2013 Chapman Et Al

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From H. David Chapman, John R. Barta, Damer Blake, Arthur Gruber, Mark Jenkins,
Nicholas C. Smith, Xun Suo, Fiona M. Tomley, A Selective Review of Advances in
Coccidiosis Research. In D. Rollinson, editors: Advances in Parasitology, Vol. 83,
Amsterdam, The Netherlands: Academic Press, 2013, pp. 93-171.
ISBN: 978-0-12-407705-8
Copyright 2013 Elsevier Ltd.
Elsevier
Author's personal copy
CHAPTER TWO
A Selective Review of Advances

in Coccidiosis Research
H. David Chapman*,1, John R. Barta, Damer Blake{, Arthur Gruber},
Mark Jenkins}, Nicholas C. Smith||, Xun Suo#, Fiona M. Tomley{
*Department of Poultry Science, University of Arkansas, Fayetteville, Arkansas, United States
Department of Pathobiology, Ontario Veterinary College, University of Guelph, Guelph, Ontario, Canada
{
Royal Veterinary College, Hatfield, United Kingdom
}
Department of Parasitology, Institute of Biomedical Sciences, University of Sao Paulo, Sao Paulo, Brazil
}
Animal Parasitic Diseases Laboratory, Agricultural Research Service, USDA, Beltsville, Maryland, United
States
||
Queensland Tropical Health Alliance Laboratory, Faculty of Medicine, Health and Molecular Sciences, James
Cook University, Cairns, Queensland, Australia
#
National Animal Protozoa Laboratory & College of Veterinary Medicine, China Agricultural University,
Beijing, China
1
Corresponding author: e-mail address: dchapman@uark.edu
Contents
1. Introduction
2. Taxonomy and Systematics
2.1 The genus Eimeria Schneider 1875: A melting pot of biologically diverse
coccidia
2.2 Molecular identification and characterization of Eimeria and related
coccidia
2.3 Conclusions
3. Genetics
3.1 Markers employed in genetic studies
3.2 Cross-fertilization and genetic recombination
3.3 Genetic linkage analyses
4. The Omics Technologies
4.1 Genomics
4.2 Transcriptomics
4.3 Proteomics
4.4 The future
5. Transfection
5.1 Transfection construct design
5.2 Transient transfection
5.3 Stable transfection
5.4 PiggyBac-based forward genetic system
5.5 Stably transfected Eimeria as a vaccine vector and beyond
Advances in Parasitology, Volume 83

ISSN 0065-308X
http://dx.doi.org/10.1016/B978-0-12-407705-8.00002-1
2013 Elsevier Ltd

All rights reserved.
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6. Oocyst Biogenesis
6.1 Veil and WFBs
6.2 Oocyst wall proteins
6.3 Formation of the oocyst wall
7. Host Cell Invasion
7.1 Parasite surface proteins
7.2 MIC proteins are adhesins and many function as multi-protein
complexes
7.3 Host glycan recognition by MIC proteins contributes to host
and tissue tropism
7.4 Regulated secretion of microneme and rhoptry organelles
7.5 AMAs and formation of the MJ
8. Immunobiology
8.1 Innate responses to primary infection
8.2 Acquired immunity
8.3 Maternal immunity
8.4 Immunological research
9. Diagnosis and Identification
9.1 Traditional methods
9.2 Early molecular methods
9.3 Methods based on DNA amplification by PCR
9.4 LAMP
9.5 Morphological diagnosis revisited
9.6 Conclusions
10. Control
10.1 Chemotherapy
10.2 Vaccination
10.3 Strategies for the control of coccidiosis
10.4 Natural products
11. Conclusions
Acknowledgement
References
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Abstract
Coccidiosis is a widespread and economically significant disease of livestock caused by
protozoan parasites of the genus Eimeria. This disease is worldwide in occurrence and
costs the animal agricultural industry many millions of dollars to control. In recent years,
the modern tools of molecular biology, biochemistry, cell biology and immunology
have been used to expand greatly our knowledge of these parasites and the disease
they cause. Such studies are essential if we are to develop new means for the control
of coccidiosis. In this chapter, selective aspects of the biology of these organisms, with
emphasis on recent research in poultry, are reviewed. Topics considered include taxonomy, systematics, genetics, genomics, transcriptomics, proteomics, transfection, oocyst
biogenesis, host cell invasion, immunobiology, diagnostics and control.

Review of Coccidiosis Research
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1. INTRODUCTION
Coccidiosis is caused by protozoan parasites of the apicomplexan
genus Eimeria that occur in many vertebrate and invertebrate hosts. This disease is a major cause of mortality, poor performance and lost productivity in
domestic livestock. The parasites have an oral-faecal life cycle involving
three phases: schizogony (also known as merogony), gametogony and sporogony (or sporulation). The infective transmission stage is the oocyst which
contains, when sporulated, four sporocysts each containing two sporozoites.
Following ingestion, the sporozoites are released and penetrate epithelial
cells of the intestine. This is followed by schizogony, an asexual phase of
multiplication involving several repeated generations, and gametogony,
which results in the production of a new generation of oocysts that are passed
out in the faeces. The third phase of the life cycle, sporogony, occurs in the
external environment and results in the formation of a new generation of
oocysts. Seven species are recognized in the fowl that vary according to
the number of generations of schizogony, physical characteristics such as
the size of the oocyst, and biological characteristics such as site of development in the intestine, pathogenicity and immunogenicity. A similar number
of species have been described from the turkey. In poultry, the life cycle is
completed in about 7 days but in ruminants it may be longer. Both schizogony and gametogony can cause pathology because the cells in which the parasites develop are functionally impaired and eventually destroyed. The
extent of such destruction is determined by the numbers of infective oocysts
ingested, which in turn depends upon the extent to which sporulation is successful. This requires warmth, oxygen and moisture, factors often not lacking in commercial livestock production.
It is in the poultry industry that coccidiosis is of the greatest economic
significance because modern production methods involve the rearing of
large numbers of birds in confinement at high stocking densities, often on
built-up litter. For example, a modern broiler house may contain as many
as 2050,000 birds under one roof at a stocking density of one bird per
0.08 m2 and as many as 10 houses may be present at one farm. Furthermore,
both the broiler industry and the turkey industry tend to be restricted geographically; for example, in the United States, one of the largest areas of production is confined to a few counties in northwest Arkansas. Thus the
commercial conditions under which poultry are raised provide ideal conditions for parasite transmission. Fortunately, control of coccidiosis can be

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achieved either by the inclusion of drugs in the feed (prophylactic chemotherapy) or by vaccination and, because of such control measures, coccidiosis
is less a problem today than in the past (Chapman, 2003). Nevertheless, the
resilience of the oocyst ensures the continued presence of these organisms
wherever poultry are reared. The expansion of the poultry industry has provided a major source of protein to feed the growing human population and
any disease that limits this production, such as coccidiosis, will have the
potential to affect human health. There is a continued need, therefore,
for both basic and applied research into all aspects of the biology of these
organisms (Shirley and Lillehoj, 2012).
An example of basic research is the utilization of genomics and proteomics to elucidate molecular aspects of invasion of host cells by sporozoites.
The apical complex of coccidia, found in motile stages of the life cycle (sporozoites and merozoites), contains several sub-cellular organelles including
paired rhoptries, micronemes and dense bodies that secrete proteins
involved in the invasion process. Another example is the investigation of
biochemical events involved in the formation of the wall of the oocyst. Progress has also been made in understanding the role of CD4/CD8 lymphocytes and cytokines in inflammatory responses to infection; cell-mediated
immune mechanisms have been studied utilizing murine Eimeria as a model.
Molecular techniques have been utilized to develop new methods for identifying the species that infect the fowl. Much applied research has been
undertaken to demonstrate efficacy of various products for the control of
coccidiosis but in general this has not been published in peer-reviewed scientific journals. In this review, some aspects of coccidiosis research in poultry is provided by the following contributors: taxonomy and systematics
(Barta); genetics (Blake); omics technologies (Blake, Tomley, Gruber);
transfection (Suo, Blake); oocyst biogenesis (Smith); host cell invasion
(Tomley); immunology (Smith); diagnostics ( Jenkins, Gruber) and control
(Chapman).
2. TAXONOMY AND SYSTEMATICS

The taxonomy and systematics of eimeriid coccidia and Eimeria species
infecting vertebrates is, at best, problematic. This confounding state has
arisen because of numerous poorly described species, lack of type deposition
(even of micrographs or drawings) and improper taxonomic methods.
Misidentification is a general difficulty in studies concerned with the biology
of Eimeria. Classical original descriptions of the species that infect poultry

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were based upon isolates that have since been lost and, thus, there are no
surviving type specimens. A few defined laboratory strains still exist
(although not type specimens in the taxonomic sense), for example, the
Houghton and Weybridge strains of various species, but others, such as
most Beltsville strains, have been lost. Ideally, molecular studies should
be based upon strains derived from a single oocyst and identified using
key parasitological characters. Difficulties with the interpretation of DNA
analysis from inadequately described strains of Eimeria have been addressed
by Williams et al. (2010).
Confounding this taxonomic history is frequent application of the
different-hostdifferent-parasite mindset. For Eimeria species in some host
groups, this principle may be justified because of relatively strict host specificities of these parasites; however, in some coccidia that infect passerine
birds (Isospora species in particular), host specificity may not be nearly as strict
and there may be many Isospora species that may ultimately be synonymized.
To further complicate matters, parasites in the genus Atoxoplasma Garnham
1950 may actually all be members of either the genus Isospora (and perhaps
synonymized with previously described Isospora species) or the genus Lankesterella (e.g. Barta et al., 2005; Merino et al., 2006).
2.1. The genus Eimeria Schneider 1875: A melting pot

of biologically diverse coccidia
The apicomplexan suborder Eimeriorina (Leger, 1911) is home to the
eimeriid coccidia (family Eimeriidae Minchin 1903) that includes the
Eimeria species and related monoxenous or facultatively heteroxenous
coccidia infecting vertebrates. A recent taxonomic definition of the family
Eimeriidae (see Upton, 2000) included apicomplexan parasites with the following features: Homoxenous or facultatively homoxenous; merogony,
gamogony and formation of oocysts all in the same host; in vertebrates or
invertebrates. Clearly this is a broadly applicable definition for inclusion
of parasites into this family. The definition of the genus is likewise permissive: oocysts with four sporocysts, each with two sporozoites. Not surprisingly, the number of Eimeria species that have been described to date exceeds
1200 and this number continues to grow. The frequently observed strict host
specificity of many Eimeria species and the infection of many hosts with multiple Eimeria species means that there remain probably tens of thousands of
undescribed Eimeria species infecting birds, herbivorous or omnivorous
mammals, reptiles, amphibia and, perhaps, even fish.

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Whilst taxonomically useful (and conservative), this rather broad definition of the genus bears part of the responsibility for one of the fundamental
taxonomic difficulties with the genus Eimeriait is not a monophyletic taxonomic group. There is considerable agreement that species currently
assigned to the genus Eimeria do not form a monophyletic group (e.g.
Jirku et al., 2002, 2009) whether assessed systematically using morphological
or molecular characters. Instead, the genus Eimeria is paraphyletic or polyphyletic. When analyzed using complete or partial nuclear 18S rDNA
sequences, the phylogenetic analysis generated a consensus tree (Fig. 2.1)
in which members of the Toxoplasmatinae (species of Cystoisospora,
Neospora, Hammondia and Toxoplasma) formed a well-supported monophyletic group that had a sister group relationship with the other coccidia,
including Eimeria species. The large clade of eimeriid coccidia that includes
all Eimeria species for which 18S rDNA sequence data have been obtained
includes many genera other than Eimeria, including Goussia, Caryospora,
Lankesterella, Atoxoplasma, Isospora and Cyclospora. Even the genera currently
included in the large family Eimeriidae are not found within a monophyletic
grouping; members of the heteroxenous family Lankesterellidae are found
within the clade that contains all species belonging to the Eimeriidae.
The tissue coccidia (Toxoplasma and its relativesToxoplasmatinae) and
all of the early branching coccidia (Hyaloklossia and Goussia spp. plus
E. tropidura) possess valvular sutures on their sporocysts and do not have
Stieda bodies (see Fig. 2.2). Eimeria arnyi from a snake and E. ranae from frogs
were found in a well-supported branch that arose near the base of the
coccidia that possess Stieda bodies in their oocysts (when sporocysts are
formed). The next branching clade consisted of: (1) Eimeria species of marsupials (E. trichosuri); (2) Eimeria spp. of cranes (E. gruis and E. reichenowi) and
(3) a clade containing Caryospora spp. and Lankesterella spp. This sister group
to these parasites was a large collection of Eimeria species from a wide variety
of hosts interrupted by the inclusion of a well-supported clade of
Cyclospora spp.
The genus Eimeria was observed to be polyphyletic with at least four
independent lineages of Eimeria species. Caryospora and Lankesterella species
formed a monophyletic clade as has been described previously (Barta et al.,
2001). Avian Isospora and Atoxoplasma species formed a well-supported
monophyletic clade; the mixing of Atoxoplasma and Isospora species gives
additional support for the questioning the validity of the genus Atoxoplasma.
Giving some support to the concept of host specificity, Eimeria species frequently formed well-supported clades of parasites that parasitized the same

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or closely related definitive hosts such as Eimeria spp. infecting swine, rabbits
or galliform birds. Among the Eimeria species infecting galliform birds, the
cecal coccidia of chickens and turkeys (i.e. E. necatrix, E. tenella and
E. adenoeides) are frequently found to form a monophyletic group of parasites
(e.g. Miska et al., 2010; Fig. 2.1) to the exclusion of the other Eimeria species
infecting the chicken. This repeated observation, and the failure to find
E. necatrix or E. tenella in wild jungle fowl (Fernando and Remmler,
1973), present the possibility that these two species of Eimeria infecting
domestic chickens may have arisen from a host transfer from some other galliform host (Barta et al., 1997).
2.2. Molecular identification and characterization of Eimeria

and related coccidia
Multiple, distinct rDNA copies were described decades ago for the malarial parasites, Plasmodium species (e.g. McCutchan et al., 1988; Nishimoto
et al., 2008). In these haemosporinid parasites, up to three independently
evolving paralogous rDNA copies were described that frequently demonstrated more sequence variation between paralogous within a single parasite species than between homologues among different species.
Interestingly, paralogous rDNA copies were expressed differentially in different life cycle stages. Until recently, the presence of paralogous rDNA
copies had not been observed in Eimeria species and thus the nuclear
18S rDNA locus was considered a relatively useful and stable genetic target
for species-level identification and for molecular phylogenetics. However,
Vrba et al (2011) recently showed that a single-oocyst-derived line of
E. mitis contained two paralogous types of nuclear 18S rDNA that had
considerable sequence variation (i.e. 1.31.7% sequence diversity between
paralogous compared with 0.30.6% sequence diversity among homologues). Recent work with a number of single-oocyst-derived lines of several Eimeria species infecting turkeys indicates that paralogous 18S rDNA
copies exist within the nuclear genome of at least some of these parasites as
well. For example, single-oocyst clonal lines of E. meleagrimitis, from
which polymerase chain reaction (PCR)-amplified, near-complete nuclear
18S rDNA amplicons were cloned and sequenced, demonstrated considerable sequence diversity. Two paralogous groups of sequences were
obtained; each group of sequences had mean intraspecific sequence diversities of about 0.5% but the mean interspecific variation between these
paralogous exceeded 2.7%. By the way of comparison, the mean interspecific variation for the 18S rDNA between the widely accepted species
Figure 2.1 Maximum likelihood consensus tree resulting from the analysis of complete or near complete nuclear 18S rDNA sequences. For
each taxon in the tree, the definitive host (DH), if known, and the GenBank Accession number for the sequence used to generate the tree are
included. Percentage bootstrap supports for major clades are presented as numbers located at nodes on the tree. Horizontal branch lengths
are proportional to hypothesized evolutionary change with the scale indicating 10% hypothesized sequence variation.

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Figure 2.2 Photomicrographs of sporulated oocysts of coccidia that each possess two
sporocysts, containing four sporozoites. (A) An avian Isospora sp. that possess Stieda
bodies at the apex of each sporocyst in the oocyst (arrows) and that belongs to the family Eimeriidae. (B) Cystoisospora felis, a coccidium infecting felids that has an oocyst with
no Stieda bodies on its sporocysts and that belongs to the family Sarcocystidae.
E. tenella and E. necatrix is only 1.1%. Clearly, such paralogous loci can
confound both molecular phylogenetics and the taxonomic pursuits of
identification and characterization.
Recognition of intragenomic polymorphisms among 18S rDNA in
Eimeria has important ramifications for use of other regions of the rDNA
gene array for molecular diagnostics and/or characterization. For example,
the internal transcribed spacer (ITS) regions have been used to characterize
species and strains of Eimeria. However, the ITS-1 and ITS-2 regions are
likely subject to both intragenomic (e.g. Blake et al., 2006; Vrba et al.,
2011) and intraspecific (e.g. Barta et al., 1998) variations that may limit
the utility any resulting ITS sequences or molecular diagnostic methods
based on these sequences.
Ogedengbe et al. (2011a) have recently demonstrated that another
genetic locus, the mitochondrial cytochrome c oxidase subunit I gene
(cox-1, COI), is a much more reliable gene for molecular species delimitation
of Eimeria species and other coccidia than nuclear 18S rDNA sequences. The
recognition of intragenomic polymorphism of the 18S rDNA locus (e.g.
Vrba et al., 2011) may explain the superiority of the COI locus for species
delimitation and, potentially, identification. The COI genetic target has
additional features that argue for its use in the molecular characterization
and identification of coccidia: (1) COI is found in multiple copies within
the mitochondria of coccidia making it a good PCR target; (2) COI

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demonstrates sufficient DNA sequence variability (23% sequence diversity between Eimeria spp.) that 500800 bp fragments will provide sufficient
data for most identification purposes; (3) COI is sufficiently divergent from
the COI of most hosts that parasite-specific PCR primers can be readily
developed, permitting the use of this locus for parasites located within host
tissues; (4) COI is located on a genome derived from an endosymbiotic prokaryote meaning that the resulting COI protein coding sequences are free of
introns and can be assessed relatively easily for contiguous open reading
frames (ORFs) as an internal check for correct PCR amplification
(Ogedengbe et al., 2011a) and finally, (5) intraspecific variation among species is relatively modest (usually <0.2%) compared with interspecific variation (usually >1.5%). However, the use of COI sequences for parasite
identification and/or species delimitation is not without its problems. Compared with the 18S rDNA locus, relatively few COI sequences have been
deposited to public sequence databases, limiting the use of this locus for
identification purposes until more reference sequences become available.
In addition, PCR amplification of COI fragments from mixed parasite
DNA templates (such as litter or mixed species samples, e.g. Schwarz
et al., 2009) can generate hybrid amplicons (e.g. GenBank Accession number FJ236441). In our experience, up to 5% of COI fragments amplified by
PCR from DNA samples containing multiple Eimeria spp. may be hybrid
molecules generated during the PCR process. For Eimeria spp. of chickens
or other well-sampled taxonomic groups (such as Plasmodium spp. and
related haemosporinid parasites), this is not a major issue. In such cases,
hybrid sequences are easily detected using a simple BLAST search because
of the availability of reliable reference sequences in the public sequence databases. Examining pairwise alignments of the top BLAST hits will show
sequence divergence restricted to only one portion of the new sequence
when aligned with sequences from one Eimeria sp. and with a different portion of the molecule when aligned with a second Eimeria species. However,
for Eimeria or Isospora species from less well-sampled hosts, hybrid sequences
may be problematic. To avoid the influence of such hybrids, direct sequencing of PCR products is recommendedmessy or ambiguous sequences
resulting from such direct sequencing likely indicates multiple species within
the sample and biological purification should be attempted (biological
enrichment or cloning of the parasite). Alternately, after cloning PCR fragments from such a sample, multiple clones must be sequenced so that both
repeated COI sequences (likely valid amplicons) and potential hybrids can
be identified.

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For the well-accepted Eimeria species infecting chickens it was frequently

assumed that lack of immunological cross-protection in the host was evidence in support of identification of particular species. For example, chickens
immunized against Eimeria maxima through natural infection would be
expected to be protected against subsequent challenge with the same species.
However, it has been shown conclusively that immunologically distinct
strains of E. maxima can arise both spontaneously and in response to selective
immunological pressure. Despite a near complete lack of immunological
cross-protection, the Guelph and M6 strains of E. maxima have identical
COI sequences (Ogedengbe et al., 2011a) demonstrating again the utility
of this genetic locus for species identification, even when classical methods
of species identification such as immunological cross-protection can fail.
2.3. Conclusions
The taxonomic mess that is the current genus Eimeria is likely to be resolved
slowly through the application of appropriate nuclear or mitochondrial
sequence analysis coupled with phenotypic characters. Ideally, monotypic
reference strains of Eimeria species and other coccidia of veterinary or medical importance should be characterized biologically (minimally oocyst
dimensions as described by Bandoni and Duszynski, 1988), but optimally
including solid descriptions of endogenous development during experimental infections as well as molecularly (preferentially obtaining both nuclear
18S rDNA and mitochondrial COI sequences).
Such combined morphological and molecular characterizations have
been accomplished with single Isospora sp. oocysts (Dolnik et al., 2009).
However, even with both molecular and morphological data available, taxonomic decisions are ultimately opinions of the various authors involved.
For example, although there were significant differences for two strains of
turkey Eimeria in oocyst dimensions and 2.3% sequence divergence at the
COI locus over 767 bp, Poplstein and Vrba (2011) concluded that these parasites were simply variants of a single species, E. adenoeides because of some
immunological cross-protection of these parasites in turkeys. Clearly further
studies are warranted even for parasites that have been described for many
years and that infect agriculturally important animals.
Finally, the taxonomic breadth of the genus Eimeria is clearly too broad.
It is anticipated that the genus Eimeria will be divided into several genera,
each containing fewer, but biologically more homogenous, species with
each new genus.

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3. GENETICS
Studies with Eimeria genomes can be divided into physical and nonphysical categories including genomic, transcriptomic and proteomic or
fundamental and applied genetics, respectively. Key among the features that
underpin such studies is the haploid state of the eimerian genome throughout the majority of the life cycle. Thus, clonal parasites can be isolated by
passage of a single sporozoite or sporocyst and each cloned parasite line
may be considered homologous at all loci prior to fertilization and zygote
formation (Chapman and Rose, 1986; Shirley and Harvey, 1996). Nonetheless, the brief sexual phase of the life cycle facilitates chromosomal segregation and genetic recombination ( Jeffers, 1976) and supports classical genetics
studies where a departure from the anticipated Mendelian phenotypic ratio
of inheritance informs on genotype (Sturtevant, 1913). More recently,
molecular biology has revolutionized our understanding of genomes and
their associated biology, providing new tools for genetics-led research and
creating the omics disciplines.
3.1. Markers employed in genetic studies

Variation between and within Eimeria species has been investigated using
several phenotypic and genotypic tools as reviewed elsewhere (Beck
et al., 2009). Briefly, phenotypic tools have included differential isoenzyme
migration through starch gels or by isoelectric focusing, resistance or susceptibility to defined chemotherapeutic exposure, precocious development and
the ability to escape strain-specific immune killing (Blake et al., 2005; Jeffers,
1976; Shirley et al., 1989; Smith et al., 1994ad). Genotypic tools, including
random amplification of polymorphic DNA polymerase chain reaction
(RAPD-PCR), restriction fragment length polymorphism (RFLP), amplified fragment length polymorphism (AFLP) and gross chromosomal size
polymorphism as revealed by pulsed field gel electrophoresis (PFGE), have
also been used to define inter- and intraspecific variation (Blake et al., 2011a;
Fernandez et al., 2003a; Shirley, 2000).
3.2. Cross-fertilization and genetic recombination

Early studies with coccidial parasites suggested the absence of genetic recombination, however, molecular, genetic and microscopic studies have now
demonstrated numerous crossover events and recombination nodules,

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respectively (Blake et al., 2011b; Canning and Anwar, 1968; del Cacho
et al., 2005; Shirley and Harvey, 2000). Almost 40 years ago, the first evidence of genetic exchange during multi-clonal infection revealed a capacity
for cross-fertilization, independent segregation and the consequential production of hybrid progeny ( Jeffers, 1974). In this example, concurrent infection of E. tenella strains resistant to the anticoccidial drugs amprolium or
decoquinate yielded a population resistant to both compounds. However,
the efficiency of cross-fertilization remains unclear, as the proportion of
hybrid progeny within specific crosses has been calculated to vary between
0.05% and 39.5% (Blake et al., 2004; Joyner and Norton, 1975). Proportions
of hybrid progeny, based on comparison of oocyst output in the presence or
absence of deleterious selection, depend on the underlying genetic complexities of the trait (i.e. the number of contributing genetic loci). In addition,
proportions are affected by the timing of selection within the life cycle and
the magnitude of the doses of oocysts given, which can result in parasite
crowding within the intestine (Blake et al., 2004; Williams, 2001).
3.3. Genetic linkage analyses

The inheritance of polymorphic genetic markers can be used to infer a
genetic map based upon comparisons of recombination frequencies between
markers. Key assumptions include a random (independent) association
between markers on separate chromosomes and a correlation between
recombination rate and physical distance for markers linked on the same
chromosome. For the Eimeria species, early linkage studies included the creation and selection of multi-drug resistant hybrid lines by cross-fertilization,
in which it was noted that close physical linkage of contributing loci may be
an explanation for incompatible resistance combinations ( Joyner and
Norton, 1978). The development of sequence-based genetic markers permitted the development of more sophisticated linkage analyses, including
the derivation of genetic linkage maps and whole genome association
(WGA) studies.
Genetic linkage maps have been produced for two Eimeria species. In the
first genome-wide linkage study, a cross was made between an attenuated
E. tenella Wisconsin line (selected for precocious development within
125 h) and a Weybridge line selected to become resistant to the anticoccidial
drug arprinocid (Shirley and Harvey, 2000). The hybrid component of the
progeny of this cross, capable of replication within 125 h in the presence of
arprinocid, were recovered and amplified by selective in vivo passage and

107
used to derive a panel of 22 hybrid clonal progeny lines by single sporocyst

cloning. Using RAPD-PCR, RFLP, AFLP and full karyotype PFGE, 443
parent-specific genetic markers were generated and mapped against all
22 clones. Using the free linkage software Map Manager QT (Manly and
Cudmore, 1997), 16 linkage groups were created, representing a genetic
genome size of 653 centimorgans (cM). Intriguingly, 53% of all the
polymorphic markers scored were clustered into just three linkage groups,
indicating the absence of dividing recombination events within all 22 cloned
progeny. At the time of publication, this was attributed to possible bias introduced by AFLP, however, the subsequent description of a segmented
genome structure for E. tenella now suggests pronounced hot and cold spots
of genetic recombination (Blake et al., 2011b; Ling et al., 2007). More
recently, a genetic map was created for E. maxima using a similar strategy
with the selectable traits resistance to the drug robenidine and escape from
strain-specific immune killing (Blake et al., 2011b). Using a larger panel of
647 genetic markers, generated exclusively by AFLP, linkage analysis created a map made up of 13 major linkage groups representing a genetic
genome size of 2883.9 cM.
The first WGA study on E. tenella (Shirley and Harvey, 2000) made the
key assumption that each parent-specific genetic marker is inherited in a 1:1
ratio in the absence of deleterious selection. Thus, a genetic marker distantly
linked to a locus under selection, or located on a separate chromosome,
should persist within the hybrid progeny irrespective of selection. Representation of a more closely linked marker should be reduced within the progeny
that survive selection, while a marker that is linked most intimately should be
severely under-represented or lost altogether. Clonal lines drawn from a
hybrid population under selection should conform to the same rules,
resulting in linkage disequilibrium. Following combined selection for resistance to arprinocid and the ability to reproduce within 125 h, all genetic
markers incorporated into the E. tenella genetic map were found to persist
in the anticipated 1:1 ratio with the exception of linkage groups mapped to
chromosomes 1 and 2, whose inheritance correlated with drug resistance
and precocious ability, respectively (Shirley and Harvey, 2000). The number
and size of loci mapped in this study would have been influenced by the biological factors described above, as well as practical factors including the number of genetic markers and independent clones.
While the number of genetic markers available can be adjusted with relative ease, the effort required to isolate and amplify clonal lines of Eimeria
places an inherent limit on the number of clones that can be handled in

108
any one laboratory. Working with an uncloned hybrid progeny population

under selection, instead of a finite panel of clones, can provide a massive
increase in mapping power. Working on this hypothesis, hybrid
E. maxima populations, selected for resistance to robenidine and the ability
to escape strain-specific immune killing, were used in the first populationbased WGA strategy with Eimeria species to map loci that encode strainspecific immunoprotective antigens (Blake et al., 2011a). Briefly,
E. maxima isolates are commonly characterized by antigenic diversity such
that immunization with one strain can induce apparently complete immune
protection against homologous challenge but incomplete protection against
challenge by an antigenically distinct strain (Smith et al., 2002). In one
extreme example, immunization of inbred Line C White Leghorn chickens
(maintained at the Institute for Animal Health, UK) with the Houghton (H)
or Weybridge (W) E. maxima strains yields no statistically significant crossprotection. WGA scrutiny of this phenotype, using the uncloned progeny of
multiple crosses between the H and W E. maxima strains before and after
W strain-specific immune selection, identified six distinct genetic loci whose
inheritance correlated absolutely with the immune phenotype (Blake et al.,
2011a). Subsequent fine mapping of two of these loci identified immune
mapped protein-1 and confirmed apical membrane antigen 1 (AMA1) as
partially immunoprotective antigens. The immunoprotective capacity of
three of the remaining four loci was also demonstrated using bacterial artificial clones (BACs) that covered each mapped W strain locus. Transient
transfection with whole BAC DNA was used to genetically complement
the H strain, and transferred the ability to induce a protective immune
response against the W strain (Blake et al., 2011a).
4. THE OMICS TECHNOLOGIES

4.1. Genomics
Three distinct DNA genomes have been defined in eimerian parasites, localized to the nucleus, the mitochondrion and the apicoplast organelle. Additionally, a double stranded RNA genome associated with virus-like particles
has been commonly found in Eimeria species (Han et al., 2011; Lee and
Fernando, 2000; Shirley, 2000). Using PFGE, the nuclear genomes of all
avian Eimeria species investigated to date have been estimated to contain
between 50 and 60 Mb DNA (Blake et al., 2011b; Shirley, 2000). The
E. tenella genome is the best characterized to date, featuring a GC content

109
of 53% and two major ribosomal gene clusters on chromosomes 10 and

12 (500 copies of the 5S rDNA and 140 copies of the 18S5.8S28S
rDNA, respectively) (Shirley, 2000). Complete mitochondrial genome
sequences have been assembled for all seven species that infect the chicken,
and comprise 6200 bp (61486407 bp) DNA with 65% A T content
(Lin et al., 2011; Liu et al., 2012). The relatively high A T content influences codon usage and, consequently, amino acid content of the three protein coding genes cox-1, cox-3 and cytB. Sequencing of the larger plastid
genome identified 35 kb DNA with a similarly high A T content
(Cai et al., 2003). Double stranded RNA virus genomes have been identified within many isolates of chicken Eimeria species ranging in size from
1.7 to >7.4 kb. Hybridization studies suggest the existence of multiple,
genetically distinct viral strains or species (Lee and Fernando, 2000;
Lee et al., 1996).
4.1.1 Nuclear karyotype

Oocysts of the Eimeria species that infect poultry are characterized by
extremely tough walls which are resistant to chemical, detergent,
enzymatic- and temperature-based disruptions, in contrast to those that
infect mammalian hosts such as the rat (Kurth and Entzeroth, 2008). Until
recently, the oocyst wall has prevented morphological analysis of eimerian
karyotypes and chromosome replication, hindering microscopic characterization (del Cacho et al., 2001). However, the recent development of
a protocol based upon oocyst incubation in a hydrochloric acid/ethanol
solution followed by multiple freezethaw cycling has permitted the
release and spread of intact chromosomes, confirming previous PFGEbased estimates of a 14 chromosome karyotype for all species that have
been investigated (del Cacho et al., 2005; Shirley, 1994a). Using PFGE,
the E. tenella and E. maxima karyotypes have been shown to range from
1 to >7 Mb and 2 to >6 Mb, respectively (Blake et al., 2011b;
Shirley, 2000). Comparison between E. tenella strains has revealed variable
sizes for chromosomes 14 and 11 (Sheriff et al., 2003; Shirley, 2000). Variation by as much as 5% for chromosome 1 between extreme examples has
been used as a genetic marker in linkage analyses (Shirley and Harvey,
2000). The molecular basis for the observed polymorphism remains
unclear, although the highly repetitive nature of the E. tenella genome is
likely to encourage non-homologous genetic recombination, resulting
in length polymorphisms.

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4.1.2 Genome sequencing

The E. tenella Houghton strain was the first eimerian to be subjected to
nuclear genome sequencing. A combination of Sanger, 454 and Illumina
sequencing technologies was used to create a whole genome assembly representing 94% of the complete nuclear genome in 4682 contiguous
sequences (contigs). While high frequency of repetitive sequences has hindered further assembly of the genome, sequencing large insert genomic
DNA libraries has permitted assembly of 90% of the genome into 1720
supercontigs. Public access to the consensus assembly is available through
the Wellcome Trust Sanger Institute website, GeneDB (www.genedb.
org) and EUPathDB (www.eupathdb.org). Additionally, chromosome 1
of E. tenella has been fully sequenced and assembled. The identification of
telomeric-like repeats at each end of the assembly suggests representation
of the majority of the chromosome, including 85% of the predicted
1.05 Mb sequence with a small number of sequence gaps (Ling et al.,
2007). Comparison with a chromosome 1 HAPPY map (mapping based
on the analysis of approximately HAPloid DNA samples using PCR) supported the validity of the assembly and provided a tool to anchor and order
the sequence contigs (Ling et al., 2007). More recently, a cloned line derived
from the E. maxima Houghton strain has been sequenced, providing the first
genomic resource for a species that parasitises the mid-intestine (Blake et al.,
2012). Sanger sequencing combined with 454 sequencing yielded 13-fold
genome coverage and a consensus assembly representing 74% of the
nuclear genome in 12,852 contigs (publically available through EmaxDB,
www.emaxdb.org). Nuclear genome sequences for the Houghton strains
of all seven species of Eimeria of the chicken, and for additional strains of
E. tenella, are now being generated using multiple next-generation sequencing technologies and during 2013 all of these sequences will be available for
public scrutiny within both the GeneDB and the EuPathDB databases.
Gross genomic comparison among apicomplexan parasites reveals a relatively conserved genome size for coccidial parasites, significantly larger than
the haemosporids and piroplasms (Table 2.1). While genome size varies significantly among the Apicomplexa, the predicted number of protein coding
genes varies less dramatically (from largest to smallest fold difference:
genome eightfold, gene number twofold; Table 2.1). Broader comparison with most other apicomplexan parasites reveals a negatively correlated association between gene density and genome size, likely to be
underpinned by a core gene set essential to parasite function and survival
irrespective of genome size. Furthermore, the multi-host lifestyles of the
Table 2.1 Apicomplexan parasites: A current genetics and genomics summary

Genome size Chromosome Predicted
Gene density
Organism
Strain
(Mb)
no.
proteins
(genes Mb1)
Recombination rate
(kb cM1)
Eimeria tenella
264b
Blake et al.
(2011b)
Houghton 55.0
14
8786
Eimeria maxima Houghton 57.5
14
Not known Not known
60120
Blake et al.
(2011b)
Toxoplasma
gondii
ME49
63.0
14
7993
127
104
Khan et al.
(2005)
Neospora
caninum
NCLiv
61.0
14
7082
116
na
na
Plasmodium
falciparum
3D7
23.3
14
5538
238
17
Su et al. (1999)
Babesia bovis
T2Bo
8.1
3706
458
na
na
Theileria parva
Muguga
8.3
4082
492
4.6
Katzer et al.
(2011)
9.1
3805
418
10-56
Tanriverdi et al.
(2007)
Cryptosporidium Iowa II
parvum
a
160
Referencea
Reference used to derive the rate of genetic recombination.

Expected to drop should additional markers be included as described previously for T. gondii (Khan et al., 2005; Sibley et al., 1992).
na, not available.
Data derived from EuPathDB (http://eupathdb.org/eupathdb/, accessed 3 July 2012).
b

112
hemoparasites and piroplasms might indicate a requirement for a larger core

gene set. While total chromosome number remains stable within the larger
genomes, the relative rate of genetic recombination exhibits a strong negative correlation (Table 2.1), possibly indicating a minimum requirement
for recombination, irrespective of genome size.
4.1.3 Genome structure

The Eimeria species are protozoan eukaryotic organisms. Each eimerian
genome is represented by a series of chromosomes including telomeres
and centromeres (del Cacho et al., 2005). Transmission electron microscopy
studies have revealed a constant telomere length of 32 nm for E. tenella and a
consistent centromere index per chromosome among several strains,
although figures vary between chromosomes, thereby providing distinctive
identifiers (del Cacho et al., 2005). Intronic sequences are common within
most coding regions and there is proteomic evidence for alternative splicing
(Lal et al., 2009). Unusually, among apicomplexan parasites, sequences
related to eukaryotic transposable elements are readily identified. It has
been suggested that these are most similar to non-LTR LINE-like
retrotransposons (long terminal repeat and long interspersed nuclear
elements) (Ling et al. 2007). Most strikingly, the sequencing and assembly
of E. tenella chromosome 1 revealed an unusual segmented structure to
the chromosome, which contains three repeat-rich segments flanked by four
repeat-poor segments (GenBank Accession number AM269894). The
repeat-rich segments contain large numbers of simple sequence repeats
and LINE-like elements as well as highly variable A T content, CpG to
GpC ratio and second-order Markov entropy, prompting the identifier
feature-rich (R-segment) (Ling et al., 2007). In contrast, the repeat-poor
segments exhibited less variation in all measures, prompting the identifier
feature-poor (P-segment). Whole genome HAPPY mapping now suggests
that the segmental organization described from chromosome 1 is also present
throughout the rest of the E. tenella genome (Lim et al., 2012).
Preliminary characterization of predicted gene structures within the
R- and P-segments suggests shorter coding sequences with larger exons
and less numerous introns in the latter. RFLP comparison of E. tenella genomic DNA revealed polymorphism between the Houghton, Weybridge and
Wisconsin strains for all four of the R-segment probes tested, but none of the
four P-segment probes, suggesting a higher rate of genome evolution within
the feature-rich regions. Comparative studies with the E. maxima genome

113
suggests that R and P regions are also present throughout the genome of this
species (Blake et al., 2011b).
4.1.4 Repetitive sequences
Eimerian genes are known to feature large numbers of repetitive DNA
sequences ( Jenkins, 1988) and early reports of numerous trinucleotide
GCA repeats (and alternative frame permutations CAG, AGC, TGC,
GCT and CTG) are supported by more systematic studies of published
EST data and the E. tenella chromosome 1 assembly. Almost 3000 simple
repeat units were identified on chromosome 1 and more than 60% of these
were GCA-based (Ling et al., 2007). Other common repeats included a
telomere-like heptamer AGGGTTT, representing nearly 20% of the repeats
on chromosome 1, and the palindromic octamer TGCATGCA, which has
been described previously within several other apicomplexan genomes. As
noted above, simple sequence repeats were confined largely to the
R-segments in the E. tenella chromosome 1 assembly, where triplet repeats
and AGGGTTT represent 14% of the sequence. Both EST and genomic
analyses identified frequent triplet repeats in putative coding regions, a feature confirmed by recent proteomic analysis (Lal et al., 2009; Ling et al.,
2007; Shirley, 2000). Importantly, triplet repeats do not interfere with
the coding frame in expressed sequences, unlike heptamer and octomer
repeats. To date, no functional association has been made with these translated repetitive sequences, although they have been hypothesized to play a
role in genome evolution and diversification as possible hotspots of recombination (Ling et al., 2007).
Other repetitive sequences include multiple putative transposable
sequences and arrays of tandemly repeated 5S and 18S5.8S28S ribosomal
genes (both discussed above). The high copy number of the rDNA arrays
and the ITS regions (ITS-1 and ITS-2) has promoted their use as targets
for molecular diagnostics and phylogenetic analyses, although it is important
to note the existence of polymorphism between copies within a single
genome (Blake et al., 2006; Vrba et al., 2011).
4.2. Transcriptomics
Systematic descriptions of four Eimeria species transcriptomes have been
reported in addition to small numbers of targeted cDNA sequences from
the same and other species. By far the most thoroughly characterized has
been E. tenella, with more than 50,000 publically available expressed
sequence tag (EST) sequences (Amiruddin et al., 2012; Chen et al., 2008;

114
Klotz et al., 2007; Novaes et al., 2012; Wan et al., 1999). E. acervulina and
E. maxima are both well represented (e.g. Dong et al., 2011; Miska et al.,
2008; Novaes et al., 2012; Schwarz et al., 2010) and E. brunetti has been sampled (Aarthi et al., 2011). Sequences have frequently been derived from the
most easily accessed oocyst and sporozoite life cycle stages, although secondgeneration merozoites have commonly been prioritized given their relevance to coccidiosis caused by E. tenella (Amiruddin et al., 2012; Miska
et al., 2008; Novaes et al., 2012; Schwarz et al., 2010). Key stages in the
eimerian life cycle waiting to be sampled include the gametocytes and developing intracellular schizonts.
4.2.1 Full-length cDNA sequences
Analysis of 433 full-length cDNA sequences from E. tenella Houghton strain
second-generation merozoites has provided the most detailed study of transcript structure for the Eimeria species. At 1647 bp the average transcript
length was longer than described for Toxoplasma gondii or Cryptosporidium
parvum (range 4413083 bp), including average 50 untranslated region
(UTR), ORF and 30 UTR sizes of 342, 867 and 438 bp, respectively
(Amiruddin et al., 2012). The longer transcript length was possibly
influenced by the high frequency of simple sequence repeats. Alignment
of translation initiation sequences proximal to each predicted start codon
identified a consensus Kozak sequence of (G/C) AAAATGG. Usage analysis
identified 10 under-represented codons, UAU, UGU, GUA, CAU, AUA,
CGA, UUA, CUA, CGU and AGU, in line with previous reports for
E. tenella (Amiruddin et al., 2012; Ellis et al., 1993). Simple sequence repeats
were again found to be common throughout many of the full-length
sequences, most commonly translating as poly-glutamine tracts (CAG) or
related equivalents following frameshifts and repeat degradation.
4.2.2 Transcript identification and inter-species comparison
Significant EST and open reading frame expressed sequence tag
(ORESTES) cDNA datasets exist for E. tenella, E. acervulina and
E. maxima in the public domain. In a recent study, 48,361 ORESTES
and EST sequences derived from a series of E. tenella zoite and oocyst stages
at multiple developmental time points were collated and assembled into
8700 contiguous and singleton sequences (Novaes et al., 2012; Rangel
et al., 2013). Comparison with the 8786 putative protein coding sequences
predicted from the E. tenella genome sequence (http://www.genedb.org;
Table 2.1) suggested a good coverage, although the absence of sequences

115
derived from schizont and gametocyte stages indicates distinct gaps imposed
by the difficulty in obtaining suitable parasite material. Comparison with
other coccidian parasites including Neospora caninum and T. gondii
highlighted a conserved genome-wide gene density (as discussed above;
Table 2.1). Consideration of the individual sequence read distribution across
these assemblies prompted the authors to hypothesize that each life cycle
stage is likely to be characterized by a small number of highly expressed
genes, supplemented by a larger number of genes expressed at a much lower
level (Novaes et al., 2012). The application of RNAseq technologies is now
starting to significantly improve transcriptome coverage and gene prediction, as has been described for N. caninum (Reid et al., 2012), and may confirm this hypothesis.
Equivalent ORESTES analyses for E. acervulina and E. maxima resulted
in 3413 and 3426 assembled cDNAs, respectively (Novaes et al., 2012;
Rangel et al., 2013), overlapping with many of the 1029 and 1380 unique
contiguous and singleton EST sequences derived in other notable studies for
these species (Miska et al., 2008; Schwarz et al., 2010). E. brunetti is at present
the only other eimerian parasite whose transcriptome has been systematically
sampled, being represented by 269 unique contiguous and singleton EST
sequences (Aarthi et al., 2011). Comparison with E. tenella cDNA sequences
identified putative homologues for between 19% and 32% of these unique
expressed sequences, with more than 47% of all sequences sharing no significant similarity to currently available annotated cDNA sequences derived
from any organism. The appearance of such a large number of unknown
putative coding sequences was anticipated and has been a common feature
of many apicomplexan genomes (Reid et al., 2012; Schwarz et al., 2010).
The number of putatively genus- and species-specific expressed sequences
is consistent with the specialized life cycle and exquisitely restricted host
and tissue range of these parasites. Nonetheless, transcripts encoding homologues of key apicomplexan invasion-relevant proteins including several
microneme proteins (MICs) and glideosome components have been readily
identified in at least three Eimeria species (Novaes et al., 2012; Schwarz et al.,
2010). Similarly, surface antigen (SAG) transcripts have been identified
within the E. tenella, E. acervulina and E. maxima transcriptomes.
Hierarchical clustering of transcriptomic data derived from each of the
Eimeria species sampled to date has revealed a highly conserved expression
profile between life cycle stages and a strong correlation with stage order
within the life cycle. Thus, transcripts derived from sporozoites were most
likely to be conserved within the sporulated oocyst transcriptome

116
(Novaes et al., 2012). Similarly, close associations have been identified

between the sampled zoite stages and between oocyst datasets collected at
different stages of sporulation.
Functional transcriptomic studies using custom designed cDNA arrays
with or without a suppression subtractive hybridization step have been
undertaken with E. tenella and E. maxima. Examples include the association
of monensin resistance with up-regulation of transcripts involved with cytoskeletal rearrangement and energy metabolism and a panel of 32 differentially
expressed transcripts associated with precocious parasite development (Chen
et al., 2008; Dong et al., 2011). Screening a cDNA panel derived from multiple purified zoite and unpurified chicken intestine/schizont samples against
a genome tiling array has been used to identify coding sequences within
genetic loci mapped by association with susceptibility to strain-specific
immune killing in E. maxima (Blake et al., 2011a).
4.3. Proteomics
Before the advent of genome sequencing and annotation, the processes by
which specific proteins could be identified, characterized and analyzed
were time-consuming, laborious and low throughput, usually limited to
the study of one or two individual proteins at a time. Methods for direct
analysis of known polypeptide sequences often utilized mass spectrometry
(MS), for example, to identify specific features of the protein such as proteolytic cleavage sites. However, generation of de novo protein sequence
was most commonly achieved by chromatographic analyses of peptides
following chemical treatment of purified protein to progressively remove
amino acids, usually from the N-terminus. Over the past decade, huge
advances in MS instrumentation and the availability of well-annotated
genomes has allowed the burgeoning of high-throughput proteomics
technologies. Complex mixtures of proteins can be fragmented into small
peptides by enzymatic digestion or chemical degradation, and subjected in
parallel to high-energy MS that generates large numbers of individual spectra from which peptide sequences can be directly inferred. These experimentally derived sequences are then mapped in silico onto databases of
predicted proteins derived from annotated genomes, allowing the
unequivocal identification of full coding sequences. This type of approach
is well suited to high-throughput experiments in which hundreds or thousands of individual proteins can be identified. While it is possible to analyze
very complex mixtures, such as a whole cell lysate, it is usual to carry a

117
protein separation technique prior to MS to reduce sample complexity and

aid the downstream in silico analysis. For parasites, a number of approaches
have been taken including one- or two-dimensional gel electrophoresis,
and in-line liquid chromatography (LC; recently reviewed by Wastling
et al., 2012).
4.3.1 Studies using MALDI-MS
High-throughput proteomics technologies emerged in parasitology at the
beginning of the twenty-first century and were readily adopted by the
coccidian research community. Initial studies focused on polypeptide spots
excised from polyacrylamide gels following their separation by twodimensional electrophoresis (2DE). Spots were subjected to in-gel digestion,
usually with trypsin, and protein identifications made by generating peptide
mass fingerprints, acquired by matrix-assisted laser desorption/ionization
(MALDI) MS. Initial proteomes were derived for tachyzoites of T. gondii
(Cohen et al., 2002) and N. caninum (Lee et al., 2003) and for sporozoites
and second-generation merozoites of E. tenella (de Venevelles et al.,
2004; Liu et al., 2009). A major disadvantage of MALDI-MS is, however,
that unambiguous protein identifications can be made only if high-quality
gene annotations are available, which was not the case for these parasites
at the time. Therefore, MALDI data were generally supplemented by
re-analysis of selected protein spots using tandem MS to generate de novo
peptide sequence data. As an adjunct to proteomics studies, de Venevelles
et al. (2004) and Liu et al. (2009) probed 2DE blots of sporozoite or merozoite proteins with hyperimmune chicken sera and identified 50 and
85 spots, respectively, that were immunogenic, numbers that are in agreement with several earlier studies using Western blotting (Sutton et al., 1989;
Tomley, 1994; Xie et al., 1992).
A different proteomics approach, aimed at elucidating the proteome of
purified microneme organelles from E. tenella sporozoites (Bromley et al.,
2003), used a post-source modification of MALDI, termed chemically
assisted fragmentation, which improves fragmentation efficiency and simplifies interpretation of the spectrum. Briefly, a negatively charged group
is coupled to the N-terminus of tryptic peptides so that formation of a positively charged ion requires the introduction of two protons, one of which
resides in the peptide backbone, where it can resonate and assist fragmentation. After fragmentation, only y-ions retain a positive charge, which simplifies the spectrum and allows it to be used to generate de novo peptide
sequences. Using this method, 37 of 96 spots excised from 2DE gels were

118
successfully identified, which included proteins known to reside in the

micronemes (EtMIC1, 2, 3) as well as several novel proteins that had not
previously been linked to this organelle. In a similar type of study, de
Venevelles et al. (2006), used a combination of MALDI and tandem MS
to analyze peptides derived from partially purified refractile bodies (RB)
of E. tenella sporozoites. As well as two proteins known to reside in the
RB (the aspartyl proteinase Eimepsin and the antigen SO7), 30 additional
putative RB proteins were identified including a hydrolase, a subtilisin
and a lactate dehydrogenase.
4.3.2 Studies using high-energy MS
Advances in MS instrumentation, especially the use of high-energy,
collision-induced dissociation-based, tandem MS, has revolutionized the
field of proteomics, allowing high-throughput identification of very large
numbers of tryptic peptides obtained from gel spots, gel slices or LC fractions. Using a range of complementary approaches (2DE, gel-LC linked
to tandem MS and multi-dimensional protein identification technology,
MuDPIT), whole cell proteomes with a high level of coverage have now
been obtained for four developmental stages of E. tenella (Lal et al.,
2009). In addition, there are also high-quality proteomes available for various sub-cellular fractions including the rhoptry organelles of E. tenella
(Oakes et al., 2013).
The most comprehensive proteomics study of an eimerian is that of Lal
et al. (2009) who generated proteomes for four life cycle stages of E. tenella
(unsporulated oocysts, sporulated oocysts, sporozoites and secondgeneration merozoites) resulting in the unequivocal identification of 1868
proteins, which represents almost 30% of the likely total number of
E. tenella proteins. A total of 288 proteins were conserved between sporozoites and merozoites, but not found in unsporulated oocysts, suggesting
these are linked to zoite-specific functions such as attachment, invasion
and egress. These included proteins known to localize to the microneme,
and rhoptry secretory organelles and proteins associated with the
glideosome, which drives zoite motility and the moving junction (MJ)
structure that is formed at the hostparasite interface during invasion.
Importantly, it was noted that stage-specific variants of key molecules
involved in the formation of the MJ are expressed, such as AMAs and
rhoptry neck proteins (RONs), suggesting strongly that sporozoites and
merozoites assemble the MJ in a stage-specific manner, something
that has since also been shown to occur in T. gondii (Fritz et al., 2012a,b)

119
and has been confirmed again in the rhoptry sub-cellular proteome of

E. tenella (Oakes et al., 2013). Additional protein differences between
the zoite stages included differential expression of the GPI-linked families
of SAGs, as also shown in the transcriptome analyses (Novaes et al., 2012).
The multi-stage proteome indicates that energy production throughout
most of the developmental cycle is linked strongly to gluconeogenesis
and glycolysis, with the mannitol cycle present in oocysts and sporozoites,
but not in merozoites, consistent with in-depth biochemical analysis of this
pathway throughout the E. tenella life cycle (Allocco et al., 1999). It was
also apparent that proteins linked to oxidative phosphorylation were
expressed at the highest levels in the merozoite, suggesting a metabolic
shift to use oxygen to mobilize energy production during the asexual
phases of growth and replication. There is also a higher abundance of
proteins linked to transcription, protein synthesis and nucleotide metabolism in the merozoite, compared to the sporozoite, which is consistent
with observations in the transcriptome and likely to be related to the
massive replication that has taken place within the schizont, just prior to
merozoite release.
4.4. The future

Progress in the development of novel molecular technologies applicable to
nucleotide sequencing, epigenetics and proteomics support increased
understanding of eimerian parasites, their close relatives and their host interactions. Improved sequencing technologies facilitating cheaper, more effective, genome sequencing and assembly are now supporting the extension of
such studies to a rapidly increasing range of coccidial parasites. Nextgeneration DNA and RNA sequencing strategies also have the potential
to revolutionize genetic mapping and our biological understanding of selectable parasite phenotypes. Importantly, many such resources have been made
publically available for the Eimeria species. Genome sequences and predicted
protein datasets are freely available through EUpathDB (http://eupathdb.
org/eupathdb/), GeneDB (http://www.genedb.org/Homepage/Etenella)
and EmaxDB (http://www.genomemalaysia.gov.my/emaxdb/). Complementary resources including annotated transcript assemblies and genetic
maps can be accessed through the Eimeria Transcript database (Rangel
et al., 2013) (http://www.coccidia.icb.usp.br/eimeriatdb/) and NCBI
Map Viewer (http://www.ncbi.nlm.nih.gov/projects/mapview/), respectively. The production of novel and improved datasets will impact upon

120
the development of new diagnostic and anticoccidial control strategies and

provide tools with which questions of evolutionary and population biology
may be interrogated.
5. TRANSFECTION
Transfection refers to the introduction of exogenous DNA or RNA
into cells by chemical, biological or physical means. Through transfection,
the recipient cell can gain a new genetic trait, and some of the introduced
DNA can be integrated into the genome of the recipient cell. In
apicomplexan parasites, such as Toxoplasma and Plasmodium, plasmidmediated transient and stable transfection systems were established in the
early 1990s, but owing to the difficulty of completing the life cycle of Eimeria
in vitro, and the lack of regulatory DNA sequences, genetic manipulation has
lagged behind that of other protozoan parasites (Hao et al., 2007; Kelleher
and Tomley, 1998; Shi et al., 2008). The first stable transfection system was
developed in Eimeria in 2008, 10 years after the first report of a transient
transfection system in this genus of parasite (Clark et al., 2008; Kelleher
and Tomley, 1998).
5.1. Transfection construct design

Transfection constructs are usually based on pre-constructed, commercially
available plasmids, and contain elements including regulatory and signal
sequences. Promoters of both constitutive genes and stage-specific genes,
together with their homologous or heterologous 30 UTR sequences, have
been used successfully to drive expression of reporter genes in eukaryotic
cells, including Eimeria. Using enhanced yellow fluorescent protein (eyfp
marker gene) as a reporter, it was shown that the E. tenella microneme protein 1 (EtMIC1) promoter drives EYFP expression in sporulated but not
unsporulated oocysts, as would be expected (Yin et al., 2011). Another study
showed that three different promoter sequences originating from E. tenella
could function effectively not only in other species of Eimeria but also in
T. gondii (Kurth and Entzeroth, 2009; Zou et al., 2009). Similarly, promoters
of the housekeeping tubulin gene and the differentially regulated surface
antigen gene (sag1) of T. gondii, were effective in driving the expression
of the EYFP marker gene in E. tenella (Zou et al., 2009). As genetic tools
are well developed for T. gondii, the mutual recognition of these promoter
sequences in Eimeria and Toxoplasma suggests that some promoter sequences
from T. gondii could be utilized directly in Eimeria and that T. gondii could be

121
used as a novel transfection system for Eimeria-rooted vectors. This has the
potential to help improve our understanding of Eimeria spp. through the
development of both forward and reverse genetic technologies.
Several signal sequences are known to exhibit conserved activity in
apicomplexan parasites. For example, parasitophorous vacuole targeting signal sequences of T. gondii GRA8 and Plasmodium falciparum repetitive interspersed family proteins have been found to function effectively in transfected
E. tenella and successfully target EYFP to the parasitophorous vacuole in
E. tenella (Shi et al., 2009; Yin et al., 2011). Furthermore, the nucleustargeting signal of the H5N1 subtypic avian influenza virus nuclear protein
also exhibits conserved functionality in eukaryotic cells, supporting nuclear
targeting when incorporated into an E. tenella transfection construct (Yin
et al., 2011).
5.2. Transient transfection

In transient transfection, introduced exogenous DNA does not integrate
into the genome of the cell, but is transcribed into mRNA and is subsequently translated to protein. Transient transfection is an efficient tool to
identify regulatory and signal sequences of genes and to screen for genes
associated with certain phenotypes. For Eimeria, transfection of sporozoites
has been achieved by electroporation with plasmids; PCR amplified DNAs
or fragmented genomic templates that encode the exogenous DNA, flanked
by Eimeria-specific regulatory sequences (Hao et al., 2007; Kelleher and
Tomley, 1998; Liu et al., 2008). The efficiency of transient expression is usually low but this has been overcome by the use of restriction enzymemediated integration (REMI), which boosts transfection efficiency about
200-fold (Liu et al., 2008). Very high transfection efficiency has been
achieved in E. tenella sporozoites by using cytomix-buffered REMI and
the AMAXA nucleofection system (Clark et al., 2008).
5.3. Stable transfection

Stable transfection refers either to the permanent expression of the gene of
interest through the integration of the transfected DNA into the nuclear
genome, or the maintenance of a transfected plasmid as an extra chromosomal replicating episome within the cell. Stable transfection has been difficult to achieve in Eimeria because of the inability to transfect oocysts or
sporocysts, the absolute requirement for in vivo amplification and selection,
and the poor survival of sporozoites in the acidic environment of the host

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stomach. The latter has been overcome by gavaging birds with sodium
bicarbonate to neutralize the acidic barrier (Clark et al., 2008). Stable transfection systems have also been established for E. tenella by cloacal inoculation
of sporozoites, combined with in vivo drug selection and/or fluorescence
activated cell sorting (FACS) (Clark et al., 2008; Yan et al., 2009). To date,
the mutated dihydrofolate reductasethymidylate synthase gene is the only
drug-mediated selection marker available for the transfection of Eimeria
(Clark et al., 2008; Yan et al., 2009). The mutated gene confers resistance
to pyrimethamine, a drug used to potentiate the action of the sulphonamides. Drug selection, together with FACS of fluorescence reporter
proteins and the high transient transfection efficiency using REMI, contributed to the success in establishing stable transfection in Eimeria.
Integration of a transfection construct into the Eimeria genome seems to
occur at random during the production of stably transfected Eimeria as
detected by Southern blotting and plasmid rescue (Yan et al., 2009). Quantitative real-time PCR analysis of insertion rate post-transfection showed an
average persistence of four copies of the tandem YFP reporter cassette per
genome from the first round of replication after electroporation and REMI.
After two further cycles of in vivo amplification with both FACS and pyrimethamine selection, an average of 10 copies per genome were detected and
remained relatively stable through five further unselected generations. In
contrast, when REMI was not used, only a single copy of the relevant
reporter gene was detected per genome in first or second-generation transfected parasite populations (Clark et al., 2008). Serial selection of fluorescent
mCitrine-transfected oocysts by FACS did not notably increase copy number, although tightened gating and FACS with sporocysts in place of oocysts
increased both the expression rate and the copy number to 23 per genome
(Clark et al., 2008).
5.4. PiggyBac-based forward genetic system

PiggyBac is a cut-and-paste transposon that is useful for transgenesis and insertional mutagenesis and has been used for stable transfection in a wide variety of
organisms. This new molecular technology has been used successfully to
achieve targeted insertional mutagenesis in Eimeria (Su et al., 2012). Using
REMI, E. tenella sporozoites were electroporated with a mix containing
the restriction enzyme AscI, an AscI-linearized helper plasmid containing
the transposase gene, and an uncut donor plasmid containing the eyfp gene.
The eyfp gene was flanked by Eimeria-specific regulatory sequences that

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were further flanked by piggyBac inverted terminal repeats (ITRs). Subsequently, electroporated sporozoites were inoculated into chickens via the
cloacal route and transfected progeny oocysts expressing eyfp were sorted
by flow cytometry. A stable eyfp expressing population was obtained by successive in vivo passaging and FACS selection (Su et al., 2012). Locus-specific
PCR and genome walking revealed that the ITR-restricted sequence was
successfully targeted into TTAA sites, with about seven copies per genome
(Su et al., 2012). Both reverse and forward genetic tools will hopefully allow
an in-depth analysis of Eimeria basic biology. PiggyBac-mediated efficient
TTAA targeted mutations should be an attractive tool for genetic manipulation of Eimeria.
5.5. Stably transfected Eimeria as a vaccine vector and beyond

The feasibility of using genetically modified Eimeria as a vaccine vector has
been studied using model antigens such as EYFP. It was found that
E. tenella expressing EYFP stimulated both humoral and cell-mediated
immunity to the expressed protein, and that antigen compartmentalization
affects the magnitude of the immune response with microneme-targeted
EYFP stimulating a higher IgA response than cytoplasm-targeted EYFP
(Huang et al., 2011). In another study, vaccination of specific pathogenfree chickens with a population of E. tenella expressing Campylobacter
jejuni antigen A caused a significant reduction in bacterial load following
challenge with C. jejuni compared with unvaccinated and wild-type
E. tenella vaccinated controls (Clark et al., 2012). Thus, it has been demonstrated that transfected Eimeria parasites can successfully express foreign
antigens that may stimulate immunity against a target pathogen. However,
to provide complete protection, co-expression of adjuvant antigens and/or
cytokines may be necessary (Guangwen Yin and Xun Suo, unpublished
observations).
Transfection of Eimeria species is still limited by the inability to transfect
oocysts and sporocysts, the difficulty of obtaining single-sporocyst-derived
recombinant clones, and the obligate requirement of in vivo amplification
and selection of stably transfected parasites (Clark et al., 2008; Shi et al.,
2008; Yan et al., 2009). It is difficult to maintain a large number of mutated
clones as a mutated Eimeria library, which needs manpower, facilities and
financial support. Nevertheless, high transfection efficiencies (Clark et al.,
2008; Hanig et al., 2012) will boost the advance of both reverse and forward genetic systems in this important group of parasites. The interchange

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and development of Eimeria transfection constructs between laboratories in

countries including China, France, Germany, Japan and the UK promises
rapid development over the coming years. More advanced genetic tools
established in other protozoa, such as Toxoplasma, Plasmodium, Leishmania
and Trypanosoma may eventually be applied to research with Eimeria.
6. OOCYST BIOGENESIS
One of the defining features of the coccidia is the oocyst. There are
three crucial milestones in oocyst production: first, merozoites undergo rapid,
asexual division within the intestine, amplifying dramatically the total number
of parasites poised to develop into microgametes or macrogametes; second,
microgametes fertilize the macrogametes and third, the macrogametes mobilize specialized organelle wall forming bodies (WFBs) to generate the
oocyst wall, one of the most remarkable biological structures known.
The oocyst wall encapsulates and protects coccidian parasites as they exit
their definitive host in faeces and, subsequently, in the harsh, external world,
while they undergo meiosis to produce infectious sporozoites. Thus, the
oocyst is the endpoint of sexual reproduction. It is also notoriously resilient,
resisting both mechanical and chemical damage and tolerating changes in
humidity and temperature for months, if not years (reviewed by Belli
et al., 2006; Fritz et al., 2012a). This resilience is critical for transmission
of coccidian parasites from host to host, via ingestion of contaminated food
or water.
6.1. Veil and WFBs

The formation of the oocyst wall proceeds via an orderly release of the contents of: first, the veil forming bodies; second, wall forming bodies type 1
(WFB1) and third, wall forming bodies type 2 (WFB2) (Ferguson et al.,
2003). The contents of the veil forming bodies are undescribed but form
a loose outer veil that appears to provide a temporary scaffold or frame
around the developing oocyst wall (Ferguson et al., 2000, 2003). It is lost
before the oocyst is excreted in the faeces and, therefore, plays no role in
protecting the parasite in transit from host to host (Ferguson et al., 1975;
Pittilo and Ball, 1980).
The release of the contents of the WFBs appears to be controlled by the
rough endoplasmic reticulum/Golgi apparatus (Ferguson et al., 2003). Once
a zygote has formed, WFB1 migrate to the periphery of the parasite, align
and disaggregate rapidly, before appearing to merge together to form the

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Figure 2.3 Immunofluorescent images of macrogametocytes and early oocysts of

E. maxima within the intestine of a chicken 144 h post-infection. (A) An early-stage
macrogametocytetype 1 and type 2, wall forming bodies (WFBs) are indistinguishable. (B) A mid-stage macrogametocytetype 1 and type 2, WFBs are distinguishable
by size (WFB1s are larger). (C) A cluster of mature gametocytes showing peripheral
alignment of WFBs, with disaggregation of some type 1 WFBs evident. (D) Early
oocyststhe outer wall has formed and disaggregation of type 2 WFBs is evident. Host
nuclei are stained with 40 ,6-diamidino-2-phenylindole (blue); WFBs and oocyst walls are
stained with antibodies to affinity-purified gametocyte antigens (green). Image supplied
by Professor D.J.P. Ferguson (University of Oxford, UK).
outer layer of the bi-layered oocyst wall (Fig. 2.3). This outer layer may initially be as thick as 600 nm but quickly compacts to 200 nm or less (Ferguson
et al., 2003). Not long after the outer layer forms, WFB2 are also transferred
to the parasites surface, by the endoplasmic reticulum, and also align, disaggregate and also appear to fuse together to form the inner layer of the oocyst
wall (Ferguson et al., 2003). This layer is less electron-dense than the outer
layer and more consistent in size, being around 40 nm in most species examined (reviewed by Belli et al., 2006). The inner and outer layers are, at first,
separated by a 40 nm zone, which shrinks as the wall compacts. However,
the two layers never fuse together and are readily separated in the laboratory
(Monne and Honig, 1954).

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6.2. Oocyst wall proteins

Gas chromatography and MS analyses of the oocyst walls of E. maxima and
E. tenella indicate that both layers are dominated by protein (>90%) with
surprisingly low levels of carbohydrate and lipids (Mai et al., 2009). Thus,
an understanding of the structure and characteristics of proteins that comprise the oocysts wall is essential for genuine understanding of how the wall
forms and why it is so robust. Only a small number of oocyst wall proteins
have been identified and the origin of all of these can be traced back to the
WFBs in macrogametes (reviewed in detail by Mai et al., 2009). These proteins can be grouped into, essentially, three groups.
First, there is a 22 kDa antigen in the macrogametocytes of E. tenella,
which is found in WFB2 and the inner layer of the oocyst wall (Krucken
et al., 2008). This 22 kDa protein is dominated by histidine and proline residues. As yet, no information is available about if or how this protein is
incorporated into the oocyst wall, though its involvement in stabilizing
the oocyst wall via cross-links between histidine and catechols, as seen in
insect cuticles, is a distinct possibility (Krucken et al., 2008).
Second, there is a family of nine large (174190 kDa), cysteine-rich proteins that localize to WFB1 of macrogametocytes and the outer wall of the
Cryptosporidium oocyst (Spano et al., 1997; Templeton et al., 2004). It is
thought that these OWPs form disulphide bridges and matrices within
the oocyst wall (Spano et al., 1997). The recent discovery of seven OWPs
in Toxoplasma, with at least some of these localized to the outer oocyst wall
(Possenti et al., 2010), supports the idea that OWPs are involved in wall formation. Recently, two OWP homologues have been found in Eimeria, at
least one of which appears to localize to WFB1 in macrogametes, further
evidence that OWPs are destined for the outer wall (Walker, 2009).
Third, there are numerous tyrosine-rich proteins, ranging in size from
8 to 31 kDa, in the inner wall of the Eimeria oocyst; all of these are derived from
precursor proteins of 56 and 82 kDa (GAM56 and GAM82) from WFB2 in
macrogametes of several species of Eimeria (Belli et al., 2003a, 2009). It has also
been discovered very recently that, although the Toxoplasma genome contains
no direct homologues of either GAM56 or GAM82, the oocyst wall of Toxoplasma contains up to six tyrosine-rich proteins (Fritz et al., 2012a).
6.3. Formation of the oocyst wall

The role of tyrosine-rich proteins in the formation of the Eimeria oocyst wall
has been studied in some depth, the result being the proposal of a two-step

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model: in step 1, precursor proteins found in WFBs are processed by

gametocyte-specific proteases into smaller, tyrosine-rich proteins and in step
2, peroxidases and/or oxidoreductases catalyze cross-linking of these proteins via their tyrosine residues, resulting in extensive dityrosine matrices
within the oocyst wall. There is some evidence for both of these proposed
reaction steps.
It has been known for more than two decades that two proteins
GAM56 and GAM82 dominate the protein profile of gametocytes
(Wallach et al., 1989). Both GAM56 and GAM82 are processed into small,
tyrosine-rich proteins, as demonstrated in two ways: (i) antibodies to
GAM56 and GAM82 react with proteins of these sizes in gametocytes
but react with proteins of 831 kDa in oocysts (Belli et al., 2003a,b,
2009); and (ii) N-terminal sequencing of these wall proteins shows that they
are cleaved from GAM56 or GAM82 at specific points (Belli et al., 2003a).
It has been discovered recently, using an in vitro assay, that the degradation of
GAM56 into smaller proteins is largely dependent on subtilase-like serine
protease activity (Katrib et al., 2012). There are at least six subtilase-like
enzymes in the genome of E. tenella, and at least three of these are expressed
specifically in gametocytes. Thus, assembly of the oocyst wall may follow a
mechanism that is similar to that involved in the assembly of the cuticle of
nematodes (Page and Winter, 2003; Thacker et al., 2006).
After formation of the numerous, small, tyrosine-rich derivatives of
GAM56 and GAM82, peroxidases or oxidoreductases are predicted to catalyze their cross-linking via dityrosine bond formation. There is substantial
circumstantial evidence indicating that oocyst walls are rich in dityrosine
bonds. First, the oocysts exhibit a vivid blue autofluorescence between
the ultraviolet excitation wavelengths of 330 and 385 nm (Fig. 2.4), which
is characteristic of dityrosine cross-linking (Belli et al., 2006). Second,
dityrosine levels have been measured in the oocyst wall of E. maxima and
found to be remarkably high (Belli et al., 2003a), begging the conclusion
that their generation within the oocyst wall is a deliberate, enzymatically
catalyzed process, initiated by the parasite (Belli et al., 2006). It has been
shown that the WFBs of E. maxima embody a highly focused region of
peroxidase activity (Belli et al., 2003a, 2006) and, while neither an endogenous peroxidase or oxidoreductase has yet been isolated from Eimeria, it
has been established that exogenous peroxidases can induce dityrosine
cross-linking of a truncated version of GAM56 in vitro (Mai et al., 2011).
And, an oxidoreductase has been found in the oocyst wall of T. gondii
(Fritz et al., 2012a).

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Figure 2.4 Sporulated and unsporulated oocysts of E. maxima showing characteristic

UV autofluorescence (blue).
The concept that dityrosine cross-linking constitutes a critical feature of

the structure of the oocyst wall helps to explain the resilience of oocysts
dityrosine cross-linking, leading to the formation of structural matrices,
sclerotization and quinone tanning, is widespread in nature, almost always
in association with the construction of protective coatings such as invertebrate egg shells, cuticles, cell walls, glues and cements (reviewed by Belli
et al., 2006). Moreover, it might be predicted that interfering with this process is a way to limit the transmission of coccidian parasites. The subunit vaccine, CoxAbic, may be an example of this. This vaccine contains GAM56
and GAM82 from E. maxima and laboratory experiments have shown that
immunization of broiler breeder hens with this vaccine stimulates the production of protective IgY (IgG) antibodies that are transferred to offspring
chicks via the egg yolk (Wallach et al., 2008). There are two potential explanations for this: (1) the antibodies protect GAM56 and GAM82 from proteolysis and, thereby, deprive the parasite of the tyrosine-rich building
blocks it needs to form the oocyst wall; and/or (2) the antibodies interfere
with dityrosine bond formation (Sharman et al., 2010).
7. HOST CELL INVASION

Apicomplexans, including all species of Eimeria, are highly successful
obligate intracellular parasites. Unlike many microorganisms that rely on
host-cellular pathways such as phagocytosis or pinocytosis for invasion,

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apicomplexans invade host cells rapidly and forcefully in a highly regulated,

parasite-driven process (reviewed by Santos and Soldati-Favre, 2011). Initial
interaction with host cells can occur with the parasite in any orientation but
commitment to invasion requires that the apical pole of the parasite makes
irreversible contact with the host cell surface. This initiates formation of a
MJ, a tight focus of constriction between the parasite and host cell membrane, which migrates towards the posterior end of the parasite as invasion
proceeds (reviewed by Besteiro et al., 2011). The parasite synthesizes specialized molecular complexes at the parasitehost interface, which are essential for gliding movement (the actinomyosin-dependent glideosome) and
formation of the MJ. These complexes are assembled by regulated secretion
of microneme (MIC) and rhoptry (RON/ROP) proteins, and interact with
the parasite motor, localized in the pellicle, and with specific receptors on
the host cell surface. As the parasite propels itself forwards, surface-bound
adhesion complexes are released by proteolysis. Other proteins, derived
largely from the rhoptries, are secreted into the host cell where they contribute to the formation of a parasitophorous vacuole and its associated membrane, and modify the host intracellular environment (reviewed by
Boothroyd and Dubremetz, 2008).
7.1. Parasite surface proteins

In common with many groups of protozoa, the surfaces of Eimeria sporozoites and merozoites are coated with glycosylphosphatidylinositol (GPI)anchored proteins that are collectively referred to as surface antigens or SAGs
(Gurnett et al., 1990; Tabares et al., 2004). In T. gondii and species of Plasmodium, GPI-anchored proteins are implicated in the early stages of parasite
attachment, prior to apical re-orientation. This requires interaction with
sulphated glycosaminoglycans on the surface of host cells. Preliminary data
indicate that several E. tenella SAGs are able to bind a variety of cultured cells
(F. Tomley and C. Subramaniam, unpublished observations) suggesting that
they too are involved in this initial non-specific binding step.
Examination of EST sequences from E. tenella identified 37 potential
GPI-linked variant SAGs encoded by multi-gene families and differentially
expressed between sporozoites and second-generation merozoites (Tabares
et al., 2004). GPI-anchored proteins in higher eukaryotes are often found
within membrane structures called lipid rafts, which are detergent-resistant
microdomains involved in signal transduction, membrane trafficking and
molecular sorting. Potential lipid rafts were identified on the surface of

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Eimeria invasive stages by staining for the lipid-raft marker flotillin-1 (del
Cacho et al., 2007). However flotillin-1 was most prominent at the apical
end of sporozoites, whereas E. tenella SAGs are expressed over the entire
sporozoite surface (Tabare et al., 2004).
Recent transcriptome, proteome and genome data indicate that E. tenella
expresses up to 80 different SAG proteins, and confirms that these are differentially regulated during the life cycle such that second-generation merozoites are coated with more complex mixtures of SAGs than either
sporozoites or first-generation merozoites (Lal et al., 2009; Novaes et al.,
2012; A. Reid, Wellcome Trust Sanger Institute, personal communication;
F. Tomley, unpublished observations). Their surface location suggests that
SAGs may induce potent immune responses that can, for example, block
sporozoite invasion of cultured cells (Brothers et al., 1988). The
co-expression by merozoites of highly polymorphic SAGs could render
anti-SAG immune responses ineffective against these stages. A study of
10 E. tenella merozoite-expressed SAGs showed that three of these induced
an increase in nitric oxide production, IL-1b and IL-10 transcription, and
induced a decrease in IL-12 and interferon-g (IFN-g) transcription in
chicken macrophages (Chow et al., 2011). This indicates that at least a subset
of SAGs has the ability to modulate chicken innate and adaptive immune
responses, which may suppress cell-mediated immunity and also contribute
to the marked pro-inflammatory responses and associated pathology seen
during E. tenella infection.
7.2. MIC proteins are adhesins and many function

as multi-protein complexes
The repertoire and broad functions of coccidian MIC proteins, including
those from Eimeria species, has been reviewed extensively, and the reader
is referred to recent articles for more details (Carruthers and Tomley,
2008; Cowper et al., 2012). Most MICs comprise modular arrangements
of protein domains that share homology with adhesins from higher eukaryotes and it is the specific binding of these domains to host cell glycans that
establish irreversible apical attachment. Across the Apicomplexa there are
many orthologous MIC proteins although the precise arrangement of
domains is not always conserved and there are some, such as the sialic
acid-binding MAR domains and galactose-binding Apple/PAN domains,
discussed below, that are restricted to the coccidia.
MIC proteins often associate to form multivalent heteromeric complexes, which assemble within the endoplasmic reticulum before being

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trafficked to the micronemes. Each MIC complex contains an escorter protein that possesses a transmembrane domain and a short cytoplasmic tail. This
facilitates targeting to the micronemes and allows the microneme complex
to interact with the underlying glideosome ( Jewett and Sibley, 2003). Some
MIC complexes are conserved across different genera, whereas others are
not. The T. gondii complex of TgMIC2/MIC2AP, which is essential for
gliding motility, host cell attachment and invasion (Huynh and
Carruthers, 2006), is orthologous to the E. tenella complex of EtMIC1/
MIC2. The introduction of the E. tenella complex into tachyzoites of
T. gondii can partially complement for loss of endogenous TgMIC2/
M2AP, indicating conservation of function (Huynh et al., 2004). However,
the T. gondii TgMIC1/4/6 complex, in which TgMIC6 is the escorter for
the MAR-domain containing TgMIC1 and Apple-domain containing
TgMIC4, is not replicated in E. tenella. The MAR-domain containing
EtMIC3 (Labbe et al., 2005) has not been found in a complex, but instead
is secreted directly from the micronemes onto the host cell surface (Lai et al.,
2011). The Apple-domain containing EtMIC5 forms a complex with
EtMIC4, which is presumed to act as an escorter, but which also bears
adhesive thrombospondin-like domains that have the potential to bind host
receptors (Periz et al., 2005, 2007).
7.3. Host glycan recognition by MIC proteins contributes

to host and tissue tropism
One of the most intriguing biological questions is why there are such huge
differences in host and tissue tropisms across members of the coccidia.
T. gondii, for example, invades virtually any nucleated cell and infects almost
all warm-blooded vertebrates, whereas each species of Eimeria infects only a
single host, replicates only in epithelial cells, and is often restricted to very
specific regions of the intestine. Recent studies on the binding of coccidian
MIC proteins to host glycans, particularly sialic acid and galactose, are now
shedding light on this issue (Cowper et al., 2012; Lai et al., 2011; Marchant
et al., 2012). Generally, there is a direct correlation between host range and
the possession of a wide repertoire of MIC proteins expressing variant
domains that are capable of binding a broad range of oligosaccharide epitopes. MAR domains, which are found in MICs across the coccidia, bind
a range of sialyl groups but evidence from carbohydrate microarrays, atomic
structure, and cell binding studies, reveals that those from T. gondii and
E. tenella are differentially equipped for binding. Thus E. tenella MAR
domains bind a limited range of structures, with a strong in vivo preference

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for a2,3-linked sialic acid and an absence of binding to any N-glycolated

sialyl structures (which are not found in the chicken). In contrast, MAR
domains from T. gondii MICs are more divergent, and bind a variety of oligosaccharides including a2,9-linked sialic acid and N-glycolylated derivatives (Lai et al., 2011). Very recent studies on carbohydrate recognition
by Apple domains, also found in MICs across the coccidia, indicates an additional contribution to host range and tissue tropism conferred by differential
recognition of galactose, another sugar that is widely distributed in animal
tissues, commonly forming b1,3 or b1,4 linkages to a preceding glucose
or galactose. While both T. gondii and E. tenella Apple domains bind galactosylated structures, there is a marked preference by T. gondii for
Galb1,3GalNAc, commonly found on gangliosides which are prevalent
on many host cell surfaces (Marchant et al., 2012). The precise binding preferences of E. tenella Apple domains have not yet been elucidated, but preliminary data indicate that these bind predominantly to b1,4-linked
galactose and do not recognize the more common b1,3 linkages (Cowper
et al., 2012).
7.4. Regulated secretion of microneme and rhoptry organelles

MIC proteins are discharged onto the parasite surface at an early stage in
invasion. The physiological trigger that induces microneme secretion is
not known, but treating parasites with agents that cause a rise in intracellular
free calcium induces rapid secretion and this can be inhibited by treatment
with intracellular calcium chelating agents (Bumstead and Tomley, 2000;
Carruthers and Sibley, 1999; Wiersma et al., 2004). Blocking calcium release
channels by treating parasites with IP3 inhibitors or ryanodine, or blocking
activity of cyclic GMP-dependent kinase or calcium-dependent protein
kinase, all interrupt the regulated exocytosis of MICs and prevent parasite
attachment and invasion of host cells (Dunn et al., 1996; Lourido et al.,
2010; Schubert et al., 2005; Wiersma et al., 2004).
ROP proteins are also secreted in a regulated manner and, while the
exact mechanisms are unknown, it is hypothesized that the initial signal
comes via the cytoplasmic tails of membrane-bound MIC complexes, following their interaction with a host cell receptor. The process is complicated
because rhoptry secretion occurs in two separate waves. Proteins that reside
within the anterior neck portion of the rhoptry (RONs) are secreted early in
invasion and are critical for the formation and maintenance of the MJ
(Alexander et al., 2005; Lebrun et al., 2005). However, proteins from the

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posterior bulb of the rhoptry (ROPs) are secreted slightly later, once the parasitophorous vacuole is formed. In T. gondii, several ROP proteins are
known to be potent virulence factors that modify and subvert host cell signalling pathways (Bradley and Sibley, 2007). The current model for differential secretion of RONs and ROPs in T. gondii is that a membrane-bound
MIC complex containing the escorter protein TgMIC8 triggers release of
the RONs (Kessler et al., 2008). Interaction of RONs with AMA1 then
triggers the release of ROPs (Tyler and Boothroyd, 2011). Regulation of
secretion of ROP/RON proteins in Eimeria, where there is no defined
orthologue of TgMIC8, has not been determined.
7.5. AMAs and formation of the MJ

The MJ was visualized many years ago (Aikawa, 1978) and is a key structure
that provides an anchor, against which the parasite can generate a force that
allows forward movement into the parasitophorous vacuole. A key component is AMA1, a transmembrane protein secreted by the micronemes onto
the parasite surface, where it interacts with secreted RONs to initiate formation of the MJ (Alexander et al., 2005; Lebrun et al., 2005). Collaboration
between proteins that are secreted from different sub-cellular organelles
requires a remarkable degree of orchestration. It has emerged recently that
while AMA1 remains anchored on the parasite surface, a RON complex is
secreted into the host cell and then one of them, RON2, becomes inserted
into the host cell membrane and interacts directly with AMA1 (Tyler and
Boothroyd, 2011). Both AMA1 and the RON protein repertoire are conserved in Eimeria indicating that the mechanism for forming the MJ is likely
to be conserved in different coccidia (Blake et al., 2011a,b; Jiang et al., 2012;
Lal et al., 2009; Oakes et al., 2013). It is worthy of note, however, that
E. tenella expresses stage-specific variants of AMA1 and several of the
RON proteins, which suggests that each zoite stage assembles a different
set of gene products with which to build the MJ (Lal et al., 2009; Oakes
et al., 2013).
8. IMMUNOBIOLOGY
It is 50 years since Elaine Rose and Peter Long published, Immunity
to four species of Eimeria in fowls, sparking a seminal year in research into
the immunology of poultry coccidiosis. By the end of 1962, it was known
that: (a) even a single infection with various species of Eimeria confers solid

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resistance to reinfection and this is increased further after a second infection

(Rose and Long, 1962); (b) immunity is expressed against the very early
asexual stages of infection such that, although penetration of epithelial cells
by sporozoites may occur, subsequent development is blocked (Pierce et al.,
1962); (c) immunity is not confined to early stages of parasite development
but also affects later stage merozoites and sexual stages (Rose, 1963); (d) in
general, immunity to one species confers no protection against other species
(Rose and Long, 1962), though it was shown subsequently that some degree
of cross-protection can occur between closely related pairs of Eimeria, such as
E. maxima and E. brunetti (Rose, 1967a), and E. tenella and E. necatrix (Rose,
1967b) and (e) B cells and the antibodies they produce play little, if any, role
in resistance to reinfection, implicating cell-mediated responses in acquired
immunity (Long and Pierce, 1963). However, in reviewing this remarkable
year, Rose (1963) noted that, The way in which an animal which has experienced an infection with a species of Eimeria subsequently prevents the
development of that species within its body is not yet understood. That
statement is equally valid today.
Immunity to Eimeria is complex, multifactorial and influenced by host
and parasite, with different elements playing greater or lesser roles in three
different types or stages of immunity: innate resistance to primary infection;
acquired immunity to reinfection and maternal immunity. Many host/
parasite combinations have been used to dissect the immunobiology of coccidiosis, with significant insights being gained through the use of murine
models due to advantages connected with the availability of murine immunological reagents, in-depth fundamental understanding of the murine
immune system and technologies to disrupt immune response genes in mice.
In this review, key similarities rather than differences in the immunobiology
of coccidial infections will be emphasized.
8.1. Innate responses to primary infection

Primary infections with the majority of Eimeria species, in poultry and
rodents, are self-limiting; asexual reproduction proceeds via a pre-set number of cycles of schizogony prior to differentiation into gametocytes, subsequent sexual reproduction and production of oocysts. This can make it
challenging to demonstrate a role for the immune system in resistance to primary infection. Nevertheless, it has been shown that increased oocyst excretion, by different Eimeria species, is a consistent feature of primary infection
in immunodeficient hosts (Klesius and Hinds, 1979; Long and Rose, 1970;

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Mesfin and Bellamy, 1979; Rose, 1970; Rose and Hesketh, 1979, 1986;
Rose and Long, 1970; Schito and Barta, 1997; Schito et al., 1996;
Stockdale et al., 1985). Moreover, there is one parasite and host pairing
E. vermiformis in the mouse where the patent period of primary infection
is clearly increased in susceptible mice, almost certainly due to a relaxation of
immune pressure on the parasite that allows additional generations of schizonts to develop (Rose and Millard, 1985; Rose et al., 1984, 1985; Schito
et al., 1996). Thus, the E. vermiformis murine model of coccidiosis has proved
particularly significant for our understanding of immunological resistance to
primary infection.
Studies with E. vermiformis have demonstrated that resistance to primary
infection is associated with more rapid inflammatory responses including
increased granulocyte numbers (Ovington et al., 1990), enhanced generation
of free oxygen radicals (Ovington et al., 1990), increased Natural Killer (NK)
cell activity (Smith et al., 1994a), earlier production of pro-inflammatory
cytokines such as IFN-g, tumour necrosis factor (TNF) and others
(Ovington et al., 1995; Wakelin et al., 1993), and faster T cell responses
(Rose et al., 1990; Wakelin et al., 1993). Corollaries of these observations
also exist for other rodent (Dkhil et al., 2011; Rausch et al., 2010; Rose
and Hesketh, 1982; Rose et al., 1979a; Schito and Barta, 1997), chicken
(Hong et al., 2006a,b; Kim et al., 2008, 2010; Rose et al., 1979a;
Rothwell et al., 1995, 2000, 2004; Yun et al., 2000) and turkey (Gadde
et al., 2011) coccidioses. However, of all these immunological parameters,
only two lymphocytes and IFN-g appear to be indispensible for resistance.
Severe combined immunodeficient mice, which are deficient in both
T and B cells, are highly susceptible to primary infection with
E. vermiformis, producing many more oocysts and harbouring parasites for
far longer than immunocompetent mice (Schito et al., 1996). Studies in
B cell-deficient mice (Smith and Hayday, 1998) and bursectomized chickens
(Long and Pierce, 1963; Rose and Hesketh, 1979), suggest that B cells play a
minor, though consistent, role in this resistance. Since deficiencies in antigen
presentation also increase susceptibility (Smith and Hayday, 1998, 2000),
this relatively minor role may be via the ability of B cells to act as antigen
presenting cells rather than anything to do with antibodies. Experiments
with congenitally athymic (nude) mice, however, show that T cells play a
critical role in resistance to primary infections with E. vermiformis; infected
nude mice excrete many more oocysts over a much extended patent period
(Rose et al., 1984, 1985). Other murine Eimeria (Klesius and Hinds, 1979;
Mesfin and Bellamy, 1979; Rose and Hesketh, 1986; Stockdale et al., 1985),

136
as well as E. nieschulzi in rats (Rose et al., 1979b), also produce many more
oocysts in athymic animals but without any affect on patency. Experiments
with thymectomized chickens have generated inconsistent data, probably
because of the difficulty in completely removing all T cells (Rose and
Long, 1970).
Depletion of specific T cell subsets, either via antibodies with appropriate, rigorous, confirmatory adoptive transfer experiments (Rose et al., 1988,
1992), or via the deletion of specific genes from mice (Roberts et al., 1996;
Smith and Hayday, 1998, 2000), show that CD4 , and not CD8, T cells
are the critical T cell subset mediating resistance to primary infection with
E. vermiformis. Moreover, protective effects appear to be ab T cell-specific
(Roberts et al., 1996; Smith and Hayday, 1998), though gd T cells may play
an important role in preventing immunopathology (Roberts et al., 1996)
rather than in contributing to the control of the parasite (Roberts et al.,
1996; Rose et al., 1996). Immune CD8 mesenteric lymph node cells have
also been shown to be capable of suppressing immunopathology in
E. falciformis infections (Pogonka et al., 2010). However, similar effects
are not seen in infections with Eimeria papillata (Schito et al., 1998) and
depletion studies in chickens infected with E. acervulina or E. tenella are
somewhat equivocal, possibly confounded by relatively small numbers of
experimental chickens in each treatment group and by inefficient depletion
of CD4 T cells (Trout and Lillehoj, 1996).
The most important role for CD4 T cells in mediating resistance to
primary infection with E. vermiformis is most likely as the source of IFNg. Mice treated with an antibody to IFN-g (Rose et al., 1991a) or IFN-g
gene-knockout mice (Smith and Hayday, 2000) are highly susceptible to
E. vermiformis, suffering prolonged patency, high levels of excretion of
oocysts and increased mortality. This is not so evident in infections of
IFN-g knockout mice with E. papillata where patency is not affected; in
this case, NK cells are the likely source of IFN-g (Schito and Barta,
1997). How IFN-g is controlling the parasite is not known; generation of
free oxygen radicals (Ovington et al., 1995), reactive nitrogen intermediates
(Ovington et al., 1995; Smith and Hayday, 2000) and interference with
tryptophan metabolism (Schmid et al., 2012) can all be ruled out. However,
it is known that the effects of IFN-g are mediated via the host cell rather than
a direct effect on parasites (Rose et al., 1991b).
Infections with E. pragensis or E. falciformis indicate an additional role for
IFN-g in the immunobiology of coccidiosis. Depletion of IFN-g using

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monoclonal antibodies has little apparent effect on parasite load but has a
significant affect on weight loss during primary and secondary infection with
E. pragensis (Rose et al., 1991a). Similarly, IFN-g receptor knockout mice
infected with E. falciformis suffer severe intestinal immunopathology and
weight loss mediated via Th17 pathways, involving the cytokines, IL17
and IL-22 (Stange et al., 2012). Thus, IFN-g may have an important immunoregulatory role in response to infection with Eimeria, helping to keep
intestinal inflammation in check.
Mice deficient in granulocyte and NK cell function are more susceptible
to primary infection with E. vermiformis (Rose et al., 1984). However, T cellmediated control of infection with E. vermiformis does not require
co-operation with granulocytes (Rose et al., 1989) and experiments with
E. papillata indicate that increased susceptibility to primary infection may
be due more to participation of NK cells than granulocytes in resistance
(Schito and Barta, 1997). However, the effects on oocyst excretion and patent period are relatively modest compared to those of lymphocyte deficiency
(Schito et al., 1996) and a role for NK cells in innate resistance is not
supported by results obtained with E. vermiformis (Rose et al., 1995;
Smith et al., 1994a).
Free oxygen radicals appear to play no role in resistance to E. vermiformis
since quenching of their activity in vivo actually leads to reduced, not
enhanced, oocyst activity (Ovington et al., 1995). Moreover, treatment
of mice with agents designed to enhance macrophage activity, including free
oxygen radical generation, leads to increased oocyst excretion (Smith and
Ovington, 1996), as does treatment with TNF (Ovington et al., 1995). Similarly, reactive nitrogen intermediates, despite their temporal association
with resistance to poultry coccidia (Allen and Fetterer, 2002), also enhance
oocyst production in E. vermiformis (Ovington et al., 1995). These are, at first
glance, puzzling results in light of the well-established anti-protozoal effects
of TNF, free oxygen radicals and reactive nitrogen intermediates (see
Ovington and Smith, 1992). However, with our current knowledge about
involvement of an oxidative reaction in oocyst wall assembly (Belli et al.,
2006; Mai et al., 2009, 2011) it makes some sense, indicating that perhaps
Eimeria actually subjugates the hosts oxidative burst to assist it in construction of its oocysts. Intriguingly, a related proposal has been put forward
recently it appears that E. falciformis also subverts IFN-g-induced
indoleamine 2,3-dioxygenase activity to help drive microgamete development (Schmid et al., 2012).

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8.2. Acquired immunity

Acquired immunity to Eimeria is even more enigmatic than innate resistance
to primary infections. All that can be said with any certainty is that immunity
to reinfection with Eimeria is remarkably effective and is T cell dependent
(this has been realized for more than 30 years; Rose et al., 1979b), and
that B cells (and, therefore, antibodies) are not involved in acquired immunity since bursectomized birds (Rose and Hesketh, 1979) and mice lacking
B cells (Rose et al., 1984; Smith and Hayday, 1998) are perfectly capable of
developing immunity to reinfection. It has proven almost impossible to
correlate any immune parameter with immunity to reinfection because
the expression of that immunity in experimental settings, at least, is so rapid
and efficient. However, studies using gene-knockout mice have proved
extremely useful in determining which factors may play a role. Thus,
as for primary infection, CD4 ab T cells are crucial for immunity to
reinfection with E. vermiformis (Roberts et al., 1996; Smith and Hayday,
1998, 2000). Similar, though less definitive data were also obtained for
secondary infections with E. papillata (Schito et al., 1998). However, in contrast to primary infection, IFN-g plays no role in this acquired immunity
(Rose et al., 1991a; Schito and Barta, 1997; Smith and Hayday, 2000).
Contrarily, some studies demonstrate that CD8 T cells can be used
to transfer immunity (e.g. to E. falciformis; Pogonka et al., 2010) or that
depletion of CD8 T cells can increase, very slightly, susceptibility to
E. vermiformis (Rose et al., 1992). Evidence from poultry experiments
(Trout and Lillehoj, 1996) is more difficult to interpret because experiments
showing an increase in oocyst excretion in secondary infection of birds
depleted of CD8 T cells did not include a concomitant primary infection
control, making it hard to assess how significant the increased oocyst production really was. More, and more sophisticated, analyses of acquired
immunity to Eimeria are required to resolve the mechanism(s) that are
operating.
8.3. Maternal immunity

The immune system of young animals is uneducated rendering them more
susceptible to infectious disease. Protection against infection during this vulnerable period is provided via transfer of antibodies from mother to young.
In chickens, this occurs via the egg yolk; indeed, the ability of hens to transfer remarkable quantities of IgY (IgG) antibodies to their hatchlings has
long been appreciated, including in regard to the transfer of antibodies that

139
protect chicks from infection with E. tenella (Rose and Long, 1971) or
E. maxima (Rose, 1972). In many of the progeny from hens deliberately
infected with high doses of E. maxima, this maternal immunity can be absolute (i.e. result in the complete absence of oocysts in the faeces of chicks), at
least during the first week post-hatching (Smith et al., 1994b). Maternal antibody levels (in egg yolk or chicks) are correlated with protection (Smith
et al., 1994b). Moreover, maternal immunity induced by E. maxima confers
partial protection against E. tenella, possibly via cross-recognition of conserved proteins (or, at least, epitopes) in different Eimeria species (Smith
et al., 1994c), an idea lent further credibility by the ability of maternal immunization with conserved macrogametocyte proteins to protect hatchlings
against multiple species of Eimeria (Wallach et al., 1995, 2008).
The effectiveness of maternal, antibody-mediated immunity to Eimeria
appears contradictory to the body of evidence, reviewed above, indicating
that antibodies play only a minor role in resistance to Eimeria. However,
there is actually no shortage of (often overlooked) evidence, dating back
over 40 years, showing that antibodies can protect against infection with
Eimeria. Thus, for example, sera taken 2 weeks after infection with
E. maxima can be transferred to nave birds and, as for maternal immunity,
protect some of them almost completely against infection (Rose, 1972,
1974). The protection conferred by these convalescent sera was later demonstrated to be correlated tightly with levels of parasite-specific IgG
(Wallach et al., 1994). Additionally, it was demonstrated 40 years ago
(Rose, 1972) and, again, more recently (Lee et al., 2009a,b) that Eimeriaspecific antibodies purified from egg yolks of immunized hens can be used
to transfer passive immunity against several species of poultry coccidia; the
protection can be achieved via injection or oral delivery of the antibodies.
Immune sera can even partially protect highly susceptible T cell-deficient
animals (Rose and Hesketh, 1979). Thus, antibodies certainly can protect
against Eimeria but the effect must be described as variable from absolute
to negligible even if similar immunization regimens are used (Wallach et al.,
1994). Why this is so is anything but clear. Maternal immunization,
however, does appear to be a phenomenon that can be harnessed to control
poultry coccidiosis (Smith et al., 1994b; Wallach et al., 2008).
8.4. Immunological research

Lamentably, in the decade since the retirement of Elaine Rose, research
into the immunobiology of coccidiosis has declined significantly

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(notwithstanding the efforts of researchers at the United States Department

of Agriculture to understand the role of various feed additives and Eimeria
profilin in boosting immune responses to Eimeria; reviewed by Lillehoj
and Lee, 2012). This has been exacerbated by the declines, even disappearance, of the coccidiosis research programmes at the UKs Institute for Animal Health and at the University of Technology, Sydney in Australia, as well
as a distinct lack of new commercial research into vaccines. It is doubly
lamentable because immunity to Eimeria, whether innate, acquired or
maternal, is remarkable among all parasites in its effectiveness. Moreover,
coccidiosis appears to be an excellent model to study the molecular basis
of gut immunopathology. The gut epithelium is the first point of contact
with the host for many pathogens but studies of this interaction have proven
challenging experimentally, with many models employing delivery of pathogens via subcutaneous, intravenous or intraperitoneal injection. Infection
with poultry or murine Eimeria, being largely confined to the intestine is,
therefore, an exceptional model for genuine study of gut immunology.
Hopefully, recent promising and innovative insights into unravelling the
complexity of the host/Eimeria inter-relationship (Blake et al., 2011a,b;
Stange et al., 2012) will be followed up and exploited fully.
9. DIAGNOSIS AND IDENTIFICATION

9.1. Traditional methods
Diagnosis of coccidiosis in poultry flocks continues to rely on necropsy and
the examination of birds for intestinal lesions in different areas of the gut.
Because of the site-specificity of invasion, the presence of lesions can provide
insight into which species of coccidia is/are responsible for clinical symptoms. Diagnosis may be corroborated by microscopic analysis of shape
and size of Eimeria oocysts shed in faeces from infected birds. Additional
criteria classically used to characterize Eimeria species include pre-patent
period, minimum sporulation time, tissue location of parasitic forms and
immunological specificity. However, definitive identification of a particular
Eimeria species based on morphological and pathological criteria can be
tedious, requires highly qualified personnel and may be confounded by
the overlapping features observed in different Eimeria species (Long and
Joyner, 1984).
While lesion site and aspect, and oocysts size and shape, are features often
sufficient to corroborate clinical signs of coccidiosis, there are instances
when knowing precisely which Eimeria species is/are present would be

141
helpful in managing the disease. For example, a preponderance of

E. acervulina in a litter sample might indicate increased drug resistance in this
species. This information would be useful in choosing alternative strategies,
such as switching to another anticoccidial compound known to be effective
against E. acervulina, or to a live Eimeria oocyst vaccine. If, on the other hand,
E. mitis or E. brunetti were present, then using a vaccine that contains only
E. acervulina, E. maxima and E. tenella would not be a particularly useful control strategy.
9.2. Early molecular methods

9.2.1 Starch gel electrophoresis
In the 1970s, a biochemical approach to identification of Eimeria spp. was
developed that involved starch gel enzyme electrophoresis of enzymes, such
as lactate dehydrogenase and glucose phosphate isomerase obtained from
oocyst homogenates (Rollinson, 1975; Shirley, 1975; Shirley and
Rollinson, 1979). The technique was employed to examine field isolates
of E. tenella obtained from around the world and was used to distinguish
mixtures of at least three species when present in one sample (Chapman,
1982; Shirley et al., 1989). The former study showed for the first time that
E. praecox, a species difficult to identify because of the absence of diagnostic
lesions, had a high incidence (74% of samples) in broiler flocks. While an
interesting laboratory tool for investigation of phenomena such as intraspecific variation, starch gel electrophoresis is a time-consuming technique that
requires large numbers of oocysts. In addition, protein variability is limited
by evolutionary constraints, thus limiting the observed phenotypic polymorphism. Due to these limitations, this technique has been superseded
by DNA-based methods for identification of Eimeria spp. in the field.
9.2.2 DNA hybridization
DNA hybridization was the first DNA-based technique proposed for the
molecular discrimination of Eimeria parasites (Shirley, 1994b). A typical protocol consisted of genomic DNA digestion with different restriction
enzymes, separation through agarose gel electrophoresis, blotting and
hybridization with DNA probes composed of repetitive regions. The final
result was a DNA fingerprinting comprising multiple band profiles. Similarly
to enzyme variation detection, this approach also required large numbers of
parasites and was highly time demanding. Also, the method was inherently
unable to deal with mixed samples, since overlapping band profiles are not
informative.

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9.3. Methods based on DNA amplification by PCR

Molecular techniques, primarily the PCR, have been developed to overcome the limitations of morphological examination and the aforementioned
molecular techniques. Since primers can be designed to specifically amplify
DNA of any single Eimeria species, samples containing multiple species can
be properly diagnosed. Also, the high level of amplification permits the use
of low numbers of oocysts. An excellent review of early efforts to develop
PCR assays for Eimeria is available (Morris and Gasser, 2006); here we provide an update of work in this area.
9.3.1 RAPD fingerprinting
The first PCR-based diagnostic assays developed for Eimeria relied on the
use of RAPD. This technique is based on DNA amplification using arbitrary
primers under low stringency. Under this condition, primers can anneal in
multiple sites of the target genomic DNA, thus producing DNA fingerprints
that permit differentiation of polymorphic populations. Because no previous
knowledge of the nucleotide sequence is required, RAPD was employed
largely for parasite discrimination at a time when very few genomic
sequences were available. Several groups succeeded in developing RAPD
assays for species and strain differentiation of poultry Eimeria (MacPherson
and Gajadhar, 1993; Procunier et al., 1993; Shirley and Bumstead, 1994).
However, given the low stringency of the reaction, RAPD typically suffered
from a low reproducibility, especially among different laboratories, and was
superseded by more reliable and specific PCR assays. Two main approaches
have been employed by different groups to develop such specific assays:
(1) the use of ITS1 and ITS2 and (2) the conversion of anonymous RAPD
markers into Sequence-Characterized Amplified Region (SCAR) markers.
9.3.2 PCR assays directed to specific targets
A specific PCR assay directed to E. tenella 5S rDNA was reported over
20 years ago (Stucki et al., 1993). This pioneer work was followed by assays
capable of detecting and differentiating the seven Eimeria species of domestic
fowl using ITS1 (Schnitzler et al., 1998, 1999) and ITS2 rDNA (Gasser et al.,
2005) as targets. ITS1 and ITS2 are intervening sequences that are posttranscriptionally excised from the rRNA precursor. Unlike 18S, 5.8S and
28S rRNAs, ITS1 and ITS2 are not subjected to an appreciable selective
pressure, and have undergone sufficient divergence among Eimeria species
to allow design of species-specific primers (Lew et al., 2003). A number

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of assays based on amplification of ITS1 and ITS2 rDNA have been developed, and used to determine the species of Eimeria present in poultry litter
(Hamidinejat et al., 2010; Haug et al., 2007, 2008; Jenkins et al., 2006a; Lew
et al., 2003). As a word of caution in using ITS sequences, Lew et al. (2003)
found sufficient variation on ITS1 of different E. maxima isolates to require
the design and use of two distinct sets of specific primers for this species.
Combined with rapid techniques for extracting high-quality DNA from
oocysts, ITS1-specific PCR was found to provide a more accurate picture
of Eimeria distribution at poultry farms than traditional morphometric analysis (Hamidinejat et al., 2010; Haug et al., 2008).
Primers directed to conserved ribosomal DNA sequences (18S, 5.8S,
28S), flanking either the ITS1 or ITS2 regions, have also been used in combination with denaturing polyacrylamide gel electrophoresis or capillary
electrophoresis (CE) for species discrimination (Cantacessi et al., 2008;
Gasser et al., 2005; Morris et al., 2007a,b). The latter method relies upon
identifying species-specific peaks in CE chromatograms that have been
established using pure cultures (Gasser et al., 2005). Using this approach,
one group has identified a genetic variant of E. maxima, and new operational
taxonomic units in oocysts isolated from poultry operations (Cantacessi
et al., 2008; Morris et al., 2007b). By employing primers directed to conserved regions (28S, 5.8S) flanking the ITS1 sequence, these authors identified genetic variants that would have gone unnoticed using ITS1-specific
primers. Although assays based on ITS1 and ITS2 are highly sensitive due to
the large number of rDNA repeats (Vrba et al., 2010), variation in the
sequence may prevent primer binding. Nevertheless, ITS1 is still being used
as a target for the development of diagnostic assays for Eimeria parasites of
other hosts, including 4 species pathogenic for turkeys (Cook et al.,
2010) and 11 species that infect the domestic rabbit (Oliveira et al., 2011).
As an alternative to ITS1 and ITS2, Fernandez et al. (2003a) used RAPD
to develop SCAR markers for each Eimeria species of domestic fowl. In
developing this assay, the DNA sequences of individual RAPD markers
were determined and used to design longer primers, which were then tested
under highly stringent conditions for species-specific amplification of
Eimeria DNA. By combining a set of seven SCAR markers, Fernandez
et al. (2003b) developed a multiplex PCR assay that permits the simultaneous discrimination of all Eimeria species infecting chickens in a single-tube
reaction (Fig. 2.5). An Eimeria SCAR database containing 151 SCARs is
publicly available on the web (Fernandez et al., 2004; http://www.
coccidia.icb.usp.br/eimeriaScardb), and SCAR markers have been used

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Figure 2.5 Agarose gel electrophoresis of multiplex PCR products using DNA samples
of E. acervulina (lane 1), E. brunetti (lane 2), E. tenella (lane 3), E. mitis (lane 4), E. praecox
(lane 5), E. maxima (lane 6), E. necatrix (lane 7), a mixture of the seven Eimeria species
(lane 8) and a control with no starting DNA (lane 9). Molecular size markers (lane M) in
base pairs are indicated on the left Reproduced from Fernandez et al. (2003b) with permission from Cambridge University Press.
by other groups to determine the Eimeria species composition on poultry

farms in different regions of the world (Carvalho et al., 2011a,b;
Ogedengbe et al., 2011b), and for the development of quantitative PCR
assays (Blake et al., 2008; Vrba et al., 2010). The advantage of SCAR marker
technology, unlike assays based on ITS1- and ITS2-PCR, is that highly conserved SCAR marker sequences are available as targets of amplification
(Blake et al., 2008; Fernandez et al., 2004; Vrba et al., 2010). This avoids
the problem of false negative reactions due to poor annealing of primer
to target DNA because of variation in the target sequence. The drawback
to SCAR technology is that it may be less sensitive than assays based on
ITS1 and ITS2, which are found in multiple copies in the Eimeria genome
(Vrba et al., 2010).
Finally, strain differentiation still lacks the variety of molecular markers
already available for other apicomplexan parasites. In this direction, it is
worth mentioning that species-specific sets of microsatellite markers for
E. acervulina, E. maxima and E. tenella have been developed in Brazil
(A. Gruber and S. Fernandez, unpublished observations). This work has
been deposited as a patent (Espacenet, patent BRPI0702051). These

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microsatellite markers allowed for the differentiation of both field samples

and commercial vaccines lines.
Despite the enormous impact of PCR-based methods to detect and discriminate Eimeria species, some drawbacks still persist. Oocysts are the most
accessible stage of the life cycle, and the obvious choice as a source of Eimeria
DNA. Since the oocyst wall is remarkably resistant to chemical agents,
mechanical disruption with glass beads is the most common method to
extract DNA. However, DNA yield does not correlate linearly with the
number of oocysts (Fernandez et al., 2003b), due to decreased efficacy of
mechanical disruption in low-concentration suspensions. Despite some
authors having proposed alternative chemical treatments to disrupt the
oocyst wall (Haug et al., 2007; Zhao et al., 2001), this step remains the most
important limiting factor for good sensitivity. Beside faeces, litter is another
important source of Eimeria samples; however, a drawback of utilizing poultry litter as a source of Eimeria DNA is the presence of PCR inhibitors.
Although different extraction techniques have been developed to recover
DNA from oocysts (Carvalho et al., 2011a; Haug et al., 2007; Zhao
et al., 2001), the adequate removal of inhibitory substances from litter is
often difficult. With the goal of controlling for false negative reactions
due to PCR inhibition, a technique has been developed based on
co-amplification of ITS1 sequences and an Eimeria species-specific internal
standard ( Jenkins et al., 2006b). Using gel electrophoresis, the target and
internal standard PCR products can easily be distinguished from each other
by acrylamide gel electrophoresis (Fig. 2.6). In the event of inhibition, a second extraction of DNA is undertaken with the goal of removing inhibitors.
9.3.3 Quantitative PCR assays
Early assays using ITS1, ITS2 or SCAR markers relied on qualitative assays
in which identification of amplification products has been obtained by agarose or polyacrylamide gel electrophoresis. Over the past 5 years, a number
of assays using quantitative real-time PCR (qPCR) amplification of ITS1,
ITS2 or SCAR markers have been developed. Blake et al. (2008) utilized
primers that were specific for SCAR markers of E. acervulina, E. necatrix
and E. tenella, and for a mic gene of E. maxima. The authors reported a sensitivity varying from 1 to 10 genomes. Another group utilized ITS1 targets
to amplify E. acervulina, E. brunetti, E. maxima, E. necatrix and E. tenella DNA
isolated from pure cultures and field samples, and achieved an assay sensitivity that was between 10 and 100 oocysts (Kawahara et al., 2008). A qPCR
assay using primers conserved among various protozoa and melting curve

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Figure 2.6 Detection of Eimeria species oocysts using ITS1-PCR and internal standard (IS).
Ea, E. acervulina; Eb, E. brunetti; Ema, E. maxima; Emi, E. mitis; En, E. necatrix; Et, E. tenella. kbp,
fX174 HaeIII DNA standards. *Target band for each species of Eimeria. Reproduced from
Jenkins et al. (2006a,b) with permission from the American Association of Avian Pathologists.
analysis could detect E. acervulina in a mixture of oocysts (Lalonde and

Gajadhar, 2011).
Multiplexing real-time PCR, using one of four different upstream
primers conserved between two species, and a conserved downstream
primer, in combination with species-specific TaqMan probes, was used to
analyze all Eimeria species in poultry litter (Morgan et al., 2009).
A greater number of species were identified than those revealed by microscopy. Another group reported the development of FAM-labelled TaqMan
species-specific probes, targeted to microneme 1 gene of E. maxima and
SCAR markers of the remaining species (Vrba et al., 2010). An assay sensitivity of a single sporulated oocyst has been claimed. In a study using only
E. acervulina, qPCR directed to SCAR markers was applied to DNA
extracted from oocysts obtained from cloacal swabs, and compared with
oocyst counts from individual faecal droppings (Velkers et al., 2010). The
authors concluded that qPCR of cloacal swabs might be useful for determining the prevalence and identity of Eimeria oocysts in litter. A similar
approach using high-resolution melting curve analysis and qPCR directed
to ITS1 sequences was found capable of identifying all seven Eimeria species
in pure oocysts cultures (Kirkpatrick et al., 2009).
9.4. LAMP
While qPCR is superior to conventional PCR in that it eliminates the need
for gel electrophoresis and provides quantitative results, samples must be run

147
on a fairly expensive real-time apparatus. In fact, the complexity of DNA

extraction from the oocyst, associated with the need for expensive
thermocycling and electrophoresis equipment, severely limit the use of
molecular assays in poultry farms. To overcome these limitations, an
Eimeria-specific PCR-based technique that utilizes loop-mediated isothermal amplification (LAMP) technology has been developed (Barkway et al.,
2011). Since the enzyme is isothermal, the reaction can be performed in a
simple heat block or water bath, without the need for thermocyclers. Also,
detection can be made with intercalating dyes using the naked eye for observation (Fig. 2.7), thus eliminating the requirement for electrophoresis.
Finally, instead of using oocysts, the proposed protocol employs mucosal tissues collected post-mortem as samples, and DNA extraction by a simple
boiling method. Altogether, the method addresses the several limitations
Figure 2.7 Loop-mediated isothermal amplification (LAMP) specific for E. tenella.

Application to a purified genomic DNA dilution series revealed a limit of detection
of between 1 and 10 E. tenella genomes using agarose gel electrophoresis (A) or
hydroxynaphthol blue as a visual indicator (B, blue: positive, pink: negative). ve,
no template negative control.

148
of conventional molecular assays and may become a mainstream costeffective tool for the diagnosis of Eimeria infection in poultry flocks.
9.5. Morphological diagnosis revisited

One of the key features of morphological diagnosis based on oocyst shape
and size is the inherent subjectivity of the method and the requirement of
skilled personnel. An early attempt to differentiate species of Eimeria in
the fowl utilized a computerized image-analysis system (Kucera and
Reznicky, 1991). The method used two measurements, length and width
of oocysts, which restricted the ability to differentiate all seven species
due to the overlap of these characters. To address these limitations,
Castanon et al. (2007) have reported the development of COCCIMORPH,
a system that implements a framework for feature extraction, shape characterization and automated classification of chicken Eimeria oocyst images.
The system employs a classifier trained with thousands of oocyst images
of all species of chicken Eimeria. COCCIMORPH provides a public web
frontend (www.coccidia.icb.usp.br/coccimorph) that permits users to
upload oocyst images and obtain a reliable diagnosis in real-time. The
Bayesian classifier showed an overall correct species assignment of 85.7%,
with individual rates varying from 74.9% for E. necatrix to 99.2% for
E. maxima. While this system still has many limitations to be widely used
as a mainstream diagnostic system, it represents a proof of principle that morphology may have gained a revival in the era of digital image processing and
pattern recognition methods. COCCIMORPH breaks a classical paradigm,
as it does not require sample transportation to a reference laboratory, and
photomicrographs sent through the Internet are sufficient to obtain species
diagnosis. While not competing with the accuracy, sensitivity and quantitative nature of modern PCR-based methods, morphological diagnosis based
on digital image processing might represent a near-zero cost alternative to be
used by technicians in poultry farms that do not harbour expensive molecular biology facilities.
9.6. Conclusions
Field diagnosis of Eimeria infection in poultry will continue to rely on the
identification of intestinal lesions and microscopic examination of faecal
droppings and litter for Eimeria oocysts. However, several molecular assays
that can detect and differentiate all seven Eimeria species of the chicken are
now available, and are being used either in a research setting to study the

149
epidemiology of avian coccidiosis, or by private companies to monitor the

purity of vaccine lines. Information gleaned from molecular assays can assist
in managing disease by allowing informed decisions on which anticoccidial
compounds or live oocyst vaccines should be used in particular poultry
farms. The development of real-time qPCR represents a step forward
towards quantitative diagnosis of Eimeria. Also, isothermal amplification
assays with colorimetric detection (e.g. LAMP), that avoids a requirement
of expensive equipment, and the development of more robust image
processing software, may provide low-cost alternatives for species diagnosis
in poultry farms.
10. CONTROL
10.1. Chemotherapy
A truly landmark contribution to poultry science was the demonstration, in
1948, that it was possible to control coccidiosis by the continuous inclusion
of an anticoccidial drug (sulphaquinoxaline) in the feed of chickens
(Grumbles et al., 1948; reviewed by Chapman, 2009). The principle
involved (prevention or prophylaxis) has had a profound impact on our ability to grow chickens and turkeys under intensive conditions. Indeed it is
possible that the modern poultry industry could never have developed to
its present extent without the advent of drugs to control coccidiosis. Today,
anticoccidial drugs are incorporated routinely into the feed of broiler
chickens and turkeys for this purpose (Chapman, 2001, 2008). For example,
data available for the United States indicates that the use of anticoccidial
drugs in broiler flocks varied from 70% to 98% depending upon the season
(AgriStats, Inc., Fort Wayne, IN, USA). In Western Europe, 91% of complexes use an anticoccidial drug (C. Bostvironnois, Elanco Animal Health,
personal communication). Drug usage is similarly extensive in other major
poultry producing regions around the world. Although there may be seasonal variation in the use of drugs, it is clear that chemotherapy as a means
of control is widespread. The long-term outlook for such a heavy reliance
upon chemotherapy is often stated to be uncertain because of the widespread
development of drug resistance, a problem first recognized in the 1950s, and
a concomitant lack of new drug discovery. Furthermore, some anticoccidials
have been banned (in the EU) and others are said to be under threat
(McDonald and Shirley, 2009). So far, however, such considerations do
not seem to have led to a decline in drug use.

150
Anticoccidial drugs fall into two categories, the synthetic compounds

(produced by chemical synthesis and popularly known as chemicals) and
the ionophore antibiotics, which are by-products of bacterial fermentation. Synthetic drugs were the first to be discovered and comprise a diverse
array of molecules that are absorbed into the blood stream of the host and
kill developing parasites in epithelial cells of villi in the intestine. One of
the oldest synthetic drugs, nicarbazin, is also one of the most successful and
is still used widely today (Chapman, 1994a). Ionophores have a different
mode of action from synthetic drugs since they are able to destroy motile
stages of the Eimeria life cycle (sporozoites and merozoites) in the gut
lumen or following cell penetration (Smith and Strout, 1979). Since the
introduction of monensin in 1972, ionophores have been the most widely
used anticoccidial drugs for the control of coccidiosis (Chapman, 2001).
The mode of action and discovery of monensin, together with matters
of importance to the poultry industry such as toxicity, pharmacology,
residues and resistance to this drug, has been reviewed recently
(Chapman et al., 2010).
Despite the availability of a dozen or so anticoccidial drugs it may be surprising to know that for the majority the mode of action against coccidia is
not known (Chapman, 1997). In one case, the discovery of a biochemical
pathway unique to Eimeria (the mannitol cycle) enabled the mode of action
of nitrophenide, a drug briefly used in the 1950s, to be elucidated (Schmatz
et al., 1989). Unfortunately, resistance to this drug developed quickly, a fate
shared by most other synthetic compounds. Diclazuril and decoquinate are
synthetic drugs to which resistance can also develop. Diclazuril has recently
been shown to induce ultrastructural changes in merozoites and cause
disruption of transmembrane potential in the mitochondrion (Zhou et al.,
2010). It is not clear if this reflects a true mode of action or is just a
consequence of cell death. Decoquinate, like other members of the quinolone family, is known to act against the electron transport chain of coccidia
(reviewed by Chapman, 1997). This drug has recently been shown to cause
chromosomal rearrangements during meiosis in oocysts of E. tenella
(Del Cacho, et al., 2006). The mode of action of ionophores involves disruption of ion transport across the parasite cell membrane (reviewed by
Chapman, et al., 2010) and resistance has been much slower to develop.
Evidence has been obtained that resistance may be due to changes in the fluidity of the cell membrane of sporozoites (Wang, et al., 2006). A recent study
suggests that monensin is also able to interrupt invasion of host cells by sporozoites (del Cacho et al., 2007).

151
Most drugs are no longer as effective as when they were first introduced
due to the development of drug resistance. For example, one recent report
indicated that 68% and 53% of field isolates of E. acervulina from chicken
flocks in the EU were resistant to the synthetic drug diclazuril, and the ionophore monensin, respectively (Peek and Landman, 2006). Similar reports
of resistance have been reported worldwide. In the turkey, drug resistance
has also been shown to be widespread (Rathinam and Chapman, 2009).
Details of the emergence of resistance in the 1970s to decoquinate have been
provided retrospectively (Williams, 2006). Although many surveys have
been published indicating the extent of resistance, little research has been
conducted on the mechanisms involved. Biochemical, genetic and applied
aspects of resistance have been reviewed (Chapman, 1997).
An early insight was that use of low concentrations of certain drugs in the
feed did not necessarily prevent the acquisition of immunity (Grumbles
et al., 1948). It is now known that most drugs are effective in the field
because they only partially suppress parasite development, allowing birds
to acquire natural immunity as a consequence of exposure to parasites that
escape drug action (Chapman, 1999). An advantage of immunity development is that it allows the safe withdrawal of drugs several weeks before the
birds are sold with considerable savings in the cost of medication and reduction of the risk of potential drug residues in poultry meat.
10.2. Vaccination
Vaccination as a means to control coccidiosis has a long history (see
Chapman, 2003; Williams, 2002a) but it is only in the past 20 years or so
that this has proved a practical method for the control of coccidiosis in commercial broiler flocks, principally because it has proved feasible to vaccinate
chicks in the hatchery by spraying birds with controlled numbers of oocysts
within enclosed cabinets. This involves considerable cost savings compared
with traditional methods of vaccination which were carried out on the farm
by trained personnel. Most commercially available vaccines comprise live
oocysts and vary according to the number of species of Eimeria included,
the numbers of oocysts present, and whether or not they are attenuated.
Vaccines containing all species that infect the chicken are used mainly to
immunize egg laying stock whereas vaccines containing fewer species (usually E. acervulina, E. maxima and E. tenella) are used in broilers. The first vaccines comprised populations of wild-type oocysts that were potentially
pathogenic, but more recently, vaccines containing attenuated parasites

152
which have reduced pathogenicity but retain immunogenicity, have been

introduced.
The purpose of vaccination with live oocysts is to provide an early initial
stimulus of the immune response. After placement of birds on litter, new
vaccinal oocysts are shed in the faeces and, following sporulation, these
are capable of re-infecting the flock. Secondary exposure to vaccinal oocysts
and wild-type oocysts present in the litter is thought to induce protective
immunity. Development of immunity takes several weeks and some cases
of vaccination failure occur because birds are overwhelmed with exposure
to wild-type virulent oocysts before they have had time to develop an
immune response. It is obviously important that vaccination is undertaken
carefully because any chicks that are not exposed to vaccinal oocysts may be
vulnerable to potentially high numbers of virulent oocysts when placed on
litter. The objective of vaccination is to induce sufficient immunity to prevent chronic infestation while still allowing sufficient Eimeria to accumulate
such that a full immune response to the local Eimeria species will develop.
Several reviews have been published that are concerned with various aspects
of vaccination (Chapman, 2000; Chapman et al., 2002; Shirley et al., 2005;
Vermeulen et al., 2001; Williams, 2002b). Guidelines have been developed
to facilitate the worldwide adoption of consistent standard procedures
for determining the efficacy and safety of live anticoccidial vaccines
(Chapman et al., 2005).
Vaccination can also be achieved by in ovo injection of sporulated oocysts
into the embyronating egg (Weber et al., 2004). This is carried out in the
hatchery using complex machines that are also able to deliver other poultry
vaccines. Surprisingly, little research has been published that explains how
the oocysts, when injected into the egg, are able to establish a patent infection in the gut of the developing embryo. It is possible that infection results
from exposure to oocysts present in the eggshells post-hatch. As with other
methods of vaccination, secondary exposure to infection via the litter following placement of birds on the litter is necessary for a protective immune
response.
Another approach to vaccination involves immunizing hens with
affinity-purified antigens from the WFBs of macrogametocytes of
E. maxima (Sharman et al., 2010; Wallach et al., 2008). The nature of these
antigens has already been described in the section on oocyst biogenesis
above. Maternal antibodies pass via the egg to the newly hatched chick
and provide passive protection of limited duration. This is the only subunit
vaccine currently employed against any protozoan parasite. As in the case of

153
live oocyst vaccines, however, full protective immunity requires exposure to

potentially pathogenic coccidia in the litter.
Considerable research has been undertaken utilizing molecular technologies to identify antigens capable of inducing an immune response but in most
cases only partial protection has been achieved and none of the vaccine candidates have been proven in commercial applications (Shirley and Lillehoj, 2012).
10.3. Strategies for the control of coccidiosis

Meetings aimed at poultry producers, poultry veterinarians and other professional groups with an interest in the poultry industry, often include presentations concerned with strategies for the control of coccidiosis.
Unfortunately, data that can be tested rigorously are rarely presented, partly
due to the difficulty in designing reproducible trials under field conditions.
Thus the almost universal presence of Eimeria in poultry flocks negates the
inclusion of an uninfected group in any controlled study. Strategies may
involve drugs, vaccines or both. In the case of drugs, different products,
often with different modes of action, may be used in the different feeds that
are provided during the life of a flock of birds. Often referred to as shuttle
programmes, one such employed in the United States involves inclusion of
nicarbazin or a nicarbazin/narasin mixture in the first feed and monensin or
salinomycin in the second; however, there are numerous variations
(Chapman, 2001). In subsequent flocks, different drugs may be used, for
which the term rotation programme has been coined. Use of such
programmes is widespread and may be considered an appropriate strategy
for the control of coccidiosis although rarely is evidence of long-term efficacy (compared with other approaches) available.
Vaccination, and its integration with chemotherapy in control
programmes, is an alternative strategy. Evidence has been obtained that partial restoration of sensitivity to drugs may occur following the use of vaccines
comprising drug-sensitive strains of Eimeria. This phenomenon has been
demonstrated for the ionophores, monensin and salinomycin, and the synthetic drug, diclazuril (Chapman, 1994b; Jenkins et al., 2010; Peek and
Landman, 2006). Based upon these observations, a yearly rotation programme has been proposed in which use of ionophores is alternated in successive flocks with vaccination (Chapman et al., 2010); such programmes are
commonly used in the United States. There is a need for more evidence to
support the notion that rotation programmes involving vaccines and drugs
prolong the life of the latter in the field.

154
10.4. Natural products

There is considerable current interest in the use of so-called natural products which may include plant extracts, probiotics and so on, to reduce problems caused by coccidiosis (e.g. Allen, 2003, 2007; Faber et al., 2012;
Giannenas et al., 2012; Lee et al., 2011). For example, the antimalarial
artemisinin, a product extracted from the herb Artemisia annua, was shown
to have a deleterious effect upon macrogametocytes of E. tenella by affecting
the expression of an enzyme sarcoplasmicendoplasmic reticulum calcium
ATPase (del Cacho et al., 2010). Improved resistance to E. acervulina was
observed when the diet of chickens was supplemented with garlic metabolites (Kim et al., 2012). There are many other examples in the literature.
Most natural products, however, are said not to specifically target the parasite
but improve gut health or act as enhancers of some aspect of immune system function. All natural products contain undefined chemicals that, necessarily, will have to be evaluated for safety and toxicity before being
acceptable for registration authorities. There is often very little scientific
literature that supports the claims made for these products and, as far as is
known, none are used commercially at present probably because of unrealistic dietary inclusion rates and failure to demonstrate efficacy under controlled conditions.
11. CONCLUSIONS
In this review, we have considered selective aspects of research concerned with the apicomplexan parasites of the genus Eimeria which cause the
disease, coccidiosis, of domestic livestock. The emphasis has been on poultry, where coccidiosis has been shown to have an enormous economic
impact. Fortunately, control of coccidiosis in poultry has been achieved,
by a combination of improved management, the prophylactic use of drugs,
and vaccination. Nevertheless, we should not be complacent because the
parasite has not been eradicated from commercial facilities where animals
are reared and is still capable of causing production losses.
In recent years, many research projects, and publications that result, have
used the modern tools of molecular biology, biochemistry, cell biology and
immunology to expand greatly our knowledge of these parasites and the disease they cause. Such studies are essential if we are to develop new means for
the control of coccidiosis. Past success was achieved by research funded and
conducted by universities, government agencies and private industry.

155
In recent years, however, a number of government funded organizations

have terminated their coccidiosis programmes and private industry has been
reluctant to allocate funds to the animal health sector due to the enormous
costs involved in drug discovery and vaccine development. Few university
departments have the facilities to undertake costly coccidiosis research. It is
evident, therefore, that both from a practical and research perspective control of coccidiosis is at a crossroads. In the future, the ever expanding human
population will become increasingly dependent upon a source of cheap protein of which poultry will necessarily be an important component. Hopefully, this will not be compromised by the ubiquitous parasites of the
genus Eimeria.
ACKNOWLEDGEMENT
We would like to thank Thilakar Rathinam for help in preparing the figures.
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A Selective Review of Advances

*Department of Poultry Science, University of Arkansas, Fayetteville, Arkansas, United States

Advances in Parasitology, Volume 83

2013 Elsevier Ltd

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H. David Chapman et al.

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2. TAXONOMY AND SYSTEMATICS

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2.1. The genus Eimeria Schneider 1875: A melting pot

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2.2. Molecular identification and characterization of Eimeria

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For the well-accepted Eimeria species infecting chickens it was frequently

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3.1. Markers employed in genetic studies

3.2. Cross-fertilization and genetic recombination

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3.3. Genetic linkage analyses

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used to derive a panel of 22 hybrid clonal progeny lines by single sporocyst

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any one laboratory. Working with an uncloned hybrid progeny population

4. THE OMICS TECHNOLOGIES

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of 53% and two major ribosomal gene clusters on chromosomes 10 and

4.1.1 Nuclear karyotype

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4.1.2 Genome sequencing

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Table 2.1 Apicomplexan parasites: A current genetics and genomics summary

Eimeria maxima Houghton 57.5

Not known Not known

Reference used to derive the rate of genetic recombination.

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hemoparasites and piroplasms might indicate a requirement for a larger core

4.1.3 Genome structure

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(Novaes et al., 2012). Similarly, close associations have been identified

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protein separation technique prior to MS to reduce sample complexity and

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