A NEW PCR ASSAY FOR SIMULTANEOUS STUDIES OF LEUCOCYTOZOON,

PLASMODIUM, AND HAEMOPROTEUS FROM AVIAN BLOOD

Author(s): Olof Hellgren, Jonas Waldenström, Staffan Bensch
Source: Journal of Parasitology, 90(4):797-802. 2004.
Published By: American Society of Parasitologists
DOI: http://dx.doi.org/10.1645/GE-184R1
URL: http://www.bioone.org/doi/full/10.1645/GE-184R1

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 J. Parasitol., 90(4), 2004, pp. 797–802
q American Society of Parasitologists 2004

A NEW PCR ASSAY FOR SIMULTANEOUS STUDIES OF LEUCOCYTOZOON,

PLASMODIUM, AND HAEMOPROTEUS FROM AVIAN BLOOD
Olof Hellgren, Jonas Waldenström, and Staffan Bensch
Department of Animal Ecology, Ecology Building, Lund University, SE-22362 Lund, Sweden. e-mail: olof.hellgren@zooekol.lu.se

ABSTRACT: Many bird species host several lineages of apicomplexan blood parasites (Protista spp., Haemosporida spp.), some
of which are shared across different host species. To understand such complex systems, it is essential to consider the fact that
different lineages, species, and families of parasites can occur in the same population, as well as in the same individual bird, and
that these parasites may compete or interact with each other. In this study, we present a new polymerase chain reaction (PCR)
protocol that, for the first time, enables simultaneous typing of species from the 3 most common avian blood parasite genera
(Haemoproteus, Plasmodium, and Leucocytozoon). By combining the high detection rate of a nested PCR with another PCR step
to separate species of Plasmodium and Haemoproteus from Leucocytozoon, this procedure provides an easy, rapid, and accurate
method to separate and investigate these parasites within a blood sample. We have applied this method to bird species with
known infections of Leucocytozoon spp., Plasmodium spp., and Haemoproteus spp. To obtain a higher number of parasite lineages
and to test the repeatability of the method, we also applied it to blood samples from bluethroats (Luscinia svecica), for which
we had no prior knowledge regarding the blood parasite infections. Although only a small number of different bird species were
investigated (6 passerine species), we found 22 different parasite species lineages (4 Haemoproteus, 8 Plasmodium, and 10
Leucocytozoon).

Species of the apicomplexans Haemoproteus, Plasmodium,
and Leucocytozoon comprise a diverse group of vector-trans-
mitted parasites that infect red blood cells (in the case of Leu-
cocytozoon spp., also white blood cells) and other organs within
their vertebrate hosts (Atkinson and Van Riper, 1991; Valkiu-
nas, 1993). Species of these parasite genera share several char-
acters with human malaria parasites, and all 3 (but most often
only Plasmodium spp.) are referred to as avian malaria. These
parasites have served as model organisms for studies on many
aspects of parasite–host interactions, including parasite–host
evolution (Perkins and Schall, 2002; Ricklefs and Fallon, 2002),
host life-history trade-offs (Richner et al., 1995; Nordling et al.,
1998), and sexual selection (Hamilton and Zuk, 1982). The fre-
quent use of these parasites in evolutionary and population
ecology studies is based on the relative ease with which in-
fected birds can be distinguished from uninfected ones and the
fact that the intensity of infection can be estimated for each
host using blood smears (Valkiunas, 1993; Richner et al., 1995;
Rintamäki et al., 1998). Thus, both quantitative and qualitative
methods can be applied to examine the costs of infection.
Species of Haemoproteus, Plasmodium, and Leucocytozoon
are closely related genetically but differ in life-history traits.
The main body of published work has focused on species of
the more easily detected Haemoproteus and Plasmodium, with
relatively few studies on Leucocytozoon spp. (Atkinson and Van
Riper, 1991). The scarcity of investigations on Leucocytozoon
spp. infections is not because the infection is itself rare but
because the life stages of Leucocytozoon spp. are detectable in
peripheral blood for only very short time periods, which makes
the infection difficult to detect and accurately identify using
traditional ocular methods (Fallis and Desser, 1977; Valkiunas,
1997). Despite the methodological difficulties, Leucocytozoon
spp. have been found to be a common (sometimes the most
common) parasite in some bird populations, primarily in the
temperate regions of the Northern Hemisphere (Rintamäki et
al., 1998; Deviche et al., 2001).
Some of the problems associated with the traditional typing
of blood parasites can now be solved with molecular methods,
Received 2 July 2003; revised 24 November 2003; accepted 24 No-
vember 2003.
which are generally much more sensitive than traditional mi-
croscopic procedures (Perkins et al., 1998; Richard et al., 2002).
Furthermore, polymerase chain reaction (PCR)–based methods
can provide sequence information that allows for the identifi-
cation of the specific parasite lineage below the species level
(Bensch and Åkesson, 2003), which is not possible using par-
asite morphology alone. There are several published PCR-based
methods (Bensch et al., 2000; Perkins and Schall, 2002; Rick-
lefs and Fallon, 2002; Fallon et al., 2003) and 1 serological
technique (Atkinson et al., 2001) for the detection and identi-
fication of Plasmodium spp. and Haemoproteus spp. from birds.
However, until now, there has been no general protocol avail-
able for the detection of Leucocytozoon spp.
The cytochrome b gene of the apicomplexan parasite mito-
chondrial genome has been found to have conserved regions
for construction of primer sites with variable sections of DNA
between the conserved regions, which have made it suitable for
detection and identification of Haemoproteus and Plasmodium
lineages (Waldenström et al., 2004).
In this article, we describe a nested-PCR assay, which targets
the cytochrome b gene of the parasites, thus enabling the
screening and typing of Leucocytozoon species in parallel with
species of Haemoproteus and Plasmodium in avian blood sam-
ples. The protocol involves a first PCR step, modified from
Waldenström et al. (2004), which amplifies parasite DNA from
all 3 genera, and then a choice of 2 primer pairs to either am-
plify Leucocytozoon spp. singly or to amplify Haemoproteus
spp. and Plasmodium spp. together (Haemoproteus–Plasmodi-
um). This new method appears highly repeatable and reliable
and provides an important tool for simultaneous studies of spe-
cies of Haemoproteus, Plasmodium, and Leucocytozoon in
birds.
Moreover, this method also provides the possibility of se-
quence-based identification of different mitochondrial lineages
within the parasite genera.
MATERIALS AND METHODS
Primer design
Primers were designed within the 59 end of the cytochrome b gene
of the parasite mitochondrial genome by using the published sequences
of avian Haemoproteus, Plasmodium, and Leucocytozoon mitochondrial

797
798 THE JOURNAL OF PARASITOLOGY, VOL. 90, NO. 4, AUGUST 2004
FIGURE 1. Schematic illustration of the directions and combinations
of the different primers. 1. Primers for amplification of the initial step
in the nested PCR. 2. Primer combination for amplification of Hae-
moproteus and Plasmodium lineages. 3. Primer combination for ampli-
fication of Leucocytozoon lineages. Note that the primer HaemR2/R2L/
NR3 is the reversed compliment and is shown as it should be ordered
(59–39). Underlined letters indicate the primer sequences not shared be-
tween HaemF–HaemFL and HaemR2–HaemR2L. I stands for a univer-
sal base, inosine.
DNA (mtDNA) (Perkins and Schall, 2002). Primer pairs that amplify
the conserved regions of the Haemoproteus spp. and Plasmodium spp.
cytochrome b genes have previously been developed at Lund (Bensch
et al., 2000; Waldenström et al., 2004). In the original protocol (Bensch
et al., 2000), the HaemF and HaemR2 primers were used to amplify a
480-bp fragment (excluding primers) with a single PCR. The perfor-
mance of this PCR was significantly improved, especially for the de-
tection of low-intensity Plasmodium sp. infections, by extending the
single PCR to a nested PCR (for full details see Waldenström et al.,
2004). HaemNF and HaemNR2 primers were used to initially amplify
a 617-bp large fragment (including the primers), to which the HaemF
and HaemR2 primers of Bensch et al. (2000) could be internally nested
in a second PCR step.
Based on sequence homology among aligned sequences of the par-
asite genera, initial primers were constructed (HaemNFI [59-CATATAT-
TAAGAGAAITATGGAG-39] [I 5 a universal base, inosine] and
HaemNR3 [59-ATAGAAAGATAAGAAATACCATTC-39]) to amplify
parasite mtDNA from species of Haemoproteus, Plasmodium, and Leu-
cocytozoon. For the second PCR, we used HaemF–HaemR2 primers
(Bensch et al., 2000) for Plasmodium spp. and Haemoproteus spp. and
constructed 2 new primers, HaemFL (59-ATGGTGTTTTAGATACTT
ACATT-39) and HaemR2L (59-CATTATCTGGATGAGATAATGGIG
C-39) for Leucocytozoon spp. (Fig. 1).
The first PCR was performed in volumes of 25 ml, which included
50 ng of total genomic DNA, 1.25 mM of each deoxynucleoside tri-
phosphate, 1.5 mM MgCl2, 13 PCR (Applied Biosystem, Foster City,
California), 0.6 mM of each primer, and 0.5 units Taq DNA polymerase.
The PCRs including HaemNFI–HaemNR3 were conducted using the
following conditions: 30 sec at 94 C, 30 sec at 50 C, and 45 sec at 72
C for 20 cycles. The samples were incubated before the cyclic reaction
at 94 C for 3 min and after the cyclic reaction at 72 C for 10 min. We
used 2 ml of the first PCR reaction as the template for the second PCR,
1 ml for Leucocytozoon spp. (HaemFL–HaemR3L) and 1 ml for Hae-
moproteus spp.–Plasmodium spp. (HaemF–HaemR2). These PCRs were
performed separately in 25-ml volumes with the same proportions of
reagents as in the initial PCR reactions. The thermal profile of the PCR
was identical to the initial PCR but performed for 35 cycles instead of
20 cycles. To check if the PCRs had been amplified successfully, we
ran 1.5 ml of the final PCR product on a 2% agarose gel and used
samples with positive amplification for further evaluation.
Evaluation
To evaluate the ability of the method to detect Leucocytozoon spp.
infections, we used samples from birds with known infections of dif-
ferent parasites, i.e., where infection had been confirmed morphologi-
cally using Geimsa-stained blood smears. The test panel included birds
with single infections of Leucocytozoon spp. (2 juvenile bramblings,
Fringilla montifringilla) and birds with simultaneous infections of both
Haemoproteus spp. and Leucocytozoon spp. (1 bluetit, Parus caereu-
leus; 1 crossbill, Loxia curvirostra; 1 robin, Erithacus rubicula; and 1
siskin, Carduelis spinus).
To further test the new method, we applied the nested-PCR assays
on a collection of blood samples (n 5 86) from adult bluethroats for
which we had no prior parasite infection knowledge. Each blood sample
contained 5–20 ml of blood and had been stored in SET buffer (0.015
M NaCl, 0.05 M Tris, 0.001 M ethylenediaminetetraacetic acid, pH 8.0)
at ambient temperature in the field and later at 220 C. DNA was ex-
tracted using a standard chloroform–isoamylalcohol method (Sambrook
et al., 2002), and diluted genomic DNA was used as template in the
PCR assays.
Samples showing positive amplification were selected for sequencing
using procedures as described by Bensch et al. (2000). Fragments were
first sequenced from the 59 end with either HaemF (in the case of Hae-
moproteus-Plasmodium spp.–positive samples) or HaemFL (in the case
of Leucocytozoon spp.–positive samples), and the obtained sequences
were edited and aligned using the program BioEdit (Hall, 1999). Sam-
ples with positive amplification yielded PCR products of 478 bp (ex-
cluding primers) for Leucocytozoon spp. and 480 bp for Haemoproteus
spp. and Plasmodium spp.
All unique haplotypes, i.e., sequences differing by 1 or more base
pair from any of the other obtained sequences, were sequenced from
the 39 end using HaemR2 (Haemoproteus-Plasmodium spp.–positive
samples) or HaemR2L (Leucocytozoon spp.–positive samples) for se-
quence validation. In cases where mixed infections occurred (n 5 3),
observed as ‘‘double base calling’’ in the electropherogram, fragments
were cloned and separated using a TA-cloning kit (Invitrogen, Carlsbad,
California) according to the manufacturer’s instructions. We amplified
the inserted DNA from 10 colonies per plate using standard M13 prim-
ers and sequenced from 1 direction using the forward primer.
We used the program MEGA, version 2.1 (Kumar et al., 2001), and
the neighbor-joining method with a Kimura 2-parameter distance matrix
to investigate the genetic relationship of the parasite sequences obtained
in this study with published sequences. The tree was rooted with Thei-
leria annulata (Piroplasmidia).
Repeatability and detection
The repeatability of the method was estimated by running 30 blue-
throat DNA samples 3 times for both the Leucocytozoon and the Plas-
modium–Haemoproteus samples. The outcomes of the different runs
were compared using a 1-way analysis of variance to calculate among-
and within-individual variation of scored prevalence (Lessells and Boag,
1987). To further evaluate the success of the amplification of Haemo-
proteus–Plasmodium spp. and to determine if the performance differed
between the 2 nested-PCR methods, the same samples were tested using
primers developed for the exclusive amplification of Plasmodium spp.
and Haemoproteus spp. (Waldenström et al., 2004). The detection suc-
cess of the method was tested with a dilution series as in Fallon et al.
(2003). Three dilution series stemming from each of 2 birds with single
infections of Haemoproteus and Plasmodium and 1 with Leucocytozoon
spp. were tested for positive amplification. The samples were diluted
with uninfected bird DNA, keeping the total concentration at 25 ng/ml,
in steps of 10, down to a 1,000,000-fold dilution (Table I). The original
intensity of the infection was scored under a light microscope as in
Fallon et al. (2003).
RESULTS
Detection of apicomplexan parasite infections
The nested PCR for the detection of Leucocytozoon spp. cor-
rectly amplified all samples in the test panel where Leucocy-
tozoon or Leucocytozoon and Haemoproteus parasites had been
recorded by microscopy. Among the test panel of birds with
unknown infections, the PCR assays gave an overall prevalence
of apicomplexan parasites in 59% (n 5 86) of birds, with vary-
ing genus-specific prevalences, i.e., 24% of 8 lineages for Plas-
modium, 48% of 5 lineages for Leucocytozoon, and 1.2% of a
single lineage for Haemoproteus (Fig. 2). The method also
identified several individuals with simultaneous infections with
 HELLGREN ET AL.—SIMULTANEOUS STUDY OF 3 PARASITE GENERA 799

TABLE I. Dilution series of malaria-infected birds. Each infected bird was tested for positive PCR amplification in three dilution series. Limits of
infection intensities for consistent scoring (all positive) and minimum detection (at least one positive) are calculated based on smear intensities.
Prefix in the lineage name corresponds to h 5 Haemoproteus, p 5 Plasmodium, and l 5 Leucocytozoon.

Smear Number of positive amplifications (max 3) Consistent Minimum

intensity scoring detection
Lineage (%) 1 1021 1022 1023 1024 1025 1026 limit (%) limit (%)

hGRW1 3.75 3 3 3 3 1 0 0 0.00375 0.00038

hGRW1 2.4 3 3 3 3 0 0 0 0.0024 0.00024
pGRW2 1.16 3 3 3 3 2 0 0 0.00116 0.00012
pGRW2 0.022 3 3 2 0 0 0 0 0.0022 0.00022
1Bram1 0.03 3 3 2 1 0 0 0 0.003 0.00003

species from 2 parasite genera (10% with Leucocytozoon–Plas-
modium infections and 1.2% with Leucocytozoon–Haemopro-
teus) or with 2 lineages from the same parasite genus (2 indi-
viduals with 2 lineages of Leucocytozoon and 1 individual with
2 lineages of Plasmodium).
The repeatability of both methods was high. Of the 30 sam-
ples tested with the Plasmodium and Haemoproteus primers, 17
were negative and 9 positive in all 3 runs, whereas 4 samples
showed mixed positive and negative results (R 5 0.814, F1,89
5 14.147, P , 0.0001; Fig. 3). The Leucocytozoon spp. primers
gave identical results in all 3 runs for 25 of the 30 tested sam-
ples (14 consistently negative and 11 positive), whereas 5 sam-
ples showed mixed positive and negative results (R 5 0.740,
F1,89 5 9.546, P , 0.0001; Fig. 3). The amplification using the

FIGURE 2. Genetic relationship (neighbor joining and a Kimura 2-parameter distance method) of blood parasites. Bootstrap values presented
on the clade branches. Shaded lineages are obtained from previous studies or from GenBank. Underlined lineages were found in a sample set of
bluethroats (Luscinia svecica) with unknown infections. † indicate lineages found in control birds with known infections. The tree was rooted
with Theileria annulata (Piroplasmidia).
800 THE JOURNAL OF PARASITOLOGY, VOL. 90, NO. 4, AUGUST 2004
FIGURE 3. Number of positive or negative amplification of blood
parasites in 3 runs of 30 test individuals. Each individual bird was tested
3 times. Black, individuals tested for Leucocytozoon spp.; white, indi-
viduals tested for Plasmodium spp.–Haemoproteus spp.
primers from Waldenström et al. (2004) did not yield any new
positive samples among the 30 tested.
Our nested-PCR method provided positive amplifications in
all cases down to dilutions corresponding to 1 parasite per
100,000 host blood cells for Haemoproteus, Plasmodium, and
Leucocytozoon (Table I). In dilutions corresponding to 1 para-
site per 1,000,000 host blood cells, 50% of the samples in the
Haemoproteus spp. dilution series, 67% in the Plasmodium spp.
series, and 67% in the Leucocytozoon spp. series showed pos-
itive amplification. The method did not show any positive am-
plification for less than 1 parasite per 1,000,000 host blood cells
except 1 positive case for Leucocytozoon at the dilutions cor-
responding to 1 parasite per 10,000,000 host blood cells (Table
I).
Phylogenetic relationships
The sequenced PCR products of the 2 PCR assays were ed-
ited and aligned, and the resulting alignment of 480 bp was
analyzed. The sequences showed high variability, and a neigh-
bor-joining tree clustered them into 3 distinct groups with high
confidence (bootstrap values from 88 to 98 for the different
genera and branches; Fig. 2). These 3 clusters corresponded to
species of Haemoproteus, Plasmodium, and Leucocytozoon.
Each cluster was confirmed using published genus-identified
sequences, together with the tested samples of known infections
from this study. All sequences obtained with the Leucocytozoon
spp. primer combination grouped in the Leucocytozoon cluster,
and all the sequences obtained with the Haemoproteus spp. and
Plasmodium spp. primer pair grouped with known Haemopro-
teus and Plasmodium sequences. Furthermore, apart from pro-
viding generic identification of tested samples, the obtained
fragment of the cytochrome b gene was also phylogenetically
informative within genera, as observed in large lineage varia-
tion in all 3 obtained clusters. Among the sequences included
in this analysis, the deepest split was 6.3% within Haemopro-
teus spp., 19.2% within Plasmodium spp., and 22.8% within
Leucocytozoon spp.
DISCUSSION
Detection of parasite lineages
Several PCR-based methods for studies of Haemoproteus
spp. and Plasmodium spp. have been published recently
(Bensch et al., 2000; Ricklefs and Fallon, 2002; Fallon et al.,
2003), but the present investigation reports the first protocol for
a general detection of Leucocytozoon spp. PCR-based methods
are more sensitive than traditional microscopy (Richard et al.,
2002); however, performance differs markedly between meth-
ods. In a comparative study, Richard et al. (2002) concluded
that PCR assays targeting parasite mtDNA had superior per-
formance over those targeting nuclear ribosomal genes. In our
laboratory, we have developed a nested-PCR assay (Walden-
ström et al., 2004), based on the primer pairs published in
Bensch et al. (2000), which further improved the detection ca-
pability, especially for low-intensity Plasmodium spp. infec-
tions. In this study, we have further developed the methods of
Bensch et al. (2000) and Waldenström et al. (2004) by making
initial primers that include Leucocytozoon spp. mtDNA in the
amplification and an additional set of primers for the second
step that allow for separation of Leucocytozoon spp. infections.
Thus, from extracted blood samples, only 3 PCR runs are need-
ed to identify individuals infected with species of Plasmodium–
Haemoproteus or Leucocytozoon in a study population of birds.
The new method correctly identified all samples with known
infections, and the repeatability was high for the detection of
Plasmodium spp., Haemoproteus spp., and Leucocytozoon spp.
Because the methods are performed on DNA extracted from
blood samples, the prerequisite for successful amplification is
the presence of parasites in the circulating blood of the host.
Because results are obtained as the presence or absence of
bands in an agarose gel, a validation of the total DNA quality,
e.g., by first using molecular sexing of the hosts, is needed
before trusting negative scores in the malaria PCR. In the case
of the bluethroat with unknown infections, the blood samples
had been previously used for host microsatellite and amplified
fragment length polymorphism typing (data not shown). Al-
though the repeatability of parasite detection was high, some
variation was observed between trials. The obtained variation
was most likely associated with low parasitemia because vari-
ation also occurred at very low intensities when the method
was tested for intensity dependence. However, the method al-
lows detection of infections without failures in the dilutions
corresponding to 1 parasite per 100,000 host blood cells, and
inconsistent results were obtained initially in dilutions corre-
sponding to 1 parasite per 1,000,000 host blood cells (Table I).
Hence, the method successfully identified infections with inten-
sities of $0.001% infected blood cells, with the possibility of
identifying infections with intensities of $0.0001% infected
blood cells. The occurrence of the variation at very low inten-
sities is likely to be due to chance events during the first PCR
cycles that determine whether a fragment will be amplified or
not. Even though the repeatability is high and the detection rate
is roughly twice that of ocular investigation (Waldenström et
al., 2004), studies of processes (fitness) at the individual level
should consider using repeated screening to include and in-
crease the reliability of birds with the lowest infection intensi-
ties.

Phylogenetic analysis
The apicomplexan cytochrome b gene is phylogenetically in-
formative and has been used previously to establish the evo-
lution and divergence of avian malarial parasites in different
host families (Perkins and Schall, 2002; Ricklefs and Fallon,
2002) and to establish the areas in which these parasites are
transmitted (Waldenström et al., 2002). This study clearly
shows that the cytochrome b gene is also suitable for construct-
ing well-supported phylogenies for Leucocytozoon species. Our
method provided highly variable sequences with the largest ob-
served sequence divergence of 23% between Leucocytozoon
dubreuli (AY099064) and Leucocytozoon sp. (AY465560) (Fig.
2). The largest divergence between lineages found in the same
host species was 8.4% (between lBT1 and lBT2). This is a
deeper divergence than that between Plasmodium falciparum
(found in humans) and P. reichenowi (found in chimpanzee)
(3.3%), which are supposed to have diverged approximately 5
million yr ago (Escalante et al., 1998). Thus, the most divergent
lineages of Leucocytozoon spp. encountered in the same host
species most likely reflect different parasite species. On the oth-
er hand, 2 of the obtained Leucocytozoon lineages, lBT5 and
lBT4, differed by only 1 and 2 substitutions, respectively, from
2 other lineages (lBT1 and lBT2, respectively), possibly indi-
cating intraspecies mtDNA variation (Fig. 2).
The construction of a powerful method for the detection and
identification of Leucocytozoon spp., in parallel with the meth-
od for Plasmodium spp. and Haemoproteus spp., will hopefully
open the way for studies on the occurrence and effect of these
parasites in natural populations of birds. To neglect the contri-
bution of fitness effects stemming from Leucocytozoon spp. in-
fections could confound results, especially in northern temper-
ate areas where Leucocytozoon spp. and its main vector, the
blackfly (Simuliidae), are both common (Malmqvist, 1994;
Malmqvist et al., 1999; Deviche et al., 2001; Ojanen et al.,
2002). The relative importance of Leucocytozoon spp. infec-
tions is illustrated by the bluethroats screened in this study, i.e.,
Leucocytozoon spp. infections were twice as common as Plas-
modium spp. (48% and 24% prevalence, respectively). There-
fore, a study focusing only on Haemoproteus spp. and Plas-
modium spp. parasites might give a biased result because it
would leave out the most common blood parasite species.
Moreover, combinations of parasites may have different effects
on host fitness because they may, positively or negatively, in-
teract with each other (Riche, 1988).
ACKNOWLEDGMENTS
The study was supported by grants from Stiftelsen Lunds Djurskydds-
fond and Olle och Signild Engkvist Stiftelser. Thanks to Charlotte Val-
ind for excellent help during the sampling trip, Åke Lindström for mak-
ing the field trip possible, Kjell Grimsby, Thomas Holmberg, Trond
Amundsen, and Nils-Åke Andersson for help during the field season.
We thank Dennis Hasselquist for scientific input, Gediminas Valkiunas
for providing blood samples, and Martin Stjernman for evaluating in-
fection intensities. David Richardson, Gediminas Valkiunas, and Rav-
inder Seghal provided insightful comments on the manuscript.
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BOOK REVIEW . . .
Parasites and Diseases of Wild Birds in Florida, by D. J. Forrester
and M. G. Spalding. University Press of Florida, Gainesville, Florida.
2003. 1132 p. Cloth cover, ISBN: 0-8130-2560-5.
At first glance, a book with the above title might not seem to be of
great general interest. However, Florida has a highly diverse bird fauna,
the state is a major migration site, and the authors have worked with
the Florida Fish and Wildlife Conservation Commission for decades,
assessing the effect of parasites and disease on both birds and mammals.
As a result, this book is one of the most comprehensive, easily used,
and data-rich resources available to parasitologists. It will be especially
relevant, indeed virtually a required reference volume, for those in any
area of conservation biology or wildlife management, and it will be
very useful to teachers and researchers who are working with natural
host–parasite systems involving birds.
Parasites and Diseases of Wild Birds in Florida is organized more
or less along classical taxonomic lines, with chapters on bird families
beginning with loons and grebes and ending with passerines. ‘‘Parasite’’
and ‘‘disease’’ are both interpreted broadly, thus information is provided
not only on viruses, bacterial infections, fungi, protozoa, helminths, and
arthropods but also on chemical residues, e.g., lead poisoning and or-
ganophosphates, and trauma, e.g., injury resulting from contact with
structures. The book is heavily referenced (even the Preface has a lit-
erature cited section!) and as a result is a virtual window revealing a
vast body of literature, not typically accessible to the average academic
or government employee. For example, the 22-page chapter on kites
has nearly 4 pages of references, with citations ranging from those in
major journals to ones in relatively obscure commission reports. Each
chapter also is generously supplied with tables, including data sources,
WALDENSTRÖM, J., S. BENSCH, D. HASSELQUIST, AND Ö. ÖSTMAN. 2004.
A new nested polymerase chain reaction method very efficient in
detecting Plasmodium and Haemoproteus infections from avian
blood. Journal of Parasitology 90: 191–194.
———, ———, S. KIBOI, D. HASSELQUIST, AND U. OTTOSSON. 2002.
Cross-species infection of blood parasites between resident and mi-
gratory songbirds in Africa. Molecular Ecology 11: 1545–1554.
J. Parasitol., 90(4), 2004, p. 802
q American Society of Parasitologists 2004
an excellent example being a 4-page table on ticks reported from pas-
serines, with collection sites, dates, tick stages represented, and preva-
lence. There are 23 tables listing similar data for helminth parasites of
wild turkeys. The tabular data and Literature Cited alone make this
single volume an exceptionally valuable resource, potentially saving
enormous amounts of time for anyone who has any reason whatsoever
to recover information on bird diseases or parasites.
This book is also unusual in that it mentions both negative data and
gaps in our knowledge, although the information applies mainly to Flor-
ida. For example, in the chapter on Anserinae (whistling ducks, swans,
and geese), we are told there is no information on neoplasia, viruses,
bacteria, fungi, or blood protozoa in these (wild) birds, but we are also
told of reports on some of those parasites in captive flocks or in nearby
states. Ecological data are included when available and relevant, a good
example being seasonal dynamics of nematodes in bobwhites. The book
provides a reasonable number of photographs of pathological condi-
tions, some of which are quite dramatic, thus useful in teaching. Tech-
niques are also illustrated, and although some readers might question
why these photographs are included, it is not always obvious to biolo-
gists in general, especially in a molecular age, how data on wild animals
are acquired. A good example of this material is the series of photo-
graphs on how to make and use a lard-can bait trap to get information
on avian malaria vectors.
The text is well written. The David Maehr pen and ink bird drawings
are a nice touch for a scientific book, and the originals of these drawings
are probably collectibles because of their subtle quality.
John Janovy, Jr., School of Biological Sciences, 348 Manter Hall, Uni-
versity of Nebraska–Lincoln, Lincoln, Nebraska 68588-0118.