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Advances in
PARASITOLOGY
VOLUME
77
SERIES EDITORS
D. ROLLINSON S. I. HAY
Department of Zoology, Spatial Epidemiology and Ecology Group
The Natural History Museum, Tinbergen Building, Department of Zoology
London, UK University of Oxford, South Parks Road
d.rollinson@nhm.ac.uk Oxford, UK
simon.hay@zoo.ox.ac.uk
EDITORIAL BOARD
M. G. BASANEZ R. E. SINDEN
Reader in Parasite Epidemiology, Immunology and Infection Section,
Department of Infectious Disease Department of Biological Sciences,
Epidemiology, Faculty of Medicine Sir Alexander Fleming Building, Imperial
(St Marys campus), Imperial College College of Science, Technology and
London, London, UK Medicine, London, UK
S. BROOKER D. L. SMITH
Wellcome Trust Research Fellow and Johns Hopkins Malaria Research Institute
Reader, London School of Hygiene and & Department of Epidemiology, Johns
Tropical Medicine, Faculty of Infectious Hopkins Bloomberg School of Public
and Tropical, Diseases, London, UK Health, Baltimore, MD, USA
R. B. GASSER R. C. A. THOMPSON
Department of Veterinary Science, Head, WHO Collaborating Centre for
The University of Melbourne, Parkville, the Molecular Epidemiology of Parasitic
Victoria, Australia Infections, Principal Investigator, Envi-
ronmental Biotechnology CRC (EBCRC),
N. HALL School of Veterinary and Biomedical
School of Biological Sciences, Bios- Sciences, Murdoch University, Murdoch,
ciences Building, University of Liverpool, WA, Australia
Liverpool, UK
R. C. OLIVEIRA X. N. ZHOU
Centro de Pesquisas Rene Rachou/ Professor, Director, National Institute
CPqRR - A FIOCRUZ em Minas Gerais, of Parasitic Diseases, Chinese Center
Rene Rachou Research Center/CPqRR - for Disease Control and Prevention,
The Oswaldo Cruz Foundation in the Shanghai, Peoples Republic of China
State of Minas Gerais-Brazil, Brazil
Advances in
PARASITOLOGY
VOLUME
77
Edited by
D. ROLLINSON
Department of Zoology
The Natural History Museum
Cromwell Road
London, UK
S. I. HAY
Spatial Epidemiology and Ecology Group
Tinbergen Building, Department of Zoology
University of Oxford, South Parks Road
Oxford, UK
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11 12 13 10 9 8 7 6 5 4 3 2 1
CONTENTS
Contributors vii
1. Coinfection of Schistosoma (Trematoda) with Bacteria,

Protozoa and Helminths 1
Amy Abruzzi and Bernard Fried
1.1. Introduction 3
1.2. Coinfection of Species of Schistosoma and Plasmodium 4
1.3. Coinfection of Schistosoma Species with Protozoans other
than in the Genus Plasmodium 24
1.4. Coinfection of Schistosoma Species with Salmonella 36
1.5. Coinfection of Schistosoma Species with Bacteria other
than Salmonella 43
1.6. Coinfection of Schistosoma and Fasciola Species 50
1.7. Coinfection of Schistosoma Species and Helminths other
than the Genus Fasciola 59
1.8. Concluding Remarks 74
References 75
2. Trichomonas vaginalis Pathobiology:

New Insights from the Genome Sequence 87
Robert P. Hirt, Natalia de Miguel, Sirintra Nakjang, Daniele Dessi,
Yuk-Chien Liu, Nicia Diaz, Paola Rappelli, Alvaro Acosta-Serrano,
Pier-Luigi Fiori, and Jeremy C. Mottram
2.1. Introduction 88
2.2. Surface and Secreted Molecules 89
2.3. Peptidases 111
2.4. Membrane Trafficking and Cell Signalling 117
2.5. The Transcriptome and the RNAi Machinery 126
Acknowledgements 130
References 130
v
vi Contents
3. Cryptic Parasite Revealed: Improved Prospects for

Treatment and Control of Human Cryptosporidiosis
Through Advanced Technologies 141
Aaron R. Jex, Huw V. Smith, Matthew J. Nolan, Bronwyn E.
Campbell, Neil D. Young, Cinzia Cantacessi, and Robin B. Gasser
3.2. Cryptosporidium Species and Genotypes Known to
Infect Humans 144
3.3. The Life Cycle of C. Parvum and C. Hominis 145
3.4. Cryptosporidiosis: Pathogenesis and Immunity 147
3.5. Genomics and Transcriptomics of Cryptosporidium 149
3.6. Improved Insights into Cryptosporidium Using In Vitro
Techniques 158
References 162
4. Assessment and Monitoring of Onchocerciasis in Latin America 175

Mario A. Rodrguez-Perez, Thomas R. Unnasch, and
Olga Real-Najarro
4.2. The Pathology and Clinical Manifestations
Produced by O. volvulus Infection in Latin America 185
4.3. Genetic Variation of O. volvulus and the Simulium Vector 187
4.4. The Control of Onchocerciasis
(with Emphasis on Programmes in Latin America) 189
4.5. Advantages and Disadvantages of Treatment with Ivermectin 195
4.6. Development of Other New Drugs 197
4.7. Monitoring and Evaluation of Control of Onchocerciasis 199
4.8. Entomological Parameters for Monitoring the
Transmission in Latin America (with Emphasis in Areas
where Transmission has been Interrupted) 209
4.9. Future Developments 213
References 215
Index 227
Contents of Volumes in This Series 233
CONTRIBUTORS
Amy Abruzzi
Skillman Library, Lafayette College, Easton, Pennsylvania; and
Epidemiology, University of Medicine and Dentistry of New Jersey
(UMDNJ), Piscataway, New Jersey, USA
Alvaro Acosta-Serrano
Liverpool School of Tropical Medicine, Liverpool, United Kingdom
Bronwyn E. Campbell
Department of Veterinary Science, The University of Melbourne,
Werribee, Victoria, Australia
Cinzia Cantacessi
Natalia de Miguel
Laboratorio de Parasitologia Molecular, Instituto de Investigaciones
Biotecnologicas-Instituto Tecnologico de Chascomus, Chascomus,
Argentina
Daniele Dessi
Department of Biomedical Sciences, and Centre for Biotechnology
Development and Biodiversity Research, University of Sassari, Sassari,
Italy
Nicia Diaz
Development and Biodiversity Research, University of Sassari,
Sassari, Italy
Pier-Luigi Fiori
Development and Biodiversity Research, University of Sassari,
Sassari, Italy
vii
viii Contributors
Bernard Fried
Department of Biology, Lafayette College, Easton, Pennsylvania, USA
Robin B. Gasser
Robert P. Hirt
Institute for Cell and Molecular Biosciences, Newcastle University,
Newcastle upon Tyne, United Kingdom
Aaron R. Jex
Yuk-Chien Liu
Wellcome Trust Centre for Molecular Parasitology, Institute of Infection,
Immunity and Inflammation, College of Medical, Veterinary and Life
Sciences, University of Glasgow, Glasgow, United Kingdom
Jeremy C. Mottram
Wellcome Trust Centre for Molecular Parasitology, Institute of Infection,
Immunity and Inflammation, College of Medical, Veterinary and Life
Sciences, University of Glasgow, Glasgow, United Kingdom
Sirintra Nakjang
Institute for Cell and Molecular Biosciences, Newcastle University, New-
castle upon Tyne, United Kingdom
Matthew J. Nolan
Paola Rappelli
Development and Biodiversity Research, University of Sassari, Sassari,
Italy
Olga Real-Najarro
Facultad de Medicina, Universidad Autonoma de Nuevo Leon; and
Centro de Estudios Asiaticos, Universidad Autonoma de Nuevo Leon,
Monterrey, Nuevo Leon, Mexico
Contributors ix
Mario A. Rodrguez-Perez
Centro de Biotecnologa Genomica, Instituto Politecnico Nacional,
Ciudad Reynosa, Tamaulipas; and Facultad de Medicina, Universidad
Autonoma de Nuevo Leon, Monterrey, Nuevo Leon, Mexico
Huw V. Smith
Scottish Parasite Diagnostic Laboratory, Stobhill Hospital, Glasgow,
United Kingdom
Thomas R. Unnasch
Department of Global Health, College of Public Health, University of
South Florida, Tampa, Florida, USA
Neil D. Young
Intentionally left as blank
CHAPTER 1
Coinfection of Schistosoma
(Trematoda) with Bacteria,
Protozoa and Helminths
Amy Abruzzi*, and Bernard Fried
Contents 1.1. Introduction 3

1.2. Coinfection of Species of Schistosoma and
Plasmodium 4
1.2.1. Animal studies 21
1.2.2. Human studies 23
1.3. Coinfection of Schistosoma Species with Protozoans
other than in the Genus Plasmodium 24
1.3.1. Leishmania 32
1.3.2. Toxoplasma 32
1.3.3. Entamoeba 34
1.3.4. Trypanosoma 35
1.4. Coinfection of Schistosoma Species with Salmonella 36
1.5. Coinfection of Schistosoma Species with Bacteria
other than Salmonella 43
1.5.1. Mycobacterium 43
1.5.2. Helicobacter pylori 49
1.5.3. Staphylococcus aureus 50
1.6. Coinfection of Schistosoma and Fasciola Species 50
* Skillman Library, Lafayette College, Easton, Pennsylvania, USA

{
Epidemiology, University of Medicine and Dentistry of New Jersey (UMDNJ), Piscataway, New Jersey, USA
{
Department of Biology, Lafayette College, Easton, Pennsylvania, USA
Advances in Parasitology, Volume 77 # 2011 Elsevier Ltd.

ISSN 0065-308X, DOI: 10.1016/B978-0-12-391429-3.00005-8 All rights reserved.
1
2 Amy Abruzzi and Bernard Fried
1.7. Coinfection of Schistosoma Species and Helminths

other than the Genus Fasciola 59
1.7.1. Echinostoma 59
1.7.2. Hookworm 70
1.7.3. Trichuris 70
1.7.4. Ascaris 71
1.7.5. Strongyloides and Trichostrongyloides 72
1.7.6. Filarids 73
References 75
Abstract This review examines coinfection of selected species of Schisto-

soma with bacteria, protozoa and helminths and focuses on the
effects of the coinfection on the hosts. The review is based mainly
on tables that contain the salient information on the coinfecting
organisms in vertebrate hosts. Further explanation and clarification
of the tables are given in the text. A table is also provided that gives
synoptic information on the 37 species in the 19 genera considered
in this review. Coinfection studies with Schistosoma species and
the other organisms were considered in six tables plus the accom-
panying text. Considerations of the Schistosoma interactions with
another species of organism include studies on coinfection with
Plasmodium, with protozoa other than Plasmodium; with Salmo-
nella, with bacteria other than Salmonella; and with Fasciola, with
helminths other than Fasciola.
Numerous factors were found to influence the effects of
coinfection on the vertebrate host, including organisms and hosts
used in the studies, order and time interval between the first and
the second infection, studies on natural versus experimental hosts,
dosage of the infectious agents, strains and pedigrees of the para-
sites, age of hosts at time of exposure to the infectious agents and
age of hosts at the time of necropsy. Overall, a prior infection with
Schistosoma, particularly a patent infection, often has an effect on
the subsequent infection by a protozoan, bacterium or other hel-
minth. In relatively few cases, a prior infection with Schistosoma
decreased the severity of the subsequent infection as with Helico-
bacter pylori, Fasciola hepatica, Echinostoma or Plasmodium, the
latter only exhibiting this behaviour when coinfected with Schisto-
soma haematobium. More often, however, a prior infection with
Schistosoma increased the severity of the second infection as with
Leishmania, Toxoplasma gondii, Entamoeba histolytica, Staphylo-
coccus aureus or Salmonella. In some of these coinfection studies,
the increased severity of the subsequent infection was associated
with a specific, prolonged form of the disease in humans, which has
implications for patient treatment and recovery. Additional
research is needed, particularly on Schistosoma coinfections
which currently have a small body of research and are current
Coinfection of Schistosoma (Trematoda) with Bacteria, Protozoa and Helminths 3
problems in human populations. Examples of such Schistosoma

interactions include the genera of Mycobacteria, Leishmania,
Staphylococcus, Necator and Strongyloides. Hopefully, future stud-
ies will elucidate valuable new information on the interesting
subject of coinfection of Schistosoma with other organisms.
1.1. INTRODUCTION
This review examines coinfection of selected species of Schistosoma with
various other organisms, that is, helminths, protozoa and bacteria. We
originally intended to examine coinfection interactions of schistosomes
with viruses, but because of the voluminous literature on that topic, we
have excluded such information from this review. The schistosomes are
water-borne digeneans of global concern. Species in the genus Schistosoma
have been well studied in terms of single infections in their vertebrate
hosts, but less information on schistosomes coinfected with other organ-
isms is available. In this review, we examine the salient studies that link
species of schistosomes with protozoa, bacteria and other helminths.
Areas of concern in our review include infections in the wild and also
experimental infections in the laboratory. Important aspects of our review
include the interactions of the schistosome of concern with the coinfecting
organism in terms of physiological, immunological, ecological and epide-
miological consequences. Important to these studies are factors such as
the order of infection, that is, was the host first infected by the schistosome
or the other organism. The time sequence when known between the first
and the second infection is given. Results of coinfection within the host
are considered to determine the effects of such infection on the pathoge-
nicity of the host. We also address the issue of whether the coinfection
increased, decreased or had no effect on the severity of the infection in the
host. The implications of the above concerns are important in both human
and veterinary medicine.
Our review has numerous tables as in Fried and Abruzzi (2010), and
tabular information is followed by text to clarify and extend the informa-
tion in the tables. We emphasize coinfection events between the schisto-
some of interest and a single other organism, that is, another helminth,
protozoan or bacterium. Numerous studies exist on polyparasitism
(multiparasitism) in which naturally infected hosts are infected with
three or more parasites. Such studies are not included in this review
unless they relate directly or tangentially to an examination of the rela-
tionship of two coinfecting organisms in a host. Case reports, when
relevant, are also referred to in the text. The effects of larval parasite
dosage on coinfection are not included in this review. In experimental
studies, the larval dosage cannot always be correlated with the number of
parasites recovered in the host; also, many authors have failed to provide
such information in their original papers. The effect of parasite dosage is
usually unknown in natural coinfection studies.
The literature in our tables ranges from January 1, 1972 to March 1,
2011. Helminthological Abstracts (1972to date) and ISI Web of Science
(1975to date) were searched in multiple ways: firstly, broadly using the
general terms with the truncation symbol * to pick up variant endings:
(interact* or coinfect* or co-infect* or concomitant* or concurrent* or mixed
infect* or double infect*) and schistosom*. Additional studies were iden-
tified by searching for pairs, such as schistosom* and fasciol*. Studies
identified this way were checked for references to other publications
within our time period. All English language papers were examined in
full. Some earlier reviews on coinfections helped us decide what should
be covered in our review; the most important earlier reviews were those
of Graham (2002), Cox (2001), Chieffi (1992) and Christensen et al. (1987).
Additional reviews were noted as relevant to the section under review.
All specific entries in each table except Table 1.1 are in reverse chronolog-
ical order and are numbered accordingly beginning with number 1. The
text of our chapter refers to the entry numbers and summarizes major
trends in the coinfection interactions between the organisms of interest.
We concentrated on the major effect of each pairing on the vertebrate host
in comparison to the relevant control group. Coinfection studies that were
mainly serologic, chemotherapeutic or used for vaccine development
were not included in our review; we have not included studies that
examined the effects of worm self- versus cross-fertilization in coinfected
hosts.
Most readers will be familiar with the organisms discussed in this
review. However, Table 1.1 provides a brief synopsis of the highlights of
organisms covered in Tables 1.21.7. Further information on these organ-
isms can be obtained from introductory texts on parasitology and micro-
biology or by using pertinent web sites.
1.2. COINFECTION OF SPECIES OF SCHISTOSOMA AND

PLASMODIUM
This section is concerned with coinfection studies on species of Schisto-

soma and Plasmodium. A total of 32 papers were selected for inclusion in
Table 1.2. Of these, 13 were experimental studies using mice as hosts
(entry numbers 1, 5, 8, 11, 18, 2426, 2832) and the remaining 19 papers
described naturally occurring coinfections in human populations (entry
numbers 24, 6, 7, 9, 10, 1217, 1923, 27). The organisms studied in these
papers were two species of Schistosoma and five species of Plasmodium.
Both Schistosoma species, S. mansoni and S. haematobium, and three of the
TABLE 1.1 A guide to species considered in Tables 1.21.7
Genus Species included in tables Remarks (based on single infections) Table(s)
Schistosoma S. bovis, S. douthitti, S. haematobium, Mainly three species concerned with human infection, 1.21.7
S. intercalatum, S. japonicum, that is, S. mansoni, S. japonicum, and S. haematobium;
S. mansoni dioecious adults live in blood vessels with hepatic
portal and intestinal vessels as the main sites for
S. mansoni and S. japonicum and venous blood vessels
of the urogenital system for S. haematobium; also listed in
this column are the animal forms S. bovis, S. douthitti,
and S. intercalatum
Protozoa
Entamoeba E. histolytica E. histolytica is a causative agent of amoebic dysentery and 1.3
intestinal and extraintestinal amoebiasis; the organism
spreads by oral-faecal contamination
Leishmania L. donovani, L. donovani infantum, Infective stages are transmitted to humans by the bite of 1.3
L. major, L. mexicana mexicana sandflies in the genus Phlebotomus, and invade and
develop in selected macrophages of vertebrate hosts
Plasmodium P. falciparum, P. malariae, P. berghei, These vector-borne sporozoans are transmitted to humans 1.2
P. chabaudi, P. yoelii and animals by the bite of anopheline mosquito; the last
three species listed in column 2 are mainly murine
forms
Toxoplasma T. gondii This apicomplexan (sporozoan) species is transmitted to 1.3
humans mainly by animal and faecal contact; it invades
many cell types including macrophages and myocytes
(continued)
TABLE 1.1 (continued)
Trypanosoma T. brucei, T. cruzi These are blood and tissue flagellates; T. brucei is 1.3
transmitted by the bite of the tsetse fly (Glossina sp.) and
T. cruzi, the intracellular myocardial form, is
transmitted by the bite of triatomid bugs
Bacteria
Helicobacteria H. pylori H. pylori is a gram-negative, microaerophilic bacterium. 1.5
It inhabits various regions of the stomach and is linked
to duodenal and gastric ulcers; in some cases, it induces
stomach cancer
Mycobacteria M. avium, M. bovis, Several of these species are in the M. tuberculosis 1.5
M. paratuberculosis, M. ulcerans complex including M. paratuberculosis, M. bovis, and
M. bovis-BCG; Buruli ulcer is associated with
M. ulcerans; organisms in this complex are aerobic,
non-motile, acid-fast, and gram-positive
Salmonella S. enterica (numerous serotypes These are gram-negative facultative rod-shaped bacteria 1.4
as discussed in Table 1.4) usually referred to as enteric bacteria, with many
strains, subspecies and variants. Major diseases
associated with these bacteria are salmonellosis and
typhoid fever
Staphylococcus S. aureus This species has many variants that are gram-positive and 1.5
form grape-like clusters; toxins associated with some
S. aureus strains cause food poisoning
Trematodes other than schistosomes
Echinostoma E. caproni, E. paraensei Echinostomes infect the intestinal tract of humans and 1.7
cause intestinal distress; seriousness of the infection
often relates to worm burdens. This is mainly a food-
borne infection for humans, although some infections
occur following ingestion of water-borne cercariae
Fasciola F. hepatica, F. gigantica F. hepatica is a liver fluke of humans and animals and is 1.6
transmitted to hosts that eat contaminated (mainly raw)
vegetation, for example, watercress. Adults develop in
the liver and bile duct of the host and can induce severe
pathology
Nematodes
Ancylostoma Unidentified species A genus of hookworm with species that infect human 1.7
and non-human hosts via the skin; larvae wander
through many organs prior to final entry in the
intestines where they develop to adult worms.
Infection is associated with anaemia in the host
Ascaris A. lumbricoides, A. suum These species infect hosts (human or pig, respectively) 1.7
when they swallow eggs in contaminated soil; larvae
migrate through the body and eventually develop as
adults in the intestine where they may cause intestinal
blockage and other types of pathology
Brugia B. pahangi A type of filarid introduced into humans via the bite of 1.7
mosquitoes. Adult filarids live in lymph nodes; larvae
in the blood and lymphatics
(continued)
Heligomosomoides H. polygyrus A common rodent trichostrongyloid with a direct life 1.7

cycle; it is often referred to as Nematospiroides dubius; it is
used as a model nematode in laboratory mice, often for
immunological studies
Necator N. americanus The best known human species is N. americanus. This is the 1.7
predominant hookworm in the tropics and is also a
cosmopolitan species. Larvae enter by penetrating the
skin of the host and take a circuitous route through the
body before colonizing in the small intestines; adult
worms can cause anaemia and other severe pathologies
in hosts
Strongyloides S. venezuelensis A rodent form of strongyloid often used as a model to 1.7
study human strongyloidiasis. An important model for
studies on the human pathogen S. steralis
Trichuris T. muris, T. trichuris The main species of concern is T. trichuris or human 1.7
whipworm, which is usually a mild pathogen causing
lower intestinal damage depending on the number of
worms in the host. T. muris is a mouse strain often used
in experimental studies
TABLE 1.2 Coinfection studies on species of Schistosoma and Plasmodium
Experimental (E) or
Entry Species of Species of coinfect- natural (N) infections Time between
number Reference trematode ing organism in vertebrate hosts coinfections Comments
1 Bucher et al. Schistosoma Plasmodium berghei (E) C57BL/6 mice Sm followed by Coinfected (Co) mice had
(2011) mansoni Pb 89 wk decreased malarial brain
later pathology compared to mice
with single Pb infection; pre-
existing infection by Sm did not
prevent severe malaria or death
but influenced the course of
malarial pathology; outcomes
were unrelated to cerebral
malaria (CM)
2 Courtin et al. S. haematobium P. falciparum (N) 7- to 19-year-old Unknown Coinfection had an additive effect
(2011) human on cytokine levels; Co hosts had
higher IL-10 levels than
individuals with single
infections and may increase risk
of Pf disease or death
3 Midzi et al. S. mansoni or P. falciparum (N) 5- to 15-year-old Unknown Co children had lower
(2010) S. haematobium humans haemoglobin levels and a higher
prevalence of anaemia than
single or non-infected children;
Co aggravates anaemia
(continued)
Experimental (E) or
4 Sangweme S. mansoni or P. falciparum (N) 6- to 17-year-old Unknown Co hosts had higher overall
et al. (2010) S. haematobium humans prevalence of malaria parasites
with greater incidence and
densities of gametocytes than
children with single Pf
infections; Co may have
implications for malaria disease
severity and transmission
dynamics
5 Waknine- S. mansoni P. berghei (E) ICR mice Sm followed by Infection with Sm followed by Pb
Grinberg Pb 4 or 7 wk 7 wk later led to reduction in CM
et al. (2010) later and was correlated with a Th2
response; no malarial reduction
after 4 wk of coinfection;
protection from CM appeared to
be a function of Sm parasite load
and timing
6 Mouk et al. S. mansoni P. falciparum (N) 8- to 10-year-old Unknown Co children had a lower mean % of
(2009) humans HLA-DR() Tact and a lower
mean level of memory Treg cells
than children with single
Sm infections; imbalances in
T lymphocyte subsets may be
related to differential morbidity
or course of infection in Co hosts
7 Nmorsi et al. S. haematobium P. falciparum (N) 1- to 15-year-old Unknown Co children had lower
(2009) humans parasitaemia and higher
haemoglobin levels than
children with single Pf infection;
concentrations of IL-4, IL-5, IL-8,
and IFN-y were elevated in Co
children compared with the Pf
group; Co altered Th1/Th2
profile, which may have
protected against severe malarial
attacks or death
8 Sangweme S. mansoni P. yoelii (E) BALB/c mice Sm followed by Hosts with patent Sm infection had
et al. (2009) Py 14 days a delayed response to Py
later infection with increased Py peak
parasitaemia and mortality in
typically self-resolving Py
infections; hepatosplenomegaly
was more marked in Co than
single infected mice; timing of Py
infection after Sm infection may
be critical to disease outcome
and pathology
(continued)
Experimental (E) or
9 Wilson et al. S. mansoni P. falciparum (N) 4- to 17-year-old Unknown Co children had higher plasma
(2009) humans levels of sTNF-RII and IL-5 than
non- or single Pf infected
children; IL-10 levels were
higher in Co than non-infected
children; elevated levels of
IL-12p70, IL-10, IL-13 and
sTNF-RII were associated with
hepatosplenomegaly and
malaria infection; levels may be
due to augmentation of the
inflammatory response in liver
and spleen
10 Faye et al. S. mansoni P. falciparum (N) 1- to 15-year-old Unknown In children aged 114 years, Co
(2008) and some older hosts had higher Pf densities
humans than children with single Pf
infection; highest malarial
densities occurred in Co children
less than 5 years old; in children
aged 15 years and older, Co had
lower Pf densities than children
with single Pf infection
11 Laranjeiras S. mansoni P. berghei (E) BALB/c mice Sm followed by Co mice had increased malarial
et al. (2008) Pb infection parasitaemia and decreased
24 wk later survival compared to single Pb
infected mice. Skewed immune
profile induced by chronic Sm
infection might affect the course
of the Pb infection and the
acquisition of malarial immunity
12 Okafor and Schistosoma sp. Plasmodium sp. (N) Newborn14- Unknown Co children had lower
Elenwo year-old humans concentrations of haemoglobin
(2007) than single infected or non-
infected children; concentrations
were lowest among children
aged 1014 years than other age
groups
13 Lyke et al. S. haematobium P. falciparum (N) 4- to 14-year-old Sh followed by Co children, 48 years old, had
(2006) humans acute Pf lower IL-6 and IL-10 levels
infection compared to children with single
Pf infection; IL 4 levels were
inversely correlated with time to
malaria infection in all 4- to 8-
year-old children; children with
underlying Sh infection had
polarized Th2 response which
may have modulated the
incidence and severity of
subsequent infection with Pf
(continued)
Experimental (E) or
14 Arinola S. haematobium P. malariae or (N) 6- to 14-year-old Unknown Co children had lower malaria
(2005) P. falciparum humans parasite density and severity,
and higher levels of leukocyte
migration inhibitory factor and
reactive oxygen species than
single Pm or Pf infected children
15 Briand et al. S. haematobium P. falciparum (N) 3- to 15-year-old Unknown Children with light Sh infection
(2005) humans had lower Pf densities than
children with single Pf infection;
parasite density decreased with
age and was lower in girls than
boys; immune responses varied
according to the stage and
intensity of infection
16 Lyke et al. S. haematobium P. falciparum (N) 4- to 14-year-old Sh followed by Children aged 48 years with
(2005) humans Pf infection asymptomatic Sh infection
showed delayed time to clinical
malaria infection with fewer
number of malarial episodes and
lower mean parasite densities
than comparably aged children
with single Pf infection; no
protective association seen in
9- to 14-year-old children;
underlying Sh infection was
associated with protection
against clinical malaria for
younger children only
17 Diallo et al. S. haematobium P. falciparum (N) 715 years-old Unknown Co children had higher levels of
(2004) and 30 years and IFN-gamma and TNF-RII than
older humans children with single Pf infection;
Co adults showed an increase in
IL-10, IFN-gamma, TGF-beta
and sTNF receptors; coinfection
appeared to unbalance the
regulation of inflammatory
factors that played a key role
during malaria infection in an
age-dependent manner
18 Legesse et al. S. mansoni P. berghei (E) Swiss albino Sm followed by Mice with Sm had increased
(2004) mice Pb 7 wk later parasitaemia and mortality from
Pb compared to mice with single
Pb infection; delayed reduction
and or clearance in parasitaemia
was also noted in Co hosts;
mortality from Pb in Co mice
was 67% compared to 20% in
single Pb mice
(continued)
Experimental (E) or
19 Sokhna et al. S. mansoni P. falciparum (N) 6- to 15-year-old Unknown Children with the highest Sm egg
(2004) humans loads (>1000 epg) had a greater
incidence of malarial attacks
than children without or with
lower Sm infections; malaria
attacks were higher in children
with the lowest egg load than in
children with medium Sm egg
burden; parasite load of Sm may
affect Pf infection, but this may
not be a simple linear
relationship
20 Mwatha et al. S. mansoni P. falciparum (N) 8- to 16-year-old Unknown Sm-infected children with
(2003) humans hepatosplenomegaly had higher
levels of antimalarial antibodies
than Sm-infected children
without hepatosplenomegaly; in
particular, antimalarial IgG1 and
IgG3 levels were higher in Sm
positive hepatosplenic children;
antimalarial antibodies
appeared to be associated
with the development of
hepatosplenomegaly in
Sm-infected children
21 Egwunyenga S. mansoni Plasmodium sp. (N) near-term Unknown In two of three study areas, Co
et al. (2001) pregnant human pregnant females were more
females likely to have severe
splenomegaly than those with
single malaria infection
22 Friis et al. S. haematobium Plasmodium sp. (N) 7- to 11-year-old Unknown Co children were less likely to have
(2000) humans splenomegaly than those
infected with single malaria
infection; malaria-induced
splenomegaly may have
impaired the establishment of Sh
infection or Sh infection may
have modified the affect of
malaria infection on the
development of splenomegaly
23 Mutapi et al. S. haematobium P. falciparum (N) 5- to 17-year-old Unknown Co children produced more anti-
(2000) humans schistosome IgE and IgG3
antibodies than single infected
Sm children; malaria infection
influenced cytokine
environment and the production
of both isotypes
24 Yoshida et al. S. mansoni P. chabaudi (E) C57/BL6 and Sm followed by C57/BL6 mice with coinfection
(2000) A/J mice Pc 8 wk later showed greater susceptibility,
parasitaemia and mortality than
mice with single Pc infection;
(continued)
Experimental (E) or
A/J mice with coinfection had a

higher parasitaemia than Pc-
infected C57/BL6 mice, but Sm
infection protected A/J mice
from mortality through
induction of increased IFN-
gamma production
25 Helmby et al. S. mansoni P. chabaudi (E) C57BC/6 mice Sm followed by Co mice developed greater Pc
(1998) Pc 8 wk later parasitaemia, showed lower
RBC counts and lower TNF-a
production than mice with single
Pc infection; Co mice had 813%
mortality compared with no
mortality among single Pc-
infected mice; coinfection altered
the immune responses to
existing and new infections
26 Rahman S. mansoni P. chabaudi (E) male CBA mice Sm followed by Co mice had lower blood
(1990) Pc 4 or 7 wk parasitaemia than single
later Pc-infected mice; Co mice had
greater parasitaemia when
coinfected at 4 wk compared to 7
wk; antibody response to Pc was
delayed in mice coinfected at 4
wk while mice coinfected at 7 wk
had consistently higher
response; timing of coinfection
affected the malaria response
27 Kassim and S. haematobium P. falciparum (N) 7- to 14-year-old Unknown Co children showed no association
Ejezie humans with frequency of blood group
(1982) types compared to children
infected with single Sh or Pf
infection
28 Lwin et al. S. mansoni P. chabaudi or (E) CBA/Ca mice Sm followed by Co mice with patent (8 wk) Sm
(1982) P. yoelii or Pc, Py, or Pb infection had lower Pc
P. berghei 412 wk later parasitaemia than mice with
single Pc infection; Co mice with
patent (8 wk) Sm infection had
higher Py parasitaemia than
mice with single Py infection; Co
mice with 8 wk Sm infection had
Pb parasitaemia comparable to
mice with single Pb infection;
pre-patent 4-wk Sm infection
had no effect on Pc, Py, or Pb
parasitaemia
29 Long et al. S. mansoni P. chabaudi (E) CBA/Lac mice Sm followed by When Sm was followed by Pc, Co
(1981) Pc 49 days mice had lower maximum
later; Pc malaria parasitaemia than mice
followed by singly infected with Pc; when Pc
Sm 5 days was followed by Sm, Co mice
later had fewer parasitized
erythrocytes than Pc only
infected mice
(continued)
Experimental (E) or
30 Lewinsohn S. mansoni P. berghei (E) Swiss mice Sm followed by Co mice had comparable levels of
(1975) infection parasitaemia and reticulocytosis
with Pb 3 or 5 compared to mice singly infected
wk later with Pb
31 Moore et al. S. mansoni P. berghei (E) mice (unknown) Sm followed by Pigments from Sm and Pb in
(1975) Pb 6 wk later endothelial cells were very
distinguishable after coinfection,
though most cells tended to
contain only one pigment type
32 Abdel-Wahab S. mansoni P. berghei (E) Swiss albino Concurrently Co mice had suppressed
et al. (1974) mice infected with granuloma formation of Sm eggs
Sm and Pb in the lungs compared to singly
infected mice; effect observed by
day 4 and peaked at day 16; no
differences observed in antibody
levels between coinfected and
mice singly infected with Sm
Co, coinfected; CM, cerebral malaria; Pb, P. berghei; Pc, P. chabaudi; Pf, P. falciparum; Pm, P. malariae; Py, P. yoelii; Sm, S. mansoni; Sh, S. haematobium; unknown, not specified in original
paper; wk, week or weeks.
Plasmodium species, P. falciparum, P. malariae and P. chabaudi, infect

humans. The remaining two malaria species, P. berghei and P. yoelii, are
causative agents of murine malaria and were used in the mouse studies in
addition to P. chabaudi. No papers were found on other species of Schis-
tosoma or Plasmodium such as S. japonicum or P. vivax as coinfective agents.
1.2.1. Animal studies

All experimental studies were conducted on mice and used S. mansoni,
and all but two experimental studies (entry numbers 29, 32) examined a
species of Schistosoma followed by coinfection with a Plasmodium species.
Typically, mice with a single Plasmodium infection served as controls.
Overall, the differences in the effect of the coinfection on the host
appeared to depend upon the species of Plasmodium, location of the
malarial infection within the host and strain of mouse used.
The five studies using P. chabaudi infections were done on either C57 or
CBA mice. In the two studies using C57 mice (entry numbers 24, 25),
coinfection clearly increased the malaria parasitaemia and mortality of
the host. In the three studies using CBA mice (entry numbers 26, 28, 29),
parasitaemia was decreased in the host. Two of six studies using P. berghei
(entry numbers 11, 18) showed that the interaction increased malaria
parasitaemia and mortality when a patent or chronic infection of
S. mansoni was followed by P. berghei. In one study (entry number 30),
the author concluded that there was no effect of the coinfection, but
examination of the data presented in the authors tables indicated that
the coinfected hosts ended the experiment with increased splenomegaly
and decreased haemoglobin levels compared to the mice infected singly
with Plasmodium. Alternatively, two studies noted that coinfected mice
had reduced severity of cerebral malaria (entry numbers 1, 5) (see Fig. 1.1,
from Bucher et al., 2011), both of which followed patent infection with
S. mansoni. This protective effect did not prevent severe disease or death
(entry number 1) from other aspects of malaria in the coinfected animals
and appeared to be correlated with a Th2 response (entry number 5).
In accord with some of the findings on P. berghei, results from work on
P. yoelii (entry numbers 8, 28) found that coinfection increased parasitae-
mia and mortality in the experimental hosts. An increase in spleen size
was also observed (entry number 8). A variety of mouse strains were used
in the S. mansoniP. berghei studies and also in the S. mansoniP. yoelii
studies, and no clear patterns in terms of possible interactions with the
hosts were apparent. Interestingly, the only study (entry number 28) that
found no effect on the host from a S. mansoniP. berghei coinfection used a
CBA mouse strain as the host, though the same study found an effect with
a S. mansoniP. yoelii interaction using the same CBA mouse strain. Two
studies showed that there was no effect on the host in regard to
FIGURE 1.1 Histomorphology of brain sections of Plasmodium berghei ANKA-infected

C57BL/6 mice on day 6 after challenge. (A) Brain section of a Schistosoma mansoniP.
berghei coinfected mouse showing a healthy uninfected blood vessel. (B and C) Brain
sections of a P. berghei mono-infected animal showing (B) mononuclear cell accumula-
tion and sequestration and (C) a microhaemorrhage (arrow). Sections stained with H&E.
Magnification, 400. Reproduced with permission from Bucher et al. (2011).
parasitaemia or cerebral malaria when coinfection with P. chabaudi,

P. berghei or P. yoelii occurred following a pre-patent S. mansoni infection
(entry numbers 5, 28). One study (entry number 26) indicated a delayed
antibody response and higher parasitaemia in mice coinfected with
P. chabaudi following pre-patent S. mansoni infection when compared with
mice coinfected with P. chabaudi following a patent S. mansoni infection,
though parasitaemia levels in the coinfected hosts were still lower than in
mice with single Plasmodium infection (entry number 26).
Several papers concluded that timing (entry numbers 5, 8, 26) with
respect to the establishment of the S. mansoni infection as well as the
malaria parasite load (entry number 5) was important. When infection
with S. mansoni followed P. chabaudi in CBA mice (entry number 29), a
decrease in parasitaemia was found. One study (entry number 32) exam-
ined mice concurrently infected with P. berghei and S. mansoni and found
that coinfected hosts had suppressed granuloma formation in the lungs
compared to mice with a single S. mansoni infection.
1.2.2. Human studies

All studies that examined naturally occurring coinfection in humans
indicated that coinfection with schistosome and malaria organisms has
an effect on the host, both in terms of pathology and in terms of immuno-
logical response. The direction of this response seems to depend on the
species of schistosome and the worm burden, host age and malaria para-
sitaemia. Little can be inferred based on the order of coinfection since
such information was unknown in all but one study (entry number 13).
Eight papers examined the cytokine response with P. falciparum coinfec-
tion: five with S. haematobium (entry numbers 2, 7, 13, 17, 23) and three
with S. mansoni (entry numbers 6, 9, 20). All but two (entry numbers 17,
21) of these studies were done on children 19 years or younger, with most
children under 15 years of age.
All but 1 (entry number 22) of the 10 studies on S. haematobium in
humans (entry numbers 2, 7, 1317, 22, 23, 27) examined the effect of
coinfection with P. falciparum. Studies that examined the pathological
effects on the host found that coinfected hosts had decreased parasitaemia,
deferred time to malaria attacks or decreased severity, and higher haemo-
globin counts than those without an underlying S. haematobium infection
(entry numbers 7, 1416, 22). One study noted that the effect was only
found in young children (entry number 16) or those children with a
relatively light worm burden (entry number 15). Though the malaria
species was not identified, decreased splenomegaly was also observed in
coinfected humans in another study (entry number 22). Five of the
S. haematobiumP. falciparum studies examined immunological effects
(entry numbers 2, 7, 13, 16, 17). Three of these studies noted an age effect
on the immune response, with young children (typically between 4 and
8 years old) showing different patterns than older children in their cyto-
kine responses (entry numbers 13, 16, 17). Overall, the studies indicated
that coinfection with S. haematobium probably mediated the incidence
and severity of infection with P. falciparum (entry numbers 7, 1317, 22),
possibly in an age-dependent manner (entry numbers 13, 16, 17).
Five of 19 human studies examined the effects of S. mansoni coinfection

with P. falciparum (entry numbers 6, 9, 10, 19, 20). One additional study
used an undetermined species of Plasmodium (entry number 21). All stud-
ies with S. mansoni found a detrimental effect of the coinfection on the host,
with increased malaria attacks (entry numbers 10, 19) or increased host
hepatomegaly and splenomegaly (entry numbers 9, 20, 21). An age effect
was also observed in one study (entry number 10), in which children
under the age of five had higher levels of parasitaemia than older children.
Increased egg load of S. mansoni was also associated with increased
malaria attacks, but not in a simple linear relationship (entry number 19).
Aspects of the cytokine response were examined in two studies, which
concluded that lower T lymphocyte subsets may be related to differential
morbidity or the course of infection (entry number 6) and higher levels of
IL10, IL12p70, IL 13 and sTNFR11 may be associated with inflammatory
responses in the liver and spleen (entry number 9).
The remaining studies examined S. mansoni and S. haematobium coin-
fections with P. falciparum (entry numbers 3, 4) or failed to identify the
species of Schistosoma or Plasmodium (entry number 12) used. These stud-
ies showed similar results to the S. mansoniP. falciparum findings noted
above, with coinfected hosts experiencing increased anaemia and para-
sitaemia. Several reviews discussed immunological and pathological
aspects of helminth interactions with Plasmodium (e.g. Brooker et al.,
2007; Hartgers and Yazdanbakhsh, 2006; Helmby, 2007; Nacher, 2008).
Some prevalence studies and case reports documented this coinfection in
children (e.g. Mazigo et al., 2010; Midzi et al., 2008), particularly the
exacerbation of hepatosplenomegaly when coinfected with S. mansoni
and Plasmodium (e.g. Wilson et al., 2007, 2010).
1.3. COINFECTION OF SCHISTOSOMA SPECIES WITH

PROTOZOANS OTHER THAN IN THE GENUS
PLASMODIUM
This section covers coinfection with species of Schistosoma and protozoans
other than those in the genus Plasmodium. Twenty nine studies were
included in Table 1.3; they examined coinfection between species of
Schistosoma and four protozoans other than Plasmodium. The studies
examined were Leishmania (entry numbers 19), Toxoplasma (entry num-
bers 1018), Entamoeba (entry numbers 1925) and Trypanosoma (entry
numbers 2629). Although most studies involved S. mansoni (entry num-
bers 120, 2229), two also examined S. haematobium (entry number 11)
and S. japonicum (entry number 21), and one examined S. bovis (entry
number 27), a non-human form. The coinfection pairs are arranged in the
table in the order listed above and discussed in the following sections.
TABLE 1.3 Coinfection studies of Schistosoma species and protozoans other than Plasmodium
Experimental (E)
Species of or natural (N)
Entry Species of coinfecting infections in Time between
number Reference trematode organism vertebrate hosts coinfections Comments
Leishmania
1 Hassan et al. Schistosoma Leishmania (E) C57BL/6 Sm followed by Co mice had similar Sm parasite burden and egg-
(2006) mansoni donovani mice Ld 8 wk later induced granulomatous response than mice with
single Sm infections; Co mice had greater Ld
parasite burden in liver and spleen than mice
with single Ld infections, despite delayed but
functional anti-Ld Th1 response; granulomatous
tissue responses to Sm formed a discrete niche
facilitating survival of intracellular Ld pathogens
2 La Flamme S. mansoni L. major (E) C57BL/6 Sm followed by Pre-infection with Sm delayed the development
et al. mice Lm 2 wk and resolution of Lm lesions; Lm infection had
(2002) later no impact on the course of Sm infection in
coinfected mice; pre-establishment of a strong
Th2 response can modulate Th1 cytokine
responses and result in exacerbation of
Th1-controlled infections
3 Yoshida S. mansoni L. major (E) BALB/c Sm followed by Despite any differences between groups during
et al. and C57BL/ Lm 8 wk course of infection, after 6 wk of infection Co
(1999) 6 mice later mice had comparable footpad thickness to mice
with single Ld infection; footpad thickness was
greater in Lm susceptible BALB/c mice than Lm
resistant C57BL/c mice
4 Mangoud S. mansoni L. d. infantum (E): Syrian Sm followed by Renal changes in Co hosts were comparable to
et al. golden Ldi 4 wk animals with either single infection, but infection
(1998a) hamster later due to Ldi occurred earlier and were more
obvious; Ldi may have modified the severity of
previous infection with Sm
(continued)
Experimental (E)
5 Mangoud S. mansoni L. d. infantum (E) Syrian Sm followed by Sm granulomas were smaller and less frequent in
et al. golden Ld 4 wk later Co hamsters compared to Sm-infected controls;
(1998c) hamster Ldi caused early appearance of cell necrosis and
fatty change; Ldi infection on top of Sm
suppressed Sm infection and accelerated
fibrosis, while infection due to Ldi became more
pronounced
6 Mangoud S. mansoni L. d. infantum (E) hamster Sm followed by Heart and lungs of Co hosts presented leishmanial
et al. Ld 4 wk later cardiac granulomas at 12 wk; pulmonary
(1998b) granulomas appeared earlier in Co hosts than in
controls
7 Morsy et al. S. mansoni L. d. infantum (E) Syrian Sm followed by Co hamsters had delayed appearance of Sm and
(1998) hamster Ldi 4 wk Ldi granulomas in small intestine compared to
later controls with either single infection
8 Mangoud S. mansoni L. d. infantum (E) Syrian Sm followed by Co hamsters had greater IgG, IgA, IgE responses
et al. golden Ldi 4 wk and greater decrease in C3 and C4 than animals
(1997) hamster later with either single infection
9 Coelho et al. S. mansoni L. mexicana (E) mice, type Sm followed by Lmm lesions appeared in all Co mice, but in only
(1980) mexicana not specified Lmm 60 days one Lmm control animal; incubation period for
later Lmm was shorter in animals with underlying Sm
infection
Toxoplasma
10 Araujo et al. S. mansoni Toxoplasma (E) C57BL/6 Sm followed by Co IL-12-deficient mice had decreased liver
(2001) gondii B6 and Tg 7 wk later damage, prolonged time to death and higher
Swiss- levels of Tg in their livers compared to controls;
Webster B6 production of inflammatory mediators was
Interleukin defective in IL-12-deficient animals; IL-12
(IL) 12(/) promoted liver damage during coinfection
mice
11 Afifi et al. S. haematobium T. gondii (N) humans, Unknown Levels of soluble intracellular adhesion molecule 1
(2000) or S. mansoni age not (sICAM-1) were correlated with disease severity
specified and pathogenesis; Co patients had higher levels
of SICAM-1 molecule compared to either single
infection; response in Co humans was similar to
infection with hepatosplenic Sm, indicating a
weak Th1 response in Co patients
12 Marshall S. mansoni T. gondii (E) C57BL/6 Sm followed by Co mice had increased morbidity and mortality
et al. mice Tg 7 wk later compared to mice with Tg alone; moribund Co
(1999) mice displayed severe liver disease including
steatosis and coagulative necrosis in areas
adjacent to egg granulomas; prior infection with
Sm increased sensitivity to Tg infection
13 Hammouda S. mansoni T. gondii (E) Swiss Tg followed by Co mice had increased spleen weights but no
et al. albino mice Sm 1 wk to difference in mean liver weights compared to
(1994a) 2 months mice with single Sm infection; Co mice had
later lower Sm worm loads than other groups; prior
infection with Tg increased resistance to Sm
14 Hammouda S. mansoni T. gondii (E): Swiss Tg followed by Co mice had increased B-lymphocytes, decreased
et al. albino mice Sm 1 wk to levels of anti-Sm antibodies and cellular immune
(1994b) 2 months responses, and reduced granuloma size
later compared with single Sm-infected controls;
toxoplasmosis induced humoural and cellular
immunosuppression to Sm
(continued)
Experimental (E)
15 Fayad et al. S. mansoni T. gondii (N): Human, Unknown Progression of liver disease in Co patients with Sm
(1992) ages not and latent Tg infection comparable to patients
specified with liver disease from single Sm infection
16 Kloetzel S. mansoni T. gondii (E) Albino Sm followed by Mice infected with Sm followed by Tg had massive
et al. mice Tg 59 days mortality during the acute stage of infection,
(1977) later; Tg great weight loss and pronounced splenomegaly
followed by compared with controls; relatively few notable
Sm 47 days effects when Tg preceded Sm
later
17 Mahmoud S. mansoni T. gondii (E) Swiss Sm followed by Co hosts had smaller hepatic granulomas and
et al. albino mice Tg 4 wk later; lower mean portal pressure compared to mice
(1977) Tg followed with single Sm infection; compared to other
by Sm 1 day timing and order sequences, mice infected with
or 4 wk later Sm followed by Tg 4 wk later had increased
spleen weight; mice infected with Tg 1 day
before Sm had reduced body weight and greatly
increased mortality
18 Mahmoud S. mansoni T. gondii (E) Swiss Sm followed by Mice infected with Sm followed by Tg at 4 wk had
et al. albino mice Tg 4 wk later; similar worm burdens and mean liver egg
(1976) Tg followed counts compared to controls; mice infected with
by Sm 1 day Tg followed by Sm at 1 day or 4 wk had reduced
or 4 wk later worm burdens and liver egg counts (43% and
35%, respectively) compared with controls
Entamoeba
19 Dolabella S. mansoni Entamoeba (E) Syrian Sm followed by Co hosts had increased morbidity and mortality
et al. histolytica hamsters Eh 70 days compared to animals with either single infection;
(2007) later adhesion of Eh trophozoites on Sm granulomas
not observed in histological sections, but Co
hosts displayed severe wasting and greater
number of amoebic lesions in livers; Sh
aggravated the course of the Eh infection
20 Mansour S. mansoni E. histolytica (N) Human, Unknown Prevalence of Eh was higher in the Sm endemic
et al. ages not village compared to the non-Sm village;
(1997) specified detection of Eh was higher by stool samples than
serologic tests; Sm may suppress immune
response of the host and increase susceptibility
to Eh infection
21 Liu et al. S. japonicum E. histolytica (E) Mongolian Concurrently Sj promoted caecal amoebiasis and stimulated
(1991) gerbil infected with symbiotic Eh infection to invasive caecal
Sj and Eh amoebiasis; trophozoites of Eh adhered to egg
shell of Sj at tissue necrosis site; affinity between
trophozoites of Eh and ova of Sj was noted
22 Abo-Shady S. mansoni E. histolytica (N) 24- to 56- Unknown Eh coinfected 47.8% of patients with Sm colonic
and year-old polypsis; 29.9% of patients with simple colonic
Yossef humans Sm lesions; 11.9% of non-Sm-infected controls;
(1986) severity of colonic Sm lesions directly correlated
with higher prevalence and level of invasiveness
of hematophagous trophozoites due to Eh
coinfection
23 Ali et al. S. mansoni E. histolytica (N) 3- to 64- Unknown Patients with Sm infection had higher Eh
(1984) year-old coinfection (53.32%) than non-Sm-infected
humans patients (13.78%); damage by Sm ova in
intestinal mucosa may have promoted
proliferation and invasion of Eh into mucosa
(continued)
Experimental (E)
24 El Raziky S. mansoni E. histolytica (N) 13- to Unknown Eh coinfected 37% of the patients with Sm colonic
et al. 50-year-old polypsis; 15% of the patients with Sm without
(1983) humans polypsis; 11% of the patients without Sm
infection; high correlation between colonic
polypsis and amaeobiasis noted
25 Knight and S. mansoni E. histolytica (E) Swiss Sm followed by Coinfection increased the infectivity of the Eh
Warren albino mice Eh 513 wk inoculum and the subsequent amoebic tissue
(1973) later invasion; some correlation existed with the
worm load; infectivity of Eh strain matters
Trypanosoma
26 Fagbemi S. mansoni Trypanosoma (E) albino mice Sm followed by Co mice had a lower faecal egg count per worm
(1987) brucei Tb 2 wk later; pair in faeces and small intestines compared to
Sm followed mice with single Sm infections; infection with Tb
by Sm may suppress immune response to Sm
challenge 6
wk later
27 Fagbemi S. mansoni, T. brucei (E) albino mice Tb followed by Co mice had a lower frequency of granulomatous
et al. S. bovis Sm or Sb 7 response and reduced diameter of granuloma
(1987) days later compared to mice with single Sm or Sb infection;
a similar response was obtained with Sm or Sb;
Tb infection had an immunosuppressive effect
on the host infections with Sm or Sb
28 Genaro et al. S. mansoni T. cruzi (E) Swiss Sm followed by When Sm followed by Tc, Co had 49% reduction in
(1986) albino mice Tc 43 days diameter of hepatic granuloma size compared to
later; Tc Sm controls; when Tc was followed by Sm 68 and
followed by 185 days later, Co had 47% and 37% reduction
Sm 68 and (respectively) in diameter of hepatic granulomas
185 days compared to Sm controls; Tc depresses Sm
later granuloma size, delaying hypersensitive
immune response during acute and chronic
phase of Tc infection
29 Kloetzel S. mansoni T. cruzi (E) albino mice Sm followed by Coinfection enhanced Tc parasitaemia in all
et al. Tc 66 days experiments; when Sm preceded Tc, Co mice
(1973) later; Tc had increased splenomegaly and higher
followed by mortality compared with controls; longer
Sm 4 to 63 duration of parasitaemia noted in Co mice that
days later had been exposed percutaneously but not
subcutaneously infected with Sm; when Tc
preceded Sm, Co mice had higher average peaks
than controls
Co, coinfected; Eh, E. histolytica; Ld, L. donovani; Lm, L. major; Ldi, L. donovani infantum; Lmm, L. mexicana mexicana; Sm, S. mansoni; Sb, S. bovis; Tc, T. cruzi; Tg, T. gondii; Tb, T. brucei;
unknown, not specified in original paper; wk, week or weeks.
1.3.1. Leishmania
All studies on SchistosomaLeishmania coinfections (entry numbers 19)
were experimental studies on mice (entry numbers 13, 9) or hamsters
(entry numbers 48) and examined a schistosome infection followed by
coinfection with Leishmania. Three species of Leishmania were used: two
with L. major (entry numbers 2, 3), one with L. mexicana (entry number 9),
and six with L. donovani (entry numbers 1, 48). The studies differed in
that they examined 2- (entry number 2), 4- (entry numbers 48), or 8-week
(entry numbers 1, 3, 9) intervals between coinfections.
In most studies, prior infection with S. mansoni allowed the subsequent
coinfection with Leishmania to develop earlier, become more pronounced,
or persist longer than in hosts with single Leishmania infection (entry
numbers 1, 2, 46, 9). These effects appeared to depend on the interval
between coinfection. The greatest pathological effects occurred when
Leishmania coinfection followed a patent schistosome infection, increasing
the parasite burden from L. donovani in both the liver and the spleen (entry
number 1); changes were also observed in the liver, lungs and heart when
coinfection occurred after a 4-week interval (entry numbers 5, 6). One
study hypothesized that the granuloma response by S. mansoni formed a
discrete niche that facilitated the intracellular survival of Leishmania organ-
isms (entry number 1). Another study proposed that the effect was due to
the early establishment of a strong Th2 cytokine response by prior infec-
tion with S. mansoni, which modulated the later Th1-based response to
Leishmania (entry number 2). There have been a few case reports of this
coinfection in human populations in China and East Africa that appear to
support either of these findings, where coinfection with helminths delayed
the resolution of leishmaniasis (Muigai et al., 1989; ONeal et al., 2007).
Similarly, the effect of Leishmania on an underlying infection with
Schistosoma may also depend upon the interval between coinfection.
There was no observed effect on the course of the Schistosoma infection
when S. mansoni was followed by Leishmania at 2- or 8-week intervals
(entry numbers 2, 1, respectively), but the severity of the schistosome
infection was reduced when Leishmania followed Schistosoma by 4 weeks
(entry numbers 4, 5). The host species may have played a role in this since
both the 2- and 8-week studies were done on mice and the 4-week studies
on hamsters. In addition, most of the mice studies used the C57 strain
(entry numbers 13) which was found to be more resistant to Leishmania
than the Balb/C strain (entry number 3).
1.3.2. Toxoplasma
The nine studies in this section examined coinfection of S. mansoni with
Toxoplasma gondii. One study also included S. haematobium, in addition to
S. mansoni (entry number 11). Seven studies were done on mice (entry
numbers 10, 1214, 1618) and examined the effect of infection with
S. mansoni followed by coinfection with T. gondii (entry numbers 10, 12,
1618) as well as infection with T. gondii followed by coinfection with
S. mansoni (entry numbers 13, 14, 1618). There were also two studies on
humans, and in both, the order and timing of the coinfection were not
reported (entry numbers 11, 15).
1.3.2.1. Animal studies

Overall, two distinct patterns were noted: coinfection that followed a
patent S. mansoni infection appeared to increase the sensitivity or severity
to subsequent infection with T. gondii (entry numbers 12, 16), while
infection with T. gondii modulated or decreased the severity of the
subsequent infection with S. mansoni (entry numbers 13, 14, 17, 18). All
studies used either C57 or Swiss albino mice, and no strain effect was
apparent. Studies on infection with S. mansoni followed by T. gondii
examined the coinfection effect of T. gondii at 4- (entry numbers 17, 18),
7- (entry numbers 10, 12) or 8-week (entry number 16) intervals. The most
consistent findings were noted at 7- and 8-week intervals, at which time
coinfected mice showed greater mortality, more severe liver damage,
greater weight loss and increased splenomegaly than mice with a single
T. gondii infection (entry numbers 12, 16). This effect was particularly
noted during the acute stage of toxoplasmosis (entry numbers 12, 16).
One study that examined the role of inflammatory mediators found that
IL-12 contributed to the increased liver damage observed in coinfected
hosts (entry number 10). Mice coinfected with T. gondii at 4-week intervals
had increased spleen weight in one study (entry number 17) but not in
another (entry number 18).
Studies on T. gondii followed by S. mansoni examined worm pairing at
1- to 28- (entry numbers 17, 18), 47- (entry number 16), or 7- to 56-day
intervals (entry numbers 13, 14). Most studies noted that coinfected hosts
had reduced worm burdens and liver egg counts, smaller granulomas
and decreased levels of S. mansoni antibodies than mice with single
S. mansoni infections (entry numbers 13, 14, 17, 18). An exception was
noted when mice were infected with T. gondii followed by S. mansoni 1
day later, which resulted in higher mortality and a reduced body weight
in coinfected hosts (entry number 17). Few effects of coinfection were
noted in the one study that used a 47-day interval (entry number 16).
1.3.2.2. Human studies

Two studies were done on humans (entry numbers 11, 15). The timing
and order of infection in these studies were unknown, but results showed
similarities to the findings observed in the previous animal studies. One
study found that coinfected patients had higher levels of an immune
substance that was correlated with disease severity and pathology than
in humans with either single infection (entry number 11). This study also
noted that responses of coinfected subjects were similar to patients with
hepatosplenic S. mansoni and may have indicated a weakened Th1
response (entry number 11). This finding is in accord with the effect
observed in mice that were coinfected with T. gondii following a patent
S. mansoni infection (entry numbers 12, 16). The other study found that the
progression of liver disease in coinfected patients was comparable to that
seen in single S. mansoni infections (entry number 15), which is similar to
the effect observed when S. mansoni infection preceded T. gondii by 4
weeks (entry number 18).
1.3.3. Entamoeba
Seven studies examined the effect of coinfection of Schistosoma and Ent-
amoeba: of these, three were done on animals (entry numbers 19, 21, 25)
and four on humans (entry numbers 20, 2224). Most studies (entry
numbers 19, 20, 2225) examined S. mansoni and E. histolytica coinfections.
One study done in China examined coinfection of S. Japonicum and
E. histolytica (entry number 21).

The three studies done on animals each used rodent hosts (entry numbers
19, 21, 25). Two examined an infection with S. mansoni followed by
E. histolytica at 10 weeks in the hamster (entry number 19) and 513
weeks in albino mice (entry number 25). In both cases, prior infection
with S. mansoni increased the subsequent infection with E. histolytica,
increased amoebic tissue invasion, mortality and caused severe wasting
compared to animals with a single E. histolytica infection. The same effect
was noted when the Mongolian gerbil was simultaneously infected with
S. japonicum and E. histolytica, which stimulated invasive caecal
amoebiasis (entry number 21). This paper suggested an affinity between
the trophozoites of E. histolytica and the eggs of S. japonicum that may
serve to stimulate E. histolytica infection to produce invasive amoebiasis
(entry number 21).

The four human studies (entry numbers 20, 2224) were done on subjects
ranging in age from 3 to 64 years, and the order and timing of infection
were unknown. The results were similar to those of the animal studies
noted above, in that patients with S. mansoni had higher levels of coinfec-
tion with E. histolytica. The severity of S. mansoni colonic polypsis was
directly associated with increased levels of E. histolytica infection with
amoebic invasiveness (entry numbers 2224). One study hypothesized
that damage by S. mansoni eggs in the intestinal mucosa may promote
the proliferation and invasion of E. histolytica into the mucosa (entry

number 23), whereas another suggested that infection with S. mansoni
may suppress the immune response and increase susceptibility to
E. histolytica (entry number 20). For further discussion of immunological
aspects of coinfection with S. haematobium, E. histolytica and other organ-
isms in children, see Hamm et al. (2009).
1.3.4. Trypanosoma
Four studies were done on coinfection of Trypanosoma and Schistosoma
(entry numbers 2629); all were done in albino mice. S. mansoni was used
in all studies and one study also included S. bovis (entry number 27). Two
species of Trypanosoma were studied, T. brucei (entry numbers 26, 27) and
T. cruzi (entry numbers 28, 29), both of which infect humans and animals.
Two studies examined the effect of the coinfection when Schistosoma
preceded Trypanosoma as well as when Trypanosoma preceded Schistosoma
(entry numbers 28, 29). Three of the four studies suggested that infection
with T. cruzi or T. brucei suppressed coinfection with S. mansoni or S. bovis
regardless of the order of infection (entry numbers 26, 27, 28).
Three studies examined infection with S. mansoni followed by T. brucei
2 weeks later (entry number 26) or T. cruzi 43 days later (entry number 28)
or T. cruzi 66 days later (entry number 29). In the first two studies, the
coinfected hosts had smaller hepatic granulomas and decreased egg
counts and worm burdens compared to those with single S. mansoni
infections (entry numbers 26, 28). The last study, which examined the
longest interval between coinfections, showed contrary results. In this
study, coinfected mice had increased splenomegaly and higher mortality
compared with controls, as well as a higher T. cruzi parasitaemia (entry
number 29).
Three studies also examined the effects of a prior Trypanosoma infec-
tion followed by an infection with Schistosoma. In the first study, T. brucei
was followed by S. mansoni or S. bovis at 7 days (entry number 27); in the
second study, T. cruzi was followed by S. mansoni at 68 and 185 days
(entry number 28); and in the last study, T. cruzi was followed by
S. mansoni 463 days later (entry number 29). The first two studies
found that coinfected mice had smaller hepatic granulomas than mice
with single Schistosoma infection (entry numbers 27, 28). No differences in
response based on S. mansoni or S. bovis were observed, though the
protective effect appeared to have weakened with increased time between
coinfection (entry number 28). The third study focused on the effect of the
T. cruzi infection and found higher average peak parasitaemia in the
coinfected hosts when compared to mice with a single T. cruzi infection;
the duration of the infection was longer in mice infected percutaneously
than mice infected subcutaneously (entry number 29).

SALMONELLA
A total of 16 studies were included in Table 1.4, which covers the inter-
actions between four species of Schistosoma and a number of Salmonella
enterica serotypes or subspecies. The four species of Schistosoma are
S. mansoni, S. haematobium, S. intercalatum and S. japonicum. Both typhoidal
(serotypes Typhi and Paratyphi A, B or C) and non-typhoidal (other
serotypes including Typhimurium, Enteritidis and subspecies arizonae)
Salmonella were included. In addition, a few studies compared strains of
Salmonella that were piliated, that is, bacteria with hair-like surface appen-
dages known as pili, along with non-piliated forms (entry numbers 79).
Pili occur on some bacteria and may have increased the ability of the
bacteria to attach to and colonize in a host; the piliated types have been
associated with increased virulence (Engelkirk et al., 2011).

Ten of 16 studies in this section were animal experiments: eight were done
on various types of mice (entry numbers 2, 69, 12, 15, 16) and two on the
Syrian Golden Hamster (entry numbers 13, 14). Most animal studies were
consistent in finding that a prior Schistosoma infection enhanced and
prolonged a subsequent infection with Salmonella. In particular, coin-
fected hosts had greater bacteremia, increased virulence, higher mortality
and more persistent local infection in the liver or spleen compared with
hosts with single Salmonella infection (entry numbers 2, 68, 1216). Sev-
eral interesting details were highlighted in these studies. The effect of the
coinfection was greatest when the Salmonella infection followed a 6- or 8-
week Schistosoma infection (entry numbers 2, 7, 8, 14) and also when
Salmonella was introduced into the host by the oral route rather than
intravenously (entry number 16). Although piliated strains of bacteria
were often associated with higher virulence, in these studies, both the
piliated and non-piliated strains were equally virulent in coinfected hosts
(entry numbers 79). One study also reported the effect of the coinfection
on Schistosoma and found that coinfected hosts had a greater hepatic
worm burden than hosts with a single Schistosoma infection (entry num-
ber 13).
Several studies reported that Salmonella bacteria multiplied in and
adhered to the schistosome worms, suggesting that these worms may be
the vehicle through which the infectivity of Salmonella is enhanced and
prolonged (entry numbers 9, 13, 15). In particular, adult female schisto-
somes were identified as being more frequently positive than males in
harbouring Salmonella (entry numbers 9, 13). Coinfected hosts were also
TABLE 1.4 Coinfection studies with Schistosoma and Salmonella
Experimental (E)
Entry Species of coinfecting infection in Time between
number Reference trematode organisma vertebrate hosts coinfections Comments
1 Nwaugo S. haematobium Unidentified (N) humans, Unknown Patients with Sm infection were more likely to
et al. Salmonella sp. mainly < 51 have concurrent typhoid fever than patients
(2005) years of age without Sm infection (4651% vs. 10%);
individuals aged 1030 years had higher
infection rates than older patients; males
and female subjects were equally coinfected
2 Njunda and S. mansoni S. Typhi (E) albino mice Sm followed by Co mice infected with ST 8 wk after Sm
Oyerinde ST at 2, 4 or infection had greater bacteremia, more
(1996) 8 wk persistent local infections in internal organs
and higher mortality than mice infected
with ST at 2 or 4 wk post-Sm or than mice
with single ST infection; adult male
schistosomes harboured more ST bacteria
than adult females; Sm enhanced the
bacterial virulence of ST
3 Abdul- S. mansoni S. Typhi (N) humans, Unknown Glomerulopathy in coinfected subjects was
Fattah age not mainly due to ST infection; Sm had a minor
et al. specified additive effect on the coinfected patients;
(1995) once hepatic fibrosis was established,
glomeruli development appeared to be
affected by circulating immune complexes
from either infection
(continued)
Experimental (E)
4 Gendrel S. intercalatum S. Typhimurium, (N) 2- to Unknown Children with Si and non-typhoidal

et al. S. Enteritidid, 16-year-old salmonellosis had symptoms of septicemia
(1994) S. galiema, humans comparable to children with single ST
S. arizonae, infection; prolonged fever (up to 26 days),
S. Typhi, swollen spleen and severe diarrhoea were
S. Paratyphi noted; underlying infection with Si
interacted with non-typhoidal Salmonella
5 Martinelli S. mansoni Various (N) humans Unknown Patients with hepatosplenic schistosomiasis
et al. unidentified (mean ages and prolonged Salmonella bacteremia
(1992) Salmonella 18 and 23 coinfection had comparable renal
species years) histopathological findings to patients with
including schistosomal glomerulonephritis without
S. Typhi Salmonella infection; pronounced
glomerular hypercellularity and interstitial
mononuclear cell infiltration were noted in
Co patients
6 Muniz- S. mansoni S. Typhimurium (E) Charles Sm followed by Co mice had reduced phagocytosis and
Junqueira River mice STy 46 intracellular destruction of the bacteria
et al. months later compared to mice infected only with STy;
(1992) underlying Sm infection altered the
function of macrophages and may have
played a role in the development of chronic
salmonellosis
7 Tuazon et al. S. japonicum S. Enteritidis, (E) Swiss mice Sm followed by Co mice showed more rapid mortality (100%
(1986) S. Typhimurium SE or STy 6 in 24 h) than mice with single Sm infection
(piliated and wk later (100% in 915 days) or mice with single
non-piliated) Salmonella infection (piliated 75% to
non-piliated 86% by day 15)
8 Tuazon et al. S. mansoni S. Typhimurium (E) Swiss mice Sm followed by Co mice had greater mortality than mice with
(1985a) (piliated and STy 6 wk single STy or Sm infections; after 7 days, Co
non-piliated) later mice with piliated STy (81%) or non-piliated
STy (73%) had higher mortality than mice
with single Sm infection (no mortality)
or single piliated STy (27%) or single
non-piliated STy (4%) infections
9 Tuazon et al. S. japonicum S. Typhimurium (E) Swiss mice Sm followed by Worms were harvested from mice 1618 h
(1985b) (piliated and STy 6 wk after coinfection; female schistosomes were
non-piliated) later more frequently positive for STy than male
schistosomes; non-piliated STy bacteria
adhered to more female than male
schistosomes; no difference observed in
adherence of piliated STy bacteria
10 Carvalho Schistosoma sp. Salmonella sp. (N) 8- to 66- Unknown Patients with schistosomiasis and chronic
et al. year-old Salmonella bacteremia had higher
(1983) humans circulating immune complexes than
patients with schistosomiasis alone; Co
patients had increased C1q-binding
complex; mean c3 levels were lower in
patients without renal involvement
(continued)
Experimental (E)
11 Gendrel S. intercalatum S. Typhi, (N) 3- to 18- Unknown Co patients with typhoid or paratyphoid fever
et al. S. Paratyphi year-old were more likely to relapse if underlying Si
(1984) strains C, B humans infection was not treated; Si prolonged the
infection with Salmonella sp.
12 Bonfim de S. mansoni S. Typhimurium (E) white mice Sm and STy at Mice coinfected at same time had much
Lima et al. same time; greater mortality than mice with single STy
(1982) Sm followed infection; mice coinfected at 120 days had
by STy at 120 slightly increased mortality than controls;
or 180 days mice coinfected at 180 days had mortality
equivalent to controls
13 Mikhail S. mansoni S. Paratyphi (E) Golden Sm followed by Co hamsters had higher percentage of Sm
et al. strain A hamsters SPa 6 wk worms in the hepatic veins than did
(1982) later hamsters with single Sm infection (83% vs.
48%); adult Sm worms were the major sites
of SPa adherence and colonization and
nutritional factors may have been involved;
higher bacteria counts were also noted in
the female worms
14 Mikhail S. mansoni S. Paratyphi (E) Golden Sm followed by Co hamsters had prolonged bacteremia,
et al. strain A hamsters SPa 6 wk diffuse visceral involvement and higher
(1981) later mortality than hamsters with single SPa
infection; Sm prolonged and enhanced SPa
infection
15 Rocha et al. S. mansoni S. Typhi (E) mice, strain Sm followed by Co mice retained ST bacteria in blood, liver
(1971) not specified ST 4050 and spleen longer than mice with single ST
days later infections; ST bacteria multiplied within Sm
worms in first week but were not present
after 2 wk
16 Collins et al. S. mansoni S. Enteritidis (E) CF-1 and Sm followed by Mice coinfected with SE orally had higher
(1972) CD-1 mice SE 18 wk levels of bacteria in the liver and spleen, and
later greater mortality than mice with single
orally administered SE infection; mice
coinfected with SE orally had more severe
systemic infection than mice with SE
administered intravenously; CF-1 mice
were used for most experiments
a
Co, coinfected; SE, S. Enteritidis; Sm, S. mansoni; Sh, S. haematobium; Si, S. intercalatum; Sj, S. japonicum; Spa, S. Paratyphi a; ST, S. Typhi; Sty, S. Typhimurium; unknown, not specified in
original paper; wk, week or weeks.
found to have altered macrophage activity, which may have played a role
in the development of chronic salmonellosis (entry numbers 7, 15, and
Lambertucci et al., 1998). No other patterns seemed apparent between
Schistosoma species and Salmonella serotypes, or by the type or strain of
rodent most used in these studies.

Four of the six studies done on humans appeared to be in agreement with
the animal studies discussed above (entry numbers 1, 4, 10, 11). Two
studies done on children infected with S. intercalatum found that coinfec-
tion prolonged the infection from Salmonella Typhi or Paratyphi, and that
subjects who were coinfected with Schistosoma and non-typhoidal Salmo-
nella strains had increased disease severity comparable to subjects with
typhoidal forms of the disease (entry number 4) and prolonged disease
(entry number 11). Similarly, a study examining S. haematobium found
that the coinfection may have increased the risk of typhoid fever (entry
number 1). Madbouly et al. (1993) reported that patients with Schistosoma
were more likely to have concurrent infection with Salmonella than
patients without schistosomes, and that tegumental tubercles (also
known as bosses) infected with Salmonella bacteria were found in several
coinfected hosts (see Fig. 1.2 from LoVerde et al., 1980). An extensive case
report of patients in the Sudan provided additional clinical data on the
coinfection, indicating that coinfected patients had prolonged fever,
severe anaemia and hepatosplenic involvement (Salih et al., 1977). Other
case reports provided similar findings and indicated that the Salmonella
infection could not be resolved without treating the underlying Schisto-
soma infection (e.g. Botterel et al., 1996; Bouree et al., 2002; Friedland and
Loubser, 1990; Gendrel et al., 1986; Lambertucci et al., 1987, 1988).
Boss
Salmonella
FIGURE 1.2 Scanning electron micrograph of the interaction of Salmonella Typhimur-

ium LT2 and Schistosoma mansoni, Puerto Rice, showing salmonellae associated with a
tegumental boss (1600). Bar units mm. Reproduced with permission from LoVerde
et al. (1980).
Several studies have examined the surface interactions between species of

Schistosoma and Salmonella (e.g. LoVerde et al., 1980; Melhem and
LoVerde, 1984; Miegeville et al., 1986). Although the significance of the
surface interaction is not known, several of these studies have proposed
that it may have contributed to prolonged infection with Salmonella
(LoVerde et al., 1980; Melhem and LoVerde, 1984). For a recent review
discussing the mechanisms and pathological features of this interaction,
see Muniz-Junqueira et al. (2009).

BACTERIA OTHER THAN SALMONELLA
Table 1.5 covers the interactions of Schistosoma spp. and bacteria other
than Salmonella spp., including various Mycobacterium spp. (entry num-
bers 15), Helicobacter pylori (entry numbers 69), and Staphylococcus
aureus (entry numbers 1012). Most studies examined coinfection with
S. mansoni (entry numbers 1, 47, 912); however, two studies examined
coinfections with S. haematobium (entry numbers 3, 4) and one included
S. japonicum bacteria interactions (entry number 8).
1.5.1. Mycobacterium
Three animal studies examined coinfection of Schistosoma and Mycobacte-
rium species: two of these were experimental studies with mice (entry
numbers 1, 5) and one studied a natural infection that occurred in sheep
(entry number 2). Each study examined a different species of Mycobacte-
rium as follows: M. bovis, M. paratuberculosis and M. avium. The two
human studies examined coinfection with Schistosoma and M. ulcerans
(entry numbers 3, 4).

The two animal studies (entry numbers 1, 2) examined interactions
between Schistosoma and M. bovis or M. paratuberculosis. These studies
found that coinfected hosts had more severe bacterial infections and
showed greater mortality than singly infected hosts. The study on
M. bovis (entry number 1) found that hosts coinfected with S. mansoni
had an increased number of bacteria in the lungs, liver and spleen com-
pared to animals with a single M. bovis infection; the authors hypothe-
sized that prior infection with schistosomes may have impaired the Th1
immune response, thereby increasing susceptibility to the subsequent
bacterial infection. The natural infection study in sheep showed greater
mortality in the coinfected hosts, although pathological changes in the
liver, small intestines and lungs and accompanying respiratory distress
TABLE 1.5 Coinfection studies of species of Schistosoma and bacteria other than Salmonella
Experimental (E)
Mycobacterium
1 Elias et al. S. mansoni Mycobacterium (E) BALB/c Sm followed Co mice had higher levels of bacteria
(2005) bovis, BCG mice by Mb in lungs, liver and spleen 6- to
strain (BCG 15-wk post-challenge; Co mice had
strain) greater lung pathology compared to
8 wk later controls with single Mb infection;
Sm increased susceptibility to Mb-
BCG infection and impaired Th1
type response to mycobacterial
antigen
2 Kataria Schistosoma sp. M. paratuberculosis (N) adult sheep Unknown Co hosts had 71% mortality; lung, liver
et al. and intestines infiltrated with
(2004) schistosome eggs; lymphocytosis
and leukocytosis in the sheep were
indicative of chronic infection;
severe respiratory distress
attributed to underlying infection
with schistosomiasis
3 Scott et al. S. haematobium M. ulcerans (N) humans, Unknown Patients with osteomyelitis were more
(2004) ages not likely to have Sh infection than
specified patients without osteomyelitis;
infection with Sh may have
increased the severity of infection
with Mu; no difference in detection
rates between Mu in patients with
and without Sh was noted
4 Stienstra S. haematobium, M. ulcerans (N) 2- to Ma followed Patients with Mu had comparable
et al. S. mansoni 53-year-old by Sm 60 levels of serum anodic antigens to
(2004) humans days later schistosomes as controls without
Mu; worm burdens from Sh or Sm
were also comparable between
those with and without Mu
infection; Sh or Sm appeared not to
increase susceptibility to Mu
5 Sacco et al. S. mansoni M. avium (E) BALB/ Ma followed Co mice developed morphologically
(2002) cAnN mice by Sm 60 distinct hepatic granulomas; spleens
days later of coinfected mice had granulomas
with mycobacteria but not
schistosome eggs; Co mice with
prior Th1 response induced by Ma
infection developed a Th2 response
to infection by Sm but modulated
subsequent coinfection with Sm
(continued)
Experimental (E)
Helicobacter pylori
6 Elsaied S. mansoni Helicobacter pylori (E) albino mice Sm followed Co mice showed increased gastric
et al. by Hp 5 pathological alterations compared
(2009) wk later to those with single Hp infection
and higher mean total of worms
than mice with single Sm infection;
severity of Hp was exacerbated by
coinfection with Sm
7 Abou S. mansoni H. pylori (N) humans, Unknown Co patients had less severe gastritis
Holw age not and lower serum malondialdehyde
et al. specified (MDA) levels, a lipid peroxidation
(2008) indicator, than patients with single
Hp infection; higher MDA levels
may be associated with
carcinogenesis in gastric mucosa
8 Du et al. S. japonicum H. pylori (N) 4- to Unknown Co subjects had a modified IgG
(2006) 73-year-old serologic response to Hp compared
humans to subjects with single Hp infection,
with reductions noted in certain
subclasses; modifications in Co
subjects may have reduced the
probability of developing gastric
atrophy
9 Elshal et al S. mansoni H. pylori (N) humans, Unknown Co subjects had reduced DNA
(2004) ages not damage, reduced proliferation
specified activity and reduced apoptosis
compared with Hp patients alone
indicating a reduction in gastric
mucosal injury; infection with Sm
may have modified an
inflammatory response to Hp
Staphylococcus aureus
10 Teixeira S. mansoni Staphylococcus (E) albino mice Sm followed 50% of mice coinfected during acute
et al. aureus by Sa 60 (60 days) phase and 47% of mice
(2001a) and 120 coinfected during chronic (120 days)
days later phase of Sm infection developed
liver abscesses; no abscess
formation occurred in mice with
either single infection or in
uninfected controls during
comparable time period; granuloma
formation was seen in coinfected
and single Sm infection groups
11 Mahmoud S. mansoni S. aureus (E) Swiss Sm followed Co mice developed pyogenic liver
and albino mice by Sa at 9 abscesses compared with no
Awad or 16 wk development of abscesses in mice
(2000) with either single infection; abscess
formation highest in mice infected
with Sa at 9wk after Sm infection
(85%) versus mice infected with Sa
at 16 wk (35%); abscess contained
granulomas with Sm egg ova as well
as Sa bacterial colonies
(continued)
Experimental (E)
12 Teixeira S. mansoni S. aureus (E) albino mice Sm followed Seventy-seven percent of coinfected
et al. by Sa 60 mice developed multiple hepatic
(1996) days later abscesses; no abscesses present in
single Sm or single Sa infections, or
in uninfected controls; granuloma
formation noted in Co mice and
mice with single Sm infection; no
pathological changes were noted in
the livers of mice with single Sa
infection or uninfected controls
Co, coinfected; Hp, H. pylori; Ma, M. avian; Mb, M. bovis; Mb-BCG, M. bovis-BCG; Mu, M. ulcerans; Sa, S. aureus; Sm, S. mansoni; Sh, S. haematobium; Sj, S. japonicum; unknown, not
specified in original paper; wk, week or weeks.
were attributed to an infiltration by eggs of the Schistosoma species (entry

number 2). The third study examined infection with M. avium followed by
S. mansoni 60 days later; this study found that spleens of the coinfected
mice with granulomas contained mycobacteria but not schistosome eggs.
The authors hypothesized that based on the order of infection, the
immune response in coinfected hosts was possibly modulated by the
subsequent coinfection with S. mansoni (entry number 5).

Two of three human studies that examined interactions between S. haema-
tobium or S. mansoni and M. ulcerans also suggested that coinfection with
Schistosoma spp. did not increase the susceptibility to the Mycobacterial
infection (entry numbers 3, 4). The findings of one study, however, sug-
gested that coinfection with Schistosoma spp. may have increased the sever-
ity of M. ulcerans since osteomyelitis occurred more frequently in the
coinfected patients than those with only the bacterial infection (entry num-
ber 3). Osteomyelitis resulted from severe infection with M. ulcerans and
often required the amputation of the affected limb. The other study exam-
ined the effect of M. ulcerans on a Schistosoma species infection and found
comparable worm burdens in coinfected patients when compared to those
with single S. mansoni or S. haematobium infections. For a further discussion
of risk factors and immunological aspects of this coinfection, see the review
of Stienstra et al. (2001), which also discussed the increasing problem of
Schistosoma and Buruli ulcer infections in West African countries.
There have been several reports of hosts coinfected with schistosomi-
asis and gastrointestinal tuberculosis, including a recent account of a
Laotian female immigrant in Australia presenting with clinical and his-
topathological characteristics similar to Crohns disease (Kwan et al.,
2009); an earlier report noted the difficulties in diagnosis of this coinfec-
tion in a patient from China (Labay et al., 1975). Cases of coinfection with
schistosomiasis and pulmonary tuberculosis have also been noted (e.g.
Olds et al., 1981; Sarwat et al., 1986; Gui et al., 1996, 1997). A number of
studies have indicated that schistosomiasis in humans reduces the effi-
cacy of the BCG vaccination; for recent reviews of helminths and myco-
bacteria, see Elias et al. (2007) and Sandor et al. (2003).
1.5.2. Helicobacter pylori

Three of four studies (entry numbers 79) were done on humans and
indicated that an infection with Schistosoma species may have a protective
effect on infection with H. pylori, since coinfected patients had less severe
gastritis (entry number 7), reduced gastric mucosal injury (entry number 9)
or modifications in serologic responses associated with lowered risk of
developing gastric atrophy (entry number 8) than patients with single
H. pylori infection. The results were consistent with H. pylori coinfection

studies and either S. mansoni (entry numbers 7, 9) or S. Japonicum (entry
number 8). Two recent studies examined aspects of immune response
between helminth infections and H. pylori in Colombian or African chil-
dren; both found increased Th2 responses, which may be associated with
decreased gastric cancer risk later in life (e.g. Cherian et al., 2010; Whary
et al., 2005).
The findings of the experimental animal study (entry number 6) were
in contrast to those on humans, with coinfected mice showing increased
gastric pathological changes than mice with single H. pylori infection. The
coinfected mice also had greater S. mansoni worm burdens than mice with
single schistosome infections.
1.5.3. Staphylococcus aureus

These studies (entry numbers 1012) were done on albino mice and
examined infection with Schistosoma species followed by S. aureus at 60
63 or 112120 days. In these studies, coinfected mice developed liver
abscesses containing eggs of Schistosoma species and S. aureus bacteria,
whereas no abscesses were found in singly infected hosts. Two studies
(entry numbers 10, 12) compared abscess formation when S. aureus infec-
tion occurred during an acute (6063 days) or chronic (112120 days)
Schistosoma species infection. One study (entry number 10) found no
difference in the mean percent abscess formation between interval groups
(50% vs. 47%), while the other (entry number 12) found a greater percent-
age of abscess formation when coinfection with S. aureus occurred with an
acute S. mansoni infection (85%) rather than a chronic infection (35%).
Numerous reports (i.e. Goldani et al., 2005; Lambertucci et al., 1990,
1997; Sanchez-Olmedo et al., 2003; Teixeira et al., 1996, 2001b) have docu-
mented cases of pyogenic liver abscesses in children and adults coinfected
with Schistosoma species and S. aureus; these reports are consistent with the
results of the animal experiments reported above. For further discussion of
these cases, see the review of Lambertucci et al. (1998, 2001).
1.6. COINFECTION OF SCHISTOSOMA AND

FASCIOLA SPECIES
There are a total of 18 studies presented in Table 1.6, which examined

coinfections with S. mansoni, S. bovis or S. douthitti and a Fasciola species,
typically F. hepatica or F. gigantica. Fourteen of 18 studies were experiments
done on a wide range of animals including mice, rats, cattle or calves, rabbits,
lambs and goats (entry numbers 3, 59, 1118); the remaining four studies
were natural infections in human populations (entry numbers 1, 2, 4, 10).
TABLE 1.6 Coinfection studies on species of Schistosoma and Fasciola
Experimental (E)
1 Abou Holw Schistosoma Fasciola sp. (N) humans, Unknown Co patients had serum gastrin levels
et al. sp. age not that were 3161% higher than
(2007) specified patients with either single infection;
alkaline phosphate activity was
associated with higher egg counts
from either parasite in all infected
patients and with higher serum
gastrin levels in the coinfected
2 Abou-Basha S. mansoni Fasciola sp. (N) humans, Unknown Co hosts had greater levels of
et al. age not precollege III peptide markers, an
(2000) specified indicator of active or established
fibrosis, than individuals with either
single infection; children aged 514
years had more coinfections than
adults as well as higher PIIIP levels;
Co hosts had greater marked
periportal fibrosis (23%) than those
with single Sm (11%) or single
Fasciola sp. (0%) combined
(continued)
Experimental (E)
3 Ferreras S. bovis F. hepatica (E) Castellana Sb followed by Co Sb/Fh hosts had greater
et al. lambsb Fh 6 wk later pathological changes in the liver
(2000)a (Sb/Fh); Fh and small intestines than hosts with
followed by Fh/Sb, or single Sb or Fh infections;
Sb 10 wk Co Fh/Sb hosts had fewer Sb egg
later (Fh/Sb) granulomas in the small intestine
and fewer globular leukocytes but
showed greater liver pathology than
hosts with single Sb infection
4 Shousha S. mansoni Fasciola sp. (N) 12- to 30- Unknown Co hosts had high levels of the free
et al. year-old radicals super oxide and nitric oxide
(1999) humans that were attributed to increased
antigen stimulation with the dual
infection; free radical production
was lower in hosts with single
Fasciola sp. infections than in hosts
with single Sm infections
5 Rodriguez- S. mansoni F. hepatica (E) lambs Sm followed by Co hosts had half the Fh worm burden
Perez and Fh 10 wk than hosts with single Fh infections
Hillyer later
(1995)
6 Rodriguez- S. bovis F. hepatica (E) Castellana Sb followed by Co Sb/Fh hosts had higher Fh worm
Osorio lambsb Fh 6 wk later burdens than hosts with single Fb
et al. (Sb/Fh); Fh infections and comparable Sh worm
(1993)a followed by burdens to single Sb-infected hosts;
Sb 10 wk conversely, Co Fh/Sb hosts had a
later (Fh/Sb) lower Sb worm burden than hosts
with single Sb infections
7 Haroun and S. mansoni F. hepatica (E) lambs Sm followed by Co hosts had a 51% reduction in Fh
Hillyer Fh 10 wk worm burden than hosts with single
(1988) later Fh infections; Co hosts also had
higher total leukocyte and
eosinophil counts, than controls, but
showed less hepatic damage
8 Ford et al. S. mansoni F. hepatica (E) PVG rats Sm followed by Co rats with prior Sm infection had
(1987) (irradiated and Fischer Fh 29 days 2833% reduction in Fh than rats
and non- F344 rats, later; Fh with single Fh infection; Co rats
irradiated) New followed by (both strains) with prior Fh infection
Zealand Sm 28 days had 6669% reduction in Sm burden
white later than rats with single Sm infection;
rabbitsb exposure to metacercariae or
juvenile worms stimulated
resistance, while irradiated Sm
cercariae and adults worms did not
9 El Sanhouri S. bovis F. gigantica (E) Nubian Sb followed by Co hosts with prior Sb infection from
et al. (irradiated) goatsb Fg 8 wk later irradiated cercariae had comparable
(1987) Fg worm burden to hosts with
single Fg infection; prior infection
with irradiated cercariae from Sb
did not reduce the subsequent
worm burden from Fg
(continued)
Experimental (E)
10 Salem et al. S. mansoni Fasciola sp. (N) humans, Unknown Co patients with fascioliasis had
(1987) age not higher IgM and lower IgG levels
specified than patients with single Fasciola
sp. infections; IgE levels were
comparable in both single and
double infection groups;
immunoglobin levels were not
correlated with egg counts
11 Yagi et al. S. bovis F. gigantica (E) Zebu cattleb Sb followed by Co cattle with Sb had 83% reduction in
(1986) (irradiated Fg 8 wk later; Fg compared to hosts with single Fg
and non- Fg followed infection; non-irradiated Sb
irradiated) by Sb 8 wk cercariae produced resistance, but
later irradiated Sb cercariae did not; Co
cattle with Fg had 92% reduction in
Sb compared to hosts with single Sb
infection
12 El-Azazy S. mansoni F. hepatica (E) rats, strain Sm followed by Co rats with prior Sm infection had
and Van not specified Fh 8 wk later fewer Fh worms and less
Veen pathological changes associated
(1985) with Fh than rats with single Fh
infection
13 Hillyer S. mansoni F. hepatica (E) GP albino Sm followed by Mice coinfected with Sm at 5 or 7 wk
(1981) miceb Fh at 3, 5 or 7 had 62% or 7189% reduction in Fh
wk worm burden compared to mice
with single Fh infection; no
difference between single and
double infection groups noted at
3-wk interval; Fh had no effect on
existing Sm infection
14 Monrad S. bovis F. hepatica (E) lambs Sh followed by Sheep coinfected with Fh at 23 or
et al. Fh at 23, 78 wk had 93% and 70% fewer Fh
(1981) 78 or 1617 worms than sheep with single Fh
wk later infection; sheep coinfected with Fh
at 1617 wk had comparable Fh
worm burden to the controls; Co
hosts had reduced Fh-induced liver
damage compared to the controls;
Sm egg burden was comparable
between single and double
infections
15 Sirag et al. S. bovis F. hepatica (E) Jersey Sb followed by Co calves had 30% reduction in worm
(1981) calvesb Fh 10 wk burden and less sever hepatobiliary
later damage compared with calves with
single Fh infection
16 Christensen S. mansoni F. hepatica (E) SVS albino Sm (single sex) Co hosts with a single sex Sm (male or
et al. miceb followed by female) infection had comparable
(1980) Fh 2276 Fh worm burdens to hosts with
days later; single Fh infection; Co hosts with
Sm (mixed mixed sex Sm infection had 61%
sex) reduction in Fh worms compared to
followed by controls; hosts infected
Fh 46 days simultaneously or up to 48 h apart
later; Sm had a reduction in Sm worms
(mixed sex)
(continued)
Experimental (E)
followed by (4043%) but not in Fh burden

Fh compared to the controls
concurrently
or 48 h later
17 Christensen S. mansoni F. hepatica (E) SVS albino Sm followed by Hosts with pre-patent Sm infections
et al. miceb Fh 728 or had comparable Fh worm burden to
(1978) 5465 days controls, while hosts with patent Sm
later; Fh infection had 3476% reductions in
followed by Fh worms; similarly, hosts with pre-
Sm 723, or patent Fh infection had comparable
2850 days Sm worm burdens to controls; hosts
later with patent Fh infection had 4150%
reductions in Sm worms
18 Maldonado- S. douthitti F. hepatica (E) albino mice Sd followed by Livers of Co mice observed 55 days
Moll Fh 25 days after initial Sd infection (followed
(1977) later; Fh by Fh) had decreased Fh
followed by unembryonated eggs and increased
Sd 20 days dead eggs compared to controls;
later similarly, livers of Co mice observed
45 days after initial Fh infection
(followed by Sd) had a greater
number of dead Sd eggs compared
to controls
Co, coinfected; Fg, F. gigantica; Fh, F. hepatica; Sb, S. bovis; Sd, S. douthitti; Sj, S. japonicum; Sm, S. mansoni; unknown, not specified in original paper; wk, week or weeks.
a
These two studies use the same study group and are discussed as one in our text.
b
See original papers for more information on the breeds and strains of hosts used.

An infection with a Schistosoma species followed by an infection with
Fasciola was examined in all 14 studies, with the interval between coinfec-
tion ranging from 2 to 17 weeks later depending in part on the species of
host animal used in the study. Most of the rodent studies used S. mansoni
for the Schistosoma species (entry numbers 8, 12, 13, 16, 17), with one study
using the murine schistosome S. douthitti (entry number 18). These studies
were consistent in findings that mice or rats with a 5- to 8-week Schisto-
soma infection had reduced (up to 89%) Fasciola worm burdens than singly
infected hosts (entry numbers 12, 13, 16, 17). One study reported that rats
with a 4-week Schistosoma infection had some reduction in Fasciola worm
burden (up to 33% reported, entry number 8); no differences in worm
burdens were reported at 14 weeks in mice (entry numbers 13, 17). Mice
that were concurrently infected or infected with Fasciola up to 48 h after
being infected with Schistosoma had 4043% reductions in Schistosoma
worm burden, indicating that the interval between coinfection may not
have a simple linear association (entry number 16). The apparent protec-
tive effect may only be found in mixed sex schistosome infections, since
single sex Schistosoma failed to induce any reductions in Fasciola worm
burdens during the expected time period (entry number 16).
The two studies done on lambs also found reductions (up to 51%) in
Fasciola worm burdens when an infection with Fasciola followed a patent
S. mansoni infection (entry numbers 5, 7). The other two lamb studies,
however, examined an infection S. bovis followed by an infection with
F. hepatica 217 weeks later with conflicting results (entry numbers 3, 6,
14). Contrary to the findings of the rodent studies, one study (entry
number 14) found that hosts with pre-patent Schistosoma infections had
greater reductions in Fasciola burden (up to 93%) than hosts with a patent
infection (70%), with no reductions observed at all in hosts with chronic
(1617 week) Schistosoma infections. Based on these findings, the authors
hypothesized that pre-patent schistosomes were responsible for the resis-
tance to Fasciola rather than the adult schistosome worm (entry number
14). Contrary to all other reported animal studies, one study found
increased Fasciola burdens when coinfection followed a 6-week Schisto-
soma infection in lambs (entry numbers 3, 6). Two large animal studies
examined an S. bovis infection followed by Fasciola were largely consis-
tent with most rodent reports, indicating reductions in Fasciola worm
burden in cattle (83% for an 8-week interval, entry number 11) and calves
(30% reduction for a 10-week interval, entry number 15). Reductions of
worm burden in pre-patent infections were not examined in these studies.
The above studies indicate that the timing interval between coinfec-
tions is important as well the species of Schistosoma and/or the host.
In studies using S. mansoni, the highest reductions in Fasciola occurred
when it followed a patent infection with S. mansoni; these studies mainly

involved mice, rats and lambs. Whereas in studies involving S. bovis, the
greatest reductions occurred in pre-patent infections; these studies
involved calves, cattle and lambs. Some of these studies reported that
the reductions in Fasciola burden were accompanied by fewer pathological
changes in the coinfected host (entry numbers 12, 14, 15, 18), including a
decrease in F. hepatica unembryonated eggs in the liver (entry number 18)
and less severe Fasciola-induced liver hepatobiliary damage (entry num-
bers 14, 15). The one study that reported an increase in worm burden also
noted greater pathological changes in the liver and small intestines in
coinfected hosts (entry numbers 3, 6). Several studies discussed above
reported that prior infection with irradiated Schistosoma cercariae failed to
produce a reduction in Fasciola burden (entry numbers 8, 9, 11).
The seven experimental studies that examined hosts Fasciola infections
followed by an infection with Schistosoma found reduced worm burdens
(up to 92%) for the subsequent infection (entry numbers 3, 6, 8, 11, 17, 18),
particularly when following a patent Fasciola infection (entry numbers 3,
6, 11, 17). Two studies also reported fewer S. mansoni eggs in the small
intestines of lambs (entry numbers 3, 6) and a greater number of dead
S. douthitti eggs in the liver of the mouse (entry number 18). Reductions in
S. bovis worm and egg burden in the lambs were also accompanied by
greater liver pathology in these coinfected hosts, indicating that reductions
in worm and egg burden may not be associated with fewer pathological
changes in the coinfected host (entry numbers 3, 6).

The four studies examining natural infections on human populations (entry
numbers 1, 2, 4, 10) each studied different aspects of the effect of coinfec-
tion, but findings generally indicated increased immunological and patho-
logical responses in the coinfected host. One study found that coinfected
hosts had greater periportal fibrosis as well as higher associated levels of
procollagen III peptide markers than single hosts, with the highest levels
occurring in children 514 years of age (entry number 2). Similarly, other
studies found that coinfected patients had higher egg counts which was
accompanied by higher serum gastrin levels (entry number 1) and higher
levels of free radicals associated with the inflammatory response (entry
number 4). Coinfected hosts with fascioliasis had higher IgM and lower
IgG levels than patients with single Fasciola levels, and these levels were not
correlated with Fasciola egg counts (entry number 10).
The few prevalence reports (e.g. Curtale et al., 2007; Esteban et al.,
2003) of coinfection with Schistosoma and Fasciola in hyperendemic areas
such as Egypt indicate that such coinfections may be rare in human
populations; reports in the veterinary literature indicate that such
coinfections may be common in cattle in some parts of the world, for

example, Zambia (Yabe et al., 2008) and that the clinical presentation in
other large game such as the Zebu in Senegal includes anaemia and
weight loss (Kaboret et al., 1993).
1.7. COINFECTION OF SCHISTOSOMA SPECIES AND

HELMINTHS OTHER THAN THE GENUS FASCIOLA
Table 1.7 presents coinfection studies on Schistosoma and various hel-

minths other than Fasciola, including echinostomatids in the genus Echi-
nosoma (entry numbers 15) and various nematodes including hookworm
species, that is (entry numbers 611) Necator americanus and Ancylostoma
spp., Trichuris (entry numbers 1215), Ascaris spp. (entry numbers 1618),
Strongyloides and Trichostrongyloides (entry numbers 1922) and filarids
(entry numbers 2325) including Brugia pahangi.
1.7.1. Echinostoma
All five coinfection studies on Schistosoma and Echinostoma were done in
Balb/C or other strains of albino mice; one study used the water rat,
Nectomys squamipes (entry number 1). These studies examined the effect of
coinfection on both S. mansoni (entry numbers 15) or S. bovis (entry number
5) and E. caproni (entry numbers 25) or E. paraensei (entry number 1).
Two of five studies examined the effect when Schistosoma was followed
by an infection with Echinostoma at intervals of 114 weeks. Mice with a
patent or chronic Schistosoma infection had a 73% or a 100% reduction
(respectively) in the Echinostoma infection compared to controls (entry
number 5), while mice with a pre-patent Schistosoma infection had no com-
parable reduction in infection (entry numbers 3, 5). Two studies (entry
numbers 1, 5) examined the effect when Echinostoma was followed by an
infection with Schistosoma at 2- to 6-week intervals. Here, mice with a
6-week-old Echinostoma infection had a reduced Schistosoma infection com-
pared to the controls (entry number 1), while mice and rats with a 2- or
3-week infection showed no comparable reduction in Schistosoma (entry
number 5). Increased Schistosoma worm burdens were noted when the
schistosome infection followed that of Echinostoma at 2 weeks (entry number
1) or 33 days (entry number 4). These results suggest that only patent
schistosome infections confer protection against Echinostoma or could be
attributed in part to differences in the strain of Schistosoma used in the experi-
ments. In regard to possible strain differences, one of the studies found that
rats had a heavier early worm burden when the wild RJ strain of Schistosoma
was used rather than the BH lab strain (entry number 1). Finally, one study
examined the effect on pregnant mice concurrently infected with S. mansoni
TABLE 1.7 Coinfection studies on species of Schistosoma and helminths other than Fasciola
Experimental (E)
Echinostoma
1 Maldonado S. mansoni Echinostoma (E) Nectomys Rat: Ep Rats showed different
et al. (2001) (wild RJ and paraensei squamipes followed by susceptibility depending on
BH lab (water rat)a; Sm at 4 wk; Sm strain; Co rats with RJ
strain)a Swiss-Webster mouse: Ep strain of Sm had greater Sm
albino mice followed by worm burdens than controls,
Sm at 2 or 6 but parasitism was not
wk affected with Bh strain;
reduction in Sm (either strain)
was noted in coinfected mice
when followed by Sm at 6 wk;
Ep infection appeared to
interfere with development of
Sm in a strain-dependent
manner in some rodents;
earlier increase in Sm was
noted when mice were
coinfected at 2 wk
2 Bindseil et al. S. mansoni Echinostoma (E) BALB/ Concurrently Co pregnant mice had a lower
(1989)b caproni cABom mice coinfected number of live foetuses than
pregnant mice without
coinfection; mean foetal
weight was lower in
coinfected hosts than in
controls
3 Christensen S. mansoni E. caproni (E) albino SS and Sm followed Expulsion of low-level Ec was
et al. SVS mice by Ec 4 wk impaired in mice harbouring
(1985)b later pre-patent Sm infection
compared to mice with single
Ec infection; timing of
expulsion was dependent on
strain and age of mice
4 Christensen S. mansoni E. caproni (E) SVS albino Ec followed Co mice had increased Sm
et al. mice by Sm worm burdens compared to
(1981)b 2033 days mice with a single Sm
later infection; the increase in Sm
worm burden was greatest in
mice with 33-day-old Ec
infections (91%) compared to
controls
5 Sirag et al. S. mansoni, E. caproni (E) SVS albino Sm or Sb Mice infected with Sm followed
(1980)b S. bovis mice followed by by Ec 43 days later had a 73%
Ec 799 reduction in Ec infection
days later; compared to mice with a
Ec followed single Ec infection; mice
by Sm 14 or coinfected with Ec at 79 or 99
21 days days after Sm had 100%
later reduction in Ec compared to
controls; Sb infection or
pre-patent Sm infection had
no effect on subsequent Ec
infection; prior infection with
Ec had no effect on
subsequent infection with Sm
(continued)
Experimental (E)
Hookworm
6 Wu et al. S. japonicum Necator (E) golden Concurrently Co hosts had comparable worm
(2010) americanus hamster coinfected burdens to hamsters with
single infection but showed
altered metabolic profiles
including depleted amino
acids and glucose in sera, and
gut-related metabolites;
changes may induce
pathological changes (e.g.
liver damage, anaemia) in Co
hosts
7 Pullan et al. S. mansoni N. americanus (N) children and Unknown There was no evidence that host
(2010) adult humans, genetics modulated the
ages not intensity of coinfection in
specified endemic areas; a high positive
correlation between Sm and
Na egg counts was noted in
coinfected hosts; a strong
correlation between intensity
of infections in Co hosts was
noted
8 Ezeamama S. japonicum Hookworm (N) 7- to 18-year- Unknown Co hosts had higher levels of
et al. (2008) (species not old humans anaemia than subjects with
designated) either single infection;
coinfection results were
suggestive of multiplicative
effects on anaemia in hosts
9 Fleming et al. S. mansoni N. americanus (N) mostly <40 Unknown Subjects with heavy hookworm
(2006) years humans infections also showed heavy
coinfections with Sm; Co
subjects had higher Sm egg
burdens than subjects with
single Sm infection; infections
appeared heaviest in
individuals aged 10 years and
older
10 Keiser et al. S. mansoni Hookworm (N) human Unknown High positive correlation
(2002) (species not children between intensity of infection
designated) with Sm and coinfection with
hookworm in study subjects,
especially among older
children; repeated faecal egg
counts were necessary to
determine the extent of
coinfection
(continued)
Experimental (E)
11 Chamone S. mansoni Ancylostoma sp. (N) 18- to 50- Unknown More than half of the patients
et al. (1986) year-old with chronic Sm infection
humans were also coinfected with
Ancylostoma; Co hosts had
higher mean Sm egg output
than patients with single Sm
infections
Trichuris
12 Bickle et al. S. mansoni Trichuris muris (E) AKR mice Tm followed Co hosts had altered cytokine
(2008) by Sm 40 response to Sm in their lungs
days later and livers; greater number of
Sm worms and eggs found in
the liver of Co hosts; results of
the study suggest that pre-
existing Tm infection
facilitated survival and
increased migration of Sm to
the hepatic portal system
13 Ezeamama S. japonicum T. trichiura (N) 7- to 18-year- Unknown Co hosts showed higher levels
et al. (2008) old humans of anaemia than subjects with
either single infection;
coinfection induced additive
effects on anaemia in hosts
14 Curry et al. S. mansoni T. muris (E) AKR micea Sm followed Mice with an established Sm
(1995) by Tm 56 infection had lower Tm worm
days later; burdens and an increased
Tm ability to resolve a subsequent
followed by infection by Tm compared to
Sm 3, 7, 21 mice with single Tm infection;
and 31 days this response was linked to a
later stronger Th2 and lower Th1
response in Co hosts;
alterations in Th1 profile were
also found in mice infected
with Tm followed by Sm
15 Parraga et al. S. mansoni Trichuris sp. (N) 7- to 15-year- Unknown Subjects with Sm were more
(1996) old humans likely to be coinfected with
Trichuris than subjects
without Sm infection; Co
subjects were more prone to
malnutrition than subjects
without coinfection
Ascaris sp.
16 Fleming et al. S. mansoni Ascaris (N) less than Unknown Fewer subjects were coinfected
(2006) lumbricoides 1- to over with Sm and Al, than were
40-year-old infected by Sm or Al as single
humans infections; Co hosts had lower
Sm egg burdens than single
infected subjects with Sm; no
difference in rate of infection
was noted between age
groups
(continued)
Experimental (E)
17 Parraga et al. S. mansoni Ascaris sp. (N) 7- to 15-year- Unknown No significant effects of the
(1996) old humans coinfection on the nutritional
status of the hosts were noted;
Co boys and girls had Ascaris
burdens that were
comparable to children
without Schistosoma infections
18 Helwigh and S. japonicum A. suum (E) Danish cross- Sj followed by Co pigs had fewer As induced
Bogh (1998) bred pigsa As 11 or 16 white spots on the liver than
wk later pigs with single As infection;
Sj may have suppressed gross
pathological changes from As
or inhibited host immune
response to As; Co pigs had
comparable levels of As
larvae in lungs, liver and
small intestines as the
controls
Strongyloid/Trichostrongyloid nematodes
19 Bazzone et al. S. mansoni Heligmosomoides (E) CBA and BL/ Hp at 4 wk Co CBA mice had smaller
(2008) polygyrus 6 mice and at hepatic granulomas and a
2 days prior reduced immune response to
to Sm Sm infection but showed
infection comparable Sm worm and
egg burdens compared to
mice with a single Sm
infection; prior infection with
Hp reduced the severity of
the Sm infection
20 Gazzinelli S. mansoni Strongyloides (E) AKR/J mice Sm followed Co mice had fewer Sv larvae in
and Melo venezuelensis by Sv 45 their lungs and fewer female
(2008) days later worms in their intestines
compared to mice with single
Sv infection; female Sv worms
in Co hosts were smaller and
less fertile than Sv worms
recovered from mice with
single Sv infection; prior
infection with Sm decreased
subsequent infection by Sv
21 Maruyama S. japonicum S. venezuelensis (E) C57BL/6 Sj followed by Mice infected with Sj showed
et al. (2000) mice Sv 6 wk strong protective immune
later; other response to subsequent
times tested infection with Sv; Co mice
but not eliminated intestinal Sv
specified infection faster than mice
with single Sv
(continued)
Experimental (E)
22 Yoshida et al. S. mansoni S. venezuelensis (E) BALB/c and Sm followed Co mice had fewer Sv larvae in
(1999) C57BL/6 mice by Sv 3, 6, 9 their lungs and far fewer
or 12 wk adult worms in the small
later intestines compared to mice
with single Sv infection; no
worms were recovered in Co
mice 9 wk after Sm infection;
immune response from prior
infection with Sm appeared to
protect from subsequent
infection with Sv
Filarid nematodes
23 Sato et al. S. mansoni Brugia pahangi (E) Mongolian Bp followed Co girds with acute (17 days) Bp
(1989) jirda by Sm 17 infection showed comparable
days or 39 Sm worm recovery to girds
wk later with single Sm infection;
some Co girds with chronic
(39 days) Bp had increased
number of Sm eggs in the
stool but comparable level of
worms than girds with single
Sm infection; infection with
Bp appeared not to enhance
the susceptibility of hosts to
subsequent infection with Sm
24 el-Hawey Schistosoma sp. Unidentified (N) humans, age Unknown Co subjects had higher fever,
et al. (1986) filarid species not specified abdominal distention, joint
pains, dysentery and anaemia
than subjects with either
single infection; Co hosts
were more likely to have
elephantiasis than subjects
with single filiarial infections;
Co had lower egg counts and
was less likely to have liver or
spleen involvement than
subjects with single
Schistosoma infections
25 Mohamed S. haematobium, Unidentified (N) humans, age Unknown Co subjects with microfilariae
et al. S. mansoni larval and not specified had higher serum C3 levels
(1983a), adult filarid than subjects with either
Mohamed species single infection; Co subjects
et al. with adult filarids had higher
(1983b) serum C4 levels than subjects
with either single infection;
Co subjects were more likely
to have elephantiasis than
singly infected subjects, with
highest prevalence among
females and subjects under 30
years of age
Co, coinfected; Al, A. lumbricoides; As, A. suum; Bp, B. pahangi; Ec, E. caproni; Ep, E. paraensei; Hp, H. polygyrus; Na, N. americanus; Sb, S. bovis; Sh, S. haematobium; Sj, S. japonicum;
Sm, S. mansoni; Sv, S. venezuelensis; Tm, T. muris; Tt, T. trichuris; unknown, not specified in original paper; wk, weeks.
a
See original papers for more information about the breeds and strains of hosts used.
b
Echinostoma caproni is referred to in this study as Echinostoma revolutum.
and E. caproni and found that coinfected mice had a lower number of live
foetuses than pregnant mice without the coinfection; the mean foetal weight
was also lower in the coinfected hosts (entry number 2).
In summary, a patent infection with either Schistosoma or Echinostoma
appeared to reduce the burden of the subsequent infection by the other
trematode, whereas there was no effect or possibly an increased burden of
the subsequent infection when worms were introduced during the pre-
patent phase of the first infection. In several studies, the authors noted
that the age and strain of the rodent host used was an important factor in
these coinfection studies (entry numbers 1, 3).
1.7.2. Hookworm
Five of six studies were natural infections in children and adults: four
examined hookworm coinfection with S. mansoni (entry numbers 7, 911)
and one studied hookworm and S. japonicum (entry number 8). These
studies included various hookworm species, identified in three studies as
N. americanus (entry numbers 7, 9) and as a Ancylostoma sp. in one study
(entry number 11). One study (entry number 6) examined an experimen-
tal coinfection between S. japonicum and N. americanus in the Golden
Hamster. The studies on humans consistently found a strong association
between the schistosome and the hookworm infections, with greater
Schistosoma egg counts and worm burdens reported mainly in hosts
with the heaviest hookworm infections (entry numbers 7, 911). In two
studies, children aged 10 or older had some of the heaviest coinfection
burdens (entry numbers 9, 10). Coinfected children also had an increased
anaemia compared to singly infected children; the possibility of a multi-
plicative schistosomehookworm interaction, that is, the combined effect
of the coinfection being greater than the sum of either infection was
suggested by several studies (entry numbers 8, 9, 11). The sole experimen-
tal schistosomehookworm study (entry number 6) found that coinfected
hamsters had comparable worm burdens to singly infected control hosts;
the coinfected hosts also had altered metabolic profiles that may have
been associated with pathological changes in the liver or anaemia.
The interval between coinfection was unknown in the human studies,
while the hamsters were infected concurrently in the experimental
schistosomehookworm study.
1.7.3. Trichuris
Coinfection interactions with Schistosoma were examined in two experi-
mental mouse studies using T. muris (entry numbers 12, 16) and two natural
infection studies in humans with T. trichiura (entry numbers 13, 15).
Both murine studies examined the immune response and the effect when T.
muris was followed by S. mansoni (entry number 12, 14); one study also
focused on S. mansoni followed by T. muris (entry number 14). The two
human studies examined the effects of coinfection in which the interval
between the first and the second infection was not given; one study exam-
ined T. trichiura with S. mansoni (entry number 15) and the other T. trichiura
with S. japonicum (entry number 13).
In entry number 12, mice with a chronic (40 days) T. muris infection
had an increased burden of Schistosoma worms and eggs in their livers
compared to singly infected mice. An immune response to T. muris
dominated the coinfected animals and an altered response to S. mansoni
was found in both the lungs and the livers. The prior infection with
T. muris appeared to enhance the migration of both male and female
schistosomes to the hepatic portal system, thus increasing the severity of
the Schistosoma infection. The other study (entry number 14) found that
coinfection altered the parasite-specific immune response, with the great-
est effect noted on the second infection. In this study, mice infected with
S. mansoni followed by infection with T. muris 56 days later showed an
established Th2 response that increased the ability of these mice to resolve
the secondary infection. In this study, coinfected hosts had lower T. muris
worm burdens than singly infected T. muris controls which had a domi-
nant Th1 response.

The two natural infection studies on children found that coinfected hosts
had more severe anaemia (entry number 13) and malnutrition (entry
number 15) than singly infected subjects. The severity of the anaemia
appeared additive in nature, that is, equal to that of singly infected
children with either infection added together (entry number 13).
In entry number 15, children with S. mansoni were more likely to have
T. trichuris coinfection than subjects without the Schistosoma infection,
suggesting that susceptibility to trichuris infection may be increased by
coinfection.
1.7.4. Ascaris
The three studies in this table examined the coinfections between Schisto-
soma species and Ascaris species: two were natural infection studies in
humans (entry numbers 16, 17) and one was an experimental study in
pigs (entry number 18). The two human studies examined different
aspects of coinfection between S. mansoni and Ascaris species, identified
in one of study as A. lumbricoides. One study (entry number 16) reported
that coinfected hosts had lower S. mansoni egg burdens than subjects with
single Schistosoma infections; it also reported that coinfection with
Schistosoma and Ascaris occurred less often than single infections with
either species in this population. The other study (entry number 17)
focused on the Ascaris worm burden, finding comparable burdens of
Ascaris infection in those with and without Schistosoma. Both papers
noted that there was no difference in the percent infected between age
groups (entry number 16) or between males and females (entry number
17). Both the order and the interval between infections were unknown in
either study. The experimental study examined the effect of an infection
with S. japonicum followed by an infection with A. suum 11 or 16 weeks
later in Danish cross-bred pigs (entry number 18). In this study, the
coinfected pigs had fewer A. suum-induced white spots on the liver but
comparable levels of Ascaris larvae in the lungs, liver and small intestines
as controls. The authors concluded that it was possible that a prior
infection with S. japonicum may have suppressed gross pathological
changes associated with Ascaris or in some way inhibited the host
immune response.
A number of prevalence studies have examined the worm burden of
coinfection with schistosomes and Ascaris, Trichiura or hookworm and
reported results that are largely consistent with those discussed above.
Coinfection with hookworm and Schistosoma in children is common in
many locations including Brazil, China, Kenya and Tanzania, while coin-
fection with Schistosoma and Trichiura or Schistosoma and Ascaris occurs
less often in endemic areas (e.g. Brito et al., 2006; Brooker and Clements,
2009; Brooker et al., 2000; Chamone et al., 1990; Ellis et al., 2007; Hamm
et al., 2009; Lwambo et al., 1999; Nguhiu et al., 2009). For a recent review
discussing these studies and additional immunological factors, see Geiger
(2008).
1.7.5. Strongyloides and Trichostrongyloides

The four experimental studies (entry numbers 1922) involving strongyloid
and trichostrongyloid nematodes examined coinfection with S. mansoni
or S. japonicum and either Strongyloides venezuelensis or Heligmosomoides
polygyrus in various strains of mice. Three of four studies (entry
numbers 2022) examined a Schistosoma infection followed by an infection
with S. venezuelensis 312 weeks later; these studies found that the first
infection decreased the severity of the subsequent Strongyloides infection.
These studies also found that coinfected hosts had decreased numbers
of strongyloid larvae in their lungs and a decreased adult worm burden
in the small intestines (entry numbers 2022), with smaller and less fertile
S. venezuelensis females (entry number 20). The greatest reduction in worm
burden in the Strongyloides infection occurred when infection followed a 6- to
9-week Schistosoma infection (entry numbers 21, 22). A similar effect was
observed for both S. mansoni and S. japonicum and did not seem to vary with
mouse strain. One study examined a prior infection with H. polygyrus

followed by infection with S. mansoni 2 days and 4 weeks later; in this study,
infection with H. polygyrus reduced the severity of the subsequent
Schistosoma infection, with a reduced schistosome worm burden and smaller
hepatic lesions in the coinfected animals compared with the controls
(entry number 19).
In this interaction, infection with one helminth in an experimental
setting seemed to confer protection from a subsequent infection with the
other helminth; as observed in other helminth interactions, worm age
associated with the first infection is important, with patent infections
tending to confer the greatest protection. Natural coinfections in human
populations do occur. A recent prevalence survey found that over 9% of
Sudanese refugees were coinfected with Schistosoma and Strongyloides
(Brodine et al., 2009), and several case reports illustrate a variety of clinical
presentations from coinfection (e.g. Fairweather et al., 2010; Olson and
Domachowske, 2006; Tzanetou et al., 2005).
1.7.6. Filarids
The past three studies in this table examined coinfections of Schistosoma
species and filarids: one was an experiment done on girds with B. pahangi
(entry number 23); the other two studies were natural infections in
humans with unidentified filarids (entry numbers 24, 25). Two studies
identified the schistosomes as S. mansoni (entry numbers 23, 25) or
S. haematobium (entry number 25); the other did not identify the schisto-
some species (entry number 24). The findings of the two human studies
were consistent, indicating that coinfected subjects had altered immune
responses and pathological changes compared to subjects with single
filarid infections. Coinfected subjects were more likely to have elephanti-
asis (entry numbers 24, 25) and increased fever, anaemia, joint pains and
other clinical symptoms associated with filariasis (entry number 24) than
subjects with a single filarid infection; one of the studies (entry number
24) also examined the effect of the coinfection on the Schistosoma infection
and found that the coinfected were less likely to have liver and spleen
involvement from than singly infected Schistosoma controls. While coin-
fection appeared to increase the severity of filariasis, it may be associated
with a reduction in the severity of the Schistosoma infection.
The experimental study (entry number 23) examined the effect of a
prior infection of B. pahangi followed by subsequent infection with
S. mansoni 17 or 39 days later; it did not find much difference in the
comparison groups other than a modest increase in the number of S.
mansoni eggs in the stool of coinfected girds with chronic B. pahangi
infection. The coinfected girds had a comparable number of S. mansoni
worms as the controls, regardless of the time interval between coinfection.
1.8. CONCLUDING REMARKS
Numerous factors influence the effects of coinfection on the vertebrate

host with Schistosoma and protozoa, bacteria or other helminths. Some of
these factors are as follows: organisms and hosts used in the studies, order
and time interval between the first and the second infection, studies on
natural versus experimental hosts, dosage of the infectious agents, strains
and pedigrees of the parasites, age of hosts at time of exposure to the
infectious agents and age of hosts at the time of necropsy.
Broad generalizations on the effects of coinfection on vertebrate hosts
are difficult to make. However, some trends based on our review for the
most studied aspects of coinfection are worth noting. A prior infection
with Schistosoma, particularly a patent infection, often has an effect on the
subsequent infection by a protozoan, bacterium or other helminth. In
relatively few cases, a prior infection with Schistosoma decreased the
severity of the subsequent infection as with H. pylori, F. hepatica, Echinos-
toma or Plasmodium, the latter only exhibiting this behaviour when coin-
fected with S. haematobium. More often, however, a prior infection with
Schistosoma increased the severity of the second infection as with Leish-
mania, T. gondii, E. histolytica, S. aureus or Salmonella. The severity of a
Plasmodium infection was also increased when it followed a prior infection
with S. mansoni. In some of these coinfection studies, the increased sever-
ity of the subsequent infection was associated with a specific, prolonged
form of the disease in humans, for example, osteomyelitis associated with
M. ulcerans, elephantiasis associated with certain filarids, non-typhoidal
salmonellosis appearing with typhoidal severity associated with Salmo-
nella and pyogenic liver abscesses associated with S. aureus. The severity
of this effect may be due to the subsequent coinfecting organism being
contained within the Schistosoma worms or associated with schistosome-
induced granuloma, as in the case of coinfection with Salmonella, S. aureus
or Leishmania. It may also be due to the subsequent coinfecting organism
taking advantage of prior physical damage caused by the Schistosoma, as
in the case of coinfection with E. histolytica. Finally, the severity of the
subsequent coinfection could also be due to the absence of a strong
immune response in the host due to an ongoing immune response to
Schistosoma, as in coinfection with E. histolytica, Leishmania or Plasmodium.
As many of these studies suggest, the subsequent infection may progress
or resist standard treatment if the underlying coinfection with Schistosoma
is not properly diagnosed and treated.
Only 3 of the 18 Schistosoma coinfection interactions reviewed herein
have a considerable body of literature, that is, between 16 and 32 papers
per interaction. These Schistosoma interactions involve Plasmodium, Salmo-
nella and Fasciola. The other Schistosoma coinfections we reviewed have a
much smaller body of literature (between three and nine papers per
interaction). Because of the common occurrence of Schistosoma with Myco-

bacteria, Leishmania, Staphylococcus, Necator and Strongyloides, additional
work is needed on coinfection with Schistosoma and these genera. Finally,
future studies should take into consideration the factors mentioned in the
first paragraph of Section 1.8.
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CHAPTER 2
Trichomonas vaginalis
Pathobiology: New Insights from
the Genome Sequence
Robert P. Hirt,* Natalia de Miguel, Sirintra Nakjang,*
Daniele Dessi, Yuk-Chien Liu,,1 Nicia Diaz, Paola
Rappelli, Alvaro Acosta-Serrano,} Pier-Luigi Fiori,
and Jeremy C. Mottram

2.2. Surface and Secreted Molecules 89
2.2.1. Surface proteins 90
2.2.2. Proteomics data for surface proteins 97
2.2.3. The glycocalyx 100
2.2.4. Secreted proteins: The trichopores 108
2.3. Peptidases 111
2.3.1. Aspartic peptidases 113
2.3.2. Cysteine peptidases 113
2.3.3. Serine peptidases 115
2.3.4. Metallopeptidases 115
2.3.5. Threonine peptidases 116
2.3.6. Peptidase inhibitors 116
2.3.7. Non-peptidase homologues 117
* Institute for Cell and Molecular Biosciences, Newcastle University, Newcastle upon Tyne, United Kingdom
{
Laboratorio de Parasitologia Molecular, Instituto de Investigaciones Biotecnologicas-Instituto Tecnologico
de Chascomus, Chascomus, Argentina
{
Department of Biomedical Sciences, and Centre for Biotechnology Development and Biodiversity Research,
University of Sassari, Sassari, Italy
}
Wellcome Trust Centre for Molecular Parasitology, Institute of Infection, Immunity and Inflammation,
College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow, United Kingdom
}
Liverpool School of Tropical Medicine, Liverpool, United Kingdom
1
Current address: Division of Infectious Diseases, Department of Internal Medicine, Stanford University
School of Medicine, Stanford, USA

ISSN 0065-308X, DOI: 10.1016/B978-0-12-391429-3.00006-X All rights reserved.
87
88 Robert P. Hirt et al.
2.4. Membrane Trafficking and Cell Signalling 117

2.4.1. Selected GTPases, ESCRT and ATG 118
2.4.2. Protein kinases 123
2.5. The Transcriptome and the RNAi Machinery 126
References 130
Abstract The draft genome of the common sexually transmitted pathogen

Trichomonas vaginalis encodes one of the largest known proteome
with 60,000 candidate proteins. This provides parasitologists and
molecular cell biologists alike with exciting, yet challenging, oppor-
tunities to unravel the molecular features of the parasites cellular
systems and potentially the molecular basis of its pathobiology.
Here, recent investigations addressing selected aspects of the para-
sites molecular cell biology are discussed, including surface and
secreted virulent factors, membrane trafficking, cell signalling, the
degradome, and the potential role of RNA interference in the
regulation of gene expression.
2.1. INTRODUCTION
The protist Trichomonas vaginalis is the most common non-viral, curable,

sexually transmitted disease (STD) agent globally, being at least equal to
the combined prevalence of the most common bacterial STD agents Chla-
mydia trachomatis and Neisseria gonorrhoeae (Hobbs et al., 2008). It is
increasingly recognised as being associated with important health
sequelae including pelvic inflammatory disease, cervical cancer, preterm
delivery and low birth weight and is in particular strongly associated with
promoting HIV sexual transmission, notably this was recently shown not
to be restricted to high-risk individuals ( Johnston and Mabey, 2008;
McClelland, 2008; Van der Pol, 2007). More recently, T. vaginalis infections
were also shown to be associated with aggressive prostate cancers
(Sutcliffe, 2010). The latter observation might contribute to broaden the
interest of the medical and scientific community as T. vaginalis is often
referred to as a female nuisance in a world where hierarchy and power
inequality usually favour the male (Buve et al., 2008; Carlton et al., 2010).
The identification of T. vaginalis, and other trichomonads including spe-
cies of zoonotic origins, in the lung of adult patients with various pulmo-
nary diseases and the rare cases of lung neonatal infections transmitted
during birth from infected mothers (Carter and Whithaus, 2008), suggest
that this parasite might be medically relevant beyond being an STD
(Duboucher et al., 2008).
Trichomonas vaginalis Pathobiology: New Insights from the Genome Sequence 89
The publication of the draft genome sequence for the G3 T. vaginalis

clinical isolate (Carlton et al., 2007) was a landmark in the study of this
common parasite, by providing an important platform for molecular and
cellular investigations. There is, for instance, little known about T. vaginalis
surface antigens and hence currently very little considerations for the
development of a potential vaccine (Cudmore and Garber, 2010). Similarly,
the development of alternative antimicrobial strategies targeting virulence
factors or based on immunotherapeutic approaches (Brotz-Oesterhelt and
Sass, 2010) would also depend on detailed knowledge of the pathogen
pathobiology and the host defence mechanisms. Very little is also known
on how the parasite senses external cues and mediates signal transduction,
transforms into an amoeba upon contact to host tissues or internalises
bacterial and host cells through phagocytosis, all-important processes
underlying the parasites pathobiology (Hobbs et al., 2008). The annotation
of the T. vaginalis genome sequence identified a plethora of candidate genes
for surface molecules mediating interaction with host tissues, membrane
trafficking and signalling (e.g. Carlton et al., 2010; Cui et al., 2010; Hirt et al.,
2007). In addition to its medical importance, T. vaginalis is also increasingly
attracting interest as a model system for evolutionary and comparative
genomics (e.g. Alsmark et al., 2009; Chen et al., 2008; Feschotte and
Pritham, 2007; Pritham et al., 2007; Zubacova et al., 2008) and molecular
cell biology investigations (e.g. Mentel et al., 2008; Morada et al., 2011;
Simoes-Barbosa et al., 2010; Smid et al., 2008; Smith et al., 2011).
Here, we present updated analyses of the existing genome sequence
data and review published material on selected topics of T. vaginalis
molecular cell biology that benefited directly from the genome sequenc-
ing project, some of which are likely to provide important cues into
the parasites pathobiology. Several of these analyses took advantage of
the TrichDB database dedicated to T. vaginalis genome-scale data
(Aurrecoechea et al., 2009; Carlton et al., 2010). Advances in one of the
most intensively investigated T. vaginalis organelle, the hydrogenosome,
have been reviewed elsewhere (e.g. Hjort et al., 2010; Tachezy, 2008) and
will not be covered here.
2.2. SURFACE AND SECRETED MOLECULES
Surface and secreted molecules of T. vaginalis are at the forefront of the

parasites interactions with its environment including human epithelial
cells, immunocytes and extracellular proteins of the urogenital tract (Hirt
et al., 2007). T. vaginalis also interacts with the human microbiota and can
phagocytose both its prokaryotic and eukaryotic (i.e. yeasts) members
(Pereira-Neves and Benchimol, 2007; Rendon-Maldonado et al., 1998).
The parasite can also phagocytose human cells including vaginal
epithelial cells, immunocytes and spermatozoids (Benchimol et al., 2008;

Midlej and Benchimol, 2010; Rendon-Maldonado et al., 1998). This pro-
vides the parasite with important sources of nutrients as well as contri-
buting to its defensive responses to attacks from the immune system. In
well-studied systems, phagocytosis is dependent on surface receptors and
a host of proteins from the cytoskeleton, membrane trafficking and signal
transduction systems (Boulais et al., 2010), but in T. vaginalis, this cellular
process has been so far only characterised at the morphological level (e.g.
Pereira-Neves and Benchimol, 2007; Rendon-Maldonado et al., 1998). No
surface receptors and very few of the other proteins known in model
systems (Boulais et al., 2010) to orchestrate phagocytosis have been iden-
tified experimentally in T. vaginalis, currently limited to some elements of
the cytoskeleton and membrane trafficking (for an overview, see Carlton
et al., 2010; Elmendorf et al., 2010). The parasite also internalises material,
such as lactoferrin and haemoglobin through receptor-mediated endocy-
tosis (Sutak et al., 2008). Endocytosis of several human viruses, including
HIV, and their accumulation in late endosomal-like structures (Pindak
et al., 1989; Rendon-Maldonado et al., 2003), might contribute to the
transmission of viruses when the parasite is transferred from dually
infected patients to new hosts (Lal et al., 2006). This mechanism could
contribute to T. vaginalis facilitating sexual transmission of HIV (Carlton
et al., 2010; Johnston and Mabey, 2008).
The annotation of the genome sequence identified a plethora of candi-
date surface proteins likely to mediate important functions in the parasite
interactions with its environment (Carlton et al., 2007; Hirt et al., 2007).
The analysis also indicated that T. vaginalis does not produce glycosyl-
phosphatidylinositol (GPI) anchors (Carlton et al., 2007), which is
surprising as GPIs are important and abundant glycolipids used by
many parasitic protists to anchor molecules on the surface, including
virulent factors.
2.2.1. Surface proteins

This section will discuss selected surface protein families with potential
adhesion functions or other binding activities relevant for the parasites
life style in the urogenital tract. Surface peptidases will be discussed in the
context of the degradome (Section 2.3).
2.2.1.1. TvBspA
The largest gene family in T. vaginalis encoding candidate surface proteins
are the TvBspA, with the initial annotation identifying over 650 entries
(Carlton et al., 2007; Hirt et al., 2007). BspA-like proteins (BspA stands for
Bacteroides surface protein A; Sharma, 2010) are characterised by a spe-
cific type of leucine-rich repeats (LRRs) named TpLRR (Tp is for
Treponema pallidum) (Kobe and Kajava, 2001), with the LRR of extracellu-
lar proteins typically involved in proteinprotein interactions and
demonstrated in different systems to be important for hostmicrobe inter-
actions (Kedzierski et al., 2004; Kobe and Kajava, 2001). For two bacterial
oral pathogens, Treponema denticola (11 BspA-like genes) and Tannerella
forsythensis (six BspA-like genes), one or more BspA proteins were shown
to be expressed on their cell surface and to be important for the bacteria
pathobiology, contributing to the two bacteria co-aggregation, binding to
extracellular matrix (ECM) proteins, biofilm formation and in the adher-
ence to, and invasion of, their target human epithelial cells (reviewed in
Sharma, 2010). In addition, one bacterial BspA protein was also shown to
stimulate pro-inflammatory cytokines through the toll-like receptor
2 (TLR2) (Sharma, 2010). The BspA TpLRR is thought to interact with
the TLR2 LRR domain (Sharma, 2010), highlighting the importance of
host LRRs, which play key roles in mucosal innate immune responses by
recognising various pathogen molecules (Lavelle et al., 2010). These dif-
ferent observations for the bacterial BspA-like proteins suggest that
TvBspA proteins could play important roles in T. vaginalis pathobiology.
More sensitive data mining combining pattern and profile searches
identified a total 911 TvBpsA genes including likely pseudogenes and
gene fragments (Noel et al., 2010). These analyses also further confirmed
that BspA-like genes are found in microbes from all three domains of life
(Hirt et al., 2002), currently the only type of LRR known with such a global
distribution (Noel et al., 2010). Of particular interest was the restricted
distribution of BspA-like genes to taxa thriving in animal hosts and on
mucosal surfaces, in particular including both mutualists and pathogens
(Noel et al., 2010). This suggests that BspA-like genes might have been
shared via lateral gene transfers (LGTs) between organisms occupying
animal host niches. In the case of the human mucosal protists T. vaginalis
(911 TvBspA genes), and to a lesser extent Entamoeba histolytica (124 BspA-
like genes) and E. dispar (298 BspA-like genes), LGT was followed by
numerous gene duplications, further indicating that these proteins are
functionally important and might represent adaptive traits for a mucosal
life style. In contrast, prokaryotes encode fewer BspA-like proteins (119
genes) (Noel et al., 2010).
A combination of clustering and inspection of a global alignment of
the 911 TvBspA proteins identified several subfamilies with distinct
structural features including transmembrane domains (TMDs), other
repetitive sequences (such as proline-rich repeatsPRRs and serine-rich
repeatsSRRs), sorting signal for endocytosis in the cytoplasmic tail (CT)
of two TvBspA subfamilies with TMD (one with a dileucine [DE]XXXL
[LI]-like motif and one with a tyrosine NPXY-like motif) and other specific
protein domains (Noel et al., 2010).
One TvBspA with a TMD and a PRR (TvBspA625: TVAG_073760) was

shown by indirect immunofluorescence analyses (IFA) to be expressed on
the cell surface of isolate G3 and to induce an antibody response in the
great majority of tested male and female patients, indicating that
TvBspA625 is expressed in vivo during infections of both sexes (Noel
et al., 2010). In addition, a proteomics survey of T. vaginalis surface
proteins identified 11 TvBspA proteins in six T. vaginalis isolates
(Table 2.1) (de Miguel et al., 2010) (discussed in Section 2.2.2). Notably,
although IFA detected TvBpsA625 on the cell surface of isolate G3 (Noel
et al., 2010), the proteomics survey did not detect it in any of the six
investigated isolates, including G3, highlighting the importance of using
several methods to detect surface proteins. Only one protein, TvBspA329
(TVAG_240680), was identified in all six investigated isolates, which also
corresponds to the gene with the highest number of ESTs among these 11
genes (Table 2.1). The protein TvBspA805 (TVAG_154640 in Table 2.1) is a
close paralogue to TvBspA625 with a longer TpLRR, but a shorter PRR,
and both are inferred to possess a TMD and a CT (Noel et al., 2010).
This protein was detected in the isolate SD7 by proteomics (de Miguel
et al., 2010). Interestingly, only seven TvBspA proteins detected by
TABLE 2.1 TvBspA identified through cell surface proteomics
G3
protein G3 G3 HA HA HA LA LA LA EST-
Accession length TMD SP PA SD7 B7268 G3 SD10 T1 Microarray
TVAG_240680 329 1 Yes 28 43 24 46 17 26 14

TVAG_131910 963 1 No 8 4 3 7 9 0 0
TVAG_169970 1008 1 No 11 0 7 0 11 2 3
TVAG_169980 1122 1 No 8 0 5 0 4 2 2
TVAG_131950 1066 1 No 6 7 0 0 12 0 1
TVAG_154640a 805 1 No 0 5 0 0 0 0 2
TVAG_268070b 788 1 No 0 0 13 0 0 0 6
TVAG_186310 1363 0 No 0 8 9 0 0 5 7
TVAG_186300 1434 0 Yes 6 2 6 0 0 5 7
TVAG_162010 816 0 Yes 6 0 2 0 0 0 0
TVAG_186290 1474 0 No 2 10 11 0 0 0 1
Information about the protein length, transmembrane domains (TMDs) and signal peptide (SP) data are from
isolate G3 (Carlton et al., 2007; Noel et al., 2010). EST and microarray data are derived from various isolates
(Noel et al., 2010). The shown numbers in columns 510 are the MudPIT spectral counts for the listed TvBspA
proteins across six T. vaginalis isolates, three highly adherent (HA) isolates to human vaginal epithelial cells
(PA, SD7 and B7268) contrasted with three low adherent isolates (LA) (G3, SD10 and T1) (de Miguel et al.,
2010) (Section 2.2).
a
Entry TVAG_154640 is related to TvBspA625 discussed in the text (Noel et al., 2010).
b
Entry TVAG_268070 was significantly up-regulated () in a microarray experiment in which cells were
exposed to high iron level (Noel et al., 2010).
proteomics were predicted to possess a TMD, including TvBspA329

(identified by TMHMM, PHOBIUS and SPLIT4; Noel et al., 2010), so
four of the TvBspA proteins identified apparently lack a TMD. This
could be explained in at least several ways. Firstly, the proteins could
have a cryptic TMD not identified by bioinformatics analyses; secondly,
the proteins could be anchored to the plasma membrane in an unknown
fashion (possibly by association with other proteins or via direct attach-
ment with a lipid anchor, see Section 2.2.3) or the genes might have
undergone recombination events in other T. vaginalis isolates that lead
to new gene variants encoding proteins with a TMD. Interestingly, the
proteomics survey did not detect in isolate G3 any of the four TvBspA
without a TMD, with only two of these detected in T1, whereas the three
highly adherent isolates expressed either three or four of the TvBspA
without TMDs (Table 2.1) (de Miguel et al., 2010). Comparative genomics
of these T. vaginalis isolates will be useful to test these possibilities along
with experimental work characterising the anchoring of the TvBspA
proteins to the plasma membrane. The observed differential TvBspA
expression could possibly reflect their involvement in surface antigen
variation (Noel et al., 2010), an important mechanism thought to be
crucial for evading the host adaptive immune responses mediating
long-term survival, and permitting reinfection (Deitsch et al., 2009).
2.2.1.2. TvPmp
Initial annotations of the G3 genome sequence data, based on protein
domain hits with RPS-Blast (Pfam profile PF02415: Chlamydia_PMP)
identified 27 T. vaginalis proteins possessing the multirepeats of the motifs
GGA[ILV] and FXXN characteristic of Chlamydia spp. polymorphic mem-
brane proteins (Pmp, hence TvPmp) (Carlton et al., 2007; Hirt et al., 2007).
The Chlamydia spp. are obligate intracellular bacteria with two major
cellular forms, the infectious elementary bodies (EBs) and the replicating
intracellular reticulate bodies (Schachter and Stephens, 2008). C. tracho-
matis, in addition to being an important STD agent, is also responsible for
trachoma leading to blindness and has attracted interest from clinicians,
immunologists and microbiologists (Schachter and Stephens, 2008). The
Pmp family were identified as candidate virulent factors in Chlamydia
spp. and were shown to be expressed as variant surface antigens and to be
important adhesins mediating the binding of the EB to human epithelial
cells via their GGA[ILV] and FXXN repeats (Molleken et al., 2010; Tan
et al., 2009). There is also evidence for a role of Pmp in tissue tropism
( Jeffrey et al., 2010).
Using TrichDB 48 TvPmp entries positive for the InterPro profile
IPR003368: Chlamydia_PMP were identified. In the Chlamydia Pmp pro-
teins, the repeats GGA[ILV] and FXXN are located close to the N-terminus
whereas the C-terminus is characterised by bacterial autotransporter
domains, which are not found in TvPmp proteins. Of the 48 TvPmp

identified, 31 possess one or two TMD and of these 11 also have an inferred
signal peptide. A visual inspection of a TMD-CT alignment identified 19
entries with shared features among which 13 possessed an [FY]NPX[FY]-
like motif for clathrin-dependent endocytosis (for a recent review on such
motifs, see Traub, 2009). Interestingly these 13 TvPmp-TMD-CT are clearly
related to the TvBspA-TMD-CT, which also possess an [FY]NPX[FY]-like
motif (Noel et al., 2010) (Fig. 2.1). This indicates that distinct extracellular
domains of T. vaginalis transmembrane proteins, with independent evolu-
tionary origins (Pmp-like vs. BspA-like), can share similar TMD-CT. This
also raises the possibility that some gene families encoding transmem-
brane proteins were generated via gene fusion between TMD-CT and
extracellular domain gene fragments (Kaessmann, 2010).
The proteomics survey of T. vaginalis surface proteins identified five
TvPmp proteins all with a TMD, three of which possess the [FY]NPX[FY]-
like signal for endocytosis (Table 2.2) (de Miguel et al., 2010) (Section 2.2.2).
Interestingly, two TvPmp were observed to be 27- and fivefold, respec-
tively, more abundant in adherent when compared to less adherent isolates
of the parasite (Table 2.2), suggesting an involvement of these proteins in
the pathogenesis of T. vaginalis (de Miguel et al., 2010).
2.2.1.3. Other candidate surface proteins

Several additional gene families encoding candidate surface proteins
have been identified in silico (Hirt et al., 2007) and by proteomics (de
Miguel et al., 2010) (see Section 2.2.2). A family of 11 legume-like lectin
receptors is of interest as lectins have been implicated in the uptake
by endocytosis of HIV by vaginal dendritic cells (de Witte et al., 2008),
so they might be important for binding and endocytosis of HIV by
T. vaginalis (Rendon-Maldonado et al., 2003).
2.2.1.3.1. A novel type of surface Zn-metallopeptidase? One T. vaginalis

gene family encoding candidate surface proteins was annotated as an
immuno-dominant surface antigen based on its Blast hits to the M17
proteins of E. histolytica (Carlton et al., 2007; Hirt et al., 2007). The M17
protein of E. histolytica is a GPI-anchored surface immuno-dominant anti-
gen against to which 70% of patients with liver abscess raise an antibody
response (Edman et al., 1990). The M17 protein was also shown by proteo-
mics to be present in phagosomes (Marion and Guillen, 2006; Okada et al.,
2006) and uropods (Marquay Markiewicz et al., 2011). Interestingly, the
N-terminal domain (approximately first 500 residues) is shared across a
broad range of microbial eukaryotes and prokaryotes (Nakjang, 2011) with
a similar taxonomic distribution to the BspA-like proteins (Noel et al.,
2010). Over 90% of the M17-like sequences possess the pattern HEXXHX
(8,28)E, a motif reminiscent of gluzincins (Hooper, 1994). Further, an
FIGURE 2.1 Comparison of the transmembrane domain (TMD) and cytoplasmic tail (CT)
of selected TvBspA and TvPmp sharing an NPXY-like motif for endocytosis. Entries with
partial or divergent versions of CT related to the shown sequences were removed,
leaving the shown selection. The TrichDB locus tags are indicated with BspA and Pmp
added to differentiate the nature of the extracellular domain for each protein.
The position of the [FY]NPX[YF]-like motif for endocytosis (Traub, 2009) is indicated.
TABLE 2.2 TvPmp identified through cell surface proteomics
G3 G3 G3 HA HA HA LA LA LA
Accession length TMD SP PA SD7 B7268 G3 SD10 T1
TVAG_212570a 624 1 No 0 19 43 4 0 2
TVAG_152680 590 1 No 3 6 13 0 0 0
TVAG_140850 613 1 No 0 77 33 0 0 0
TVAG_299640a 812 1 No 0 0 2 0 0 0
TVAG_528820a 652 1 Yes 0 0 0 0 0 2
Information about the protein length, transmembrane domains (TMDs) and signal peptide (SP) data are from
isolate G3 (Carlton et al., 2007). The shown numbers in columns 510 are the spectral counts for the listed
TvPmp proteins across six T. vaginalis isolates, three highly adherent (HA) isolates to human vaginal epithelial
cells (PA, SD7 and B7268) contrasted with three low adherent (LA) isolates (G3, SD10 and T1) (de Miguel
et al., 2010).
a
Entries with the [FY]NPX[FY]-like signal for endocytosis.
in silico analysis shows that the great majority of the microbial sequences
possess sequence features, suggesting they localise to the cell surface or are
secreted (Nakjang, 2011). In addition, several animals also encode M17-
related proteins, but none of these possess sequence features indicating
membrane anchoring or secretion. Detailed analyses of protein alignments
(where all the HEXXHX(8,28)E were all co-aligned to each other) were used
to generate a profile for a new domain that was given the Pfam accession
PF13402 (68 sequences and 387 columns) (Nakjang, 2011). In profileprofile
comparisons, this new domain was significantly related to the domain
PF03272 corresponding to the MA Clan M60-enhancin family. Hence, this
new domain was named M60-like/PF13402 to differentiate it from its
related M60-enhancin/PF03272 domain. A total of 25 TvM60-like/
PF13402-containing proteins were identified in T. vaginalis and 11 of these
possess the HEXXHX(8,28)E motif and among them six have one TMD
with only three of these entries possessing a complete M60-like/PF13402
domain. Indeed, a sequence alignment suggested that most entries are
likely to represent gene fragments or truncated versions of longer proteins.
Of the most likely three complete TvM60-like/PF13402-containing pro-
teins, two were detected on the cell surface by proteomics, with one protein
identified in all six tested isolates and the other exclusively in the G3 isolate
(de Miguel et al., 2010). It remains to be determined if the TvM60-like/
PF13402 proteins have peptidase activity.
The approximate position of the TMD is boxed, as is the position of a potential cluster of
acidic residues motif for endocytosis (Traub, 2009). The shadings correspond to identical
residues (black) or conservative changes (grey) with a 60% conservation threshold across
the alignment.
2.2.2. Proteomics data for surface proteins

2.2.2.1. Overview and proteins with known homologues
Proteomic studies have been used to better define the surface proteins of
T. vaginalis (de Miguel et al., 2010). Isolation of surface proteins using cell
surface biotinylation and comparison using multidimensional protein
identification technology (MudPIT) resulted in the identification of a
total of 411 proteins (de Miguel et al., 2010). Most of the families predicted
to encode membrane proteins based on the genome sequence annotation
(Carlton et al., 2007; Hirt et al., 2007) were identified: TvBspA proteins
(Table 2.1, Section 2.2.1.1), GP63-like metallopeptidases, subtilisin-like
serine peptidases, calpain-like cysteine peptidases (CPs), Chlamydia poly-
morphic membrane-like proteins (Table 2.2, Section 2.2.1.2), immuno-
dominant variable surface antigen (see Section 2.2.1.3) and the P270
surface immunogen (de Miguel et al., 2010). Many of these families
contain multiple paralogues, yet only a subset of members within any
protein family was reproducibly identified in the membrane proteome
when using six different isolates (de Miguel et al., 2010). The detected
proteins identified through MudPIT are likely to be the most abundant
and/or accessible, so this does not preclude the expression of additional
family members (see TvBspA625 in Section 2.2.1.1). Differential expres-
sion might be playing an important role in mediating surface antigen
variation, allowing the pathogen to avoid adaptive immune responses
directed against a given set of gene family members. In addition, given
the high percentage of pseudogenes observed for some gene families in
T. vaginalis (e.g. Cui et al., 2010), proteomic analyses is likely to be
valuable in identifying functional genes (expressed at the protein level)
versus pseudogenes/gene fragments (no protein detected) for further
studies. Novel protein families that have yet to be examined in T. vaginalis
were also revealed in this surface membrane proteome (de Miguel et al.,
2010). These include tetraspanins (Hemler, 2003), a rhomboid-like serine
peptidase (Freeman, 2008) (see Section 2.3.3), and nicastrin precursor (Xia
and Wolfe, 2003), among others. In mammalian cells, tetraspanins are
known to form complexes that regulate diverse processes such as cellcell
and cellECM adhesion, cell migration and intracellular signalling
(Hemler, 2003), processes important for the colonisation by T. vaginalis
of the female and male urogenital tracts. Pathogenic roles for rhomboid
peptidases have been established in both apicomplexan parasites and
Entamoeba (Baxt et al., 2008; Freeman, 2008; Kim, 2004). These intramem-
brane serine peptidases are known to cleave adhesins involved in host cell
invasion by apicomplexan parasites (Freeman, 2008). In E. histolytica, a
rhomboid host cell invasion has been demonstrated to cleave the major
surface Gal-GalGNc lectin involved in host cell attachment, phagocytosis
and immune evasion (Baxt et al., 2008). Although important roles for
surface intramembrane peptidases are anticipated in T. vaginalis, bio-

chemical analyses are necessary to define their function.
In addition to investigating qualitative changes in protein expression
patterns, proteomic studies are also important to measure quantitative
levels of proteins and substantial variation in the level of protein expres-
sion between isolates (Cuervo et al., 2008; De Jesus et al., 2009; de Miguel
et al., 2010) (Tables 2.1 and 2.2). Of the 411 proteins identified, 11 were
found to be between 5 and 42 times more abundant in at least two of the
three adherent parasite strains (de Miguel et al., 2010). The proteins
include two Chlamydia polymorphic membrane-like proteins (Table 2.2),
a serine/threonine protein phosphatase, an adenylate/guanylate cyclase,
a subtilase-like serine peptidase and six hypothetical proteins (see
Section 2.2.2.2).
Proteomics also provide the opportunity to analyse the relation
between expression at the mRNA and protein levels, providing a useful
insight into the regulatory mechanisms mediating differential expression
of genes at the level of transcription, mRNA stability (see Section 2.5),
translation and/or protein stability. As an example, Fig. 2.2 illustrates the
contrast of mRNA level measured by qPCR and corresponding protein
quantifications for the two hypothetical surface proteins TVAG_166850
and TVAG_244130 discussed in Section 2.2.2.2. In this case, a strict
TVAG_166850 TVAG_244130
200 1400
180
1200
160
Fold increase
140 1000
mRNA
120
800
100
80 600
60 400
40
200
20
0 0
T1 G3 SD10 PA B7268 SD7 T1 G3 SD10 PA B7268 SD7
1400 800
1200 700
1000 600
Protein
800 500
NSAF
400
600
300
400
200
200
100
0 0
T1 G3 SD10 PA B7268 SD7 T1 G3 SD10 PA B7268 SD7
FIGURE 2.2 Contrasting quantifications of gene transcripts and corresponding proteins

detected on the cell surface for two cell surface hypothetical proteins. The expression
at the mRNA level was measured by qPCR and is contrasted with the MudPIT data
(de Miguel et al., 2010). These two proteins are member of a large gene family (Table 2.3).
The TrichDB locus tags are indicated.
relative correlation was observed between the analysed isolates, suggest-

ing a transcriptional/mRNA stability regulation for these two genes.
Similar correlations were also observed for three additional and unrelated
genes/proteins (de Miguel et al., 2010).
Surface proteins of T. vaginalis are expected to be important for initiat-
ing and sustaining infections, whilst intracellular proteins are likely to be
critical to alter cell shape, process nutrients and for other important
cellular processes. Following the same criteria of isolate comparison,
Cuervo et al. performed a small scale comparative analysis of soluble
proteins expressed in a laboratory adapted, low-virulence T. vaginalis
isolate versus a fresh clinical isolate exhibiting a virulent phenotype
(Cuervo et al., 2008). These analyses identified both quantitative and
qualitative differences in protein expression profiles, including a number
of proteins involved in carbohydrate and energy metabolism, cytoskeletal
structure and proteolysis. Further, De Jesus et al. identified six CPs that
were differentially expressed between high- and low-virulence pheno-
types (De Jesus et al., 2009). As only two isolates were compared in both of
these studies, the differences observed may result from non-virulence
related genetic changes or alterations in protein expression due to pro-
longed in vitro cultivation. To demonstrate that heterogeneity in protein
expression is related to pathogenesis, an extended proteomic analysis
involving a greater number of isolates with differing degrees of virulence
will be required.
2.2.2.2. Hypothetical proteins

An important fraction of the 411 proteins identified in the surface prote-
ome (37%) were annotated as hypothetical, meaning that no functional
inferences could be made based on sequence homology. Some of these are
likely to carry out Trichomonas-specific functions. The ability to identify
these novel surface proteins provides a foundation for future studies with
the potential of uncovering novel hostparasite interactions. Several
hypothetical proteins were detected in multiple strains and a fraction of
those were observed to be differentially expressed, potentially mediating
surface antigen variation (de Miguel et al., 2010). One could aim at
identifying a protein that has a crucial, so far unknown function in
virulent strains of the parasite that has no homologue in the human host;
these could be a good starting point for selecting an effective and safe
drug or a vaccine target. A very interesting example is provided with the
two hypothetical proteins, TVAG_244130 and TVAG_166850, which were
among the most highly expressed surface proteins in the adherent strains.
They were either 20 or 42 times more abundant in highly adherent para-
sites (Fig. 2.2) and over-expression of either of these in a non-adherent
strain increased attachment of transfected parasites to vaginal epithelial
cells in vitro by 2.2-fold (de Miguel et al., 2010). These data revealed a
role in adhesion for these novels, abundant surface proteins. Through

comparative genomics it was observed that they belong to a large
T. vaginalis gene family with over 150 members. They share sequence
features with hypothetical candidate surface proteins of a collection of
bacteria known to thrive on human mucosal surfaces, either as mutualists,
commensals or pathogens (Table 2.3). This restricted taxonomic distribu-
tion suggests that these genes were also shared through LGT as proposed
for the TvBspA. These data highlight interesting T. vaginalis, as well as
bacterial, candidate surface proteins to investigate humanmutualist/
pathogen interactions as discussed for the TvBspA (Section 2.2.1.1).
Revealing novel proteins on the surface of the parasite has provided a
foundation for future studies with the potential of uncovering unprece-
dented hostparasite interactions that may be important for T. vaginalis
pathobiology. The next few years will hopefully see the assessment of
some of these new membrane proteins.
2.2.3. The glycocalyx

The components and the function(s) of the T. vaginalis glycocalyx have
been poorly studied. Most research has focused on elucidating the struc-
ture and function of a major lipid-anchored phosphosaccharide known as
lipophosphoglycan (Bastida-Corcuera et al., 2005; Singh et al., 2009), but
little is known about the contributions from other types of glycoconju-
gates, like glycoproteins or glycolipids. Unlike all parasitic protists stud-
ied so far, T. vaginalis does not express surface glycoconjugates via a GPI
anchor, as most of the genes involved in making GPIs are absent in this
organism (Carlton et al., 2007) (see Section 2.2.3.2). In comparison to
higher eukaryotes, this organism has also a truncated N-glycosylation
pathway (Kelleher et al., 2007). However, several different families of
surface proteins (Carlton et al., 2007; de Miguel et al., 2010; Hirt et al.,
2007) are predicted to be glycosylated and thus they are likely compo-
nents of T. vaginalis glycocalyx. In addition, this organism is able to
modify proteins with O-glycans, although the nature and localisation of
the acceptor proteins have yet to be determined (Grabinska et al., 2008).
2.2.3.1. The T. vaginalis lipophosphoglycan (TvLPG)

TvLPG is perhaps the most abundant component of the T. vaginalis
surface glycocalyx (Bastida-Corcuera et al., 2005; Singh et al., 2009) and
is an important virulence factor that is expressed on the trophozoite
surface. Similar to the well-characterised group of Leishmania LPGs
(McConville and Ferguson, 1993), it has been proposed that TvLPG archi-
tecture is also comprised of an oligosaccharidic cap attached to a pho-
sphate-saccharide-rich backbone (Singh et al., 2009). However, the nature
and the organisation of the different sugar residues that are present in
TABLE 2.3 The proteins related to the hypothetical protein TVAG_244130 identified by PSI-Blast
Total number of
Taxa Habitat and relationship with host Top bit score Top e-value related proteins
Trichomonas vaginalis G3 Urogenital tractpathogen 627a 1.00 10 177a 155

(Parabasalia)
Clostridium bolteae ATCC Digestive tractnormal flora 154 3.00 10 35 3
BAA-613 (Firmicutes)
Bacteroides sp. 2_1_33B Digestive tractnormal flora 123 8.00 10 26 1
(Bacteroidetes)
Anaerofustis stercorihominis Digestive tractnormal flora 115 2.00 10 23 1
DSM 17244 (Firmicutes)
Clostridium hathewayi DSM Digestive tractnormal flora 82 2.00 10 13 1
13479 (Firmicutes)
Clostridiales bacterium Digestive tractisolate from the 79 2.00 10 12 1
1_7_47FAA (Firmicutes) colon of a patient with Crohn
disease
Treponema denticola ATCC Human mouthpathogen 78 2.00 10 12 1
35405 (Spirochaetes)
The shown bit scores and e-values are derived from a PSI-Blast (e-value 1 10 10) at the NCBI Blast server using the T. vaginalis protein TVAG_244130 as query with two
iterations. Only the values for the top hit for each taxa are shown. All proteins were annotated as hypothetical proteins. The highest taxonomic names, below the domain level, are
indicated along the species names.
a
Values for the non-self-hit TVAG_335250.
TvLPG appear to be quite different to that expressed by Leishmania

(Bastida-Corcuera et al., 2005). The only experimental evidence for the
presence of phosphosaccharide repeats in TvLPG is its susceptibility to
mild acid hydrolysis (Singh et al., 2009), but the presence of such struc-
tures remains to be corroborated by biophysical methods. Unique to
TvLPG is the substitution of the polysaccharidic backbone with several
N-acetyllactosamine (LacNAc, a disaccharide composed of Galb1-
4GlcNAcb1-) repeats (Singh et al., 2009), which are suggested to be
involved in the binding to human cells (Okumura et al., 2008). Further,
although T. vaginalis is unable to synthesise GPIs, TvLPG appears to be
anchored to the parasite membrane via a modified version of the canoni-
cal GPI anchor, which contains inositolphospho-ceramide (IPC) as the
lipid moiety (Singh et al., 2009) (Fig. 2.3). Apart of Gal and GlcNAc
residues, which are arranged as part of the LacNAc repeats (Singh et al.,
2009), TvLPG is rich in rhamnose (Rhm), xylose (Xyl), GalNAc and Glc
(Bastida-Corcuera et al., 2005; Singh et al., 2009). Rhm is a deoxysugar
commonly found in bacteria and plants, but not in mammalian cells
(Dong et al., 2003). This makes the T. vaginalis Rhm biosynthetic pathway
an attractive drug target, as this sugar is suggested to be part of the
polysaccharidic backbone of TvLPG (Carlton et al., 2007).
Oligosaccharide Polysaccharide backbone Glycan

cap ? (Rhm, Xyl) core ?
IPC
LacNAc repeats
(Gal1-4GlcNAc-)
FIGURE 2.3 Schematic representation of the putative overall structural organisation of

TvLPG. TvLPG is a lipopolysaccharide anchored to the parasite membrane by an
inositolphospho-ceramide (IPC) (Bastida-Corcuera et al., 2005; Liu, 2011, and references
herein). It has been suggested that the TvLPG oligosaccharidic backbone comprises
phosphosaccharides probably rich in Rhm and Xyl. The exact primary sequence of the
oligosaccharide backbone is still unknown and the presence of phosphoglycans remains
to be experimentally determined. However, recent evidence suggests that it contains a
series of N-acetyllactosamine (LacNAc) repeats, which probably branch off from the
main oligosaccharidic backbone (Singh et al., 2009). The presence of an oligosaccharidic
cap and a glycan core (bridging the backbone to the lipid tail) are hypothetical, although
the glycan core main contain non-acetylated GlcN linked to the inositol moiety
(Liu, 2011). The IPC molecule is mainly composed of long-chain base sphinganine
(d18:0 dihydrosphingosine) and a C16:0 acyl group (Liu, 2011; Singh et al., 1994).
The precise function(s) of the TvLPG is unknown. In Leishmania, pho-

sphoglycans (PGs) as part of LPG and proteophosphoglycans (PPGs) are
the main virulence factors linked to attachment to the fly midgut and
survival in macrophages (Pimenta et al., 1992; Spath et al., 2003). In
E. histolytica, PPGs also seem important for adherence to the intestinal
epithelial cells (Moody et al., 1998). In the case of T. vaginalis, mutant cells
deficient in the glycosylation of TvLPG showed reduced adherence and
cytotoxicity to human ectocervical cells (EVCs), highlighting TvLPG as an
important virulence factor (Bastida-Corcuera et al., 2005). As TvLPG is a
ligand that binds to human galectin-1 expressed on the surface of EVCs
(so far the only human receptor identified for this parasite) via its terminal
Gal residues (Okumura et al., 2008), the reduction in parasite binding seen
in the TvLPG mutants is probably a direct correlation with their lower
content of LacNAc repeats (Bastida-Corcuera et al., 2005). However, it
cannot be ruled out that T. vaginalis normally expresses other surface
LacNAc-rich glycoconjugates that not only participate in the binding to
EVCs, but also have altered glycosylations as a result of the mutation(s).
As differentiation from the trophozoite into the amoeboid form is trig-
gered following binding to the host cells or ECM (Arroyo et al., 1993; Lal
et al., 2006), the binding of TvLPG to human galectin-1 may be important
to trigger parasite differentiation. It remains to be determined whether
there are differences in the levels of expression and structure of TvLPG
between the trophozoite and amoeboid stages. In addition, when purified
TvLPG is incubated in vitro with human endocervical, ectocervical and
vaginal epithelial cells deficient in the expression of toll-like receptor
4/MD2, this molecule induces an up-regulation of chemotactic cytokines
leading to inflammatory responses (Fichorova et al., 2006; Singh et al.,
2009), thus suggesting that the immune response triggered by TvLPG is
contributing to the pathogenicity of T. vaginalis.
The complete primary structure of TvLPG is still unknown; however,
based on its overall composition and structural domains, one can predict
from the T. vaginalis genome several genes (and pathways) that may
be involved in its synthesis. These include genes involved in both the
synthesis and metabolism of nucleotide sugars, like UDP-Gal and
UDP-GlcNAc (to make LacNAc repeats), UDP-GalNAc, UDP-Glc and
UDP-Xyl. In addition, although the nucleotide donor of Rhm is unknown,
T. vaginalis contains genes encoding for the four enzymes (RmlA-D)
necessary to produce dDTP-L-Rhm (Carlton et al., 2007). The same path-
way of Rhm synthesis can also be found in bacteria, fungi and plants
(Dong et al., 2003). It is of particular interest to identify the transporter
responsible for the import of dDTP-L-Rhm (or any other nucleotide-Rhm
donor) into the Golgi of T. vaginalis. Since T. vaginalis lacks GPIs, it is
unknown whether there is an equivalent glycan core linking the polysac-
charide backbone with the IPC lipid tail (Fig. 2.3). As for the synthesis of
IPC, T. vaginalis contains all the enzymes for de novo biosynthesis of

ceramide (Carlton et al., 2007) but it also may be capable of environmental
scavenging ceramide from glycosylated ceramide molecules based on the
presence of b-galactosidase, a-galactosidase, exo-a-sialidase and arylsul-
fatase. Further, T. vaginalis also contains all the enzymes for de novo
synthesis of phosphatidylinositol (PI) (inositol-3-phosphate synthase,
inositol-phosphate phosphatase and CDP-diacylglycerol-inositol 3-phos-
phatidyltransferase) (Carlton et al., 2007), but can also scavenge PI (but
not inositol) from its environment (Beach et al., 1991).
2.2.3.2. Absence of GPI biosynthesis: Unmasking novel types

of lipid anchors?
One of the most interesting discoveries that arose from sequencing the
genome of T. vaginalis was that despite the large size of its genome and the
high degree of redundancy, this parasite lacks the entire repertoire of
conserved genes involved in GPI synthesis (Carlton et al., 2007). The
biosynthesis and addition of GPI anchors to nascent proteins is a post-
translational modification that was previously considered ubiquitous to
all eukaryotic organisms. GPIs are synthesised in the ER and, depending
on the cell type or the organism, more than 15 gene products are involved
in this pathway (Eisenhaber et al., 2003; Orlean and Menon, 2007)
(Table 2.4).
The production of GPI-anchored proteins and GPI-glycoconjugates is
characteristic of many parasitic protists, where they play important roles
as Pathogen Associated Molecular Patterns (PAMPs) (Gazzinelli and
Denkers, 2006).
Using BlastP analyses and sequences of known genes involved in GPI
synthesis from three evolutionary distant organisms (i.e. Homo sapiens,
Saccharomyces cerevisiae and Trypanosoma brucei) only two of the 15 genes
necessary to make and transfer a GPI anchor were identified after inter-
rogating the T. vaginalis genome (Table 2.4). One highly significant hit
relates to the GPI8 gene product (catalytic component of the GPI:protein
transamidase) (Table 2.4). However, this protein can be classified as a
member of the Clan CD cysteine peptidase Family C13, which includes
asparaginyl endopeptidases, as well as the GPI:protein transamidases
(Mottram et al., 2003). No homologues for the other conserved compo-
nents of the transamidase complex (i.e. gene products of GAA1, GPI17,
GPI16 and GAB1) were detected. The other significant hit was DPM1,
which encodes for the enzyme that makes dolicholphosphate-Man (the
only Man donor in the formation of the GPI glycan core; Menon et al.,
1990) (Table 2.4). However, a closer inspection into the potential TvDPM1
homologue suggests that it probably encodes for a protein that is more
related to an UDP/CDP-dependent glycosyltransferase than DPM1 gene
TABLE 2.4 Comprehensive search for GPI anchor biosynthetic genes in the T. vaginalis genome
Comparison of T. vaginalis with other organisms
H. sapiens T. brucei S. cerevisiae

Gene
Biosynthetic step
a
namea Other name b
Gene ID e-Value Gene ID e-Value Gene ID e-Value
GPI-GlcNAc transferase pig-A GPI3 TVAG_372200 0.012 None None

pig-C GPI2 None None None
pig-H GPI15 TVAG_372000 0.014 None None
pig-P YDR437W/ TVAG_393940 0.00076 None None
GPI19
pig-Q GPI1 None None None
DPM2 None None Absent in Sc
pig-Y ERI1 None Absent in Tb None
GlcNAc de-N-acetylase pig-L GPI12 None None None
Acyl-transferase pig-W None Absent in Tb Absent in Sc
Mannosyltransferases pig-M GPI14 None None None
pig-B GPI10 None None None
pig-Z SMP3 None Absent in Tb None
pig-V GPI18 None Absent in Tb TVAG_27000 0.044
pig-X PBN1 None Absent in Tb None
Phosphoethanolamine pig-N None Absent in Tb Absent in Sc
transferases GPI7/LAS21 None Absent in Tb None
pig-O GPI13 None Absent in Tb None
pig-F GPI11 None Absent in Tb None
Transamidase pig-K GPI8 TVAG_068410 7.50 10 26 TVAG_185540 6.40 10 16 TVAG_426660 1.10 10 26
GPAA1 GAA1 None None None
pig-S GPI17 None Absent in Tb TVAG_051350 0.0048
pig-T GPI16 None None None
(continued)
Comparison of T. vaginalis with other organisms
H. sapiens T. brucei S. cerevisiae

Gene
Biosynthetic stepa namea Other nameb Gene ID e-Value Gene ID e-Value Gene ID e-Value
pig-U GAB1/ TVAG_407470 0.0056 Absent in Tb None

CDC91
TTA1 None None Absent in Sc
TTA2 None None Absent in Sc
Dol-P-Mannose formation DPM1 DPM1 TVAG_174740 8.10 10 11 None TVAG_358380 0.001
a
Names for mammalian GPI genes. Only the minimal numbers of genes involved in the formation of protein GPI-precursors are included, starting with the formation of GlcNAc-PI. Genes
involved in PI biosynthesis, lipid remodelling or in the addition of extra Man or EtN-P residues were not included.
b
Names for yeast and parasitic protozoa GPI genes. A set of GPI biosynthetic enzymes from Homo sapiens (Hs), Saccharomyces cerevisiae (Sc) and T. brucei (Tb) were blasted against the
annotated proteins of the TrichDB 1.1 database to look for the presence of GPI biosynthetic enzymes in T. vaginalis. Some genes do not have homologues in other species and therefore could
not be compared to the T. vaginalis genome database. These genes are labelled as absent in Tb or absent in Sc. e-Values below 10 5 were considered to be significant. For entries with hits
(in bold), the T. vaginalis gene identifier and the corresponding e-value were recorded.
product. This is further substantiated by the previously reported absence

of DPM1 in the T. vaginalis genome (Samuelson et al., 2005).
Previous reports have shown evidence that TvLPG may contain non-
acetylated GlcN linked to inositol, as normally found in GPI molecules
(Singh et al., 2009, and references therein). In all eukaryotes studied to
date, the formation of GlcNa1-6Ino-P-lipid first involves the transfer of
GlcNAc to a PI acceptor (where up to six genes have been identified)
followed by a de-N-acetylation step, which is mediated by pig-L/GPI12
(Table 2.4). However, none of the genes involved in the formation of this
intermediate are present in T. vaginalis, which suggest that if GlcN is
linked to inositol it must originate from a pathway yet to be elucidated.
Thus, T. vaginalis represents the first example of a eukaryotic cell lacking
the entire suite of genes involved in the GPI biosynthetic pathway.
In addition, it is the first example of a eukaryotic human pathogen that
does not use GPI-anchored glycoconjugates as virulence factors (Gazzinelli
and Denkers, 2006). What are the main implications of these findings?
First, it would be interesting to determine whether T. vaginalis has
gradually lost the genes in its GPI biosynthetic pathway. This secondary
loss may have happened during the environmental adaptation from the
human intestine to the urogenital tract (Carlton et al., 2007). A secondary
loss in T. vaginalis is supported by several considerations: Parabasalids are
relatively closely related to the diplomonads and more generally are
members of the Excavata that include the kinetoplastids (Hampl et al.,
2009), which possess GPI anchors; GPI anchors are also thought to have
originated in the Archaea (Eisenhaber et al., 2001). It will be important to
compare enzymes of the GPI pathway from a variety of other eukaryotes,
including the genomes of Archaea, to gain a greater understanding of the
evolution of GPI anchors biosynthesis.
A second implication relates to the type of lipid anchor present in
TvLPG. It is likely that this glycoconjugate is anchored to the parasite
membrane via an IPC molecule (Liu, 2011; Singh et al., 1994). Interest-
ingly, despite the lack of a mannosylated glycan core like that is present in
all protein GPI anchors, TvLPG is susceptible to degradation by bacterial
PI-PLC (Singh et al., 1994) which is an enzyme typically used to detect the
presence of a GPI in a given molecule or cell type. This finding may imply
an evolutionary relationship between the TvLPG anchor and eukaryotic
GPIs and also suggests that both types of anchors may be structurally
related. The topology of TvLPG synthesis is unknown, but if it is similar to
that of Leishmania LPGs (or other surface GPI-anchored glycoconjugates
from kinetoplastid parasites), it is tempting to postulate that this molecule
originates from a precursor containing an IPC anchor, which is then further
extended during its transit through Golgi. This hypothesis is partially
supported by the detection of ethanolamine phosphate-substituted
inositol phosphosphingolipids expressed in the trophozoite stage
(Costello et al., 1993), which may represent a precursor or a metabolic

intermediate during TvLPG synthesis.
Finally, the third major implication resulting from the absence of GPIs
in T. vaginalis is that some of its surface proteins may be attached to the
membrane via novel types of lipid anchors. Indirect evidence for such
alternative lipid anchors comes from a recent proteomic study, which
described several surface TvBspA, and other proteins, which appear to
lack a hydrophobic C-terminal domain (de Miguel et al., 2010) (but see
Section 2.2.1.1, for an alternative explanation). Analogous implications
have been deduced from analysing the E. histolytica EhLRRP1 protein, a
member of a family of EhBspA proteins expressed on the cell surface of
this parasite (Davis et al., 2006). EhLRRP1 lacks a TMD but contains
(together with other members of the EhBspA family) a predicted prenyla-
tion site (CaaX, where a is an aliphatic amino acid) at the C-terminus.
Thus, it remains to be biochemically determined whether members of the
T. vaginalis BspA-like family (and/or other surface proteins) could be
anchored to lipids, which may differ in nature to the IPC lipid anchor
present in TvLPG. In contrast to some of the EhBspA proteins, none of the
TvBspA entries possess a CaaX motif (Noel et al., 2010). Further studies
are necessary to determine exactly how non-TMD containing T. vaginalis
surface molecules are tethered to the membrane. Understanding this
mechanism could unravel novel ways in which cell surface virulent
factors are expressed and displayed by T. vaginalis.
2.2.4. Secreted proteins: The trichopores

T. vaginalis exerts an important cytopathic effect on vaginal cells, which is
thought to be mediated by both contact-dependent and contact-independent
mechanisms and to contribute to the clinical symptoms of the infection and
to be mediated by several secreted and cell surface proteins including
peptidases (see Section 2.3; reviewed by Hobbs et al., 2008). Following
adhesion to target cells, the parasite transforms from the typical pyriform
shape into a flattened amoeboid form, tightly adhering to host epithelial cells
(Fiori et al., 1999; Hobbs et al., 2008).
The cytolytic effect of T. vaginalis has been characterised using human
red blood cells (RBCs) (Fiori et al., 1993) and epithelial cells (reviewed by
Hobbs et al., 2008). In fact, T. vaginalis-mediated RBC lysis also occurs
in vivo, where it is thought to represent a means of acquiring lipids and
iron. The haemolytic activity and the consequent acquisition of nutrients,
especially iron, have been suggested to be responsible for the exacerba-
tion of symptoms observed during and following menstruation (Lehker
et al., 1990). Haemolysis is dependent on contact, temperature, and Ca2
and is tightly regulated by environmental pH, with a peak of activity at
pH 5.8 (Fiori et al., 1996). These characteristics strongly suggested the

involvement of pore-forming proteins (PFPs) (Fiori et al., 1996). The PFPs
are molecules able to insert into the lipid bilayer of target cells, forming
transmembrane channels that lead to cell death by osmotic lysis (Ojcius
and Young, 1991). Several pathogens, from bacteria to protists, use PFPs
for lysing target host cells; PFPs are also a weapon used by cytotoxic T
lymphocytes (Leippe and Herbst, 2004). The use of osmotic protectants
led to the demonstration that T. vaginalis haemolysis is mediated by the
functional formation of pores on RBC membranes and that the size of the
pores formed on target cell membrane is between 1.14 and 1.34 nm (Fiori
et al., 1993). Electron microscopy studies have shown that during infec-
tion a close contact takes place between T. vaginalis and the target cell
(Bastos Furtado and Benchimol, 1998). The intimate adhesion of tricho-
monads to target cells could contribute to create a microenvironment
where the parasite might control local pH and where high concentrations
of pore-forming factors could be reached in order to obtain an efficient
lysis (Fiori et al., 1999). Even if the pore-forming activity of T. vaginalis has
been characterised to some extent, the molecular effectors involved have
never been identified.
The parasitic protists E. histolytica and Naegleria fowleri (Hecht et al.,
2004; Herbst et al., 2004) produce PFPs (amoebapores and naegleriapores,
respectively) that are secreted (Espinosa-Cantellano and Martinez-
Palomo, 2000). Amoebapores and naegleriapores belong to the conserved
family of saposin-like proteins (SAPLIPs), which can be found in phylo-
genetically distant organisms (e.g. protists and mammals). Human pro-
teins such as saposins A to D, granulysin, NK-lysin, and pulmonary
surfactant protein B also belong to the SAPLIP family. Members of the
SAPLIP family have the most diverse functions, yet with a common
feature: the interaction with lipids. All these proteins share the same
75 amino acid SAPLIP domain characterised by the invariable 6-cyste-
ine pattern and an abundance of hydrophobic residues, resulting in a
common folding characterised by 5 alpha-helices interconnected by three
disulfide bridges between the conserved cysteine residues (Bruhn, 2005;
Munford et al., 1995; Zhai and Saier, 2000).
Twelve SAPLIP predicted genes (TvSaplip112) have been identified in
T. vaginalis. RT-PCR experiments performed with reference isolates G3
and SS22 showed that all TvSaplip genes, except TvSaplip9, are transcrip-
tionally active. TvSaplip1, TvSaplip2, and TvSaplip4 have multiple
domains with seven, six and four different SAPLIP motifs, respectively
(Fig. 2.4). This organisation is similar to that of naegleriapores, where a
precursor protein composed of several SAPLIP domains is cleaved to
obtain the biologically active 75-residue polypeptides (Herbst et al.,
2004). The other nine TvSaplips comprise a single unique SAPLIP
TvSaplip1 (TVAG_388060)
FIGURE 2.4 Structural organisation of TvSaplip112. The TrichDB locus tags of

respective genes are shown. Grey rectangles represent the conserved SAPLIP domains.
TvSaplip1, 2 and 4 display multiple domains. Putative signal peptides are represented as
black rectangles at the N-termini (left hand side).
domain. A putative N-terminal signal peptide is present in TvSaplip16

and in TvSaplip11 (Fig. 2.4), consistently with the hypothesis that these
putative cytolytic effectors might be secreted.
The mechanism of pore formation has been extensively studied in
E. histolytica (Hecht et al., 2004). Acidic pH causes the protonation of the
basic His75 residue that in turn triggers amoebapore dimerisation as the
result of the interaction of histidine with a negatively charged residue.
The interaction of three amoebapore dimers leads to the formation of a
hexameric ring-like structure with a hydrophobic external surface and a
hydrophilic inner channel (Leippe et al., 2005). T. vaginalis-mediated
haemolysis is also triggered by an acidic pH and several TvSaplip
domains show a basic His or Lys residue in the same key position of
His75 of amoebapores, suggesting a conserved pH-dependent mecha-
nism driving oligomerisation.
All the features displayed by TvSaplips make this new family of
predicted proteins good candidates as effectors contributing to the cyto-
lytic effect of T. vaginalis, named the trichopores. However, given the
heterogeneous nature of SAPLIP functions, it seems unlikely that all
TvSaplips are directly involved in the cytopathogenicity of the parasite.
It is well known that in different biological systems SAPLIPs can have
functions other than cytolytic activity. In fact, while in N. fowleri naegle-

riapores A to B are the only known SAPLIPs, the complete sequencing of
E. histolytica genome (Loftus et al., 2005) leads to the annotation of 12 new
putative proteins with conserved SAPLIP domains, but unknown func-
tions (Bruhn and Leippe, 2001). Accordingly, it can be hypothesised that
some TvSaplip genes may be involved in other cellular processes, such as
lipid metabolism (Zhai and Saier, 2000) rather than pathogenicity.
2.3. PEPTIDASES
Peptidases perform important roles in many biological processes and are

potential virulence factors, vaccine candidates and drug targets in
T. vaginalis and other parasitic protists (Klemba and Goldberg, 2002). Pepti-
dases are enzymes that hydrolyse peptide bonds and constitute a structur-
ally and functionally diverse set of proteins that can be grouped into distinct
Clans and Families based on intrinsic evolutionary relationships (Rawlings
et al., 2009). The MEROPS database (http://merops.sanger.ac.uk, release
9.3, September 2010) was used to define the T. vaginalis degradome (the
protein degradation capacity of the cell). In this analysis, predicted proteins
are classified as peptidases if they contained a peptidase unit incorporating
the expected amino acids required for catalysis and non-peptidase homo-
logues if they lack one or more catalytic residues. Using these stringent
criteria T. vaginalis is estimated to contain in the order of 310 peptidases,
which comprise 5 aspartic peptidases, 156 cysteine peptidases, 89 metallo-
peptidases, 44 serine peptidases and 17 mixed peptidases (summary in
Tables 2.5 and 2.6). This represents 0.5% of the predicted protein coding
capacity of the genome, but is likely to be an underestimate as the
TABLE 2.5 Distribution of peptidases in different species
Catalytic type
Species Total number Asp Cys Metallo Ser Mixed
T. vaginalis 310 5 156 89 44 16

E. histolytica 89 5 33 21 11 19
L. major 153 2 63 54 13 21
P. falciparum 92 10 33 20 16 13
Human 561 21 148 186 178 28
Mouse 641 27 163 198 227 26
S. cerevisiae 100 15 29 36 17 3
Values for listed species were derived from different sources: L. major (Ivens et al., 2005), P. falciparum (Wu et al.,
2003), Human and mouse (Puente et al., 2003) and E. histolytica and S. cerevisiae (http://merops.sanger.ac.uk/).
TABLE 2.6 Clans and families of T. vaginalis peptidases
Catalytic
type Clan Family Number Example from family
Aspartic AA A1 1 Cathepsin D
AD A22 4 Presenilin
Cysteine CA C1 42 Papain
C2 4 Calpain
C12 2 Ubiquitin hydrolase
C40 9 NlpC/P60
C54 5 ATG4
C88 1 OTU1 deubiquinylating enzyme
CD C13 9 Legumain
C14 6 Metacaspase
C50 1 Separase
CE C48 6 Ulp1 peptidase
CF C15 1 Pyroglutamyl peptidase I
CO C40 9 PgpA peptidase
Serine SB S8 11 Subtilisin
SC S9 8 Acylaminoacyl peptidase
S28 8 Pro-Xaa carboxypeptidase
S33 13 Prolylaminopeptidase
SE S12 1 Carboxypeptidase B
SF S26 1 Signal peptidase I
ST S54 2 Rhomboid
Metallo MA M1 3 Aminopeptidase N
M3 1 Oligopeptidase A
M8 42 Leishmanolysin
M41 1 FtsH peptidase
M48 4 CaaX prenyl protease 1
ME M16 7 Hydrogenosomal processing
peptidase
MG M24 8 Methionyl aminopeptidase 2
MH M18 5 Aspartyl aminopeptidase
M20 10 Peptidase T
M28 1 Aminopeptidase S
MK M22 1 Sialoglycoprotein endopeptidase
MP M67 4 Jab1 peptidase
M- M49 1 Dipeptidyl peptidase III
M- M79 1 CaaX prenyl protease 2
PB T1 11 Proteasome subunits
Catalytic
type Clan Family Number Example from family
Mixed PB T2 1 Isoaspartyl dipeptidase

(C, S, T) (theronine type)
PC C26 1 g-Glutamyl hydrolase
PB C44 2 Amidophosphoribosyltransferase
precursor
PB C69 1 Dipeptidase A
Trichomonas genome is fragmentary and is also characterised by many

divergent genes. Indeed, a more general annotation that included all anno-
tated genes (including potential gene fragments and/or divergent proteins)
identified 469 peptidases and related entries (Carlton et al., 2007). The
T. vaginalis degradome is more complex than S. cerevisiae (100 peptidases),
E. histolytica (89), Leishmania major (153) or Plasmodium falciparum (92), but
less complex than mammals such as human (561) or mouse (641) (Table 2.5).
2.3.1. Aspartic peptidases

T. vaginalis has only two families of aspartic peptidase (AD). There are
four Clan AD, Family A22 peptidases with sequence similarity to pre-
senilin 1, which is a multi-pass membrane peptidase that cleaves type I
membrane proteins, such as the amyloid precursor protein of Alzheimers
disease and the Notch receptor that is involved in signalling during
differentiation and development (Xia and Wolfe, 2003). Presenilin-like
peptidases have been described in mammals, worms, trypanosomatids
and Dictyostelium, but not in yeast or other microbial eukaryotes such as
Plasmodium. T. vaginalis has a single A1 Family cathepsin D-like aspartic
peptidase. This family is abundant in apicomplexan parasites, for exam-
ple, the plasmepsins, which are so abundant in Plasmodium and other
apicomplexan (Coombs et al., 2001), but are lacking in trypanosomatids.
2.3.2. Cysteine peptidases

T. vaginalis contains many distinct cysteine peptidases. Papain family
enzymes have been extensively characterised in trichomonads and have
been implicated as virulence factors, in adherence, in nutrition and hae-
molysis, as well as ability to induce apoptosis in human vaginal epithelial
cells (Alvarez-Sanchez et al., 2000; Arroyo and Alderete, 1989; Mallinson
et al., 1994; Mendoza-Lopez et al., 2000; Sommer et al., 2005). Forty-two
distinct papain-like cysteine peptidase genes of Clan CA, Family C1 were

identified, which highlights the diversity and importance of this family of
enzymes in T. vaginalis (De Jesus et al., 2009; Hernandez-Gutierrez et al.,
2003; Ramon-Luing et al., 2010), as well as other parasitic protozoa such as
E. histolytica (He et al., 2010; Irmer et al., 2009). Calpains (Clan CA, Family
C2 cysteine peptidases) are involved in signal transduction pathways in
mammals, as well as in the function of the cytoskeleton and are particu-
larly abundant in the trypanosomatids and other protists. By way of
comparison, S. cerevisiae has a single calpain whereas T. vaginalis has
four candidates. T. vaginalis is particularly abundant in a group of cyste-
ine peptidases that are involved in ubiquitylation; the ubiquitin C-termi-
nal hydrolases (59 members of Clan CA, Families C12 and C19
representing 20% of the degradome). The presence of threonine pepti-
dases that form the core structure of the proteasome in T. vaginalis,
together with the presence of many ubiquitin and ubiquitination
enzymes, confirms the importance of cytosolic protein degradation in
the parasite. The presence of Clan CA, Family C54 cysteine peptidases
with similarity to yeast ATG4, an enzyme which is involved in formation
of autophagosomes, suggests that autophagy occurs in T. vaginalis and
that the core molecular mechanisms involved are similar, at least to an
extent, to those of yeast and mammals. The presence of other ATG genes
in Trichomonas supports this view (Duszenko et al., 2010; Rigden et al.,
2009) (see Section 2.4.1.3).
T. vaginalis has a number of Clan CD cysteine peptidases. These
include nine members of Family C13, which contain the GPI:protein
transamidase and legumain-like enzymes. Previous work has shown
that some Family C13 members form a new subfamily within the legu-
main-like family in T. vaginalis (Leon-Felix et al., 2004; Ramon-Luing et al.,
2010). In the absence of a metabolic pathway for GPI-anchor formation
(see Section 2.2.3.2), it is unlikely that the C13 Family members are part of
a GPI:protein transamidase. The organism also has six metacaspases,
which are caspase-like cysteine peptidases present in yeast, protists and
plants, but not worms, mammals or flies. The metacaspases of yeast and
plants have been reported to be involved in cell death (Madeo et al., 2004;
Suarez et al., 2004), but a similar apoptosis-like process has not been
described in T. vaginalis. The parasite also has a single separase, a highly
conserved, essential, peptidase involved in chromosome segregation.
T. vaginalis also has representatives of Clans CE, CF, PB(C), PC(C)
showing the wide diversity peptidases that utilise an active site cysteine
in the organism and highlighting the fact that it is not just the papain
family enzymes that contribute to cysteine peptidase activity in this
organism. For example, T. vaginalis has nine PgpA-like members of Clan
CO, Family C40. These cysteine peptidases have a role in the degradation
of bacterial cell wall components and the T. vaginalis orthologues have the
highest level of similarity to bacterial proteins. However, none of the

T. vaginalis proteins appear to contain a signal peptide, so they may
have intracellular roles, rather than extracellular roles.
2.3.3. Serine peptidases

One of the most abundant families of serine peptidases in metazoa com-
prises the trypsin/chymotrypsin family. No members of this particular
family appear to be present in T. vaginalis, although there are representa-
tives of seven serine peptidase families. T. vaginalis has 11 subtilisin-like
serine peptidases in MEROPS and 33 subtilisin-like entries annotated in
the genome (Carlton et al., 2007; Hernandez-Romano et al., 2010; Hirt
et al., 2007), some of which might play a role in processing secreted or
surface proteins. One (SUB1) appears to be on the T. vaginalis cell surface
and is also dispersed in vesicles throughout the cytosol (Hernandez-
Romano et al., 2010) and the surface proteomics survey identified seven
subtilisin-like serine peptidase (de Miguel et al., 2010). T. vaginalis has two
rhomboid-like proteins annotated in MEROPS (nine in the genome anno-
tation; Carlton et al., 2007), which are intramembrane serine peptidases
whose signalling function is conserved in eukaryotes and prokaryotes
(Koonin et al., 2003). Rhomboids have been shown to be important for
cleavage of cell surface adhesins in Toxoplasma and thus are crucial for
invasion (Brossier et al., 2005). One entry was identified in the surface
proteomics survey (de Miguel et al., 2010); hence a potential role in
processing T. vaginalis adhesins can be envisaged.
2.3.4. Metallopeptidases
There are 14 families of metallopeptidases represented in the T. vaginalis
genome. Among them, the most pre-eminent family comprises 42 leish-
manolysin-like (or GP63-like) members of Family M8 in MEROPS and a
total of 77 GP63-like entries were annotated in the genome (Carlton et al.,
2007; Hirt et al., 2007). Leishmanolysin is the main surface peptidase of
L. major and appears to contribute to its virulence and pathogenicity
through diverse functions, possibly through promoting resistance to
complement-mediated lysis or facilitating the invasion of the host cell
(Yao et al., 2003). An M8 Family member has been reported to play a role
in T. vaginalis infection (Ma et al., 2011) and the surface proteomics survey
identified 16 GP63-like proteins (de Miguel et al., 2010) of which only 10
are listed in the MEROPS database. Other metallopeptidases found in
T. vaginalis can be assigned to intracellular roles, such as turnover of
abnormal proteins (Families M41, M48), peptide degradation (Families
M18, M20) or protein processing (Families M16, M24), indicating a com-
plex array of metallo-enzymes in the parasite. One member of the Clan ME
Family M16 (so-called inverzincin with motif HXXEH rather than the
more common zincin motif HEXXH of the catalytic site) is involved in
processing N-terminal hydrogenosomal-targeting signals (hydrogenoso-
mal processing peptidaseHPP) and is a homologue of the catalytic b
subunit of the mitochondrial processing peptidases (MPPs) (Smid et al.,
2008). It was initially thought that the bHPP (TVAG_233350) would func-
tion as a monomer (Brown et al., 2007). However, it was subsequently
demonstrated that a divergent aHPP (TVAG_119710) is also encoded
by the T. vaginalis genome and it is required for a functional abHPP
heterodimer, as known for the abMPP (Smid et al., 2008).
T. vaginalis contains two CaaX prenyl peptidase homologues. These
enzymes cleave the tripeptide aaX following prenylation of target pro-
teins on cysteine by farnesyl transferase and geranylgeranyl transferase
proteases (Wright and Philips, 2006). Both CaaX prenyl proteases are
metallopeptidases (Clan MA, Family M48 and Clan M(), Family M79).
The presence of these peptidases indicates that prenylation is important
in Trichomonas. A total of 93 proteins annotated in T. vaginalis G3 possess a
CaaX motif. Among these 12 small GTPases are listed including Rho and
Ras-like annotated proteins, some of the best-known substrate for the
CaaX prenyl peptidase (Wright and Philips, 2006). However, there are
currently no experiment data on prenylation in T. vaginalis.
2.3.5. Threonine peptidases

In T. vaginalis 11 a and b threonine peptidase subunits of the 20S unit of
the proteasome complex can be identified, showing that the parasite
possesses the core machinery necessary for its intracellular protein turn-
over. Another threonine peptidase can be found, an isoaspartyl dipepti-
dase. This type of peptidase (Hsieh et al., 2003) has been found to be
involved in various metabolic processes in organisms ranging from bac-
teria to mammals, but have not yet been characterised in any protists.
2.3.6. Peptidase inhibitors

The human genome contains 183 genes encoding natural peptidase inhibi-
tors, such as the serpins (inhibitors of serine peptidases) and cystatins
(inhibitors of cysteine peptidases). Only three cystatin-like inhibitors
could be identified in T. vaginalis; these may regulate the activity of the
abundant endogenous Clan CA, Family C1 peptidases. T. vaginalis does not
appear to contain members of the ICP (for inhibitor of cysteine peptidase)
family, which have been shown to be potent inhibitors of Family C1 pepti-
dases in some parasitic protists, including E. histolytica, and pathogenic
bacteria (Monteiro et al., 2001; Riekenberg et al., 2005; Sanderson et al., 2003).
2.3.7. Non-peptidase homologues

Trichomonas has a remarkable number of proteins that have peptidase-like
sequences but appear to lack the essential residues for catalytic activity
(Table 2.7). These pseudo-peptidases may play roles in regulating pepti-
dase activity or mediate binding functions that no longer require a pepti-
dase activity. For those entries that represent transmembrane peptidases,
they might be involved in binding activities such as adhesins functions as
suggested for some human metallopeptidases (Bauvois, 2001).
2.4. MEMBRANE TRAFFICKING AND CELL SIGNALLING
Membrane trafficking and cell signalling are often considered as separate

cellular systems but are now increasingly recognised as being intimately
interlinked (Sorkin and von Zastrow, 2009). They both play central roles
in orchestrating the dynamic interactions of all eukaryotic cells with their
environment, modulating the repertoire and amount of surface exposed
and secreted molecules and, conversely, that of internalised material
TABLE 2.7 Clans and families of T. vaginalis non-peptidase homologues
Catalytic type Clan Family Number Example from family
Cysteine CA C1 3 Papain
C2 1 Calpain
C66 1 IdeS peptidase
CD C13 1 Legumain
Serine SB S8 12 Subtilisin
SC S28 5 Pro-Xaa carboxypeptidase
S33 3 Prolylaminopeptidase
SE S12 1 Carboxypeptidase B
SF S26 1 Signal peptidase I
ST S54 3 Rhomboid
Metallo M8 15 Leishmanolysin
M48 1 CaaX prenyl protease 1
MG M24 1 Methionyl aminopeptidase 2
MO M23 2 b-Lytic metallopeptidase
MP M67 1 Jab1 peptidase
M- M49 1 Dipeptidyl peptidase III
Mixed (C, S, T) PB T1 4 Proteasome subunits
PC C56 5 PfpI peptidase
PC S51 1 Dipeptidase E
through endocytosis and phagocytosis (Boulais et al., 2010; Sorkin and

von Zastrow, 2009). These two systems contribute at detecting and trans-
ducing external cues to intracellular systems to optimise cellular func-
tions in relation to the external conditions. Secretion and internalisation,
through endocytosis and phagocytosis, are intimately linked with
T. vaginalis pathobiology as discussed earlier. Here selected candidate
GTPases mediating membrane trafficking and signalling in model sys-
tems are considered, along with a selection of their potential functional
partners. A brief discussion of the endosomal sorting complex required
for transport (ESCRT) proteins, involved in late endosomal trafficking,
and the ATG proteins mediating autophagy, which are now two systems
recognised to be functionally interlinked (Fader and Colombo, 2009), are
also provided. An overview of major signalling pathways identified in the
genome is depicted in Fig. 2.5. Selected aspect of signalling and mem-
brane trafficking are discussed below.
2.4.1. Selected GTPases, ESCRT and ATG

2.4.1.1. Heterotrimeric G-proteins and GPCR-RGS proteins
A heterotrimeric G-protein a subunit (Ga) was initially identified in
T. vaginalis in an EST survey, encoding Ga402 (TVAG_452120) (Hirt
et al., 2003). Biochemical evidence for additional Ga proteins (Hirt et al.,
2003) was eventually confirmed by the identification of seven Ga encod-
ing genes (Carlton et al., 2007). The genome also encodes 10 candidates
surface G-protein coupled receptors (GPCRs) of which five have a cyto-
plasmic GTPase-activating protein (GAP) made of a conserved regulator
of G-protein signalling (RGS) domain (Anantharaman et al., 2011; Carlton
et al., 2007) (Fig. 2.6). All but one TvGa can potentially function as
GTPases (Anantharaman et al., 2011), as TVAG_453150 does not possess
all the critical residues required for GTPase activity and has an unusual
C-terminal extension of 113 residues possibly due to a frame shift. The
Ga402 (TVAG_452120) protein was shown to locate by IFA to internal
membranes and identified a very dynamic behaviour of multi-vesicular
body (MVB)-like structures during amoeboid transformation upon the
parasite binding to ECM in vitro (Lal et al., 2006) that could lead to the
secretion of MVB cargo (Fig. 2.7) (Carlton et al., 2010).
Surprisingly, no orthologues for Gb and Gd could be identified in
T. vaginalis (Anantharaman et al., 2011). The Gbd heterodimer is the
classic functional partner of Ga in well-studied model systems acting as
a GDP dissociation inhibitor for the Ga-GDP. Upon the action of an
activated GPCR, which functions as a GDP exchange factor (GEF), the
GDP of the Ga-GDP is changed to a GTP leading to a structural switch
that dissociates Ga-GTP from Gbd, which can now both interact with
downstream effector proteins (Anantharaman et al., 2011, and references
GPCR (10): Cyclases cAMP/cGMP (103):

- 7 TMD - Membrane cyclases with 116 TMD
- 5TMD - Soluble cyclases
5 GPCR
5 GPCR-RGS
ATP/
GTP Soluble
RGS Cyclase cyclase
Ga(7) cAMP/
cGMP
Ras/Rho/Rab/ARF
GTPase (354)
Cyclic
PIP2 PLC-like nucleotides
phosphodiesterase PDE (44)
IP3 (2)
Protein Protein
RRR
kinases phosphatases
(9)
(188)
Protein kinases (795) Histidine kinases (7)

- No evidence for kinases - Receptors with 12 TMD
with TMD - Soluble
FIGURE 2.5 Signalling modules identified in T. vaginalis genome annotation. A combi-

nation of published data (Anantharaman et al., 2011; Fritz-Laylin et al., 2010; Kutuzov and
Andreeva, 2008) and searches at TrichDB (using Pfam or InterPro domains accessions)
were used to establish the presence of selected entries implicated in various signalling
pathways. There are, in particular, a large number of kinases (see Section 2.4.2), protein
Ser/Thr phosphatases (Kutuzov and Andreeva, 2008), small GTPases (see Section 2.4.1.2),
cyclases (PF00211 domain at TrichDB; see also Cui et al., 2010) and cyclic nucleotide
phosphodiesterases (PDE) (PF00233 domain at TrichDB; see also Cui et al., 2010). Both
cytosolic and membrane cyclases (with one to 16 TMD) were identified. Also notable is
the presence of G-protein coupled receptor (GPCR) and cognate Ga (see Fig. 2.6 and
Section 2.4.1.1) as well as histidine kinases (PF0512 domain at TrichDB) and cognate
response regulators receiver (RRR, PF00072 domain at TrichDB) proteins, absent from
several parasitic protists (Fritz-Laylin et al., 2010). Two candidate PLC-like PDE that
convert phosphatidylinositol 4,5 bisphosphate (PIP2) into D-myo-inositol-1,4,5-trispho-
sphate (IP3) and diacylglycerol (not illustrated) were also identified (entries with domain
PF00388 at TrichDB). Numbers in brackets indicate the number of annotated proteins at
TrichDB, derived from our analyses (see text), or in published material.
therein). T. vaginalis is currently the only organism known to encode Ga

proteins and no Gbd (Anantharaman et al., 2011; Fritz-Laylin et al., 2010).
The absence of Gbd suggests that a simpler signalling cycle might func-
tion in T. vaginalis (Fig. 2.6). It will be important to establish the nature
of the external cues for the 10 GPCR-like proteins (including the 5 GPCR-
RGS) and if/how the different GPCR interact with the seven TvGa.
A
N RGS C 426-435 (3)
352-361 (2)
184-285 (3)
B GPCR-RGS (GEF) C
N
Name (TMD) Accession EST

(1) TvRGS435 (7) TVAG_094600 4
RGS
TvRGS428 (7) TVAG_302510 8
Ga GDP GTP (GAP)
(2) TvRGS426 (7) TVAG_478870 4
C
? TvRGS361 (5) TVAG_009990 1
TvRGS352 (5) TVAG_360180 1
TvRGS285 (0) TVAG_396300 1
TvRGS197 (0) TVAG_259390 1
Effectors TvRGS184 (0) TVAG_261480 1
FIGURE 2.6 Structural organisation of candidate TvGPCR-RGS proteins. (A) Structural

organisation of the eight identified TvRGS proteins with the N-termini on the left. The
TMD are shown in dark grey boxes and the RGS domains in black boxes. Three RGS
domains are linked to proteins containing seven TMD and could represent GPCRs. The
range of amino acid length is shown with the number of proteins shown in brackets.
There are two RGS that are characterised by five TMDs. Three RGS are without TMD as
commonly found in animals. (B) Cartoon illustrating a basic model of how the GPCR-RGS
could mediate signalling from external cues with a Ga protein in the absence of a Gbd
heterodimerthe question mark indicates the potential presence of a functional ana-
logue of a Gbd heterodimer. A potentially simplified cycle could mediate signal trans-
duction with the membrane bound RGS acting as a GAP stimulating the recycling to the
Ga-GDP state and the GPCR acting as a GEF. (C) Table listing the eight TvRGS-containing
proteins depicted in (A) with the name indicating the protein length and values in
brackets the number of TMD. All entries have EST support with the higher number of
ESTs among proteins with seven TMD.
As Gas have been implicated in both phagocytosis (Boulais et al., 2010)

and exosome secretion via secretory MVB (Lal et al., 2006, and references
therein), it will be of interest to investigate the potential role of the TvGa
into these processes in T. vaginalis (Lal et al., 2006).
2.4.1.2. Small GTPasesRas and ARF superfamily

One of the surprising discoveries of the genome annotation was the
presence of a vast gene family of small GTPases of the Ras- and ARF-
like families with over 300 entries (Carlton et al., 2007, 2010). A combina-
tion of InterPro domains for small GTPases (IPR003579: GTPase_Rab,
IPR003578: GTPase_Rho, IPR006689: ARF/SAR) identified a total of 382
related sequences. Removing the most divergent (longer on either ends
and missing key residue of the G-domains) and partial sequences still left
354 Ras-like/ARF-like sequences. Interestingly, not all subfamilies of
Endocytosis: Autophagocytosis
- Host proteins (e.g. lactoferrin) - Organelles
- Viruses (e.g. HIV, HSV) - Proteins and RNAs
- Receptors for signalling? - Bacteria and viruses?
Rab11 RE Endosomes Atg8

Phagocytosis: ARF ARF Rab5 Rab7
- Bacteria, yeast
- Host cells Rab5 Phagophore HYD
(+viruses) EE
Rab11 Autophagosome
Rho ESCRT
ARF
Phagosomes LE
HYD
Rab11
Rab7
Ga MVB HYD
Rab5 Lysosomes Amphisome
Rab7 Ga402
Rho
Lysosomes
Rab7 HYD
Rab11 Secretory
Ga402
Phagolysosomes MVB Autolysosome
Exosomes and viruses?
FIGURE 2.7 Schematic representation of three interlinked membrane trafficking

routes: phagocytosis, endocytosis and autophagy. Endosomes are linked with both
phagocytosis and autophagy, providing membranes for the development and matura-
tion of these two cellular processes. The material inside these organelles will eventually
be degraded upon organelles fusion with lysosomes. The phagocytosis of a bacteria and
autophagocytosis of a hydrogenosome (HYD) and protein complexes (black disks) are
illustrated. Important selected effectors identified in the T. vaginalis genome are
indicated: small GTPases Rab, Rho and ARF, ESCRT proteins of the multi-vesicular bodies
(MVBs) and the G-proteins Ga. The Ga402 (TVAG_452120) protein is likely to localise to
MVB-like structures (Lal et al., 2006).
small GTPases were represented in large numbers (only two Ran, for
example, were identified), consistent with some preferential gene dupli-
cation processes, in particular, for the Rab subfamily (Carlton et al., 2007,
2010). This is extraordinary compared to what is known from the best
investigated model systems of mammals, yeast and plants (Carlton et al.,
2010; Lal et al., 2005), where the complexity of small GTPase gene families
was thought to correlate with organism complexity expressed in terms of
cell diversity (Brighouse et al., 2010). Since most of these small TvGTPases
have all of the key features expected for functional GTPases, and assum-
ing that they correspond to expressed and functional proteins, this will
represent an interesting challenge to rationalise in terms of functional
relevance for the parasite. The working hypothesis is that the large num-
ber of small GTPases (TvRab, TvRho and TvARF in particular) contributes
to the functioning of a highly dynamic and complex endomembrane sys-

tem and endosomal/phagosomal system, in particular (Carlton et al.,
2010). This was recently suggested for ciliates (Paramecium tetraurelia 229
Rabs and Tetrahymena thermophila 88 Rabs) where a large number of Rab
proteins (18 Rab) were shown to be associated with phagocytosis in
T. thermophila (Bright et al., 2011; Saito-Nakano et al., 2010).
2.4.1.3. ESCRT and ATG proteins

Consistent with T. vaginalis possessing a complex endomembrane system,
two additional sets of protein complexes support the presence of MVB,
suggested by the localisation of TvGa402 and ultrastructural data (e.g. Lal
et al., 2006; Rendon-Maldonado et al., 2003), and autophagy, suggested by
ultrastructural data (Benchimol, 1999).
The ESCRT is made of up to five protein complexes, of which four are
ubiquitous across eukaryotes (Leung et al., 2008). The genome of T. vagi-
nalis encodes homologues for ESCRT complexes IIII and the ESCRT-III
associated complex, which are required for the formation of intraluminal
vesicles (ILVs) (Leung et al., 2008). The ESCRT complex mediates lyso-
somal targeting of ubiquitylated proteins, which play an important role in
down regulating surface receptors and other membrane proteins follow-
ing their endocytosis. These proteins are incorporated into the ILV and are
eventually degraded upon fusion of MVBs to lysosomes (Leung et al.,
2008; Sorkin and von Zastrow, 2009). Hence, the MVB contributes directly
to signal transduction regulation (Sorkin and von Zastrow, 2009).
Autophagy is a distinct lysosomal-mediated degradation pathway,
which focuses on the cells own components (organelles, protein com-
plexes), but that can also target pathogens (bacteria, viruses) located in
the cytosol (Brennand et al., 2011; Duszenko et al., 2010). Proteins mediat-
ing autophagy have been named autophagy-related proteins (ATG) and
different types of autophagy have been described including selective and
non-selective forms (Brennand et al., 2011). In all cases the structures to be
degraded become entrapped in a double membrane, called the autopha-
gosome, which upon fusion to lysosomes leads to the degradation of the
internal membrane and its content for eventual recycling to the cytosol
(Brennand et al., 2011). Over 30 ATG proteins have been described with
some of these characterised as having broad distribution across eukar-
yotes. Seven successive steps are defined for autophagy with specific
proteins involved in each of these and T. vaginalis encode at least one
protein for each step (Brennand et al., 2011). Some of the best-conserved
ATG homologues in T. vaginalis include Atg1, Atg7 and Atg9, with the
latter possessing the characteristic 6 TMDs (Carlton et al., 2010; Rigden
et al., 2009). Notably there are also five Atg4 homologues (see
Section 2.3.2), cysteine peptidases involved in cleavage of Atg8 of which
there are two paralogues in T. vaginalis. Autophagy is known to be
important for overall cell homeostasis, during cell starvation and cell
differentiation (Brennand et al., 2011). During its life cycle T. vaginalis is
exposed to important changes in its environment, including during shift
from male to female hosts and vice versa, changes due to the menstrual
cycle and the effect of the innate and adaptive immune responses and it is
also undergoing cellular differentiation into amoeboid forms and pseu-
docysts (Hobbs et al., 2008). Hence, autophagy is likely to play important
roles in all these different contexts and represent attractive drug targets to
control T. vaginalis and other parasites (Brennand et al., 2011).
Autophagy and MVBs have been shown to be functionally
interconnected, with the MVB representing the main fusion partners to
autophagosomes, forming the so-called amphisomes (Fader and
Colombo, 2009). Similarly, phagocytosis also requires MVB for matura-
tion with the ESCRT proteins being part of the phagosome protein net-
work (Boulais et al., 2010). The potential functional interplay between
phagocytosis, endocytosis and autophagy in T. vaginalis are illustrated
in Fig. 2.7. As each of these cellular processes underline important aspects
of T. vaginalis pathobiology future work dissecting the molecular basis of
these processes will be of great interest and possibly contribute to the
design of new control strategies.
2.4.2. Protein kinases

Nothing is known about protein kinases of T. vaginalis and their role in the
parasites cell signalling pathways. Very little is also known about
T. vaginalis protein phosphatases, the functional partners of protein
kinases in regulating the phosphorylation status of phosphorylated pro-
teins. Protein phosphatases are not discussed here. A recent survey across
selected parasites identified the largest gene set encoding serine and
threonine phosphatases (188 entries) in T. vaginalis among the analysed
species (Kutuzov and Andreeva, 2008) (Fig. 2.5). The draft T. vaginalis
genome allows some predictions to be made on the protein kinase reper-
toire. T. vaginalis has 795 genes encoding distinct eukaryotic protein
kinases (ePKs), and 34 atypical protein kinases (aPKs). This is one of the
largest kinomes known to exist in the eukaryotic domain, being 1.6
larger than humans (> 500 ePKs; Manning et al., 2002), almost 2 larger
than the amoeba E. histolytica (270 ePKs; Loftus et al., 2005) and 3.4
larger than L. major (El-Sayed et al., 2005; Ivens et al., 2005; Parsons et al.,
2005). One hundred and thirty-two of the 795 ePKs are predicted to be
catalytically inactive, as they lack key amino acid residues thought be
essential for enzyme activity. Bioinformatics analyses place all 795
T. vaginalis ePKs into the eight major groups defined for the human
kinome (Manning et al., 2002; Martin et al., 2004; http://www.compbio.
dundee.ac.uk/kinomer/index.html) and all the T. vaginalis protein kinase

genes are unique, with many paralogues but each with distinct sequences.
Whilst T. vaginalis has many metabolic adaptations to its micro-anaer-
obic environment that are similar to E. histolytica, the two species would
appear to differ significantly in their mechanisms of cell signalling. Whilst
E. histolytica has 90 receptor serine/threonine ePKs of three types, all of
which contain an extracellular domain, a single transmembrane helix and
a cytosolic tyrosine kinase-like (TKL) domain (Loftus et al., 2005),
T. vaginalis has none. T. vaginalis has one potential tyrosine kinase, but it
is predicted to have an amino acid substitution in one of the key catalytic
residues, making it an apparent inactive homologue. No other receptor
protein kinases were identified that could act to transduce extracellular
signals. Indeed none of the 795 ePKs in T. vaginalis have a TMD; however,
the parasite has some GPCR proteins and Ga subunits (see Section 2.4.1.1),
as well as ePKs of three component MAPK pathway, suggesting signal
transduction mechanisms that may be more similar to yeast than other
protists, such as the trypanosomatids or Plasmodium. Another unusual
feature of the T. vaginalis kinome is the presence of 105 cytosolic TKL
genes, of which 21% are predicted to be inactive. This family of ePKs,
which includes human Src and Jak and are important for activating cyto-
solic signalling molecules, are found in E. histolytica, but are absent from
the trypanosomatids (Parsons et al., 2005) and P. falciparum (Ward et al.,
2004). In comparison to the human kinome, the CAMK and CK1 ePK
families are greatly over-represented in T. vaginalis, whereas the STE
family is significantly under-represented having just 37 members and
this includes the highest proportion of predicted inactive kinases (30%).
Some ePK domains are known to lack protein kinase activity experi-
mentally, and these have been postulated to act as kinase substrates and
scaffolds for assembly of signalling complexes (Anamika et al., 2009;
Manning et al., 2002; Pils and Schultz, 2004). The percentage of inactive
kinases among families can vary substantially (Table 2.8). For instance,
30% of STE ePKs (11/37) are predicted to be inactive catalytically,
whereas only 3% of CK1 ePKs are inactive (2/62). The overall rate of
catalytically inactive kinases was found to be 16.6%, considerably higher
than that found in the human genome (9.6%; Manning et al., 2002), or the
T. brucei genome (8%; Berriman et al., 2005; El-Sayed et al., 2005; Parsons
et al., 2005). Of note is the finding that many of the predicted inactive
kinases form phylogenetic clusters, for example, four STE kinases are
closely related, suggesting that the mutations arose in a progenitor ePK,
but that the function of the inactive kinase was maintained when the
genes duplicated and evolved.
Accessory domains on ePKs have been shown to be important for
regulating signalling pathways, principally through regulation of
proteinprotein interactions or modulation of protein kinase activity.
TABLE 2.8 The kinome of T. vaginalis
ePKs aPKs
Total Total
Family AGC CAMK CK1 CMGC STE TK TKL ePK PIKK RIO aPK
T. vaginalis 146 328 62 116 37 1 105 795 32 2 34

T. vaginalis 31 57 2 8 11 1 22 132
inactive
% Inactive 21% 17% 3% 7% 30% 100% 21% 17%
ePKs, eukaryotic protein kinases; aPKs, atypical protein kinases.
TABLE 2.9 The accessory domains of T. vaginalis ePKs
Pfam accession T. vaginalis ePKs Domain name
PF00433 37 Protein kinase C-terminal domain

PF00169 30 Pleckstrin-homology domain
PF00515 1 Tetratricopeptide repeat (TPR)
PF00023 6 Ankyrin repeat
PF00786 4 PAK-box/P21-Rho-binding, CRIB
(Cdc42/Rac interactive binding)
PF00659 6 POLO box
PF02207 1 Putative zinc finger in N-recognin
(UBR box)
PF00478 4 IMP Dehydrogenase/GMP reductase
PF01468 1 GA (protein G-related albumin-
binding) Module
Almost 50% of human ePKs contain one or more of 83 types of Pfam

accessory domains (Manning et al., 2002), whereas only nine types of
Pfam domain could be identified in 11% of the T. vaginalis ePKs
(Table 2.9). This suggests that regulation of protein kinase function and
cell signalling in general is less complex than in higher eukaryotes and
may provide an explanation for the abundance of ePKs in T. vaginalis.
Four of the accessory domains are found in seven parasite ePKs but not in
humans. These include the TPR domain, which is a short all-helical
structural motif known to be responsible for specific proteinprotein
interactions. It functions in a variety of different proteins that are involved
in a plethora of cellular processes (DAndrea and Regan, 2003).
The diversity of functions and arrangements of this motif makes
the prediction of the binding partner difficult. It is found not only in
microbial eukaryotes but also in many metazoan proteins, including

human, but not in human protein kinases. The zf_UBR1 domain is a
short Zn-finger recognition motif found in all metazoan species studied,
but not in human protein kinases, involved in targeting proteins for
ubiquitin-dependent proteolysis. The IMP-dehydrogenase/GMP reduc-
tase domain, found in two T. vaginalis ePKs maintains the intracellular
balance of adenosine and guanosine nucleotides. This protein domain is
common to all Domains of life, but has not been found associated with
ePKs before. The GA domain represents a group of Clan AD, Family A24
aspartic peptidases found in bacteria, but rarely in eukaryotes.
Five accessory domains were found in combination with the kinase
catalytic domain both in the parasite and in human ePKs. The C-terminal
domain of protein kinase is found in 40 human protein kinases and in 37
protein kinases of T. vaginalis. Since this domain is thought to be involved
in domaindomain interactions and is found in a variety of cellular
signalling contexts, little information can be gleaned as to the substrate
specificity, signalling route, or mode of regulation of the parasites
kinases harbouring this domain. However, it is interesting to note that
all human kinases harbouring this domain belong to the AGC family, and
so do all the 37 parasite kinases having this domain. The Pleckstrin-
homology (PH) domain is typically found in proteins involved in intra-
cellular signalling. Twenty-one human kinases were found to contain the
PH domain, split in several families (AGC, CAMK, and TK), whereas all
the 30 parasite kinases with the PH domain belong to the AGC family. The
ankyrin repeat is a rather common domain mediating proteinprotein
interactions and is found in six TKL family ePKs in T. vaginalis. The
Cdc42/Rac Interactive Binding (CRIB) domain is only found in the PAK
subfamily of the human STE ePKs. There are four T. vaginalis ePKs
harbouring the CRIB domain all of which are STE and potential homo-
logues of human PAK. Other conserved domains include the p21-acti-
vated kinases and the POLO box, a domain important for cell cycle
regulation of the POLO kinases.
T. vaginalis contains examples of two of the four well-characterised
aPK families (PIKK and RIO), whilst Alpha and PDHK family members
appear to be absent. There is an abundance of PIKK aPKs, potentially
involved in genome surveillance, response to growth signals and nutrient
availability, as well as one member each of the RIO1 and RIO2 subfamilies
that are required for ribosome biogenesis.
2.5. THE TRANSCRIPTOME AND THE RNAI MACHINERY
The enormous coding capacity of T. vaginalis for proteins, with nearly

60,000 candidate proteins, must represent a formidable challenge for the
regulation of gene expression during the life cycle of the parasite.
The spatial and temporal coordination of the complex series of events

that take place at the hostpathogen interface is likely to involve a sophis-
ticated, finely tuned programme of gene expression, with genes and/or
group of genes expressed during the different stages of infection. The
different mechanisms involved in the transcriptional regulation of gene
expression in T. vaginalis have been intensively studied (reviewed in
Smith and Johnson, 2011), whilst the possibility that small RNA-mediated
mechanisms of gene silencing were present in the parasite only became
apparent after the genome was sequenced (Carlton et al., 2007).
Besides providing an invaluable tool to specifically and potently
manipulate gene expression in eukaryotes, RNAi and related RNA
silencing pathways have emerged as new mechanisms for the regulation
of the gene expression (Fire et al., 1998; Moazed, 2009). The composition
of the RNAi machinery displays a high degree of variability among
different organisms. Nevertheless, two conserved gene families are the
universal genetic hallmarks of RNAi and reflect on the two main steps
of the post-transcriptional gene silencing (PTGS) process: the Dicer
family of RNaseIII endonucleases (Carmell and Hannon, 2004) and
the Argonaute (AGO) family (Hutvagner and Simard, 2008). Briefly,
dsRNA is processed to 2030nt long dsRNA (small interfering
RNAsiRNA) by the endoribonuclease Dicer. siRNAs are then directed
to the RNAi-induced silencing complex (RISC), which mediates the
recognition and cleavage of homologous mRNA. AGO proteins are
key components of RISC, where they mediate siRNA binding and
mRNA cleavage activity, acting as the catalytic engine of this multi-
protein complex (Carmell et al., 2002; Hammond et al., 2001; Liu et al.,
2004; Meister and Tuschl, 2004).
Among pathogenic protists, RNAi has been described in African
trypanosomes (Ngo et al., 1998), E. histolytica (Kaur and Lohia, 2004;
Vayssie et al., 2004; Voinnet, 2005; Zhang et al., 2011), Giardia intestinalis
(Prucca et al., 2008) and some Leishmania species (Lye et al., 2010). No
experimental evidence for an RNAi in T. vaginalis has yet been obtained;
however, analysis of the genome sequence allowed the identification of
an RNaseIII gene (TvdsRNase) and two AGO family genes (TvAgo1 and
-2), that were all shown to be transcriptionally active (Carlton et al.,
2007). Figure 2.8 shows the domain organisation of TvdsRNase com-
pared to that of Dicer proteins from Drosophila melanogaster and Caenor-
habditis elegans, G. intestinalis and T. brucei Dicer-like proteins, and
E. histolytica dsRNase. TvdsRNase, similarly to other protist RNaseIII,
differs from Dicers of higher eukaryotes in that it lacks a dsRNA-
binding, PAZ and helicase domains, while it has the same domain
organisation as T. brucei Dicer-like protein. Indeed, T. brucei and
G. intestinalis Dicer-like proteins show a siRNA-producing activity and
are essential for dsRNA-mediated PTGS in these microorganisms
(Macrae et al., 2006; Patrick et al., 2009).
TvdsRNase RNaseIII RNaseIII
GiDicer PAZ RNaseIII RNaseIII
TbDcl1 RNaseIII RNaseIII
EhdsRNase RNaseIII
dsRNA
Dm Dicer-1 helicase PAZ RNaseIII RNaseIII binding
Ce Dicer-1 DExD helicase PAZ RNaseIII RNaseIII

dsRNA
binding
FIGURE 2.8 Comparison of the domain organisation of selected Dicer proteins.

Drososphila melanogaster (DmDicer) and Caenorhabditis elegans (CeDicer) Dicers;
Trichomonas vaginalis TvdsRNase (TVAG_481260); Giardia intestinalis Dicer (GiDicer);
Trypanosoma brucei Dicer-like-1 (TbDcl1); Entamoeba histolytica dsRNase (EhdsRNase).
Cartoons are not to scale.
TvAgo1 PAZ PIWI
TvAgo2 PAZ PIWI
TbAgo1 PAZ PIWI
EhAgo1 PAZ PIWI
EhAgo2 PAZ PIWI
GiAgo-like PIWI
FIGURE 2.9 Argonaute proteins domain organisation in selected pathogenic protists,

displaying the characteristic PAZPIWI domains. Solid lines represent sequences with no
detectable domains. Abbreviations: TvAgo1 and -2, T. vaginalis AGO1 and -
2 (TVAG_453810 and TVAG_411040). TbAgo1, Trypanosoma brucei AGO1; EhAgo1 and -2,
Entamoeba histolytica AGO1 and -2; GiAgo-like, Giardia intestinalis Argonaute-like.
Cartoons are not to scale.
E. histolytica dsRNase was shown to have Dicer-like activity, but it

lacks the typical Dicer domain organisation and shows a typical prokary-
otic organisation with a unique RNaseIII domain (Abed and Ankri, 2005).
Unlike EhdsRNase, and like Dicers, TvdsRNase has a typical two tandem
RNaseIII domain organisation. TvAGO1 and -2 amino acid sequences
show the typical AGO family domain organisation (Fig. 2.9), with a
PAZ and a PIWI domain. In particular, the piwi-box sequence, which
has been shown to interact with Dicer and to bind siRNA (Collins and
Cheng, 2005), appears to be highly conserved in T. vaginalis (Fig. 2.10).
FIGURE 2.10 Sequence alignment of the PIWI-BOX from AGO proteins of pathogenic
protists depicted in Fig. 2.9 contrasted with Arabidopsis thaliana (At) and Drosophila
melanogaster (Dm) sequences. Protein lengths are indicated after their name.
A total of 41 DEAD/DEAH-box helicase genes were predicted in the

T. vaginalis genome sequence, all of which have been confirmed as tran-
scriptionally active either through searching EST databases or by RT-PCR.
Studies in several organisms showed that a number of RNA helicases are
involved in PTGS mechanisms (Tabara et al., 2002). In particular, DEAD/
DEAH-box helicases are thought to be involved in siRNA unwinding in
RISC (Meister and Tuschl, 2004). The DEAD-box helicase Armitage is
essential for RISC assembly in D. melanogaster (Tomari et al., 2004).
Another important component of the RNAi pathway is an RNA-
dependent RNA polymerase (RdRP), a protein that catalyses the synthesis
of secondary siRNA from cleaved target mRNA (Pak and Fire, 2007). This
activity amplifies the RNAi effect, while ensuring that the RNAi machin-
ery focuses on the target mRNA (Sijen et al., 2001). A genomic survey did
not lead to the identification of RdRP orthologues in T. vaginalis.
siRNAs are one of many small non-coding RNAs with regulatory
functions. microRNAs (miRNA) are another class of small RNA duplexes
involved in PTGS, which originate in the nucleus. A nuclear ribonuclease
named Drosha processes long stem-loop transcripts (primary-miRNA
pri-miRNA) into pre-miRNA, which are in turn translocated into the
cytoplasm by Exportin5. Pre-miRNAs are then cleaved by Dicer to 21 nt-
long miRNA, that act on gene expression by translational repression,
specifically interacting with 30 UTRs of target mRNAs (Filipowicz et al.,
2008). While Blast analysis could not detect any Drosha or Exportin5
orthologues in the T. vaginalis genome, two publications describe 9
and 11 predicted miRNA candidates, respectively (Chen et al., 2009;
Lin et al., 2009).
Interestingly, several different dsRNA viruses can infect T. vaginalis
(T. vaginalis virusTVV) (Khoshnan and Alderete, 1993; Wang and Wang,
1986). This could be an indication that the RNAi machinery is actually not
functional in T. vaginalis and that the hypothetical machinery is lacking
some fundamental component(s). Alternatively, it could be hypothesised
that TVVs have evolved a means to evade the antiviral activity of RNAi.
Indeed, several animal and plant viruses encode proteins that inhibit RNAi
by sequestering both dsRNA and siRNAs, thus preventing the action of
Dicer and RISC (Voinnet, 2005). The detection of a dsRNase, two AGO, and
41 DEAD/DEAH-box helicase genes in the T. vaginalis genome sequence,
together with the identification of miRNA candidates, strongly suggest that
this divergent protist is RNAi-competent or, at least, that it possesses a
sophisticated dsRNA processing machinery. Experimental evidence of
dsRNA-mediated PTGS is needed to set the stage for a reliable, specific
and reproducible tool for the genetic dissection of T. vaginalis.
It was recently demonstrated that G. intestinalis uses an RNAi machin-
ery to regulate the differential expression of variant surface proteins
(VSPs), mediating antigenic variation allowing the parasite to evade the
mammalian hosts adaptive immune responses (Prucca et al., 2008).
Generating a mutant cell line deficient in RNAi activity resulted in the
expression of numerous variants of VSP that lead to the vaccination of an
animal model (Rivero et al., 2010). It would be of great interest to test
whether the expression of surface antigen variants in T. vaginalis is
mediated by RNAi or RNAi-related processes. This could lead to impor-
tant new insights into the molecular basis of T. vaginalishuman host
interactions, providing leads to develop novel strategies to limit and
control trichomoniasis.
ACKNOWLEDGEMENTS
R. P. H. was supported by a Wellcome Trust University Award. A. A. S., J. C. M. and Y.-C. L.
were supported by the Wellcome Trust. The Ph.D. project of S. N. was funded by the Faculty
of Medical Sciences and the School of Computing Science at Newcastle University and an
Overseas Research Students Awards Scheme. P. L. F. was supported by Legge 7/2007
Regione Autonoma Sardegna. D. D. was supported by Fondazione Banco di Sardegna.
NOTE ADDED IN PROOF

Two publications relevant for the discussion of the T. vaginalis glycocalix (section 2.2.3) were
recently published. In the paper by Ryan et al. (2011), no evidence was found for the presence
of phosphoglycans in the polysaccharidic backbone. Therefore, this glycoconjugate has been
renamed lypoglycan. In the paper by Paschinger et al. (2011) it was shown that the total
population of N-glycans from T. vaginalis trophozoites is mainly composed of high-mannose
type oligosaccharides and also glycans containing pentose residues, phosphoethanolamine
and terminal N-acetyllactosamine units.
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CHAPTER 3
Cryptic Parasite Revealed:
Improved Prospects for
Treatment and Control of
Human Cryptosporidiosis
Through Advanced
Technologies
Aaron R. Jex,* Huw V. Smith, Matthew J. Nolan,*
Bronwyn E. Campbell,* Neil D. Young,*
Cinzia Cantacessi,* and Robin B. Gasser*

3.2. Cryptosporidium Species and Genotypes Known to
Infect Humans 144
3.3. The Life Cycle of C. Parvum and C. Hominis 145
3.4. Cryptosporidiosis: Pathogenesis and Immunity 147
3.5. Genomics and Transcriptomics of Cryptosporidium 149
3.6. Improved Insights into Cryptosporidium Using
In Vitro Techniques 158
References 162
Abstract Cryptosporidium is an important genus of parasitic protozoa of

humans and other vertebrates and is a major cause of intestinal
disease globally. Unlike many common causes of infectious enteritis,
This review is dedicated to the memory of our colleague and friend, Huw V. Smith.
* Department of Veterinary Science, The University of Melbourne, Werribee, Victoria, Australia
{
Scottish Parasite Diagnostic Laboratory, Stobhill Hospital, Glasgow, United Kingdom
Advances in Parasitology, Volume 77 #2011 Elsevier Ltd.

141
142 Aaron R. Jex et al.
there are no widely available, effective vaccine or drug-based inter-

vention strategies for Cryptosporidium, and control is focused
mainly on prevention. This approach is particularly deficient for
infections of severely immunocompromised and/or suppressed,
the elderly or malnourished people. However, cryptosporidiosis
also presents a significant burden on immunocompetent individuals,
and can, for example have lasting effects on the physical and mental
development of children infected at an early age. In the last few
decades, our understanding of Cryptosporidium has expanded sig-
nificantly in numerous areas, including the parasite life-cycle, the
processes of excystation, cellular invasion and reproduction, and the
interplay between parasite and host. Nonetheless, despite extensive
research, many aspects of the biology of Cryptosporidium remain
unknown, and treatment and control are challenging. Here, we
review the current state of knowledge of Cryptosporidium, with a
focus on major advances arising from the recently completed
genome sequences of the two species of greatest relevance in
humans, namely Cryptosporidium hominis and Cryptosporidium par-
vum. In addition, we discuss the potential of next-generation
sequencing technologies, new advances in in silico analyses and
progress in in vitro culturing systems to bridge these gaps and to
lead toward effective treatment and control of cryptosporidiosis.
3.1. INTRODUCTION
Cryptosporidium species represent a genus of faecal-orally transmitted
parasitic protozoa that infect the epithelial tissues of the alimentary or
respiratory tract of vertebrates (Fayer, 2004; Xiao et al., 2004). Infection
occurs following the ingestion of viable and resistant oocysts (see Korich
et al., 1990; Peeters et al., 1989), through direct host-to-host contact or via
food or water (Caccio, 2005; Caccio and Pozio, 2006). In humans, cryp-
tosporidial infection may be transmitted via anthroponotic (human-to-
human) or zoonotic (animal-to-human) pathways, depending on the
species of parasite (Xiao et al., 2004). Although cryptosporidial infection
can be asymptomatic (Checkley et al., 1997; Hellard et al., 2000), com-
mon clinical signs of the intestinal disease (cryptosporidiosis) include
diarrhoea, abdominal pain, headache, nausea, vomiting, dehydration
and/or fever (Kosek et al., 2001; Tzipori and Ward, 2002). Cryptosporid-
ium infections are often short-lived (days to weeks: Chappell et al., 1996;
Okhuysen et al., 1999) and eliminated following the stimulation of an
effective host immune response (Riggs, 2002). However, in high-risk
host groups, such as neonatal or young animals, infants, the elderly and
immunocompromised or -suppressed patients, an infection can become
chronic and sometimes fatal in the absence of supportive and
Cryptic Parasite Revealed 143
chemotherapeutic treatments (Amadi et al., 2001; Colford et al., 1996;

Mwachari et al., 1998). Estimating the global disease burden for cryp-
tosporidiosis is challenging due to a lack of detailed epidemiological
data for many countries ( Jex and Gasser, 2010; Lim et al., 2010;
Putignani and Menichella, 2010). In 2004, the World Health Organiza-
tion (Anonymous, 2004a,b) estimated the global impact of all diarrhoeal
diseases to represent approximately 62 million disability adjusted life
years (DALYs; second only to HIV among the infectious diseases).
Cryptosporidium is considered to be amongst the most common causes of
diarrhoea in regions of the world for which substantial epidemiological
data are available (Karanis et al., 2007; Leoni et al., 2006; PHLSSG, 1990);
thus, although its specific contribution to this burden is difficult to esti-
mate, this genus is a major and potentially underestimated contributor
(Guerrant et al., 1999; Ricci et al., 2006).
Because effective chemo- or immunotherapeutics for cryptosporidio-
sis are limited (Armson et al., 2003; Caccio and Pozio, 2006; Zardi et al.,
2005), the control of this infection relies on prevention. Many of these
preventative measures are behavioural and include the adherence to
appropriate hygienic and sanitation practices (e.g. proper disposal of
wastes, frequent hand-washing and suitable water treatment practices;
Anonymous, 2002, 2004a,b; HSE, 2000a,b; Kaye, 2001). Although preven-
tative measures aid in limiting the spread of human cryptosporidiosis,
there is a pressing need for improved chemotherapeutics or prophylactic
vaccines, particularly for use in impoverished nations. In the absence of
an effective anti-Cryptosporidium vaccine, there has been a considerable
focus on developing chemotherapeutic compounds (Armson et al., 2003;
Smith and Corcoran, 2004; Zardi et al., 2005). Specific treatment strategies
seem to be improving, and there are case reports describing effective
reductions in oocyst excretion levels and the alleviation of clinical signs
of cryptosporidiosis in immunocompromised patients upon treatment
with paromomycin and/or azithromycin, following effective highly
active antiretroviral therapy (HAART) intervention (Palmieri et al.,
2005; Zardi et al., 2005). Other evidence also suggests that nitazoxanide
reduces the duration of diarrhoea associated with cryptosporidiosis in
immunocompetent (Rossignol et al., 2001) and malnourished children
(Amadi et al., 2002). Although this latter compound is now licensed for
the treatment of cryptosporidiosis in immunocompetent children in
the USA (Rossignol, 2006), it is not licensed in Europe and is not
widely available in developing countries. As a result, in most situations,
the treatment of cryptosporidiosis relies solely on maintaining the hydra-
tion of the patient and with an expection that the immune response
mounted will eliminate the parasite. Further exploration and develop-
ment of effective anti-cryptosporidial chemotherapeutics and/or vaccines
is urgently needed.
The present chapter reviews key aspects of the biology of the known
species of Cryptosporidium, describes the significance of cryptosporidiosis
in humans and summarizes recent advances in our knowledge of these
parasites. Together with the use of advanced genomic and bioinformatic
technologies, an improved understanding of Cryptosporidium should pro-
vide better insights into the complexities of the interplay between differ-
ent genotypes/species and their hosts, with new prospects for the
development of improved diagnostic tests, anti-cryptosporidial drugs
and vaccines.
3.2. CRYPTOSPORIDIUM SPECIES AND GENOTYPES KNOWN

TO INFECT HUMANS
Currently, based primarily on molecular data, approximately 20 Crypto-

sporidium species and more than 40 unclassified genotypes have been
recorded among all classes of vertebrates (Xiao and Feng, 2008; Xiao
et al., 2004). In humans, the two major Cryptosporidium species recognized
as being associated with infection and disease are Cryptosporidium hominis
and Cryptosporidium parvum (Caccio, 2005; Leoni et al., 2006; Xiao and
Ryan, 2004). C. hominis (Morgan-Ryan et al., 2002) is considered to be
transmitted by anthroponotic pathways only and, with few exceptions
(Giles et al., 2001; Morgan et al., 2000; Smith et al., 2005b), is reported to
be human-specific. In contrast, C. parvum has a broad reported host range
(Fayer et al., 2000; Xiao et al., 2004), including numerous mammalian
species which might act as zoonotic reservoirs (Hunter and Thompson,
2005; Smith and Nichols, 2006; Xiao and Feng, 2008). Based on available
data, there is strong evidence that cattle represent a major source for
zoonotic transmission of C. parvum (see Starkey et al., 2005; Xiao and
Feng, 2008). The potential contribution of other animals as reservoirs of
zoonotic Cryptosporidium is less certain. Considering their large population
numbers in many countries and importance as major livestock animals,
small ruminants (e.g. sheep and goats) appear likely candidates
(Robertson, 2009). However, the extent to which these animals pose a
risk to the public health is not well established, with some research indi-
cating a low-prevalence of C. parvum in sheep (Ryan et al., 2005b) and
others (Alves et al., 2001; Caccio et al., 2000, 2001; Morgan et al., 1998;
Santin et al., 2007) identifying this species with significant frequency.
Molecular methods have provided evidence of C. parvum also in wild
ruminants (Alves et al., 2003; 2006), including deer (Hajdusek et al., 2004;
Ryan et al., 2005a; Siefker et al., 2002), and canids (Giangaspero et al., 2006;
Matsubayashi et al., 2004). Such findings reinforce that further study of the
breadth of the host range for C. parvum is required using molecular tools
( Jex and Gasser, 2009; 2010; Jex et al., 2008). Other species/genotypes
(e.g. C. meleagridis, C. felis, C. canis, C. muris, C. suis and cervine and

monkey genotypes of Cryptosporidium) have been reported to infect
humans, but are much less common (Chalmers et al., 2002; Leoni et al.,
2006; Xiao et al., 2001) and are likely to be of lesser zoonotic significance.
However, the impact of these latter species/genotypes on immunocom-
promised persons, particularly in developing countries, has not been
examined in detail and thus warrants further study.
Illness and oocyst excretion patterns can vary significantly among
infected individuals due to host factors, including immune status
(Goodgame et al., 1993; Lazar and Radulescu, 1989) as well as parasite
factors, such as the origin and age of the oocysts, the species/genotype,
virulence and/or infective dose (Cama et al., 2007; Okhuysen et al., 1999).
Although asymptomatic infections can occur (Biggs et al., 1987; Lacroix
et al., 1987), clinical signs of disease usually commence 112 days after the
ingestion of infective oocysts and usually coincide with the excretion of
oocysts in the faeces ( Jokipii and Jokipii, 1986; Jokipii et al., 1983). How-
ever, oocyst excretion may continue for up to 2 months after clinical signs
disappear ( Jokipii and Jokipii, 1986; Soave and Armstrong, 1986). Con-
versely, intermittent faecal oocyst excretion has been observed in patients
with clinical signs of cryptosporidiosis ( Jokipii and Jokipii, 1986). Infected
individuals often defaecate between 2 and > 20 times a day, producing
watery, light-coloured, stools containing mucus (Casemore, 1987). Illness
usually has a mean duration of approximately 13 weeks, with a range of
144 days (Elsser et al., 1986; Jokipii and Jokipii, 1986). Although chronic
Cryptosporidium infections have been reported to occur in otherwise
healthy humans (Lazzari et al., 1991; Rey et al., 2004), they are usually
eliminated through an effective immune response. In contrast, infections
in immunocompromised patients can develop into chronic disease
(Blanshard et al., 1992; Flanigan et al., 1992; Hayward et al., 1997) and
spread from the intestine to the hepatobiliary and pancreatic ducts, caus-
ing cholangiohepatitis, cholecystitis, choledochitis and/or pancreatitis
(Current and Owen, 1989; Current et al., 1983; Soave and Armstrong,
1986). In immunocompromised or -deficient persons, clinical cryptospo-
ridiosis can be fatal (Soave and Armstrong, 1986), causing intractable
diarrhoea with severe dehydration, malabsorption, malnutrition and
wasting, often in association with infections by other opportunistic patho-
gens (Huh et al., 2008; Scaglia et al., 1994; Soave and Johnson, 1998).
3.3. THE LIFE CYCLE OF C. PARVUM AND C. HOMINIS

Both C. parvum and C. hominis are transmitted via the faecal-oral route
and have a direct life-cycle. In brief, an infective dosage of sporulated
oocysts (containing four naked, sporozoites) is ingested by the host.
Upon encountering specific environmental cues, which, for C. parvum and

C. hominis, appear to include salt (particularly bile salt) concentration, pH
and temperature (Smith et al., 2005a), the oocysts excyst in the small
intestine; this process appears to involve the activation of several serine
proteases (Rosenthal, 1999). Upon excystation, each sporozoite migrates
along the gut epithelium (e.g. microvilli of enterocytes in the small intes-
tine) by gliding motility, powered by an intracellular actinomyosin
motor (Forney et al., 1998; Wetzel et al., 2005). Cell-surface glycoproteins
on the sporozoite, including P23 and the 15-kDa glycoprotein (GP15),
appear to be involved in this process (Boulter-Bitzer et al., 2007). Upon
finding a suitable site for invasion, the sporozoite forms an attachment
zone between its apical complex and the host cell membrane (Valigurova
et al., 2008). Various proteins associated with the apical complex have
been identified to be involved in this process (Boulter-Bitzer et al., 2007;
Smith et al., 2005a), including the 40 and 900 kDa cell-surface glycopro-
teins (Barnes et al., 1998; Bonnin et al., 2001; Cevallos et al., 2000; Strong
et al., 2000) as well as the thombrospondin-related attachment proteins
(TRAPs; Lally et al., 1992; Spano et al., 1998).
Upon attachment, the host cell membrane envelopes the sporozoite,
encasing it in an epicellular parasitophorous vacuolar membrane (PVM);
(Valigurova et al., 2008). The stimuli that initiate this process and the
molecular mechanisms by which it proceeds are not well understood,
but various cysteine proteases appear to play a critical role (Rosenthal,
1999). Within the PVM, the trophozoite transforms and then undergoes
asexual reproduction (called merogony or schizogony; longitudinal
binary fission) to produce type 1-meronts (schizonts). Each of these type
1-meronts contains 16 merozoites, which are released from the PVM. Each
merozoite infects a new enterocyte (reforming the PVM), then replicates
and develops into a new type 1-meront to repeat the cycle, or enters into
the reproductive phase to replicate and develop into type 2-meronts, each
of which contains four merozoites. After infecting a host cell, each type
2-merozoite initiates the sexual reproductive cycle (gametogony) and
develops either into a microgametocyte (containing 1216 microgametes)
or a macrogametocyte (maturing into a macrogamete). Microgametes
(male) are released and fertilize macrogametes (female) to form zygotes,
which ultimately develop into oocysts. In another asexual reproductive
phase (called sporogony), the oocyst sporulates to produce, internally,
four naked sporozoites. Two types of oocyst are produced; thin-walled
oocysts remain in the alimentary tract and have the ability to sustain an
autoinfection, whereas thick-walled oocysts are passed in the faeces. The
thin-walled oocysts and/or type 1-meronts are of particular relevance in
immunocompromised, -deficient or -suppressed individuals, as they can
perpetuate chronic cryptosporidiosis (Arenas-Pinto et al., 2003; Certad
et al., 2005; Chhin et al., 2006; Lebbad et al., 2001) due to autoinfection
within the gut (Sun and Teichberg, 1988). Unlike the motility, attachment
and invasion phases, very little is known about the molecules involved in
the endogenous phases of the Cryptosporidium life-cycle because of practi-
cal limitations in isolating these stages from infected hosts and culturing
developmental stages in vitro.
3.4. CRYPTOSPORIDIOSIS: PATHOGENESIS AND IMMUNITY

In humans, Cryptosporidium most directly and severely affects the small
intestine (Xiao et al., 2004), although, in rare instances, and usually in
relation to immunocompromised individuals, gastric cryptosporidiosis
has been reported (Lumadue et al., 1998; Ventura et al., 1997). In such
infections, combined endoscopic/histopathological examination of the
gastric epithelial tissues reveals hyperplasia of the epithelial cells, inflam-
mation in the lamina propria and non-specific lesions and oedema (Rivasi
et al., 1999). In contrast, Cryptosporidium infection in the intestine is rela-
tively well characterized. In humans, such infections are initiated when
activated zoites attach to the vicinal enterocytes and endogenous forms
spread to the enterocytes of both the villi and crypts (Current et al., 1983).
The infective dose, extent of spread, the sites involved and the immune
response induced appear to be involved in determining whether an
infection is clinical or subclinical (Tzipori and Ward, 2002).
The site of infection within the intestine can be associated with the
severity of clinical signs. Infection of the proximal small intestine is mainly
related to symptoms of severe and watery diarrhoea, whereas infections
confined to the distal ileum and/or the large bowel tend to be associated
with intermittent diarrhoea or can be asymptomatic (Tzipori and Ward,
2002). The endogenous stages of Cryptosporidium disrupt the microvillus
border, leading to a loss of mature enterocytes, shortening and/or fusion
of the villi and lengthening of the crypts due to increased cell division (see
Inman and Takeuchi, 1979; Tzipori et al., 1981). These changes lead to the
loss of membrane-bound digestive enzymes and diminish the effective
surface area of the intestine, leading to the decreased uptake of fluids,
electrolytes and nutrients (Adams et al., 1994; Argenzio et al., 1990;
Griffiths et al., 1994). Inflammation also occurs to a significant degree
and is linked to the hosts immune response against the parasite.
Although less commonly reported than gastrointestinal infections,
extra-gastrointestinal cryptosporidiosis does occur, and can affect both
immunocompetent (Westrope and Acharya, 2001) and, with greater fre-
quency, immunocompromised, -suppressed or -deficient individuals
(Bonacini, 1992; Dowsett et al., 1988; Vakil et al., 1996). Such infections
can be categorized as pulmonary (Clavel et al., 1996) as well as biliary or
pancreatic (Forbes et al., 1993; Goodwin, 1991; Vakil et al., 1996), and often
appear to result from the systemic spread of an initial infection from the
gastrointestinal tract (e.g. Edwards et al., 1990). Ultrasonic examinations
of patients with biliary (Dolmatch et al., 1987; Teixidor et al., 1991) or
pancreatic (Cappell and Hassan, 1993) infections indicate a notable dila-
tion of the gall bladder and/or bile or pancreatic ducts, respectively, and
an increase in pericholecystic fluid and thickening of the epithelial layers.
The pathophysiology of diarrhoea is multifactorial and linked, to a
significant extent, to a loss of the intestinal surface area due to carpeting
of the luminal surface by parasites, as well as villus fusion and atrophy
(Buret et al., 2003; Inman and Takeuchi, 1979) and enterocytic destruction,
following merogony and gametogony. Enterotoxins produced by the par-
asite have been proposed as playing a possible role in diarrhoeal illness
(Guarino et al., 1994, 1995). The recent detection of a neurokinin-1 receptor
antagonist, dubbed Substance P, produced by the parasite supports this
hypothesis (Robinson et al., 2003; Sonea et al., 2002). The presence of this
substance during an infection with Cryptosporidium appears to correlate
with the severity of diarrhoea (Robinson et al., 2003; Sonea et al., 2002).
Experimental data have indicated that this receptor antagonist is directly
linked to glucose malabsorption and the increased secretion of chloride
ions in the host intestinal tract (Hernandez et al., 2007), which has been
demonstrated to be an important factor in the inducement of diarrhoeal
illness (Kapel et al., 1997). The induction of diarrhoea may also relate to the
attachment of C. parvum sporozoites to the apex of enterocytes. This
complex process appears to involve multiple parasite ligands and host
receptors, and induces a reorganisation of the host cell actin cytoskeleton
(Elliott et al., 2001; Smith et al., 2005a; Tzipori and Ward, 2002). Such large
changes to the cellular skeletal structure likely impact on host cell function.
Indeed, significant negative effects on the integrity and function of enter-
ocytes linked to cryptosporidial infections have been reported (Argenzio
et al., 1990), and may, at least in part, be linked to the activation/perturba-
tion of apoptotic pathways in these cells (Chen et al., 1998, 1999).
A reduced permeability of the intestinal epithelia, due to a Cryptosporid-
ium-induced disruption, may also play a role in the pathogenesis of diar-
rhoea. Such changes in permeability have been linked to various intestinal
disorders, including inflammatory bowel disease (IBD), Crohns disease
and ulcerative colitis (Fiocchi, 1998). With respect to cryptosporidiosis,
reduced permeability of the intestinal barrier is proposed to relate partly to
the disruption of zonula-occludens (ZO)-1, a 220-kDa cytoperipheral pro-
tein, which acts as a physical bridge between tight junction occludin and
cytoskeletal F-actin (Balda and Anderson, 1993; Fanning et al., 1998). This
hypothesis is supported by observations of the affects of Cryptosporidium
andersoni on in vitro cultures of human and bovine intestinal epithelial cells
in which significant disruptions of the tight junctions and apoptosis of the
enterocytes were both noted (Buret et al., 2003). However, when apical
epidermal growth factor (which inhibits the induction of apoptosis and

the disruption of ZO-1, but does not kill the parasite) was added to the
culture, these effects were reduced (Buret et al., 2003).
During the endogenous phases of the Cryptosporidium life-cycle,
regardless of the organ being infected (e.g. stomach, intestine, lung,
liver or pancreas), a common histopathological manifestation is hyperpla-
sia of the affected epithelial tissues (e.g. Blagburn et al., 1987; Rivasi et al.,
1999). This thickening of the epithelia of infected organs might be, in part,
the result of scarring (or fibrosis) following the infection of the host cells
and the effect of Cryptosporidium-induced host cell mitosis (Hatkin et al.,
1990; Masuno et al., 2006). Studies of the nuclear genome of C. parvum and
C. hominis conducted to date (Abrahamsen et al., 2004; Xu et al., 2004)
have indicated that these parasites have greatly reduced metabolic path-
ways and are heavily dependent on the host for resources (e.g. nucleo-
tides and amino acids) that they cannot produce themselves. One
hypothesis is that the parasites role in the induction of hyperplasia and
mitotic division is to satisfy these resource requirements by eliciting
increased production in the host cell. However, the mechanism by
which this may occur and the parasite derived signals and/or molecules
involved are not yet known. In addition to the major role that the parasite
plays in the pathogenesis of disease, various host-related factors, includ-
ing inflammatory and immunological responses are also of critical impor-
tance (Savioli et al., 2006).
Although much of the knowledge of the immune responses to cryptos-
poridial infections relates to studies of mice, key insights have been made
also through investigations of humans and cattle (Deng et al., 2004; Gomez
Morales and Pozio, 2002; Riggs, 2002) as well as in vitro explorations of
cultured monolayers of mammalian cell lines infected with C. parvum
(Current and Haynes, 1984). A recent review of the literature ( Jex et al.,
2011) reveals that the immunological control of cryptosporidial infection is
associated with both innate and adaptive host responses. Epithelial cells
and NK cells appear to be central to innate immunity, whereas adaptive
immunity required for elimination of the parasite is coordinated by CD4
T cells. IFN-g expressed by both T cells and NK cells could be central to
immunity early in infection (reviewed by Jex et al., 2011).
3.5. GENOMICS AND TRANSCRIPTOMICS OF

CRYPTOSPORIDIUM
A major advance in our understanding of the molecular biology of Crypto-
sporidium has arisen from the sequencing of the genomes of C. parvum and
C. hominis (Abrahamsen et al., 2004; Xu et al., 2004). The genomes of these
closely related species are similar in size ( 9.19.2 Mbp), content ( 4000
genes among 8 chromosomes) and sequence ( 9697% identity;

Abrahamsen et al., 2004; Xu et al., 2004). These genomes are substantially
smaller than those reported for other apicomplexans, such as Eimeria tenella
( 60 Mbp; Shirley, 1994, 2000) and Plasmodium falciparum ( 23 Mbp;
Gardner et al., 2002). This size difference is consistent with Cryptosporidium
having fewer genes (e.g. 4000 vs. 5300 for Plasmodium), fewer introns
and shorter non-coding regions (Abrahamsen et al., 2004; Xu et al., 2004).
The small genome of C. parvum and C. hominis reveals a reduced
complement of genes associated with both anabolic and catabolic phases
of metabolism. Energy generation (i.e. ATP) appears to be dependent
exclusively upon the degradation of simple sugars via anaerobic glycoly-
sis, with no evidence of a mitochondrial genome or many of the nuclear
genes associated with the Krebs cycle or electron transport chain
(Abrahamsen et al., 2004; Xu et al., 2004). In addition, there is no evidence
for the presence of genes associated with energy production via the diges-
tion of fatty acids or proteins (Abrahamsen et al., 2004; Xu et al., 2004). The
substantial reduction in these energy production pathways significantly
reduces the ability of both C. parvum and C. hominis to synthesize a variety
of essential building blocks (e.g. some nucleotides and amino acids). This
deficiency is further accentuated by an absence of the genes encoding the
enzymes involved in the urea and nitrogen cycles (i.e. for amino acid
synthesis) and the shikimate pathway (Abrahamsen et al., 2004; Xu et al.,
2004). The absence and/or substantial depauperacy of enzymes associated
with these major metabolic pathways indicates that Cryptosporidium spe-
cies are highly reliant on the host cell for building blocks (Abrahamsen
et al., 2004; Xu et al., 2004). Consistent with this hypothesis is the finding
that the nuclear genomes of both C. parvum and C. hominis contain numer-
ous amino acid transporter genes, which are hypothesized to be involved
in amino acid salvaging (Abrahamsen et al., 2004; Xu et al., 2004). Com-
plementing these apparent salvage pathways are a variety of enzymes
necessary for the conversion of the amino acids and nucleotides (e.g.
pyrimidines to purines and purines to pyrimidines; Abrahamsen et al.,
2004; Striepen et al., 2004; Xu et al., 2004). Interestingly, there is no evidence
of redundancy in these pathways, such that, for example, a single enzyme
(inosine 50 monophosphate dehydrogenase) appears to be responsible for
the conversion of adenosine monophosphate (AMP) to guanosine mono-
phosphate (GMP). Such metabolic and catabolic bottle-necks are likely to
represent significant targets for the development of new and specific anti-
cryptosporidial drugs (e.g. Chaudhary and Roos, 2005).
In silico drug target prediction, docking and screening represent signif-
icant areas of interest in current research of a range of neglected infectious
diseases (Fig. 3.1). An emerging array of online resources, such as the
Braunschweig Enzyme Database (BRENDA: Chang et al., 2009),
CHEMBL (accessible via http://www.ebi.ac.uk/chembl/) and the
Sample
collection
In vitro
screening
Culture
purification
NGS
Clinical
Bioinformatics trials
In silico Structural
BLAST docking modelling
homology
Essentiality
Lethality
Model
organisms
Chemical
inhibitor
databases
FIGURE 3.1 An approach to the in silico prediction of novel drug targets and drugs. The
diagram outlines the collection of parasite material or production with culturing (in vitro
or in a surrogate host), followed by next-generation sequencing (NGS). Following
sequencing, bioinformatic analyses allow the rapid assembly and annotation of data.
BLAST homology comparisons of sequence data with those from model organisms (such
as Drosophila melanogaster, Saccharomyces cerevisiae, Xenopus ranitans, and/or
Caenorhabditis elegansclockwise) allow the prediction of essential genes linked to
lethal phenotypes. Peptides encoded by these genes can be screened in silico for
potential inhibitors (drugs) in curated chemical databases (e.g. CHEMBL, BRENDA, TDR
targets) that bind to them. Structural modelling of the predicted drug targets, supported
by crystal structures, and subsequent in silico docking experiments can assist in the
prediction of compounds and their analogues. Compounds designed can then be tested
in vitro and in vivo for safety and efficacy.
CHEMBL-Neglected Tropical Disease databases (accessible via http://

www.ebi.ac.uk/chemblntd) as well as the Tropical Disease Research
(TDR) targets database (accessible via http://www.tdrtargets.org/;
Aguero et al., 2008) provide a tremendous amount of information to
facilitate such research. A study by Crowther et al. (2010) represents a
recent example of the application of the in silico prediction of drug targets
for a range of key parasitic protists, including Leishmania major, Trypano-
soma cruzi and P. falciparum. In this study, peptide sequences inferred from
genomic data for each species were assessed for their suitability as poten-
tial drug targets using a variety of priority-weighted selection criteria,
including essentiality (based on lethal RNA interference phenotypes),
their absence from the host organism and the availability of a predicted
protein and/or a solved (crystallized) structure. Using this approach,
these authors (Crowther et al., 2010) inferred 31 high priority druggable
molecules for P. falciparum. Seventeen of these molecules have homolo-
gues in C. parvum and C. hominis (see Table 3.1). Conspicuous among these
homologues is a dihydrofolate reductase (DHFR). Dihydrofolate reduc-
tase, which plays a critical role in purine synthesis, is a known target of
pyrimethamines (Crowther et al., 2010; Sirichaiwat et al., 2004), which
have been used historically for the clinical treatment of malaria and toxo-
plasmosis (Amin, 1992; Roberts et al., 1998; Rosenblatt, 1992; Watkins,
1995). Early in vitro trials of these compounds as anti-cryptosporidial
medications were unsuccessful (Lemeteil et al., 1993), suggesting that
pyrimethamines were not effective against Cryptosporidium infection.
However, such a conclusion could be premature. A more recent study,
using recombinant Cryptosporidium DHFR expressed in yeast (Saccharomy-
ces cerevisiae), identified several pyrimethamine structural analogues that
had significant inhibitory effect (Lau et al., 2001). Although the rapid
emergence in Plasmodium of resistance against pyrimethamine (Mita,
2010; Mita et al., 2009; Talisuna et al., 2007) may limit its appeal as a
compound to treat cryptosporidiosis, the blind prediction of dhfr as a
potential drug target (Crowther et al., 2010) supports the assertion that in
silico methodologies do yield genuine targets worthy of pursuit. In addi-
tion, the contrasting results reported by Lemeteil et al. (1993) and Lau et al.
(2001) highlight that the difference between a successful drug and a failed
compound can be small, as subtle changes to the molecular structure of a
drug candidate can alter or maximize bioavailability, efficacy and/or
safety (Lipinski, 2001; Lipinski et al., 1997; van de Waterbeemd and
Gifford, 2003).
For novel drug targets whose protein structures have been solved or
may be confidently inferred from close homologues (de Beer et al., 2009;
Wieman et al., 2004), advances in the predictive modelling of molecular
interactions can assist significantly in the design and subsequent synthe-
sis of structural analogues of a particular compound as candidate
TABLE 3.1 Cryptosporidium hominis and C. parvum genes (identification ID) inferred to encode peptides with high sequence homology to
prioritized drug targets in Plasmodium falciparum predicted using the TDR targets database (www.tdrtargets.org; Crowther et al., 2010)
C. hominis BLASTp homology C. parvum BLASTp homology P. falciparum

gene ID (e-value) gene ID (e-value) gene ID Gene description
118 120
Chro.10305 1.00 10 cgd1_2700 1.00 10 PF10_0150 Methionine aminopeptidase, putative
Chro.10107 2.00 10 41 cgd1_870 2.00 10 41 PF11_0164 Peptidyl-prolyl cistrans isomerase
Chro.70577 3.00 10 44 cgd7_5170 1.00 10 44 PF11_0282 Deoxyuridine 50 -triphosphate
nucleotidohydrolase, putative
Chro.30215 1.00 10 146 cgd3_40 1.00 10 112 PF11_0377-b Casein kinase 1, PfCK1
Chro.30013 1.00 10 112 cgd3_1810 1.00 10 146
Chro.60090 1.00 10 118 cgd6_690 1.00 10 121 PF14_0053 Ribonucleotide reductase small subunit
Chro.70303 1.00 10 159 cgd7_2670 1.00 10 159 PF14_0142 Serine/threonine protein phosphatase
Chro.20263 1.00 10 131 cgd2_2480 1.00 10 131 PF14_0327 Methionine aminopeptidase, type II, putative
Chro.10337 3.00 10 65 cgd1_3040 4.00 10 64 PF14_0378 Triosephosphate isomerase
Chro.10335 1.00 10 119 cgd1_3020 1.00 10 120 PF14_0425 Fructose-bisphosphate aldolase
Chro.40038 2.00 10 89 cgd4_240 1.00 10 106 PFC0525c Glycogen synthase kinase 3
Chro.50038 4.00 10 56 cgd2_4120 1.00 10 60 PFC0975c Peptidyl-prolyl cistrans isomerase
Chro.20441 1.00 10 60 cgd5_3350 2.00 10 55
Chro.40506 1.00 10 113 cgd4_4460 1.00 10 113 PFD0830w Bifunctional dihydrofolate reductase-
thymidylate synthase
Chro.30017 1.00 10 147 cgd3_80 1.00 10 147 PFE1050w Adenosylhomocysteinase (S-adenosyl-L-
homocystein e hydrolase)
Chro.60435 8.00 10 71 cgd6_3800 5.00 10 69 PFF1155w Hexokinase
Chro.70113 1.00 10 145 cgd7_910 1.00 10 149 PFI1105w Phosphoglycerate kinase
Chro.60524 8.00 10 71 cgd6_4570 9.00 10 71 PFI1110w Glutamine synthetase, putative
Chro.20464 1.00 10 159 cgd2_4320 1.00 10 159 PFI1170c Thioredoxin reductase
inhibitors (Fig. 3.1). Thus, armed with a suite of novel drug targets, for
which structural models are available, and having identified classes of
inhibitors based on information in current literature or databases (i.e.
BRENDA or CHEMBL), in silico prediction and docking can assist in the
prioritisation of structural analogues for synthesis, subsequent safety and
efficacy testing in vitro (in cultured cells or pathogens) and in vivo (in
animals). Some examples of open-source tools available in silico docking
include MolDock (Thomsen and Christensen, 2006) and Lidaeus (Taylor
et al., 2008) as well as Patchdock and Symmdock (Schneidman-Duhovny
et al., 2005). Such an integrated approach to drug design and discovery
provides substantial scope to improve the efficiency and reduce the costs
associated with the research and development of new drugs (e.g.
Campbell et al., 2010; Taylor et al., 2008; Wu et al., 2003; Yang et al., 2007).
In the present review, we examined the druggability of the genomes of
Cryptosporidium spp. and predicted, on a global scale, selective targets for
known chemicals. We selected the sequences for all annotated coding
genes common to C. parvum and C. hominis (accessible via http://www.
CryptoDB.org), conducted homology searches (BLASTx) against the
S. cerevisiae (yeast) genome and discovered > 1400 homologues for Cryp-
tosporidium genes, 536 of which are associated with lethal phenotypes
based on gene perturbation experiments (see http://www.yeastgenome.
org). Recently, Doyle et al. (2010) demonstrated that functional genomic
data for a range of eukaryotic model organisms could be used to assist in
the prediction of the essentiality of conserved genes that represented
prime targets for anti-parasitic drugs. Thus, genes in Cryptosporidium
that are linked to homologues that display lethal phenotypes in S. cerevi-
siae, if their function/s is/are perturbed, could represent candidate tar-
gets for anti-cryptosporidial drugs. The collation of such genes and the
corresponding interrogation of publicly available databases for known
protein inhibitors (e.g. available via the BRENDA database; Schomburg
et al., 2002) revealed 313 molecules in Cryptosporidium that may be inhib-
ited by chemical compounds that are known to have activity against
homologues in other organisms and/or in vitro. Conspicuous among
these proteins are 61 GTPases, all of which contain a domain consistent
with a protein-synthesizing GTPase (EC:3.6.5.3) and 21 of which also
contain domains consistent with heterotrimeric G-protein (EC:3.6.5.1),
small monomeric (EC:3.6.5.2) and signal-recognition-particle (EC:6.5.3.4)
GTPases. In recent years, GTPases have received significant attention as
druggable targets for anti-cancer therapies (Saxena et al., 2008; Thomas
et al., 2008; Williams et al., 2008). Although, we are not aware of this
specific family of GTPases having been suggested or tested as druggable
targets in parasites, several GTPases have been proposed as playing an
important functional role in key biological pathways in parasitic proto-
zoa, including T. cruzi (Barrias et al., 2010; Yokoyama et al., 2008),
Toxoplasma gondii (Caldas et al., 2009), Entamoeba histolytica (Welter and

Temesvari, 2009) and P. falciparum (Zhou et al., 2009). Furthermore, recent
studies have indicated that compounds that actively inhibit these targets
may indeed represent new treatments for trypanosomiasis (Barrias et al.,
2010) and toxoplasmosis (Caldas et al., 2009), highlighting the potential of
GTPase inhibitors to be used specifically against cryptosporidiosis.
The link between GTPases and anti-cancer therapies has undoubtedly
contributed to an abundance of information for compounds that inhibit/
bind these enzymes. For example, we find that there are currently 37, 9, 7
and 4 known inhibitors of protein-synthesizing, signal-recognition-
particle, small monomeric and heterotrimeric G-protein GTPases in the
BRENDA database (Schomburg et al., 2002). Strikingly, many of these
inhibitors represent a variety of common, commercially available,
antibiotics, including chloramphenicol, fusidic acid, streptogramin and
tetracyclin. Notable among these inhibitors are mycins, including dihy-
drostreptomycin, hygromycin, neomycin, pulvomycin, ribostamycin,
sparsomycin and viomycin, all of which are listed as having known
activity against protein-synthesizing GTPases. The finding of a significant
number of enzymes inhibited/bound by mycins is interesting. Various
mycins have been investigated for activity against Cryptosporidium,
including spiramycin (Portnoy et al., 1984; Saez-Llorens et al., 1989),
salinomycin (Lindsay et al., 1987), clarithromycin (Cama et al., 1994),
roxithromycin (Sprinz et al., 1998; Uip et al., 1998) and, most frequently,
paromomycin and azrithromycin (Palmieri et al., 2005; Zardi et al., 2005).
However, none of them are predicted to have activity against GTPases. Of
the mycins that have been evaluated for efficacy against Cryptosporidium,
only paromomycin is listed in the BRENDA database as having activity
against an essential Cryptosporidium gene-product. Furthermore, based
on available data, only four enzymes are inhibited/bound by this com-
pound; they are the PAP1P poly A polymerase (encoded by cgd4_930),
the Po1 beta superfamily nucleotidyltransferase (cgd2_2730), a putative
RNA binding protein (cgd4_3410) and a conserved hypothetical protein
of unknown function (cgd3_2820) which is linked to ribonuclease P based
on gene ontology (see http://www.Cryptodb.org). Of these molecules, only
PAP1P is predicted to be encoded by an essential gene (i.e. has a lethal
phenotype in S. cerevisiae). One hypothesis could be that the variable efficacy
of paromomycin in field studies (Hewitt et al., 2000; Zardi et al., 2005) is
the result of differences in temporal expression of this gene throughout the
life-cycle or linked to microenvironmental factors within the lumen of
the intestine. Although some aminoglycosides (e.g. mycins) have been
reported to be toxic to mammalian cells (Guthrie, 2008; Martinez-Salgado
et al., 2007; Rizzi and Hirose, 2007), this can be reduced through careful
management (Murakami et al., 2008; Pannu and Nadim, 2008) and/or
improved formulations that incorporate, for example, low-molecular
weight proteins (Tugcu et al., 2006) and/or other compounds ( Jeyanthi and
Subramanian, 2009; Nagai and Takano, 2010). Moreover, with current tech-
nologies, it should be possible to synthesize analogues with optimum
bioavailability and parasite-specificity (supported by chemical, structural
and in silico docking studies) but negligible toxicity to host tissues. Given
the essentiality of GTPases in Cryptosporidium, there appears to be
considerable scope for the design of relatively specific, safe and effective
anti-cryptosporidial compounds.
An enhanced understanding of the biology of known species and
genotypes of Cryptosporidium should support the prediction of a larger
and/or better panel of potential drug targets. The genomic sequencing of
species of Cryptosporidium other than C. parvum and C. hominis, combined
with transcriptomic and proteomic studies, is greatly needed to improve
our understanding of these important parasites. In addition to directly
revealing potential drug targets, such studies could explore, for example,
the genomic characters linked to parasite virulence and pathogenicity
as well as host-specificity and infection-site. Furthermore, investigating
and understanding the temporal and spatial changes in transcription
and expression in these parasites, as they progress through their life-
cycle, is of paramount importance. Specific alterations associated with
excystation, cellular invasion, development into and existence as the tro-
phozoite, reproduction (asexual and sexual) as well as development into
type-1 or -2 merozoites and thin- or thick-walled oocysts are particularly
pertinent. To this end, the sequencing and draft assembly of the genome of
a distinct species of Cryptosporidium, such as C. muris, which infects the
stomach of mice (but not humans; Xiao et al., 2004), appears to be nearing
completion (accessible http://www.ncbi.nlm.nih.gov; genome sequenc-
ing accession AAZY02000000). This sequence, along with the C. parvum
and C. hominis genomes, will provide significant, new insights into the
molecular biology of Cryptosporidium, is likely to assist in elucidating the
molecular basis of host- and site-specificity of the parasites and provide a
wealth of new genetic markers for the development of molecular-diagnos-
tic tools. The sequencing of a range of species and genotypes of Cryptospo-
ridium would greatly assist in both fundamental and applied areas. In
particular, C. meleagridis, which is the only species of Cryptosporidium
known to infect hosts of multiple taxonomic classes (i.e. birds and mam-
mals; Xiao et al., 2004) and displays a significant plasticity in infection-site
(i.e. respiratory tract in birds and intestinal tract in humans; Xiao et al.,
2004) would be an interesting candidate. In addition, the exploration of
genomic variation within species (e.g. C. parvum and C. hominis) would
also be of significant interest. For example, a recent review of the global
variation in a key population marker, the 60 kDa glycoprotein gene
( gp60), has revealed that, despite the substantial sequence variation
recorded for this locus, there are five sequence types (three representing
C. parvum and two representing C. hominis) that account for approximately

70% of all reports associated with human infections ( Jex and Gasser, 2010).
A link between genotypic identity based on gp60 sequence and that based
on the complete sequence of the nuclear genome has not been explored.
However, there is some evidence of a relationship between identity based
on gp60 sequence and the clinical signs associated with infection in some
humans (Cama et al., 2007; 2008). Large-scale re-sequencing of the gen-
omes of these and other genetic types of C. parvum and C. hominis would
allow the testing of the hypothesis that gp60 sequence type does correlate
with overall genomic sequence, which would indicate that, despite the
richness and diversity of genetic types of Cryptosporidium reported to date,
a small number of sub-specific genotypes are linked to the vast majority
of human infections. Insights into the genetics governing the mechanisms
(e.g. virulence, infectivity and pathogenicity) that might give rise to such
an association would likely have major relevance toward developing new
strategies to prevent and control Cryptosporidium and cryptosporidiosis.
A major limitation to genomic and genetic research of Cryptosporidium
has been access to sufficient quantities of material for next-generation
sequencing (NGS). For genomic sequencing, it may be possible to over-
come this limitation, to an extent, through the use of whole genomic
amplification (WGA) systems (Pinard et al., 2006; Sorensen et al., 2007),
which allow the synthesis of microgram quantities of total genomic DNA
from minute quantities of starting material (e.g. nanograms). These sys-
tems are particularly attractive for re-sequencing projects, wherein the
overall structure of the genome is largely established (e.g. for C. parvum
and, to a lesser extent, C. hominis; see Abrahamsen et al., 2004; Xu et al.,
2004). Although, like any enzymatic amplification, WGA approaches
have the potential to introduce artefacts into the genomic DNA, studies
directly comparing the sequencing of WGA-amplified and non-ampli-
fied templates have not detected substantial errors (e.g. Pinard et al.,
2006; Sorensen et al., 2007), particularly when approaches relying on
multiple displacement amplification (e.g. using y29 DNA polyermase)
are employed (see Burtt, 2011). Although WGA can assist in genomic
research, which can take advantage of DNA isolated from purified
oocysts, the challenges associated with isolating RNA needed for the
study of transcription are not so readily overcome. Although some tran-
scriptomic data are available for oocysts (Ortega-Pierres et al., 2009), little
is known about transcription occurring in other life-cycle stages (e.g.
trophozoites, merozoites and gametocytes) or linked to important aspects
of the parasite biology, including invasion, nutrient uptake and reproduc-
tion/development. The acquisition of sufficient amounts of pure parasite
material for transcriptomic and genomic studies has been the major
obstacle preventing such research. Because the purification of endoge-
nous stages of these parasites from infected animals is challenging, the
development of in vitro cultivation technologies for any species of

Cryptosporidium would represent a major advance.
3.6. IMPROVED INSIGHTS INTO CRYPTOSPORIDIUM USING

IN VITRO TECHNIQUES
Although some Cryptosporidium species can be maintained in experimen-

tal animals, this approach does not allow for the isolation or analysis of the
intracellular parasite life-cycle stages. In addition, due to the broad genetic
richness and diversity of cryptosporidia infective to humans ( Jex and
Gasser, 2010; Leoni et al., 2006; Xiao et al., 2004), the maintenance of
experimental infections as reference lines for each known variant is
impractical and costly. The establishment of a diverse range of Cryptospo-
ridium isolates in in vitro culture would greatly aid a number of areas of
research of these important parasites. For example, in vitro culturing has
led to the first examination of the genes expressed and/or transcribed
during infection of the host cell ( Jakobi and Petry, 2006) and in response to
host defences (Zaalouk et al., 2004). Improved culturing techniques may
also enable the investigation of changes in transcription during intracellu-
lar replication and development, and/or changes in response to external
stimuli (e.g. host molecules, immune cells and/or drugs) under well-
controlled conditions. In any practical sense, such information cannot be
obtained using experimental infections in animals.
The culturing of Cryptosporidium in vitro has been a challenging pros-
pect and the subject of substantial research. Current and Long (1983) were
the first to complete the Cryptosporidium life-cycle in vitro and used
oocysts from humans and calves to infect chicken embryos (chorioallan-
toic membrane). These authors reported the successful completion of the
parasite life-cycle and normal epicellular development. However, oocyst
yields were low, the endogenous stages of the parasite life-cycle were
difficult to isolate, and the results could not be reproduced in subsequent
investigations (Arrowood, 2002; Hijjawi, 2010). Numerous studies have
explored the use of various cell lines for the cultivation of C. parvum from
sporozoites (reviewed by Arrowood, 2002). These lines include human
rectal tumour (HRT), human foetal lung (HFL), primary chicken (PCK),
porcine (PK-10), baby hamster (BHK), Madin-Darby bovine (MDBK) and
Madin-Darby canine (MDCK) kidney as well as HT29.74 human colon
adenocarcinoma, RL95-2 human endometrial carcinoma and Caco-
2 human colon adenocarcinoma cells. A major deficiency of many of
these cell lines is that the life-cycle of Cryptosporidium does not complete
or accurately reflect that in the host animal, and the yield of oocysts is
usually low (Arrowood, 2002). More recent publications have reported
the successful in vitro cultivation of C. parvum in human ileocaecal
adenocarcinoma 8 (HCT-8; Hijjawi et al., 2001) and VELI (rabbit chondro-

cyte; Lacharme et al., 2004) cell lines, resulting in the production of
infective oocysts. In particular, the HCT-8 cell line is gaining usage
( Jakobi and Petry, 2006; Sifuentes and Di Giovanni, 2007; Wu et al.,
2009). A recent study (Woods and Upton, 2007) has reported that oocyst
yields from culture in HCT-8 cells can be enhanced further using serum-
free media, with MDCK (Sigma) and PC-1, UltraCHO, UltraCulture and
UltraMDCK (BioWhittaker) being most successful. Despite the success of
some previous studies, the isolation of purified parasites from cells,
particularly specific endogenous stages remains a challenge.
Cell-free cultures have been evaluated as an alternative approach to
the culturing of Cryptosporidium, greatly facilitating the isolation of para-
site stages for subsequent experimentation. One of the first attempts at
culturing C. parvum in cell-free medium (Hijjawi et al., 2004) utilized
RPMI-1640 (Sigma-Aldrich) containing coagulated new born calf serum
(NBCS) inoculated with oocysts. Using this approach, the authors
reported the completion of the parasite life-cycle in vitro, resulting in the
production of new oocysts. Excitingly, the cultures were maintained
successfully for 2 months (after which the experiment was terminated),
with the parasites seemingly self-propagating (Hijjawi et al., 2004). The
promise of these results, however, were somewhat blunted by
subsequent, unsuccessful attempts to replicate the initial findings (e.g.
Girouard et al., 2006; Karanis et al., 2008). A publication (Woods and
Upton, 2007) has suggested that some of photomicrographs taken of the
developing parasitic stages reported in the original study (Hijjawi et al.,
2004) appear to have been budding yeasts and/or fungi rather than stages
of Cryptosporidium, and the authors questioned the possibility of culturing
an obligate, intracellular parasite in vitro without host cells. Queries
about the identity of the trophozoites and merozoites described by
Hijjawi et al. (2004) and also observed by Karanis et al. (2008) were also
raised in a recent study (Petry et al., 2009). In the latter study (Petry et al.,
2009), it was suggested that the stages observed by Hijjawi et al. (2004)
were aged sporozoites that had become misshapen as a result of nutrient
deficiencies due to the lack of a host-cell in cell-free cultures. Unfortu-
nately, it is not possible to confirm the identity of any of the stages from
the original study (Hijjawi et al., 2004), as the results were not verified by
detailed ultrastructural examinations (including the use of nucleic acid or
specific antibody probes).
However, a subsequent study (Zhang et al., 2009), attempting to repli-
cate the cell-free culturing of C. parvum, appears to have provided the first
independent support of the original findings of Hijjawi et al. (2004). In this
study, three distinct Cryptosporidium-specific monoclonal antibodies were
used to successfully immunolabel various morphologically distinct cell
types detected in host-cell free culture and interpreted them to represent
distinct phases of the parasites life-cycle. Zhang et al. (2009) also used
quantitative real-time PCR targeting the glyceraldehyde 3-phosphate
dehydrogenase gene employing primers reported to be specific to
Cryptosporidium and measured a fivefold increase in genomic DNA over
the course of the cell-free culturing. On the basis of these results, the
authors concluded that C. parvum could indeed be cultured in vitro in
cell-free media, albeit with modest yields. The validity of this finding
appears to be further supported by a recent report describing the success-
ful culturing of C. hominis in cell-free medium (Hijjawi et al., 2010). Here
also, fluorescent labelling was utilized to support the morphological
identification of each Cryptosporidium life-cycle stage and quantitative
PCR was used to estimate the production of new parasite cells. As
observed by Zhang et al. (2009), there was a five- to sixfold increase
in DNA in a cell-free culture. In order to control for the potential contami-
nation of their cell-cultures with non-cryptosporidial organisms, as sug-
gested previously by Woods and Upton (2007), cell-free cultures were
inoculated with heat-deactivated sporozoites also, with no measureable
evidence of cellular proliferation, indicating that the culturing of
Cryptosporidium cells could be achieved in a cell-free medium.
The recent advances in in vitro culturing are intriguing and provide
the prospect that problems associated with the inadequate supply of
Cryptosporidium stages for molecular, immunological or biochemical
investigations might be overcome in the future. Certainly, if the genome
of Cryptosporidium were to remain entirely stable in vitro, the culturing of
large quantities of parasite material would be a substantial step forward
for exploring the genomics of this genus. This has been demonstrated to
be a challenge for the culturing of other parasites, such as some genetic
types of Giardia (Upcroft and Upcroft, 1994). Despite this, the successful
in vitro culturing of Cryptosporidium would allow the exploration of iso-
lates displaying a variety of phenotypes and could facilitate the genera-
tion of transgenic lines, as has be achieved for other apicomplexans,
including for species of Plasmodium (Fairhurst, 2007; Kocken et al.,
2009), and/or well-controlled gene knockout experiments. Further opti-
mization of the proliferation of parasite material through culture and/or
the development of an approach to purify specific stages in vitro or in vivo
represent some of the last remaining obstacles to broad-scale transcrip-
tomic and proteomic investigation of these parasites. Such research may
prove a boon to our understanding of this group. However, extensive
experimentation would be required to characterize, and, if possible,
account for the impacts of culturing on the parasite (e.g. development,
transcription and expression) and to determine the extent to which iso-
lates of Cryptosporidium cultured in vitro reflect their natural phenotype
in vivo. Such factors should be considered carefully when interpreting
transcriptional or proteomic data derived from cultured parasites.
3.7. CONCLUDING REMARKS
Cryptosporidium derives its name from the small mysterious (hidden or

cryptic) spores within its resilient and microscopic oocysts and was
dubbed so by Edmund Tyzzer in the early years of the last century. In
the many years since, and despite substantial research, this has proven a
particularly apt moniker, as the species comprising this genus of infec-
tious protozoa have remained, in many respects, cryptic. Expansion of
our knowledge of these organisms has progressed with increasing rapid-
ity, from the first description in the early 1900s, to the detailed observation
and description of the endogenous stages of the life-cycle, leading, in the
early 1980s to the first real recognition of cryptosporidia as parasites
rather than commensalists. The onset of the global HIV pandemic led to
the first considerations of these organisms as troublesome opportunistic
pathogens of immunocompromised or -suppressed people. However, the
massive waterborne outbreak of cryptosporidiosis in Milwaukee in the
earlier 1990s and the discovery that the resistant, transmissive stage of
these parasites are not killed by common water treatment practices led to
the revelation of the enormity of the adverse impact that Cryptosporidium/
cryptosporidiosis has on global health.
The introduction and application of molecular tools (including the PCR)
further accelerated the expansion of the collective knowledge of these organ-
isms, leading to new insights into the mechanisms associated with infection
and the disease. This information provided us with an improved under-
standing of the zoonotic potential, systematics and molecular detection
of Cryptosporidium, leading to a range of new species descriptions and the
determination that the genus is made up of a number of, in many cases, host-
adapted lineages with varying levels of host-specificity and significantly
different levels of relevance to human health. These advances have led to
substantial expansions in the availability of a range of molecular-diagnostics
tools to detect, characterize and identify these parasites in clinical and
environmental samples. Within the last decade, we have seen the sequenc-
ing of the complete genomes of two key members of this genus (C. parvum
and C. hominis) and anticipate the completion of the sequencing of another,
systematically and biologically distinct, species (e. g., C. muris) in the very
near future. Interestingly, through all of this, Cryptosporidium species have
remained enigmatic, their basic biology has remained controversial, and
perhaps most significantly, our ability to actively kill these parasites in
infected individuals, through drug or vaccine, has remained noticeably
absent. Recent advances in drug development have heralded new promise
for treating and controlling these pathogens, which cause major human
suffering and disease. However, the treatments adopted to date have had
limited or ephemeral efficacy or are sometimes toxic. The magic bullet has
not been found.
The purpose of the present chapter was to review the present state of
our knowledge in the mechanisms behind the biology of these parasites
wherein novel forms of treatment may be found. Our knowledge to date
reveals parasites that are highly dependent upon specific cues within the
host and a cascade of peptides and chemical reactions to successfully
conduct the exquisite symphony of their life-cycle. The genomic
sequences completed in the early 2000s revealed a genus of parasite
with a highly streamlined metabolism, minimal modes of energy produc-
tion, and a complex, but critically important, armada of transport pro-
teins, allowing it to salvage essential nutrients and building blocks from
its host. Much of what was once hidden is now exposed. Herein we see,
for example, a variety of molecular mechanisms that are predicted to be
essential for the parasites survival and could potentially be disrupted by
a range of common, commercially available, antimicrobial compounds.
These compounds are as yet untried, but the tools with which to test them
are readily available. What is more, new and exciting advances in NGS
technologies provide real prospects to delve deeper beneath the surface of
these cryptic parasites to better understand their biology and further
exploit their weaknesses. The advent of these platforms coupled with
advances in in vitro culturing provide the means of exploring gene func-
tion and critical changes in the cellular biology of the parasite at key
moments in its life-cycle, as well as, the prospects to identify, test and
optimize novel targets for drug development. Broad-scale genomic, tran-
scriptomic and proteomic research of Cryptosporidium coupled to the
ability to test the findings of this research in vitro and in vivo provides
the means to ultimately know this enemy and the potential to finally
develop efficacious therapies against it.
ACKNOWLEDGEMENTS
Support through the Melbourne Water Corporation, the National Health and Medical
Research Council (Career Development Award Level 1 Industry FellowshipARJ) and the
Australian Research Council (LP0989137) is gratefully acknowledged.
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CHAPTER 4
Assessment and Monitoring of
Onchocerciasis in Latin America
Mario A. Rodrguez-Perez,*, Thomas R. Unnasch,
and Olga Real-Najarro,

4.1.1. The infection with the filarial nematode
Onchocerca volvulus 178
4.1.2. Distribution of onchocerciasis in
Latin America 178
4.1.3. The new world onchocerciasis vectors 182
4.2. The Pathology and Clinical Manifestations
Produced by O. volvulus Infection in Latin America 185
4.2.1. Onchocercomata (nodules) 185
4.2.2. Dermatological lesions (dermal pathology) 186
4.2.3. Ocular lesions (ocular pathology) 187
4.3. Genetic Variation of O. volvulus and the
Simulium Vector 187
4.4. The Control of Onchocerciasis (with Emphasis on
Programmes in Latin America) 189
4.4.1. Control through nodulectomy 193
4.4.2. Control through vector control 194
4.4.3. Control through chemotherapy 194
4.5. Advantages and Disadvantages of Treatment with
Ivermectin 195
4.6. Development of Other New Drugs 197
* Centro de Biotecnologa Genomica, Instituto Politecnico Nacional, Ciudad Reynosa, Tamaulipas, Mexico
{
Department of Global Health, College of Public Health, University of South Florida, Tampa, Florida, USA
{
Facultad de Medicina, Universidad Autonoma de Nuevo Leon, Monterrey, Nuevo Leon, Mexico
}
Centro de Estudios Asiaticos, Universidad Autonoma de Nuevo Leon, Monterrey, Nuevo Leon, Mexico

175
176 Mario A. Rodrguez-Perez et al.
4.7. Monitoring and Evaluation of Control

of Onchocerciasis 199
4.7.1. Parasitological diagnosis of O. volvulus
infection 200
4.7.2. Immunological and molecular diagnosis of
O. volvulus infection 200
4.7.3. Development of immunological tests for the
diagnosis of onchocerciasis 201
4.7.4. Development of DNA probes for the
diagnosis of onchocerciasis 206
4.8. Entomological Parameters for Monitoring the
Transmission in Latin America (with Emphasis in
Areas where Transmission has been Interrupted) 209
4.8.1. The intensive epidemiological surveillance
programme in Latin America during the post
treatment era 212
4.9. Future Developments 213
4.9.1. Basic research 213
4.9.2. Applied research 214
References 215
Abstract Onchocerciasis has historically been one of the leading causes of

infectious blindness worldwide. It is endemic to tropical regions
both in Africa and Latin America and in the Yemen. In Latin America,
it is found in 13 foci located in 6 different countries. The epidemio-
logically most important focus of onchocerciasis in the Americas is
located in a region spanning the border between Guatemala and
Mexico. However, the Amazonian focus straddling the border of
Venezuela and Brazil is larger in overall area because the Yanomami
populations are scattered over a very large geographical region.
Onchocerciasis is caused by infection with the filarial parasite
Onchocerca volvulus. The infection is spread through the bites of
an insect vector, black flies of the genus Simulium. In Africa, the
major vectors are members of the S. damnosum complex, while
numerous species serve as vectors of the parasite in Latin America.
Latin America has had a long history of attempts to control
onchocerciasis, stretching back almost 100 years. The earliest pro-
grammes used a strategy of surgical removal of the adult parasites
from affected individuals. However, because many of the adult
parasites lodge in undetectable and inaccessible areas of the
body, the overall effect of this strategy on the prevalence of
infection was relatively minor. In 1988, a new drug, ivermectin,
was introduced that effectively killed the larval stage (microfilaria)
of the parasite in infected humans. As the microfilaria is both the
stage that is transmitted by the vector fly and the cause of most of
the pathologies associated with the infection, ivermectin opened
Onchocerciasis in Latin America 177
up a new strategy for the control of onchocerciasis. Concurrent

with the use of ivermectin for the treatment of onchocerciasis, a
number of sensitive new diagnostic tools were developed (both
serological and nucleic acid based) that provided the efficiency,
sensitivity and specificity necessary to monitor the decline and
eventual elimination of onchocerciasis as a result of successful
control. As a result of these advances, a strategy for the elimination
of onchocerciasis was developed, based upon mass distribution of
ivermectin to afflicted communities for periods lasting long
enough to ensure that the parasite population was placed on the
road to local elimination. This strategy has been applied for the
past decade to the foci in Latin America by a programme overseen
by the Onchocerciasis Elimination Program for the Americas
(OEPA). The efforts spearheaded by OEPA have been very success-
ful, eliminating ocular disease caused by O. volvulus, and eliminat-
ing and interrupting transmission of the parasite in 8 of the 13 foci in
the region.
As onchocerciasis approaches elimination in Latin America,
several questions still need to be addressed. These include defining
an acceptable upper limit for transmission in areas in which trans-
mission is thought to have been suppressed (e.g. what is the
maximum value for the upper bound of the 95% confidence inter-
val for transmission rates in areas where transmission is no longer
detectable), how to develop strategies for conducting surveillance
for recrudescence of infection in areas in which transmission is
thought to be interrupted and how to address the problem in areas
where the mass distribution of ivermectin seems to be unable to
completely eliminate the infection.
4.1. INTRODUCTION
During the 49th Directing Council of the Pan American Health Organiza-
tion (PAHO), 2009, the Member States adopted the resolution Elimina-
tion of Neglected Diseases and Other Infections Related to Poverty. The
resolution expresses the commitment of PAHOs Member States to elimi-
nate or reduce the neglected diseases and other infections related to
poverty for which tools exist, to levels so that these diseases will no longer
be considered as public health problems in Latin America and the
Caribbean by 2015.
One of the commitments of the State members is to provide support for
the promotion of research and scientific development related to new and
improved tools, strategies, technologies and methods to prevent and control
neglected diseases. This review is based on an evidence report that was
requested by the Communicable Disease Research/Health Surveillance
and Disease Prevention and Control/PAHO to support research action for

neglected infectious diseases related to poverty in Latin America.
4.1.1. The infection with the filarial nematode

Onchocerca volvulus
Onchocerciasis is a disease produced by the infection with the parasitic
nematode O. volvulus and transmitted through the bite of the black flies of
the genus Simulium. Infection may result in symptoms varying from
dermatological changes to blindness. Onchocerciasis is generally a cumu-
lative infection, where the severity of clinical features depends on the
length of exposure to Simulium fly bites and the density of mf in the skin.
Onchocerciasis is a chronic disease causing ever-increasing disability to
those afflicted. It has serious socio-economic consequences for the most
heavily affected communities in endemic areas.
Onchocerciasis is one of the major causes of blindness in the world.
Historically, it ranks among the top five causes of blindness and has
ranked second as a cause of infectious blindness, after trachoma
(Etyaale, 2008; Hotez et al., 2008). For this reason, onchocerciasis has
been the subject of much research and many control efforts under the
umbrella of the World Health Organization (WHO) and other interna-
tional organizations. Official estimates suggest that about 123 million
individuals are at risk of infection, approximately 17.7 million are
infected, of whom some 270,000 are blind; in addition, a further half a
million are severely visually disabled (WHO, 1995). These figures are
almost certainly an underestimate and do not fully reflect the importance
of the disease and its implications. Thus, despite resounding control
achievements in some areas of West Africa and the Americas, human
infection with O. volvulus still constitutes an important public health
problem, with a recent estimate indicating that 37 million people remain
infected, mostly in Africa (APOC, 2005; Basanez et al., 2006).
4.1.2. Distribution of onchocerciasis in Latin America1

Onchocerciasis tends to affect people living in poor rural areas. By far, the
highest prevalence is in Africa, and its distribution extends over broadly
continuous regions in sub-Saharan Africa, particularly West Africa. It is
also found in more isolated foci in East and South-Central Africa and in
one country of the Middle East (Yemen). There are also relatively small
and scattered foci in six countries of Central (Mexico and Guatemala) and
South America (Ecuador, Colombia, Venezuela and Brazil). Older
1
WER 2011 was available during the final production stage of this book chapter. Therefore, figures are based
on data published until 2010.
estimates by WHO (1995) reported that in the Americas, over 140,000

individuals were infected and 4.7 million were at risk. Current estimates
in the Americas by Onchocerciasis Elimination Program for the Americas
(OEPA) (Sauerbrey, 2008; WER, 2009, 2010, 2011) suggest that as a result of
effective control measures over the past decade and a half, the population
at risk has been reduced by approximately 90% from that previously
reported (from 4.7 million to 470,222 inhabitants). In terms of endemicity
levels, the Amazonian focus comprises a large number of scattered com-
munities with more than half of them being hyperendemic, that is, an
onchocerciasis prevalence equal or greater than 60% (Botto et al., 2005).
In Guatemala, active foci are concentrated on the Western slopes of the
volcanic mountain range. There is also a focus in the North-West (Hue-
huetenango) near to the border with Mexico. However, migrant workers
who cross the MexicoGuatemala border may spread the disease to other
areas. Almost 450,000 people lived in the foci in Guatemala of whom 62,961
were infected and 600 individuals were blind as a result of onchocerciasis
(WHO, 1995). The central focus of Guatemala currently accounts for 22% of
the population at risk (124,498 individuals). As this region contains many
migrant workers, human migration may be a significant factor to consider
when targeting at risk populations for treatment. The largest and most
intensively infected area in Guatemala is the Central focus, comprising the
departments of Chimaltenango, Solola and Suchitepequez (WER, 2011).
The population at risk in the other focus of Huehuetenango in Guatemala
is 30,239 individuals (Sauerbrey, 2008; WER, 2009, 2010, 2011). In Vene-
zuela, three main foci of the disease have been detected: one in the North-
Central region, other in the North-East, and another in the South region. In
the Northern Venezuelan regions, the number of infected people identified
between 1959 and 1970 was above 41,000. There were just 3000 infected
individuals reported in the South region (part of the Amazonian focus),
although it is believed that these numbers are underestimated (WHO,
1995). The current estimation by OEPA (Sauerbrey, 2008; WER, 2009,
2010, 2011) of the population at risk in Venezuela is as follows: North-
Central focus: 14,385; North-East focus: 93,239; and South focus: 9,168
inhabitants (Table 4.1; Fig. 4.1). However, it is in the South focus where
most of the hyperendemic communities of the Amazonian focus are found.
In Mexico, there were originally estimated 630,000 people at risk, with
286,000 inhabitants residing in the endemic areas of which 25,645 were
infected (Martn-Tellaeche et al., 1998; WHO, 1991, 1995). Fortunately,
although 112 cases of blindness as a result of infection with O. volvulus
were detected in 1989, no new cases of blindness have been detected
recently (Martn-Tellaeche et al., 1998; Rodrguez-Perez et al., 2008b).
Onchocerciasis-endemic areas in Mexico are located in the Southern states
of Oaxaca and Chiapas. The most extended endemic area is found in
Southern Chiapas, which is contiguous to the Huehuetenango focus of
TABLE 4.1 Number of endemic communities for onchocerciasis and current endemic population in Latin Americaa
Mexico Guatemala Brazil Colombia Venezuela Ecuador All countries
Number of endemic foci 3 4 1 1 3 1 13

Number of hyperendemic communities 39 42 7 0 41 42 171
Number of hypoendemic communities 411 461 6 0 279 54 1211
Number of mesoendemic communities 220 15 9 1 200 23 468
Endemic population at risk 158,943 154,737 12,521 1366 116,792 25,863 470,222
Endemic population no longer at riskb 7,125 74,798 81,923
Endemic population UESPTc 44,919 30,239 0 1366 14,385 25,863 116,772
a
Onchocerciasis Elimination Program for the Americas. (2010). URL: http:///www.oepa.net. XX Inter- American Conference on Onchocerciasis. (2010). Guatemala, Guatemala,
10-12th November. (WER, 2009, 2010, 2011). All data were provided by the Onchocerciasis Programmes (Mexico, Guatemala, Ecuador, Colombia, Venezuela, and Brazil) in
coordination with the Onchocerciasis Elimination Program for the Americas (OEPA).
b
Population in the foci where transmission has been eliminated.
c
UESPT = under epidemiological surveillance post-treatment.
Transmission status of focus
Eliminated
Interrupted
Suppressed
N
Ongoing
Oaxaca Northern Chiapas
44,919 0 North-Central
14,385
Huehuetenango North-east
Mexico 30,239 93,239
Guatemala South
9168
Santa Rosa Venezuela
0 Amazonans
Southern Chiapas 12,521
114,024
Escuintla-Guatemala
0 Colombia
Central
124,498
Ecuador Brazil
Lpez de Micay
1366
Esmeraldas
25,863 1000 km
FIGURE 4.1 Distribution of human onchocerciasis endemic areas and current status of transmission in Latin America. The dark- and medium-
grey areas represent the foci where transmission has been eliminated and interrupted, respectively, and where mass treatments with
ivermectin were halted; light grey areas depict the foci where transmission has been suppressed or is ongoing with mass ivermectin
distribution. The current population at risk is also presented per each focus in the inset boxes. Map credit: designed by Javier Alfonso Garza-
Hernandez, Genomics Center and Biotechnology-Mexico. All data were provided by the Onchocerciasis Programmes (Mexico, Guatemala,
Ecuador, Colombia, Venezuela, and Brazil) in coordination with the Onchocerciasis Elimination Program for the Americas (OEPA).
Guatemala. These endemic areas and the principal endemic area around
Lake Atitlan in Guatemala make up the MexicoGuatemala zone. The
endemic areas in Oaxaca belong to 30 municipalities in 4 districts: Ixtlan,
Villa Alta, Tuxpepec and Cuicatlan, in a 4250 km2 area, representing 4.2% of
the total land area in Oaxaca. Eleven localities are mesoendemic and 87
hypoendemic (Table 4.1). OEPA (Sauerbrey, 2008; WER, 2009, 2010, 2011)
estimates that 44,919 individuals are at risk in the Oaxaca focus. In Chiapas
State, the affected localities belong to 22 municipalities, in an area of
12,640 km2 where there were over 18,000 registered cases. In 1990, the
population at risk in this area was estimated in 183,643 inhabitants but
current estimates by OEPA (Sauerbrey, 2008; WER, 2009, 2010, 2011) indi-
cate an at-risk population of 114,024 for the Southern focus (Table 4.1;
Fig. 4.1). The lack of accurate epidemiological data had hampered the
stratification of the onchocerciasis endemicity in Chiapas, but it has been
estimated that the majority of the onchocerciasis-endemic communities in
Chiapas are either mesoendemic or hypoendemic, with rather few hyper-
endemic localities (WHO, 1991). Current estimates of endemic levels in the
whole Chiapas area indicated 39 hyperendemic, 209 mesoendemic and 324
hypoendemic localities (Sauerbrey, 2008; WER, 2009, 2010, 2011) (Table 4.1).
The other three affected countries in South America contain minor
foci: Ecuador, Brazil and Colombia combined accounted for just over
7,000 registered total cases. The major onchocerciasis foci in these
countries are located in the North-Western coastal province of Esmeral-
das in Ecuador, the Northern part of Amazon state of Brazil, which
borders Venezuela (and is part of the Amazonas focus), and the Lopez
de Micay area on the Pacific coast of Colombia (WHO, 1995). Current
estimations by OEPA (Sauerbrey, 2008) indicate that Colombia has only
1,366 individuals at risk, followed by the Amazon state of Brazil with
12,521 individuals. In the Ecuador, the Esmeraldas, and the main satellite
focus, constituted by Rio Santiago, Cayapas and Onzole, have 19,735
inhabitants at risk, whereas Rio Canande and the other satellite foci
have 6,128 inhabitants at risk (Table 4.1; Fig. 4.1).
4.1.3. The new world onchocerciasis vectors

The insect vectors of O. volvulus have a cosmopolitan distribution. The
African and the Southern Arabian Peninsula vectors of O. volvulus are
members of the S. damnosum sensu lato (s.l.) complex (subcomplexes:
S. damnosum, S. sanctipauli, S. squamosum), the S. neavei complex and
S. albivirgulatum. The American vectors of O. volvulus are members of
the S. ochraceum, S. metallicum, S. exiguum, S. oyapockense S. guianense
complexes, and other species including S. callidum, S. incrustatum,
S. quadrivittatum and S. limbatum (WHO, 1995) (Fig. 4.2). S. ochraceum s.l.
vector capacity is an intrinsic function of the size of the community
Vector species (Simulium spp.)
S. ochraceum s.l.
S. exiguum s.l.
S. metallicum s.l.
S. quadrivittatum
S. guianense s.l.
N
S. oyapockense s.l.
S. incrustatum
Northern Chiapas
North-Central
Oaxaca Huehuetenango North-East
Mexico
South
Guatemala Santa Rosa Amazonas
Venezuela
Southern Chiapas
Central Lpez de Micay Colombia

EscuintlaGuatemala
Ecuador Brazil
Esmeraldas
1000 km
FIGURE 4.2 Distribution of human onchocerciasis endemic areas and vector species in Latin America discussed in the text. Map credit:
designed by Javier Alfonso Garza-Hernandez, Genomics Center and Biotechnology-Mexico. All data were provided by the Onchocerciasis
Programmes (Mexico, Guatemala, Ecuador, Colombia, Venezuela, and Brazil) in coordination with the Onchocerciasis Elimination Program for
the Americas (OEPA).
microfilarial load, and its biting rates on humans are sufficiently large to
overcome a generally poor vector competence. S. ochraceum s.l. is respon-
sible for transmitting over 50% of all cases of onchocerciasis reported in
the region. Hence S. ochraceum s.l. has been subject of much research
(Rodrguez-Perez et al., 2006c). S. exiguum s.l. in Ecuador is a highly
efficient vector even when compared to the most efficient vector known,
the savannah-dwelling sibling species of the S. damnosum s.l. complex in
West Africa (Collins et al., 1995; Shelley and Arzube, 1985; Wetten et al.,
2007), S. guianense s.l. in the Amazonian focus is also important in terms of
its vector competence and the intense transmission it is associated with
(Basanez et al., 1988, 1995; Takaoka et al., 1984).
The simuliid females of most species require a blood meal from a
warm-blooded vertebrate in order to complete their egg development.
This provides an opportunity to encounter skin or blood-dwelling verte-
brate pathogens. This process has to be repeated for each gonotrophic
cycle. In addition, females and males of Simulium flies feed on plant juices
in order to obtain energy for dispersion and host-seeking activities.
Simulium flies lay their eggs on trailing vegetation in fast-flowing water.
Each egg batch may contain 100900 eggs and are laid with communal
oviposition under pheromonal control (McCall, 1995; McCall and
Cameron, 1995; McCall et al., 1994, 1997a,b). Thus, oviposition attractants
and pheromones have potential as surveillance-trap baits, for black flies
and other vectors. Ovitraps represent one possible method of routinely
sampling a vector population safely, obviating the need for human-bait
catches, and the efficiency of such traps may be improved significantly
through the use of oviposition attractants, such as pheromone-baited light
traps and/or pheromone-baited bucket traps (Moore and Noblet, 1974;
Reiter et al., 1991; Rodrguez-Perez et al., 2003).
Under field conditions, only a small percentage of Simulium popula-
tions are infected with O. volvulus. In Mexico and Guatemala, the figure
averages ca. 1% for S. ochraceum s.l. (Rodrguez-Perez and Reyes-
Villanueva, 1994; Shelley, 1988), S. guianense s.l. and S. oyapockense s.l. in
Venezuela (Basanez et al., 1988; Botto et al., 2007), S. exiguum s.l. and
S. quadrivittatum in Ecuador (Vieira et al., 2005) and the vectors in Brazil
(Marchon-Silva et al., 2007). S. ochraceum s.l. biting activity exhibits
marked seasonality following a defined wet and dry season cycle. As a
consequence, transmission of O. volvulus is also seasonal and is greatest
when parity, infection and infectivity rates are at their highest in the biting
population (Grillet et al., 2001; Porter and Collins, 1988; Rodrguez-Perez
and Reyes-Villanueva, 1994; Vieira et al., 2005). However, it is not known
if these seasonal patterns will remain stable over time, or if other factors
such as global warming have any effect on the ecology of the Simulium
Onchocerca system. For example, in a study performed in Southern
Chiapas, Mexico, during 19971999, the proportions of parous and infected
flies were higher during MayJuly than during NovemberFebruary sea-

sons (Rodrguez-Perez et al., 2007). This finding was contrary to the predic-
tion that the older flies in the host-seeking population would enhance
transmission during the latter part of the season, a prediction that arose as
a result of studies conducted in Guatemala in the late 1980s/early 1990s
(Rodrguez-Perez et al., 2007). Similarly, the data of the study in Southern
Chiapas indicate that the parous rates seem to have increased over the past
few years. In the early 1990s, parous rates ranged between 25% (168/674;
95% CI 2228%) in September 1990 and 54% (452/838; 95% CI 5057%)
in June 1991 before widespread ivermectin administration (Basanez
et al., 1998). In contrast, after 1013 treatment rounds with ivermectin, the
parity rates appeared to increase, ranging from 63% (12,431/19,784; 95%
CI 6263%) in November 1998February 1999 to 86% (10,345/12,097; 95%
CI 8586%) in MayJuly 1997 (Rodrguez-Perez et al., 2007). This finding
suggests that a reduced microfilarial reservoir in the human population
may result in increased vector survival. This issue has been investigated
through fly-feeding experiments (Basanez et al., 1996) but not in natural
populations. In addition, no correlation between our field observations and
fly-feeding experimental data has yet been proved.
The intrinsic capacity of Simulium black flies to transmit O. volvulus
(vector competence) varies from species to species. For a recent review on
all the aspects of the OnchocercaSimulium interactions, see Basanez et al.
(2009). Some Simulium vectors (like S. ochraceum s.l.) possess a well-developed
buccopharyngeal armature. These teeth can lacerate the microfilariae (mf)
as they pass towards the midgut, rendering them incapable of further
development, thus reducing the vector competence of these species. Thus,
the ability to sustain local transmission by simuliids with buccopharyngeal
armature generally depends on high human-biting rates, and the availability
of infected humans with high microfilarial densities in their skin, thereby
providing the vector with a large number of mf per blood meal. S. ochraceum
s.l., is thus probably able to maintain transmission in mesoendemic to hyper-
endemic onchocerciasis, probably because of its high intrinsic susceptibility,
high vector densities and strong anthropophagy (Basanez et al., 2002, 2009).
4.2. THE PATHOLOGY AND CLINICAL MANIFESTATIONS

PRODUCED BY O. VOLVULUS INFECTION
IN LATIN AMERICA
4.2.1. Onchocercomata (nodules)

Adult worms are encapsulated in nodules which are often located over
bony prominences on the head, scapular girdle, ribs, pelvic girdle,
trochanters, knees and ankles. The nodules can be removed surgically,
and this process (nodulectomy) historically formed the core strategy of
control programmes in Mexico and Guatemala (Rodrguez-Perez and

Rivas-Alcala, 1991).
However, the efficacy of the long-term nodulectomy campaigns on the
dynamics of parasite populations, and the development of morbidity has
scarcely been evaluated. A study conducted to relate the estimated num-
bers of adult worms in the body to the total load of mf in the skin (Schulz-
Key, 1990) has suggested that considerable numbers of female worms are
hidden in deep-lying, non-visible nodules. These deep nodules may be
sufficient to maintain a large population of mf in the skin and eyes (Duke,
1990), and the removal of visible nodules may not therefore reduce the mf
load significantly enough to improve the individuals condition or affect
the intensity of transmission of the parasite.
4.2.2. Dermatological lesions (dermal pathology)

Dermal pathology (onchocercal dermatitis) is almost exclusively asso-
ciated with the presence of skin mf. The severity of this pathology will
depend mainly on the hosts response to infection, and possibly on differ-
ences in exposure levels between individuals. Early skin changes include
papular eruptions reflecting intra-epithelial abscesses, while in Mexico
and Guatemala, an acute inflammatory reaction of the face known as
erisipelas de la costa has been observed (Robles, 1919). After the passage of
time, skin chronic lesions give the appearance of premature ageing asso-
ciated with the following features: lichenoid change, hyperkeratosis and
exaggerated wrinkling of the skin; atrophy of the epidermis, with loose,
redundant, thin and shiny skin and skin depigmentation. A grading
system for recording the cutaneous changes of onchocerciasis in Africa
was devised by Murdoch et al. (1993). As the pathology and cutaneous
lesions caused by O. volvulus infection are difficult to distinguish from
those caused by other skin diseases, studies on the natural history of skin
lesions as well as the impact of control programmes on them have
been difficult and the results variable. In Africa, skin lesions caused by
O. volvulus infection are common. A rapid method of epidemiological
assessment of infection intensity has been widely used which is based on
the assessment of the number of individuals within a community who
have pre-tibial skin depigmentation (Carme et al., 1993; Edungbola et al.,
1987). However, the value of this method as a rapid assessment tool in
Latin America seems to be less useful due to similar skin changes that are
induced by treponemal infection (Guderian et al., 1991).
Typical onchocercal dermatitis is generalized and symmetrical.
However, some patients develop an unusual localized and asymmetrical
dermatitis termed sowda. Sowda is usually limited to one limb, but it
can affect more than one limb or even the trunk. The involved skin is
itchy, swollen, darkened and covered with scaling papules, and regional
lymphadenopathy is present. Sowda is found in Yemenites, and less

commonly in patients from Sudan, West Africa, Guatemala and Ecuador.
Schwartz and co-workers (Schwartz et al., 1983) documented the
occurrence of sowda type in two Guatemalan patients.
4.2.3. Ocular lesions (ocular pathology)

The most serious clinical feature of onchocerciasis is visual damage. It has
been reported that onchocerciasis has caused blindness in 270,000 people
through 1995, with almost 99% of these cases in Africa. However, APOC
epidemiological mapping studies indicate that some 37 million people
were infected in 1995. This estimate corresponds to an estimated 1.99
million disability-adjusted life years lost as a result of onchocerciasis
through 1995 (Remme et al., 2006). As the death of mf in eyes causes
ocular pathology, ocular lesions are usually seen in individuals with
moderate or heavy mf loads. Little et al. (2004a) found a direct relation-
ship between microfilarial load and the incidence of blindness in the
Onchocerciasis Control Programme (OCP) area. It was also shown that
mortality of the human host has a direct association with increasing mf
burden, but not with blindness (Little et al., 2004b). Microfilarial death,
resulting in the release of components of endosymbiotic (Wolbachia) bac-
teria, and the resulting induction of proinflammatory cytokines are con-
tributory factors to onchocerciasis-related pathology (Saint Andre et al.,
2002). In early and light infections, a few mf may invade the cornea and
may cause an acute inflammatory exudate surrounding dead and dying
mf, producing snowflake opacities that resolve without sequelae. How-
ever, many individual acute reactions to mf in the cornea may produce
chronic inflammation with fibrovascular pannus formation that starts at
the inferior or medial and lateral margins of the cornea and slowly
becomes confluent (sclerosing keratitis). This may lead to irreversible
visual damage and blindness if it encroaches on the visual axis (Bradley
et al., 2005). Complications of anterior uveal tract involvement (e.g. papil-
lary deformity) are particularly prominent relative to other causes of
impaired vision in Central America (Woodruff et al., 1966). Atrophy of
the optic nerve can produce constriction of the visual fields, keyhole
vision or even total loss of light perception.
4.3. GENETIC VARIATION OF O. VOLVULUS AND THE

SIMULIUM VECTOR
O. volvulus is widespread, occupying much of the African tropical belt,

and a variety of evidence suggests that population genetic variation exists
among the different foci of onchocerciasis worldwide. For example,
in West Africa, two strains of the parasite inhabiting the savannah and
rainforest bioclimes of the continent seem to exist, which may be differ-
entiated by biochemical and molecular methods (Bradley and Unnasch,
1996; Unnasch and Williams, 2000). These population variations appear to
affect the biology of the parasites as well. For example, the forest and
savannah-dwelling parasites appear to develop with different efficiencies
in the S. damnosum s.l. sibling species that serve as vectors for O. volvulus
in West Africa (Duke et al., 1966). For example, under experimental
conditions, the O. volvulus isolates from the rainforest develop poorly or
not at all in the savannah-dwelling vectors (S. sirbanum and S. damnosum
sensu stricto (WHO, 1995). Similarly, De Leon and Duke (1966) observed
that S. ochraceum, S. metallicum and S. callidum ingested 1020 times more
mf of Guatemalan O. volvulus than either West African strain, and that
poor parasite development took place in Guatemalan flies fed on African
O. volvulus carriers. A recent review on the OnchocercaSimulium interac-
tions by Basanez et al. (2009) concluded that the parasite and the vector
exert reciprocal effects on each others survival at various stages of the
parasites life cycle within the black fly, and these may have adapted to
minimize deleterious effects on fitness and maximize transmission. How-
ever, the role that such adaptation may play in transmission of the
parasite in natural conditions remains unclear. For example, Toe et al.
(1997b) demonstrated that in naturally infected flies collected from areas
in West Africa where the savannah and forest strains were co-endemic,
there was no preferential transmission of the forest strain and savannah
strain of O. volvulus by the different sibling species of S. damnosum s.l.
This result suggests that although the different strains of the parasite
may have adapted to maximize their developmental efficiency in the
black fly sibling species with which they are sympatric, the effect of this
advantage on parasite transmission under natural conditions may be
insignificant.
It is also evident that the strains of the parasite differ in their pathoge-
nicity and distribution in the human body (Duke et al., 1966). The West
African savannah form is especially associated with severe blinding
lesions in the anterior segment of the eye (WHO, 1995). Prost (1980)
found that severe complications such as dermal, ocular and lymphatic
clinical manifestations were more common in savannah villages than in
forest communities. Anderson et al. (1974) showed that concentrations of
dermal mf were 50% higher in the savannah than in the forest, but
conversely, that the number of palpable nodules in the forest was 50%
higher than in the savannah.
Molecular methods have been developed to distinguish between the
West African rain forest and the savannah strains of the parasite, which
are based upon an analysis of a highly polymorphic sequence family
(Unnasch and Williams, 2000; Zimmerman et al., 1993). A similar analysis
was applied to isolates from Latin America, which suggested that the
isolates from Latin America were indistinguishable from the West African
Savannah strain (Zimmerman et al., 1994a,b). This finding supported the
hypothesis that onchocerciasis was introduced to the New World as a
result of the trans-Atlantic slave trade in the eighteenth and nineteenth
centuries (Mouchet and Teppaz, 1993).
Together, the population-based studies have suggested that the para-
site has evolved somewhat independently in each of its isolated foci,
adapting to maximize its ability to survive in both the endemic Simulium
species and the local human population. However, the exact genetic
mechanisms underlying these adaptations remain to be elucidated.
4.4. THE CONTROL OF ONCHOCERCIASIS (WITH EMPHASIS

ON PROGRAMMES IN LATIN AMERICA)
As the Simulium flys breeding requirements restrict them to rivers with
well-oxygenated waters, the transmission of infection usually takes place
near the black fly breeding sites. Because onchocerciasis causes blindness
and is associated with rivers, it is commonly known as river-blindness.
In Africa, several programmes to control onchocerciasis exist. The OCP in
West Africa was the largest and longest running of these programmes,
maintaining active control operations from 1975 to 2002. Older pro-
grammes in Africa and Latin America were those implemented in
Kenya during 1946; Abuja, Nigeria during 1955; and Mexico during
1932 (Davies, 1994). The control strategy of the OCP was based on weekly
aerial larviciding of all breeding sites in the rivers of the programme area.
The OCP virtually eliminated transmission throughout 90% of its original
area (Ba et al., 1987; Hougard et al., 2001). In the area where OCP has
interrupted transmission, only 37 of over 8000 children examined har-
boured onchocercal infection. It was predicted that 652 children would
normally have become infected in such a sample if there had not been
larvicidal treatments of the OCP (Ba et al., 1987).
The advent of the microfilaricidal drug ivermectin resulted in the
establishment of two new programmes: the OEPA (Sauerbrey, 2008) and
the APOC (APOC, 2001; Boatin, 2008; WHO, 1995). APOC is active in the
countries of sub-Saharan Africa and integrates the former OCP (Boatin,
2008), of ca. 18 million infected individuals. This represents roughly 99%
of the total number of people infected with O. volvulus in the African
continent (Amazigo et al., 2007; APOC, 2001; Remme, 1995). Information
from rapid epidemiological mapping of onchocerciasis allowed the
APOC to more precisely delineate the distribution of the parasite in
Africa. APOCs strategy relies upon establishing sustainable commu-
nity-directed ivermectin delivery systems in the endemic countries of
Africa (Amazigo et al., 2007; APOC, 2001; Boatin et al., 1997; Dadzie et al.,
2003). It has been suggested that elimination of onchocerciasis in Africa
would not be feasible with current chemotherapy. Thus, APOC uses an
annual ivermectin treatment approach with a purpose of disease control,
and elimination has not yet been promulgated by the programme as its
ultimate goal. In contrast, in Latin America, OEPAs overall goal is the
complete elimination of onchocerciasis in the 13 foci in the 6 endemic
countries in the region, namely, to eliminate the O. volvulus reservoir and
not just the public health burden. The strategy employed by OEPA relies
upon semi-annual or in some instances quarterly treatments of the eligible
population with ivermectin. OEPA has made substantial progress
towards the goal of eventual elimination. Transmission has been
interrupted in 8/13 of the foci in Latin America to date, including Lopez
de Micay in Colombia, Esmeraldas in Ecuador, Escuintla-Guatemala,
Santa Rosa and Huehuetenango foci in Guatemala, North-Central
in Venezuela, and North Chiapas and Oaxaca in Mexico (Gonzalez
et al., 2009; Lindblade et al., 2007; Rodrguez-Perez et al., 2008a,b, 2010a,
b; Sauerbrey, 2008; Vieira et al., 2007; WER, 2009, 2010, 2011) (Fig. 4.1;
Table 4.2). Hence, the other five endemic foci of four countries presently
undergo mass ivermectin administration (Fig. 4.3).
In order to more efficiently allocate the resources in the OCPs and to
assist them in measuring their impact, it was necessary to develop strate-
gies to accurately measure epidemiological parameters such as preva-
lence, incidence and intensity of infection in the human population, as
well as the vector infection/infectivity rate, and the annual transmission
potential (ATP). The latter is an estimate of the number of L3s received by
a person who deliberately exposes himself and remains stationary at the
collection site for a complete year. It grossly overestimates the number
received by someone going about their normal activities. This is calcu-
lated from estimations of the number of black fly bites a person will
receive in a given year, the prevalence of infected flies in the vector
population and the average number of larvae carried by an infected fly
(Duke et al., 1966). OCPs in Africa and, in particular, the OCP have paid
special attention to monitoring blinding onchocerciasis in both the human
and the vector populations. In order to accurately measure these indica-
tors, a test must have the ability to differentiate strains of the parasite that
exist in the forest and savannah bioclimes of West Africa. The test must
also distinguish the cattle parasite O ochengi, which is sympatric with
O. volvulus throughout much of West Africa. O. ochengi is transmitted
by the same vector S. damnosum s.l. that serves as the vector of O. volvulus
(Omar et al., 1979; Wahl and Schibel, 1998; Wahl et al., 1988) and
can therefore complicate the estimation of the ATP for O. volvulus. The
ability to distinguish O. volvulus from other animal filariae and to further
distinguish the forest and savannah strain of the parasite has important
TABLE 4.2 Ocular morbidity and prevalence of infective flies in Latin Americaa
Prevalence of infective flies

Year of Prevalence Year of expressed as rate per 2000
Country Endemic focus ophthalmologic study (%) of MfACb entomologic study flies examinedc
Brazil Amazonas 2007 2.20 No data No data

Colombia Lopez de Micay 2006 0.00 2004 0.19 (0.0060.98)
Ecuador Esmeraldas 2008 0.00 2008 0.0 (UCI 0.08)
Guatemala Central 2009 0.30 2010 0.0 (UCI 0.10)
Guatemala EscuintlaGuatemala 2006 0.00 2010 0.0 (UCI 0.30)
Guatemala Huehuetenango 2008 0.00 2008 0.0 (UCI 0.40)
Guatemala Santa Rosa 2005 0.00 2010 0.0 (UCI 0.60)
Mexico Southern Chiapas 2008 0.00d 2010 0.20 (0.020.30)
Mexico Northern Chiapas 2006 0.00 2010 0.0 (UCI 0.30)
Mexico Oaxaca 2008 0.00 2008 0.0 (UCI 0.07)
Venezuela North-Central 2010 0.00 2009 0.0 (UCI 0.36)
Venezuela Northeast 2009 0.40 2007 0.0 (UCI 0.10)
Venezuela South 2008 5.80 2010 0.0 (UCI 0.70)
a
a Onchocerciasis Elimination Program for the Americas. (2010). URL: http://www.oepa.net. XX Interamerican Conference on Onchocerciasis. (2010). Guatemala, Guatemala,
10-12th November. (WER, 2009, 2010, 2011). All data were provided by the Onchocerciasis Programmes (Mexico, Guatemala, Ecuador, Colombia, Venezuela, and Brazil) in
coordination with the Onchocerciasis Elimination Program for the Americas (OEPA).
b
MfAC = Microfilariae in anterior chamber of the eye.
c
Value represents point estimate and values in parentheses represent 95% lower and upper limit confidence interval surrounding point estimate. UCI = 95% upper limit
confidence interval.
d
Study in three extra-sentinel communities.
Colombia Brazil
3.0% Mexico
Venezuela 0.0%
32.9%
29.2%
N Ecuador
0.0%
112,388 94,235
(91.2%97.7%) (94.6%95.1%)
Guatemala
34.8%
Mexico 9,839
(87.4%93.4%)
Guatemala
Venezuela
106,615
(93.9%94.9%) Colombia
Ecuador Brazil
1000 Km
FIGURE 4.3 Distribution of human onchocerciasis endemic countries in Latin America presently undergoing mass ivermectin administration.
The current eligible population for treatment with ivermectin is presented in the pie chart (the value represents the eligible population and
values in parentheses represent the coverage in percent of the first and second rounds in 2010). All ivermectin coverage rates are above the
85%, the minimum coverage needed in a sustained fashion to interrupt transmission. The percent of the eligible population for treatment per
country on the total eligible population in Latin America is depicted in the upper right inset. Map credit: designed by Javier Alfonso Garza-
Hernandez, Genomics Center and Biotechnology-Mexico. All data were provided by the Onchocerciasis Programmes (Mexico, Guatemala,
Ecuador, Colombia, Venezuela, and Brazil) in coordination with the Onchocerciasis Elimination Program for the Americas (OEPA).
applications for disease control in Africa. However, this has not been the
case for the Latin America region as strain differences in the parasite itself
have not been documented. Usually, the Simulium vector species in this
hemisphere do not appear to be vectors for other filarial species. How-
ever, there is an unknown filaria commonly found in S. metallicum in
Guatemala, and a report of Ornithofilaria in S. antillarum in the Amazo-
nian focus (Basanez et al., 1988). In addition, S. exiguum and S. metallicum
are zoophilic vectors in some locations in South America and could
transmit animal Onchocerca species, hence the need for accurate diagnosis.
The zoophily of S. metallicum is reflected in low natural infection rates
with O. volvulus and infection with filariae of suspected animal origin. In
many endemic localities where S. metallicum is the primary vector, cattle
are parasitized by O. gutturosa and horses with O. cervicalis. S. callidum is
also a largely zoophilic species (Shelley, 1988).
4.4.1. Control through nodulectomy

A therapeutic measure to control onchocerciasis is the removal of the
adult worm in the nodule. In Latin America, the efficacy of this therapeu-
tic measure varied as a function of onchocerciasis prevalence (Guderian
et al., 1987). In hypoendemic areas, nodulectomy had a positive effect in
reducing the intensity of infection, but although this effect was also
observed in hyperendemic areas, new nodules developed rapidly
(Guderian, 1988).
Individuals that undergo nodulectomy receive beneficial effects as
their mf loads and skin pathology are reduced. However, palpable
nodules do not represent the majority of active parasites, and deep
nodules can occur in chronic infections. In addition, nodulectomy may
lessen numbers of mf entering the eye, though the evidence for preven-
tion of blindness is not strong (Aoki et al., 1983).
Nodulectomy campaigns have been undertaken in Mexico since 1932.
However, given that the frequency and coverage of these campaigns have
been variable, that not all nodules are palpable, and that the number of
worms per nodule constitutes only a proportion of the total worm burden,
it has not been feasible to accurately estimate the impact of this interven-
tion on the parasite populations. Basanez and Ricardez-Esquinca (2001)
simulated what would be the effect of a combination of nodulectomy and
chemotherapy with ivermectin on the ratio of palpable nodules per
person over time and predicted that a reduction in this ratio from one
palpable nodule per person in 1980 would reach values near to zero in
2000. It is also strong evidence that the nodulectomy campaigns have
reduced the size of nodules (and numbers of worms contained) over
time, but this has not been precisely quantified.
4.4.2. Control through vector control

Reducing transmission and preventing new cases can be attained by the
use of vector control. Davies (1994) published the most updated review of
60 years of vector control of onchocerciasis. In brief, in 1975, the OCP in
Africa initiated vector control by weekly aerial spraying of the breeding
sites of savannah S. damnosum s.l. with the organophosphate temephos
(Abate). After the appearance of resistance to temephos in 1980, chloro-
phoxin was evaluated but became rapidly ineffective, and it was replaced
with the biocide Bacillus thruringiensis serotype H-14. These actions, which
were supplemented with ivermectin treatment, protected some 20 million
people from onchocerciasis by the end of the programme (Bradley et al.,
2005). It has been estimated that nearly half a million people will have been
prevented from going blind as a result of the OCP (WHO, 1997).
In Latin America, the use of vector control has been limited to some
foci in Guatemala (Shelley, 1991). The Japan International Cooperation
Agency based in Guatemala performed studies on the biology and the
ecology of the vectors for their control (Takaoka, 1981). Following these
studies, during 19791989, the San Vicente Pacaya area in Guatemala was
subjected to larviciding with temephos which produced excellent control
of onchocerciasis in that area (Ochoa et al., 1997). In Mexico, vector control
has not been accepted as part of the integral onchocerciasis control, and
there have been few scientific research activities towards the search of
new options for vector control. In onchocerciasis areas in South America,
the use of vector control has been extremely difficult to implement as a
strategy for the control and elimination of onchocerciasis given the ele-
vated operational costs in areas where communities are dispersed on
mountain ranges and with high numbers of large and small rivers serving
as breeding sites (Shelley, 1991; Vivas-Martnez et al., 2007).
4.4.3. Control through chemotherapy

Microfilarial death, and the resulting release of products derived from
the endosymbiotic (Wolbachia) bacteria, and the consequential release
of proinflammatory cytokines are believed to be responsible for
most of the onchocerciasis pathology (Saint Andre et al., 2002). Because
the severity of skin and eye lesions is mainly related to the worm
burden, a reduction in mf number is associated with a lower incidence
of disease manifestations. For many years (until the 1990s), the microfi-
laricidal drug diethylcarbamazine (DEC) was the only choice of treatment
to onchocerciasis. DEC, however, produced numerous, frequent and
serious side effects. As a result of therapy with DEC, many instances of
optic nerve damage were recorded (Greene, 1990; Rodrguez-Perez and
Rodrguez-Lopez, 1994; Taylor and Greene, 1981; Thylefors, 1978). DEC is
a potent microfilaricide, and as a result of the sudden death of numerous

mf, DEC frequently caused adverse skin allergic reactions (Mackenzie
and Kron, 1985). In particularly heavily infected individuals, severe side
effects such as headache, musculoskeletal pains, fever, tachycardia, hypo-
tension and vertigo among others occurred frequently (WHO, 1987).
These side effects were so common that it was suggested that an adverse
reaction to a single dose of DEC could be used to diagnose onchocerciasis
(Mazzotti, 1948). Because of the dangers associated with these adverse
reactions, this test was limited to those individuals suspected of having
onchocerciasis but with no detectable mf in skin snips.
A less severe skin diagnostic patch test with DEC was developed (Stingl
et al., 1984). The patch test consisted in the administration of 10% DEC in
Nivea cream over a 5-cm area of skin and could detect 92% of mf-positive
individuals in the Sudan. Although initially promising as a tool for repla-
cing skin biopsies, the patch test varied according to the study area (Boatin
et al., 2002; Kilian, 1988; Newland et al., 1987). With the advent of ivermec-
tin, the cornerstone of current therapy for onchocerciasis, treatment with
DEC has become obsolete. Unlike DEC, ivermectin does not precipitate or
exacerbate optic neuritis in the period shortly after treatment. Even though
some manifestations in the posterior segment of the eye, such as chorior-
etinitis, do not improve with treatment, long-term therapy with ivermectin
is effective in preventing visual impairment (Meredith and Dull, 1998).
4.5. ADVANTAGES AND DISADVANTAGES OF TREATMENT

WITH IVERMECTIN
Ivermectin is a semi-synthetic product, being an 80:20 mixture of avermec-

tins B1a and B1b, which are macrocyclic lactones synthesized by the actino-
mycete Streptomyces avermectilis (later renamed S. avermectinius). Originally,
it was formulated as 6-mg tablets and given orally, though later ivermectin
was delivered in three-mg tablets. The recommended dose is 150 mg/kg
weight taken annually or biannually. Initially, children under five years of
age, or less than 15 kg body weight, or 90 cm height were not eligible for
treatment. Since 2004, they may be treated once they are 5 years old.
Pregnant women, breast-feeding mothers within 1 week of delivery and
individuals with neurological disorders or severe intercurrent disease are
excluded from treatment with ivermectin.
Ivermectin acts on the receptor complex of the inhibitory neurotransmit-
ter, gamma-aminobutyric acid (GABA) (Estambale and Howells, 1989; Goa
et al., 1991). It activates glutamate-gated chloride channels that contain
alpha-type subunits, resulting in a hyperpolarization of the neuronal mem-
brane, leading to a flaccid paralysis of Caenorhabditis elegans and Haemonchus
contortus (Ardelli et al., 2009; Forrester et al., 2003). In a study, it has been
shown that ivermectin is a competitive inhibitor of specific 3H-GABA sites of

nematodes (Ros-Moreno et al., 1999). Thus, the interaction of ivermectin with
specific GABA receptors in O. volvulus paralyses the mf and incidentally
suppresses reproduction. Ivermectin is well tolerated by humans because
GABA is only one of many such inhibitory neurotransmitters. Additionally,
ivermectin does not cross the bloodbrain barrier and cannot interfere with
GABA production in the central nervous system (Campbell et al., 1983;
Greene et al., 1989). Ivermectin exhibits potent microfilaricidal activi-
ty against many major filarial parasites in humans, including Wuchereria
bancrofti, Brugia malayi, Loa loa and Mansonella ozzardi, but not against Man-
sonella perstans. Ivermectin is also active against Ascaris lumbricoides, Trichuris
trichiura, Enterobius spp. and Strongyloides stercoralis. It also has potent activ-
ity against cutaneous larva migrans, for which treatment is not available, and
has the potential to become the drug of choice for ectoparasitic infestations
(mites and lice) in humans (Omura, 2008).
Ivermectin is a safer microfilaricidal drug than DEC, but it does not kill
adult worms at the standard dose which is a single annual dose aimed at
disease control. However, multiple treatments a year with ivermectin have
effects on adult worms. Data on the impact of three repeated doses of
ivermectin on ocular microfilarial loads indicated a reduction by about
70% (Rodrguez-Perez et al., 1995), which may result in beneficial effects
like regression of early lesions in the anterior segment of the eye, oncho-
cercal optic nerve disease and visual field loss. Treatment with ivermectin
has also been shown to reduce skin mf levels to undetectable levels within
days (Duke, 1990; Greene et al., 1989). A meta-analysis of the effect of a
single standard dose of ivermectin on O. volvulus showed that after treat-
ment, microfilaridermia would be reduced by half after 24 h, by 85% after
72 h, by 94% after 1 week, and by 9899% after 12 months (Basanez et al.,
2008). Histological studies of onchocercal nodules from Mexico, Guate-
mala and Ecuador from individuals with recurrent ivermectin treatments
have shown dramatic effects on adult female viability and fertility and
marked reductions in the frequencies of male worms (Cupp et al., 2004),
leading to the conclusion that long-term ivermectin treatment has a pro-
found effect on survival and reproduction of this species (Cupp and Cupp,
2005). Unfortunately, the adult worms, which have an average estimated
lifespan of 10 years (Bradley et al., 2005), are not killed by the treatment
when a single annual dose is used for disease control. Studies conducted in
Guatemala, Sierra Leone and Cameroon indicated that using the standard
therapeutic dose on a 6-monthly basis results in significant mortality of
adult worms. Severe adverse neurological reaction risks, including deaths,
have been reported during mass treatment with ivermectin in loiasis-
endemic areas, and this has seriously curtailed treatment programmes in
areas potentially co-endemic for L. loa (Chippaux et al., 1996). It can be
argued that programmes should be curtailed only where O. volvulus is
hypoendemic and co-endemic with L. loa from a risk-benefit perspective.
The efficacy of ivermectin has varied in Latin America somewhat from

one geographical area to another. Some field and experimental studies in
Guatemala showed that ivermectin can be used to interrupt transmission
(Cupp, 1992; Cupp et al., 1986, 1989, 1992). In the Rio Santiago river basin
of Ecuador, mass distribution of ivermectin twice a year resulted in
dramatic declines in the prevalence of infected and infective black flies
(S. exiguum and S. quadrivittatum) (Guevara et al., 2003). However, in other
areas of Ecuador where the prevalence and transmission of infection was
persisting, more frequent treatments with ivermectin were required to
achieve the objective of elimination of the infection (Vieira et al., 2007).
There was a similar impact of ivermectin on the infection of the flies in
communities from Amazonian Brazil where it decreased from 8.6% to
0.3% in Balawau and from 4% to 0.1% in Toototobi (Marchon-Silva et al.,
2007). However, the nomadic Yanomami populations from the Amazon
rainforest are one of the most severely affected groups given that they are
at continuous risk of exposure to infected black flies. They represent only
2.6% of the total population at risk in the Americas, but they all reside in a
vast single focus (the AmazonasRoraima focus), bordering between
Venezuela and Brazil (Moraes, 1991; Vivas-Martnez et al., 2007). Given
this situation, active transmission is believed to continue in the two
cross-border foci of the Yanomami area (South Venezuela and
Amazonas). Ongoing transmission is also present in the North-East
focus in Venezuela, Fig. 4.1); however, 8 out of 13 foci have eliminated
new cases of eye disease attributable to onchocerciasis (Table 4.2). In
Mexico, repeated mass treatment administered twice per year alleviated
clinical manifestations, but in hyperendemic communities of the Southern
Chiapas focus had little influence on onchocercal transmission after the
first rounds of treatment (Rodrguez-Perez et al., 1995).
Afterwards, given the progress of the Mectizan programme that
achieved higher levels of coverage and compliance, the parasite transmis-
sion was nearly blocked (Rodrguez-Perez et al., 2004, 2006a,b). A rapid
suppression of residual transmission in this area was completed by quar-
terly treatments with Mectizan (Rodrguez-Perez et al., 2008b). Thus it
appears that the value of long-scale-repeated treatments with ivermectin
at individual and community control of onchocerciasis is effectively
higher than that of a short scheme of treatment.
4.6. DEVELOPMENT OF OTHER NEW DRUGS

Elimination of onchocerciasis may be possible through interruption of
transmission through long-term ivermectin treatments in Africa (Diawara
et al., 2009) and in most foci from Latin America (Cupp and Cupp, 2005;
Rodrguez-Perez et al., 2008b; Vieira et al., 2007). Currently, the only
approved drug available for mass treatment is ivermectin. However, as

ivermectin does not kill the adult worm with a single annual treatment,
new therapeutic targets and agents are needed to treat and cure this
devastating disease in other endemic foci where elimination will not be
feasible. Therefore, the development of a macrofilaricidal drug and/or
vaccine with similar qualities as ivermectin would be of significant impact
in the control of onchocerciasis. The only true macrofilaricidal drugs are
suramin, which is too toxic for general use (Whitworth, 1988), and amo-
carzine (Poltera, 1998). Amocarzine, a piperazinyl derivative of moscanate
(CGP 6140), showed to have both good macrofilaricidal and microfilarici-
dal activity in some studies in Latin America (Nutman et al., 1996; Poltera
et al., 1991a,b; Zak et al., 1991) and Africa (Awadzi et al., 1997). However,
the optimal time-point to assess the adulticidal efficacy of amocarzine in
onchocerciasis was not well established, and more clinical trials need to be
performed. It has been suggested that dual therapy (amocarzine preceded
by ivermectin) would possibly permit the introduction of longer intervals
for distribution in mass chemotherapy (Poltera, 1998). Many other chemicals
(e.g. pyrimidinylguanidines, amidine derivatives, the imidazolinylhydra-
zones, thiosemicarbazone derivatives, and thiadiazole derivatives) have
been evaluated with O. volvulus using in vitro culture systems, but only
those within three different chemical classes had significant activity on
adult O. volvulus. However, these new promising macrofilaricidal com-
pounds still require assessment in in vivo models to provide evidence for
the usefulness of the in vitro system and for the efficacy of the compounds
tested (Strote et al., 1998). Later, other promising chemicals also appeared
such as nafuredin (Omura et al., 2001). The discovery of a new chemical class
of synthetic anti-helminthics (the amino-acetonitrile derivatives) has been
reported. These seem to have a novel mode of action involving a unique,
nematode-specific clade of acetylcholine receptor subunits. The new drugs
were efficacious against various species of livestock-pathogenic nematodes,
well tolerated, and of low toxicity to mammals (Kaminsky et al., 2008).
Recently, Closantel, a veterinary anthelmintic, was identified as a potent
and specific inhibitor of the chitinase OvCHT1 from O. volvulus. Closantel
was found also to inhibit the moulting of O. volvulus infective L3 stage
larvae, another important biochemical processes essential to this filarial
parasite (Gloeckner et al., 2010).
In a study aimed to discover macrofilaricidal drugs of potential
human benefit (Langworthy et al., 2000), cattle infected with the intrader-
mal parasite O. ochengi were treated with the antibiotic oxytetracycline
over a period of 6 months. All O. ochengi adult worms were killed as a
consequence of the death of numerous Wolbachia bacteria, which are
endosymbionts of the female parasite. Wolbachia bacteria are intracellular
symbionts that are species specific for each filarial species. The adult
female worm can transmit those microorganisms to its progeny, the mf.
In the absence of these organisms, all larval stages die, but the role of the
symbionts and the precise mechanisms involved in the bacteria/worm
relationship are unknown (Bandi et al., 1998, 1999; Genchi et al., 1998).
Available antibiotics, such as tetracyclines, rifampicin, chloramphenicol
and doxycycline, deplete Wolbachia and can interrupt embryogenesis (i.e.
permanent sterilization of adult worm). In addition, partial or complete
macrofilaricidal activity has also been reported (Hoerauf, 2008; Hoerauf
et al., 2008a,b, 2009; Langworthy et al., 2000; Specht et al., 2008). However,
the treatment regimens are long (i.e. 6 week/100 mg doxycycline per day
has shown to be the most efficient regimen), and shorter courses with
rifampin and/or azithromycin (5 days of treatment) will not likely clear
Wolbachia from O. volvulus (Richards et al., 2007). More efficient antibiotic
treatment may be further improved by using novel bioinformatic
proteomic approaches in the near future. New drug targets could also be
developed for O. volvulus when the genome is complete. At present, antibi-
otic treatment is useful for individual patients who seek treatment because
of symptoms, the treatment of travellers and immigrants from filarial-
endemic regions. It can also be used for the treatment of selected individuals
identified to be reluctant to take ivermectin or cannot take ivermectin for
medical reasons, particularly in areas where transmission has been inter-
rupted. With the aim of eliminating the residual reservoir of infection, an
efficient antibiotic treatment of remaining infected individuals may be
potentially useful during the final stages of a successful ivermectin-based
elimination programme, and in areas with intensive epidemiological sur-
veillance where ivermectin has been halted.
As long as a macrofilaricidal drug remains unavailable, ivermectin
remains the only safe and well-tolerated drug for the treatment of oncho-
cerciasis. The most significant effects of ivermectin given repeatedly, on an
annual basis, may be summarized as follows: a 90% decrease in the preva-
lence of ocular mf loads after 24 years; 50% and 30% reductions, respec-
tively, in the prevalence of early iridocyclitis and sclerosing keratitis after
the same period; a less marked impact on posterior segment lesions and no
significant benefit in terms of visual acuity or blindness (WHO, 1995).
Clinical and community trials involving more than 70,000 people showed
that annual ivermectin treatment was safe, prevented ocular and dermal
morbidity and significantly reduced transmission (Remme et al., 2006).
4.7. MONITORING AND EVALUATION OF CONTROL

OF ONCHOCERCIASIS
The next sections will describe the most current strategies utilized in the
surveillance and monitoring of OCPs. The development of different
methods of diagnosis of onchocerciasis has proved to be useful for the
programmes, but it has been equally important to calibrate and evaluate

them according to specific epidemiological situations and the constant
development of new drugs and strategies of control.
4.7.1. Parasitological diagnosis of O. volvulus infection

The gold standard diagnosis method for onchocerciasis is the bloodless
skin snip (WHO, 1987). Typically, 26 skin snips or biopsies are taken from
both iliac crests (African patients) and/or from shoulders (Guatemalan
Mexican patients) and/or from lower part of the body in individuals of the
Amazonian focus (Vivas-Martnez et al., 2007) using either a corneoscleral
biopsy instrument (an obsolete ophthalmological surgical instrument) or a
scalpel blade. These biopsy punches enable snips to be taken easily and
rapidly, but are expensive, need regular setting and sharpening and should
be sterilized between patients. The skin biopsy is then weighed and incu-
bated overnight in 0.2 ml of buffer (normal saline or culture medium) in a
microtitre plate. About 60% of mf contained in the biopsy will emerge after
30 min of incubation, rising to over 75% after 24 h. Mf are counted by
microscopy and the species can be distinguished from other species of mf
after staining with Giemsa or Mayers haemalum (Bradley et al., 2005). In
communities with high infection intensity and prevalence, this method is
both sensitive and specific. However, the sensitivity of the assay depends
on the intensity of infection within the community (Taylor et al., 1987). Two
skin snips are sufficient to determine high infection intensities, but when
two extra skin snips are taken, the accuracy of the technique improves
(Taylor et al., 1987). However, in those communities with low infection
intensities (mf densities less than 3.5 mf/mg of skin), even taking six snips
may result in false negatives (Taylor et al., 1989). As long as the impact of
control programmes is based mainly on reducing mf loads, the use of this
technique may lead to an overestimation of the efficacy of such control
measures. Thus, the skin biopsy method is not useful for determining the
infection rates in Latin America because of the success of the ivermectin
distribution programmes in reducing the skin mf loads. Currently, skin
snips are only used to further investigate infection status in individuals that
have borderline positive results in one of the serological individuals. In
these studies, the snips are tested using the O-15 PCR, which is an
extremely sensitive and specific test for residual parasite DNA, and do
not require the presence of viable or intact parasites to give a positive result.
4.7.2. Immunological and molecular diagnosis of

O. volvulus infection
Given that there is an urgent requirement for monitoring accurately
the impact of control programmes, the application of very sensitive
and specific immunological and molecular techniques is essential.
A particular requirement of the OCP was to be able to rapidly detect

reinfection in an area where control strategies had broken down or after
vector control has stopped. In these situations, an assay capable of detect-
ing either pre-patent infections or exposure to the parasite is desirable. In
other situations, such as Latin America, the detection of specific antibo-
dies in sera may be ideal since low mf loads as a result of the widespread
distribution of ivermectin have made the skin biopsy a limited technique.
DNA-based assays rely on the detection of specific sequences in the
parasites genome, so the presence of the parasite is obligatory and they
cannot be used in the detection of pre-patent infections. The detection of
parasite products or antigens in the circulation of other body fluids such
as urine and blood provides conclusive evidence of an ongoing infection.
Even if a test detecting specific antigens would be more efficient defining
current infection, the development of such an assay has proved to be
particularly difficult. This could be because the anatomical locations of
the mf and adults in onchocerciasis (in the skin and subcutaneous tissues,
respectively) do not allow reliable release of antigen into the circulation
(Bradley et al., 2005). This is in contrast to lymphatic filariasis where
antigen-based immunochromatographic tests (although less sensitive
than an antibody test) have been developed for its control and elimination
through mass drug distribution (P. Lammie, personal communication)
(Lammie et al., 2004). As a consequence of the intervention with ivermec-
tin in Latin America, transmission has been interrupted in 8 of 13 foci,
thus there is an urgent need for new more sensitive antibody and/or
antigen tests to help guide programmes on when to stop treatment cam-
paigns and have an accurate-sensitive posttreatment surveillance system
(Molyneux, 2009).
The next sections will describe two of the most sensitive and specific
techniques for the diagnosis of onchocerciasis that have been applied by
OEPA, namely, the detection of parasite-specific antibody in human
blood fluids using a single or combination of recombinant proteins as
antigen, and the application of a PCR assay based on repetitive DNA to
detect O. volvulus infections in flies and humans.
4.7.3. Development of immunological tests for the diagnosis

of onchocerciasis
The most practical and sensitive serological test for the detection of
parasite-specific antibody is the enzyme-linked immunosorbent assay
(ELISA). This is a simple indirect binding assay, detecting antibody
bound to a parasite extract with visualization with a secondary antibody
conjugated to an enzyme. Using the ELISA format, in the 1970s and 1980s,
research was directed to investigate a variety of O. volvulus and other
nematodes extracts for the diagnosis of onchocerciasis, and although
these antigens proved to be highly sensitive, most of the preparations

gave cross-reactivity with other filarial infections.
Early in the 1990s, the use of monoclonal antibodies to define
onchocercal-specific antigens also became popular (Cabrera et al., 1989;
Engelbrecht et al., 1992; Lucius et al., 1988b; Wandji et al., 1990). The use of
monoclonal antibodies provided advantages for their production with
reproducible quality. However, their diagnostic use remained dependent
on an inhibition assay system that still required a supply of parasite
material (Bradley and Unnasch, 1996). The problem associated with the
production of antigens in sufficient quantities was resolved by the advent
of recombinant DNA technology.
The first report of the cloning of an immunodominant antigen (Ov33)
of O. volvulus was by Lucius et al. (1988a). Later, other diagnostic antigens
including Ov16 (Lobos et al., 1990) and the overexpression of the full
length Ov33 molecule in pGEX2T and PCG808, which yielded fusion
proteins with glutathione S-transferase and the maltose-binding protein
(MBP) of Escherichia coli, were described (Lobos et al., 1990; Lucius et al.,
1992). The approach followed by Lucius et al. (1988a,b) was to immunize
mice with affinity-purified antigen using a monoclonal antibody anti-
Ov33. The sera from immune mice were used to screen a gt11 expression
cDNA library, and a cDNA clone encoding 239 amino acids was isolated.
They found that the predominant response to this antigen by ELISA was
the lgG4 isotype, which gave a sensitivity and specificity of 93% and 96%,
respectively, when assayed with a panel of sera from individuals with
onchocerciasis and other filarial infections. Similarly, Lobos et al. (1990)
isolated a 22- to 30-kDa glycoprotein antigen (Ov16) by immunoscreening
a cDNA library with human antibody that had been affinity purified on a
low-molecular-weight fraction of O. volvulus extract. The recombinant
antigen (Ov16) was recognized by antibodies in sera of individuals
infected with O. volvulus but not by antibodies in those infected with
W. bancrofti, B. malayi, M. ozzardi, M. perstans and L. loa. Moreover, Ov16
was able to detect antibody up to 1 year before patency in humans and
infected chimpanzees, and as a consequence, it was considered as an early
marker of infection (Lobos et al., 1991).
In a similar manner, several groups approached the cloning of specific
diagnostic antigens by differentially screening libraries (Donelson et al.,
1988) with onchocerciasis serum and serum from individuals with other
potentially cross-reactivity infections (Bradley et al., 1991; Chandrashekar
et al., 1991; Garate et al., 1990; Maizels et al., 1990). Bradley et al. (1991)
took the approach of screening with onchocerciasis and bancroftian fila-
riasis serum pools. The bancroftian sera were taken from a non-onchocer-
ciasis-endemic area. Subsequently, 31 specific clones were isolated and
screened again by a microplaque lysis technique with either O. volvulus-
or W. bancrofti-infected sera. In this study, two recombinant antigens
(Ov22/31 M and Ov20/36 M) were subcloned into a plasmid vector,

pHGS8, and purified for use in ELISA. These recombinant peptides
were then assayed using the individual antigen (Ov22/31 M or Ov20/
36 M) and a combination of the two against 31 O. volvulus- and 11
W. bancrofti-infected sera. In the ELISA test, both peptides retained the
100% specificity evident from spot lysis; however, the level of sensitivity
was reduced to 74% and 45% for Ov22/31 M, Ov20/36 M, respectively.
The investigators mentioned that this was probably due to the fact that the
sera had not been pre-absorbed to remove antibacterial antibodies, a step
that would be neither desirable nor practical for a test designed for field
conditions in endemic countries. Interestingly, when both recombinant
antigens were used in combination, 90% sensitivity was achieved, indi-
cating that maximum sensitivity would be attained if a second fraction
step were introduced in the purification of the fusion proteins from E. coli
lysates and with the addition of other peptides. As a consequence, other
recombinant peptides were assessed with a broader number of individual
patients sera.
As a result of the studies mentioned above, in a multi-centre study
(Ramachandran, 1993), the specificity and sensitivity of 34 recombinant
proteins were evaluated using sera of individuals from different geo-
graphical areas. Three of those proteins (Ov29, Ov11 and Ov7) were
found to give sufficient sensitivity in an ELISA test without the loss of
specificity when used as fusion proteins (Bradley et al., 1991; Trenholme
et al., 1994). This cocktail of peptides was able to detect antibodies in
onchocerciasis individuals from most endemic areas and both forest and
savannah regions where other filarial parasites were also present, and
gave a sensitivity between 84% and 100%, but was 100% specific (Bradley
et al., 1993b). The same combination of antigens in an ELISA was also
used to assess the effects of vector and ivermectin control on antibody
responses (Bradley et al., 1993a; Gillespie et al., 1994), evaluate areas of
potential recrudescence of transmission (WHO, 1995) and detect a new
focus of transmission (Maia-Herzog et al., 1999). It also had the potential
as an epidemiological tool to provide a comparative index of community
exposure to infection as accurately as the community microfilarial load
estimated by skin snip (Bradley et al., 1998). However, in all of the studies
mentioned above, antibodies were still detectable even in those indivi-
duals who had become mf negative, suggesting that the assay would not
be capable of distinguishing new infections from historical ones. How-
ever, this problem can be overcome, if children born after the initiation of
a successful vector control or ivermectin programme are tested or used as
indicators of prevalence of exposure to infection. For example, Botto et al.
(1999) described onchocerciasis endemicity levels in the Unturan Moun-
tains of Venezuela using the cocktail in the ELISA test, indicating that
high seroprevalence in children could serve as an indicator of local and
intense transmission, and that this may aid the selection of communities
for further epidemiological surveys.
Other groups undertook a similar approach employing other combina-
tions of recombinant proteins (Chandrashekar et al., 1991; Ogunrinade
et al., 1992, 1993). These groups described two clones: OC 3.6 and OC 9.3.
The first clone was used in an immunoblot assay to diagnose onchocercia-
sis children in Africa. The test proved to be useful for pre-patent diagnosis
as 24% of mf-negative children had antibody reactive to this protein. Both
recombinants (OC 3.6 and OC 9.3) were also used in an IgG4 ELISA. The
test gave a sensitivity of 95% and 81%, respectively, and was negative
when tested with sera from individuals infected with different species of
filariasis or gastrointestinal nematodes.
Based on a WHO initiative to find the best antigen formulation for a
diagnostic test to monitor OCPs, and as indicated previously,
Ramachandran (1993) reported the assessment of 34 recombinant anti-
gens, which were used on an ELISA format to determine their reactivity
with sera collected in different epidemiological situations. The specificity
and sensitivity of these proteins varied from 75% to 100% and from 11% to
96%, respectively. Specificity was an absolute requirement for the test but
as sensitivity could be increased by using more than one antigen the
resolution was to take up the cocktail approach (Bradley et al., 1991;
Bradley et al., 1993a,b). Selected antigens were also tested for their ability
to detect early and pre-patent infections. Of these, four antigens (Ov16,
Ov7, Ov11 and OC 3.6) were also easily overexpressed using the protein
expression and purification system of New England Biolabs protocol
and selected for their highest specificity. As a result, new studies were
performed using a combination of these recombinant proteins (Bloch
et al., 1998).
Bloch et al. (1998) tested individual antigen and cocktails using sera
from patients infected with O. volvulus, W. bancrofti and Dracunculus
medinensis. All sera from patients infected with O. volvulus responded
positively to all three antigens; however, some immunological cross-
reactivity of sera from patients infected with D. medinensis was observed.
When individual O. volvulus recombinant proteins were used, the highest
specificity was obtained for clone Ov10 (60%) and the lowest for clone
Ov16 (40%). However, when the cocktail was used in the ELISA test, the
specificity was 95%. Bloch et al. (1998) assumed that the individual
recombinant proteins had a positive response to D. medinensis and
W. bancrofti, as the sera were from a highly W. bancrofti endemic area,
in opposition to a low transmission area studied by Bradley et al. (1993b).
To improve the specificity of each individual clone, a higher cut-off level
was approached; therefore, when the cut-off value was the mean plus
seven standard deviations, estimated from control sera, the specificity for
Ov10 and Ov16 was 100% and 95%, respectively (Bloch et al., 1998).
If any of the previous ELISA tests using any combination of three

recombinant antigens were used to monitor populations rather than
individuals, the test must be extremely specific, but within reasonable
limits and need not be 100% sensitive (Bradley and Unnasch, 1996). In a
study, the particular combination of antigens Ov29, Ov11 ( Ov20) and
Ov10 could distinguish between different levels of endemicity (Bradley
et al., 1998) as accurately as the skin biopsy. Thus, regular monitoring
using such tests could evaluate whether endemicity is declining in a
successful control programme.
An ELISA using three recombinant antigens (Ov16, Ov7 Ov10 and
Ov11) was assessed for its application with finger prick blood samples
collected on filter papers as it was previously used with venous serum.
The finger prick blood test is a relatively non-intrusive method; thus the
individuals participate more voluntarily during blood collection when
they are informed that it would take only a few blood drops from a finger
and that no venous blood is required (Rodrguez-Perez et al., 1999a). This
finger prick blood ELISA was compared with that of the skin biopsy to
estimate incidence of infections in a sentinel cohort of individuals living
in an endemic community of southern Mexico and used also to determine
the effect of 7 years of semi-annual mass administration with ivermectin
on the incidence of exposure to infection, and indirectly, on transmission
(Rodrguez-Perez et al., 1999b). All individuals in the community became
positive to both tests simultaneously, indicating that seroconversion
assessed infection incidence as accurately as skin biopsy in the sentinel
group. Thus, if a group of individuals is initially identified to be seroneg-
ative and, therefore not infected, they may be used to investigate the
presence of transmission in a community if followed for seroconversion
at later times. The ELISA was also compared with a test designed to
operate under field conditions (Rodrguez-Perez et al., 2003). The rapid-
format antibody card test (immunochromatographic test) using an indi-
vidual antigen, the Ov16, was less sensitive than the ELISA (86% in
comparison to 97%; Rodrguez-Perez et al., 2003) but more specific as it
only detects antibodies of the IgG4 subclass (Weil et al., 2000). If other
filarial infections such as D. medinensis, W. bancrofti, M. perstans and
M. streptocerca do not occur in onchocerciasis-endemic areas, the ELISA
based upon Ov16 may be engineered to be 100% specific, although the
sensitivity of an Ov16 ELISA designed to have a specificity of 100% is just
60%. However, the sensitivity of the assay may be increased by using the
cocktail in the initial screening process (Rodrguez-Perez et al., 2003).
At present, the ELISA using an individual antigen to detect antibodies
anti-IgG4 of Ov16 O. volvulus (one of the specific diagnostic antigens used
in the cocktail) is being used as a surveillance tool by the programmes in
Latin America. Tests based on the detection of antibody have the problem
that it is difficult to distinguish between current and historical infection
(Bradley et al., 2005). The problem of detecting current infection has at

least been partially overcome by using anti-immunoglobuin IgG4
reagents to detect the reactivity in the serum because this antibody iso-
type has been shown to be related to active infection (Lucius et al., 1992;
Weil et al., 1990). In a study carried out in Santa Rosa focus of Guatemala,
no IgG4 antibody positives to recombinant antigen OV16 were found in a
sample of 3232 school children (95% CI, 00.009%). The interpretation of
this datum, together with other historical and epidemiological data, sug-
gested that transmission of O. volvulus is permanently interrupted in
Santa Rosa, Guatemala, and that ivermectin treatment there could be
halted (Lindblade et al., 2007). A similar study was performed in the
focus of Escuintla in Guatemala where successful interruption of the
parasite transmission has also been achieved (Gonzalez et al., 2009). The
Ov16 ELISA was used, in a large-scale study, to determine incidence of
parasite exposure in the Oaxaca and Chiapas foci in Mexico (Rodrguez-
Perez et al., 2008a, 2010a,b). Thus, the Ov16 ELISA has become the test of
choice of the programmes for evaluating the progress of the Mectizan
programme throughout of Latin America.
In parallel to the use of the Ov16 ELISA, studies are performed to
evaluate other antibodyantigen tests. For example, two hybrid con-
structs, OvH2 (composed of Ov20 fused to Ov33) and OvH3 (composed
of C-terminus of Ov20 linked to Ov3), when tested with sera from healthy
individuals and patients of onchocerciasis showed a sensitivity and spec-
ificity of 98.5% and 97.7% for OvH2, and 98.5% and 95.35% for OvH3.
The authors concluded that the test based on OvH2 should prove suitable
for monitoring OCPs and individual diagnosis in endemic areas (Nde
et al., 2002), but OvH3 may be also useful for diagnosis of individuals in
non-endemic areas (Andrews et al., 2008).
4.7.4. Development of DNA probes for the diagnosis

of onchocerciasis
Attempts to improve diagnosis of onchocerciasis have been generally
based mostly on the detection of anti-O. volvulus antibodies or in a few
other studies on detection of antigens of the parasite. The use of DNA
probes has become useful for the detection of the parasite. As a DNA
probe can detect any parasite life cycle stage, its application has been
useful for detecting either of the two strains of the parasite in the human
and the vector populations. In addition, a liquid chromatographymass
spectrometry (LCMS)-based parasite metabolomics has been recently
developed to identify potentially diagnostic biomarkers for onchocercia-
sis (Denery et al., 2010).
A number of Onchocerca genomic DNA probes were isolated in the
mid-1980s. Most were initially isolated by a strategy of screening of
genomic libraries with total genomic DNA preparations. Some of the

DNA probes recognized all parasites of the genus Onchocerca (Perler
and Karam, 1986; Shah et al., 1987). However, two DNA probes were
identified that were specific for O. volvulus (Harnett et al., 1989; Meredith
et al., 1989), as well as specific probes for the rain forest strain (Erttmann
et al., 1987) and savannah strain (Erttmann et al., 1990). All these DNA
probes consisted of specific members of a variable tandemly repeated
DNA sequence family with a unit length of 150 bp found in the Onchocerca
genome. For example, the O. volvulus-specific probe pOVS134 contained
12 tandemly linked monomers of the O-150 family (Meredith et al.,
1989). This sequence family was designated as O-150 family (Meredith
et al., 1989).
Based upon these findings, and the discovery that variation within the
sequence family was apparently constrained by the mechanisms of con-
certed evolution, a PCR-based assay able to classify parasites of the genus
Onchocerca was developed (Meredith et al., 1991; Zimmerman et al., 1993).
The method involved amplification of the entire O-150 repeat sequence
family using degenerate primers, followed by characterization of the
resulting PCR products by hybridization to a variety of species and
strain-specific oligonucleotide probes. The use of degenerate primers to
amplify the entire O-150 repeat family presented various practical advan-
tages. Firstly, the process permitted the production of amplicons from any
sample containing DNA from Onchocerca parasites. This supplied a useful
internal control to monitor DNA isolation from an individual parasite
sample. Secondly, the degenerate PCR products can be easily subdivided
for simultaneous analysis by a number of different oligonucleotide probes
in order to provide greater specificity (Unnasch and Meredith, 1996).
The structure and organization of the O-150 repeat family were
explored by DNA sequence analysis of a number of amplicons produced
from a variety of parasite isolates (Zimmerman et al., 1993). These studies
revealed that the sequence family from any given parasite isolate could
be arranged into several distinct clusters, within which the sequence of
the monomers was identical or nearly identified. Some of these clusters
were found to be species or strain specific, explaining the varying degrees
of specificity afforded by the O-15 DNA probes originally isolated by
genomic library screening.
The O-150 PCR has proven to be useful in a number of applications for
the surveillance of O. volvulus infection. One of the first applications of the
O-150 assay was to classify parasite isolates from larvae collected by the
OCP field fly dissection teams throughout the entire OCP area in Africa
(Toe et al., 1994, 1997a,b). As the identification of these larvae was at the
species and strain levels, it provided the OCP with much more precise
estimates of the transmission levels of blinding onchocerciasis throughout
the programme area (Toe et al., 1994). Results from this monitoring effort
were also used to obtain an accurate estimate of the ATP for O. volvulus in
the presence of the zoonotic species and to target control measures in
areas where O. volvulus transmission potential was high (Barker, 1994).
Although valuable information was obtained by the adaptation of the
O-150 PCR to identify individual larvae, a significant disadvantage to this
process was that the method involved testing of individual parasite-
infected vector S. damnosum s.l. identified by traditional dissection meth-
ods. This was extremely labour intensive and became increasingly ineffi-
cient in areas where the control programme was effective, and the
prevalence of infected flies was therefore very low. Thus, it was necessary
to develop a method capable of determining the prevalence of infection in
the vector population without resorting to examination of individual files.
The O-150 DNA probe-based assay was therefore adapted to detect a
single O. volvulus-infected S. damnosum s.l. in pools of 100 uninfected
files. An algorithm was also developed that permitted one to calculate
the point estimate of the prevalence of infection (and confidence limits
surrounding that estimate) in the vector population from the results
obtained from pool screening (Katholi et al., 1995). This O-150 PCR
assay was applied under field conditions in Africa and Mexico with the
purpose of comparing its outcomes with the traditional method of detec-
tion of O. volvulus by the dissection of large numbers of flies (Rodrguez-
Perez et al., 1999c; Yameogo et al., 1999). In both cases, the results
demonstrated that the estimates of the prevalence of infection in the fly
populations produced by the two methods (dissection and pool
screening) were not significantly different.
The O-150 PCR assay was employed to monitor the impact of the
onchocerciasis programme in Ecuador on infection levels in vector popu-
lations (Guevara et al., 2003). In parallel, a large-scale entomologic study
employing the pool screen PCR based on an ELISA was performed in
Mexico (Rodrguez-Perez et al., 2004, 2006a). At present, the PCR-ELISA
is being employed as a reliable approach to estimate parasite transmission
levels in some foci of Africa and the six affected countries of Latin
America (Adjami et al., 2004; Lindblade et al., 2007; Marchon-Silva et al.,
2007; Rodrguez-Perez et al., 2006b, 2008a,b, 2010a,b; Vieira et al., 2007).
Thus, the O-150 PCR coupled to an ELISA has been a useful approach for
monitoring the progress of the OEPA which have led to the declaration of
interruption of transmission in the eight foci to date (which were listed in
Section 4.4, p. 189; Table 4.2; Figs. 4.1 and 4.3).
Another practical application of the O-150 PCR-based assay was to
detect the presence of patent O. volvulus infections in skin snips. The
O-150 PCR was found to detect infection with a higher sensitivity than
the standard parasitological technique (Freedman et al., 1994;
Zimmerman et al., 1994a,b). In Zimmermans study, skin snips from 94
patients in an onchocerciasis-endemic region of Ecuador were examined.
The results were then compared with those attained by using the O-150
PCR-based assay. All 60 patients mf positive on skin snip examination
were positive in the PCR. In addition, 13 of 34 who were mf negative by
skin snip were also positive in the PCR (Zimmerman et al., 1994a). It is not
likely that these represented false-positive test results, as additional
experiments demonstrated that none of 97 samples collected from indivi-
duals never exposed to O. volvulus were positive in the PCR (Zimmerman
et al., 1994b). These conclusions were confirmed in a study with 10 mf-
negative individuals of Africa, in which 9 of 10 were found to be positive
by O-150 PCR assay (Freedman et al., 1994). Thus, the O-150 PCR assay
has been of relevance in areas where the skin infection levels are very low
as a consequence of the administration of more frequent treatments with
ivermectin (Rodrguez-Perez et al., 2008b). The O-150 PCR assay can also
be employed to determine the strain of parasite in infected humans and in
vector black flies. In Southern Mexico, the strain of parasite in a group of
coffee workers in Chiapas was determined to belong to the non-blinding
forest strain (Rodrguez-Perez et al., 2004). This result is in contrast to
Africa, where the severe strain of O. volvulus predominated in the savan-
nah and forest/transition zones (Adjami et al., 2004). It would be interest-
ing to expand investigation on the occurrence of the different strains of
parasite in other foci in Latin America.
4.8. ENTOMOLOGICAL PARAMETERS FOR MONITORING

THE TRANSMISSION IN LATIN AMERICA (WITH
EMPHASIS IN AREAS WHERE TRANSMISSION
HAS BEEN INTERRUPTED)
The overall goal of the OEPA is to first eliminate onchocerciasis as a

public health problem and then to completely eliminate the reservoir of
infection in the six endemic countries of Latin America. To assist in
this process, the WHO developed a series of guidelines to certify that an
area is free of onchocerciasis (WHO, 2001). Two different measures of
transmission suppression were recommended by WHO. In areas where
pretreatment data were available, WHO defined suppression of infectiv-
ity as a 99% reduction in transmission from pretreatment rates. Where
pretreatment data are not available, transmission suppression was defined
by the WHO guidelines as an absence or near absence of L3 infection in
the vector population and the absence of infection in humans. The WHO
did not specify quantitative metrics to the term near absence, but inves-
tigators from the regional initiative at OEPA recently concluded that an
infectivity rate of < 1 infective fly per 2000 (i.e. < 0.05% of infective flies in
the vector population) would meet this criterion. This measure was
derived from a similar measure developed by the OCP in West Africa,
which developed a cut-off of 1 infective fly in 1000 parous files carrying

infective larvae (Dadzie et al., 2003). In the Americas, a parity rate of 50%
was assumed due to the uncertainty in the estimate and a desire to err on a
conservative side of any estimate. Thus, an examination of 2000 flies will
mean that approximately 1000 parous flies will be examined.
The basic reproduction ratio (R0) is a fundamental concept in both
within-host and epidemiological models of pathogen dynamics. R0,
a dimensionless parameter, is the average number of female offspring
produced throughout the lifetime of a mature female parasite, which
themselves achieve reproduction maturity in the absence of density-
dependent constraints on the parasite establishment, survival or repro-
duction. Effective reproductive ratio (Re) is similar to R0 within which
density-dependent constraints limit parasite population growth
(Anderson et al., 1991). Under conditions of stable endemic infection,
Re 1, namely, when on average each female worm only replaces itself
(regardless of the value of the R0, which would have been greater than one
for introduction and persistence of the infection). Endemic equilibrium is
not guaranteed because it depends on the age structure and infection
pattern of the communities not having been perturbed (i.e. before intro-
duction of vector- or ivermectin-based control). Once control starts, the
parasite population is moved away from this endemic equilibrium and
density-dependent constraints are relaxed. This relaxation may make the
Re increase to greater than one initially, but the ratio will decrease in the
face of an effective control regimen, eventually becoming less than one,
namely, when each female worm is unable to replace itself (Basanez and
Boussinesq, 1999; Basanez and Ricardez-Esquinca, 2001; Basanez et al.,
2007; Churcher et al., 2005, 2006; Duerr et al., 2005, 2006).
In order for transmission to be significant from an epidemiological
perspective, therefore, it must occur at a level that maintains the R0 at a
value equal to or greater than one. If a control programme can succeed in
bringing transmission to a level that results in a reduction of the R0 to
below one and if it can maintain this until a point where the parasite
population is no longer able to increase transmission to a level where the
R0 equals or surpasses one when control ends, the parasite population
will eventually become extinct in the area under control. The R0 provides
a threshold criterion, which will be determined by the force of infection,
that is, the rate at which susceptible individuals become infected by an
infectious disease which may be measured indirectly by the ATP.
Unfortunately, the exact relationship between the ATP and the R0 is not
precisely known, and the threshold ATP necessary to maintain the R0
below one depends on many factors such as vector competence, biting
rates, degrees of vector anthropophagy, strength of positive and negative
density dependence and parasite intensity and distribution among other
heterogeneities. However, previous deterministic modelling studies
using data derived from West Africa have suggested that threshold prob-
ably lies somewhere between 5 and 20 L3s per person per year (Basanez
et al., 2007; Duerr et al., 2006). However, both authors discuss the caveats
inherent in their estimations concluding that in any case, any observed
ATP in a pre-control situation refers to an endemic ATP, not an ATP that
may correspond to an unstable equilibrium or breakpoint density.
In particular, where pretreatment data for the level of transmission do
not exist, the upper bounds for the 95% confidence intervals for both the
prevalence of infective flies and the biting rate, that is, the ATP should be
calculated. If the maximum transmission potential is shown to be within
that range referred to as the threshold ATP, then the parasite popula-
tion is likely on the path to elimination. This datum when taken together
with other epidemiological information can conclusively suggest that
transmission may have been suppressed in the communities under
study (Rodrguez-Perez et al., 2008b). It must be emphasized that even
when transmission has been suppressed, treatment cannot be discontin-
ued immediately. Transmission may be suppressed by treatment, but it
may rebound if the pressure on the population is removed. Thus, it is
necessary to maintain control activities until the level of transmission is so
low that any rebound in transmission that occurs when control activities
end will not reach a level that will cause the reproduction R0 ratio to
increase above the breakpoint. Unfortunately, it is difficult to predict to
what extent transmission will increase once control activities are ended.
This is because the degree of the increase will depend in part upon the
competence of the vector, which may, in turn, depend upon microfilarial
skin densities, with vectors that lack a cibarial armature, such as
S. damnosum s.l., S. exiguum s.l. and S. guianense s.l., being quite competent
at low densities (Basanez and Rodriguez, 2004; Duerr et al., 2005; Vieira
et al., 2005) while vectors possessing an armature such as S. ochraceum s.l.
being less competent at low densities (Basanez et al., 2002, 2009). Basanez
et al. (2009) pointed out that, in onchocerciasis, the higher the vector biting
rates, the lower the threshold ATP, making O. volvulus harder to elimi-
nate. Hence, vector control may be of significance for parasite elimination.
Individuals also differ in compliance and responsiveness to treatment,
which may also lead to an aggregated or overdispersed distribution of
parasites (with a few hosts harbouring the majority of the parasites). In
lymphatic filariasis, the aggregation enhances transmission because it
increases the probability that female and male worms will mate, and it
increases with higher worm burdens (Churcher et al., 2005, 2006; Stolk
et al., 2006), which again make it difficult to predict with certainty when
treatment may be safely stopped. These issues can be explored with
relevant mathematical models, which will have to be individually tailored
to the ecology of each focus in the Americas (Rodrguez-Perez et al.,
2008a). Once the parasite transmission is interrupted, a country can
decide when mass drug administration can be stopped and develop a

sensitive posttreatment surveillance system.
4.8.1. The intensive epidemiological surveillance programme in

Latin America during the post treatment era
The computer simulation model for onchocerciasis in the Americas
(SIMON-a) is a microsimulation model designed to emulate the transmis-
sion dynamics of onchocerciasis in Latin America. It mimics any chosen
community and the vectors associated with it and follows the progress of the
disease in that community as it is subjected to variable regimes of ivermectin
distribution (Dadzie et al., 2003; Davies, 1993). All versions of the SIMON-a
model indicate that at the end of the treatment regime, there will be a period
of 35 years during which very few members of a community will continue
to be infected at a low parasite level. These members are the residue of the 2
3% who are not eligible for treatment plus chronic refusers and absentees
and the occasional individual who does not respond to treatment. Since they
will not have received a proper regime of ivermectin, they will remain
infected (and theoretically infective to vectors) for a period of some
10 years, the average estimated lifespan of the adult worms (Bradley et al.,
2005), from the time that transmission is effectively stopped. It is likely that
such individuals will remain infectious to the vector population, meaning
that a very low level of transmission might continue, and perhaps even
increase somewhat once treatment is halted (Dadzie et al., 2003). This even-
tuality has some important strategic implications for the control pro-
grammes in the posttreatment era. Firstly, because the infected individuals
are likely to represent a small segment of the overall population, the number
of flies carrying parasites of any stage is likely to be very low. It will thus be
important to screen as many flies as possible to monitor this low level of
parasitevector contact and therefore detect any uptick in transmission at an
early stage. This may require the development of new more efficient meth-
ods to collect and process flies. Secondly, it will be important to identify and
monitor any such infected members of each community and to attempt to
convince them to undergo treatment if possible. While their number and
intensity of infection may not pose a risk to recrudescence, the disease cannot
be claimed to be permanently eliminated while they are in the community,
and they may represent a significant threat for transmission to redevelop.
Therefore, someone in each treated community or district should be given
the responsibility to keep track of these potentially infected people and also
to record the arrival of any incomers who might be infected (e.g. old resi-
dents returning from cities who were infected on departure and who have
escaped regular treatment. Itinerant workers are a special case). This moni-
toring should continue for at least 5 years. Again, this may require the
development of new tests capable of detecting patently infected individuals.
Thirdly, increases in the vector density or seasonal changes in parous vector

density (Rodrguez-Perez et al., 2007) during the post treatment era may
allow the parasite to re-establish itself in the host population; thus, measures
aimed at reducing the vector population, in particular, high risk endemic
communities could play a significant role in aiding parasite elimination.
If infective stage parasites are found to exist in the vector population, it
will be necessary to determine if this represents a level of transmission that
is a threat to the goal of eventual elimination. In any infectious disease, the
R0 must be kept at a level of one or above for the infectious agent to
maintain itself. If transmission occurs, but the rate is insufficient to main-
tain the R0 at one or above, the infectious agent is on the path to extinction.
In the case of onchocerciasis, if the ATP is maintained at a level that results
in a R0 of less than one, the parasite population will never recover and will
eventually go extinct. Although it has not been possible to determine
precisely what level of ATP is necessary to maintain the R0 at one, the
ABR from some African and Central American vectors has been correlated
with R0. Seven thousand six hundred and sixty-five S. ochraceum s.l. bites
per person per year was estimated to be the minimum below which human
onchocerciasis would be unable to become endemic in Central America,
that is, the R0 would be < 1. In contrast, the critical ABR for higher compe-
tent vectors such as those from the savannah members of the S. damnosum
complex ranged from 720 in Northern Cameroon and 288 in Burkina Faso
and Cote dIvoire. In localities with endemic equilibrium, R0 values ranged
from 5.3 in the Amazonian focus to 7.7 in Northern Cameroon (R0 value in
Guatemala was estimated to be of 7.3). The 95% CI of these R0 values
ranged from 4.1 to 8.9 (Filipe et al., 2005). Studies to further explore the
relationship between R0 and ATP are, therefore, necessary to determine
whether any residual transmission detected following the end of treatment
will represent an existential threat to the goal of elimination.
4.9. FUTURE DEVELOPMENTS
4.9.1. Basic research

Future developments should include more sensitive diagnostic tools that
may supplement or supplant the antibody detection technique such as
novel antigen detection systems (Molyneux, 2009), new vector trapping
tools for monitoring of transmission avoiding the use of human baits,
finding a macrofilaricide and developing a system to detect drug resis-
tance (Churcher and Basanez, 2009; Taylor et al., 2009). In the end, the
most effective control method would be the formulation of a drug that can
destroy adult worms and can be distributed via community-based
schemes (Alley et al., 2001). In the meantime, it is imperative to
understand about the parasite and its relation with the host, the nature of
the systemic effects of O. volvulus infection (Pion et al., 2009), the natural
history of skin disease and a better appreciation of the economic and
social consequences of this disease. When the O. volvulus genome and
those of its vectors, the black flies, are completed, several basic research
studies are likely to be developed. The following are highlighted: identi-
fication of DNA microsatellite/SNPs to map transition zones of transmis-
sion in Africa; genetics of vector competence, insecticide resistance,
anthropophilic attraction and salivary proteins, and odour binding pro-
teins; phylogenetics of nuclear and mitocondrial genes; PCR diagnosis of
DNA inversion breakpoints; and vector-parasite interactomes.
4.9.2. Applied research

At present, the goal of the OEPA is to stop transmission in areas with
ongoing transmission such as the bi-national Amazonian focus and
completely eliminate the reservoirs of infection in other areas where trans-
mission has been interrupted. In theory, onchocerciasis elimination may be
achieved by interrupting transmission over a 10-year period, the average
estimated lifespan of the adult worms (Bradley et al., 2005). However, for
various reasons, it has not been possible to stop parasite transmission in
some endemic areas in Latin America. It is necessary to establish sustainable
long-term distribution programmes of ivermectin in areas with ongoing
transmission, to develop effective control strategies using ivermectin
treatment alone and in combination with other drugs. Vector control pro-
grammes in Latin America have been extremely difficult to sustain in remote
endemic areas with high numbers of breeding sites which are the targets
from larviciding. Hence, it is not realistic to call for vector control in such
areas given the elevated operational costs. To investigate more targeted
approaches such as the use of doxycycline would be a more sustainable
and cost-effective approach. The presence of potentially infected itinerant
migrants which are not treated regularly could jeopardize the attainment of
the 85% coverage which is the present target for elimination of the disease
(Rodrguez-Perez et al., 2007). Thus, new strategies to improve drug Mecti-
zan coverage and compliance in areas with residual transmission that will
include the treatment of temporary settlers should be beneficial for the
attainment of the elimination goals of the regional initiative (Rodrguez-
Perez et al., 2007). In general, it is also recommended the incorporation of
other means to stop transmission, such as interventions aimed at reducing
the vector population (Basanez et al., 2009; Bradley et al., 2005); however, in
particular, this approach is not sustainable in the bi-national Amazonian
focus. Any measures of control implemented should be carefully evaluated
with the most sensitive and specific diagnostic tests currently available
(Burbelo et al., 2009; Lipner et al., 2006). Yet, it is extremely urgent to
incorporate more accurate, sensitive and non-invasive diagnostic tests for

overcoming the problems associated with the onchocerciasis control, elimi-
nation and post-control surveillance. The first step taken towards this direc-
tion has been done by a group of the Scripps Research Institute in La Joya.
California, USA which has used LCMS-based parasite metabolomics to
discover diagnostic biomarkers for onchocerciasis. The diagnostic approach
consists of a set of 14 metabolomics biomarkers that may be useful in
identifying individuals with active infection (Denery et al., 2010). The utility
and accuracy of this test as well as its general applicability remain to be
validated.
ACKNOWLEDGEMENTS
This project was supported by the Pan American Health Organization (PAHO)/World
Health Organization (WHO) and TDR/UNICEF/UNDP/WORLD BANK/WHO/(Contract
No. ME/CNT/0800238.001). We are grateful to the Onchocerciasis Programmes in Latin
America (Mexico, Guatemala, Ecuador, Colombia, Venezuela, and Brazil) and the Oncho-
cerciasis Elimination Program for the Americas (OEPA) for the report herein of unpublished
and published data.
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INDEX
A anabolic and catabolic phases,

metabolism, 150
AD. See Aspartic peptidases (AD)
BRENDA, 150155
Ascaris, Schistosoma coinfection, 60, 7172
CHEMBL-Neglected Tropical Disease
Aspartic peptidases (AD), 113
databases, 150152
and genetic research, limitations,
B
157158
Bacteria, Schistosoma coinfection GTPases, 154156
Helicobacter pyroli, 44, 4950 in silico drug target prediction,
Mycobacterium, 4349 150152
Staphylococcus aureus, 44, 50 NGS, 157158
Braunschweig Enzyme Database potential drug targets prediction,
(BRENDA), 150155 156157
BRENDA. See Braunschweig Enzyme sequencing, 156158
Database (BRENDA) TDR targets database, 150152, 153
WGA systems, 157158
C and genotypes, 144145
in vitro culturing techniques, 158160
Cdc42/Rac Interactive binding (CRIB) illness and oocyst excretion
domains, 126 patterns, 145
CHEMBL-Neglected Tropical Disease infections, 142143
databases, 150152 preventative measures, 143
Cryptic parasite. See Cryptosporidium quantitative real-time PCR tools,
Cryptosporidium 159160, 161
cell-free cultures, 159160 transcriptomics, 149158
C. hominis Cysteine peptidases, 111
anthroponotic pathways, 144145
cell-free cultures, 159160 D
genomes, 149150
genomic sequencing, 156157 Dermatological lesions (dermal pathology),
life cycle, 145147 186187
C. parvum DNA probes development
anthroponotic pathways, 144145 (LCMS)-based parasite metabolomics,
cell-free cultures, 159160 206
genomes, 149150 O-150 PCR, 207208
genomic sequencing, 156157 PCR-based assay, 207
life cycle, 145147
E
molecular methods, 144145
zoonotic transmission, 144145 Echinostoma, Schistosoma coinfection, 5970
cryptosporidiosis (see Human Entamoeba, Schistosoma coinfection
cryptosporidiosis) animal studies, 25, 34
genomics human studies, 25, 3435
227
228 Index
F ELISA format, 201202

finger prick blood test, 205
Fasciola species, Schistosoma coinfection
IgG4 antibody positives, 205206
animal studies, 51, 5758
OC 3.6 and OC 9.3, 204
human studies, 51, 5859
Ov33 molecule, 202
Filarids, Schistosoma coinfection, 60, 73
Ov16, Ov7, Ov11 and OC 3.6 antigens, 204
specificity and sensitivity, 34 recombinant
G
proteins, 203204
Glycocalyx W. bancrofti, 204
GPI biosynthesis, 104108 Intensive epidemiological surveillance
TvLPG, 113 programme, 212213
Glycosylphosphatidylinositol (GPI) Ivermectin treatment, onchocerciasis
biosynthesis vs. DEC, 196
anchor biosynthetic genes, 105 efficacy, 197
EhBspA proteins, 108 glutamate-gated chloride channels,
Golgi, 107108 195196
Pathogen Associated Molecular Patterns
(PAMPs), 104 K
secondary loss, 107
60 kDa glycoprotein gene (gp60), 156157
TvDPM1 homologue, 104107
GTPases L
and anti-cancer therapies, 155156
anti-cryptosporidial compounds, design, Lateral gene transfers (LGTs), 91
155156 Leishmania, Schistosoma coinfection
biological pathways, parasitic protozoa, pathological effects, 32
154155 time interval, 32
LGTs. See Lateral gene transfers (LGTs)
H
M
Helicobacter pyroli, Schistosoma coinfection,
44, 4950 Membrane trafficking and cell signalling
Helminths, Schistosoma coinfection GTPases, ESCRT and ATG
Ascaris, 60, 7172 ESCRT and ATG proteins, 122123
Echinostoma, 5970 heterotrimeric G-proteins and
Filarids, 60, 73 GPCR-RGS proteins, 118120
hookworm, 60, 70 small GTPasesRas and ARF
Strongyloides and Trichostrongyloides, 60, superfamily, 120122
7273 protein kinases
Trichuris, 60, 7071 accessory domains, 125
Hookworm, Schistosoma coinfection, 60, 70 aPK families, 126
Human cryptosporidiosis bioinformatics analyses, 123124
epithelial tissues, hyperplasia, 149 Cdc42/Rac Interactive binding (CRIB)
extra-gastrointestinal, 147148 domains, 126
gastric cryptosporidiosis, 147 ePK domains, 124
immune responses, 149 kinome, 125
innate and adaptive host response, 149 whilst E. histolytica, 124
pathophysiology, diarrhoea, 148149 Metallopeptidases, 115116
small intestine, infection, 147 Monitoring and evaluation, onchocerciasis
DNA probes development
I (LCMS)-based parasite
metabolomics, 206
Immunological tests O-150 PCR, 207208
bancroftian sera, 202203 PCR-based assay, 207
Index 229
immunological and molecular diagnosis, through chemotherapy, 194195

O. volvulus infection, 200201 through nodulectomy, 193
immunological tests through vector control, 194
bancroftian sera, 202203 distribution in Latin America
ELISA format, 201202 endemic communities, 180, 181
finger prick blood test, 205 in Guatemala, 178179
IgG4 antibody positives, 205206 in Mexico, 179182
OC 3.6 and OC 9.3, 204 prevalence in Africa, 178179
Ov33 molecule, 202 stratification, 179182
Ov16, Ov7, Ov11 and OC 3.6 antigens, drugs development, 197199
204 entomological parameters, 212213
specificity and sensitivity, 34 genetic variation, O. volvulus, 187189
recombinant proteins, 203204 infection, filarial nematode Onchocerca
W. bancrofti, 204 volvulus, 178
prasitological diagnosis, O. volvulus insect vectors
infection, 200 American vectors, 182184
Mycobacterium, Schistosoma coinfection, intrinsic capacity, 185
4349 simuliid females, 184185
monitoring and evaluation
N DNA probes development, 206209
immunological and molecular
Next-generation sequencing (NGS), diagnosis, O. volvulus infection,
157158, 162 200201
Non-peptidase homologues, 117 immunological tests, 201206
parasitological diagnosis, O. volvulus
O infection, 200
Ocular lesions (ocular pathology), 187 pathology and clinical manifestations
Onchocerca volvulus dermatological lesions (dermal
genetic variation, 187189 pathology), 186187
immunological and molecular diagnosis, ocular lesions (ocular pathology),
200201 187
infection, filarial nematode, 178 onchocercomata (nodules), 185186
pathology and clinical manifestations Onchocercomata (nodules), 185186
dermatological lesions (dermal
pathology), 186187 P
ocular lesions (ocular pathology), 187 Peptidases, T. vaginalis
onchocercomata (nodules), 185186 AD, 113
parasitological diagnosis, 200 clans and families, 112
Onchocerciasis cysteine, 111
advantages and disadvantages, distribution, 111
ivermectin treatment, 195197 inhibitors, 116
applied research, 214215 metallopeptidases, 115116
basic research, 213215 non-peptidase homologues, 117
control serine, 115
distribution, human onchocerciasis threonine, 116
endemic countries, 192 Plasmodium falciparum, in silico drug target
microfilaricidal drug ivermectin, prediction, 150152, 153
189190 Plasmodium, Schistosoma coinfection
OCPs in, 190193 animal studies, 9, 2123
ocular morbidity and prevalence, 191 human studies, 9, 2324
Simulium flys breeding Protozoans, Schistosoma coinfection
requirements, 189 Entamoeba, 25, 3435
230 Index
Protozoans, Schistosoma coinfection (cont.) T

Leishmania, 25, 32
Toxoplasma, 25, 3234 Threonine peptidases, 116
Trypanosoma, 25, 35 Toxoplasma gondii, Schistosoma coinfection
animal studies, 25, 33
S human studies, 25, 3334
Transcriptome and RNAi machinery
Salmonella, Schistosoma coinfection argonaute protein domains, 128
animal studies, 3642 Dicer-like protein, 127, 128
human studies, 37, 4243 dsRNA viruses, 129130
Schistosoma coinfection E. histolytica dsRNase, 128
with bacteria enormous coding capacity, 126
Helicobacter pyroli, 4950 sequence alignment, PIWI-BOX from
Mycobacterium, 4349 AGO proteins, 129
Staphylococcus aureus, 50 siRNA binding and mRNA cleavage
factors, 3 activity, 127
with Fasciola species Trematoda. See Schistosoma coinfection
animal studies, 51, 5758 Trichomonas vaginalis
human studies, 51, 5859 accessory domains, 125
and helminths cellular process, 8990
Ascaris, 60, 7172 description, 88
Echinostoma, 5970 as female nuisance, 88
Filarids, 60, 73 genome-scale data, 89
hookworm, 60, 70 glycosylphosphatidylinositol (GPI)
Strongyloides and Trichostrongyloides, 60, anchors, 90
7273 kinome, 125
Trichuris, 60, 7071 11 legume-like lectin receptors, 94
organisms, synopsis of, 4, 5 medical importance, 89
and Plasmodium membrane trafficking and cell signalling
animal studies, 2123 ESCRT and ATG proteins, 122123
human studies, 2324 GTPases, ESCRT and ATG, 118123
and protozoans protein kinases, 123126
Entamoeba, 3435 small GTPasesRas and ARF
Leishmania, 32 superfamily, 120122
Toxoplasma, 3234 peptidases
Trypanosoma, 35 AD, 113
with Salmonella cysteine, 113115
animal studies, 3642 inhibitors, 116
human studies, 37, 4243 metallopeptidases, 115116
Serine peptidases, 115 non-peptidase homologues, 117
Staphylococcus aureus, Schistosoma serine, 115
coinfection, 44, 50 threonine, 116
Strongyloides and Trichostrongyloides, phagocytose human cells, 8990
Schistosoma coinfection, 60, 7273 secreted proteins, 108111
Surface proteins, T. vaginalis surface proteins
glycocalyx, 100108 glycocalyx, 100108
proteomics data proteomics data, 97100
hypothetical proteins, 99100 TvBspA, 9093
with known homologues, 9799 TvPmp, 9394
TvBspA, 9093 Zn-metallopeptidase, 9496
TvPmp, 9394 transcriptome and RNAi machinery,
Zn-metallopeptidase, 9496 126130
Index 231
Trichopores putative overall structural organisation,

amoebapores and naegleriapores, 109 102
haemolysis, 108109 TvBspA
pore formation, 110 BspA-like proteins, 9091
SAPLIP domains, 109110 identification, cell surface proteomics, 92
structural organisation, TvSaplip112, LGTs, 91
110 TpLRR, 9091
TvSaplip genes, 110111 transmembrane domains (TMDs), 91, 93
Trichuris, Schistosoma coinfection TvBspA805, 9293
animal studies, 60, 7071 TvLPG. See T. vaginalis lipophosphoglycan
human studies, 60, 71 (TvLPG)
Tropical Disease Research (TDR) targets TvPmp
database, 150152, 153 Chlamydia spp., 93
Trypanosoma, Schistosoma coinfection, 25, 35 proteomics survey, 94
T. vaginalis lipophosphoglycan (TvLPG) TrichDB 48, 9394
GPI biosynthesis
anchor biosynthetic genes, 105 W
EhBspA proteins, 108
Whole genomic amplification (WGA)
Golgi, 107108
systems, 157158
Pathogen Associated Molecular
Patterns (PAMPs), 104
Z
secondary loss, 107
TvDPM1 homologue, 104107 Zn-metallopeptidase
Leishmania LPGs, 100102 M17 protein, 9496
precise function(s), 103 25 TvM60-like/PF13402-containing
primary structure, 103104 proteins, 9496
CONTENTS OF VOLUMES
IN THIS SERIES
Volume 41 M. Albonico, D.W.T. Cromption, and

L. Savioli
Drug Resistance in Malaria Parasites of
Animals and Man DNA Vaocines: Technology and
W. Peters Applications as Anti-parasite and
Anti-microbial Agents
Molecular Pathobiology and Antigenic J.B. Alarcon, G.W. Wainem and
Variation of Pneumocystis carinii D.P. McManus
Y. Nakamura and M. Wada
Ascariasis in China
P. Weidono, Z. Xianmin and Volume 43
D.W.T. Crompton
Genetic Exchange in the
The Generation and Expression of Trypanosomatidae
Immunity to Trichinella spiralis in W. Gibson and J. Stevens
Laboratory Rodents
R.G. Bell The Host-Parasite Relationship in
Neosporosis
Population Biology of Parasitic A. Hemphill
Nematodes: Application of
Genetic Markers Proteases of Protozoan Parasites
T.J.C. Anderson, M.S. Blouin and P.J. Rosenthal
R.M. Brech Proteinases and Associated Genes of
Schistosomiasis in Cattle Parasitic Helminths
J. De Bont and J. Vercruysse J. Tort, P.J. Brindley, D. Knox, K.H. Wolfe,
and J.P. Dalton
Parasitic Fungi and their
Volume 42 Interaction with the Insect
Immune System
The Southern Cone Initiative Against A. Vilcinskas and P. Gotz
Chagas Disease
C.J. Schofield and J.C.P. Dias
Phytomonas and Other Trypanosomatid
Parasites of Plants and Fruit Volume 44
E.P. Camargo
Cell Biology of Leishmania
Paragonimiasis and the Genus B. Handman
Paragonimus
Immunity and Vaccine Development in
D. Blair, Z.-B. Xu, and T. Agatsuma
the Bovine Theilerioses
Immunology and Biochemistry of N. Boulter and R. Hall
Hymenolepis diminuta
The Distribution of Schistosoma bovis
J. Anreassen, E.M. Bennet-Jenkins, and
Sonaino, 1876 in Relation to
C. Bryant
Intermediate Host Mollusc-Parasite
Control Strategies for Human Intestinal Relationships
Nematode Infections H. Mone, G. Mouahid, and S. Morand
233
234 Contents of Volumes in This Series
The Larvae of Monogenea Satellites, Space, Time and the African

(Platyhelminthes) Trypanosomiases
I.D. Whittington, L.A. Chisholm, and D.J. Rogers
K. Rohde Earth Observation, Geographic
Sealice on Salmonids: Their Biology Information Systems and
and Control Plasmodium falciparum Malaria in
A.W. Pike and S.L. Wadsworth Sub-Saharan Africa
S.I. Hay, J. Omumbo, M. Craig, and
R.W. Snow
Volume 45
Ticks and Tick-borne Disease Systems in
The Biology of some Intraerythrocytic Space and from Space
Parasites of Fishes, Amphibia S.E. Randolph
and Reptiles
The Potential of Geographical
A.J. Davies and M.R.L. Johnston
Information Systems (GIS) and
The Range and Biological Activity of FMR Remote Sensing in the Epidemiology
Famide-related Peptides and and Control of Human Helminth
Classical Neurotransmitters Infections
in Nematodes S. Brooker and E. Michael
D. Brownlee, L. Holden-Dye, and R.
Advances in Satellite Remote Sensing of
Walker
Environmental Variables for
The Immunobiology of Gastrointestinal Epidemiological Applications
Nematode Infections in Ruminants S.J. Goetz, S.D. Prince, and J. Small
A. Balic, V.M. Bowles, and E.N.T.
Forecasting Diseases Risk for Increased
Meeusen
Epidemic Preparedness in Public
Health
Volume 46 M.F. Myers, D.J. Rogers, J. Cox, A.
Flauhalt, and S.I. Hay
Host-Parasite Interactions in
Acanthocephala: A Morphological Education, Outreach and the Future of
Approach Remote Sensing in Human Health
H. Taraschewski B.L. Woods, L.R. Beck, B.M. Lobitz, and
M.R. Bobo
Eicosanoids in Parasites and Parasitic
Infections
A. Daugschies and A. Joachim
Volume 48
The Molecular Evolution of
Volume 47 Trypanosomatidae
J.R. Stevens, H.A. Noyes, C.J. Schofield,
An Overview of Remote Sensing and and W. Gibson
Geodesy for Epidemiology and
Public Health Application Transovarial Transmission in the
S.I. Hay Microsporidia
A.M. Dunn, R.S. Terry, and J.E. Smith
Linking Remote Sensing, Land Cover
and Disease Adhesive Secretions in the
P.J. Curran, P.M. Atkinson, G.M. Foody, Platyhelminthes
and E.J. Milton I.D. Whittington and B.W. Cribb
Spatial Statistics and Geographic The Use of Ultrasound in Schistosomiasis

C.F.R. Hatz
Information Systems in
Epidemiology and Public Health Ascaris and Ascariasis
T.P. Robinson D.W.T. Crompton
Volume 49 Volume 52
Antigenic Variation in Trypanosomes: The Ecology of Fish Parasites with
Enhanced Phenotypic Variation in a Particular Reference to
Eukaryotic Parasite Helminth Parasites and their
H.D. Barry and R. McCulloch Salmonid Fish Hosts in Welsh
Rivers: A Review of Some of the
The Epidemiology and Control of Human
Central Questions
African Trypanosomiasis
J.D. Thomas
J. Pepin and H.A. Meda
Biology of the Schistosome Genus
Apoptosis and Parasitism: from the
Trichobilharzia
Parasite to the Host Immune
P. Horak, L. Kolarova, and C.M. Adema
Response
G.A. DosReis and M.A. Barcinski The Consequences of Reducing
Transmission of Plasmodium
Biology of Echinostomes Except
falciparum in Africa
Echinostoma
R.W. Snow and K. Marsh
B. Fried
Cytokine-Mediated Host Responses
during Schistosome Infections:
Volume 50 Walking the Fine Line Between
The Malaria-Infected Red Blood Cell: Immunological Control and
Structural and Functional Changes Immunopathology
B.M. Cooke, N. Mohandas, and R.L. K.F. Hoffmann, T.A. Wynn, and D.W.
Coppel Dunne
Schistosomiasis in the Mekong Region:

Epidemiology and Phytogeography
S.W. Attwood Volume 53
Molecular Aspects of Sexual Interactions between Tsetse
Development and Reproduction in and Trypanosomes with
Nematodes and Schistosomes Implications for the Control of
P.R. Boag, S.E. Newton, and R.B. Gasser Trypanosomiasis
S. Aksoy, W.C. Gibson, and M.J. Lehane
Antiparasitic Properties of Medicinal
Plants and Other Naturally Enzymes Involved in the Biogenesis of
Occurring Products the Nematode Cuticle
S. Tagboto and S. Townson A.P. Page and A.D. Winter
Diagnosis of Human Filariases (Except
Volume 51 Onchocerciasis)
M. Walther and R. Muller
Aspects of Human Parasites in which
Surgical Intervention May Be
Important
D.A. Meyer and B. Fried Volume 54
Electron-transfer Complexes in Ascaris Introduction Phylogenies,
Mitochondria Phylogenetics, Parasites and the
K. Kita and S. Takamiya Evolution of Parasitism
D.T.J. Littlewood
Cestode Parasites: Application of In Vivo
and In Vitro Models for Studies of the Cryptic Organelles in Parasitic Protists
Host-Parasite Relationship and Fungi
M. Siles-Lucas and A. Hemphill B.A.P. Williams and P.J. Keeling
Phylogenetic Insights into the Evolution The Mitochondrial Genomics of Parasitic

of Parasitism in Hymenoptera Nematodes of Socio-Economic
J.B. Whitfield Importance: Recent Progress, and
Implications for Population Genetics
Nematoda: Genes, Genomes and the
and Systematics
Evolution of Parasitism
M. Hu, N.B. Chilton, and R.B. Gasser
M.L. Blaxter
The Cytoskeleton and Motility in
Life Cycle Evolution in the Digenea: A
Apicomplexan Invasion
New Perspective from Phylogeny
R.E. Fowler, G. Margos, and G.H. Mitchell
T.H. Cribb, R.A. Bray, P.D. Olson, and
D.T.J. Littlewood
Progress in Malaria Research: The Case Volume 57
for Phylogenetics Canine Leishmaniasis
S.M. Rich and F.J. Ayala J. Alvar, C. Canavate, R. Molina, J.
Phylogenies, the Comparative Moreno, and J. Nieto
Method and Parasite Evolutionary Sexual Biology of Schistosomes
Ecology H. Mone and J. Boissier
S. Morand and R. Poulin
Review of the Trematode Genus Ribeiroia
Recent Results in Cophylogeny Mapping (Psilostomidae): Ecology, Life
M.A. Charleston History, and Pathogenesis with
Inference of Viral Evolutionary Rates Special Emphasis on the Amphibian
from Molecular Sequences Malformation Problem
A. Drummond, O.G. Pybus, and A. P.T.J. Johnson, D.R. Sutherland,
Rambaut J.M. Kinsella and K.B. Lunde
Detecting Adaptive Molecular Evolution: The Trichuris muris System: A Paradigm

Additional Tools for the of Resistance and Susceptibility to
Parasitologist Intestinal Nematode Infection
J.O. McInerney, D.T.J. Littlewood, and L.J. Cliffe and R.K. Grencis
C.J. Creevey Scabies: New Future for a Neglected
Disease
Volume 55 S.F. Walton, D.C. Holt, B.J. Currie, and
D.J. Kemp
Contents of Volumes 2852
Cumulative Subject Indexes for Volumes
2852
Contributors to Volumes 2852
Volume 58
Leishmania spp.: On the Interactions they
Establish with Antigen-Presenting
Volume 56 Cells of their Mammalian Hosts
J.-C. Antoine, E. Prina, N. Courret, and
Glycoinositolphospholipid from
T. Lang
Trypanosoma cruzi: Structure,
Biosynthesis and Immunobiology Variation in Giardia: Implications
J.O. Previato, R. Wait, C. Jones, for Taxonomy and Epidemiology
G.A. DosReis, A.R. Todeschini, N. R.C.A. Thompson and P.T. Monis
Heise and L.M. Previata
Recent Advances in the Biology of
Biodiversity and Evolution of the Echinostoma species in the
Myxozoa revolutum Group
E.U. Canning and B. Okamura B. Fried and T.K. Graczyk
Human Hookworm Infection in the Volume 61

21st Century
S. Brooker, J. Bethony, and P.J. Hotez Control of Human Parasitic
Diseases: Context and Overview
The Curious Life-Style of the David H. Molyneux
Parasitic Stages of Gnathiid Isopods
N.J. Smit and A.J. Davies Malaria Chemotherapy
Peter Winstanley and Stephen Ward
Insecticide-Treated Nets
Volume 59 Jenny Hill, Jo Lines, and Mark Rowland
Genes and Susceptibility to Control of Chagas Disease
Leishmaniasis Yoichi Yamagata and
Emanuela Handman, Colleen Elso, and Jun Nakagawa
Simon Foote
Human African Trypanosomiasis:
Cryptosporidium and Cryptosporidiosis Epidemiology and Control
R.C.A. Thompson, M.E. Olson, G. Zhu, E.M. Fevre, K. Picozzi, J. Jannin,
S. Enomoto, Mitchell S. Abrahamsen S.C. Welburn and I. Maudlin
and N.S. Hijjawi
Chemotherapy in the Treatment and
Ichthyophthirius multifiliis Fouquet and Control of Leishmaniasis
Ichthyophthiriosis in Freshwater Jorge Alvar, Simon Croft, and
Teleosts Piero Olliaro
R.A. Matthews
Dracunculiasis (Guinea Worm Disease)
Biology of the Phylum Nematomorpha Eradication
B. Hanelt, F. Thomas, and A. Schmidt- Ernesto Ruiz-Tiben and Donald
Rhaesa R. Hopkins
Intervention for the Control of Soil-
Volume 60 Transmitted Helminthiasis in the
Community
Sulfur-Containing Amino Acid
Marco Albonico, Antonio Montresor, D.W.
Metabolism in Parasitic Protozoa T. Crompton, and Lorenzo Savioli
Tomoyoshi Nozaki, Vahab Ali, and
Masaharu Tokoro Control of Onchocerciasis
Boakye A. Boatin and Frank O. Richards,
The Use and Implications of Ribosomal Jr.
DNA Sequencing for the
Discrimination of Digenean Species Lymphatic Filariasis: Treatment, Control
Matthew J. Nolan and Thomas H. Cribb and Elimination
Eric A. Ottesen
Advances and Trends in the Molecular
Systematics of the Parasitic Control of Cystic Echinococcosis/
Platyhelminthes Hydatidosis: 18632002
Peter D. Olson and Vasyl V. Tkach P.S. Craig and E. Larrieu
Wolbachia Bacterial Endosymbionts of Control of Taenia solium Cysticercosis/
Filarial Nematodes Taeniosis
Mark J. Taylor, Claudio Bandi, and Achim Arve Lee Willingham III and
Hoerauf Dirk Engels
The Biology of Avian Eimeria with an Implementation of Human
Emphasis on their Control by Schistosomiasis Control: Challenges
Vaccination and Prospects
Martin W. Shirley, Adrian L. Smith, and Alan Fenwick, David Rollinson, and
Fiona M. Tomley Vaughan Southgate
Volume 62 Targeting of Toxic Compounds to the

Trypanosomes Interior
Models for Vectors and Vector-Borne Michael P. Barrett and Ian H. Gilbert
Diseases
D.J. Rogers Making Sense of the Schistosome
Surface
Global Environmental Data for Patrick J. Skelly and R. Alan Wilson
Mapping Infectious Disease
Distribution Immunology and Pathology of
S.I. Hay, A.J. Tatem, A.J. Graham, Intestinal Trematodes in Their
S.J. Goetz, and D.J. Rogers Definitive Hosts
Rafael Toledo, Jose-Guillermo Esteban, and
Issues of Scale and Uncertainty in Bernard Fried
the Global Remote Sensing of
Disease Systematics and Epidemiology of
P.M. Atkinson and A.J. Graham Trichinella
Edoardo Pozio and K. Darwin Murrell
Determining Global Population
Distribution: Methods, Applications
and Data
D.L. Balk, U. Deichmann, G. Yetman,
Volume 64
F. Pozzi, S.I. Hay, Leishmania and the Leishmaniases:
and A. Nelson A Parasite Genetic Update and
Defining the Global Spatial Limits of Advances in Taxonomy,
Malaria Transmission in 2005 Epidemiology and Pathogenicity
C.A. Guerra, R.W. Snow and S.I. Hay in Humans
Anne-Laure Banuls, Mallorie Hide and
The Global Distribution of Yellow Fever Franck Prugnolle
and Dengue
D.J. Rogers, A.J. Wilson, S.I. Hay, and Human Waterborne Trematode and
A.J. Graham Protozoan Infections
Thaddeus K. Graczyk and Bernard Fried
Global Epidemiology, Ecology and
Control of Soil-Transmitted Helminth The Biology of Gyrodctylid
Infections Monogeneans: The Russian-Doll
S. Brooker, A.C.A. Clements and Killers
D.A.P. Bundy T.A. Bakke, J. Cable, and P.D. Harris
Tick-borne Disease Systems: Mapping Human Genetic Diversity and the

Geographic and Phylogenetic Space Epidemiology of Parasitic
S.E. Randolph and D.J. Rogers and Other Transmissible Diseases
Michel Tibayrenc
Global Transport Networks and
Infectious Disease Spread
A.J. Tatem, D.J. Rogers and S.I. Hay Volume 65
Climate Change and Vector-Borne ABO Blood Group Phenotypes and
Diseases Plasmodium falciparum Malaria:
D.J. Rogers and S.E. Randolph Unlocking a Pivotal Mechanism
Mara-Paz Loscertales, Stephen Owens,
James ODonnell, James Bunn, Xavier
Bosch-Capblanch, and Bernard J. Brabin
Volume 63
Structure and Content of the Entamoeba
Phylogenetic Analyses of Parasites in the histolytica Genome
New Millennium C. G. Clark, U. C. M. Alsmark, M.
David A. Morrison Tazreiter, Y. Saito-Nakano, V. Ali,
S. Marion, C. Weber, C. Mukherjee, Volume 67

I. Bruchhaus, E. Tannich, M. Leippe, Introduction
T. Sicheritz-Ponten, P. G. Foster, Irwin W. Sherman
J. Samuelson, C. J. Noel, R. P. Hirt,
T. M. Embley, C. A. Gilchrist, An Introduction to Malaria Parasites
B. J. Mann, U. Singh, J. P. Ackers, Irwin W. Sherman
S. Bhattacharya, A. Bhattacharya, The Early Years
A. Lohia, N. Guillen, M. Duchene, Irwin W. Sherman
T. Nozaki, and N. Hall
Show Me the Money
Epidemiological Modelling for Irwin W. Sherman
Monitoring and Evaluation of
Lymphatic Filariasis Control In Vivo and In Vitro Models
Edwin Michael, Mwele N. Malecela- Irwin W. Sherman
Lazaro, and James W. Kazura Malaria Pigment
The Role of Helminth Infections in Irwin W. Sherman
Carcinogenesis Chloroquine and Hemozoin
David A. Mayer and Bernard Fried
Irwin W. Sherman
A Review of the Biology of the
Isoenzymes
Parasitic Copepod Lernaeocera
branchialis (L., 1767)(Copepoda: Irwin W. Sherman
Pennellidae The Road to the Plasmodium falciparum
Adam J. Brooker, Andrew P. Shinn, and Genome
James E. Bron Irwin W. Sherman
Carbohydrate Metabolism
Irwin W. Sherman
Volume 66 Pyrimidines and the Mitochondrion

Irwin W. Sherman
Strain Theory of Malaria: The First
50 Years The Road to Atovaquone
F. Ellis McKenzie,* David L. Smith, Irwin W. Sherman
Wendy P. OMeara, and Eleanor The Ring Road to the Apicoplast
M. Riley
Irwin W. Sherman
Advances and Trends in the Molecular
Ribosomes and Ribosomal Ribonucleic
Systematics of Anisakid Nematodes,
Acid Synthesis
with Implications for their
Irwin W. Sherman
Evolutionary Ecology and
HostParasite Co-evolutionary De Novo Synthesis of Pyrimidines
Processes and Folates
Simonetta Mattiucci and Giuseppe Irwin W. Sherman
Nascetti
Salvage of Purines
Atopic Disorders and Parasitic Infections Irwin W. Sherman
Aditya Reddy and Bernard Fried
Polyamines
Heartworm Disease in Animals and Irwin W. Sherman
Humans
John W. McCall, Claudio Genchi, Laura H. New Permeability Pathways
Kramer, Jorge Guerrero, and and Transport
Luigi Venco Irwin W. Sherman
Hemoglobinases Tracking Transmission of the Zoonosis

Irwin W. Sherman Toxoplasma gondii
Judith E. Smith
Erythrocyte Surface Membrane Proteins
Irwin W. Sherman Parasites and Biological Invasions
Alison M. Dunn
Trafficking
Irwin W. Sherman Zoonoses in Wildlife: Integrating Ecology
into Management
Erythrocyte Membrane Lipids Fiona Mathews
Irwin W. Sherman
Understanding the Interaction
Invasion of Erythrocytes Between an Obligate Hyperparasitic
Irwin W. Sherman Bacterium, Pasteuria penetrans
Vitamins and Anti-Oxidant Defenses and its Obligate Plant-Parasitic
Irwin W. Sherman Nematode Host, Meloidogyne spp.
Keith G. Davies
Shocks and Clocks
Irwin W. Sherman HostParasite Relations and Implications
for Control
Transcriptomes, Proteomes Alan Fenwick
and Data Mining
Irwin W. Sherman OnchocercaSimulium Interactions and the
Population and Evolutionary Biology
Mosquito Interactions of Onchocerca volvulus
Irwin W. Sherman Mara-Gloria Basanez, Thomas
S. Churcher, and Mara-Eugenia Grillet
Volume 68 Microsporidians as Evolution-Proof
Agents of Malaria Control?
HLA-Mediated Control of HIV and HIV
Jacob C. Koella, Lena Lorenz, and Irka
Adaptation to HLA
Bargielowski
Rebecca P. Payne, Philippa C. Matthews,
Julia G. Prado, and Philip J. R. Goulder
An Evolutionary Perspective on
Volume 69
Parasitism as a Cause of Cancer The Biology of the Caecal Trematode
Paul W. Ewald Zygocotyle lunata
Bernard Fried, Jane E. Huffman, Shamus
Invasion of the Body Snatchers:
Keeler, and Robert C. Peoples
The Diversity and Evolution of
Manipulative Strategies in Fasciola, Lymnaeids and Human
HostParasite Interactions Fascioliasis, with a Global
Thierry Lefevre, Shelley A. Adamo, David Overview on Disease Transmission,
G. Biron, Dorothee Misse, David Epidemiology, Evolutionary
Hughes, and Frederic Thomas Genetics, Molecular Epidemiology
and Control
Evolutionary Drivers of Parasite-Induced
Santiago Mas-Coma, Mara Adela Valero,
Changes in Insect Life-History Traits:
and Mara Dolores Bargues
From Theory to Underlying
Mechanisms Recent Advances in the Biology of
Hilary Hurd Echinostomes
Rafael Toledo, Jose-Guillermo Esteban, and
Ecological Immunology of a Tapeworms
Bernard Fried
Interaction with its Two Consecutive
Hosts Peptidases of Trematodes
Katrin Hammerschmidt and Martin Kasny, Libor Mikes, Vladimr
Joachim Kurtz Hampl, Jan Dvorak,
Components of Asobara Venoms and their

Conor R. Caffrey, John P. Dalton, and
Effects on Hosts
Petr Horak
Sebastien J.M. Moreau, Sophie Vinchon,
Potential Contribution of Anas Cherqui, and Genevieve Prevost
Sero-Epidemiological Analysis
Strategies of Avoidance of Host Immune
for Monitoring Malaria
Defenses in Asobara Species
Control and Elimination:
Genevieve Prevost, Geraldine Doury,
Historical and Current
Alix D.N. Mabiala-Moundoungou,
Perspectives
Anas Cherqui, and Patrice Eslin
Chris Drakeley and Jackie Cook
Evolution of Host Resistance and
Parasitoid Counter-Resistance
Volume 70 Alex R. Kraaijeveld and H. Charles
J. Godfray
Ecology and Life History Evolution of
Frugivorous Drosophila Parasitoids Local, Geographic and Phylogenetic
Frederic Fleury, Patricia Gibert, Scales of Coevolution in Drosophila
Nicolas Ris, and Roland Allemand Parasitoid Interactions
S. Dupas, A. Dubuffet, Y. Carton, and
Decision-Making Dynamics in
M. Poirie
Parasitoids of Drosophila
Andra Thiel and Thomas S. Hoffmeister DrosophilaParasitoid Communities as
Model Systems for HostWolbachia
Dynamic Use of Fruit Odours to Locate
Interactions
Host Larvae: Individual Learning,
Fabrice Vavre, Laurence Mouton, and
Physiological State and Genetic
Bart A. Pannebakker
Variability as Adaptive
Mechanisms A Virus-Shaping Reproductive Strategy
Laure Kaiser, Aude Couty, and in a Drosophila Parasitoid
Raquel Perez-Maluf Julien Varaldi, Sabine Patot,
Maxime Nardin, and Sylvain Gandon
The Role of Melanization and Cytotoxic
By-Products in the Cellular Immune
Responses of Drosophila Against
Parasitic Wasps Volume 71
A. Nappi, M. Poirie, and Y. Carton
Cryptosporidiosis in Southeast
Virulence Factors and Strategies of Asia: Whats out There?
Leptopilina spp.: Selective Responses Yvonne A.L. Lim, Aaron R. Jex,
in Drosophila Hosts Huw V. Smith, and Robin B. Gasser
Mark J. Lee, Marta E. Kalamarz,
Indira Paddibhatla, Chiyedza Small, Human Schistosomiasis in the Economic
Roma Rajwani, and Shubha Govind Community of West African States:
Epidemiology and Control
Variation of Leptopilina boulardi Success in Helene Mone, Moudachirou Ibikounle,
Drosophila Hosts: What is Inside the Achille Massougbodji, and Gabriel
Black Box? Mouahid
A. Dubuffet, D. Colinet, C. Anselme,
S. Dupas, Y. Carton, and M. Poirie The Rise and Fall of Human
Oesophagostomiasis
Immune Resistance of Drosophila Hosts A.M. Polderman, M. Eberhard, S. Baeta,
Against Asobara Parasitoids: Cellular Robin B. Gasser, L. van Lieshout,
Aspects P. Magnussen, A. Olsen, N.
Patrice Eslin, Genevieve Prevost, Spannbrucker, J. Ziem,
Sebastien Havard, and Geraldine Doury and J. Horton
Volume 72 Combating Taenia solium Cysticercosis

in Southeast Asia: An Opportunity
Important Helminth Infections in for Improving Human Health and
Southeast Asia: Diversity, Potential Livestock Production Links
for Control and Prospects for A. Lee Willingham III, Hai-Wei Wu, James
Elimination Conlan, and Fadjar Satrija
Jurg Utzinger, Robert Bergquist, Remigio
Olveda, and Xiao-Nong Zhou Echinococcosis with Particular Reference
to Southeast Asia
Escalating the Global Fight Against Donald P. McManus
Neglected Tropical Diseases Through
Interventions in the Asia Pacific Food-Borne Trematodiases in Southeast
Region Asia: Epidemiology, Pathology,
Peter J. Hotez and John P. Ehrenberg Clinical Manifestation and Control
Banchob Sripa, Sasithorn Kaewkes,
Coordinating Research on Neglected Pewpan M. Intapan, Wanchai
Parasitic Diseases in Southeast Asia Maleewong, and Paul J. Brindley
Through Networking
Remi Olveda, Lydia Leonardo, Feng Zheng, Helminth Infections of the Central
Banchob Sripa, Robert Bergquist, and Nervous System Occurring in
Xiao-Nong Zhou Southeast Asia and the Far East
Shan Lv, Yi Zhang, Peter Steinmann,
Neglected Diseases and Ethnic Minorities Xiao-Nong Zhou, and Jurg Utzinger
in the Western Pacific Region:
Exploring the Links Less Common Parasitic Infections in
Alexander Schratz, Martha Fernanda Southeast Asia that can Produce
Pineda, Liberty G. Reforma, Nicole M. Outbreaks
Fox, Tuan Le Anh, L. Tommaso Peter Odermatt, Shan Lv, and Somphou
Cavalli-Sforza, Mackenzie K. Sayasone
Henderson, Raymond Mendoza, Jurg
Utzinger, John P. Ehrenberg, and Ah
Sian Tee Volume 73
Controlling Schistosomiasis in Southeast Concepts in Research Capabilities
Asia: A Tale of Two Countries Strengthening: Positive Experiences
Robert Bergquist and Marcel Tanner of Network Approaches by TDR in
the Peoples Republic of China and
Schistosomiasis Japonica: Control and Eastern Asia
Research Needs Xiao-Nong Zhou, Steven Wayling, and
Xiao-Nong Zhou, Robert Bergquist, Robert Bergquist
Lydia Leonardo, Guo-Jing Yang,
Kun Yang, M. Sudomo, and Multiparasitism: A Neglected Reality on
Remigio Olveda Global, Regional and Local Scale
Peter Steinmann, Jurg Utzinger, Zun-Wei
Schistosoma mekongi in Cambodia and Du, and Xiao-Nong Zhou
Lao Peoples Democratic Republic
Sinuon Muth, Somphou Sayasone, Health Metrics for Helminthic Infections
Sophie Odermatt-Biays, Samlane Charles H. King
Phompida, Socheat Duong, and Implementing a Geospatial Health Data
Peter Odermatt Infrastructure for Control of Asian
Elimination of Lymphatic Filariasis in Schistosomiasis in the Peoples
Southeast Asia Republic of China and the
Mohammad Sudomo, Sombat Chayabejara, Philippines
Duong Socheat, Leda Hernandez, John B. Malone, Guo-Jing Yang, Lydia
Wei-Ping Wu, and Robert Bergquist Leonardo, and Xiao-Nong Zhou
The Regional Network for Asian Studies on the Parasitology,

Schistosomiasis and Other Phylogeography and the Evolution of
Helminth Zoonoses (RNAS ): HostParasite Interactions for the
Target Diseases in Face of Snail Intermediate Hosts of Medically
Climate Change Important Trematode Genera in
Guo-Jing Yang, Jurg Utzinger, Shan Lv, Southeast Asia
Ying-Jun Qian, Shi-Zhu Li, Qiang Stephen W. Attwood
Wang, Robert Bergquist, Penelope
Vounatsou, Wei Li, Kun Yang, and
Xiao-Nong Zhou
Volume 74
Social Science Implications for Control of
Helminth Infections in Southeast The Many Roads to Parasitism: A Tale of
Asia Convergence
Lisa M. Vandemark, Tie-Wu Jia, and Robert Poulin
Xiao-Nong Zhou Malaria Distribution, Prevalence, Drug
Towards Improved Diagnosis of Zoonotic Resistance and Control in Indonesia
Trematode Infections in Southeast Iqbal R.F. Elyazar, Simon I. Hay, and
Asia J. Kevin Baird
Maria Vang Johansen, Paiboon Cytogenetics and Chromosomes of
Sithithaworn, Robert Bergquist, and Tapeworms (Platyhelminthes,
Jurg Utzinger Cestoda)
The Drugs We Have and the Drugs We Marta Spakulova, Martina Orosova, and
Need Against Major Helminth John S. Mackiewicz
Infections Soil-Transmitted Helminths of Humans
Jennifer Keiser and Jurg Utzinger in Southeast AsiaTowards
Research and Development of Integrated Control
Antischistosomal Drugs in the Aaron R. Jex, Yvonne A.L. Lim, Jeffrey
Peoples Republic of China: Bethony, Peter J. Hotez, Neil D. Young,
A 60-Year Review and Robin B. Gasser
Shu-Hua Xiao, Jennifer Keiser, Ming-Gang The Applications of Model-Based
Chen, Marcel Tanner, and Jurg Geostatistics in Helminth
Utzinger Epidemiology and Control
Control of Important Helminthic Ricardo J. Soares Magalhaes, Archie C.A.
Infections: Vaccine Development as Clements, Anand P. Patil, Peter W.
Part of the Solution Gething, and Simon Brooker
Robert Bergquist and Sara Lustigman
Our Wormy World: Genomics,
Proteomics and Transcriptomics in Volume 75
East and Southeast Asia Epidemiology of American
Jun Chuan, Zheng Feng, Paul J. Trypanosomiasis (Chagas Disease)
Brindley, Donald P. McManus, Louis V. Kirchhoff
Zeguang Han, Peng Jianxin,
and Wei Hu Acute and Congenital Chagas Disease
Caryn Bern, Diana L. Martin, and
Advances in Metabolic Profiling of Robert H. Gilman
Experimental Nematode and
Trematode Infections Cell-Based Therapy in Chagas Disease
Yulan Wang, Jia V. Li, Jasmina Saric, Antonio C. Campos de Carvalho,
Jennifer Keiser, Junfang Wu, Jurg Adriana B. Carvalho, and
Utzinger, and Elaine Holmes Regina C.S. Goldenberg
Targeting Trypanosoma cruzi Sterol Volume 76

14a-Demethylase (CYP51)
Galina I. Lepesheva, Fernando Villalta, Bioactive Lipids in Trypanosoma cruzi
and Michael R. Waterman Infection
Fabiana S. Machado, Shankar Mukherjee,
Experimental Chemotherapy and Louis M. Weiss, Herbert B. Tanowitz,
Approaches to Drug Discovery for and Anthony W. Ashton
Trypanosoma cruzi Infection
Frederick S. Buckner Mechanisms of Host Cell Invasion by
Trypanosoma cruzi
Vaccine Development Against Kacey L. Caradonna and Barbara A.
Trypanosoma cruzi and Chagas Burleigh
Disease
Juan C. Vazquez-Chagoyan, Gap Junctions and Chagas Disease
Shivali Gupta, and Daniel Adesse, Regina Coeli Goldenberg,
Nisha Jain Garg Fabio S. Fortes, Jasmin, Dumitru
A. Iacobas, Sanda Iacobas,
Genetic Epidemiology of Antonio Carlos Campos de
Chagas Disease Carvalho, Maria de Narareth
Sarah Williams-Blangero, Meirelles, Huan Huang, Milena B.
John L. VandeBerg, John Blangero, Soares, Herbert B. Tanowitz,
and Rodrigo Correa-Oliveira Luciana Ribeiro Garzoni, and
Kissing Bugs. The Vectors of Chagas David C. Spray
Lori Stevens, Patricia L. Dorn, The Vasculature in Chagas Disease
Justin O. Schmidt, John H. Klotz, Cibele M. Prado, Linda A. Jelicks,
David Lucero, and Stephen A. Klotz Louis M. Weiss, Stephen M.
Advances in Imaging of Animal Models Factor, Herbert B. Tanowitz, and
of Chagas Disease Marcos A. Rossi
Linda A. Jelicks and Herbert B. Tanowitz Infection-Associated Vasculopathy in
The Genome and Its Implications Experimental Chagas Disease:
Santuza M. Teixeira, Najib M. El-Sayed, Pathogenic Roles of Endothelin and
and Patrcia R. Araujo Kinin Pathways
Julio Scharfstein and Daniele Andrade
Genetic Techniques in Trypanosoma cruzi
Martin C. Taylor, Huan Huang, and Autoimmunity
John M. Kelly Edecio Cunha-Neto, Priscila Camillo
Teixeira, Luciana Gabriel Nogueira,
Nuclear Structure of Trypanosoma cruzi and Jorge Kalil
Sergio Schenkman, Bruno dos Santos
Pascoalino, and Sheila C. Nardelli ROS Signalling of Inflammatory
Cytokines During Trypanosoma cruzi
Aspects of Trypanosoma cruzi Stage Infection
Differentiation Shivali Gupta, Monisha Dhiman, Jian-jun
Samuel Goldenberg and Andrea Rodrigues Wen, and Nisha Jain Garg
Avila
Inflammation and Chagas Disease: Some
The Role of Acidocalcisomes in the Stress Mechanisms and Relevance
Response of Trypanosoma cruzi Andre Talvani and Mauro M. Teixeira
Roberto Docampo, Veronica Jimenez,
Sharon King-Keller, Zhu-hong Li, and Neurodegeneration and
Silvia N.J. Moreno Neuroregeneration in Chagas
Disease
Signal Transduction in Trypanosoma cruzi Marina V. Chuenkova and Mercio
Huan Huang PereiraPerrin
Adipose Tissue, Diabetes and Chagas

Xiaohua Qi, Mahalia S. Desruisseaux,
Disease
Streamson C. Chua, Philipp E. Scherer,
Herbert B. Tanowitz, Linda A. Jelicks,
and Fnu Nagajyothi
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AMSTERDAM BOSTON HEIDELBERG LONDON

First edition 2011

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1. Coinfection of Schistosoma (Trematoda) with Bacteria,

2. Trichomonas vaginalis Pathobiology:

3. Cryptic Parasite Revealed: Improved Prospects for

4. Assessment and Monitoring of Onchocerciasis in Latin America 175

Contents 1.1. Introduction 3

* Skillman Library, Lafayette College, Easton, Pennsylvania, USA

Advances in Parasitology, Volume 77 # 2011 Elsevier Ltd.

1.7. Coinfection of Schistosoma Species and Helminths

Abstract This review examines coinfection of selected species of Schisto-

problems in human populations. Examples of such Schistosoma

1.2. COINFECTION OF SPECIES OF SCHISTOSOMA AND

This section is concerned with coinfection studies on species of Schisto-

Genus Species included in tables Remarks (based on single infections) Table(s)

Genus Species included in tables Remarks (based on single infections) Table(s)

Genus Species included in tables Remarks (based on single infections) Table(s)

Heligomosomoides H. polygyrus A common rodent trichostrongyloid with a direct life 1.7

A/J mice with coinfection had a

Plasmodium species, P. falciparum, P. malariae and P. chabaudi, infect

1.2.1. Animal studies

FIGURE 1.1 Histomorphology of brain sections of Plasmodium berghei ANKA-infected

parasitaemia or cerebral malaria when coinfection with P. chabaudi,

1.2.2. Human studies

Five of 19 human studies examined the effects of S. mansoni coinfection

1.3. COINFECTION OF SCHISTOSOMA SPECIES WITH

1.3.2.1. Animal studies

1.3.2.2. Human studies

1.3.3.1. Animal studies

1.3.3.2. Human studies

the proliferation and invasion of E. histolytica into the mucosa (entry

1.4. COINFECTION OF SCHISTOSOMA SPECIES WITH

1.4.1. Animal studies

4 Gendrel S. intercalatum S. Typhimurium, (N) 2- to Unknown Children with Si and non-typhoidal

1.4.2. Human studies

FIGURE 1.2 Scanning electron micrograph of the interaction of Salmonella Typhimur-

Several studies have examined the surface interactions between species of

1.5. COINFECTION OF SCHISTOSOMA SPECIES WITH

1.5.1.1. Animal studies

were attributed to an infiltration by eggs of the Schistosoma species (entry

1.5.1.2. Human studies

1.5.2. Helicobacter pylori

H. pylori infection. The results were consistent with H. pylori coinfection

1.5.3. Staphylococcus aureus

1.6. COINFECTION OF SCHISTOSOMA AND

There are a total of 18 studies presented in Table 1.6, which examined

followed by (4043%) but not in Fh burden

1.6.1. Animal studies

when it followed a patent infection with S. mansoni; these studies mainly

1.6.2. Human studies

coinfections may be common in cattle in some parts of the world, for

1.7. COINFECTION OF SCHISTOSOMA SPECIES AND

Table 1.7 presents coinfection studies on Schistosoma and various hel-

1.7.3.2. Human studies