pasteuria ramosa in Daphnia
272
Trait(s) for which
phenotypic variation Potential for the host
Step Description of process was studied to evolve resistance Key references
1. Encounter with Filter-feeding host comes Behavioural differences Avoidance behaviour may Decaestecker
parasite spores into contact with among hosts inuence evolve, which is et al. (2002)
nonmotile parasite the likelihood of functionally linked to
spores that either rest in encountering spores predator avoidance and
pond sediment or oat foraging
in the water
2. Spore activation Spore activation upon No variation in spore Avoidance of activation is Duneau et al. (2011)
physical/chemical activation observed unlikely to evolve:
contact with the host among Daphnia clones There is no genetic
before ingestion. and species variation among hosts or
Shedding of outer spore parasites genotypes
shell (exosporium)
releases activated spore
3. Attachment of Activated spores are Attachment to host gut Impeding attachment Duneau et al. (2011)
activated spores ingested by the host and wall varies with host and and Luijckx
attach to the gut wall parasite genotype et al. (2013)
4. Penetration Penetration into the hosts Likelihood that the parasite Moulting is Duneau and
body cavity. This takes penetrates the gut wall developmentally/ Ebert (2012b)
about 12 h. Host depends on host phylogenetically
Dieter Ebert et al.

moulting within 12 h moulting constrained, preventing
post attachment change in moulting rate.
prevents penetration Variability in
permeability of gut wall
may be possible
Evolution and the stepwise infection process
5. Early within- The host immune system
Parasite clearance, spore Clearance of parasite; Hall and Ebert (2012),
host phase interacts with the counts and reducing parasite growth Hall et al. (2013),
invading parasite development, castration, and development and Ben-Ami
host body size et al. (2010)
6. Late within-host Phase of chronic infection Spore counts, host Reducing parasite growth; Hall and Ebert (2012),
phase fecundity, castration castration relief Ben-Ami and
relief, host body size Routtu (2013),
and Mageroy
et al. (2011)
7. Host death Hosts die 30e70 days after Time to host death None Hall and Ebert (2012),
infection. The cadaver Jensen et al. (2006),
breaks open and releases Ben-Ami et al.
the environmentally (2008a), and
resistant parasite Ben-Ami and
endospores Routtu (2013)
Mentioning of quantitative estimates were collected from experiments conducted at 20 C. Higher temperatures accelerate these processes. Compare to Figure 1.
273
were not due to differential susceptibility of the Daphnia genotypes, but only
to the higher exposure of the negative phototactic clones to the sediment-
borne spores. Genetic variation for phototactic behaviour is believed to be a
quantitative genetic trait and the interaction between Daphnia
clones and kairomones highlights that the encounter step is subject to
genotype environment interactions (Table 2) (De Meester, 1989; Routtu
and Ebert, 2015). Besides the correlation between phototactic behaviour
and infection, the actual uptake of spores from the sediment has so far not
received attention. Disregarding phototactic tendencies, Daphnia individuals
may differ in their propensity to dig into the sediment surface and thus to
inuence the encounter rate with spores. A negative phototactic clone
with a low propensity to dig may enjoy the combined benets of protection
from sh predation and parasitism.
In summary, exposure to free-oating parasite spores in the water is
unavoidable for the lter-feeding hosts (Hall et al., 2007), whereas exposure
to spores in pond sediments depends on host behaviour. While the former
process is density dependent, transmission in the latter type of parasite
encounter is density independent, with important consequences for the start
and the spread of epidemics (Ebert et al., 1997). Variation among host clones
for encounter rates varies strongly among populations (local adaptation),
seems to have a quantitative genetic basis, and is prone to genotype by
environment interactions.
3.2 Step 2. Activation of dormant parasite spores

Once a dormant parasite comes into contact with a potential host, it must
become active. This process has been well documented in fungal pathogens
of plants and animals and in spore-forming bacteria and has been shown to
require specic triggers associated with the host (Hu et al., 2014; Jaronski,
2010; Paredes-Sabja et al., 2014). Pasteuria spores can rest dormant for
decades in pond sediments (Decaestecker et al., 2004), but within minutes
of coming into contact with a potential host, the nearly spherical spores
shed their exosporium and assume a disc-like shape with a thick central
body (Duneau et al., 2011) (Figure 2). Only this activated spore can attach
to the host (see next step). How activation is induced is not yet known,
but requires some form of interaction between spore and host. Pasteuria
activation alone does not induce vegetative outgrowth of the germ tube
(germination), which happens only after the activated spores attach to a
susceptible host (see penetration step below).
Experiments with different combinations of parasite and host clones and

species under various environmental conditions have revealed that Pasteuria
spore activation is largely nonspecic (Duneau et al., 2011). Any tested host
genotype within species belonging to the family Daphniidae, whether sus-
ceptible or resistant, was found to activate spores of P. ramosa, while a more
distant arthropod, a lter-feeding mosquito larvae, did not (Duneau et al.,
2011) (Table 2). Thus, the activation signal seems phylogenetically
conserved. Possibly, the costs for the host of evolving a defence against acti-
vation are so high that a mutant doing so could not spread. Furthermore,
spores have been activated under a variety of test conditions, e.g. at different
temperatures, in well-fed and starved hosts, in male and female hosts and in
conventional and microbiota-free hosts (Duneau et al., 2011) (M. Sison-
Mangus et al., in prep.), suggesting that activation is insensitive to environ-
mental conditions.
Hardly anything is known about the mechanism of activation. Preheat-
ing Pasteuria spores to 99 C does not prevent subsequent activation at room
temperature, suggesting that the activation process does not depend on pro-
teins or on the viability of the spores, which are rendered incompetent by
exposure to temperatures above 70 C (Metzger, 2014). Under laboratory
conditions, activated spores have a lifespan of under 24 h (S. Gygli, unpub-
lished data), unless they are frozen (King et al., 2013), but they remain in-
fectious after passing through the gut of susceptible or resistant Daphnia
(King et al., 2013).
In summary, Pasteuria spores shed their exosporium upon receiving
a phylogenetically conserved trigger from Daphnia and closely related
Cladocera. Since neither ecological conditions nor host or parasite geno-
type measurably inuence spore activation, the host has little room for
an evolutionary adaptation at this step that would reduce the likelihood
of infection.
3.3 Step 3. Attachment of activated spores

Attachment of parasite cells to host tissue is important in many infectious dis-
eases and often requires specic adhesion molecules (Adamu et al., 2013;
Benhamed et al., 2014; Doran et al., 2013). In many systems, the contact
zone between bacterium and host epithelium marks the hosts rst line of
defence and is the subject of anti-adhesion therapy research (Krachler and
Orth, 2013). In Pasteuria, host attachment is an important step in the infec-
tion process, as variation in this step explains most of the overall variation in
the entire infection process, as we elaborate in section 4 of this article.
Table 2 Variation in disease trait expression at different steps of the infection process
6. Late
276
2. Spore 5. Early within- within-host 7. Host
Step 1. Encounter activation 3. Attachment 4. Penetration host phase phase death 1e7. All steps
Traits Likelihood Change in Attachment Penetration Likelihood Spore Time to Likelihood

measured of contact spore of activated of germ of infection, counts, parasite- of infection,
with phenotype spore to tube spore counts, host and induced host and
spores while host through host and parasite host death parasite life
being gut wall host gut parasite life life history history traits
activated wall history traits traits
Form of Quantitative None Binary (0/1) Linked to Quantitative Quantitative Quantitative Quantitative
phenotypic moulting
variation cycle*
observed
Evidence for genetic and environmental contribution to trait variation
Host genetic Yes No Yes No Yes Yes Yes Yes
variation, HG
(among host
clone variation)
Within (HGW) HGW < HGB No HGW > HGB No ? ? ? ?
versus between
population
Dieter Ebert et al.

(HGB)
genetic variation
Parasite genetic No No Yes No Yes Yes Yes Yes
variation, PG
(among parasite
clone variation)
Impact of the Yes No No Yes Yes Yes Yes Yes
environment, E
Evolution and the stepwise infection process

H G PG No No Yes No Yes No No Yes
HG E Yes No No No Yes Yes No Yes
PG E No No No No Yes No No Yes
H G PG E No No No No No No No Yes
Host maternal ? No No No Yes Yes Yes Yes
environmental
effect
Host status
Host sex Yes No No Yes Yes Yes No Yes
Host age Yes ? ? Yes Yes Yes No Yes
Constraints on trait evolution
Cost of resistance Yes NA No NA ? ? NA NA
Phylogenetic/ ? Yes ? Yes ? ? ? NA
developmental
constraint
Yes indicates evidence for signicant contribution to phenotypic variation; No and ? indicate no current evidence or unknown, although a contribution may be
discovered in future experiments; NA, not applicable; For references see Table 1 and main text.
Times given in the inner circle are approximate estimates.
* Processes inuencing the moulting frequency of Daphnia (e.g. faster at higher temperature, slower at larger body size) inuence the penetration process.
277
Figure 2 Schematic representation of Pasteuria development during the seven infec-

tion steps (in clockwise order, beginning with the encounter step at the left). The
host encounters the dormant spore, enclosed in the exosporium (step 1). Upon activa-
tion the exosporium is shed (step 2) and the activated spore attaches to the gut wall
(step 3). The attached parasite penetrates the gut wall (step 4) and soon starts to pro-
duce cauliower-like stages (step 5), which break into smaller and smaller fractions, un-
til each branch represents a single grape-seed stage spore, which further develops into
a mature spore. During the late within-host phase (step 6), the hosts entire body cavity
becomes lled with mature, dormant spores, which are released into the environment
upon host death (step 7).
Activated spores of Pasteuria need to attach to the cuticle of the foregut

wall (the oesophagus) to cause infection. Failure to do so terminates the
infection process and the activated spores are quickly degraded (Duneau
et al., 2011). Fluorescently labelled Pasteuria spores can be readily observed
in the living host when they adhere to the oesophagus wall of the transparent
host. The attachment is so strong that spores are not dislodged by mechanical
disturbance, such as food passing through the oesophagus. The attachment
process observed in the Daphnia system is similar to the attachment process
of Pasteuria penetrans to its juvenile nematode host (for a review see Davies,
2009). In both systems, attachment is to a chitin-containing cuticle of endo-
dermal origin. Since the area of the tissue to which Pasteuria spores can attach
to their host is small, interference competition among spores is likely, poten-
tially inuencing parasite evolution during host exposure to multiple parasite
genotypes.
A particularly interesting feature of P. ramosa attachment to Daphnia, is
that for a given combination of host and parasite genotypes, activated spores
either attach or do not (Duneau et al., 2011), resulting in a binary form of

variation. In laboratory and natural populations, most of this variation is
explained by pronounced genetic hosteparasite interactions (Andras and
Ebert, 2013; Duneau et al., 2011; Luijckx et al., 2011). Genetic crosses be-
tween D. magna clones with different susceptibility to attachment by
different Pasteuria clones have revealed that attachment (susceptibility) is
recessive but strongly inuenced by the presence of closely linked interact-
ing loci (epistasis) (Little et al., 2006; Luijckx et al., 2012, 2013; Metzger,
2014). Observed patterns of hosteparasite interactions show a signature of
a matching allele model, whereby a single allele substitution can reverse
the infection patterns of different parasite clones (Luijckx et al., 2013). Vari-
ation in attachment among hosts (resistotypes) and parasite (infectotypes)
varies widely within and between Daphnia populations (Andras and Ebert,
2013; Luijckx et al., 2014), suggesting evolutionary processes at work,
which maintain genetic diversity (see section on coevolution below).
A further interesting feature of the attachment step is that environmental
conditions (e.g. temperature, well-fed or starved hosts, host crowding) as
well as host sex and age do not affect attachment (Duneau et al., 2011)
(Table 2). Even host microbiota, which have been shown in other systems
to inuence hosteparasite specicity (Koch and Schmid-Hempel, 2011)
and generally inuence Daphnia biology (Qi et al., 2009; Sison-Mangus
et al., 2015), do not interfere with the attachment process: replacing the nat-
ural microbiota of resistant and susceptible D. magna clones did not inuence
the results of attachment tests (M. Sison-Mangus et al., in prep.) (Table 2).
It is not understood how the high genetic specicity in the attachment
process arises from the interaction between host and parasite genes. Proteins
are likely involved on the side of the parasite, as spores heat treated to about
70 C or above lose their ability to attach (Metzger, 2014). This has also
been supported in studies of spores of P. penetrans attaching to their nema-
tode host (Davies, 2009; Freitas et al., 1997). For both systems, it is believed
that collagen-like proteins, expressed in large numbers on the surface of the
activated spores, play a central role for attachment of Pasteuria (Davies, 2009;
McElroy et al., 2011; Mouton et al., 2009; Schaff et al., 2011). Genes coding
for these proteins are found in high abundance and variability in the P.
ramosa genome (McElroy et al., 2011; Mouton et al., 2009). The molecules
and pathways involved in attachment in the host are not known, but the
chromosomal region for one locus has been mapped with the help of a
quantitative trait loci (QTL) mapping panel (Routtu and Ebert, 2015;
Routtu et al., 2014).
In summary, the attachment step is characterized by a very high speci-

city of hosteparasite genotype combination without environmental factors
being involved. The strong hosteparasite interactions show a binary pattern
of variation (attachment or not), making it a strong candidate for a step that
may undergo hosteparasite coevolution.
3.4 Step 4. Host penetration

After attaching to the host, the parasite must enter the host cell or body. Bac-
teria often use different secretion systems to achieve penetration, while plant
fungal pathogens grow hyphae into the host tissue (Cossart and Helenius,
2014; He, 1998; Naglik et al., 2011; Underwood, 2012). Although the pro-
cess of host cuticle penetration for P. ramosa has not yet been described, for
the related pathogen P. penetrans it was suggested that the nematodes cuticle
is locally dissolved by an enzymatic process and that a germ tube penetrates
the cuticle of the hosts (Dickson et al., 2009; Sayre and Wergin, 1977).
Through this tube the parasite injects its sporoplasma into the host body.
P. ramosa may use a similar mechanism for penetration.
The Daphnia oesophagus is part of the foregut and therefore of ecto-
dermal origin (as is the hindgut, but not the midgut). Therefore, when
the Daphnia moults, it also sheds the oesophagus lining and any attached
spores (Duneau and Ebert, 2012b). When moulting occurs within 12 h of
spore attachment (at 20 C), the host sheds the attached spores with the cara-
pace, and has a high likelihood of escaping infection (Duneau and Ebert,
2012b). Moulting is essential for growth and development in arthropods
and in Daphnia continues throughout life. Juvenile Daphnia moult about
every 36e48 h, adults every 3e4 days at 20 C and at higher temperatures,
moulting is more frequent (Bottrell, 1975). Thus, a considerable proportion
of the attached spores are lost before penetration. Although, moulting seems
to be a mechanism that reduces the likelihood of disease progression, there is
so far no evidence that the host can alter this developmentally and phyloge-
netically constrained mechanism to further reduce infections (Table 2)
(Duneau and Ebert, 2012b).
An alternative unexplored mechanism by which Daphnia may achieve
resistance is by altering the thickness or strength of the cuticle thereby
reducing the likelihood of penetration. A strengthening of the cuticle has
been observed in other systems, conferring resistance against pathogens
(Cotter et al., 2008; Dubovskiy et al., 2013) and may play a role in the
Daphnia system as well.
In summary, the attached spores penetrate the hosts gut epithelium and
enter the body cavity. This process takes several hours, giving the host a
chance to repel the parasite by moulting. So far no variation has been
observed for this process among host or parasite genotypes.
3.5 Step 5. Early within-host phase

After a parasite enters the host, the actual disease develops. In most cases, the
parasites begin to proliferate and cause disease-specic harm to the host. At
the same time, the hosts immune defence may be activated and, in some
cases, eliminate the parasite. The mechanistic basis of host immune defence
has been well explained for vertebrates, plants and some invertebrates, but
little is known for less-studied taxa, like the lower crustaceans. This is true
for the DaphniaePasteuria system, where we know a lot about disease pro-
gression and ecological immunity, but next to nothing about the molecular
processes at work.
Once Pasteuria enters the host, it undergoes a rather unusual series of
developmental stages resulting in the production of the mature endospores
(Box 2, Figure 2). A clear and early external sign of infection is host castra-
tion, i.e. females stop producing eggs. We call this time period the within-
host phase (as opposed to step), as, in contrast to the other steps, it takes a
considerably longer time e about 50 days from penetration to death,
although the length of this period is highly variable (Ben-Ami et al.,
2008a; Jensen et al., 2006). The within-host phase cannot yet be easily
divided into clearly separated steps. However, because most experimental
studies terminate infection experiments after 20e30 days, we dene the
early within-host phase as the rst 25 days, and the following period until
host death as the late within-host phase. Day 25 marks the approximate
halfway point from infection to host death. As we gain more knowledge,
this somewhat arbitrary classication may be replaced with a more meaning-
ful biological classication. For example, the time until the rst mature en-
dospores are observed (about 15e18 days post infection) could be used as a
biomarker for the early within-host phase. However, to make maximal use
of the available information, we use here the halfway point to divide the
within-host phase into two parts.
After the parasite enters its host, it replicates in the hosts body cavity and
muscle tissue. For approximately the rst 7 days post-penetration (at 20 C),
the bacteria are not detectable by light microscopy. Thereafter, they are
easily distinguished as large, multicellular vegetative structures, referred to
as the cauliower stage (up to 15 mm diameter, Box 2, Figure 2), and later
Box 2 Development of Pasteuria ramosa

Individual Pasteuria cells undergo remarkable morphological development
during the infection process (Figure 2). There is no evidence of growth,
reproduction or development outside the host. The life cycle begins when
the resting endospore comes into contact with a host and sheds its exosporium
(activation step). The activated spore (i.e. the spore without the exosporium)
has a spherical central body with a ring of parasporal bres (peripheral
bres, or perisporium) around its circumference, forming a disc-like structure
(also described sombrero-like). The activated spore attaches to the host
cuticle in the gut (attachment step), and from here penetrates the hosts
body cavity (penetration step). In P. penetrans (but not yet investigated in P.
ramosa) where host penetration was studied in more detail (Dickson et al.,
2009), the spore seems to produce a germ tube (germination) that penetrates
the cuticle and hypodermis of its nematode host. Penetration seems to be
achieved by an enzymatic process (Dickson et al., 2009). The attached spore
and parasporal bres remain outside, while bacterial cells penetrate into
the host.
It is not yet known what happens to the bacterial cells in the rst few days
after entering the hosts body cavity, but after 5e8 days, the parasite is
observable by light microscopy in infected hosts, appearing as oret- or
cauliower-like microcolonies up to 15 mm diameter. These microcolonies are
composed of a dichotomously branched septate mycelium, which fragment
into branch-like structures after the colonies reach a critical size. Peripheral
cells (terminal hyphae) of the microcolony expand and give rise to sporangia.
Branches with peripheral sporangia continue to grow and fragment into
branchlets of quartet, triplet and doublet congurations, with the sporangia
attached to each other at the pointed ends. At the rounded end, each
sporangium has a small central refractile body visible by light microscopy
that will develop into the actual endospore. Eventually, branchlets develop
into single teardrop or grape-seed like sporangia. These sporangia and their
endospore continue maturation, and eventually, about 14e18 days in the
infection, assume the more spherical structure of mature spores. Mature
spores, the transmission stage of the bacterium, are nonmotile, have a
diameter of about 5e6 mm and are composed of an environmentally resistant
exosporium surrounding the endospore. Early during the within-host phase,
the development of the parasite cells is synchronous; later during the infection,
cauliower stages can be found again, and eventually all developmental
stages are present concurrently. The number of mature spores increases and
accumulates until the hosts death, when they are released in millions from
the dead host.
by their characteristic spores. The typical symptoms of a Pasteuria infection

are parasite-induced sterilization (castration) and enhanced body growth
(gigantism). Castration is sometimes detectable as early as 10 days post infec-
tion. Other traits of interest during the early within-host growth phase are
the proportion of infected hosts, the fecundity of the infected hosts before
parasitic castration, the host body growth and early parasite spore production
(Coors et al., 2008; Hall and Ebert, 2012; Hall et al., 2013; Vale and Little,
2012).
Some disease symptoms previously attributed to the early within-host
phase (e.g. reduced infection rate, host castration (Hall and Ebert, 2012))
may also be inuenced by the penetration step (step 4), as the ease of pene-
tration may determine the number and speed of parasite spores entering the
host body cavity. This in turn may be inuenced by experimental condi-
tions. Furthermore, wounding of the hosts gut wall during penetration
may induce an immune response, with consequences for the subsequent
within-host phase. So far, no study has tested whether the penetration
step inuences the expression of subsequent disease symptoms. A few exper-
imental studies, however, applied different treatments only after penetration
was complete (i.e. several days after penetration), revealing strong effects on
host and parasite traits that had to be due to processes during the early
within-host phase (Cressler et al., 2014; Ebert et al., 2004). Here we discuss
traits expressed during the early within-host phase as part of step 5, but do
not exclude the possibility that the penetration step may play a role in
shaping disease expression during the within-host step.
To exclude the large variation caused by the attachment step, experi-
ments were conducted using host and parasite genotypic combinations
known to be 100% compatible, as assessed by the attachment of spores to
the oesophagus. Assuming uninhibited penetration and no effective immune
response, one can expect 100% infection rates for these cases. However, the
proportion of hosts that progress to disease is often less than 100% depending
on the treatments, which indicates parasite clearance and the elimination of
the parasite by the hosts immune defence (Hall and Ebert, 2012), although
the mechanism for this is unknown. Such a reduction in infection rates is not
observed in the late, chronic within-host phase (see next step), as once a
parasite has established itself in the host (as judged from the presence of dis-
ease symptoms), it is not cleared anymore (Hall and Ebert, 2012).
The Daphnias immune system is complex, involving melanisation and
the typical immune pathways described in other arthropods (Brites et al.,
2008; McTaggart et al., 2009; Metchnikoff, 1884), each of which may
contribute to control infections. The production of antimicrobial peptides

has so far not been reported (McTaggart et al., 2009). Several studies have
examined physiological and immunological responses expressed during
the early within-host phase, but no strong effects were observed. Jansen
et al. (2013) reported the largest number of differentially expressed genes
4 days post exposure to Pasteuria spores. Pauwels et al. (2011) reported
that Pasteuria spore production in the rst 21 days of infection is negatively
correlated with phenol oxidase (PO) activity, but another study (Mucklow
et al., 2004) reported that the PO activity of unexposed D. magna clones was
not a good predictor of resistance. Auld et al. (2012b) reported that the
number of phagocytic cells increased upon exposure and that this increase
correlated positively with parasite dose. A report of effective immune
priming 48 h after Daphnias exposure to noninfective Pasteuria spores
(McTaggart et al., 2012) suggests that, even without attachment, the host
may sense the parasites presence. However, this nding seems to contradict
the observation that a cellular response is only observed when parasite and
host are compatible at the attachment step (Auld et al., 2012b). Taken
together, these ndings support the presence of a functional immune
defence in Daphnia, but it is not clear if this immune defence is responsible
for Pasteuria clearing, nor is it clear when during the infection process
immune induction occurs.
Excluding variation at the attachment step, a number of studies have
revealed pronounced effects of nongenetic factors inuencing host and para-
site disease traits during the early within-host phase, such as salinity, food
quantity, food C:P ratio, fatty acid composition of food and the temporal
distribution of feeding times (Cressler et al., 2014; Frost et al., 2008a,b;
Hall et al., 2013; Schlotz et al., 2013). These studies suggest, that better con-
ditions for the host are also better for the parasite. For example, better host
nutrition results in higher host and parasite tness estimates (Ebert et al.,
2004; Vale et al., 2013) (for a review see (Tseng and Myers, 2014)). Another
nongenetic factor inuencing the outcome of disease is parasite exposure
dose. Higher doses at exposure lead to more severe disease but also reduced
parasite spore counts (Ebert et al., 2000b). In addition, the environmental
conditions experienced by host mothers have strong effects on infection
outcomes for the offspring (Ben-Ami et al., 2010; Frost et al., 2010; Hall
and Ebert, 2012; Schlotz et al., 2013). Experiments including different
host and parasite genotypes (all compatible at the attachment step) have
also shown ample genetic variation in disease traits expressed during the early
within-host phase and in some cases genotype/genotype (GxG) and
genotype/environment (GxE) interactions for parasite infection success,

host fecundity before castration and early parasite spore production (Hall
and Ebert, 2012).
One of the most important features of any individual is its age. Recent
studies showed that D. magna of different ages differ in their susceptibility
to Pasteuria infections. Izhar and Ben-Ami (2015) showed that after
controlling for all other steps of the infection process, juvenile D. magna
are more susceptible to P. ramosa than older females. This difference goes
hand-in-hand with reduced parasite proliferation in older hosts, but does
not change the time until host death. Furthermore, age at exposure played
a strong role in mediating the outcome of within-host competition, with
much stronger competitive exclusion being observed in hosts exposed at
a higher age (Izhar et al., 2015). Given the strong variation in age structure
of Daphnia populations over the course of a season, these ndings have
important consequences for the epidemiology and evolution of the system.
For example, the number of new infections may be much higher in pop-
ulations dominated by young Daphnia e as is typical in spring e than in
mid-summer populations, where juveniles are much less common. On
the other hand, competition among parasite genotypes would increase
across the season.
Expression of disease symptoms depends strongly on host sex (Duneau
et al., 2012). Spore counts at different time points across the infection period
are substantially higher in females than in the much smaller males, even after
correcting for body size. Both sexes suffer from fecundity reduction (sperm
and eggs counts), but only female hosts show parasite-induced gigantism. As
during these experiments, other steps of the infection process were
controlled, these differences are primarily due to sex-specic effects at the
within-host phase (Duneau et al., 2012).
Finally, the early within-host phase is a period where intense within-
host competition takes place. High dosages of spores administered to hosts
will result in strong within-host competition, resulting in the retarded
development of the parasites endospores (Ebert et al., 2000b). Consistent
with this, within-host competition of different Pasteuria clones and isolates
is largely determined during the early within-host phase (Ben-Ami and
Routtu, 2013), although this effects varies with host age at infection (Izhar
et al., 2015).
Many other experiments that did not explicitly exclude variation at the
attachment step, reported environmental effects (direct and maternal effects)
for diverse stressors, such as pesticide, food, predator kairomones and
temperature during the early within-host phase (for example Coors and De
Meester, 2011; Coors et al., 2008; Cressler et al., 2014; Garbutt et al., 2014;
Mitchell and Read, 2005; Stjernman and Little, 2011; Vale et al., 2008).
Because environmental effects are absent in the attachment step (Table 2),
it is reasonable to assume that these environmental effects are caused by fac-
tors acting on the early within-host step, not the attachment step.
In summary, the early within-host phase is a complex step of the infec-
tion process, with the traits being expressed during this period showing
ample evidence for quantitative genetic variation, sensitivity to environ-
mental conditions, and genotype by environment interactions. Parasite evo-
lution may be shaped strongly by competition during the early within-host
phase. Several immunological pathways may act in parallel during this phase,
however, the processes governing immunity in Daphnia are still poorly
understood.
3.6 Step 6. Late within-host phase

In invertebrate taxa and plants, late infection stages are often chronic, lasting
until host death. Such is the case for the late within-host infection phase of
P. ramosa. During this phase, parasite spore production continues as before,
leading to an intensive colouration of the host, showing various shades of
yellow, red and brown (Ebert, 2005). The parasite is mainly seen in the
form of mature spore stages that eventually ll the entire body cavity,
although cauliower and pre-spore stages (grape-seed stage, Box 2; Figure 2)
can also be seen. When hosts have enough resources (i.e. sufcient food
quantity and quality), Pasteuria-induced gigantism starts to become apparent
shortly after hosts are effectively castrated (about 10e20 days post infection)
(Cressler et al., 2014; Jensen et al., 2006) but is strongest during the late
within-host phase. Clearing of infections has not been reported during
the late phase of within-host growth, but is easily achieved with antibiotics
(Little and Ebert, 2000). Antibiotic treatment of late-stage infections allows
the host to reproduce again, suggesting that parasitic castration is not caused
by physical destruction of the ovaries, but by physiological means. Consis-
tent with this, during the late within-host phase, some hosts regain the abil-
ity to produce clonal offspring (Hall and Ebert, 2012; Mageroy et al., 2011;
Schlotz et al., 2013), a trait called castration relief. During castration relief,
hosts produce one or a few, typically small, clutches about 25e40 days post
infection (at 20 C). In a comparison of ve P. ramosa clones, the number of
offspring produced during castration relief was shown to be negatively
correlated with parasite spore production (Clerc et al., 2015). So far no
evidence has been found showing variation in castration relief among host
clones. The mechanism for castration relief is not known, but it may be
linked to the reduced physiological activity on the part of the parasite, whose
physiologically inert endospores occurring at this phase exert less inuence
on the host.
Experiments have revealed that host genotype, parasite genotype and
environmental effects (direct and maternal environmental effects) all
strongly affect virulence and parasite spore production late in the infection
process (Hall and Ebert, 2012; Schlotz et al., 2013; Vale et al., 2011). This is
also true for castration relief (Hall and Ebert, 2012; Schlotz et al., 2013).
Interestingly, these genetic and environmental main effects explain most
variation in disease expression, while interaction terms between host and
parasite genotypes or between genotypes and the environment seem
much less inuential (Hall and Ebert, 2012; Vale et al., 2011). Whether
this pattern is typical for late-phase infections in general is not yet clear:
unfortunately, most experiments terminate observations before the late
phase is reached.
In summary, the late within-host phase is characterized by the chronic
nature of the infection. The host seems to have no chance of eliminating
the parasite, but may ameliorate the tness cost of infection by castration re-
lief. Genetic variation for disease-related traits is high throughout the
within-host phase of infection, but genetic interactions seem to play less
of a role.
3.7 Step 7. Host death and spore competence

As with many other invertebrate parasites, Pasteuria is an obligate killer,
whose transmission stages are only released after host death (Ebert and
Weisser, 1997). The time to host death and the quality of the parasite spores
released are the key traits of interest here. Other traits, like host body size and
spore counts, are considered in the late within-host phase.
Under optimal conditions, female hosts are killed by Pasteuria after
30e70 days (Ben-Ami et al., 2008a; Hall and Ebert, 2012; Jensen et al.,
2006). For one hosteparasite combination, it was found that an interme-
diate time to host death (about 50 days) resulted in the highest number
of P. ramosa spores ( Jensen et al., 2006). In natural ponds, the dying host
most likely sinks to the bottom of the pond where it decays, releasing
0.5 to 20 million mature spores (Ebert et al., 2004). Pasteuria spores may
also be released by infected hosts that die early from other causes, e.g. envi-
ronmental stress (starvation, intoxication) and predation. Spore counts
increase from the rst fully developed spores around 15e18 days post
infection (at 20 C) until death (Ebert et al., 2004; Hall and Ebert,
2012), although the rate of increase can vary considerably among
genotypes (Clerc et al., 2015). It is not known if Pasteuria survives the
gut passage of Daphnia predators, e.g. sh, but this seems likely, as this
was shown for at least one fungal parasite (Metschnikowia bicuspidata) of
Daphnia (Duffy, 2009). Spores may also be released into the free water as
a consequence of sloppy feeding predators on infected hosts (Auld et al.,
2014; Hall et al., 2010; Goren and Ben-Ami, 2015).
Time to parasite-induced host death differs among parasite genotypes
infecting the same host clone and among host clones infected with the
same parasite clone (Ben-Ami et al., 2008a; Ben-Ami and Routtu, 2013;
Hall and Ebert, 2012; Izhar et al., 2015; Vale et al., 2013, 2011). Time to
death also depends on environmental factors, such as food level, parasite
spore dose, the presence of other parasites and temperature (Ben-Ami
et al., 2011; Ebert et al., 2004, 2000b; Hall and Ebert, 2012; Vale et al.,
2013, 2008, 2011). As was the case for the late within-host phase, interaction
terms (G G, G E, G G E) tend to explain hardly any variation in
time to death (Table 2). Time to Pasteuria-induced host death seems not to
depend on host age at exposure or host sex: D. magna infected at different
ages died after the same number of days (Izhar and Ben-Ami, 2015; Izhar
et al., 2015) and males, which normally live about half as long as females,
are killed about twice as fast as females by the parasite (Duneau et al., 2012).
After the death of the host, spores of Pasteuria are released into the envi-
ronment. Spores as old as 30 years have been revived from sediment cores
(Decaestecker et al., 2004). Nothing is known about genotypic or environ-
mental effects on spore survival. However, experiments with spores
collected from infected females kept under different feeding regimes have
shown that the quality of spores may vary: spores from well-fed hosts
were more virulent than spores from poorly fed hosts (Little et al., 2007).
A similar effect was also found for a fungal parasite of Daphnia (Searle
et al., 2015).
In summary, the parasite produces transmission stages that are only
released when the host dies or is killed. The time to parasite-induced death
depends strongly on host and parasite genetics and environmental factors,
but not on interactions between these factors. Although mature spores are
found in infected hosts as early as 15e18 days post infection, the parasite
normally kills the host much later. Premature host death can contribute to
parasite transmission.
4. USING THE STEPWISE MODEL TO ADDRESS

EVOLUTIONARY QUESTIONS
Dividing the infection process into discrete steps allows us to more
closely examine individual processes and how they are linked to func-
tional aspects of the system. It also enables us to relate individual steps
to evolutionary models, which are typically based on simplifying assump-
tions, such as simple genetics and no environmental effects. In this section
of this review we address a number of questions using the stepwise
approach to better understand the epidemiology and evolution of the
system.
4.1 How much host variation can be explained by each step?

The overview in Table 2 illustrates the tremendous difference in the degree
to which genetic and nongenetic factors shape trait variation during the steps
of the infection process. Traits expressed during the rst step and the last
three steps (steps 1, 5, 6 and 7) show the typical signature of complex quan-
titative genetic traits: variation is quantitative, environmental factors inu-
ence trait expression and host and parasite genetic effects are apparent. In
contrast, spore activation (step 2) seems not to be inuenced by any known
factor, while penetration (step 4) seems to be inuenced only by environ-
mental factors. In these two steps, variation appears limited by phylogenetic
and developmental constraints. The spore attachment step (step 3) is gov-
erned by binary genetic variation, without any evidence for environmental
effects. While this step-by-step consideration reveals the enormous diversity
in the contribution of different steps to disease progression, it does not allow
us to assess the relative importance of variation at each step. Here we ask, to
what extent does the overall expression of a specic disease trait depend on
variation at individual steps, and how does this variation inuence the
evolution of the trait?
As a rst approximation, earlier steps tend to inuence total variation of
host traits more than later steps, as each step acts as a lter, reducing the
possible variance of later steps. However, as the amount and distribution
of variation among and within populations differs for each step, some steps
may contribute more to the total variation than their position in the chain-
like process would suggest. The list of reported signicant effects on trait
expression does not help us judge the relative importance of a given step,
as these effects are typically assessed by reducing or even excluding variation
at other steps. For example, testing which factors inuence traits expressed
during the within-host phase only makes sense if hosteparasite combina-

tions are used that are compatible at the attachment step.
To illustrate how each step contributes to variation in disease, we focus
here on the likelihood of infection, as we know most about this trait.
Although it might seem that the rst step, the host encounter step, would
exert the strongest inuence on the likelihood of infection, two factors
reduce its impact: First, parasite variation is unlikely to contribute to varia-
tion in this step, as the parasite is passively waiting to be picked up by the
lter-feeding host. Second, encounter depends in part on the spatial distri-
bution of hosts and parasites. If parasite spores are homogeneously distrib-
uted in the water (free oating spores in the planktonic phase), no
variation in encounter is expected, unless the intensity of lter feeding varies
among host genotypes. In contrast, spores located in the pond sediments
are more likely to be encountered by negative phototactic host clones
(Decaestecker et al., 2002). However, as phototactic behaviour is known
to show a signature of local adaptation in Daphnia (De Meester, 1993,
1996), it differs more between populations than within population (Table 2).
Thus, the encounter step does little to explain overall variation in disease on
a within-population level but may have a potentially high impact on varia-
tion globally. The second step, spore activation, does not contribute to the
variation in infection success, because it appears to be a xed trait common
to all hosts.
The attachment step, however, shows particularly strong variation,
both within and between populations, but without evidence for local
adaptation for infection rate (Ebert et al., 1998; Luijckx et al., 2011).
The hallmark of the attachment step is binary variation, caused by the
strong hosteparasite interactions, which may render combinations of
host and parasite genotype incompatible (no attachment) (Luijckx
et al., 2011). Approximately one-third of host cloneeparasite clone com-
binations showed attachment (Luijckx et al., 2012), leaving more than
two-thirds of the combinations incompatible. Incompatibility terminates
the infection process and thus illustrates the strong lter effect of the
attachment step.
The fourth step, penetration, has so far only been associated with varia-
tion caused by host moulting, which is more frequent in juveniles than in
adults and at higher temperatures (Bottrell, 1975; Duneau and Ebert,
2012b). Given the variation in age and temperature, and no known variation
among host and parasite genotypes, this step acts mostly as a random lter,
reducing the number of parasites that reach the next step.
After excluding variation from the attachment step, strong variation dur-
ing the early within-host phase (step 5) is due to both genetic and environ-
mental effects. However, overall, the early within-host phase explains a
much smaller proportion of the total variation in infection rate than the
attachment step (step 3), as it can only contribute to variation in the subset
of hosteparasite combinations that have passed the earlier steps. As clearance
does not seem to occur once the infection is established, the late within-host
step does not inuence infection success, but does inuence the expression
of host and parasite life history.
In summary, the lter-like nature of the stepwise infection process suc-
cessively reduces the likelihood that later steps of the host defence machinery
encounter the parasite. Thus, everything else being equal, selection for resis-
tance is strongest at the earliest host steps. However, due to the specic
biology of the DaphniaePasteuria system, we suggest that the attachment
step (step 3) explains most variation in infection within a population and
that selection would be strongest here. For other steps, e.g. host castration,
this will be different, with the within-host phase playing possibly a stronger
role. In a spatial setting, however, with different Daphnia populations
showing divergent phenotypes due to local adaptation, steps that show
spatial divergence (e.g. phototactic behaviour and thus encounter rate)
may contribute more strongly to overall variation.
4.2 Genetic basis of disease expression

What is the genetic architecture underlying each step? So far, most genetic
studies have focused on the attachment step. Breeding experiments and a
QTL study with D. magna have revealed that resistance to parasite attach-
ment is dominant, and that a few loci interact epistatically to produce an
overall pattern, which seems always binary (Luijckx et al., 2011, 2012,
2013; Routtu and Ebert, 2015). So far, three closely linked loci have
been hypothesised to be responsible for this pattern (Metzger, 2014).
The genes responsible for attachment are not known, but comparative
genomics, QTL studies, genome scans and transcriptome approaches are
in progress (Decaestecker et al., 2011; McTaggart et al., 2009; Orsini
et al., 2012; Routtu and Ebert, 2015; Routtu et al., 2010). The other steps
with a signature of among clone variation (e.g. the encounter step and
within-host steps) are all quantitative, complicating the identication of
the underlying genes.
Currently, our limited evidence indicates that genes responsible for the
variation in traits at different steps are independent of each other on a
genomic level. D. magna genotypes that differed strongly in their behaviour

and thus in their propensity to encounter Pasteuria spores from sediment did
not otherwise differ in resistance (Decaestecker et al., 2002). A sediment
core study of PasteuriaeD. magna coevolution has proposed that selection
shapes infectivity (probability of parasite establishment upon host encounter;
presumably caused mainly by variation in the attachment step) and virulence
(host tness loss due to infection; presumably mainly due to variation during
the within-host phases) differently, suggesting that they may be coded by
different genes (Decaestecker et al., 2007).
Genes for resistance to P. ramosa seem also to be different from genes for
resistance to other parasites. Evidence comes from the absence of correla-
tions between the resistance to Pasteuria and resistance to other parasites,
four microsporidian species, a virus and a fungus, suggesting that most vari-
ation in resistance is explained by different underlying genetic architectures
(Auld et al., 2012a; Decaestecker et al., 2003; Ebert, 2008; Mucklow et al.,
2004; Zbinden et al., 2008). Likewise, mapping resistance to P. ramosa and
the microsporidium Hamiltosporidium tvaerminnensis in the same QTL panel,
indicates a different genetic architecture underlying resistance to these two
diseases: P. ramosa resistance showed a single strong QTL, while H. tvaermin-
nensis showed several weak QTLs and epistasis, without any co-localization
of QTLs for the two parasites (Routtu and Ebert, 2015). However, minor
QTL inuencing resistance to both parasites may have gone undetected.
In contrast to the host, we know very little about the underlying genetics
for disease-related traits in the unculturable P. ramosa. Proteomic and
genomic analyses have suggested that collagen-like proteins (the bacterial
version of collagen) may inuence the attachment of Pasteuria genotypes
(McElroy et al., 2011; Mouton et al., 2009). This hypothesis is supported
by the fact that collagen-like genes seem to act as adhesins in pathogenic bac-
teria (McElroy et al., 2011; Mouton et al., 2009) and by studies on the nem-
atode parasite P. penetrans (Davies, 2009). The family of collagen-like genes
is vastly expanded in both Pasteuria species, far beyond what is found in any
other fully sequenced bacterium, making it unusual among bacteria (Davies,
2009; McElroy et al., 2011). However, although collagen-like proteins may
inuence the attachment step, they are not candidates for variation observed
in later steps of the infection process.
In summary, host genetic effects are seen at most steps, with the marked
exception of the activation and the penetration step (Table 2). There is no
evidence that genes with a function specically relevant at one step inuence
disease expression at other steps. However, genetic independence is not the
same as evolutionary independence, as genes at different steps contributing

to the expression of the same trait can be under selection together.
4.3 Evolution of resistance and its costs

Resistance, the hosts ability to prevent or reduce parasite growth, is related
to tolerance, where hosts minimize the tness impact of the parasite but
without the associated damage to the parasite (Raberg et al., 2007). The
evolution of resistance and tolerance are driven by selection on the host
to reduce the harmful consequences of infection. Any step during the infec-
tion process where the host shows genetic variation for the degree it is
harmed by the parasite could contribute to the evolution of resistance and
tolerance. As very little work has been done on tolerance in the
DaphniaePasteuria system (but see Vale and Little, 2012; Vale et al.,
2011), we will focus here on resistance. We suggested above that the genetic
architecture for resistance is different across steps, with no current evidence
of physical linkage. Nevertheless, partial resistance early in the infection pro-
cess inuences selection at later steps by modifying the parasite population
composition and by reducing the number of parasites arriving at the later
steps. In extreme cases, if one step evolves to prevent infection entirely,
the following steps will not be exposed to the parasite and their variation
for resistance may become neutral. Therefore, this indirect form of interac-
tion among steps creates epistasis among the genes that act in different steps
(Hall and Ebert, 2013).
Step-specic costs of resistance may modify this picture. Costs manifest
as trade-offs between the tness benets of resistance and the tness loss of
having (constitutive) or using (inducible or deployment) resistance ma-
chinery (Schmid-Hempel, 2011). If resistance costs occur at several steps,
resistance will more likely evolve at the step with the better costebenet
ratio. So far we know little about the resistance costs expressed at different
steps in the DaphniaePasteuria system. The encounter step presents a clear
case of behavioural trade-offs between avoiding sediment-borne parasites
and other tness components such as reducing protection against sh
(Decaestecker et al., 2002) and the opportunity to browse for food
resources directly over the sediment (Ebert, 2005; Horton et al., 1979).
In the attachment step, resistance comes at a cost of lost opportunity,
because possessing a certain resistance allele precludes other alleles, such
that resistance to a particular Pasteuria genotype may be traded-off against
others (Luijckx et al., 2013). There is no evidence that resistance at the
attachment step is resource intensive. In contrast, the within-host steps
(5 and 6) may show resistance costs, as immune defences may be resource

intensive. Indeed, strong environmental effects are observed. However,
studies on costs of resistance (excluding the encounter step) have yielded
mixed results (Allen and Little, 2011; Jansen et al., 2011; Labbe et al.,
2010; Little et al., 2002; Little and Ebert, 2001; Little and Killick, 2007).
In hindsight, uncontrolled variation at the attachment step may have
confounded some of these experiments, resulting in strong intra- and inter-
experiment variation in detecting costs. No studies have yet examined
resistance costs for Pasteuria at the within-host phase after excluding varia-
tion at all earlier steps.
Costs may also be paid as reduced resistance to other parasites, but this
situation seems not to be the case in the Pasteuria system as discussed above
(Auld et al., 2012a; Decaestecker et al., 2003; Ebert, 2008; Mucklow et al.,
2004; Zbinden et al., 2008). Again, however, these experiments are incon-
clusive, as they did not control for variation at the attachment step.
Additionally, while the host genes of the attachment step seem specic to
the interaction with Pasteuria, the within-host steps likely include compo-
nents that are also functionally important in defending against other parasite
species. As a consequence, in a parasite-rich environment, costly immune
functions may be maintained. However, much remains to be done to under-
stand how the Daphnia immune system functions against P. ramosa and other
parasites.
4.4 Expression and evolution of virulence

Pasteuria has severe tness costs for its host: a Daphnia infected as a juvenile
loses 90e100% of its expected lifetime reproductive success, a young adult
between 60% and 90% (Ben-Ami et al., 2008a, 2011; Decaestecker et al.,
2005; Ebert et al., 2000a). Understanding the factors that inuence the evo-
lution and expression of parasite-induced harm in the host (mortality and
morbidity virulence) is a central issue in evolutionary parasitology
(Poulin, 2007; Schmid-Hempel, 2011). Pasteuria has become a model system
for the study of the evolution of virulence, in particular with respect of para-
sitic castration, gigantism and obligate host killing (Ben-Ami et al., 2008a,
2011; Ben-Ami and Routtu, 2013; Cressler et al., 2014; Ebert et al.,
2004; Jensen et al., 2006). We have now a rather good understanding of
the evolutionary process at work, with this system having pushed forward
our insights into parasite-induced host castration and gigantism, a virulence
syndrome known to have evolved several times independently in other
hosteparasite systems (Baudoin, 1975).
Only three steps, the encounter and the two within-host steps,
contribute to the expression of virulence. As Pasteuria virulence is to some
degree dose dependent, the encounter step plays a role for disease severity:
higher exposure dose can lead to faster castration, more pronounced host
gigantism and earlier host death (Ben-Ami and Routtu, 2013; Ebert et al.,
2000b, 2004). However, since we lack theory and predictions for the evo-
lution of virulence under conditions of variable exposure doses, studies on
the evolution of Pasteuria virulence often avoid these effects by controlling
dose (but see Ben-Ami and Routtu, 2013) and thus reduce the contribution
of the encounter step to disease expression. Consequently, the hosteparasite
interactions during the within-host steps become the key players for the
expression and evolution of virulence.
The within-host steps have a strong impact on the life history traits of
both parasite (e.g. spore production, time to host death) and host (e.g.
time to castration, castration relief, gigantism), with these traits all showing
the signature of quantitative traits with genetic and environmental factors
contributing to their variation (Tables 1 and 2). The different components
contributing to virulence are correlated with each other, resulting in the
typical Pasteuria virulence syndrome characterized by host castration, host
gigantism and obligate host killing. Experimental work allowed to disen-
tangle the different disease traits by manipulating the host and parasite ma-
terial used and the experimental conditions (Ben-Ami et al., 2008a, 2011;
Ben-Ami and Routtu, 2013; Coors and De Meester, 2011; Cressler et al.,
2014; Ebert et al., 2004; Ebert and Weisser, 1997; Jensen et al., 2006; Little
et al., 2008), thereby producing a rather clear picture about optimal disease
expression in this system.
Evolutionary theory about parasitic castration (Baudoin, 1975; Ebert
et al., 2004; OKeefe and Antonovics, 2002; Obrebski, 1975) is based on
the assumption of a zero-sum-game where host and parasite are competing
for a xed amount of resources, leading to a negative correlation between
host and parasite resource allocation. The general idea is that castration serves
the parasite by channelling resources away from host reproduction to serve
the needs of the parasite. Since in the early phase of infection the parasite
does not yet have the need for the large amount of resources liberated by
host castration, it was suggested that parasite-induced host gigantism is
benecial for the parasite as it allows to store the excess resources liberated
early during an infection until they can be used by the growing parasite later
during infection (the temporal storage hypothesis, TSH) (Ebert et al., 2004).
Under the TSH, it is expected that hosts will be killed when the parasite
cannot extract more resources from the gigantic host. While earlier models
of castrating parasites predicted instantaneous and complete host castration
(OKeefe and Antonovics, 2002; Obrebski, 1975), the TSH predicts that
castration starts when the parasite reaches a sufcient biomass to exert con-
trol over the host and that the age at castration is largely independent from
the resource level because resources at early stages of the infection process
are not yet limiting (Cressler et al., 2014; Ebert et al., 2004). Finally, taking
coevolution into account, hosts are selected to resist castration as long as
possible or if possible to reverse castration during the late within-host phase.
Work on the expression of virulence in the PasteuriaeDaphnia system is
largely in agreement with the TSH, but some gaps in our knowledge are
apparent. Pasteuria has high resource requirements, as the endospores even-
tually ll the hosts body cavity entirely, reaching a substantial biomass. This
supports the TSHs assumption of a hosteparasite conict over resources.
The nding that environmental factors that reduce resource intake also
reduce both host fecundity and parasite spore counts (Cressler et al., 2014;
Ebert et al., 2004; Schlotz et al., 2013) further supports this assumption.
During the early within-host phase, Pasteuria castrates its host and shortly
later induces enhancement of host growth. Castration is not instantaneous,
but starts only after 7e20 days post infection and depends on the combina-
tion of host and parasite genotype (Ebert et al., 2004). Castration is initially
complete, but in some hosteparasite combinations the hosts may resume
reproduction (castration relief) during the late within-host phase. This trait
is not predicted by parasite-centred models on the evolution of virulence,
but can be explained by taking host evolution into account (Minchella,
1985). In agreement with the TSH, the host is killed by the parasite
when host growth slows down, consistent with the suggestion that host
death occurs when all available resources are used up.
Since models of the evolution of virulence are mainly concerned with
parasites maximizing their tness, symptoms expressed in the host must be
related to parasite tness components. For example, it was predicted
that parasites should kill their host when the transmission potential for
the parasite is maximal (Anderson and May, 1981, Ebert and Weisser,
1997). Indeed, for one sympatric D. magnaeP. ramosa combination it
was shown that parasite spore production peaked at the average time of
host death ( Jensen et al., 2006). Furthermore, fast castration and strong
gigantism were shown to benet the parasite, while both traits have
obvious costs for the host (Ebert et al., 2004), giving support for the
TSH. Cressler et al. (2014) compared the TSH to two alternative models
of resource allocation and the expression of virulence. By manipulating

food levels, they supported the TSH by showing that gigantism, but not
castration, correlates with food level and that the parasite is able to use en-
ergy put into host growth as a resource. Alternative models were not sup-
ported by this experiment (Cressler et al., 2014).
There is also evidence for the host being able to counteract the parasite
by reducing its harm. Hosts that are infected early in life mature earlier and
are thus able to produce more offspring before castration starts (Ebert et al.,
2004). Each clutch a host is able to produce before castration starts has costs
for the parasite in terms of reduced spore production, highlighting the con-
ict over the limited resources (Ebert et al., 2004). The same is true for
castration relief expressed during the late within-host phase, which is bene-
cial for the host, but has high costs in terms of spore production for the
parasite (Clerc et al., 2015; Hall and Ebert, 2012). Infections caused by
different clones of P. ramosa differ strongly in the extent of castration relief
observed (Clerc et al., 2015; Hall and Ebert, 2012). A consequence of this
strong genetic variation is that traits inuenced by it (e.g. host fecundity
and spore production) show increased levels of genetic variation during
the late within-host phase. For example, an assessment of genetic variation
for host size, fecundity and spore production during the infection period
across ve P. ramosa genotypes found no variation expressed during the
early, but strong genetic variation during the late within-host phase (Clerc
et al., 2015).
The within-host phase is clearly the virulence-determining step in
Pasteuria infections. This was also apparent in assessment of sex-specic
virulence. Two particular features of the Daphnia-Pasteuria system allow
for predictions regarding the evolution of sex-specic virulence. First, as
Daphnia populations are strongly female biased (a consequence of mostly
asexual reproduction) the parasite encounters many more female than
male hosts. Second, the strong dependence of Pasteuria on the large amount
of resources liberated by castration makes females the more protable sex.
From this it was predicted, that Pasteuria should adapt primarily to female
hosts (Duneau and Ebert, 2012a; Duneau et al., 2012). Indeed, P. ramosa
reveals sex-specic adaptive virulence (Duneau et al., 2012), with females
being more exploited than males. Since variation at other steps was
excluded, these differences are likely caused by differences during the
within-host phase.
Finally, the evolution of virulence is believed to be strongly inuenced
by the rate at which hosts become infected by multiple host genotypes.
Multiple infections are expected to lead in the long term not only to
an evolutionary increase in virulence, but also to an immediate plastic
upregulation of virulence (Frank, 1996; van Baalen and Sabelis,
1995). Data from the Pasteuria system conrms this prediction, with
multiple infections resulting in earlier host death and higher success of
the more virulent parasite genotypes (Ben-Ami et al., 2008a, 2011;
Ben-Ami and Routtu, 2013; Izhar et al., 2015). Since multiple infections
are likely to increase when the exposure to the parasite increases, the expo-
sure step can indirectly play an important role for the evolution of
virulence.
In summary, the exposure step and the two within-host phases of the
infection process determine the evolution and expression of virulence in
the DaphniaePasteuria system. Models of the evolution of virulence tailored
to castrating parasites agree with the ndings from this system, making this
one of the best understood systems in the eld of virulence research. The
role of host counter defences needs more attention both in empirical
research and in coevolutionary models of virulence.
4.5 Hosteparasite coevolution

Hosteparasite coevolution refers to evolutionary changes in host and
parasite populations that act as agents of natural selection on each other,
causing adaptive changes in both antagonists. Several genetic models for
hosteparasite coevolution have been put forth, the most prominent being
the selective sweep model, and coevolution by negative frequency-
dependent selection (NFDS), also called Red Queen dynamics (Lively,
2010; Woolhouse et al., 2002). During selective sweep coevolution, mu-
tants arise and spread in the population. Selection in this case is directional.
Any benecial mutant, regardless of its genetic background or of the gene it
affects, can spread and may reach xation. In contrast, coevolution by
NFDS operates on a specic genetic architecture based on a few loci in
the host and parasite and highly specic interactions between genotypes
or alleles of the two antagonists (so-called matching allele matrices).
Matching allele interactions can lead to NFDS, such that parasite genotype
frequencies track the frequencies of the host genotypes they are able to
infect (Lively, 2010). In this case, alleles at the host and parasite loci respon-
sible for the specic interaction engage in potentially endless cycles of
frequency changes. Coevolution by selective sweeps and by NFDS can
act simultaneously at different genes in the genome, as long as recombina-
tion exists, which is the case for Daphnia and Pasteuria, although at irregular
intervals (Andras and Ebert, 2013; Lampert, 2011). The DaphniaePasteuria

system is among the few systems with eukaryotic hosts where it is possible
to conduct experiments that explore mechanisms and test hypotheses of
coevolutionary models (reviewed in Ebert, 2008). Furthermore, the possi-
bilities of tracing coevolutionary dynamics over decades by using material
recovered from lake sediment cores make this an even more powerful
system.
Most research on coevolution in the DaphniaePasteuria system focuses
on coevolution by NFDS, where advances on the phenotypic and genetic
level under both controlled and natural conditions have been made. The
key genetic assumption of coevolution by NFDS e the matching allele
model e has so far only been conrmed in the D. magnaeP. ramosa system,
where it is visible in the attachment step (Luijckx et al., 2013) (see above).
The loci responsible for the attachment step are the likely sites for coevolu-
tion by NFDS in this system. As this step shows no sensitivity to environ-
mental variation and explains most variation in resistance, selection is
likely to be rather efcient at these loci.
Hosteparasite genetic interactions, a prerequisite for coevolution by
NFDS, are also found in the early within-host phase (Table 1) (Hall and
Ebert, 2012; Vale and Little, 2009), but the underlying genetic interaction
matrix has not been studied. Since the amount of variation explained by
this step is overall relatively low and its sensitivity to environmental factors
high, it is not a good candidate for NFDS. Using hosteparasite combina-
tions that are fully compatible at the attachment step, will allow us in the
future to explore how the hosts immune response during the early
within-host step coevolves with the parasite. Other steps of the infection
process do not show genetic hosteparasite interactions (Table 2), excluding
them as candidates for NFDS.
A study of sediment cores from a Belgium pond, in which viable P.
ramosa spores and D. magna resting stages were recovered from layers as
old as about 25 years, provided the rst evidence that their evolutionary in-
teractions for infection were indeed highly dynamic over the observation
period of about 25 years (Decaestecker et al., 2013, 2007). These results
are consistent with the idea that differential infectivity, as caused by the
attachment step, evolves by NFDS. The same experiments also suggested
that traits resulting from interactions during the within-host phase, such
as castration and production of parasite spores, are under directional selec-
tion, hinting that genes for these traits may have evolved by directional
selection.
4.6 The evolution of host range

The host range refers to the genetic compatibility of a parasite with a range
of different host species. Perfectly resistant hosts are not part of a parasites
host range. The lter function of the different steps can help to explain the
evolution of host ranges by identifying the step where resistance occurs.
Blocking of the parasite at any single step will exclude the host from the
parasites host range, no matter how permissible the other steps may be
(Antonovics et al., 2013; Combes, 2001; Poulin, 2007). By examining
individual steps of the infection process across a range of potential host
species, one can test which steps contribute to shape the host range of
the parasite.
Field surveys of P. ramosa have reported the parasite in several Daphnia
species as well as in closely related genera, such as Ceriodaphnia, Moina and
Simocephalus (Auld et al., 2012a; Goren and Ben-Ami, 2013; Green, 1974;
Sayre et al., 1977; Stirnadel and Ebert, 1997). As no molecular analyses
had been conducted, it was unclear if Pasteuria had a very broad host range
or if cryptic host races existed, infecting only one or a smaller subset of host
species. Infection experiments have indicated that P. ramosa can cause disease
in host species different from the one it was isolated from, although rarely
(Duneau et al., 2011) (F. Ben-Ami, unpublished data). A study testing
Pasteuria isolates collected from natural D. magna and D. longispina infections
with various clones of D. magna, D. pulex and D. longispina, found that the
inability of Pasteuria to progress after attachment blocks disease progression,
thus marking the penetration and/or the early within-host step as being pri-
marily responsible for determining the host range of Pasteuria (Luijckx et al.,
2014). The parasite causes disease only in the host species it was isolated
from. Therefore, it is unlikely that Pasteuria evolving in one host species
encounters and recombines with Pasteuria in another host species. Indeed
the observed genetic divergence among the D. magna and the D. longispina
derived P. ramosa (Luijckx et al., 2014) suggests that cryptic Pasteuria host
races or even species exist.
5. CONCLUSIONS
Stepwise models of complex biological processes, such as sexual selec-
tion (pre- and postcopulatory selection), speciation (pre- and post-zygotic
isolation), development (different life-history stages), cell division (two-
step meiosis) and migration (migrant production, dispersal, establishment),
can provide a deeper understanding of the evolution of these processes by

linking mechanisms to population processes. The breakdown of the infec-
tious disease process into a series of steps is also not new (Burnet and White,
1972; Cox, 1993), but applying a population perspective to these steps to
gain an evolutionary perspective has only rarely been undertaken (Combes,
2001; Schmid-Hempel and Ebert, 2003). Using this approach allows us to
explore the contributions of natural genetic and environmental factors on
variation at each step of the infection process and clarify the direct and in-
direct interactions that occur in the sequence of steps. Each step can be un-
derstood as a lter through which the parasite must pass. The step-specic
variation determines how the lter acts: Steps that reduce the likelihood
of the parasite passing on to the next step, reduce the number of hosts
who encounter the parasite at the next step, which reduces the strength
of selection for disease traits at the later steps. Interestingly, this reduction
of the intensity of selection does not apply to the parasite, which has to
pass through every step to conclude its life cycle and transmit to the next
host. Besides the direct effect of the lters at each step, each lter inuences
the evolution at later steps by reducing the effective population size and thus
making selection less efcient. Furthermore, ltering during the step-wise
process has also consequences for the evolutionary dynamics of genes inter-
acting across different steps. This is because earlier steps constrain later steps
pleiotropically, by linking the lter function of one step to trait expression at
later steps (Donohue, 2014). This effect has also emerged in models of hoste
parasite coevolution using two-step processes (Agrawal and Lively, 2003;
Fenton et al., 2012).
The example of the DaphniaePasteuria system highlighted in this review
reveals that variation at individual steps is due to a unique combination of
factors (Table 2). Some steps have no variation while others are highly sen-
sitive to host, parasite and environmental factors (Table 2). This knowledge
provides a better mechanistic picture of how hosteparasite interactions
evolve. For example, identifying the variance components in the
DaphniaePasteuria steps has revealed which step is the best candidate for
explaining coevolution between the antagonists, which steps might carry
the greatest cost of resistance, which steps limit the host range of the parasite
and at which steps adaptive evolution is most likely to occur. In human and
livestock systems, the same approach may further suggest which steps are
best for therapy or vaccine development, namely those where the parasite
is least likely to evolve resistance against our measures to control them.
This has been suggested for Helicobacter pylori associated with human gastric
cancer by He et al. (2014), but is also discussed for other pathogens such as
human immunodeciency virus (Ar et al., 2008), Picornaviridae (Koike,
2011), streptococci (Courtney et al., 2002) and several other bacterial path-
ogens (Koike, 2011).
By reducing the complexity of the infection process, we are also able to
test the (often too simple) assumptions of mathematical infectious disease
models, such as resource trade-offs, genetic architecture, and effects of envi-
ronmental factors. Parasite models relating to the evolution of sex, for
example, are often based on a matching allele model (Otto and Nuismer,
2004; Salathe et al., 2008). Close examination of Pasteurias infection process
has shown that the attachment step is indeed based on a matching allele type
model (Duneau et al., 2011; Luijckx et al., 2013), but its signature had pre-
viously been disguised by variation in other steps. Testing assumptions of
evolutionary models is an important step towards closing the gap between
empirical ndings and theory.
The ideas and concepts presented in this review are not specic to the
DaphniaePasteuria system but can be applied to any infectious disease,
although the biology of the steps will differ from system to system, and
the relative contribution of host, parasite and environmental factors will
change. In the future, we may be able to compare stepwise accounts of
the genetic and nongenetic contributions of different diseases and analyse
them for common patterns.
ACKNOWLEDGEMENTS
We thank all members of the infectious disease and symbiosis group at the Zoological Insti-
tute of Basel University for helpful discussions. In particular J
urgen Hottinger, Cesar Metzger,
Laurence Mouton, Jarkko Routtu, Marilou Sison-Mangus, Sebastian Gygli, Gilberto Bento,
Melanie Clerc, Jessica Michel, Kristina Anselm and Roberto Arbore contributed to the ideas
expressed here. We thank Dita Vizoso for the wonderful artwork. Suzanne Zweizig
improved the language of the manuscript. This work was supported with grants from the
Swiss National Science Foundation and the European Research Council.
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CHAPTER SIX
The Increase of Exotic Zoonotic

Helminth Infections: The Impact
of Urbanization, Climate Change
and Globalization
Catherine A. Gordon*, 1, Donald P. McManus*, Malcolm K. Jones*, x,
Darren J. Gray{, Geoffrey N. Gobert*
*Molecular Parasitology Laboratory, Infectious Diseases Division, QIMR Berghofer Medical Research
Institute, Brisbane, QLD, Australia
x
School of Veterinary Science, University of Queensland, Brisbane, QLD, Australia
{
Research School of Population Health, The Australian National University, Canberra, ACT, Australia
1
Corresponding author: E-mail: Catherine.Gordon@qimrberghofer.edu.au
Contents
1. Introduction 312
2. Globalization 314
2.1 Aquatic FBHs 317
2.2 Terrestrial FBHs 320
3. Urbanization 330
3.1 Echinococcosis 332
3.2 Emerging wildlife zoonoses 334
4. Climate Change 343
4.1 Zoonotic lariasis 343
4.1.1 Onchocerca spp. 343
4.1.2 Dirolaria spp. 344
4.1.3 Thelazia spp. 347
4.2 Schistosoma spp. 348
4.3 STHs: Hookworm/Toxocara/Ascaris/Trichuris 351
4.4 Hookworm 352
4.4.1 Toxocariasis 353
4.4.2 Ascaris suum and Trichuris suis 355
5. Points for Discussion 355
5.1 Health education 355
5.2 Targeting denitive hosts and vectors 357
5.3 Molecular tools 357
5.3.1 Environmental monitoring/surveillance 358
5.3.2 Species identication 358
5.3.3 Diagnosis and assessment of control programmes 360

2016 Elsevier Ltd.
j
ISSN 0065-308X
312 Catherine A. Gordon et al.
5.4 Success stories 361

5.4.1 Schistosomiasis and dracunculiasis 361
6. Conclusions 362
Supplementary data 364
References 364
Abstract
Zoonotic parasitic diseases are increasingly impacting human populations due to the
effects of globalization, urbanization and climate change. Here we review the recent
literature on the most important helminth zoonoses, including reports of incidence
and prevalence. We discuss those helminth diseases which are increasing in endemic
areas and consider their geographical spread into new regions within the framework of
globalization, urbanization and climate change to determine the effect these variables
are having on disease incidence, transmission and the associated challenges presented
for public health initiatives, including control and elimination.
1. INTRODUCTION
A zoonosis is a disease whereby a pathogen can be naturally transmitted
from animals to humans. This review covers zoonotic helminths, many of
which appear to be on the rise. Figure 1 shows a number of zoonotic hel-
minths and their main denitive hosts. Denitive hosts are hosts in which
the helminth reaches maturity, while reservoir hosts, another term used in
this review, are long-term denitive hosts for a helminth which can be
important sources of human infection. Helminth infections are generally
responsible for chronic disease and morbidity and are not commonly associ-
ated with high mortality levels in humans. In the 2013 global burden of dis-
ease (GBD) study, it was estimated that neglected tropical diseases (including
schistosomiasis, cysticercosis, echinococcosis, lymphatic lariasis, onchocer-
ciasis, foodborne helminths (FBHs) and intestinal worms) accounted for
1248.4 per 100,000 disability adjusted life years (DALYs), or 1.0% of the
GBD (Murray et al., 2015, 2012). However, many zoonotic helminths
were not specically represented in the GBD study, such as Clonorchis or
Opisthorchis species, which can lead to infection-associated morbidity and
the more serious cholangiocarcinoma.
Climate change is also playing a role in the spread of helminth zoonoses
by changing ranges of animals and habitats of helminth vectors such as
mosquitoes as well as increasing survivability of soil-transmitted helminths
(STHs) by providing the right conditions of warm, moist soil resulting
The Increase of Exotic Zoonotic Helminth Infections
Figure 1 Animal families and their associated zoonotic helminths grouped as either helminths of wildlife, wildlife and companion animals,
313
or wildlife and livestock.
from the expansion of tropical and subtropical zones due to climate change
(Genchi et al., 2011; Montarsi et al., 2015; York et al., 2014). Globalization
increases the risk of FBHs, particularly in sh and meat products, but also
vegetables and fruits, due to the large amounts of exports and imports of
food products occurring globally. Meanwhile, wild animals are increasingly
found in urban environments bringing closer contact between wildlife and
humans, which gives rise to a number of wildlife maintained parasitic infec-
tions in humans and domestic animals. Raccoons and the red fox are impor-
tant examples of wildlife hosts that exist in urban environments (Kellner
et al., 2012; Page et al., 2014; Plumer et al., 2014). Helminths of wildlife
crossing to domestic animals, which live in close contact with humans, is
another mechanism whereby a zoonosis can spread (Magwedere et al.,
2012; Jones et al., 2013; Miller et al., 2013). Many of the helminth species
discussed in this review are affected by more than one of the categories of
urbanization, climate change, or globalization (Figure 2).
Here we review the literature regarding helminth zoonoses with a focus
on recent case reports (reports usually of a single patient) and reports of inci-
dence (rate of new infections occurring in a population) and prevalence
(number of cases occurring in a population at a single time point). We will
focus on the prominent literature from the last ve years (2010ecurrent)
and discuss important transmission factors, the impact of climate change,
urbanization and globalization, and prevention and control strategies to
minimize the risk of zoonotic helminth infections.
2. GLOBALIZATION
The ease and availability of air travel, and the subsequent movement
of a large number of people around the world as tourists or immigrants, will
lead to more cases of zoonotic helminthiases occurring in nonendemic
countries (Shaw et al., 2003). For some of these species, such as the larial
worms, the presence of the appropriate intermediate hosts may lead to
exotic species becoming established in new countries, while changes in
climate to warmer temperatures, ideal for STHs, can lead to their establish-
ment in nonendemic areas. Infections occurring in tourists returning from
developing countries may not be diagnosed readily due to lack of expertise
by medical staff in countries where these helminths are not endemic and are
therefore rarely seen. Immigration and, in particular, refugee intakes are also
potential sources of new infections. Most developing countries perform
health checks of refugees which will identify and treat any infectious diseases
Figure 2 Spread of zoonotic helminths grouped by globalization, climate change and urbanization, showing the different inuences which
315
lead to increased distribution or cases of zoonotic helminths.
found (Redditt et al., 2015; Martin and Mak, 2006; Seybolt et al., 2006;
Varkey et al., 2007). For this reason any of the zoonotic helminths can be
affected by globalization, and imported cases can be identied worldwide
(Figure 2). Under the appropriate conditions some of these parasites could
become established in new areas.
Globalization is an important aspect in the foodborne transmission of
parasites. Fish and other animals produced in areas endemic for FBH are
shipped globally, as are fruit and vegetables which may contain helminths.
Depending on the food regulations in the country these products are im-
ported to, infected sh, meat or fruits and vegetables may make it to the
consumer, leading to infection in nonendemic areas. Imported live animals
can also harbour parasites either from their originating country or having
been picked up en route. In Israel, bovine cysticercosis (Cysticercus bovis)
was found in live cattle imported from Australia which may have helped
to establish this tapeworm infection in cattle herds in that country (Meiry
et al., 2013). Australia is a country with a low incidence of the zoonotic
tapeworm, Taenia saginata, although an outbreak in cattle occurred there
in a feedlot, traced back to commercially available feed, copra meal, im-
ported from Papua New Guinea (Jenkins et al., 2013).
An estimated 60 million tons of sh are shipped globally each year, pri-
marily from South East Asia (SEA), an area endemic for shborne FBH.
Introduction of rats and exotic species, such as the giant African snail, Acha-
tina fulica, by ships and in shipping containers with their successful coloniza-
tion in new countries is a concern in the spread of angiostrongyliasis, as well
as providing suitable hosts for a number of zoonotic lariasis species and
predator-prey helminths. Rats are often found in peridomestic and domestic
areas in urban and rural areas where they come into close contact with
humans and can carry a number of zoonotic helminths. A. fulica is an
extremely good colonizer and is a host for Angiostrongylus cantonensis. While
there are other molluscan hosts of A. cantonensis, the giant African snail has
spread very quickly and may help transmit the disease globally (Stockdale-
Walden et al., 2015; Thiengo et al., 2010). In Brazil the giant African snail
was thought to have been introduced in the 1980s and, as of 2013, all bar
one of the 25 Brazilian states have reported the presence of A. fulica. Angios-
trongylus cantonensis itself may have been imported to Brazil along with these
snails, or much earlier from parasitized rats which would have been intro-
duced on ships (Thiengo et al., 2013, 2007).
There have been reports of a number of exotic zoonotic helminths pre-
sent in nonendemic countries in zoo animals, or in illegally imported animals
The Increase of Exotic Zoonotic Helminth Infections 317
coming from endemics areas (Davidson et al., 2013; Widmer and Jurczynski,
2012; Luzon et al., 2010). While most zoo animals are kept separately from
wild animals in the new country, animals such as rats and birds may interact
with these animals. In the case of larial disease, the spread may occur more
quickly if the requisite mosquito host is present. In the USA alone over
37 million amphibians, birds, mammals and reptiles were illegally imported
in the period from 2002 to 2004 (Wildlife, 2007). In the same period the
number of legally imported animals was over 1 billion, including sh, and
a number of unidentied species (Wildlife, 2007).
FBH infections are a common source of zoonotic infection in humans
with 40e50 million people thought to be infected worldwide (Chai
et al., 2009), particularly in populations that consume raw meat. FBHs occur
mainly after eating raw or undercooked infected meat but can also be found
contaminating raw vegetables or fruit. Fish and other marine animals
account for a large proportion of FBH, with 59 species of FBH from sh
known (Hung et al., 2013). Here we focus on the most medically important
FBH species, for both common infections and those that are emerging. A
comprehensive review of shborne zoonoses was published in 2014 by
Waikagul and Thaenkham.
FBHs, more than any other group of helminths, have the most impact in
terms of potential geographic spread of cases and are particularly affected by
globalization e the movement of food, including live exports/imports, and
the movement of people around the world (Figure 2). The movement of
people for travel and immigration is also a major factor in globalization
and can result in exotic infections being identied in nonendemic areas
and the risk of new species becoming endemic. This aspect of globalization
can impact the spread of all helminth diseases (Figure 2).
2.1 Aquatic FBHs

Globalization has led to an increased demand for sh and other marine
foods worldwide, which requires suppliers to increase production, naturally
leading to an increase in farmed sh and other aquaculture. Increased levels
of intensive aquaculture give rise to increased infections of shborne FBH
(Nguyen et al., 2012a; DPI, 2015; Phan et al., 2010b). Freshwater sh
hatcheries and pens in streams can easily be contaminated by human and
animal excrement run-off (Phan et al., 2010a). This is of particular impor-
tance as the majority of zoonotic sh helminths have dogs, cats and birds as
reservoir hosts, complicating control of these parasites. In Vietnam, the
prevalence of FBH (Clonorchis sinensis, Haplorchis pumilio, Haplorchis taichui,
Centrocestus formosanus) in articial hatcheries was found to be more than

50% in overwintering ponds (Phan et al., 2010b).
Fish farms are a considerable source of infection for aquatic helminths.
Many animal hosts, including dogs and poultry, as well as infected humans,
that live near these farms contribute to disease transmission. Contamination
of sh ponds can occur when eggs, defecated by a denitive host, are
washed into them, such as by rain (Anh et al., 2010; Nissen et al., 2014).
Snail hosts in ponds are also a risk factor for transmission within sh farms
(Hedegaard et al., 2012). Interventions to prevent infection include snail
control through direct removal of snails and vegetation where snails live,
concreting ponds to prevent vegetation growth, and placing lters on water
pipes to prevent snail movement between ponds and entry to ponds via
pipes (Hedegaard et al., 2012). Snail control measures can also be imple-
mented as surrogate control measures for other helminths species that use
snails as an intermediate host. Wild and domestic animals can also act as
the denitive host for many shborne FBH, contributing to the ongoing
life cycle. Felids, canids, pigs, birds and rodents can act as denitive hosts
for C. sinensis (Ye et al., 2013; Lin et al., 2011). In Vietnam, poultry has
also been found to be an important reservoir host of zoonotic aquatic trem-
atodes in sh farms (Anh et al., 2010) such as C. sinensis (Ye et al., 2013; Lin
et al., 2011; Anh et al., 2010).
Other control methods relevant for all zoonotic helminths include
chemotherapy of humans and animal hosts, such as dogs, in transmission
areas (Erfe et al., 2013; Inobaya et al., 2015; Ross et al., 2014). Intervention
using chemotherapy on dogs living in sh farms has been unsuccessful for
control due to high reinfection rates (Anh et al., 2010; Nissen et al.,
2014). In sh aquaculture, fences to keep animals out and walls around
ponds to minimalize surface run-off, which may be contaminated with
infected faeces, are also used in efforts to attempt reduction in transmission
(Hedegaard et al., 2012; Pitaksakulrat et al., 2013; Phan et al., 2010a; Tesana
et al., 2014). Similar issues are also relevant for wild sh in open waterways
that may be contaminated by human and animal excrement.
Climate change also plays a role in shborne FBH. A study on the effects
of temperature, rainfall and humidity on human cases of clonorchiasis,
caused by infection with C. sinensis in China, found a positive association
between cases and increasing temperature and rainfall, while humidity had
an inverse relationship (Li et al., 2014b). With increasing global temperatures
due to global warming this may result in an increase in shborne helminths,
particularly C. sinensis (Figure 2).
Trematode families containing zoonotic species that are transmitted to

the denitive host in sh are Clinostomatidae, Echinostomatidae, Hetero-
phyidae, Opisthorchiidae and Troglotrematidae (Waikagul and Thaenkham,
2014). These families display diverse life cycle strategies; however, all species
are similar in that they require at least two hosts e a denitive and an inter-
mediate molluscan host (Doughty, 1996). Of these, the Opisthorchiidae are
important in human health due to liver disease and the association of Opis-
thorchis and Clonorchis spp. with the development of cancer.
An estimated 45 million people are infected with trematode liver ukes;
of these 35 million are infected with C. sinensis, 10 million with Opisthorchis
viverrini, and 1.6 million with Opisthorchis felineus (Keiser and Utzinger, 2009;
Hung et al., 2015). These parasites are most commonly found in Asia, partic-
ularly in developing countries, with the exception of O. felineus which is
found in humans and animals in Europe. The emergence of clinical O. felineus
in Europe can be linked both to changes in human diet, due to the popular-
ization of consuming raw sh products, and those sh products smoked or
marinated since neither process kills metacercariae contained in the muscle
of infected sh, and to cultural practices of eating raw sh that already exist
in some European countries, such as Iceland (Pozio et al., 2013).
Clonorchis sinensis and O. viverrini are also known to cause cholangiocarci-
noma in humans, while O. felineus has been implicated as a potential cause of
cancer, although this needs to be further investigated (Correia da Costa et al.,
2014; de Martel et al., 2012; Watanapa and Watanapa, 2002; Ogorodova
et al., 2015). The only other helminth currently linked to the development
of cancer in humans is Schistosoma haematobium, the cause of urogenital schis-
tosomiasis in Africa, although the underlying mechanism involved is yet to be
determined (Honeycutt et al., 2014; Thomas et al., 1990). There is some
discussion as to whether Schistosoma japonicum causes an increased risk of
developing colorectal adenocarcinoma (Peterson and Weidner, 2011). The
number of cancers caused by O. viverrini and C. sinensis infection in 2008
was estimated to be 2000, while cancer caused by S. haematobium was esti-
mated to be 6000 (de Martel et al., 2012). These helminths cause cancer
by indirect release of carcinogens or direct physical irritation, both of which
lead to chronic inammation (Sripa et al., 2012). Chronic inammation
caused by helminth or helminth depositions, for example, eggs in the case
of S. haematobium, is a key feature in helminth-induced cancers (Vennervald
and Polman, 2009). Due to the location of the adult worms and eggs,
C. sinensis and O. viverrini cause hepatic and biliary cancers while S. haema-
tobium causes bladder cancer. Cancer in C. sinensis and O. viverrini occurs
due to secretions from adult ukes into the bile, while cancer in S. haema-
tobium results from the inammatory response to eggs lodged in the bladder
wall and the induction of chronic inammation (Oh and Weiderpass, 2014).
Diphyllobothrium spp. are common cestode infections with up to
20 million people estimated to be infected worldwide with Diphyllobothrium
spp. (Scholz et al., 2009). There are 14 species of Diphyllobothrium spp. which
cause disease in humans; of these Diphyllobothrium latum and Diphyllobothrium
nihonkaiense are the most common (Table 1).
Anisakis simplex is a common nematode helminth. There are 12,000
conrmed cases of anisakiasis, although more are likely to be infected due
to under-reporting (Murrell, 2014). Allergic reactions are common with
Anisakis spp. infection (Audicana and Kennedy, 2008). People travelling
to endemic countries are also at risk due to the cultural practice of eating
raw sh products in many Southeast Asia (SEA) countries and some Euro-
pean countries, such as Iceland and the Netherlands.
From 2000 to 2006, world sh imports rose 49%, with developed coun-
tries accounting for the majority when considering the total cost (FAO,
2008b). China, Vietnam and Thailand are among the top exporters of
sh; these countries are also endemic for a number of zoonotic shborne
helminths (FAO, 2008a). In the USA in 2011 an estimated $1.3 to $2.1
billion worth of illegal sh was imported (Pramod et al., 2014).
Fishborne infections are likely to become even more prevalent due to
the global increases in food imports and exports. Cases can occur in nonen-
demic countries if the parasites have not been adequately killed (Esteban
et al., 2014; Santos and Faro, 2005; Pastor-Valle et al., 2014). This is of
particular concern given that for some shborne and terrestrial helminths
(see below), freezing for 24 h is insufcient to kill the infective stages (Lacour
et al., 2013; Pozio et al., 2013). Various sources quote freezing at different
temperatures and for different lengths of times to inactivate and kill parasites.
The most stringent recommendations recommend freezing at 20 C for
7 days, particularly for products that are eaten raw or cooked in a manner
that would not inactivate parasites, such as the smoking of food (Audicana
and Kennedy, 2008; FAO, 2001, 2008b). In terms of inactivating O. felineus
metacercariae, smoking and marinating does not kill the parasites in the
deeper tissues of the sh host (Pozio et al., 2013).
2.2 Terrestrial FBHs

There are a number of terrestrial FBHs which can be further classied
based on the nature of the food type with which they are present in.
Table 1 Human cases of Diphyllobothrium species published since 2010
Species Country Case no. Diagnosis References
Diphyllobothrium nihonkaiense USA, Korea, 24 Morphology, Sequencing Chen et al. (2014), Fang et al. (2015),
Japan, China cox1, nad3 Go et al. (2015), Ikeda et al.
(2012), Kim et al. (2014),
Nakamura et al. (2010), Ohta et al.
(2011), Ono et al. (2010), Park
et al. (2013, 2015), Shimizu et al.
(2012), Shin et al. (2014) and Soga
et al. (2011, 2014)
Diphyllobothrium pacicum Spain 3 Morphology, PCR Pastor-Valle et al. (2014)
Diphyllobothrium latum China, Spain, 15 Morphology, Sequencing cox1 Choi et al. (2012), Esteban et al.
Korea USA, (2014), Li et al. (2013b),
Nigeria Ojurongbe et al. (2011) and
Hariadi et al. (2011)
Diphyllobothrium dendriticum Switzerland 1 PCR (cox1, and 5.8S-ITS1- de Marval et al. (2013)
ITS2-18S)
Diphyllobothrium balaenopterae/ Spain 1 Morphology, PCR Pastor-Valle et al. (2014)
grandis
Diphyllobothrium species Argentina, India 2 Egg morphology Cargnelutti and Salomon (2012) and
Ramana et al. (2011)
321
Thus, some helminths are transmitted to humans with meat, while others are
transmitted on vegetables or fruits. In both cases human infection occurs by
eating infected or contaminated meat or plant material that has been
improperly cooked or washed.
Consumption of raw, unwashed vegetables is a common source of infec-
tion for FBH and is the main source of infection with Fasciola spp., Fasciolop-
sis buski and for the rarer Dicrocoelium spp., as well as a possible route of
infection with Angiostrongylus sp. (Blair et al., 2013; Yeung et al., 2013).
In developed countries, access to clean running water allows for easy
washing of vegetables and fruits; but in developing countries, particularly
in rural areas, clean water for washing food is scarce, and the consumption
of plants containing infected ants (D. dendriticum) or metacercariae (Fasciola
hepatica, Fasciola gigantica, and F. buski) is a particular concern.
Ingestion of raw water plants such as Zizania latifolia (wild rice), water-
cress, scallion or Latifolia aquatic, harbouring metacercariae is a common cause
of infection for Fasciola spp. and F. buski (Mailles et al., 2006; Croese et al.,
1982; Kumari et al., 2006; Adamu et al., 2012). Hookworm and F. buski
were identied from vegetables in Ghana, while a fatal case of fasciolopsiasis
in India was traced to consumption of raw caltrops and water chestnuts
(Duedu et al., 2014; Kumari et al., 2006; Adamu et al., 2012). Fasciolopsis
buski is the largest intestinal uke of humans and is the only recognized spe-
cies in the genus Fasciolopsis. Human infections of F. buski in SEA have
decreased from 10 million in 1984 to 1.3 million in 2009, but the parasite
may be returning to areas where it has been previously controlled (Beaver
et al., 1984; Keiser and Utzinger, 2009; Bhatti et al., 2000).
Adult Fasciola worms live in the liver of the denitive host, with the
ukes often being found in bovines but also in other animals such as
nonhuman primates (Legesse and Erko, 2004; Gray et al., 2008b). Pigs
and cattle are hosts for F. buski, and the rearing of either host is considered
a risk factor for infection (Muralidhar et al., 2000).
Fascioliasis in humans is caused by F. gigantica and F. hepatica, with the
former more common in tropical areas and the latter more prevalent in
temperate zones (Gray et al., 2008b; Chaudhry et al., 2015; Ashra et al.,
2015; Chen et al., 2013; Gu et al., 2012). Adult Fasciola ukes live in the
liver of the denitive host, with species often found in bovines, but can
also occur in other animals such as nonhuman primates (Legesse and
Erko, 2004; Gray et al., 2008b). Infection with Fasciola spp. can result in
neurofascioliasis and ophthalmofascioliasis (Mas-Coma et al., 2014), and a
review by Mas-Coma et al. (2014) provides a comprehensive list of all
published reports (80 in number) prior to 2014 of fascioliasis resulting in

minor or major neurological and ocular manifestations in Europe.
Dicrocoelium dendriticum is a rare parasite of humans that can be acquired
by ingesting raw fruits and vegetables, as well as through drinking contam-
inated water (Schweiger and Kuhn, 2008). Infections among children in
Kyrgyzstan were found to be as high as 8%, while other recent cases of
human dicrocoeliasis have also been found in Turkey, Iran, Egypt, Ghana,
USA, Italy and Spain (Jeandron et al., 2011; Cengiz et al., 2010; Ashra,
2010; El-Shae et al., 2011; Steinmann et al., 2010). Dicrocoelium dendriticum
has been identied in ruminants (including bovines, equines, cervines and
ovines), cats, dogs and nonhuman primates from countries in Europe, Africa,
the Middle East and North America (Arias et al., 2011; Bian et al., 2013;
Bolukbas et al., 2012; Borji et al., 2012; Dadak et al., 2013; Katsoulos
et al., 2011; Mahmoodi et al., 2010; Ofori et al., 2015; Gualdieri et al.,
2011; Khalil et al., 2013; El-Shae et al., 2011; Cabeza-Barrera et al.,
2011; Sammet et al., 2013). Human infections are often asymptomatic
and are thus frequently undiagnosed (Schweiger and Kuhn, 2008; Haridy
and Morsy, 2003).
Pseudo-infections, where adults are present without eggs occurring, of
dicrocoeliasis in humans are more common than true infections result
from the consumption of raw sheep liver infected with adult ukes of
D. dendriticum (Cabeza-Barrera et al., 2011; Haridy and Morsy, 2003).
Only 7 of 208 cases of dicrocoeliasis in Saudi Arabia were thought to be
true infections (El-Shiekh Mohamed and Mummery, 1990). In Lebanon,
Lebanese Halzoun syndrome (LHS) is an allergic reaction in the upper res-
piratory tract which can occur after the consumption of raw sheep or bovine
liver (a traditional Lebanese dish) (Khalil et al., 2013). Of 32 patients present-
ing with LHS, parasites were recovered from 3 and morphologically identi-
ed as D. dendriticum, indicating that this helminth may be the cause of LHS
(Khalil et al., 2013).
Trichinella spiralis and Taenia spp. also cause disease in humans due to con-
sumption of infected or contaminated and improperly cooked meat. In the
case of T. spiralis there have been 337 clinical cases, including an outbreak in
Argentina, infections identied during community surveys in the Peoples
Democratic Republic of Lao (Lao PDR), and two cases in the USA, since
2010 (Conlan et al., 2014; Calcagno et al., 2014; Holzbauer et al., 2014).
All cases were traced to eating raw or undercooked pork. The two cases
from the USA were a father and son who had hunted and eaten the inade-
quately cooked wild boar (Holzbauer et al., 2014). The outbreak in
Argentina was from undercooked commercially available pork (Calcagno

et al., 2014), while in Lao PDR there have been a number of outbreaks
over the years due to consumption of raw pork, a local delicacy (Conlan
et al., 2014).
There are three Taenia spp. which mainly cause zoonotic taeniasis or cysti-
cercosis, namely Taenia solium, Taenia saginata and Taenia asiatica. Known as
the beef tapeworm, T. saginata is found in bovines, while T. solium, the
pork tapeworm, and T. asiatica infect pigs (Ale et al., 2014). Despite having
different denitive hosts, T. asiatica is very similar genetically to T. saginata,
much closer than it is to T. solium with whom it shares a common host
(Bowles and McManus, 1994; Jeon et al., 2007; Gordon et al., 2015). To
date, there have been no reports of T. asiatica infection occurring outside
Asia (Figure 3, Suppl. Table 1).
Taenia solium causes cysticercosis in humans, a disease in which the para-
site eggs ingested singly or in gravid proglottids develop into cysticerci in
body tissues, similar to the process in a porcine host. In contrast, T. saginata
and T. asiatica cysticerci do not develop in humans and only cause intestinal
taeniasis (adult worms in the gastrointestinal system (GIT)). Ingestion of raw
or undercooked meat containing cysticerci of Taenia spp. results in adult
worms in the intestine. Poor hygiene is a key part of transmitting cysticer-
cosis, with individuals harbouring adult T. solium worms able to autoinfect
themselves by ingesting expelled eggs, or infecting others by contaminating
the environment and food products. Neurocysticercosis (NCC) occurs
when cysticerci of T. solium form in the brain of the host, causing severe
neurological complications including behavioural changes (psychosis and
depression) and death in some individuals (Almeida and Gurjao, 2010; de
Almeida and Gurjao, 2011; Verma and Kumar, 2013; Sarangi et al., 2013)
(Table 2). Largely, NCC has been reported in developing countries, partic-
ularly in India, with cases in Europe and the USA generally attributed to im-
migrants and returning travellers (Bouteille, 2014) (Figure 2). A study in
Brazil showed that 84% of patients with NCC displayed depression as a
comorbid factor (de Almeida and Gurjao, 2011). However, cysticerci can
develop in any tissue and can cause a range of symptoms (Figure 3, Table 2).
Since 2010 there have been 81 published case reports of NCC (n 81)
(Figure 3, Table 2). Seizures are the most common symptom of NCC,
together with headache and numbness (Table 2).
Taenia asiatica has never been reported as causing cysticercosis in humans
(Galan-Puchades and Fuentes, 2013b), and this is supported by the genetic
similarity of T. asiatica with T. saginata, which likewise does not cause human
Figure 3 World map showing the geographic locations of human and animal infections with Taenia asiatica, Taenia solium, and Taenia sag-
inata based on the 2010e2015 published literature. Pie graphs show the relative percentage of each species infecting humans and animals
325
based on the number of cases identied (Suppl. Table 1) from the 2010e2015 published reports.
Table 2 Human cases of cysticercosis published since 2010
326
No. Country (acquired
cases Cyst location (n) infection) Symptoms (n) References
1 Brain (1) Australia (India) Seizures (1) Britton and Chaseling (2013)
2 Brain (1), tongue (1) Brazil Racemose NCC (1), Bruns Rodrigues et al. (2012) and Alves
Disease (1), swelling (1) et al. (2011)
4 Brain (2), Spine (1), China NCC (2), Progressive weakness Yamasaki (2013), Qi et al. (2011),
SCC (1) (1), anal sphinchter and bladder Shih et al. (2010) and Song et al.
dysfunction (1), numbness (1), (2013)
headache (1), hearing
impairment (1)
1 Brain (1) Denmark (Zambia) NCC (1) Cortnum et al. (2011)
1 Leg Dubai (Nepal) Asymptomatic Hussain et al. (2013)
2 Brain (2) Ecuador Seizures (8) Del Brutto (2012, 2013a)
1 Spine (1) Egypt NCC Ahmed and Paul (2014)
1 Brain (1) France (prior travel to Asthenia (1), headache (1) LOllivier et al. (2012)
endemic areas)
1 Neck (1) Germany (India) Nodule (1) Salzer et al. (2013)
1 Spinal cord (1) Guatemala Brown-Squard syndrome (1) Noguera et al. (2015)
187 Brain (132), tongue (3), India Swelling (42), seizures (28), Singhal et al. (2013a, 2014),
facial muscles/mouth sensory loss (2), bowel and Thambiah et al. (2012), Kumar
(19), DCC (6), spine bladder dysfunction (1), fever et al. (2011), Deshmukh et al.
Catherine A. Gordon et al.

(3), SCC (3), trunk (4), drooping eye lid (2), (2011), Sacchidanand et al. (2012),
(5), eye/eye lid (9), double vision (2), behavioural Kumar et al. (2010), Yamasaki
neck (6), liver (1), abnormalities (4), memory loss (2013), Rastogi et al. (2013),
limbs (2), breast (1) (1), headache (7), back ache (1), Khare et al. (2014), Azfar et al.
numbness/weakness (4), ptosis (2011), Sinha et al. (2013), Raoot
(3), pain (4), hepatomegaly (1), (2014), Chaurasia et al. (2013),
neck stiffness (1), vomiting/ Khurana et al. (2012), Tewari
nausea (2), motor monoparesis et al. (2014), Verma and Kumar

(1), free oating scolex in eye (2013), Agrawal et al. (2013), Joshi
(2), eye drooping (1), loss of et al. (2014), Singh et al.
vision (1), periorbital cellulitis (2011a,b), Jain et al. (2012),
(1), dry cough (1) Bothale et al. (2012), Malhotra
et al. (2011), Muthyala et al.
(2015), Dysanoor and Pol (2013),
Kumar et al. (2012), Akhtar and
Agarwal, (2012), Chakravarti et al.
(2014), Patil et al. (2010),
Sathyanarayanan et al. (2011),
Mishra et al. (2014), Takkar et al.
(2014), Sarangi et al. (2013),
Chatterjee et al. (2013), Verma
and Jaiswal (2013), Giri et al.
(2013), Wanjari et al. (2013),
Singh et al. (2013b), Gokarn et al.
(2011), Ramakrishnan et al.
(2012), Chandran et al. (2012),
Ranjith et al. (2011), Netravathi
et al. (2011), Goyal et al. (2015),
Yadav et al. (2013), Joshi et al.
(2012), Bhat et al. (2015), Qazi
et al. (2014), Chaudhary (2014),
Malik et al. (2014), Jain et al.
(2014), Gupta et al. (2014), Kumar
and Subrahmanyam (2014),
Puttaraju and Hanasoge Srivathsa
327
(2013), Kumar et al. (2013),
(Continued)
Table 2 Human cases of cysticercosis published since 2010dcont'd
No. Country (acquired
328
cases Cyst location (n) infection) Symptoms (n) References
Furtado et al. (2013), T M K et al.
(2012), Suchitha et al. (2012),
Gupta et al. (2012), Prasad et al.
(2011), Karnik et al. (2011),
Ramraje et al. (2011), Kohli et al.
(2010), Madhuri et al. (2013) and
Sahu et al. (2014)
1 Eye Indonesia (Bali) Redness (1), pain (1), increased Swastika et al. (2012)
eye secretions (1)
7 Brain (7) Italy (Brazil, Burkina Loss of consciousness (1), fever Giordani et al. (2014), Raffaldi et al.
Faso, Cameroon, (2), pneumonia (1), seizure (4), (2011), Vecchio et al. (2014),
China, Ecuador, headache (1), confusion (1), Giacomet et al. (2013) and
Senegal) vomiting (1), back pain (1) Iacoangeli et al. (2013)
5 SCC (1), DCC (1), brain Japan Racemorse NCC (1), seizure (1), Yamasaki (2013), Yokota and
(3), limb (1) swelling (1) Furukawa (2012), Kobayashi et al.
(2013) and Maeda et al. (2011)
2 Spine (1) Korea Pain (1), back pain (1) Yoo et al. (2014) and Choi et al.
(2010)
30 Brain (30) Kuwait NCC (30) Del Brutto (2013b)

1 GIT (1) Myanmar Abdominal pain (1), back pain Khin et al. (2013)
(1), constipation (1)
30 Brain (6), limbs (1), Nepal Seizure (1), loss of consciousness Shoji et al. (2013), Yamasaki (2013),
tongue (2), SCC (3), (1), altered consciousness (1), Pant et al. (2011), Mesraoua et al.
breast (1), eye (6), disorientation (1), headache (2012), Chaudhary et al. (2015),
muscles (11) (3), vomiting (1), vision loss (1), Sharma et al. (2014), Labh and
swelling (18), ptosis (1) Sharma (2013) and Agrawal
(2012)
3 Brain (3) Netherlands Motor developmental delay (1), van de Pol et al. (2015)
(Cambodia, India, headache (1), vomiting/nausea
Nepal) (1), seizure (1), muscle
dysfunction (1)
8 Brain (8) Qatar Numbness (1), seizure (4) Khan et al. (2011) and Del Brutto
(2013b)
6 Eye (1), brain (6) Saudi Arabia Swelling (1), redness in eye (1), Damani et al. (2012) and Del Brutto
seizure (1), NCC (5) (2013b)
2 Brain (2), Spine (1) South Korea Headache (1), dizziness (1), Ataxia Kim et al. (2010a), Song et al. (2010)
(2), Racemose (1), Claudes and Park et al. (2011)
syndrome (1), back pain (1)
3 Brain (3) Spain (Ecuador, Seizure (3), headache (1), Tejado Lde et al. (2012) and Frieiro-
immigrants) dizziness (1) Dantas et al. (2013)
2 Brain (1), eye (1) UK (Nepal) Seizures (1), limb weakness (1), Azzopardi and Quirk, (2012) and
ptosis (1), periorbital pain (1), Ziaei et al. (2011)
diplopia (1)
14 Brain (14) USA (Burma, Headache (11), fever (1), nuchal Gardner et al. (2012), Hanak et al.
Guatemala, Haiti, rigidity (1), seizures (6), coma (2011), Weber et al. (2012),
Honduras, Mexico, (1), confusion (1), dizziness (1), ONeal et al. (2012), Rapoport
Myanmar, limb weakness (3), vomiting/ et al. (2014), Nash et al. (2014),
Philippines, nausea (3), dyesthesias (1), Naddaf et al. (2014), Purvey et al.
Nicaragua) difculty speaking (1), (2015), Shahani et al. (2015) and
difculty hearing (1), facial Mejia and Nash (2013)
twitching (1)
1 DCC (1), brain (1) Vietnam Seizures (1), headache (1), Nguyen and Thanh (2013)
swelling (1)
329
cysticercosis (Jeon et al., 2009, 2007). Molecular diagnosis of cysticerci in

humans, rather than immunodiagnosis, which does not distinguish between
T. solium and T. asiatica, is a method whereby species identity can be
conrmed. However, the morphologies of the scoleces of T. solium and
T. asiatica are sufciently distinct that species diagnosis can be made
(Galan-Puchades and Fuentes, 2013a). In contrast, T. asiatica and T. saginata
are morphologically quite similar and cases of taeniasis may thus have been
misdiagnosed (Galan-Puchades and Fuentes, 2013b; Parija and Ponnambath,
2013). Differentiating between those two species is important in an epidemi-
ological context to determine whether T. asiatica has spread beyond Asia.
Other zoonotic Taenia spp. include Taenia multiceps, Taenia serialis and
Taenia brauni, which cause coenurosis, while Taenia crassiceps, Taenia ovis,
Taenia taeniaeformis and Taenia hydatigena cause taeniasis (http://www.cdc.
gov/dpdx/coenurosis/index.html) (Webman and Gilman, 2013). These
are rare infections, however, with only small numbers of T. multiceps
(n 3) and T. crassiceps (n 4) infections reported in humans since 2010
(Ambekar et al., 2013; Mahadevan et al., 2011; Goesseringer et al., 2011;
Flammer Anikpeh et al., 2014; Ntoukas et al., 2013; Roesel et al., 2014).
These tapeworms can be found in a variety of animals including domestic
and wild canids and felids as well as ruminant animals worldwide (Image 1);
humans act only as intermediate hosts for these Taenia spp.
Taenia crassiceps may in future be an emerging zoonotic helminth, with its
emergence likely linked to immune deciency, such as that associated with
HIV AIDS, as it is more common in individuals who are HIV-positive
(Goesseringer et al., 2011; Giordani et al., 2014; Tian et al., 2012; Flammer
Anikpeh et al., 2014).
3. URBANIZATION
Cats and dogs are responsible for transmission of a number of zoonotic
helminths with Echinococcus, hookworm and Toxocara among the most well-
known. These animals have considerable contact with humans, being pop-
ular pets worldwide. In the USA, 36.5% of households own a dog and
30.4% a cat, while in the European Union, 26% of households own a cat
and 18% own a dog (FEDIAF, 2014; AVMA, 2012). In addition to the pres-
ence of these companion animals, stray cats, wild dogs and other canids, such
as foxes, are often found around human dwelling sites having adapted well to
urban and semiurban environments. There are an estimated 70 million stray
cats in the USA alone (ASPCA, 2015). Rodents are another group of ani-
mals that have adapted well to urban environments. The black rat, Rattus
rattus, has an almost global distribution due to human movement around
the world, and it may be responsible for the introduction of new species
of zoonotic helminth to previously nonendemic areas (Figure 2). Helminth
infections in wild animals, which may normally have limited human con-
tact, can be introduced to domestic animals which do have contact with
humans (Figure 4). This leads to the increased potential of human helminth
infections which were once generally considered diseases of wildlife only
(Figures 1, 2 and 4). Hunting and consumption of wild animals, such as
boar, are another mode of FBH infection in humans (see section on
Terrestial FBH) (Figure 4).
Environmental modication is the main driver of wildlife spillover into
human environments and includes deforestation, city development, min-
ing, and dams. Deforestation is a major factor in the spillover of wildlife
zoonoses. In the last 10 years, 13 million hectares of forests were cleared
per year globally, compared with 16 million hectares per year in the
1990s (FAO, 2010) (Figure 4). While deforestation has slowed, it still occurs
at a high rate, reducing the natural habitat available for native animals.
Much of this land is converted into farmland or city development. By
reducing the natural habitats, native animals are forced into closer contact
Figure 4 Venn diagram showing transmission pathways of zoonotic helminths

between wild animals, domestic animals and humans.
with domestic animals and humans, which has led to disease spillover from
wildlife to humans and domestic animals, but also from domestic animals to
wildlife. In the case of echinococcosis there are both sylvatic and domestic
life cycles whereby the tapeworm infection can be transmitted between the
two groups of animals. There are species of Echinococcus that are primarily
parasites of wildlife but which can also be found in domesticated animals
(Addy et al., 2012; de la Rue et al., 2011). Foxes, which frequent rural
and semiurban areas, are important denitive hosts of Echinococcus granulosus
and E. multilocularis.
Figure 1 shows different host groups, the parasite species covered in this
review that they can act as denitive or intermediate hosts for, and indicates
whether these groups involve wildlife, livestock or companion animals. The
majority of these parasite species overlap these three categories being either
wildlife/livestock or wildlife/domestic, with the remainder maintained in
wild animal populations. Baylisascaris procyonis is an important wildlife para-
site of raccoons which, due to the mammals rapid adaptation to urban
environments, is now found in domestic dogs and is a clear zoonotic infec-
tion risk for humans (Rudmann et al., 1996; Bowman et al., 2005; Lee et al.,
2010).
3.1 Echinococcosis
Echinococcosis is a clinically important disease and among the most preva-
lent of the zoonotic helminthiases with 2e3 million individuals worldwide
estimated to be infected with E. granulosus and 0.3e0.5 million with Echino-
coccus multilocularis (McManus, 2010; Craig et al., 2007).
Echinococcus granulosus occurs globally while E. multilocularis infections
mainly occur in the Northern hemisphere (Figure 5, Suppl. Table 2)
(McManus et al., 2012). Echinococcus multilocularis causes alveolar echinococ-
cosis (AE), while E. granulosus causes cystic echinococcosis (CE).
Denitive hosts for E. granulosus and E. multilocularis are typically dogs or
other canids including foxes, coyotes and wolves, as well as cats (McManus
et al., 2003). Feeding raw offal to dogs is an important mode of transmission
and a risk factor for human CE infection (Van Kesteren et al., 2013; Li et al.,
2014a). Intermediate hosts for E. granulosus are typically livestock such as
sheep, goats, pigs, cattle, horses and camels (Cardona and Carmena, 2013;
McManus et al., 2003); while intermediate hosts for E. multilocularis are typi-
cally small rodents (McManus et al., 2003). Echinococcus vogeli and Echinococcus
oligarthrus have rodents as intermediate hosts and bush dogs and wild felids as
denitive hosts, respectively.
Figure 5 World map showing the geographic location of human and animal infections with Echinococcus spp. based on the 2010e2015
333
published literature. Pie graphs show the relative proportion of each species infecting humans and animals based on the number of cases
recorded for the period 2010e2015 (Suppl. Table 2).
The red fox (Vulpes vulpes) is an important denitive host of E. multilo-

cularis and one that has increasing contact with humans in both urban and
rural environments. Most cases of echinococcosis occur in developing
countries where rapid urbanization is occurring in combination with poor
hygiene and food safety practices (Figure 5, Suppl. Table 2). Echinococcus
vogeli cases, resulting in polycystic echinococcosis, are rare in humans and
are primarily limited to rural areas of South and Central America (Figure 5).
However, with the population expansion and deforestation that is occurring
in South America, the interface between humans and the animals carrying
E. vogeli will only increase. Since 2010 there have been seven cases reported
in the literature of E. vogeli infection including one imported case in the
Netherlands (Figure 5, Suppl. Table 2) (Stijnis et al., 2013).
Figure 5 and Suppl. Table 2 outline details of reported human echino-
coccosis cases occurring worldwide since 2010. The majority of these cases
are assumed to be E. granulosus, although immunological or molecular diag-
nostics are rarely performed on the cysts once identied as a hydatid.
In Australia, E. granulosus is of veterinary importance with the wild life
cycle maintained in rabbits, kangaroos, wallabies, wombats, feral pigs, foxes,
dingoes and dog/dingo hybrids and the domestic life cycle involving live-
stock and domestic dogs. The presence of E. granulosus is of concern for
potential human infection through public use of rural areas for picnicking,
hiking and camping. Dingoes, foxes and wild dogs are also increasingly
encroaching into urban areas bringing the parasite closer to the human pop-
ulation (Jenkins, 2006; Jenkins et al., 2014a,b; Beveridge and Spratt, 2015).
3.2 Emerging wildlife zoonoses

Increasing habitat destruction and human encroachment on natural areas has
led to an increase in the contact of wild animals with humans and also with
companion animals (Figure 4). Animals such as raccoons and skunks are
known pests in North America and are often found in cities living off house-
hold rubbish. As indicated above, raccoons carry B. procyonis, a nematode,
which can cause severe neurological disease in humans (Cottrell et al.,
2014). In Africa and Asia, primates are often found in cities and villages
and are hunted for food, allowing close human associations from which zoo-
notic infections can occur, such as for Oesophagostomum bifurcum and Schisto-
soma mansoni (Ghai et al., 2014; Erko et al., 2001). It is inevitable that as wild
habitats are destroyed and global warming impacts on food sources that trans-
mission of wildlife zoonosis to humans will increase. Due to urbanization,
wildlife zoonoses are currently emerging as medically important diseases of
humans, O. bifurcum (Africa), B. procyonis (North America), A. cantonensis

(worldwide), Angiostrongylus mackerrasae (Australia), Gnathostoma spp. (world-
wide) and Haycocknema perplexum (Australia), among them.
Oesophagostomum bifurcum is primarily a parasite of monkeys but is also the
most common cause of oesophagostomiasis in humans. Humans become
infected by ingesting infective third-stage larvae (http://www.cdc.gov/
dpdx/oesophagostomiasis/index.html). All human and animal cases reported
in the literature since 2010 have occurred in Africa (Ghai et al., 2014)
(Table 3). Prior to this there were reports of human infections with this spe-
cies in Asia and South America (Bogers et al., 2001). Around 250,000 indi-
viduals in Ghana and Togo are thought to be infected with O. bifurcum.
Random amplied polymorphic DNA comparison of O. bifurcum from
humans and primates showed distinct clustering with the human isolates
genetically distinct to those from different primate species (de Gruijter
et al., 2004, 2006). This would indicate that an anthropogenic life cycle is
occurring and that zoonotic spillover does not occur. Similarly, while a
high prevalence of O. bifurcum was found in primates from an area of
Northern Ghana, no human cases were reported despite the fact that activ-
ities considered to promote zoonotic transmission occurred in this location
(van Lieshout et al., 2005). Cryptic species of O. bifurcum have been described
in Uganda with at least one found in humans and ve other species of
primates (Ghai et al., 2014). Experimental infection of primates with
O. bifurcum cultured from human stools resulted in infection in primates,
indicating that while zoonotic transmission may not be apparent, the biolog-
ical potential is there (Eberhard et al., 2001).
Alariosis, caused by infection with a larval trematode from the genus
Alaria, can be categorized as an FBH as human infection occurs following
the consumption of raw or undercooked intermediate hosts (Gonzalez-
Fuentes et al., 2014), much in the same way as gnathostomiasis, which is
caused by Gnathostoma spp., parasites of amphibians, reptiles and birds.
Alariosis is rare, with only one reported human case in North America,
caused by infection with Alaria americana (Fernandes et al., 1976; Freeman
et al., 1976; McDonald et al., 1994). However, there are concerns in Europe
regarding the zoonotic potential of Alaria alata, since it has been found in
animals across the continent, particularly wild boars and foxes (Table 3).
Wild boars are a common paratenic host in Europe and may be the cause
of future zoonotic infections of A. alata in humans.
Baylisascariasis is caused by the nematode B. procyonis. A survey of birds
and mammals in the USA found B. procyonis in 87 birds (18 species) and
Table 3 Recent human (and animal) cases of wildlife zoonoses
336
No. of
Country cases Symptoms (n) Diagnosis Species Animal hosts References
Uganda 9 [216] Unknown (9) PCR O. bifurcum Nonhuman primates Ghai et al. (2014)
[216]
USA 13 [548] Pica (2), neurological Intestinal extrusion, B. procyonis Raccoon [444], White- Perlman et al. (2010),
symptoms (6), incision, direct smear, footed mouse [103], Kelly et al. (2012),
lethargy/fatigue (3), saturated sugar Green-cheeked Rowley et al. (2000),
neck stiffness (1), otation, carcass amazon parrot [1] Peters et al. (2012),
upper respiratory examination, Pipas et al. (2014),
illness (2), morphology, faecal Jardine et al. (2014),
eosinophilic oation, histology Cottrell et al. (2014),
meningoencephalitis Hernandez et al.
with myelitis (4), (2013), Blizzard et al.
chorioretinitis (2), (2010a,b), Kresta et al.
headache (1), (2010), Chavez et al.
vomiting (1), ataxia (2012), Samson et al.
(2) (2012), Beasley et al.
(2013), Done and
Tamura (2014), Baird
Mets et al. (2003),

Sorvillo et al. (2002),
Chun et al., (2009),
Pai et al. (2007),
Moertel et al. (2001)
and Wise et al. (2005)
Norway [4] Sequencing ITS1, ITS2, B. procyonis Raccoon [4] Davidson et al. (2013)
cox1
China [35] Morphology, B. procyonis Raccoon [35] Xie et al. (2014)
sequencing ITS1,
cox1
Canada 1 Cardiopulmonary arrest B. procyonis Hung et al. (2012)
(1)
Australia 6 Weakness (6), weight Muscle biopsy H. perplexum McKelvie et al. (2013),
loss (3), difculty Basuroy et al. (2008)
swallowing (2), fever and Spratt et al.
(1), dysphagia (1), (1999)
muscle pain (1),
fatigue (1)
Australia 1 Carcass examination, A. mackerrasae Black ying fox Mackie et al. (2013)
histology (Pteropus alecto) [1]
USA 4 [5] Neurological symptoms qPCR, worm tracts in A. cantonensis Geoffroys tamarins Chi et al. (2014), Kwon
(3), periorbital brain, carcass (Saguinus geoffroyi) [3], et al. (2013), Howe
swelling (1), pain (1), examination, African pygmy falcon (2013), Thyssen et al.
headache (3), muscle histology, PCR, (Polihierax (2013), Kottwitz et al.
and joint pain (1), qPCR semitorquatus) [1], (2014), Burns et al.,
dizziness (1), facial orangutan (Pongo (2014) and Emerson
droop (1), ataxia (1), pygmaeus) [1] et al. (2013)
coma (1)
French 13 Eosinophilic meningitis Serology A. cantonensis Oehler et al. (2014)
Polynesia (13) (retrospectively
identied)
Canary [30] Morphology, serology A. cantonensis Rat (R. rattus) [30] Martin-Alonso et al.
Islands (2011)
(Continued)
337
Table 3 Recent human (and animal) cases of wildlife zoonosesdcont'd
338
No. of
India 3 Fever (3), headache (3), Serology CSF A. cantonensis Rai et al. (2014), Nalini
altered senses (1), et al. (2013) and Pai
vomiting (1), loss of et al. (2013)
appetite (1),
paraesthesias (1)
New 1 Headache (1), nausea/ A. cantonensis Lilic and Addison (2013)
Zealand vomiting (1)
Taiwan 1 [17] Abdominal pain (1), ELISA, morphology A. cantonensis R. norvegicus [6], R. rattus Hsueh et al. (2013) and
neurological [10], Suncus murinus Tung et al. (2013)
symptoms (1), [1]
difculty urinating (1)
Jamaica 1 Fever (1), refusal to Worms in CSF e A. cantonensis Evans-Gilbert et al.
walk/crawl (1) morphology (2014)
South [57] Morphology, PCR A. cantonensis R. norvegicus [56], Archer et al. (2011)
Africa R. rattus [1]
Japan [49] Histology, qPCR A. cantonensis Diplothrix legata [3], Okano et al. (2014) and
R. rattus [46] Tokiwa et al. (2013)
Australia 2 [98] Irritability (2), fever (1), Serology CSF, worm A. cantonensis Gang-gang cockatoo Morton et al. (2013),

vomiting (1), morphology at [4], tawny frog mouth Sinawat et al. (2013),
lethargy, (2), autopsy, carcass [90], bushtail possum Reece et al. (2013),
neurological examination [4] Ma et al. (2013) and
symptoms (2) Gelis et al. (2011)
Thailand 1 [1] Loss of vision (1), worm morphology A. cantonensis R. rattus [1] Sinawat et al. (2013) and
in eye (1) Vitta et al. (2011)
Brazil 1 [102] Headache (1), isochoric Serology 135 post onset A. cantonensis R. norvegicus [100], R Espirito-Santo et al.
(1), nuchal rigidity (1) (ealier serology rattus [2] (2013), Simoes et al.
negative), Carcass (2011, 2014),
examination, Cognato et al. (2013)
morphology, PCR, and Moreira et al.
sequencing (cox1) (2013)
China 4 [743] ELISA, morphology A. cantonensis R. norvegicus [183], Yang et al. (2012), Pan
Rattus avipectus [34], et al. (2011), Deng
Rattus species [496], et al. (2012), Hu et al.
Bandicota indica [26], (2011), Qu et al.
Rattus losea [1], Mus (2011) and Chen et al.
musculus [1], Rattus (2011a,b)
sladeni [4], Rattus
rattoides [3]
Bulgaria [2] Mesocercariae migration A. alata Wild boar (Sus scrofa) [2] Riehn et al. (2013)
technique (AMT),
PCR
China [3] DNA barcoding A. alata Red fox (Vulpes vulpes) Li et al. (2013a)
[3]
Czech [15] A. alata Wild boar (S. scrofa) [15] Paulsen et al. (2013)
Republic
Denmark [201] Sedimentation/ A. alata Raccoon dog Al-Sabi et al. (2013)
microscopy (Nyctereutes
procyonoides) [69], red
fox (V. vulpes) [132]
France [166] PCR A. alata Wild boar (S. scrofa) Portier et al. (2011,
[166] 2014)
339
Germany [35] A. alata Raccoon [35] Renteria-Solis et al.
(2013)
(Continued)
340
Table 3 Recent human (and animal) cases of wildlife zoonosesdcont'd
No. of
Hungary [446] A. alata Golden jackal (Canis Takacs et al. (2014),
aurelius) [10], red fox Szll et al. (2013) and
(V. vulpes) [426], Majoros et al. (2010)
domestic dog [10]
Ireland [105] A. alata Red fox (V. vulpes) Murphy et al. (2012)
[105]
Lithuania [393] A. alata Raccoon dog (N. Bruzinskaite-
procyonoides) [94], red Schmidhalter et al.
fox (V. vulpes) [299] (2012)
Poland [24] Flotation and A. alata European otter [1], wolf Gorski et al. (2010) and
sedimentation (Canis lupus) [23] Szafranska et al.
(2010)
Romania [1] A. alata Mink [1] Tabaran et al. (2013)
Serbia [4] A. alata Golden jackal Cirovic et al. (2013)

(C. aurelius) [4]
O. bifurcum, Oesophagostomum bifurcum; B. procyonis, Baylisascaris procyonis; H. perplexum, Haycocknema perplexum; A. mackerrasae, Angiostrongylus mackerrasae; A. cantonensis,
Angiostrongylus cantonensis; R. rattus, Rattus rattus; R. norvegicus, Rattus norvegicus; A. alata, Alaria alata.
64 mammals (8 species), including raccoons (Evans, 2002a). Raccoons pose

the most threat to humans due to the close association between human hab-
itats and these animals (Evans, 2002a). Baylisascaris procyonis has been found in
animals from the USA, Germany, Canada, China and Japan (Jardine et al.,
2014; Evans, 2002a; Kuchle et al., 1993; Popiolek et al., 2011; Xie et al.,
2014), although reported human cases are restricted to Germany, Canada
and the USA, with most occurring in the USA (Table 3). There have
been 4 human cases of B. procyonis since 2010, 23 reported since 1975,
with 21 recorded since 1993, showing that this disease is likely on the in-
crease (Table 3).
Humans are accidental hosts of infection with B. procyonis and infection
occurs from ingestion of embryonated eggs (http://www.cdc.gov/parasites/
baylisascaris/biology.html). The larval stage of the parasite migrates to
various tissues causing visceral larva migrans (VLMs), ocular larva migrans
and neural larva migrans (Boschetti and Kasznica, 1995; Chun et al.,
2009; Gavin et al., 2002; Kazacos et al., 2013). Human infections are rare
and often occur in children who accidentally ingest contaminated soil.
Angiostrongylus cantonensis is known colloquially as the rat lung worm, the
rat being the main denitive host of this parasite, and it is found worldwide
(Aghazadeh et al., 2015). Rats, much like raccoons, are common inhabitants
of urban environments. Human infections with Angiostrongylus result from
ingestion of infective larvae, either on raw vegetables or by eating an
infected molluscan host (http://www.cdc.gov/parasites/angiostrongylus/
biology.html).
Twenty species of Angiostrongylus spp. have been described but only
A. cantonensis and Angiostrongylus malaysiensis have been found to cause
meningitis in humans (Aghazadeh et al., 2015). Angiostrongylus vasorum is
commonly found in dogs in Europe but no human cases have been identi-
ed (Di Cesare et al., 2015; Guardone et al., 2013; Majoros et al., 2010).
Clinical indicators such as eosinophilic meningitis and neurological signs
are common symptoms associated with Angiostrongylus spp. infection
(Mackie et al., 2013). Molecular diagnostics have not yet been used in the
clinical setting for angiostrongyliasis although loop mediated isothermal
amplication (LAMP) and polymerase chain reaction (PCR) assays have
been developed for the detection of A. cantonensis in snails and for retrospec-
tive PCR on the cerebral spinal uid of previously identied human cases of
Angiostrongylus spp. (Chen et al., 2011; Constantino-Santos et al., 2014;
Eamsobhana et al., 2013). Morphological identication can only occur
when fully mature adult worms are available as the other life cycle stages
are virtually identical (Bhaibulaya, 1968; Aghazadeh et al., 2015). In

Australia, where two almost identical species occur, it is possible that
some human cases have been caused by A. mackerrasae, an Angiostrongylus
spp. native to Australia (Aghazadeh et al., 2015). A patent infection of
A. mackerrasae has recently been found in a ying fox in Queensland,
Australia. This is the rst report of adult A. mackerrasae in an accidental
host and demonstrates biological potential for human infections with this
species (Mackie et al., 2013). Birds (tawny frogmouths) and possums from
Sydney, Australia, have both been shown to be infected with A. cantonensis
causing neurological symptoms in these animal hosts. Infections in tawny
frogmouths follow a seasonal pattern which indicates that they could be
an important sentinel species for A. cantonensis (Ma et al., 2013). Two hu-
man infections from Sydney, occurred in autumn and winter, further indi-
cating a seasonal pattern for this helminth in Australia (Aghazadeh et al.,
2015).
Angiostrongylus (Parastrongylus) costaricensis is an intestinal parasite of rats
and has also been shown to cause intestinal or abdominal angiostrongyliasis
in humans. Up to 500 human cases are estimated to occur in Costa Rica
annually (Thiengo et al., 2013). The rst instance of patent infection of
this parasite in a dog was recently reported in South America (Alfaro-
Alarcon et al., 2015).
The worldwide distribution of A. cantonensis was likely due to the intro-
duction of infected rats or snails in ships and shipping containers. Thiengo
et al. (2013) present a distribution map of the imported African snail,
A. fulica, in Brazil, showing an almost universal spread throughout the coun-
try. While other snail species in Brazil, including native snail species, act as
intermediate host, the explosive geographical distribution of A. fulica is a
concern (Thiengo et al., 2010, 2013). In China, A. fulica and the large fresh-
water snail, Pomacea canaliculata, are both introduced snails which have
similarly spread throughout the country. Recent outbreaks of A. cantonensis
in China can be traced back to consumption of P. canaliculata (Thiengo et al.,
2013; Lv et al., 2009, 2008).
Infected slugs and/or snails may be ingested either by consumption of
raw or undercooked molluscs on purpose, or accidentally, perhaps on fresh
vegetables; snails are regularly consumed in China as a delicacy. A study on
washing vegetables focussing specically on removal of snails from lettuce
found that even after rinsing each lettuce leaf individually, some snails
remained, providing a simple mechanism for accidental infection (Yeung
et al., 2013; Ewers and Anisowicz, 2014).
Haycocknema perplexum is an emerging nematode parasite of humans in

Australia. Similar to Trichinella spp., it occurs in the muscle bres of the
host. Six cases have been reported since 1994 with the most recent case iden-
tied in 2011 (Table 3) (Basuroy et al., 2008; McKelvie et al., 2013; Spratt
et al., 1999). Progressive muscle weakness and wasting are the common signs
of infection, and all cases were formally identied after muscle biopsy
detected nematode worms in the muscle bres. Three cases each occurred
in Tasmania and in North Queensland.
The life cycle of H. perplexum is unknown, and the parasite may originate
from a vertebrate or invertebrate host. Transmission may occur from contact
with the infected animal host or from contaminated soil. The history of the
known human infections with H. perplexum suggests that the parasite is a
zoonotic species occurring in native Australian animals such as wombats,
Tasmanian devils, wallabies or kangaroos, and potentially in domestic ani-
mals such as dogs, chickens and rabbits (Basuroy et al., 2008; McKelvie
et al., 2013; Spratt et al., 1999). Native animals in Australia are increasingly
found in urban and semiurban areas and these include tawny frogmouths
and possums which are known hosts of A. cantonensis (Gelis et al., 2011;
Ma et al., 2013).
4. CLIMATE CHANGE
4.1 Zoonotic lariasis
Filarial nematodes require an insect vector in which the parasite must
undergo stages of development. The vectors for many lariids are mosqui-
toes, although blackies (family Simuliidae) transmit Onchocerca spp. Of all
the zoonotic helminths, larial nematodes are the most likely to increase
their areas of future transmission due to climate change and weather changes
which will result in the expansion of the relevant insect vectors into new
regions.
4.1.1 Onchocerca spp.

The most prevalent larial nematodes of humans are Onchocerca and Diro-
laria spp. Onchocerca spp., which cause disease in humans and originate
from wildlife are O. lupi, originally identied in wolves but now found
in domestic dogs, Onchocerca dewittei japonica in wild boars and Onchocerca
jakutensis in wild deer (Srter-Lancz et al., 2007; Egyed et al., 2001;
Otranto et al., 2013c; Koehsler et al., 2007; Burr et al., 1998; Labelle
et al., 2013; Eberhard et al., 2013). Domestic herds of deer are at risk of
infection with O. jakutensis thereby establishing a domestic life cycle.

Onchocerca gutterosa and Onchocerca cervicalis are parasites of bovines, and
horses and mules, respectively.
There have been 14 cases of zoonotic onchocerciasis reported in the
literature since 2000, 9 identied as O. lupi and 2 as O. dewittei japonica
and 3 where a species was not determined (Figure 6, Suppl. Table 3) (Bergua
et al., 2015; Chen et al., 2015; Otranto et al., 2015; Dudley et al., 2015;
Mowlavi et al., 2014; Eberhard et al., 2013; Ilhan et al., 2013; Biswas and
Yassin, 2013; Otranto et al., 2011; Uni et al., 2010, 2015; Takaoka et al.,
2005, 2004, 2001). Onchocerciasis caused by O. lupi in dogs and wolves
has been reported worldwide (Egyed et al., 2001; Labelle et al., 2013;
Otranto et al., 2013a, 2013b). The rst reported case of O. lupi in a human
originated from Turkey and was identied both morphologically and by
molecular analysis using the 12S ribosomal and cox1 mitochondrial genes
(Otranto et al., 2011). Further human cases have been identied in the
USA, Turkey, Tunisia and Iran (Mowlavi et al., 2014; Eberhard et al.,
2013; Otranto et al., 2012) (Figure 6, Suppl. Table 3).
Being primarily a canine helminth, monitoring of canine populations for
O. lupi will help inform the potential risk for human infection. Originally
identied in wolves, O. lupi is a good example of a wildlife helminth which
has transitioned from a sylvatic to a domestic life cycle.
4.1.2 Dirolaria spp.

Dirolaria spp. are more prevalent in humans than Onchocerca spp. and are
increasing both in incidence and geographical range. Dirolaria spp. have a
similar life cycle to Onchocerca spp. with Dirolaria immitis differing in that
the adults are found in the pulmonary arteries, rather than in the skin and sub-
cutaneous tissues as occurs for Onchocerca spp. (http://www.cdc.gov/dpdx/
dirolariasis/index.html; http://www.cdc.gov/parasites/onchocerciasis/
biology.html). Other Dirolaria spp., such as Dirolaria repens, are commonly
found as nodules, often migratory, in subcutaneous tissue. Vectors for Diro-
laria spp. (D. immitis, D. repens, Dirolaria tenuis and Dirolaria ursi) are mosqui-
toes, including Culex pipiens, Anopheles maculipennis and Aedes albopictus, while
D. ursi utilizes a blacky. Dirolaria immitis causes pulmonary disease in
humans while other Dirolaria spp. produce subcutaneous nodules which
can be painful and sometimes migratory; these nodules can occur anywhere
on the body (Benzaquen et al., 2015; Yaranal et al., 2015; Ilyasov et al., 2014;
Vucaj Cirilovic et al., 2014; Madi et al., 2014; Pozgain et al., 2014; Kurup
et al., 2013; Kang et al., 2013; Leccia et al., 2012; Joseph et al., 2011).
Figure 6 World map showing geographic locations of human and animal infections with zoonotic larial nematodes based on the published
345
literature from 2010 to 2015. Pie graphs showing the relative proportions of each species infecting humans and animals based on the num-
ber of cases identied (Suppl. Table 3) from reports published in 2010e2015.
Dirolaria spp. are found worldwide. Among them, D. tenuis is found in

North America and is primarily a helminth of raccoons. Only two cases of
human infection with D. tenuis have been reported since 2010, both in the
USA (Figure 6, Suppl. Table 3). The most recent human case of D. ursi
was reported in 1996 but it still occurs in bear populations of North America
(Michalski et al., 2010; Haldane et al., 1996).
Dirolaria immitis and D. repens are the most common causes of dirolar-
iasis in humans with the majority of D. repens human cases reported post-
2010 occurring in Europe (percentage of cases occurring in the literature
73.8%) and the majority of D. immitis human cases from the same period
occurring in Africa (percentage of cases occurring in the literature 54.5%)
(Figure 6, Suppl. Table 3).
In Hong Kong there is some evidence of a novel zoonotic species of
Dirolaria. The 18S-ITS1-5.8S gene sequences of larial helminths, taken
from a small number (3) of humans, were identical to each other but did
not share 100% homology with the sequences from D. repens or D. immitis
(To et al., 2012). In the same study, worms were also taken from dogs
(n 200) and cats (n 100) and the majority were identied as D. immitis
and D. repens, although 3% of the worms from dogs were identical in
sequence to those of human origin (To et al., 2012). This demonstrated a
potential zoonotic transfer from dogs to humans for this new species, which
the authors suggested the taxonomic name of Candidatus Dirolaria hongkon-
gensis (synom. Dirolaria hongkongensis) (Figure 2).
Mosquito monitoring is an important measure to determine the potential
for clinical impact of zoonotic lariasis, by specically determining which
species are circulating within a region. Such studies on mosquitoes have
documented the spread of vectors and larial nematodes throughout
Europe. Dirolaria immitis and D. repens have been found in North and South
America. In Europe there are many studies identifying these species in
mosquitoes and dogs, as well as in humans (Carleton and Tolbert, 2004;
Cuervo et al., 2013a; Eberhard, 2013; Ermakova et al., 2014; Kronefeld
et al., 2014; Salamatin et al., 2013; Cuervo et al., 2013b; Bockova et al.,
2013; McKay et al., 2013; Sassnau et al., 2013; Joseph et al., 2011; Cielecka
et al., 2012) (Figure 6, Suppl. Table 3). A 2014 study of mosquitoes in
Germany identied the presence of D. immitis, D. repens and Setaria tundra
(Kronefeld et al., 2014). This reects the introduction of exotic mosquito
species capable of carrying dirolarial helminths emerging in new areas
within Europe since 2010 (Bockova et al., 2013; Capelli et al., 2011; Ferreira
et al., 2015; Kronefeld et al., 2014; Montarsi et al., 2015; Yildirim et al.,
2011). The prevalence of Dirolaria spp. in mosquito vectors and dogs in the
same area indicates that human infections are autochthonous in Europe,
rather than originating from abroad. Due to climate change, allowing the
spread of the mosquito vectors to new regions, as well as the movement
or spread of infected hosts (i.e. dogs, both wild and domestic, transmitting
D. repens), dirolariasis is an emerging disease in Europe with potential for
human transmission. Dirolaria repens proportionally causes 90.7% of human
cases in Europe, while D. immitis has been reported in 5.9% of cases
(Figure 6, Suppl. Table 3). Human dirolariasis cases are becoming more
common in Europe (Figure 6, Suppl. Table 3) (Suzuki et al., 2015; Rossi
et al., 2015; Ferreira et al., 2015; Tasic-Otasevic et al., 2015; Sergiev
et al., 2014; Sassnau et al., 2014). Whereas there had been only 30 prior cases
of dirolariasis in Serbia, serology of 46 individuals in 2014 was Dirolaria
spp.-positive; both D. repens and D. immitis were reported (Tasic-Otasevic
et al., 2014). Similarly, seroprevalence of Dirolaria spp. in blood donors
from Russia was 10.4% (n 317) in 2011 (Kartashev et al., 2011).
Based on the results of one very large antigen-based study on Dirolaria
spp. in dogs (1.3% positive;,142,426/10,734,132) in the USA, D. immitis was
the most common zoonotic infection of dogs over the last 5 years (Figure 6,
Suppl. Table 3) (Little et al., 2014). In Europe, both D. repens and D. immitis
are responsible for a high proportion of reported cases in the literature in an-
imals (36.78% and 42.68%, respectively) (Figure 6, Suppl. Table 3) (Giudice
et al., 2014; Vichova et al., 2014; Saevik et al., 2014; Miterpakova et al.,
2013; Albanese et al., 2013; Otranto et al., 2013a; Di Cesare et al., 2013;
Iglodyova et al., 2012; Traversa et al., 2010; Miterpakova et al., 2010).
Other rare Dirolaria spp. causing human infection are Dirolaria striata,
which is a larial nematode of bobcats, although it has also been found in
dogs (Orihel and Ash, 1964; Orihel and Isbey, 1990; Pacheco and Tulloch,
1970), and Dirolaria subdermata, a larial nematode of porcupines. There are
no recent reported human cases due to these species.
4.1.3 Thelazia spp.

Thelaziasis is a rare nematode infection in humans caused by Thelazia calli-
paeda and Thelazia californiensis (http://www.cdc.gov/dpdx/thelaziasis/
index.html). While not strictly a lariid, belonging to the family Thelazioi-
dea, it has been included here as it shares many similarities with those hel-
minths in the family Filarioidea. Thelazia has the potential for increased
human prevalence, with the geographical distribution and incidence of
Thelazia spp. in animals showing an upward trend (Figure 6, Suppl. Table 3)
(Mihalca et al., 2015; Diakou et al., 2015; Wang et al., 2014a; Maia et al.,
2014; Krishnachary et al., 2014; Hodzic et al., 2014; Sargo et al., 2014;
Motta et al., 2014; Calero-Bernal et al., 2014; Soares et al., 2013; Caron
et al., 2013; Rodrigues et al., 2012; Nguyen et al., 2012b; Vieira et al.,
2012; Sohn et al., 2011). A range of animals can act as denitive hosts
with the family Canidae the most common. In all hosts the parasites reside
in the conjunctival sac, leading to eye irritation, watering and sometimes
pain. For humans, the presence of a foreign body sensation has been
described (Wang et al., 2014b; Krishnachary et al., 2014; Akhanda et al.,
2013; Fuentes et al., 2012; Hossain et al., 2011; Kim et al., 2010b; Sohn
et al., 2011; Viriyavejakul et al., 2012). Originally known as the oriental
eye worm, T. callipaeda has been increasingly reported in animals from
European countries including a recent report of canine ocular thelaziasis
in Greece, the rst report of this parasite in an animal from that country
(Diakou et al., 2015) (Diakou et al., 2015; Magnis et al., 2010; Maia
et al., 2014). Based on the high prevalence of T. callipaeda in animals and
its increasing geographical distribution, T. callipaeda human cases from other
countries are inevitable (Figure 6, Suppl. Table 3) (Calero-Bernal et al.,
2013; Caron et al., 2013; Diakou et al., 2015; Dorchies et al., 2007; Hodzic
et al., 2014; Magnis et al., 2010; Maia et al., 2014; Malacrida et al., 2008;
Mihalca et al., 2015; Miro et al., 2011; Vieira et al., 2012).
Human infections with T. californiensis are rare with no reports in the
literature since 1996, and only three case reports since 1975, all occurring
in the USA (Doezie et al., 1996; Knierim and Jack, 1975; Kirschner et al.,
1990). On the other hand, T. callipaeda infections are identied in humans
more frequently since the rst reported case in China in 1917. In China
alone, 613 cases of Thelazia spp. infection were reported between 1917
and 2013; only 48 of these were reported prior to the 1980s. This shows
increasing recognition and diagnosis of the infection as well as increasing
incidence (Wang et al., 2014a).
4.2 Schistosoma spp.

Clinical schistosomiasis occurs worldwide in the tropics and subtropics and
is caused by four main species of schistosome, S. haematobium (Africa),
S. mansoni (Africa, South America, the Middle East) and S. japonicum and
Schistosoma mekongi (SEA). Of these, only S. japonicum and S. mekongi are
traditionally considered zoonotic. However, hybridization, occurring in
Africa between S. haematobium and other species, particularly Schistosoma
bovis and Schistosoma curassoni, may give rise to a third zoonotic schistosome
(Webster et al., 2013), while natural infections of S. mansoni have also been
found in nonhuman hosts (Muller-Graf et al., 1997; Legesse and Erko,
2004). Autochthonous infections of S. haematobium have recently been
found in Europe marking a new expansion of this species (Boissier et al.,
2015).
Schistosoma japonicum is endemic in China, the Philippines and parts of
Indonesia and has been shown to parasitize 46 mammalian species as
denitive hosts (He et al., 2001). Schistosoma mekongi occurs in the Peoples
Democratic Republic of Lao (Lao PDR) and Cambodia and is currently
only thought to infect dogs and pigs, as well as humans (Khieu et al.,
2013; Matsumoto et al., 2002; Strandgaard et al., 2001). In China an esti-
mated 600 million people are at risk of infection and approximately
0.3 million people are currently infected, while in the Philippines 6.7 million
people live in endemic areas; of these, 1.8 million people are considered to be
directly exposed to infection through water contact activities (McManus
et al., 2009; Carabin et al., 2005; Riley et al., 2005; Yang et al., 2014).
A number of drug-based intervention trials have indicated that bovines
are major reservoir hosts for schistosomiasis japonica in China (Gray et al.,
2009a, 2007, 2008a, 2009b; Guo et al., 2006). The results of these interven-
tion trials, combined with mathematical modelling, found that bovines are
responsible for approximately 75% of human transmission (Gray et al.,
2009a, 2007). These animals are used as work animals, primarily on the
marshlands (China) or rice paddy elds (Philippines), where the Oncomelania
snail intermediate hosts live. As a result it is principally farmers and shermen
who are at most risk of infection with S. japonicum, although domestic
(washing) and social (swimming) activities are also important risk factors
(McManus et al., 2010; Li et al., 2000).
Thousands of livestock remain infected so this number of potential res-
ervoirs of infection means that simply treating humans with praziquantel
(PZQ) will not prevent transmission. An individual living in an endemic
area can readily become reinfected after treatment. After the implementa-
tion of the World Bank Loan Project (WBLP) in 1992e2001, relaxation
of control efforts saw an increase in infection in previously controlled areas
in China (Xianyi et al., 2005). Re-emerging schistosomiasis in areas of
Sichuan province (China) was studied with 24 counties found to be endemic
for the disease with 8 re-emerging (Liang et al., 2006). The average return
time, or time it took for the disease to be considered endemic again after
cessation of treatment, was 8.1 years with the shortest time 2 years and the
longest 15 years (Liang et al., 2006). Other reasons for reoccurrence are
unusual ooding events, environmental modication, such as the building

of dams, and relaxation in control efforts following the termination of the
WBLP (Zhou et al., 2004, 2005; Zhu et al., 2008; Zhou et al., 2005; Wu
et al., 2008). The Three Gorges Dam (TGD) upstream of the Dongting
Lake area in Hunan province, a region highly endemic for schistosomiasis,
has raised concerns about the potential redistribution of Oncomelania hupensis
in China and the potential for the spread of schistosomiasis into new areas.
An initial 5-year assessment of transmission of schistosomiasis following
construction of the TGD found no immediate impact (Gray et al., 2012).
A more recent study considered the density of oncomelanid snails in low,
medium and high elevation areas showed a decrease generally in snail pop-
ulations during the period 2003e2014, with the exception of low elevation
areas where the snail density began to increase in 2014 (Wu et al., 2015).
While the TGD in China seems to have had a limited effect on schistosomi-
asis transmission to date, this is not true for other areas where dam building
projects have been undertaken. In several African countries, the construction
of dams, such as the Gezira-Managil Dam in Sudan, the Aswan Dam in
Egypt and the Melkasadi Dam in Ethiopia, has led to increased schistosomi-
asis transmission (Gryseels et al., 2006).
Rodents have also been implicated in maintaining transmission of schis-
tosomiasis in endemic areas; however, their small size and the relatively
limited amount of faeces they produce would minimize the level of envi-
ronmental contamination with schistosome eggs (Cabrera, 1976; Fernandez
et al., 2007; Lu et al., 2010a, 2010b; Rudge et al., 2009).
Horses, donkeys, mules, pigs and dogs e which until recently were un-
common in rural areas e are susceptible to infection but are not considered
important in transmission due to their relative lack of water contact. Never-
theless, an increase in infection of humans in mountainous areas of Yunnan
Province in China has been linked to an increase in the numbers of domestic
animals in the area ( Jiang et al., 1997b). In mountainous areas with low
bovine numbers, transmission appears to involve a human-snail cycle, partic-
ularly as human faeces are used as fertilizer ( Jiang et al., 1997a). The use of
human faeces as fertilizer will likely vary from village to village, and its use
has reportedly been decreasing in China (Wang et al., 2005). From these
studies it can be concluded that humans probably act as the main hosts for
schistosomiasis transmission in areas where there are low numbers of domes-
tic animals, even though wild rodents are present.
As indicated earlier, S. mekongi is found in Lao PDR and Cambodia and
was endemic in Thailand, although this schistosome has not been reported
there since the 1980s, although the requisite snail intermediate host, Neotri-
cula aperta, can still be found in some areas (Limpanont et al., 2015; Bunnag
et al., 1986). To date only humans, dogs and pigs in Cambodia have been
found infected with S. mekongi (Matsumoto et al., 2002; Muth et al.,
2010). Bovines are thought to be involved in transmission, but this has
yet to be proven and to date S. mekongi has not been identied in bovines
(Muth et al., 2010). Recent reports of S. mekongi in humans from Lao
PDR infection indicated a prevalence of 24.3% in 2006, 0.1% in 2012
and 8.6% S. mekongi coinfection with O. viverrini from 2006 to 2007
(Sayasone et al., 2012; Laymanivong et al., 2014; Sayasone et al., 2015).
Schistosoma haematobium, the cause of urinogenital schistosomiasis, is
considered to be a parasite only of humans with no animal reservoirs involved
in its life cycle. However, recent multiloci molecular analysis of parasite sam-
ples has shown that hybridization events have occurred between S. haema-
tobium and ruminant schistosome species resulting in S. haematobium/
S. bovis and S. haematobium/S. curassoni hybrid worms (Webster et al.,
2013). These hybrid schistosomes were found in children, but not in rumi-
nants, in Senegal (Webster et al., 2013). In another worrying trend for the
emergence of new foci of schistosomiasis, S. haematobium has recently been
reported in Europe where a number of human cases in France, Italy and
Germany were linked to a popular tourist spot in France (Boissier et al.,
2015). While no snails infected with S. haematobium have been identied
in this area, laboratory infections with locally occurring Bulinus spp. snails
were successful in producing cercariae. The emergence of schistosomiasis
in Europe may be linked to climate change and globalization. Globalization,
through the movement of people from endemic areas to new areas, may have
played a role in introducing S. haematobium to the waterways while climate
change may allow the snail hosts to better survive and migrate to new areas
for longer periods of time.
4.3 STHs: Hookworm/Toxocara/Ascaris/Trichuris

The soil-transmitted helminths (STH) refers to intestinal worms which are
transmitted through soil contaminated with infectious eggs or larvae and is
generally used for the human-only helminths including hookworm (Ancylos-
toma duodenale and Necator americanus), roundworm (Ascaris lumbricoides) and
whipworm (Trichuris trichiura). These human parasites have closely related
species in animals which have zoonotic potential including hookworm
(Ancylostoma ceylanicum, Ancylostoma caninum, Ancylostoma braziliense and Unci-
naria stenocephala), roundworm (Ascaris suum) and whipworm (Trichuris suis).
Toxocara spp. (Toxocara cati and Toxocara canis) can also be transmitted via
contaminated soil. Climate change will be an important factor in worm
development in a range of helminths including Ascaris and hookworm spe-
cies. Temperature is an important factor for the embryonation of eggs in
the external environment. While A. suum egg embryonation can be
completed at 25 C, the speed of embryonation is increased at the higher
temperature of 35 C (Kim et al., 2012). Increasing global temperatures
will likely speed up development of nematode larvae in eggs, potentially
increasing the level of transmission as well as creating new habitats for egg
and larval survival.
4.4 Hookworm
Ancylostoma spp. from cats and dogs present a range of different disease states
in humans; from dermatitis caused by A. braziliense, patent infections caused
by A. ceylanicum and eosinophilic enteritis due to A. caninum (Loukas et al.,
1992; Prociv and Croese, 1996). The eggs of different Ancylostoma spp. are
morphologically identical, so denitive microscopic diagnosis based on eggs
is impossible. Precise diagnosis is important for monitoring hookworm prev-
alence in cats and dogs e both domestic and wild. Ancylostoma ceylanicum and
A. braziliense are hookworms of both cats and dogs, while A. caninum is the
dog hookworm (http://www.cdc.gov/parasites/hookworm/biology.html).
Ancylostoma braziliense is a common cause of nematode-induced cutaneous
larval migrans (CLMs) in humans, although other helminths, particularly
trematode species such as Schistosoma spp. and Trichobilharzia spp., and the
nematode U. stenocephala can also cause this condition (Le Joncour et al.,
2012). The morbidity most commonly associated with A. caninum infection
is eosinophilic enteritis and potentially unilateral subacute neuroretinitis
(Sabrosa and de Souza, 2001).
It has been suggested that many human hookworm cases caused by Ancy-
lostoma spp. may have been incorrectly diagnosed as A. duodenale when they
were actually due to A. ceylanicum (Traub, 2013; Schar et al., 2014; Ngui
et al., 2012b). As indicated above, the eggs of Ancylostoma spp. are indistin-
guishable microscopically, and larval cultures are required for precise
morphological identication based on larval anatomy. Whereas molecular
diagnosis can be specic and sensitive there is also high molecular similarity
between A. duodenale and A. ceylanicum, and PCR primers need to be care-
fully designed for species specicity. High-resolution melting (HRM) after
PCR amplication has previously been used to distinguish between hook-
worm species (Ngui et al., 2012a). Differentiation between the species in
animals is an important factor in planning public health measures due to the

differences in the pathology in humans ranging from CLM to eosinophilic
enteritis. Historically, A. ceylanicum had been thought to be a rare or
abnormal infection in humans.
A recent study in Cambodia found a high prevalence of hookworm in
both human and dog populations. Of the infected dogs, 90% harboured
A. ceylanicum while 51.6% of humans with hookworm were infected with
A. ceylanicum (Inpankaew et al., 2014; Schar et al., 2014). The remaining
48% of hookworm-positive individuals harboured A. duodenale (3.2%) and
N. americanus (44.8%) (Inpankaew et al., 2014). Hookworm is prevalent
in tropical regions such as SEA due to the prevailing environmental condi-
tions which result in damp soil conditions, a factor conducive for the survival
of hookworm larvae (Bethony et al., 2006).
Indeed, climate change may play a major role in changing the distribution
of hookworm spp. by modifying the environment through the production of
warm moist soil, thereby increasing the survival rates of hookworm larvae, or
conversely by causing soil to become too dry to support larval development.
4.4.1 Toxocariasis
Toxocara canis and T. cati are nematode parasites of dogs and cats, respec-
tively, but are also zoonotic. Toxocara spp. infections in companion animals
are a source of human transmission in developing countries due to stray dogs
and cats and in developed countries attributable to domestic pets.
Toxocariasis is caused by the migration of Toxocara larvae through tissues,
producing a condition known as visceral larva migrans (VLM). Liver ab-
scesses are normally thought to be a rare complication of human infection
with Toxocara spp.; however, there have been 13 reported cases of toxocar-
iasis resulting in this pathology since 2010, and it is therefore of medical
importance, likely representing an emerging syndrome (Table 4) ( Jackson
et al., 2010; Treska et al., 2011; Mukund et al., 2013; Ramachandran
et al., 2013). There has been a total of 150 cases of human infection with
Toxocara spp. reported since 2010 with various pathologies (Table 4) (Zibaei
et al., 2014; Grama et al., 2014; Viola et al., 2014; Antolova et al., 2015).
There are worrying trends in the prevalence of Toxocara eggs on the fur
of pets and the poor hand-washing practices of their owners (Overgaauw
et al., 2009). A study in the Netherlands found high prevalence of Toxocara
spp. eggs on the fur of 12.2% (n 152) of dogs and 3.4% (n 60) of cats
(Overgaauw et al., 2009). The same study looked at the behaviour of dog
and cat owners with 50% reporting that they allowed their animal to lick
354
Table 4 Human cases of toxocariasis
No. of cases
with liver Other reported
Country abscess malignancies (n) Diagnosis References Species
India 5 Imaging, biopsy, ELISA, Mukund et al. (2013) and Toxocara sp.,
histopathology Ramachandran et al. (2013) Toxocara canis
Korea 6 Urticaria (1) Imaging, serology Lee and Shin (2011) and Toxocara sp.,
Park et al. (2012) T. canis
Canada 1 Imaging, serology Jackson et al. (2010) Toxocara sp.
Brazil Polyarthritis (1) ELISA, serology Grama et al. (2014) and Toxocara sp.
unknown (32) Viola et al. (2014)
Iran Ocular (1) ELISA Zibaei et al. (2014) Toxocara cati
Slovakia Unknown (102) ELISA Antolova et al. (2015) Toxocara sp.
Czech Republic 1 ELISA, imaging, biopsy Treska et al. (2011) Toxocara sp.

their face. Only 15% of dog owners and 8% of cat owners always washed
their hands after touching the animal. This is a potential risk as simply patting
an infected animal and not washing hands afterwards can lead to infection
and toxocariasis. Similar studies have been performed elsewhere in Europe
including Italy, where two studies found 6.6e9.7% of stray and pet dogs
harboured T. canis infection (Simonato et al., 2015; Paoletti et al., 2015;
Nagy et al., 2011; Roddie et al., 2008).
4.4.2 Ascaris suum and Trichuris suis

Ascaris suum and T. suis are nematodes of pigs, and occasionally of humans.
Humans and pigs become infected with A. suum by ingesting eggs or food
contaminated with eggs (http://www.cdc.gov/parasites/ascariasis/biology.
html; http://www.cdc.gov/parasites/whipworm/biology.html). Ascaris
suum is found worldwide while A. lumbricoides, a human-only Ascaris spe-
cies, is restricted to the tropics, particularly in developing countries. Ascaris
suum has been linked to VLMs and eosinophilic pneumonia in humans
(Izumikawa et al., 2011; Pinelli et al., 2011).
Ascaris lumbricoides and A. suum are very similar morphologically and
genetically. There has been considerable discussion as to whether they are
in fact the same species, which may mean that ascariasis occurring in devel-
oped countries may be due to porcine ascariasis (Arizono et al., 2010; Shao
et al., 2014; Leles et al., 2012; Dutto and Petrosillo, 2013; Betson et al.,
2014). Hybridization between A. suum and A. lumbricoides has resulted in
at least one human case (Dutto and Petrosillo, 2013). Human cases of
T. suis and hybrids of T. suis and T. trichiura have been identied in Uganda
(Nissen et al., 2012; Cutillas et al., 2009). Such hybridization events will
impact the zoonotic potential of T. suis, likely increasing its infectivity to
humans.
5. POINTS FOR DISCUSSION

5.1 Health education
In 2000, unsafe water, inadequate sanitation and poor hygiene were
attributed to 3.7% of the global disease burden and disability (as measured
by DALYs), leading to 1.7 million deaths (Mathers et al., 2007). With
many parasitic helminths the same features are major risk factors for infection.
Health education packages have been developed for a number of helminth
infections. In China an education package for the prevention of human
STH infections (A. duodenale, A. lumbricoides and T. trichiura) which targets

school children has been successful in increasing hand washing, awareness
of STH and signicantly reducing the incidence of STH (McManus et al.,
2014; Bieri et al., 2013). The worms have zoonotic counterparts in A. cani-
num and A. ceylanicum, A. suum and T. suis, which could also be targeted for
control using this developed and tested education package.
A health education programme for control of taeniasis has also been tri-
alled in Tanzania. The programme consisted of a presentation by a teacher
trained in disease awareness as part of the study a video and a pamphlet
which increased knowledge of cysticercosis among the school children
(Mwidunda et al., 2015). One of the interesting outcomes of the trial in
Tanzania was an increase in the willingness of children to condemn infected
pork, although there was a reluctance to report cysticercosis in their own
pigs, partly due to the lack of effective treatment available in the study
area (Mwidunda et al., 2015). Introduction and use of latrines in endemic
areas helps prevent infection by increasing hygiene practices and preventing
pigs from eating human faeces. However, in some areas cultural taboos pre-
vail, particularly among men, relating to sharing a latrine, who were there-
fore more likely to defecate in the open than in a latrine (Thys et al., 2015).
An additional education resource is an online programme, the vicious
worm which found a signicant increase in knowledge among 79 study
participants ( Johansen et al., 2014; Ertel et al., 2015). In addition to good
hygiene practices, reducing the overall environmental contamination with
helminth eggs and larvae by animal hosts is also necessary. This involves reg-
ular deworming of animals, although wildlife populations, such as raccoons,
which are infected with B. procyonis, would be difcult to target in any
chemotherapy-based control campaigns. The use of bait containing an
anthelmintic has recently been trialled in the USA (Smyser et al., 2015).
In trials, B. procyonis infections were cleared from 9 of 12 wild raccoons
consuming 10 g of bait, and further study is being done to determine why
nematodes were not cleared from the remaining animals and to make the
bait more palatable (Smyser et al., 2015). Drug resistance is a growing vet-
erinary problem in domestic animals, so the treatment of wild populations
with anthelmintics would need to be closely controlled and monitored.
Limiting contact with potentially egg-contaminated locations through
avoiding communal raccoon defecation areas covering sand boxes and
enclosing areas, where children play to prevent animals from entering, can
also be effective preventative measures.
5.2 Targeting denitive hosts and vectors

Control programmes for fascioliasis and Asian schistosomiasis often target
animal hosts, thereby reducing the number of eggs entering the environ-
ment and limiting human exposure to infection. In the Philippines, methods
of control targeting bovines with Fasciola spp. have included keeping animals
penned, rather than tethered on rice paddies, in rivers, or close to water
holes e which are habitats of lymnaeid snail hosts (Gray et al., 2008b). Other
fascioliasis control measures include drying out bovine stool for a number of
weeks before utilizing it as fertilizer, bovine chemotherapy and snail control
(Gray et al., 2008b). Similar methods would apply for schistosomiasis con-
trol although bovine chemotherapy, bovine vaccination, snail control,
replacement of bovines with tractors and barrier farming for bovines have
been trialled in China (Gray et al., 2012, 2009a, 2007, 2008a, 2009b;
Guo et al., 2006; Hou et al., 2008). In the Philippines, human chemo-
therapy has often been the only method of schistosomiasis control, although
mollusciciding and/or environmental modications (such as removing
vegetation from along waterways) to remove snail habitats have also been
employed.
Climate change is also a potential player with regards to expansion of
certain parasites to new areas, particularly those helminths whose life cycles
involve mosquito or snail vectors, which often have specic climatic re-
quirements (Genchi et al., 2011; Lv et al., 2006; Caminade et al., 2015;
Pedersen et al., 2014). Climate change may also inhibit spread of some dis-
ease vectors due to reductions in suitable environments, an example being
A. cantonensis, for which mathematical models predict will decrease in prev-
alence as a result (Lv et al., 2006).
The control of zoonotic lariasis can involve targeting the insect vectors,
animal reservoirs and prophylaxis or treatment of humans by chemotherapy.
There are currently no human vaccines against any of the larial worms;
however, potential vaccine targets have been identied and veterinary
only vaccines are being trialled (Verma and Jaiswal, 2013; Godel et al.,
2012).
5.3 Molecular tools

Molecular techniques can be invaluable for investigating zoonotic helminths
including their environmental monitoring, diagnosis and species identica-
tion, and the assessment of control interventions, life cycle elucidation and
identication of hosts.
5.3.1 Environmental monitoring/surveillance

Monitoring the environment and animal hosts for zoonotic diseases is an
important tool in control. Due to the morphological similarity of many hel-
minths, molecular methods are ideal to correctly identify species. Speciation
in this case is important for differentiating between animal-only parasites and
those that are zoonotic. Environmental monitoring is already employed for a
number of parasite species such as B. proyonis in the USA since raccoons use
communal areas for defecation, or racoon latrines, where these areas are
examined for eggs (Evans, 2002b; Page et al., 2011, 2014). Raccoon latrines
pose a risk for transmission of B. procyonis to children and animals that may
encroach on the latrines. Infections of B. procyonis have also been found in
dogs, another crossover of a wildlife infection to a domestic animal, and kin-
kajous (honey badger) (Windsor et al., 2009; CDC, 2011; Rudmann et al.,
1996). Sentinel pigs have previously been employed for monitoring the
transmission of T. solium in Africa with some success (Devleesschauwer
et al., 2013; Gonzalez et al., 1994).
Monitoring vectors, such as snails and slugs, and mosquitoes in addition
to denitive hosts is an important way of measuring potential exposure to
infection and implementing control procedures, or for issuing health warn-
ings. The presence of vectors outside of a recognized parasite-endemic area
can be an important indicator of the potential for zoonotic spread into new
transmission zones.
5.3.2 Species identication

Unambiguous and accurate identication of a parasite species is important
when considering potential hosts, control interventions and treatment. As
mentioned above, eggs and larvae of many helminths are morphologically
similar and it is often difcult to identify a particular species. There have
been several cases where the original parasite diagnosis has been retrospec-
tively corrected after molecular analysis, including the identication of the
novel species D. hongkongensis (Suzuki et al., 2015; To et al., 2012). Mito-
chondrial genes, particularly the cox1 and nad genes, and nuclear ribosomal
genes, particularly the ITS1 and ITS2 genes, have been utilized in molecular
identication of helminth parasites infecting humans. Mitochondrial and/or
ribosomal genes for most species are available through NCBI GenBank
(http://www.ncbi.nlm.nih.gov/genbank/).
High resolution melting (HRM) qPCR can be used to distinguish be-
tween closely related parasites (Rojas et al., 2015; Li et al., 2015; Ngui
et al., 2012a). Identication of zoonotic lariasis is most commonly done
after excision of subcutaneous nodules containing the parasite (Dirolaria

spp.) or removal of parasites from eyes (Onchocerca spp.) and other tissues
(Meningonema peruzzii) for morphological examination, histopathological
characteristics and/or DNA sequencing. There are also a number of
commercially available serological tests, particularly for diagnosis of canine
lariasis (SNAP 4Dx, Uranotest Dirolaria, Witness Heartworm, RIM).
The pronounced morphological similarity of many worms, which are not al-
ways amenable for removal without tissue damage, means that morpholog-
ical identication to species is difcult and species identication based solely
on morphology is almost always not reliable or conclusive. Therefore the use
of molecular amplication and/or sequencing and immunological tech-
niques have proved invaluable.
A Russian study investigated human cases (n 8) of echinococcosis using
sequencing of the cox1 gene to differentiate the species, identied Echinococcus
canadensis (genotype G6) as the cause of CE in two of the cases while E. gran-
ulosus sensu stricto (genotype G1) was identied as the cause of CE in the other
six cases; all of the AE cases for which sequence information was obtained
were shown to be caused by Asian type E. multilocularis (Konyaev et al.,
2012). The authors noted that camels, the usual host for E. canadensis,
were not common in the area from which the patients originated (Konyaev
et al., 2012). Other potential hosts of E. canadensis include moose (Alces alces),
Siberian roe deer (Capreolus pygargus) and reindeer (Rangifer tarandus), empha-
sizing that E. canadensis is a potential concern for human CE infection in areas
where camels are absent. Similarly, E. vogeli was diagnosed in a hunter from
French Guiana using mitochondrial DNA sequencing for species differenti-
ation as serology showed patterns for both AE and CE (Knapp et al., 2009).
Further E. canadensis cases have been identied in Mongolia and China using
molecular techniques (Ito et al., 2014; Ma et al., 2015). In Mongolia, of 43
CE cases examined, 31 were due to E. canadensis, while in China a single case
of E. canadensis infection was identied. Molecular diagnosis is an important
approach for Echinococcus spp. identication. Due to the different intermedi-
ate and denitive hosts used by the different species (Nakao et al., 2013), cor-
rect identication of human cases will help with control efforts and in the
monitoring of human disease risk.
Diphyllobothrium latum (the sh or broad tapeworm) is the largest tape-
worm infecting humans, reaching up to 10 m in length (http://www.cdc.
gov/parasites/diphyllobothrium/). Several other Diphyllobothrium spp. have
been reported to infect humans; they include Diphyllobothrium pacicum,
Diphyllobothrium cordatum, Diphyllobothrium ursi, Diphyllobothrium dendriticum,
Diphyllobothrium lanceolatum, Diphyllobothrium dalliae, Diphyllobothrium nihon-

kaiense, and Diphyllobothrium yonagoensis. Although these species are consid-
ered to occur less frequently in humans than D. latum, it is possible that
misdiagnosis has resulted due to the high level of morphological similarity
and/or the poor quality of proglottids obtained for microscopy. In Korea a
retrospective study utilizing the cox1 gene found 62 cases originally identied
as D. latum were actually D. nihonkaiense (Jeon et al., 2009). Retrospective
molecular analysis in China has also found incorrect identication of
D. nihonkaiense as D. latum, although D. latum infection was also conrmed
in some cases (Chen et al., 2014). When looking at case reports of Diphyllo-
bothrium spp. infections since 2010, D. nihonkaiense has been identied more
frequently than D. latum (Table 1). The majority of Diphyllobothrium spp. in-
fections do not cause any adverse events and infected individuals only became
aware of the infection after passing worm segments.
5.3.3 Diagnosis and assessment of control programmes

Assessing the effectiveness of control interventions relies on the diagnostics
used. In the case of schistosomiasis, diagnosis using the thick faecal smear
Kato-Katz (KK) method is the gold standard for egg detection in humans.
However, it is recognized as having a low level of sensitivity, particularly in
low intensity infections (McGarvey et al., 2006; Yu et al., 2007). Similarly,
the KK has been shown to underestimate the prevalence of hookworm
infection by as much as 80% (Easton et al., 2015). Nevertheless, due to
the low cost of implementing the KK (US <4 cents per slide), it remains
a popular choice (Speich et al., 2010) but it must be emphasized that eval-
uating control interventions based on KK data may drastically underestimate
the amount of parasite infection present. The intervention may have
reduced intensity, but it may not necessarily have reduced prevalence. In
villages in China where mass drug (PZQ) administration (MDA) for S. japo-
nicum was terminated, there was a rebound of infection to the original levels
2e15 years later despite the low prevalence and intensity of infections
achieved while the MDA was ongoing (Liang et al., 2006). Nevertheless,
considerable government effort in China over many years has led to a large
reduction in both S. japonicum prevalence and intensity of infection, so that
the elimination of schistosomiasis there is now conceivable. However, the
poor sensitivity of the KK for detecting low intensity infections will
continue to be a problem in China as the control programme continues
towards the goal of elimination, and the authorities will need in future to
ensure that rebound infection does not appear. The true endemic picture
and impact of schistosomiasis control in the P.R. China can only be

measured accurately by implementing more sensitive diagnostic techniques
than are used currently. Accordingly, molecular diagnostics can provide an
essential adjunct for assessing the true picture in areas where S. japonicum
prevalence and intensity are low or where elimination is suspected.
5.4 Success stories

5.4.1 Schistosomiasis and dracunculiasis
Although discussed earlier, it is worth re-emphasizing here that the control
of schistosomiasis in China has been a major success story with the estimated
numbers of infection falling from 12 million in 1949 to about 300,000 in
2011 (Zhou et al., 2004; McManus et al., 2010). Continued surveillance us-
ing better diagnostics will be required to maintain the schistosomiasis control
efforts into the future so that the goal of elimination can be achieved.
A similar perhaps even a greater story of success is dracunculiasis, now
slated for global elimination. Dracunculus medinensis, or guinea worm, is a para-
site of humans in Africa, the Middle East and parts of Asia, primarily India.
Guinea worm disease is contracted when people consume water from stag-
nant sources contaminated with Guinea worm larvae (http://www.cdc.
gov/parasites/guineaworm/). The initiation of the Guinea Worm Eradica-
tion Program (GWEP) has led to a decrease in the number of Dracunculus
infections from 3.5 million in 1986 to 148 reported cases in 2013 (WHO,
2013) to 126 in 2014 (http://www.cartercenter.org/health/guinea_worm/
index.html), and as such is a great success. If successful, Guinea worm will
be the second human disease, after smallpox, to be eradicated. Elimination
has relied on health education due to lack of an effective drug or vaccine.
Challenges to complete eradication include potential zoonotic transmis-
sion. There have been reports of Dracunculus spp. infecting animals such as
dogs and donkeys (Bimi et al., 2005). It is unclear if the species infecting
these animals is D. medinensis or instead has been misidentied due to the
considerable morphological similarity of female worms, as diagnosis is pri-
marily based on microscopy. Male worms of different Dracunculus spp. do
differ morphologically but they are rarely available for study as they die after
copulation. Dracunculus insignis is known to occur in a range of canids (foxes,
coyotes) and felids (bob cat, opossum) in North America (Langlais, 2003;
Lucio-Forster et al., 2014). Sequencing of the 18S rRNA gene of D. insignis
and D. medinensis isolates from raccoons in North America, canids from
Africa and humans in Africa showed that each species was distinct at the
genomic level, although sequence similarity was high (Bimi et al., 2005).
Furthermore, sequences from Dracunculus spp. removed from canids and

humans in Ghana were identical, suggesting that despite being primarily a
helminth of humans, there may be instances where D. medinensis does infect
other animals (Bimi et al., 2005). Indeed, recent studies on the state of
guinea worm in Chad reported D. medinensis in dogs (Al-Awadi et al.,
2014; Eberhard et al., 2014). All dog and human specimens collected in
the Chad study were subject to sequencing using both the 18S rRNA
and cox1 genes (Eberhard et al., 2014). All isolates were identical and posi-
tively identied as D. medinensis. It is therefore possible that dogs can act as
reservoir host for D. medinensis, leading to increased transmission to humans.
Patients in the area were asked a series of dietary questions in order to access
and identify the potential of paratenic hosts in the area which might serve as
reservoirs of transmission. Generally, humans admitted to eating cooked
monitor lizards, snake skin and frogs at certain times of the year (Eberhard
et al., 2014). Dogs had been noted to scavenge and eat raw reptiles and am-
phibians and, particularly, to consume sh entrails (Eberhard et al., 2014).
Dracunculus insignis has been shown to require a paratenic host (Eberhard
and Brandt, 1995), and it is thought that the infection reported in dogs
from Chad, and some of those human infections reported outside the
endemic areas, may have been due to consumption of paratenic hosts
(Eberhard et al., 2014).
It is still unclear whether Dracunculus spp. from animals can infect
humans, and the situation will have to be monitored further.
6. CONCLUSIONS
Under-reporting or misdiagnosis of zoonotic helminths leads to an
underestimation of their importance for human health. Many of the hel-
minth diseases considered in this review are chronic in nature, and it may
be many years before a positive diagnosis is sought and obtained by an
aficted individual. For many zoonotic helminths of public health impor-
tance, appropriate and accurate surveillance of animal hosts and vectors is
essential to determine the risk for human infections and for mapping the
geographical distribution of the diseases they cause. Climate change, urban-
ization and globalization are increasing and/or changing the distribution of
zoonotic helminths. Climate change modies the environment, creating op-
timum temperatures and levels of humidity for survival of STH eggs and
larvae in soil, and for the movement of vectors including certain mosquito
species, and molluscan hosts into new areas, leading to an increased
geographical distribution. However, climate change will not always result
in increased survivability or the spread of zoonotic infections as some areas
will become drier, thereby reducing the survivability of STH and limiting
the spread of mosquitoes and molluscs. As an example, climate change is pre-
dicted to have a detrimental effect on the spread of the molluscan hosts for
Angiostrongylus spp. Based on climate change predictions a model found that
suitable habitats for A. cantonensis would decrease in the future if tempera-
tures increased as per the model (York et al., 2014). The current situation
is that the distribution and number of cases appear to be increasing world-
wide, and the spread of molluscan hosts is helping this (Morassutti et al.,
2014; Thiengo et al., 2007; Deng et al., 2011; Eamsobhana, 2014; Hu
et al., 2011; Qu et al., 2011).
FBH can be readily controlled by implementing food standards and
treatment for imported and locally grown foods, as well as education
regarding the preparation of food. Simply cooking food adequately kills
all zoonotic helminths that may be present in meat or on vegetables and
fruits. Similarly, washing vegetables and fruits before consumption will
remove up to 80% of zoonotic helminths present as eggs, larvae, or within
vectors such as slugs or snails (Adenusi et al., 2015; Duedu et al., 2014;
Yeung et al., 2013), while freezing sh will help the control of shborne
helminths at the consumer end.
Environmental monitoring of soil, vectors and denitive hosts are crucial
for measuring the potential impact of zoonotic helminthiases on human
populations. Education programmes should be undertaken or public warn-
ings should be given in endemic areas when monitoring shows high infec-
tion prevalence in surveyed animals, or during known seasonal transmission
times. Mosquito control is already undertaken in some areas for control of
viral diseases, and the same measures can be implemented for control of
larial nematodes. In the case of B. procyonis, the ability to identify a raccoon
latrine and manage the risk can help prevent human infections. FBH can be
readily controlled through food education and by implementing food stan-
dards and treatment for imported and locally grown foods. In summary, the
overall message is that while globalization, urbanization and climate change
will impact the spread of zoonotic helminths, appropriate awareness of the
risk they pose will help in the future control and prevention of the human
diseases they cause.
SUPPLEMENTARY DATA
Supplementary data related to this article can be found online at http://dx.doi.org/10.1016/
bs.apar.2015.12.002.
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INDEX
Note: Page numbers followed by f indicate gures, t indicate tables, and b indicate boxes.
A Bile duct cancer, 178179

Achatina fulica (A. fulica), 316 Bioinformatic tools, 235
Acidic terminal sequence (ATS), 45 Bovine cysticercosis (Cysticercus bovis), 316
Actin (ACT), 242243 Brazilian case series, 186
Acute hepatitis from HBV, 171t174t, 187 Brefeldin A, 2829
HBsAg, 188
carrier rate, 189 C
AE. See Alveolar echinococcosis (AE) Caenorhabditis elegans (C. elegans), 235236,
Alanine transaminase (ALT), 175 238
Alariosis, 335 Calreticulin (CRT), 242243
Alveolar echinococcosis (AE), 332 Case series, 117118, 133
Ancylostoma spp., 352 Casecontrol studies, 118
A. braziliense, 352 Castration, 296
A. ceylanicum, 352 relief, 286287
Angiostrongylus spp., 341342 Cauliower stage, 281283
A. cantonensis, 341 CD36. See Cluster of Differentiation 36
A. costaricensis, 342 (CD36)
A. vasorum, 341342 CE. See Cystic echinococcosis (CE)
Animal studies, 116117 Chaperones, 3436
Anisakis simplex, 320 Chondroitin sulphate A (CSA), 3839,
Anthelmintics, 234 4647
anti-HCV. See HCV antibodies (anti-HCV) Chronic hepatitis from HBV, 171t174t,
API. See Aspartyl protease inhibitor (API) 187
Aquatic FBHs, 317318. See also Chronic liver disease (CLD), 115. See also
Terrestrial FBHs Hepatitis C virus (HCV)
C. sinensis, 319320 subjects with, 134t140t, 175
chemotherapy of humans, 318 ALT, 175
Diphyllobothrium spp., 320 coinfected CLD patients, 177
sh farms, 318 coinfection with HCV, 176177
shborne infections, 320 liver biopsy from anti-HCV positive
trematode families, 319 patient, 178f
Ascaris lumbricoides (A. lumbricoides), 355 SchistosomaHBV coinfections,
Ascaris suum (A. suum), 355 177178
Aspartyl protease inhibitor (API), serum tests, 176
242243, 251252 CIDR1. See Cysteine-rich interdomain
ATS. See Acidic terminal sequence (ATS) region (CIDR1)
Attachment of activated spores, 275280 CLAG. See Cytoadherence-linked asexual
gene (CLAG)
B CLD. See Chronic liver disease (CLD)
Baylisascariasis, 335341 Climate change, 312314, 357
Baylisascaris procyonis (B. procyonis), 332, hookworm, 352355
335341 Schistosoma spp., 348351
399 j
400 Index
Climate change (Continued ) steps of infection process in, 269,

STHs, 351352 272t273t
zoonotic lariasis, 343348 attachment of activated spores,
CLMs. See Cutaneous larval migrans 275280
(CLMs) development of Pasteuria ramosa, 282b
Clonorchis sinensis (C. sinensis), 319320 dormant parasite spores activation,
Cluster of Differentiation 36 (CD36), 274275
3839 early within-host phase, 281286
Coat proteins (COP), 36 host death and spore competence,
Cohort study, 118119 287288
Collagen biosynthesis, 235236 host encounter with parasite
Computerized tomography scan (CT scan) transmission stages, 270274
scan, 119 host penetration, 280281
Control programmes diagnosis and infection steps in host, 270f
assessment, 360361 late within-host phase, 286287
COP. See Coat proteins (COP) Pasteuria development, 278f
Covert toxocariasis (CT), 8889 variation in disease trait expression,
CRMP. See Cysteine repeat modular 276t277t
proteins (CRMP) using stepwise model, 289
Cross-sectional studies, 118 genetic basis of disease expression,
CRT. See Calreticulin (CRT) 291293
CSA. See Chondroitin sulphate A (CSA) host range evolution, 300
CT. See Covert toxocariasis (CT) host variation, 289291
CUT-1. See Cuticlin-1 (CUT-1) hostparasite coevolution, 298299
Cutaneous larval migrans (CLMs), 352 resistance and costs evolution, 293294
Cuticle collagens, 235236 virulence expression and evolution,
Cuticlin-1 (CUT-1), 242243, 250251 294298
Cyclophilin (CYN), 242243 Dauer hypothesis, 252
Cysteine proteases, 238 DBL1. See Duffy-binding-like domain
Cysteine repeat modular proteins (DBL1)
(CRMP), 4243 2D-DIGE. See Two-dimensional
Cysteine-rich interdomain region difference gel electrophoresis
(CIDR1), 6062 (2D-DIGE)
Cystic echinococcosis (CE), 332 Denitive hosts and vectors, targeting, 357
Cysticercosis human cases, 326t329t Deforestation, 331332
Cysticercus bovis. See Bovine cysticercosis Dicrocoelium dendriticum (D. dendriticum),
(Cysticercus bovis) 323
Cytoadherence-linked asexual gene DIM-1. See Disorganised muscle protein 1
(CLAG), 6667 (DIM-1)
Diphyllobothrium spp., 320
D D. latum, 359360
DALYs. See Disability adjusted life years human cases, 321t
(DALYs) Dirolaria spp., 344346
Daphnia oesophagus, 280 D. immitis, 344346
Daphnias immune system, 283284 D. repens, 346
DaphniaPasteuria system, 268 human infection, 347
Daphnia model, 269b mosquito monitoring, 346347
Index 401
very large antigen-based study, 347 Excretory/secretory proteins (ES proteins),

Disability adjusted life years (DALYs), 312 9495
Disorganised muscle protein 1 (DIM-1), EXP2. See Exported protein 2 (EXP2)
242243 Exported parasite proteins, 3738
DnaJ domains, proteins containing, 5355 MC-associated proteins, 38
DnaJ protein. See Hsp40 less-well characterized proteins,
Dormant parasite spores activation, 274275 4243
Dracunculiasis, 361362 MAHRP1, 39
Dracunculus spp., 361362 MAHRP2, 3940
Drug resistance, 356 parasite proteins, 4344
Duffy-binding-like domain (DBL1), 6062 REX1, 4042
REX2, 42
E SBP1, 3839
Early transcribed membrane proteins tubovesicular network, 4344
(ETRAMPs), 4243 proteins exposed on surface of infected
Early within-host phase, 281286 RBCs, 5960
Ecdysis, 236 CLAG, 6667
Echinococcosis, 332334 exported proteins of sexual stage
Echinococcus spp. parasites, 6869
E. granulosus, 332 glycophorin-binding proteins, 65
E. multilocularis, 332 less-well characterized putative iRBC
human and animal infections with, 333f surface proteins, 6768
EDV. See Electron-dense vesicles (EDV) PfEMP1, 6063
Eicosanoids, 241242 RIFINs, 6364
Electron-dense vesicles (EDV), 34 STEVOR, 64
ELISA. See Enzyme-linked SURFINS, 6465
immunosorbent assay (ELISA) in RBC cytosol or RBC membrane
Endoplasmic reticulum (ER), 414 skeleton, 44
Environmental monitoring/surveillance, FIKK kinases, 5658
358 KAHRP, 4445
Enzyme-linked immunosorbent assay less-well characterized exported
(ELISA), 9091 proteins, 59
Enzymes, 96 MESA, 5556
ER. See Endoplasmic reticulum (ER) Pf332, 4647
Erythrocyte vesicle protein 1 (EVP1), PfEMP3, 4546
4344 PfPTPs, 5859
ES proteins. See Excretory/secretory PHIST proteins, 4752
proteins (ES proteins) proteins containing DnaJ domains,
ESPs. See Excretory/secretory products 5355
(ESPs) RESA, 5253
ETRAMPs. See Early transcribed Exported protein 2 (EXP2), 3233
membrane proteins (ETRAMPs) Exsheathed L3s (xL3s), 240
Evolutionary theory, 295296 Exsheathment, 237
EVP1. See Erythrocyte vesicle protein 1
(EVP1) F
Excretory/secretory products (ESPs), Fascioliasis in humans, 322323
241242 Fasciolopsis buski (F. buski), 322
402 Index
FBA. See Fructose-bisphosphate aldolase Glycophorin-binding proteins, 65

(FBA) GPD. See Glucose-6-phosphate
FBHs. See Foodborne helminths (FBHs) 1-dehydrogenase (GPD)
FIKK kinases, 5658 GPU. See UTP-glucose-1-phosphate
FIKK10.2, 57 uridylyltransferase (GPU)
FIKK12, 5758 GST. See Glutathione S-transferase (GST)
FIKK4.1, 57 Guinea Worm Eradication Program
FIKK4.2, 57 (GWEP), 361
kk7.1, 5758 GWEP. See Guinea Worm Eradication
FIKK9.3, 57 Program (GWEP)
Fish aquaculture, 318 GxE. See Genotype/environment (GxE)
Fish farms, 318 GxG. See Genotype/genotype (GxG)
Foodborne helminths (FBHs), 312. See also
Soil-transmitted helminths (STHs) H
aquatic, 317320 Haycocknema perplexum (H. perplexum), 343
infections, 317 HBsAg. See Hepatitis B surface antigen
terrestrial, 320330 (HBsAg)
Fructose-bisphosphate aldolase (FBA), HBV. See Hepatitis B virus (HBV)
242243 HCC. See Hepatocellular carcinoma
(HCC)
G HCT. See 4-hydroxybutyrate-CoA
Gametocyte erythrocyte cytosolic protein transferase (HCT)
(GECO), 6869 HCV. See Hepatitis C virus (HCV)
Gastrointestinal system (GIT), 324 HCV antibodies (anti-HCV), 120122
GBD study. See Global burden of disease HDP motif, 3435
study (GBD study) Health education, 355356
GBP130. See Glycophorin-binding Heat shock proteins (Hsps), 3435
protein 130 (GBP130) HSP-12.6, 242243
GDP dissociation inhibitor (GDI), Hsp40, 3435
242243 HSP-60, 242243
GECO. See Gametocyte erythrocyte HSP-70, 3435, 242243
cytosolic protein (GECO) Hsp90, 3435
Gender-enriched transcription, 98 Hsp101, 3233
Gene set, 9394 Hepatitis B surface antigen (HBsAg),
Genetic basis of disease expression, 120122
291293 Hepatitis B virus (HBV), 114115
Genome, 9394 SchistosomaHBV coinfections, 116117
Genotype/environment (GxE), 284285 on HBsAg seropositivity, 130131
Genotype/genotype (GxG), 284285 Studies on subjects with acute or chronic
GIT. See Gastrointestinal system (GIT) hepatitis, 171t174t
Global burden of disease study (GBD Hepatitis C virus (HCV), 114115,
study), 312 206209. See also Chronic liver
Glucose-6-phosphate 1-dehydrogenase disease (CLD)
(GPD), 242243 comparing subjects with schistosomiasis,
Glutathione S-transferase (GST), 241242 206209, 211t221t
Glycophorin-binding protein 130 SchistosomaHCV coinfections, 116117
(GBP130), 65 subjects with, 189203, 190t202t
Index 403
anti-HCV seropositivity, 203 ITS-1. See Internal transcribed spacers-1

asymptomatic anti-HCV seropositive (ITS-1)
health-care workers, 205206 ITS-2. See Internal transcribed spacers-2
cohort studies, 204 (ITS-2)
immune responses, 205
Schistosoma species, 203204 K
Hepatocellular carcinoma (HCC), 118 K-PNEPs, 3031
High-resolution melting (HRM), Kato-Katz method (KK method),
352353 360361
Hookworm, 352355 Knob-associated histidine-rich protein
A. suum, 355 (KAHRP), 2829, 4445
T. suis, 355
toxocariasis, 353355 L
Host attachment, 275 Lao PDR. See Peoples Democratic
Host death and spore competence, Republic of Lao (Lao PDR)
287288 Late within-host phase, 286287
Host encounter with parasite transmission Lebanese Halzoun syndrome (LHS), 323
stages, 270274 LIM domain protein (LIM), 242243
Host penetration, 280281 Liver-stage surface antigen 3 (LSA3),
Host range evolution, 300 6768
Host variation, 289291 Liverlung migration of larvae (L3s),
Hostparasite coevolution, 298299 9798
HRM. See High-resolution melting Lysine PEXEL, 2829
(HRM) Lysine-rich membrane-associated PHISTb
Hsps. See Heat shock proteins (Hsps) (LyMP), 50
Hydrolases, 238
4-hydroxybutyrate-CoA transferase M
(HCT), 242243 MAHRP1. See Membrane-associated
histidine-rich protein 1
I (MAHRP1)
ICC. See Intrahepatic cholangiocarcinoma Malaria in humans, 23
(ICC) Malate dehydrogenase (MDH), 242243
IFB. See Intermediate lament protein B MALDI-TOF mass spectrometry,
(IFB) 242243
Immunogenic proteins, 253 Mass drug administration (MDA),
Infection process, 267 360361
Inside out vesicles (IOVs), 50 Matching, 118
Intermediate lament protein B (IFB), Matching allele matrices, 298299
242243 Mature-parasite-infected erythrocyte
Internal transcribed spacers-1 (ITS-1), 91 surface antigen (MESA), 5556
Internal transcribed spacers-2 (ITS-2), 91 Maurers clefts (MCs), 3031, 3637
Intestine-enriched transcription, 97 MC-associated proteins, 38
Intrahepatic cholangiocarcinoma (ICC), less-well characterized proteins, 4243
178179 MAHRP1, 39
IOVs. See Inside out vesicles (IOVs) MAHRP2, 3940
iRBCs. See Parasite-infected RBCs parasite proteins, 4344
(iRBCs) REX1, 4042
404 Index
MC-associated proteins (Continued ) N

REX2, 42 NCC. See Neurocysticercosis (NCC)
SBP1, 3839 Negative frequency-dependent selection
tubovesicular network, 4344 (NFDS), 298299
MCs. See Maurers clefts (MCs) Nematode cuticle, 235
MDA. See Mass drug administration composition, 235236
(MDA) cuticlin proteins and glycoproteins,
MDH. See Malate dehydrogenase 236
(MDH) worms viability, 236
MEC domain. See MESA erythrocyte Nematodes, 234
cytoskeleton-binding domain Neurocysticercosis (NCC), 324
(MEC domain) Neurotoxocariasis (NT), 8889
Membrane-associated histidine-rich NFDS. See Negative frequency-dependent
protein 1 (MAHRP1), 39 selection (NFDS)
Membrane-associated histidine-rich NHR-23, 237238
protein 2 (MAHRP2), 3940 NHR-25, 237238
Merozoite surface protein 7 (MSP7), NHRs. See Nuclear hormone receptors
3031, 43 (NHRs)
Merozoite surface protein 7-related Nongenetic factors, 267
proteins (MSRP), 3031, 43 NT. See Neurotoxocariasis (NT)
MESA. See Mature-parasite-infected Nuclear hormone receptors (NHRs),
erythrocyte surface antigen 237238
(MESA)
MESA erythrocyte cytoskeleton-binding O
domain (MEC domain), 50 Ocular larva migrans (OLM), 8889
Metalloproteases, 238 Oesophagostomum bifurcum (O. bifurcum),
Molecular chaperones, 3435 335
Molecular groups, 9497 Oesophagostomum dentatum (O. dentatum),
Molecular techniques, 357 239
Molecular tools, 357 characterization, 245t248t
diagnosis and assessment of control elucidating aspects of moulting and
programmes, 360361 implications, 241242
environmental monitoring/surveillance, biological pathways, 244f
358 CUT-1, 250251
species identication, 358360 dauer hypothesis, 252
Mosquito monitoring, 346347 differential expression of energy
Moulting process, 236 metabolic enzymes, 249250
C. elegans, 238 downregulation of enzymes, 243249
in different nematode species, 238239 immunogenic proteins, 253
exsheathment, 237 in-depth analyses, 249
morphological changes, 237238 MALDI-TOF mass spectrometry,
in parasitic nematodes, 239 242243
sheath, 236237 molecules, 252
MSP7. See Merozoite surface protein 7 nematode moulting, 243
(MSP7) proteomic approach, 242
MSRP. See Merozoite surface protein TTL-5 and API, 251252
7-related proteins (MSRP) exsheathment process, 240241
Index 405
molecular biology and developmental SURFINS, 6465

processes, 239240 Parasitehost interactions, 266267
studying parasites development and Parasitic worms, 8889
cuticular moulting, 240 Parasitophorous vacuolar membrane
OLM. See Ocular larva migrans (OLM) (PVM), 414
Onchocerca spp., 343344 Parasitophorous vacuole (PV), 414
Opisthorchiidae, 319 Parastrongylus costaricensis (P. costaricensis).
Opisthorchis felineus (O. felineus), 319 See Angiostrongylus costaricensis (A.
costaricensis)
P Parenteral antischistosomal therapy (PAT),
P. falciparum antigen 332 (Pf332), 4647 132133
P. falciparum erythrocyte protein 1 Pasteuria ramosa (P. ramosa), 268
(PfEMP1), 2728, 60 development, 282b
KAHRP, 6263 Pasteuria spores, 274
trafcking, 6062 PAT. See Parenteral antischistosomal
P. falciparum erythrocyte protein 3 therapy (PAT)
(PfEMP3), 4546 PCC. See Propionyl-CoA carboxylase
P. falciparum N-ethylmaleimide-sensitive (PCC)
factor (PfNSF), 36 PCR. See Polymerase chain reaction
P. falciparum proteins involved in (PCR)
trafcking of PfEMP1 (PfPTPs), PDH. See Pyruvate dehydrogenase (PDH)
5859 PEBP. See Phosphatidylethanol-amine
PAM. See Pregnancy-associated malaria binding protein (PEBP)
(PAM) Peoples Democratic Republic of Lao
Parasite proteins, 4344 (Lao PDR), 323324, 349
Parasite-infected erythrocyte surface PEPCK. See Phosphoenolpyruvate
proteins (PIESP), 67 carboxykinase (PEPCK)
Parasite-infected RBCs (iRBCs), 23. Peroxiredoxin (PRDX), 242243
See also Red blood cells (RBCs) PEXEL. See Plasmodium EXport
FIKK kinases, 24t26t ELement (PEXEL)
protein interactions at iRBC membrane PEXEL-negative exported proteins
skeleton, 14t (PNEPs), 3031
protein trafcking within, 3337 Pf332. See P. falciparum antigen 332 (Pf332)
chaperones, 3436 PFA660, 5354
MC, 3637 PFB90, 5354
vesicle-mediated trafcking, 34 PfD80, 50
proteins exposed on surface of infected PFE55, 5354
RBCs, 5960 PfEMP1. See P. falciparum erythrocyte
CLAG, 6667 protein 1 (PfEMP1)
exported proteins of sexual stage PfEMP3. See P. falciparum erythrocyte
parasites, 6869 protein 3 (PfEMP3)
glycophorin-binding proteins, 65 PfHsp70x. See Presence of exported Hsp70
less-well characterized putative iRBC (PfHsp70x)
surface proteins, 6768 PfNSF. See P. falciparum N-ethylmaleimide-
PfEMP1, 6063 sensitive factor (PfNSF)
RIFINs, 6364 PfPTPs. See P. falciparum proteins involved
STEVOR, 64 in trafcking of PfEMP1 (PfPTPs)
406 Index
Phenol oxidase activity (PO activity), Polymerase chain reaction (PCR), 91

283284 Potassium antimony tartarate, 132133
PHIST proteins. See Plasmodium helical PP1. See Protein phosphatase (PP1)
interspersed sub-telomeric proteins Praziquantel (PZQ), 349350
(PHIST proteins) PRDX. See Peroxiredoxin (PRDX)
PHISTa, 4849 Pregnancy-associated malaria (PAM),
PHISTb, 4951 4950
PHISTc, 5152 Presence of exported Hsp70 (PfHsp70x),
Phosphatidylethanol-amine binding 3536, 5354
protein (PEBP), 92, 9596, Primary liver cancer, subjects with,
242243 141t145t, 178179
Phosphoenolpyruvate carboxykinase casecontrol studies, 180
(PEPCK), 242243 HBsAg marker, 179180
PI(3)P-mediated PEXEL function, 2930 liver cancer, 179
PIESP. See Parasite-infected erythrocyte SchistosomaHCV coinfection, 181
surface proteins (PIESP) Propionyl-CoA carboxylase (PCC),
Plant-parasitic nematodes, 234 242243
Plasmepsin V, 2829 Proteases, 238
Plasmepsin V-mediated PEXEL function, Protein phosphatase (PP1), 37
2829 Protein trafcking within iRBCs, 3337
Plasmodium EXport ELement (PEXEL), chaperones, 3436
414, 2728 MC, 3637
PI(3)P-mediated PEXEL function, 2930 vesicle-mediated trafcking, 34
Plasmepsin V-mediated PEXEL function, PTEX proteins. See Plasmodium
2829 translocon of exported proteins
Plasmodium falciparum (P. falciparum), 23 (PTEX proteins)
DnaJ domain, 21t23t PV. See Parasitophorous vacuole (PV)
exported proteins in P. falciparum PVM. See Parasitophorous vacuolar
blood-stage parasites, 5t13t membrane (PVM)
PHIST domain, 15t20t Pyrophosphatases, 238
putative protein export pathways, 27f Pyruvate dehydrogenase (PDH), 242243
Plasmodium helical interspersed PZQ. See Praziquantel (PZQ)
sub-telomeric proteins (PHIST
proteins), 47 Q
PHISTa, 4849 Quantitative trait loci (QTL), 279
PHISTb, 4951
PHISTc, 5152 R
Plasmodium spp., 23 RACK-1. See Receptor for activated
Plasmodium translocon of exported protein kinase C 1 (RACK-1)
proteins (PTEX proteins), 414 RBCs. See Red blood cells (RBCs)
PTEX150, 3233 Receptor for activated protein kinase C 1
PTEX88, 3233 (RACK-1), 242243
role of, 3233 Red blood cells (RBCs), 23. See also
PNEPs. See PEXEL-negative exported Parasite-infected RBCs (iRBCs)
proteins (PNEPs) alteration of host RBC proteins during
PO activity. See Phenol oxidase activity malaria infection, 69
(PO activity) modications, 34
Index 407
parasite replication within, 3 RNAi. See RNA-mediated interference

trafcking of parasite proteins into, (RNAi)
414
DnaJ domain, 21t23t S
exported proteins in P. falciparum S-PNEPs. See Soluble PNEPs (S-PNEPs)
blood-stage parasites, 5t13t SARs. See Structureactivity relationships
FIKK kinases, 24t26t (SARs)
PEXEL motif, 2730 SBP1. See Skeleton-binding protein 1
PHIST domain, 15t20t (SBP1)
PNEPs, 3031 Schistosoma species, 114115, 203204,
protein interactions at iRBC 348349
membrane skeleton, 14t animal studies, 116117
protein trafcking within iRBCs, comparing subjects with schistosomiasis
3337 and HCV, 206209, 211t221t
putative protein export pathways, 27f drug-based intervention trials, 349
role of PTEX, 3233 HCC, 118
Red fox (Vulpes vulpes), 334 HVB and HCV, 115116
Red Queen dynamics. See Negative rodents, 350
frequency-dependent selection S. haematobium, 351
(NFDS) S. japonicum, 349
Relaxed PEXEL, 5253 S. mekongi, 350351
repetitive interspersed family (rif), 6364 schistosomiasis, 115, 119
RESA. See Ring-infected erythrocyte serological markers
surface antigen (RESA) HVB and HCV, 120t121t
RESA-2, 51 in liver disease evaluation, 119t
RESA-like PHISTb proteins, 51 studies on general populations, 122,
Resistance and costs evolution, 293294 123t
Rhoptry contents, 414 HBsAg markers, 130
rif. See repetitive interspersed family (rif) HBsAg seropositivity, 131132
RIFINs, 6364 PAT, 132133
RIFIN A, 6364 Schistosoma-HBV coinfections,
RIFIN B, 6364 130131
Ring-exported protein (REX) studies on special populations, 133
REX1, 3031, 4042 on subjects with acute or chronic
REX2, 42 hepatitis, 171t174t, 187189
REX3, 59 on subjects with chronic liver disease,
REX4, 59 134t140t, 175178
Ring-infected erythrocyte surface antigen subjects with HCV, 189206
(RESA), 414, 5253 on subjects with primary liver cancer,
RNA-mediated interference (RNAi), 141t145t, 178181
99100, 237238 on subjects with schistosomiasis,
RNA-seq technology, 97 146t170t, 181187
RNA-seq technology. See RNA- study design designations, 117119
sequencing technology (RNA-seq Schistosomal liver disease, 175
technology) Schistosomiasis, 115, 119, 361362
RNA-sequencing technology (RNA-seq comparing subjects with HCV and,
technology), 93 206209, 211t221t
408 Index
Schistosomiasis (Continued ) T
subjects with, 146t170t, 181182 Taenia spp., 323324
ALT and AST liver enzyme blood T. asiatica, 324, 325f
levels, 186187 T. crassiceps, 330
Brazilian case series, 186 T. saginata, 324, 325f
casecontrol and cross-sectional studies, T. solium, 324, 325f
185 Temporal storage hypothesis (TSH),
HBsAg seromarker, 182183 295296
with HBV, 184185 Terrestrial FBHs, 320322. See also
HBV infection in liver section, 184f Aquatic FBHs
HCV, 183184 D. dendriticum, 323
SEA. See Southeast Asia (SEA) fascioliasis in humans, 322323
Sexual stage parasites, exported proteins of, human cases of cysticercosis, 326t329t
6869 pseudo-infections, 323
Sheath, 236237 Taenia spp., 324
shRNAmirs. See Small hairpin RNAs T. crassiceps, 330
(shRNAmirs) T. solium, 324
Signal peptide-PNEPs (SP-PNEPs), 3031 unwashed vegetables, 322
Skeleton-binding protein 1 (SBP1), TES. See Toxocara excretory/secretory
3031, 3839 antigen (TES)
Small hairpin RNAs (shRNAmirs), TGD. See Three Gorges Dam (TGD)
100101 Thelazia spp., 347348
Soil-transmitted helminthiases, 234 Thelaziasis, 347348
Soil-transmitted helminths (STHs), 8889, Thioredoxin (Trx2), 3233
312314, 351352. See also Three Gorges Dam (TGD), 349350
Foodborne helminths (FBHs) TM. See Transmembrane domain (TM)
Soluble PNEPs (S-PNEPs), 3031 TM-PNEPs. See Transmembrane PNEPs
Southeast Asia (SEA), 320 (TM-PNEPs)
SP-PNEPs. See Signal peptide-PNEPs TNT. See Troponin T (TNT)
(SP-PNEPs) Toxocara canis (T. canis), 8889. See also
Species identication, 358360 Toxocara spp.
Stage-enriched transcription, 9798 pathogens molecular biology, 93
Stepwise model, 289 gene set, 9394
genetic basis of disease expression, genome, 9394
291293 insights into pathogens biology, 9798
host range evolution, 300 molecular groups, 9497
host variation, 289291 Toxocara excretory/secretory antigen
hostparasite coevolution, 298299 (TES), 9091
resistance and costs evolution, 293294 Toxocara spp., 8889
virulence expression and evolution, infections, 353
294298 large-scale genomic and transcriptomic
STEVOR, 64 analyses, 9293
STHs. See Soil-transmitted helminths new intervention targets in, 98100
(STHs) signicance and diagnostic
Structureactivity relationships (SARs), considerations, 89
99100 biochemical and nucleic acid
SURFINS, 6465 methods, 91
Index 409
identication and differentiation, 90 Vesicle-like structures (VLS), 34

serological techniques, 9091 Vesicle-mediated trafcking, 34
Toxocariasis, 8889, 353355 Virulence expression and evolution,
human cases, 354t 294298
TPM. See Tropomyosin (TPM) Visceral larva migrans (VLM), 8889, 341,
Transcriptomes, pathogens molecular 353
biology, 93 VLM. See Visceral larva migrans (VLM)
genome and gene set, 9394 VLS. See Vesicle-like structures (VLS)
insights into pathogens biology, 9798 Voltagedependent anion-selective
molecular groups, 9497 channel (VDAC), 242243
Transmembrane domain (TM), 6062 VTS. See Vacuolar Transport Signal (VTS)
Transmembrane PNEPs (TM-PNEPs), Vulpes vulpes. See Red fox (Vulpes vulpes)
3031
Transporters, pores and channels, 9697 W
Transthyretin-like protein 5 (TTL-5), Wildlife zoonoses, 334335
242243, 251252 A. cantonensis, 341342
Trichinella spiralis (T. spiralis), 323324 A. costaricensis, 342
Trichostrongylus colubriformis (T. alariosis, 335
colubriformis), 253 H. perplexum, 343
Trichuris suis (T. suis), 355 human (and animal) cases, 336t340t
Tropomyosin (TPM), 242243 Within-host phase, 297
Troponin T (TNT), 242243 World Bank Loan Project (WBLP),
Trx2. See Thioredoxin (Trx2) 349350
TSH. See Temporal storage hypothesis
(TSH) X
TTL-5. See Transthyretin-like protein 5 xL3s. See Exsheathed L3s (xL3s)
(TTL-5)
Tubovesicular network (TVN), 4244 Z
TVN. See Tubovesicular network (TVN) Zoonosis, 312
TVN junction protein (TVN-JP1), 4344 Zoonotic lariasis, 343
TVN-JP1. See TVN junction protein Dirolaria spp., 344347
(TVN-JP1) geographic locations of human and
Two-dimensional difference gel animal infections with, 345f
electrophoresis (2D-DIGE), 242 Onchocerca spp., 343344
Type I DnaJs, 53 Thelazia spp., 347348
Type II DnaJs, 53 Zoonotic helminths, 312
Type III DnaJs, 5355 animal families, 313f
Type IV DnaJs, 5355 climate change
hookworm, 352355
U Schistosoma spp., 348351
UTP-glucose-1-phosphate STHs, 351352
uridylyltransferase (GPU), 242243 zoonotic lariasis, 343348
dracunculiasis, 361362
V globalization, 314316
Vacuolar Transport Signal (VTS), 414 aquatic FBHs, 317320
VDAC. See Voltagedependent anion- FBH infections, 317
selective channel (VDAC) terrestrial FBHs, 320330
410 Index
Zoonotic helminths (Continued ) transmission pathways between wild

health education, 355356 animals, domestic animals and
molecular tools, 357361 humans, 331f
schistosomiasis, 361362 urbanization, 330331
spread, 315f echinococcosis, 332334
targeting denitive hosts and vectors, emerging wildlife zoonoses, 334343
357 environmental modication, 331332
CONTENTS OF VOLUMES IN THIS SERIES
Volume 41 Volume 43
Drug Resistance in Malaria Parasites of Genetic Exchange in the Trypanosomatidae
Animals and Man W. Gibson and J. Stevens
W. Peters
The Host-Parasite Relationship in Neosporosis
Molecular Pathobiology and Antigenic A. Hemphill
Variation of Pneumocystis carinii
Y. Nakamura and M. Wada Proteases of Protozoan Parasites
P.J. Rosenthal
Ascariasis in China
P. Weidono, Z. Xianmin and Proteinases and Associated Genes of Parasitic
D.W.T. Crompton Helminths
J. Tort, P.J. Brindley, D. Knox,
The Generation and Expression of Immunity K.H. Wolfe, and J.P. Dalton
to Trichinella spiralis in Laboratory Rodents
R.G. Bell Parasitic Fungi and their Interaction with the
Insect Immune System
Population Biology of Parasitic Nematodes: A. Vilcinskas and P. Gotz
Application of Genetic Markers
T.J.C. Anderson, M.S. Blouin Volume 44
and R.M. Brech
Cell Biology of Leishmania
Schistosomiasis in Cattle B. Handman
J. De Bont and J. Vercruysse
Immunity and Vaccine Development in the
Volume 42 Bovine Theilerioses
N. Boulter and R. Hall
The Southern Cone Initiative Against Chagas
The Distribution of Schistosoma bovis Sonaino,
Disease
1876 in Relation to Intermediate Host
C. J. Schoeld and J.C.P. Dias
Mollusc-Parasite Relationships
Phytomonas and Other Trypanosomatid H. Mon, G. Mouahid, and S. Morand
Parasites of Plants and Fruit
The Larvae of Monogenea (Platyhelminthes)
E.P. Camargo
H.D. Whittington, L.A. Chisholm, and
Paragonimiasis and the Genus Paragonimus K. Rohde
D. Blair, Z.-B. Xu, and T. Agatsuma
Sealice on Salmonids: Their Biology and
Immunology and Biochemistry of Hymenolepis Control
diminuta A.W. Pike and S.L. Wadsworth
J. Anreassen, E.M. Bennet-Jenkins, and
C. Bryant Volume 45
Control Strategies for Human Intestinal The Biology of some Intraerythrocytic
Nematode Infections Parasites of Fishes, Amphibia and Reptiles
M. Albonico, D.W.T. Cromption, and A.J. Davies and M.R.L. Johnston
L. Savioli
The Range and Biological Activity of FMR
DNA Vaocines: Technology and Applications Famide-related Peptides and Classical
as Anti-parasite and Anti-microbial Agents Neurotransmitters in Nematodes
J.B. Alarcon, G.W. Wainem and D. Brownlee, L. Holden-Dye,
D.P. McManus and R. Walker
411 j
412 Contents of Volumes in This Series
The Immunobiology of Gastrointestinal Forecasting Diseases Risk for Increased

Nematode Infections in Ruminants Epidemic Preparedness in Public Health
A. Balic, V.M. Bowles, and E.N.T. Meeusen M.F. Myers, D.J. Rogers, J. Cox, A. Flauhalt,
and S.I. Hay
Volume 46 Education, Outreach and the Future of

Remote Sensing in Human Health
Host-Parasite Interactions in Acanthocephala: B.L. Woods, L.R. Beck, B.M. Lobitz, and
A Morphological Approach M.R. Bobo
H. Taraschewski
Eicosanoids in Parasites and Parasitic Infections Volume 48
A. Daugschies and A. Joachim The Molecular Evolution of
Trypanosomatidae
Volume 47 J.R. Stevens, H.A. Noyes, C.J. Schoeld, and
W. Gibson
An Overview of Remote Sensing and Geodesy
for Epidemiology and Public Health Transovarial Transmission in the Microsporidia
Application A.M. Dunn, R.S. Terry, and J.E. Smith
S.I. Hay Adhesive Secretions in the Platyhelminthes
Linking Remote Sensing, Land Cover and I.D. Whittington and B.W. Cribb
Disease The Use of Ultrasound in Schistosomiasis
P.J. Curran, P.M. Atkinson, G.M. Foody, and C.F.R. Hatz
E.J. Milton
Ascaris and Ascariasis
Spatial Statistics and Geographic Information D.W.T. Crompton
Systems in Epidemiology and Public
Health Volume 49
T.P. Robinson
Antigenic Variation in Trypanosomes:
Satellites, Space, Time and the African Enhanced Phenotypic Variation in a
Trypanosomiases Eukaryotic Parasite
D.J. Rogers H.D. Barry and R. McCulloch
Earth Observation, Geographic Information The Epidemiology and Control of Human
Systems and Plasmodium falciparum Malaria African Trypanosomiasis
in Sub-Saharan Africa J. Ppin and H.A. Mda
S.I. Hay, J. Omumbo, M. Craig, and
R.W. Snow Apoptosis and Parasitism: from the Parasite to
the Host Immune Response
Ticks and Tick-borne Disease Systems in Space G.A. DosReis and M.A. Barcinski
and from Space
S.E. Randolph Biology of Echinostomes Except Echinostoma
B. Fried
The Potential of Geographical Information
Systems (GIS) and Remote Sensing in the
Epidemiology and Control of Human
Volume 50
Helminth Infections The Malaria-Infected Red Blood Cell:
S. Brooker and E. Michael Structural and Functional Changes
B.M. Cooke, N. Mohandas, and R.L. Coppel
Advances in Satellite Remote Sensing
of Environmental Variables for Schistosomiasis in the Mekong Region:
Epidemiological Applications Epidemiology and Phytogeography
S.J. Goetz, S.D. Prince, and J. Small S.W. Attwood
Contents of Volumes in This Series 413
Molecular Aspects of Sexual Development Enzymes Involved in the Biogenesis of the

and Reproduction in Nematodes and Nematode Cuticle
Schistosomes A.P. Page and A.D. Winter
P.R. Boag, S.E. Newton, and R.B. Gasser
Diagnosis of Human Filariases (Except
Antiparasitic Properties of Medicinal Plants and Onchocerciasis)
Other Naturally Occurring Products M. Walther and R. Muller
S. Tagboto and S. Townson
Volume 54
Volume 51 Introduction d Phylogenies, Phylogenetics,
Aspects of Human Parasites in which Surgical Parasites and the Evolution of Parasitism
Intervention May Be Important D.T.J. Littlewood
D.A. Meyer and B. Fried
Cryptic Organelles in Parasitic Protists and Fungi
Electron-transfer Complexes in Ascaris B.A.P. Williams and P.J. Keeling
Mitochondria
K. Kita and S. Takamiya Phylogenetic Insights into the Evolution
of Parasitism in Hymenoptera
Cestode Parasites: Application of In Vivo and J.B. Whiteld
In Vitro Models for Studies of the
Host-Parasite Relationship Nematoda: Genes, Genomes and the
M. Siles-Lucas and A. Hemphill Evolution of Parasitism
M.L. Blaxter
Volume 52 Life Cycle Evolution in the Digenea: A New

Perspective from Phylogeny
The Ecology of Fish Parasites with Particular T.H. Cribb, R.A. Bray, P.D. Olson, and
Reference to Helminth Parasites and their D.T. J. Littlewood
Salmonid Fish Hosts in Welsh Rivers:
Progress in Malaria Research: The Case for
A Review of Some of the Central
Phylogenetics
Questions
S.M. Rich and F.J. Ayala
J.D. Thomas
Phylogenies, the Comparative Method and
Biology of the Schistosome Genus
Parasite Evolutionary Ecology
Trichobilharzia
S. Morand and R. Poulin
P. Horak, L. Kolarova, and C.M. Adema
Recent Results in Cophylogeny Mapping
The Consequences of Reducing Transmission
M.A. Charleston
of Plasmodium falciparum in Africa
R.W. Snow and K. Marsh Inference of Viral Evolutionary Rates from
Molecular Sequences
Cytokine-Mediated Host Responses during
A. Drummond, O.G. Pybus, and A. Rambaut
Schistosome Infections: Walking the Fine
Line Between Immunological Control Detecting Adaptive Molecular Evolution:
and Immunopathology Additional Tools for the Parasitologist
K.F. Hoffmann, T.A. Wynn, and J.O. Mclnerney, D.T.J. Littlewood, and
D.W. Dunne C.J. Creevey
Volume 53 Volume 55
Interactions between Tsetse and Contents of Volumes 2852
Trypanosomes with Implications for the Cumulative Subject Indexes for Volumes
Control of Trypanosomiasis 2852
S. Aksoy, W.C. Gibson, and M.J. Lehane Contributors to Volumes 2852
Volume 56 Variation in Giardia: Implications for

Taxonomy and Epidemiology
Glycoinositolphospholipid from Trypanosoma R.C.A. Thompson and P.T. Monis
cruzi: Structure, Biosynthesis and
Immunobiology Recent Advances in the Biology of Echinostoma
J.O. Previato, R. Wait, C. Jones, species in the revolutum Group
G.A. DosReis, A.R. Todeschini, N. Heise B. Fried and T.K. Graczyk
and L.M. Previata Human Hookworm Infection in the
Biodiversity and Evolution of the Myxozoa 21st Century
E.U. Canning and B. Okamura S. Brooker, J. Bethony, and P.J. Hotez
The Mitochondrial Genomics of Parasitic The Curious Life-Style of the Parasitic Stages
Nematodes of Socio-Economic of Gnathiid Isopods
Importance: Recent Progress, and N.J. Smit and A.J. Davies
Implications for Population Genetics
and Systematics
M. Hu, N.B. Chilton, and R.B. Gasser
Volume 59
Genes and Susceptibility to Leishmaniasis
The Cytoskeleton and Motility in
Emanuela Handman, Colleen Elso, and Simon
Apicomplexan Invasion
Foote
R.E. Fowler, G. Margos, and G.H. Mitchell
Cryptosporidium and Cryptosporidiosis
Volume 57 R.C.A. Thompson, M.E. Olson, G. Zhu,
S. Enomoto, Mitchell S. Abrahamsen and
Canine Leishmaniasis N.S. Hijjawi
J. Alvar, C. Ca~navate, R. Molina, J. Moreno,
and J. Nieto Ichthyophthirius multiliis Fouquet and
Ichthyophthiriosis in Freshwater Teleosts
Sexual Biology of Schistosomes R.A. Matthews
H. Mon and J. Boissier
Biology of the Phylum Nematomorpha
Review of the Trematode Genus Ribeiroia B. Hanelt, F. Thomas, and A. Schmidt-Rhaesa
(Psilostomidae): Ecology, Life History,
and Pathogenesis with Special Emphasis
on the Amphibian Malformation Problem Volume 60
P.T.J. Johnson, D.R. Sutherland, J.M. Kinsella Sulfur-Containing Amino Acid Metabolism
and K.B. Lunde in Parasitic Protozoa
The Trichuris muris System: A Paradigm of Tomoyoshi Nozaki, Vahab Ali, and Masaharu
Resistance and Susceptibility to Intestinal Tokoro
Nematode Infection The Use and Implications of Ribosomal DNA
L.J. Cliffe and R.K. Grencis Sequencing for the Discrimination of
Scabies: New Future for a Neglected Disease Digenean Species
S.F. Walton, D.C. Holt, B.J. Currie, Matthew J. Nolan and Thomas H. Cribb
and D.J. Kemp Advances and Trends in the Molecular
Systematics of the Parasitic
Volume 58 Platyhelminthes
Peter D. Olson and Vasyl V. Tkach
Leishmania spp.: On the Interactions they
Establish with Antigen-Presenting Cells Wolbachia Bacterial Endosymbionts of Filarial
of their Mammalian Hosts Nematodes
J.-C. Antoine, E. Prina, N. Courret, and Mark J. Taylor, Claudio Bandi, and
T. Lang Achim Hoerauf
The Biology of Avian Eimeria with an Emphasis Implementation of Human Schistosomiasis

on their Control by Vaccination Control: Challenges and Prospects
Martin W. Shirley, Adrian L. Smith, and Alan Fenwick, David Rollinson, and
Fiona M. Tomley Vaughan Southgate
Volume 62
Volume 61
Models for Vectors and Vector-Borne Diseases
Control of Human Parasitic Diseases: Context D.J. Rogers
and Overview
David H. Molyneux Global Environmental Data for Mapping
Infectious Disease Distribution
Malaria Chemotherapy S.I. Hay, A.J. Tatem, A.J. Graham,
Peter Winstanley and Stephen Ward S.J. Goetz, and D.J. Rogers
Insecticide-Treated Nets Issues of Scale and Uncertainty in the Global
Jenny Hill, Jo Lines, and Mark Rowland Remote Sensing of Disease
Control of Chagas Disease P.M. Atkinson and A.J. Graham
Yoichi Yamagata and Jun Nakagawa Determining Global Population Distribution:
Human African Trypanosomiasis: Methods, Applications and Data
Epidemiology and Control D.L. Balk, U. Deichmann, G. Yetman,
E.M. Fvre, K. Picozzi, J. Jannin, F. Pozzi, S.I. Hay, and A. Nelson
S.C. Welburn and I. Maudlin Dening the Global Spatial Limits of Malaria
Chemotherapy in the Treatment and Control Transmission in 2005
of Leishmaniasis C.A. Guerra, R.W. Snow and
Jorge Alvar, Simon Croft, and Piero Olliaro S.I. Hay
Dracunculiasis (Guinea Worm Disease) The Global Distribution of Yellow Fever and
Eradication Dengue
Ernesto Ruiz-Tiben and Donald R. Hopkins D.J. Rogers, A.J. Wilson, S.I. Hay, and
A.J. Graham
Intervention for the Control of
Soil-Transmitted Helminthiasis in Global Epidemiology, Ecology and Control
the Community of Soil-Transmitted Helminth Infections
Marco Albonico, Antonio Montresor, S. Brooker, A.C.A. Clements and
D.W.T. Crompton, and Lorenzo D.A.P. Bundy
Savioli Tick-borne Disease Systems: Mapping
Control of Onchocerciasis Geographic and Phylogenetic Space
Boakye A. Boatin and S.E. Randolph and D.J. Rogers
Frank O. Richards, Jr. Global Transport Networks and Infectious
Lymphatic Filariasis: Treatment, Control and Disease Spread
Elimination A.J. Tatem, D.J. Rogers and S.I. Hay
Eric A. Ottesen Climate Change and Vector-Borne Diseases
Control of Cystic Echinococcosis/Hydatidosis: D.J. Rogers and S.E. Randolph
1863-2002
P.S. Craig and E. Larrieu Volume 63
Control of Taenia solium Cysticercosis/ Phylogenetic Analyses of Parasites in the New
Taeniosis Millennium
Arve Lee Willingham III and Dirk Engels David A. Morrison
Targeting of Toxic Compounds to the T. Sicheritz-Ponten, P. G. Foster,

Trypanosomes Interior J. Samuelson, C.J. Nol, R.P. Hirt,
Michael P. Barrett and Ian H. Gilbert T.M. Embley, C. A. Gilchrist,
B.J. Mann, U. Singh, J.P. Ackers,
Making Sense of the Schistosome Surface S. Bhattacharya, A. Bhattacharya,
Patrick J. Skelly and R. Alan Wilson A. Lohia, N. Guilln, M. Duchene,
Immunology and Pathology of Intestinal T. Nozaki, and N. Hall
Trematodes in Their Denitive Hosts Epidemiological Modelling for Monitoring
Rafael Toledo, Jos-Guillermo Esteban, and and Evaluation of Lymphatic Filariasis
Bernard Fried Control
Systematics and Epidemiology of Trichinella Edwin Michael, Mwele N. Malecela-Lazaro,
Edoardo Pozio and K. Darwin Murrell and James W. Kazura
The Role of Helminth Infections in
Volume 64 Carcinogenesis
David A. Mayer and Bernard Fried
Leishmania and the Leishmaniases: A Parasite
A Review of the Biology of the
Genetic Update and Advances in
Parasitic Copepod Lernaeocera
Taxonomy, Epidemiology and
branchialis (L., 1767)(Copepoda:
Pathogenicity in Humans
Pennellidae
Anne-Laure Ba~nuls, Mallorie Hide and
Adam J. Brooker, Andrew P. Shinn, and
Franck Prugnolle
James E. Bron
Human Waterborne Trematode and
Protozoan Infections Volume 66
Thaddeus K. Graczyk and Bernard Fried
Strain Theory of Malaria: The First 50 Years
The Biology of Gyrodctylid Monogeneans: F. Ellis McKenzie,* David L. Smith,
The Russian-Doll Killers Wendy P. OMeara, and
T.A. Bakke, J. Cable, and P.D. Harris Eleanor M. Riley
Human Genetic Diversity and the Advances and Trends in the Molecular
Epidemiology of Parasitic and Other Systematics of Anisakid Nematodes, with
Transmissible Diseases Implications for their Evolutionary
Michel Tibayrenc Ecology and HostParasite
Co-evolutionary Processes
Simonetta Mattiucci and Giuseppe Nascetti
Volume 65
Atopic Disorders and Parasitic Infections
ABO Blood Group Phenotypes and Aditya Reddy and Bernard Fried
Plasmodium falciparum Malaria: Unlocking
a Pivotal Mechanism Heartworm Disease in Animals and Humans
Mara-Paz Loscertales, Stephen Owens, John W. McCall, Claudio Genchi, Laura
James ODonnell, James Bunn, H. Kramer, Jorge Guerrero, and
Xavier Bosch-Capblanch, and Luigi Venco
Bernard J. Brabin
Structure and Content of the Entamoeba Volume 67
histolytica Genome Introduction
C.G. Clark, U.C.M. Alsmark, Irwin W. Sherman
M. Tazreiter, Y. Saito-Nakano, V. Ali,
S. Marion, C. Weber, C. Mukherjee, An Introduction to Malaria Parasites
I. Bruchhaus, E. Tannich, M. Leippe, Irwin. W. Sherman
The Early Years Polyamines

Irwin W. Sherman Irwin W. Sherman
Show Me the Money New Permeability Pathways and Transport
In Vivo and In Vitro Models Hemoglobinases
Malaria Pigment Erythrocyte Surface Membrane Proteins
Chloroquine and Hemozoin Trafcking
Invasion of Erythrocytes Erythrocyte Membrane Lipids
Vitamins and Anti-Oxidant Defenses
Irwin W. Sherman
Volume 68
HLA-Mediated Control of HIV and HIV
Shocks and Clocks
Adaptation to HLA
Irwin W. Sherman
Rebecca P. Payne, Philippa C. Matthews,
Transcriptomes, Proteomes and Data Julia G. Prado, and Philip J.R. Goulder
Mining
An Evolutionary Perspective on Parasitism as a
Irwin W. Sherman
Cause of Cancer
Mosquito Interactions Paul W. Ewald
Irwin W. Sherman
Invasion of the Body Snatchers: The Diversity
Isoenzymes and Evolution of Manipulative Strategies
Irwin W. Sherman in HostParasite Interactions
Thierry Lefvre, Shelley A. Adamo, David G.
The Road to the Plasmodium falciparum Biron, Dorothe Miss , David Hughes, and
Genome Frdric Thomas
Irwin W. Sherman
Evolutionary Drivers of Parasite-Induced
Carbohydrate Metabolism Changes in Insect Life-History Traits:
Irwin W. Sherman From Theory to Underlying Mechanisms
Pyrimidines and the Mitochondrion Hilary Hurd
Irwin W. Sherman Ecological Immunology of a Tapeworms
The Road to Atovaquone Interaction with its Two Consecutive
Irwin W. Sherman Hosts
Katrin Hammerschmidt and
The Ring Road to the Apicoplast Joachim Kurtz
Irwin W. Sherman
Tracking Transmission of the Zoonosis
Ribosomes and Ribosomal Ribonucleic Acid Toxoplasma gondii
Synthesis Judith E. Smith
Irwin W. Sherman
Parasites and Biological Invasions
De Novo Synthesis of Pyrimidines and Folates Alison M. Dunn
Irwin W. Sherman
Zoonoses in Wildlife: Integrating Ecology into
Salvage of Purines Management
Irwin W. Sherman Fiona Mathews
Understanding the Interaction Between an Volume 70

Obligate Hyperparasitic Bacterium,
Pasteuria penetrans and its Obligate Ecology and Life History Evolution of
Plant-Parasitic Nematode Host, Frugivorous Drosophila Parasitoids
Meloidogyne spp. Frdric Fleury, Patricia Gibert, Nicolas Ris, and
Keith G. Davies Roland Allemand
HostParasite Relations and Implications for Decision-Making Dynamics in Parasitoids of

Control Drosophil
Alan Fenwick Andra Thiel and Thomas S. Hoffmeister
OnchocercaSimulium Interactions and the Dynamic Use of Fruit Odours to Locate Host
Population and Evolutionary Biology of Larvae: Individual Learning, Physiological
Onchocerca volvulus State and Genetic Variability as Adaptive
Mara-Gloria Basa~nez, Thomas S. Churcher, Mechanisms
and Mara-Eugenia Grillet Laure Kaiser, Aude Couty, and
Raquel Perez-Maluf
Microsporidians as Evolution-Proof Agents of
Malaria Control? The Role of Melanization and Cytotoxic
Jacob C. Koella, Lena Lorenz, and By-Products in the Cellular Immune
Irka Bargielowski Responses of Drosophila Against Parasitic
Wasps
A. Nappi, M. Poiri, and Y. Carton
Virulence Factors and Strategies of Leptopilina
Volume 69 spp.: Selective Responses in Drosophila
Hosts
The Biology of the Caecal Trematode
Mark J. Lee, Marta E. Kalamarz,
Zygocotyle lunata
Indira Paddibhatla, Chiyedza Small,
Bernard Fried, Jane E. Huffman, Shamus Keeler,
Roma Rajwani, and Shubha Govind
and Robert C. Peoples
Variation of Leptopilina boulardi Success in
Fasciola, Lymnaeids and Human Fascioliasis,
Drosophila Hosts: What is Inside the Black
with a Global Overview on Disease
Box?
Transmission, Epidemiology,
A. Dubuffet, D. Colinet, C. Anselme,
Evolutionary Genetics, Molecular
S. Dupas, Y. Carton, and M. Poiri
Epidemiology and Control
Santiago Mas-Coma, Mara Adela Valero, and Immune Resistance of Drosophila Hosts Against
Mara Dolores Bargues Asobara Parasitoids: Cellular Aspects
Patrice Eslin, Genevieve Prvost, Sebastien
Recent Advances in the Biology of
Havard, and Graldine Doury
Echinostomes
Rafael Toledo, Jos-Guillermo Esteban, and Components of Asobara Venoms and their
Bernard Fried Effects on Hosts
Sbastien J.M. Moreau, Sophie Vinchon, Anas
Peptidases of Trematodes
Cherqui, and Genevieve Prvost
Martin Kasn, Libor Mikes, Vladimr Hampl,
Jan Dvorak, Conor R. Caffrey, John P. Strategies of Avoidance of Host Immune
Dalton, and Petr Horak Defenses in Asobara Species
Genevive Prevost, Graldine Doury, Alix D.N.
Potential Contribution of
Mabiala-Moundoungou, Anas Cherqui, and
Sero-Epidemiological Analysis for
Patrice Eslin
Monitoring Malaria Control and
Elimination: Historical and Current Evolution of Host Resistance and Parasitoid
Perspectives Counter-Resistance
Chris Drakeley and Jackie Cook Alex R. Kraaijeveld and H. Charles J. Godfray
Local, Geographic and Phylogenetic Scales of Coordinating Research on Neglected Parasitic

Coevolution in DrosophilaParasitoid Diseases in Southeast Asia Through
Interactions Networking
S. Dupas, A. Dubuffet, Y. Carton, and Remi Olveda, Lydia Leonardo, Feng Zheng,
M. Poiri Banchob Sripa, Robert Bergquist, and
Xiao-Nong Zhou
DrosophilaParasitoid Communities as
Model Systems for HostWolbachia Neglected Diseases and Ethnic Minorities in
Interactions the Western Pacic Region: Exploring
Fabrice Vavre, Laurence Mouton, and Bart the Links
A. Pannebakker Alexander Schratz, Martha
Fernanda Pineda, Liberty G. Reforma,
A Virus-Shaping Reproductive Strategy in a Nicole M. Fox, Tuan Le Anh,
Drosophila Parasitoid L. Tommaso Cavalli-Sforza,
Julien Varaldi, Sabine Patot, Mackenzie K. Henderson,
Maxime Nardin, and Raymond Mendoza, Jurg Utzinger,
Sylvain Gandon John P. Ehrenberg, and
Ah Sian Tee
Volume 71 Controlling Schistosomiasis in Southeast Asia:
A Tale of Two Countries
Cryptosporidiosis in Southeast Robert Bergquist and Marcel Tanner
Asia: Whats out There?
Yvonne A.L. Lim, Aaron R. Jex, Schistosomiasis Japonica: Control and
Huw V. Smith, and Robin B. Gasser Research Needs
Xiao-Nong Zhou, Robert Bergquist,
Human Schistosomiasis in the Economic Lydia Leonardo, Guo-Jing Yang,
Community of West African States: Kun Yang, M. Sudomo, and
Epidemiology and Control Remigio Olveda
Hlen Mon, Moudachirou Ibikounl,
Achille Massougbodji, Schistosoma mekongi in Cambodia and Lao
and Gabriel Mouahid Peoples Democratic Republic
Sinuon Muth, Somphou Sayasone,
The Rise and Fall of Human Sophie Odermatt-Biays,
Oesophagostomiasis Samlane Phompida, Socheat Duong, and
A.M. Polderman, M. Eberhard, S. Baeta, Peter Odermatt
Robin B. Gasser, L. van Lieshout,
P. Magnussen, A. Olsen, N. Spannbrucker, Elimination of Lymphatic Filariasis in
J. Ziem, and J. Horton Southeast Asia
Mohammad Sudomo, Sombat
Chayabejara, Duong Socheat,
Volume 72 Leda Hernandez, Wei-Ping Wu, and
Robert Bergquist
Important Helminth Infections in Southeast
Asia: Diversity, Potential for Control and Combating Taenia solium Cysticercosis in
Prospects for Elimination Southeast Asia: An Opportunity for
Jurg Utzinger, Robert Bergquist, Remigio Improving Human Health and Livestock
Olveda, and Xiao-Nong Zhou Production Links
A. Lee Willingham III, Hai-Wei Wu,
Escalating the Global Fight Against James Conlan, and Fadjar Satrija
Neglected Tropical Diseases Through
Interventions in the Asia Pacic Echinococcosis with Particular Reference to
Region Southeast Asia
Peter J. Hotez and John P. Ehrenberg Donald P. McManus
Food-Borne Trematodiases in Southeast Asia: Towards Improved Diagnosis of

Epidemiology, Pathology, Clinical Zoonotic Trematode Infections in
Manifestation and Control Southeast Asia
Banchob Sripa, Sasithorn Kaewkes, Pewpan M. Maria Vang Johansen, Paiboon
Intapan, Wanchai Maleewong, and Sithithaworn, Robert Bergquist, and
Paul J. Brindley Jurg Utzinger
Helminth Infections of the Central Nervous The Drugs We Have and the Drugs
System Occurring in Southeast Asia and We Need Against Major Helminth
the Far East Infections
Shan Lv, Yi Zhang, Peter Steinmann, Jennifer Keiser and Jurg Utzinger
Xiao-Nong Zhou, and Jurg Utzinger
Research and Development of
Less Common Parasitic Infections in Southeast Antischistosomal Drugs in the Peoples
Asia that can Produce Outbreaks Republic of China: A 60-Year Review
Peter Odermatt, Shan Lv, and Somphou Sayasone Shu-Hua Xiao, Jennifer Keiser,
Ming-Gang Chen, Marcel Tanner,
and Jurg Utzinger
Volume 73
Control of Important Helminthic Infections:
Concepts in Research Capabilities Vaccine Development as Part of the
Strengthening: Positive Experiences of Solution
Network Approaches by TDR in the Robert Bergquist and Sara Lustigman
Peoples Republic of China and
Eastern Asia Our Wormy World: Genomics, Proteomics
Xiao-Nong Zhou, Steven Wayling, and and Transcriptomics in East and Southeast
Robert Bergquist Asia
Jun Chuan, Zheng Feng,
Multiparasitism: A Neglected Reality on Paul J. Brindley, Donald P. McManus,
Global, Regional and Local Scale Zeguang Han, Peng Jianxin, and Wei Hu
Peter Steinmann, Jurg Utzinger,
Zun-Wei Du, and Xiao-Nong Zhou Advances in Metabolic Proling of
Experimental Nematode and Trematode
Health Metrics for Helminthic Infections Infections
Charles H. King Yulan Wang, Jia V. Li, Jasmina Saric, Jennifer
Implementing a Geospatial Health Data Keiser, Junfang Wu, Jurg Utzinger, and
Infrastructure for Control of Asian Elaine Holmes
Schistosomiasis in the Peoples Republic Studies on the Parasitology, Phylogeography
of China and the Philippines and the Evolution of HostParasite
John B. Malone, Guo-Jing Yang, Lydia Interactions for the Snail Intermediate
Leonardo, and Xiao-Nong Zhou Hosts of Medically Important Trematode
The Regional Network for Asian Genera in Southeast Asia
Schistosomiasis and Other Helminth Stephen W. Attwood
Zoonoses (RNAS+): Target Diseases
in Face of Climate Change
Guo-Jing Yang, Jurg Utzinger, Shan Lv,
Volume 74
Ying-Jun Qian, Shi-Zhu Li, Qiang Wang, The Many Roads to Parasitism: A Tale of
Robert Bergquist, Penelope Vounatsou, Convergence
Wei Li, Kun Yang, and Xiao-Nong Zhou Robert Poulin
Social Science Implications for Control of Malaria Distribution, Prevalence, Drug
Helminth Infections in Southeast Asia Resistance and Control in Indonesia
Lisa M. Vandemark, Tie-Wu Jia, and Iqbal R.F. Elyazar, Simon I. Hay, and
Xiao-Nong Zhou J. Kevin Baird
Cytogenetics and Chromosomes of Advances in Imaging of Animal Models of

Tapeworms (Platyhelminthes, Cestoda) Chagas Disease
Marta Spakulova, Martina Orosova, and Linda A. Jelicks and Herbert B. Tanowitz
John S. Mackiewicz
The Genome and Its Implications
Soil-Transmitted Helminths of Humans in Santuza M. Teixeira, Najib M. El-Sayed,
Southeast AsiadTowards Integrated and Patrcia R. Araujo
Control
Aaron R. Jex, Yvonne A.L. Lim, Jeffrey Genetic Techniques in Trypanosoma cruzi
Bethony, Peter J. Hotez, Martin C. Taylor, Huan Huang, and
Neil D. Young, and Robin B. Gasser John M. Kelly
The Applications of Model-Based Geostatistics Nuclear Structure of Trypanosoma cruzi

in Helminth Epidemiology and Control Sergio Schenkman, Bruno dos Santos
Ricardo J. Soares Magalh~aes, Archie C.A. Pascoalino, and Sheila C. Nardelli
Clements, Anand P. Patil, Aspects of Trypanosoma cruzi Stage
Peter W. Gething, and Simon Brooker Differentiation
Samuel Goldenberg and Andrea
Volume 75
Rodrigues Avila
Epidemiology of American Trypanosomiasis The Role of Acidocalcisomes in the Stress

(Chagas Disease) Response of Trypanosoma cruzi
Louis V. Kirchhoff Roberto Docampo, Veronica Jimenez,
Sharon King-Keller, Zhu-hong Li, and
Acute and Congenital Chagas Disease Silvia N.J. Moreno
Caryn Bern, Diana L. Martin, and
Robert H. Gilman Signal Transduction in Trypanosoma cruzi
Huan Huang
Cell-Based Therapy in Chagas Disease
Antonio C. Campos de Carvalho,
Adriana B. Carvalho, and Regina Volume 76
C.S. Goldenberg Bioactive Lipids in Trypanosoma cruzi Infection
Targeting Trypanosoma cruzi Sterol Fabiana S. Machado, Shankar Mukherjee,
14a-Demethylase (CYP51) Louis M. Weiss, Herbert B. Tanowitz, and
Galina I. Lepesheva, Fernando Villalta, Anthony W. Ashton
and Michael R. Waterman Mechanisms of Host Cell Invasion by
Experimental Chemotherapy and Approaches Trypanosoma cruzi
to Drug Discovery for Trypanosoma cruzi Kacey L. Caradonna and Barbara
Infection A. Burleigh
Frederick S. Buckner Gap Junctions and Chagas Disease
Vaccine Development Against Trypanosoma Daniel Adesse, Regina Coeli Goldenberg, Fabio
cruzi and Chagas Disease S. Fortes, Jasmin, Dumitru A. Iacobas, Sanda
Juan C. Vazquez-Chagoyan, Iacobas, Antonio Carlos Campos de
Shivali Gupta, and Nisha Jain Garg Carvalho, Maria de Narareth
Meirelles, Huan Huang, Milena B. Soares,
Genetic Epidemiology of Chagas Disease Herbert B. Tanowitz, Luciana Ribeiro
Sarah Williams-Blangero, John L. VandeBerg, Garzoni, and David C. Spray
John Blangero, and Rodrigo Corra-Oliveira
The Vasculature in Chagas Disease
Kissing Bugs. The Vectors of Chagas Cibele M. Prado, Linda A. Jelicks,
Lori Stevens, Patricia L. Dorn, Justin O. Schmidt, Louis M. Weiss, Stephen M. Factor,
John H. Klotz, David Lucero, and Herbert B. Tanowitz, and
Stephen A. Klotz Marcos A. Rossi
Infection-Associated Vasculopathy in Assessment and Monitoring of Onchocerciasis

Experimental Chagas Disease: in Latin America
Pathogenic Roles of Endothelin and Mario A. Rodrguez-Prez,
Kinin Pathways Thomas R. Unnasch, and
Julio Scharfstein and Daniele Andrade Olga Real-Najarro
Autoimmunity
Edecio Cunha-Neto, Priscila Camillo Volume 78
Teixeira, Luciana Gabriel Nogueira, Gene Silencing in Parasites: Current Status and
and Jorge Kalil Future Prospects
ROS Signalling of Inammatory Cytokines Raul Manzano-Roman, Ana Oleaga,
During Trypanosoma cruzi Infection Ricardo Prez-Sanchez, and
Shivali Gupta, Monisha Dhiman, Jian-jun Mar Siles-Lucas
Wen, and Nisha Jain Garg GiardiadFrom Genome to Proteome
Inammation and Chagas Disease: Some R.C. Andrew Thompson and Paul Monis
Mechanisms and Relevance Malaria Ecotypes and Stratication
Andr Talvani and Mauro M. Teixeira Allan Schapira and Konstantina Boutsika
Neurodegeneration and Neuroregeneration in The Changing Limits and Incidence of Malaria
Chagas Disease in Africa: 19392009
Marina V. Chuenkova and Mercio Robert W. Snow, Punam Amratia,
PereiraPerrin Caroline W. Kabaria, Abdisalan M. Noor,
Adipose Tissue, Diabetes and Chagas Disease and Kevin Marsh
Herbert B. Tanowitz, Linda A. Jelicks,
Fabiana S. Machado, Lisia Esper, Volume 79
Xiaohua Qi, Mahalia S. Desruisseaux,
Streamson C. Chua, Philipp E. Scherer, Northern Host Parasite Assemblages:
and Fnu Nagajyothi History and Biogeography on the
Borderlands of Episodic Climate and
Environmental Transition
Volume 77 Eric P. Hoberg, Kurt E. Galbreath,
Joseph A. Cook, Susan J. Kutz, and
Coinfection of Schistosoma (Trematoda) with Lydden Polley
Bacteria, Protozoa and Helminths
Amy Abruzzi and Bernard Fried Parasites in Ungulates of Arctic North America
and Greenland: A View of Contemporary
Trichomonas vaginalis Pathobiology: New Diversity, Ecology and Impact in a World
Insights from the Genome Sequence Under Change
Robert P. Hirt, Natalia de Miguel, Susan J. Kutz, Julie Ducrocq, Guilherme
Sirintra Nakjang, Daniele Dessi, G. Verocai, Bryanne M. Hoar, Doug D.
Yuk-Chien Liu, Nicia Diaz, Colwell, Kimberlee B. Beckmen,
Paola Rappelli, Alvaro Acosta-Serrano, Lydden Polley, Brett T. Elkin, and
Pier-Luigi Fiori, and Jeremy C. Mottram Eric P. Hoberg
Cryptic Parasite Revealed: Improved Prospects Neorickettsial Endosymbionts of the Digenea:
for Treatment and Control of Human Diversity, Transmission and Distribution
Cryptosporidiosis Through Advanced Jefferson A. Vaughan, Vasyl V. Tkach, and
Technologies Stephen E. Greiman
Aaron R. Jex, Huw V. Smith, Matthew
J. Nolan, Bronwyn E. Campbell, Priorities for the Elimination of Sleeping
Neil D. Young, Cinzia Cantacessi, and Sickness
Robin B. Gasser Susan C. Welburn and Ian Maudlin
Scabies: Important Clinical Consequences Natural Acquisition of Immunity to

Explained by New Molecular Studies Plasmodium vivax: Epidemiological
Katja Fischer, Deborah Holt, Bart Currie, and Observations and Potential Targets
David Kemp Ivo Mueller, Mary R. Galinski, Takafumi
Tsuboi, Myriam Arevalo-Herrera,
Review: Surveillance of Chagas Disease William E. Collins, and
Ken Hashimoto and Kota Yoshioka Christopher L. King
Volume 80 G6PD Deciency: Global Distribution,
Genetic Variants and Primaquine Therapy
The Global Public Health Signicance of Rosalind E. Howes, Katherine E. Battle,
Plasmodium vivax Ari W. Satyagraha, J. Kevin Baird, and
Katherine E. Battle, Peter W. Gething, Simon I. Hay
Iqbal R.F. Elyazar,
Catherine L. Moyes, Maranne E. Sinka, Genomics, Population Genetics and
Rosalind E. Howes, Carlos A. Guerra, Evolutionary History of Plasmodium vivax
Ric N. Price, J. Kevin Baird, and Jane M. Carlton, Aparup Das, and
Simon I. Hay Ananias A. Escalante
Relapse Malariotherapy Insanity at the Service of
Nicholas J. White and Mallika Imwong Malariology
Georges Snounou and Jean-Louis Prignon
Plasmodium vivax: Clinical Spectrum, Risk
Factors and Pathogenesis
Nicholas M. Anstey, Nicholas M. Douglas, Volume 82
Jeanne R. Poespoprodjo, and
Ric N. Price Recent Developments in Blastocystis Research
C. Graham Clark, Mark van der Giezen,
Diagnosis and Treatment of Plasmodium vivax Mohammed A. Alfellani, and
Malaria C. Rune Stensvold
J. Kevin Baird, Jason D. Maguire, and
Ric N. Price Tradition and Transition: Parasitic Zoonoses of
People and Animals in Alaska, Northern
Chemotherapeutic Strategies for Canada, and Greenland
Reducing Transmission of Plasmodium Emily J. Jenkins, Louisa J. Castrodale,
vivax Malaria Simone J.C. de Rosemond, Brent R. Dixon,
Nicholas M. Douglas, George K. John, Stacey A. Elmore, Karen M. Gesy,
Lorenz von Seidlein, Nicholas M. Anstey, Eric P. Hoberg, Lydden Polley,
and Ric N. Price Janna M. Schurer, Manon Simard, and
R.C. Andrew Thompson
Control and Elimination of Plasmodium vivax
G. Dennis Shanks The Malaria Transition on the Arabian
Peninsula: Progress toward a Malaria-Free
Volume 81 Region between 1960-2010
Robert W. Snow, Punam Amratia, Ghasem
Plasmodium vivax: Modern Strategies Zamani, Clara W. Mundia, Abdisalan M.
to Study a Persistent Parasites Noor, Ziad A. Memish, Mohammad H. Al
Life Cycle Zahrani, Adel AlJasari, Mahmoud Fikri, and
Mary R. Galinski, Esmeralda V.S. Meyer, and Hoda Atta
John W. Barnwell
Microsporidia and The Art of Living
Red Blood Cell Polymorphism and Together
Susceptibility to Plasmodium vivax Jir Vavra and Julius Lukes
Peter A. Zimmerman, Marcelo U. Ferreira,
Rosalind E. Howes, and Odile Patterns and Processes in Parasite Co-Infection
Mercereau-Puijalon Mark E. Viney and Andrea L. Graham
Volume 83 Volume 85
IronSulphur Clusters, Their Biosynthesis, Diversity and Ancestry of Flatworms Infecting
and Biological Functions in Protozoan Blood of Nontetrapod Craniates Fishes
Parasites Raphael Orlis-Ribeiro, Cova R. Arias,
Vahab Ali and Tomoyoshi Nozaki Kenneth M. Halanych,Thomas H. Cribb,
and Stephen A. Bullard
A Selective Review of Advances in Coccidiosis
Research Techniques for the Diagnosis of Fasciola
H. David Chapman, John R. Barta, Infections in Animals: Room for
Damer Blake, Arthur Gruber, Mark Jenkins, Improvement
Nicholas C. Smith, Xun Suo, and Cristian A. Alvarez Rojas, Aaron R. Jex,
Fiona M. Tomley Robin B. Gasser, and
Jean-Pierre Y. Scheerlinck
The Distribution and Bionomics of
Anopheles Malaria Vector Mosquitoes in Reevaluating the Evidence for Toxoplasma
Indonesia gondii-Induced Behavioural Changes in
Iqbal R.F. Elyazar, Marianne E. Sinka, Rodents
Peter W. Gething, Siti N. Tarmidzi, Amanda R. Worth, R.C. Andrew Thompson,
Asik Surya, Rita Kusriastuti, Winarno, and Alan J. Lymbery
J. Kevin Baird, Simon I. Hay, and
Michael J. Bangs Volume 86
Next-Generation Molecular-Diagnostic Tools Historical Patterns of Malaria Transmission in
for Gastrointestinal Nematodes of China
Livestock, with an Emphasis on Small Jian-Hai Yin, Shui-Sen Zhou, Zhi-Gui Xia,
Ruminants: A Turning Point? Ru-Bo Wang, Ying-Jun Qian, Wei-Zhong
Florian Roeber, Aaron R. Jex, and Yang, and Xiao-Nong Zhou
Robin B. Gasser
Feasibility and Roadmap Analysis for Malaria
Elimination in China
Volume 84 Xiao-Nong Zhou, Zhi-Gui Xia, Ru-Bo Wang,
Joint Infectious Causation of Human Ying-Jun Qian, Shui-Sen Zhou, Jurg
Cancers Utzinger, Marcel Tanner, Randall Kramer,
Paul W. Ewald and Holly A. Swain Ewald and Wei-Zhong Yang
Neurological and Ocular Fascioliasis in Lessons from Malaria Control to Elimination:

Humans Case Study in Hainan and Yunnan
Santiago Mas-Coma, Veronica H. Agramunt, Provinces
and Mara Adela Valero Zhi-Gui Xia, Li Zhang, Jun Feng, Mei Li,
Xin-Yu Feng, Lin-Hua Tang, Shan-Qing
Measuring Changes in Plasmodium falciparum Wang, Heng-Lin Yang, Qi Gao, Randall
Transmission: Precision, Accuracy and Kramer, Tambo Ernest, Peiling Yap, and
Costs of Metrics Xiao-Nong Zhou
Lucy S. Tusting, Teun Bousema, David
L. Smith, and Chris Drakeley Surveillance and Response to Drive the
National Malaria Elimination Programme
A Review of Molecular Approaches for Xin-Yu Feng, Zhi-Gui Xia, Sirenda Vong,
Investigating Patterns of Coevolution in Wei-Zhong Yang, and Shui-Sen Zhou
Marine HostParasite Relationships
Gotz Froeschke and Sophie von der Heyden Operational Research Needs Toward Malaria
Elimination in China
New Insights into Clonality and Panmixia in Shen-Bo Chen, Chuan Ju, Jun-Hu Chen,
Plasmodium and Toxoplasma Bin Zheng, Fang Huang, Ning Xiao, Xia
Michel Tibayrenc and Francisco J. Ayala Zhou, Tambo Ernest, and Xiao-Nong Zhou
Approaches to the Evaluation of Malaria Andrea Bosman, Robert David Newman,

Elimination at County Level: Case Study Tambo Ernest, Michael Oleary, and
in the Yangtze River Delta Region Ning Xiao
Min Zhu, Wei Ruan, Sheng-Jun Fei,
Jian-Qiang Song, Yu Zhang, Xiao-Gang
Mou, Qi-Chao Pan, Ling-Ling Zhang, Volume 87
Xiao-Qin Guo, Jun-Hua Xu, Tian-Ming The Allee Effect and Elimination of Neglected
Chen, Bin Zhou, Peiling Yap, Li-Nong Tropical Diseases: A Mathematical
Yao, and Li Cai Modelling Study
Surveillance and Response Strategy in the Manoj Gambhir, Brajendra K. Singh, and
Malaria Post-elimination Stage: Case Edwin Michael
Study of Fujian Province Mathematical Modelling of Leprosy and Its
Fa-Zhu Yang, Peiling Yap, Shan-Ying Zhang, Control
Han-Guo Xie, Rong Ouyang, Yao-Ying David J. Blok, Sake J. de Vlas,
Lin, and Zhu-Yun Chen Egil A.J. Fischer, and Jan Hendrik Richardus
Preparation of Malaria Resurgence in China: Mathematical Models of Human African
Case Study of Vivax Malaria Trypanosomiasis Epidemiology
Re-emergence and Outbreak in Kat S. Rock, Chris M. Stone,
Huang-Huai Plain in 2006 Ian M. Hastings, Matt J. Keeling,
Hong-Wei Zhang, Ying Liu, Shao-Sen Zhang, Steve J. Torr, and Nakul Chitnis
Bian-Li Xu, Wei-Dong Li, Ji-Hai Tang,
Shui-Sen Zhou, and Fang Huang Ecology, Evolution and Control of Chagas
Disease: A Century of Neglected
Preparedness for Malaria Resurgence in Modelling and a Promising Future
China: Case Study on Imported Cases in Pierre Nouvellet, Zulma M. Cucunuba,
20002012 and Sbastien Gourbiere
Jun Feng, Zhi-Gui Xia, Sirenda Vong,
Wei-Zhong Yang, Shui-Sen Zhou, and Mathematical Inference on Helminth Egg
Ning Xiao Counts in Stool and Its Applications in
Mass Drug Administration Programmes
Preparation for Malaria Resurgence in China: to Control Soil-Transmitted
Approach in Risk Assessment and Rapid Helminthiasis in Public Health
Response Bruno Levecke, Roy M. Anderson,
Ying-Jun Qian, Li Zhang, Zhi-Gui Xia, Dirk Berkvens, Johannes Charlier,
Sirenda Vong, Wei-Zhong Yang, Brecht Devleesschauwer, Niko Speybroeck,
Duo-Quan Wang, and Ning Xiao Jozef Vercruysse, and Stefan Van Aelst
Transition from Control to Elimination: Modelling Lymphatic Filariasis Transmission
Impact of the 10-Year Global Fund and Control: Modelling Frameworks,
Project on Malaria Control and Lessons Learned and Future Directions
Elimination in China Wilma A. Stolk, Chris Stone, and Sake J. de Vlas
Ru-Bo Wang, Qing-Feng Zhang, Bin Zheng,
Zhi-Gui Xia, Shui-Sen Zhou, Lin-Hua Modelling the Effects of Mass Drug
Tang, Qi Gao, Li-Ying Wang, and Administration on the Molecular
Rong-Rong Wang Epidemiology of Schistosomes
Poppy H.L. Lamberton, Thomas Crellen,
ChinaAfrica Cooperation Initiatives in James A. Cotton, and Joanne P. Webster
Malaria Control and Elimination
Zhi-Gui Xia, Ru-Bo Wang, Duo-Quan Economic and Financial Evaluation of
Wang, Jun Feng, Qi Zheng, Chang-Sheng Neglected Tropical Diseases
Deng, Salim Abdulla, Ya-Yi Guan, Wei Bruce Y. Lee, Sarah M. Bartsch, and
Ding, Jia-Wen Yao, Ying-Jun Qian, Katrin M. Gorham
Volume 88 Volume 90
Recent Developments in Malaria Vaccinology The Importance of Fossils in Understanding
Benedict R. Halbroth and Simon J. Draper the Evolution of Parasites and Their
Vectors
PfEMP1 A Parasite Protein Family of Key Kenneth De Baets and D. Timothy J. Littlewood
Importance in Plasmodium falciparum
Malaria Immunity and Pathogenesis The Geological Record of Parasitic Nematode
Lars Hviid and Anja TR. Jensen Evolution
George O. Poinar, Jr.
Prospects for Vector-Based Gene Silencing to
Explore Immunobiological Features of Constraining the Deep Origin of Parasitic
Schistosoma mansoni Flatworms and Host-Interactions with
Jana Hagen, Jean-Pierre Y. Scheerlinck, Fossil Evidence
Neil D. Young, Robin B. Gasser, and Kenneth De Baets, Paula Dentzien-Dias,
Bernd H. Kalinna Ieva Upeniece, Olivier Verneau, and
Philip C.J. Donoghue
Chronobiology of Trematode Cercarial
Emergence: from Data Recovery to From Fossil Parasitoids to Vectors: Insects as
Epidemiological, Ecological and Parasites and Hosts
Evolutionary Implications Christina Nagler and Joachim T. Haug
Andr Thron
Trace Fossil Evidence of TrematodeBivalve
Strongyloidiasis with Emphasis on Human ParasiteHost Interactions in Deep Time
Infections and Its Different Clinical Forms John Warren Huntley and Kenneth De Baets
Rafael Toledo, Carla Mu~noz-Antoli, and
Jos-Guillermo Esteban Fossil Crustaceans as Parasites and Hosts
Adil A. Klompmaker and Geoff A. Boxshall
A Perspective on Cryptosporidium and Giardia,
with an Emphasis on Bovines and Recent A Prejudiced Review of Ancient Parasites
Epidemiological Findings and Their Host Echinoderms: CSI Fossil
Harshanie Abeywardena, Aaron R. Jex, and Record or Just an Excuse for
Robin B. Gasser Speculation?
Stephen K. Donovan
Volume 89 Differentiating Parasitism and Other

Interactions in Fossilized Colonial
Ecology of Free-Living Metacercariae Organisms
(Trematoda) Paul D. Taylor
Neil J. Morley
Palaeoparasitology Human Parasites in
Cross-Border Malaria: A Major Obstacle for Ancient Material
Malaria Elimination Adauto Araujo, Karl Reinhard, and
Kinley Wangdi, Michelle L. Gatton, Gerard C. Luiz Fernando Ferreira
Kelly, Archie CA. Clements
Human Parasites in Medieval Europe:
Development of Malaria Transmission- Lifestyle, Sanitation and Medical
Blocking Vaccines: From Concept to Treatment
Product Piers D. Mitchell
Yimin Wu, Robert E. Sinden, Thomas S.
Churcher, Takafumi Tsuboi, Vidadi
Yusibov

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(Advances in Parasitology, Volume 91) David Rollinson, Russell Stothard-Elsevier - Academic Press (2016)

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VOLUME NINETY ONE

AMSTERDAM BOSTON HEIDELBERG LONDON

First edition 2016

Copyright 2016 Elsevier Ltd. All rights reserved.

For information on all Academic Press publications

Malaria Parasite Proteins and

3.2.6 Proteins containing DnaJ domains 53

worlds population lives in malaria-endemic regions making the develop-

2. TRAFFICKING OF PARASITE PROTEINS

Malaria Parasite Proteins and the RBC

Nicholas I. Proellocks et al.

Malaria Parasite Proteins and the RBC

Nicholas I. Proellocks et al.

Nicholas I. Proellocks et al.

Malaria Parasite Proteins and the RBC

Nicholas I. Proellocks et al.

Table 2 Protein interactions at the iRBC membrane skeleton

MESA/protein 4.1 0.63e1.25 Bennett et al. (1997), Waller et al. (2003)

Malaria Parasite Proteins and the RBC

Nicholas I. Proellocks et al.

Malaria Parasite Proteins and the RBC

Nicholas I. Proellocks et al.

Malaria Parasite Proteins and the RBC

Nicholas I. Proellocks et al.

Malaria Parasite Proteins and the RBC

Nicholas I. Proellocks et al.

Nicholas I. Proellocks et al.

Nicholas I. Proellocks et al.

Figure 1 Schematic representation of putative protein export pathways in P. falciparum-

2.1 The PEXEL motif

2.1.1 Plasmepsin V-mediated PEXEL function

2.1.2 PI(3)P-mediated PEXEL function

2.2 PEXEL-negative exported proteins

2.3 The role of PTEX

2.4 Protein trafcking within iRBCs

2.4.1 Vesicle-mediated trafcking

2.4.3 MCs: an external Golgi?

It was previously thought that proteins became associated with MCs

3. EXPORTED PARASITE PROTEINS

3.1 MC-associated proteins

3.1.1 Skeleton-binding protein 1

where abnormal MCs are associated with abnormal trafcking of PfEMP1

3.1.2 Membrane-associated histidine-rich protein 1

3.1.3 Membrane-associated histidine-rich protein 2

proteins. While MAHRP2 is highly expressed in trophozoite-stage parasites

3.1.4 Ring-exported protein 1

3.1.5 Ring-exported protein 2

3.1.6 Other less-well characterized proteins associated with MCs

3.1.7 Parasite proteins and the tubovesicular network

composition or the function of the TVN but it is thought to be important

3.2 Parasite proteins in the RBC cytosol or at the RBC

3.2.1 Knob-associated histidine-rich protein

In parasites expressing truncated forms of KAHRP that still contain the

3.2.2 P. falciparum erythrocyte membrane protein 3

by a single motif within PfEMP3 (Waller et al., 2007), suggesting that it

3.2.3 P. falciparum antigen 332

3.2.4 Plasmodium helical interspersed sub-telomeric proteins

compared to laboratory-adapted lines or between clonal laboratory-adapted

invasion. Clearly, more work is required to determine the precise function

important pathophysiological roles in P. falciparum. This is highlighted by

The RESA-like PHISTb proteins are an interesting subgroup within this

(Maier et al., 2008). PF3D7_0936800 appears to be essential for parasite

3.2.5 Ring-infected erythrocyte surface antigen