CLINICAL MICROBIOLOGY REVIEWS, Jan. 2002, p. 58–65 Vol. 15, No.

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0893-8512/02/$04.00⫹0 DOI: 10.1128/CMR.15.1.58–65.2002

Recent Advances in Biology and Immunobiology of Eimeria Species

and in Diagnosis and Control of Infection with These Coccidian
Parasites of Poultry
P. C. Allen* and R. H. Fetterer
Parasite Biology, Epidemiology, and Systematics Laboratory, Animal and Natural Resources Institute, Agricultural
Research Service, U.S. Department of Agriculture, Beltsville, Maryland 20705

INTRODUCTION .........................................................................................................................................................58
LIFE CYCLE, GENETICS, AND BIOCHEMISTRY...............................................................................................58
SPECIES DETERMINATION AND DIAGNOSIS...................................................................................................59
IMMUNOBIOLOGY ....................................................................................................................................................59
CONTROL OF COCCIDIOSIS ..................................................................................................................................60

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Role of Poultry House Management ......................................................................................................................60
Prophylactic Control with Anticoccidials ..............................................................................................................60
Vaccination ................................................................................................................................................................61
Recombinant vaccines ..........................................................................................................................................61
Live vaccines..........................................................................................................................................................61
Use of cytokines as adjuvants .............................................................................................................................62
Alternative Controls Including Natural-Product Feed Additives.......................................................................62
DIRECTION OF FUTURE RESEARCH ..................................................................................................................63
REFERENCES ..............................................................................................................................................................63

INTRODUCTION development. Within the host cells, sporozoites undergo asex-

Coccidiosis is recognized as the parasitic disease that has the
greatest economic impact on poultry production. The annual
worldwide cost is estimated at about $800 million (107), and
that for the American broiler industry about $450 million.
These estimates include the costs of prophylactic in-feed med-
ication for broilers and broiler-breeders, alternative treatments
(e.g., with amprolium) if the medications fail, and losses due to
mortality, morbidity, and poor feed conversions of birds that
survive outbreaks.
LIFE CYCLE, GENETICS, AND BIOCHEMISTRY
Avian Eimeria spp. have homoxenous life cycles, which have
been well described (38). A web site (http://www.iah.bbsrc
.ac.uk/eimeria/) that clearly details the biology of coccidia has
also become available. In general, oocysts shed in feces un-
dergo sporogony (a meiotic process) in the external environ-
ment, a process requiring oxygen, taking about 24 h. The
sporulated oocysts contain four sporocysts, each containing
two sporozoites. Upon ingestion by a suitable host, oocysts
excyst within the intestinal lumen. This process is aided by
trypsin, bile, and CO2. The released sporozoites penetrate the
villous epithelial cells. Sporozoites of some species (E. brunetti
and E. praecox) develop within cells at the site of penetration.
Sporozoites of other species (E. acervulina, E. maxima, E.
necatrix, and E. tenella) are transported (1, 49, 95, 97) to other
sites, for example the crypt epithelium, where they undergo
* Corresponding author. Mailing address: Parasite Biology, Epide-
miology, and Systematics Laboratory, Animal and Natural Resources
Institute, Agricultural Research Service, U.S. Department of Agricul-
ture, Beltsville, MD 20705. Phone: (301) 504-8772. Fax: (301) 504-
5306. E-mail: pallen@ANRI.barc.usda.gov.
ual reproduction (schizogony or merogony) in which nuclear
division is followed by cytoplasmic differentiation, resulting in
merozoites that break free and penetrate other host cells.
These may carry out several more merogonic generations. Sex-
ual reproduction or gametogeny follows the last merogonic
cycle. Merozoites enter host cells and develop into either male
(microgamonts) or female (macrogamonts) forms. The mi-
crogamonts give rise to many microgametes, that exit, seek,
and penetrate (fertilize) the macrogamonts that then develop
into oocysts. Oocysts are shed with the feces. Prepatent periods
may generally range from 4 to 5 days postinfection. Maximum
oocyst output ranges from 6 to 9 days postinfection.
The biochemical and genetic mechanisms that control the
development of Eimeria spp. within host cells are not known.
However, through study of precocious and drug-resistant lines
of E. tenella (86, 87), two linkage groups associated with these
traits have been identified and mapped to parasite chromo-
somes 1 and 2. This information may help identify other ge-
netic loci involved in regulating the life cycle of E. tenella.
Genetic segregation data from this study are available through
the internet (http://www.genome.org). Other researchers (70)
have recently identified a gene (ets3a) whose expression is
developmentally regulated and which may be important in
controlling the life cycle of E. tenella. More genetic informa-
tion on Eimeria and many other parasites can be found on the
internet. Some of the websites include Eimeria (http://www
.iah.bbsrc.ac.uk/eimeria/genome.htm), GenBank (http://www
.ncbi.nlm.nih.gov), Parasitology Online (http://www.parasitology-
online.com/), and George Washington School of Medicine
(http://genome.wustl.edu/gsc/).
The metabolism of Eimeria spp. has come under new scru-
tiny with the increase in our efforts to define protective anti-

58
VOL. 15, 2002
gens for use in vaccines and to target metabolic pathways for
chemotherapy. For example, a mannitol cycle has been char-
acterized in E. tenella (12, 80) that apparently provides man-
nitol as an energy source for sporulation. This pathway may be
widespread within Eimeria spp. (12). Newly characterized en-
zymes in E. tenella and E. maxima have been reported also, as
well as a list of enzyme activities previously reported in avian
Eimeria (108).
SPECIES DETERMINATION AND DIAGNOSIS
There are seven valid species of chicken coccidia, E. acervu-
lina, E. brunetti, E. maxima, E. mitis, E. necatrix, E. praecox, and
E. tenella (83), each species developing in a particular location
within the chick digestive tract. It is common to find at least six
species (e.g., E. acervulina, E. maxima, E. tenella, E. brunetti, E.
mitis, and E. praecox) in litter samples from a single flock
during its first 6 weeks (106). Five of the species, E. acervulina,
E. brunetti, E. maxima, E. necatrix, and E. tenella, are well
known and identifiable with relative ease, because they pro-
duce characteristic gross lesions. Their pathogenicities range
from moderate to severe. On the other hand, E. praecox and E.
mitis do not kill chickens or produce pathognomic lesions and
often have been considered to be benign. However, experimen-
tal infections result in enteritis, diarrhea, and reduced feed
efficiencies (107), indicating that these two species certainly
can cause commercial losses and hence need to be controlled.
Generally, the five most pathogenic species listed above can
be differentiated in the host on the basis of clinical signs,
characteristic lesions at particular sites of infection in the
chicken intestine, and consideration of the prepatent period,
size of oocysts, and morphology of intracellular stages. How-
ever, the less pathogenic species such as E. mitis and E. praecox
might be overlooked. A very useful handbook is available (29)
which describes standard diagnostic and testing procedures for
Eimeria species and includes colored photographs of gross
lesions caused by the most commonly found species.
Shirley (82) was the first to use a molecular biological ap-
proach to differentiate species on the basis of isoenzyme pat-
terns of oocysts by starch block electrophoresis. Ellis and Bum-
stead (37) were among the first to demonstrate that rRNA and
rDNA probes could be used to identify individual species
through characteristic restriction fragment patterns. Procunier
et al. (72) used a randomly amplified polymorphic DNA assay
to differentiate E. acervulina and E. tenella and detect within-
strain differences. Recombinant DNA techniques have been
used to discriminate different strains of E. tenella (84) and
develop markers for precocious and drug-resistant strains (86),
and PCR amplification of internal transcribed spacer region 1
from genomic DNA has been used to detect and differentiate
six Eimeria species (81). Eight species (including E. hagani) are
claimed to be differentiated using a two-step PCR procedure
(98), and six Australian species have been characterized using
a PCR-linked restriction fragment length polymorphism ap-
proach (110). These PCR methods should prove very useful for
epidemiological surveys of avian coccidiosis.
IMMUNOBIOLOGY
Great progress has been made in our understanding of the
immune response to coccidial infection. Much of this has come
from work on model infections with E. vermiformis in geneti-
BIOLOGY AND IMMUNOBIOLOGY OF EIMERIA 59
cally altered mice (88). In mice, TCR␥␦⫹ CD4⫹ major histo-
compatibility complex type II (MHC-II)-restricted T cells that
produce gamma interferon (IFN-␥) are essential for antigen-
specific resistance to both primary and secondary infections.
Additionally, B cells and interleukin-6 IL-6 are minor but nec-
essary components of resistance to primary infections (88).
Other, non-class II-restricted cells appear to be involved in
effecting good “memory” responses to challenge infections.
Lack of chickens with genetic modifications of the immune
effector systems (targeted gene disruptions), as well as the
paucity of monoclonal antibody reagents, has made it more
difficult to dissect the immune responses to avian coccidiosis.
Nevertheless, the following facts are clear. There are genetic
components to resistance to primary infections (50, 114).
Chickens develop solid immunity to homologous secondary
infections (74). Immunity does not prevent sporozoite invasion
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of cells but does prevent sporozoite development (14).
The effectors of immune responses to primary and challenge
coccidial infections are primarily T cells residing in the gut-
associated lymphoid tissues (58, 59, 60). Humoral immune
responses also occur, but antibodies play a minor role in resis-
tance and immunity to coccidia (51, 74). Chicken intestinal
lymphocytes have been phenotyped using monoclonal antibod-
ies, and found to exhibit markers homologous to those of
murine and human CD3, CD4, and CD8 T cells (24, 27, 53, 60,
94). The CD3 polypeptide complex is found in association with
the antigen-specific T-cell receptor (TCR) (21) characterized
by ␣␤ or ␥␦ heterodimers (27). The CD4⫹ T (helper) cells
recognize MHC-II antigens, and CD8⫹ T (cytotoxic/suppres-
sor) cells recognize MHC-I antigens (60). In chickens,
TCR␥␦⫹ T cells can be either CD4⫹ or CD8⫹ (60).
The roles of these T cells in response to avian Eimeria
infections appear somewhat different from those in murine
Eimeria infections. Studies in which these T-cell populations in
chickens infected with E. acervulina or E. tenella have been
selectively depleted by treatment with monoclonal antibody
(96) show a different contribution of CD4⫹ and CD8⫹ T cells
to resistance and immunity, which appears, as judged by oocyst
output, to be dependent on the infecting species of coccidia. In
chickens infected with E. acervulina, CD4⫹ T-cell depletion did
not significantly increase oocyst production during either pri-
mary or challenge infection (96). Additionally, fewer oocysts
were produced during challenge than during primary infection.
These results suggest that CD4⫹ T cells are not significant
effectors of resistance or immunity to E. acervulina. On the
other hand, depletion of CD4⫹ cells in E. tenella infections
resulted in increased oocyst output during primary but not
challenge infection, indicating that CD4⫹ cells are important
effectors of resistance to primary E. tenella infections.
Depletion of CD8⫹ T cells significantly decreased oocyst
output during primary infections of both E. acervulina and E.
tenella. It is theorized that this effect is due to a reduction in the
number of CD8⫹ cells that serve as transporters for sporozo-
ites (see below). However, CD8⫹ cell depletion results in a
larger oocyst output after a challenge infection. These results
indicate CD8⫹ cells may not be effectors of resistance in pri-
mary infection but are necessary effectors in immunity to chal-
lenge infections (59, 96). The importance of CD8⫹ T cells in
protective immunity to E. tenella infections is also suggested by
the observation of a sharp transitory increase in the number of
60 ALLEN AND FETTERER
these cells in the peripheral blood (19) during primary infec-
tion.
In fact, CD8⫹ T cells seem to play dual role in the immune
response to coccidial infections in chickens, as determined by
two-color immunofluorescence staining of infected gut from
chicken strains differing genetically in susceptibility to coccid-
iosis (59, 60). First, during primary infections with E. acervulina
and E. tenella, the number of T cells coexpressing TCR␥␦ and
CD8 markers increases in the epithelium. There they appear to
be invaded by sporozoites. It is thought that these are the cells
that transport sporozoites (1, 49, 95, 97) through the lamina
propria to the crypt epithelium, where they exit and invade the
crypt cells. Populations of CD8⫹ cells also increase in the
mucosa following challenge infection, where they frequently
can be seen in close contact with infected cells. It is theorized
that they are functioning as cytotoxic cells, helping to eliminate
the parasite challenge by killing the infected cells (59).
Coccidia invade other leukocyte types in the gut mucosa. For
example, sporozoites of E. tenella have been found in het-
erophils (75) and merozoites of E. tenella have been found in
goblet cells and mast cells (34). The relevance of these obser-
vations to immune control of avian coccidiosis is not yet clear.
Populations of cells bearing markers (low-level CD8⫹ and
asialo-GM1) for natural killer (NK) cells increase early during
primary infections (28, 52). These cells exhibit NK acitivity in
vitro (23) and thus may be involved in immune surveillance.
Mucosal macrophages phagocytize sporozoites during pri-
mary and challenge infections, but this activity does not seem
enhanced in the immune chicken (97). Macrophages may also
function in sporozoite transport (99). Activated macrophages
are the source of various inflammatory cytokines that can mod-
ulate cellular immune responses.
Increases in the numbers of mucosal mast cells have been
noted early and late following primary and challenge infections
with E. acervulina (76), but the contribution of these cells to
effective immunity and resistance is not known.
Cytokines synthesized and secreted by leukocytes play im-
portant regulatory roles during the immune response to infec-
tion. In avian coccidiosis, it is clear that IFN-␥ is produced by
the host at sites of infection (78, 112), and IFN-␥ release has
been used as a measure of the T-cell response to coccidial
antigens (68, 73). Treatment of coccidia-infected chickens with
recombinant chicken IFN-␥ (89) improved weight gains with
respect to uninfected controls (55, 64). Tumor necrosis factor
(TNF), an inflammatory cytokine, is secreted by activated mac-
rophages. TNF-␣-like activity can be detected in stimulated
macrophages from infected chickens (22, 114) and in sera from
infected chickens (113). Treatment of infected chickens with
TNF-␣ exacerbated the suppression of weight gain due to
infection, whereas treatment with polyclonal antibodies to
TNF-␣ partially reversed the weight gain depression (114).
These results suggest that TNF-␣ may play an important role
in the pathophysiology of coccidiosis infections in chickens.
Free radicals, including reactive oxygen species and nitric
oxide (NO䡠), are products of activated macrophages and other
phagocytic leukocytes and are known to be toxic to bacteria
and some parasites (77). They are thus implicated as playing
significant roles in resistance and immunity to infectious dis-
eases (65, 71).
Peaks in the mucosal activity of NADPH oxidase, which
CLIN. MICROBIOL. REV.
generates superoxide (O2䡠), and in the levels of NO2⫺ ⫹
NO3⫺, stable metabolites of NO䡠, occur in a qualitatively dif-
ferent time pattern in chickens infected with E. maxima (days
1, 3, and 6 postinfection [p.i.] for NADPH oxidase, day 6 PI for
NO2⫺ ⫹ NO3⫺) (2). The differences in the time courses of
change in these parameters during primary infection suggest
the participation of separate cell types in their production.
Plasma NO2⫺ ⫹ NO3⫺ levels are significantly elevated at
about 6 days PI during primary infections with E. acervulina
and E. tenella, as well as E. maxima (3, 9), but not during
homologous challenge infections of well-immunized chickens.
This observation suggests that NO䡠 production may be associ-
ated more closely with immune responses associated with in-
nate resistance than with acquired immunity to coccidiosis.
There appears to be a genetic link between NO䡠 production
during primary infection and resistance to E. tenella (9). A
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recent report (36) of in vitro studies suggests that macrophages
stimulated by IFN-␥ produce NO, which inhibits the replica-
tion of E. tenella within these cells.
CONTROL OF COCCIDIOSIS
Role of Poultry House Management
Because coccidial oocysts are ubiquitous and easily dissem-
inated in the poultry house environment and have such a large
reproduction potential, it is very difficult to keep chickens
coccidia free, especially under current intensive rearing condi-
tions. Oocysts sporulate readily in poultry house litter. How-
ever, they can be damaged by bacteria, other organisms, and
ammonia that are also present, and their viability can begin to
diminish after 3 weeks (106). Many producers in the United
States basically remove caked litter, let the houses air out for
2 to 3 weeks, and top dress with fresh litter before placing a
new flock (H. D. Danforth, private communication). On the
other hand, it is common practice in most European countries
and Canada to do a thorough cleanout between flocks. This
practice may become more widespread in the United States as
the effectiveness of anticoccidials continues to decrease and
the use of live vaccines increases. Biocontrol measures such as
requiring caretakers to change clothes between houses can
minimize the spread of infective oocysts. The practice of strict
biosecurity is essential in the care of broiler breeders.
Prophylactic Control with Anticoccidials
The effective use of anticoccidial feed additives over the past
50 years has played a major role in the growth of the poultry
industry and has allowed the increased availability of high-
quality, affordable poultry products to the consumer. These
anticoccidials can be classified as (i) chemicals which have
specific modes of action against parasite metabolism, such as
amprolium, clopidol decoquinate, halofuginone, or (ii) poly-
ether ionophores such as monensin lasalocid, salinomycin,
narasin, and maduramycin, which act through general mecha-
nisms of altering ion transport and disrupting osmotoic bal-
ance. These latter compounds are now the mainstay of coccid-
iosis control (41). A compendium of the most frequently used
anticoccidials is available through Janssen Pharmaceutica (40).
It is quite clear, however, (25, 26, 79), that some degree of
resistance to all anticoccidial drugs, including ionophores, has
VOL. 15, 2002
developed. To minimize the effects of resistance, poultry pro-
ducers rotate the use of various anticoccidials with successive
flocks, combine chemical and ionophore treatments, or employ
shuttle programs during a flock growout. Application of these
treatment programs depends on seasonal conditions and prev-
alence of various species of coccidia (107). In recent years,
pharmaceutical companies have not brought new anticoccidials
to market. However, two potential drug targets, enzymes of the
sporozoite mannitol cycle (12, 80) and trophozoite histone
deacetylase, have been recently identified (80).
Vaccination
Avian coccidia are highly immunogenic, and primary infec-
tions can stimulate solid immunity to homologous challenges.
Therefore, it would seem obvious that vaccines could offer
excellent alternatives to drugs as a means of controlling coc-
cidiosis. Efforts to develop various types of vaccines (57, 58, 61)
have been continuous over the past several decades, but
progress has been slow. Highlights of research on vaccine de-
velopment are included here.
Recombinant vaccines. Over the past 10 years, much re-
search effort has been spent on the development of recombi-
nant vaccines. Although none are in commercial use, this re-
search has served to highlight the complex nature of the avian
host-coccidia interaction. A major hurdle to overcome in the
development of a recombinant vaccine is the lack of cross-
species immune protection. Other factors impeding the devel-
opment of a successful vaccine have been recently reviewed
(42, 100), the most important of which is the identification of
protective antigens. Many potential coccidial antigens have
been characterized and cloned. A total of 29 DNA sequences
encoding immunity-stimulating Eimeria proteins from various
species and developmental stages have been listed in a recent
review (42). Many of these antigens are surface proteins or
internal antigens associated with organelles such as mi-
cronemes (92, 93), rhoptries (91), and refractile bodies (101,
102). Recently (90) a low-molecular-weight immunogenic an-
tigen with a single immunodominant epitope was reported to
be present in all endogenous stages of E. tenella. Metabolic
antigens from developing sporozoites (44, 45, 46), merozoite
antigens (18, 100), and gamete antigens (103, 104) all elicit
various degrees of protective immunity.
A delivery mechanism for coccidial vaccines that produces
optimum resistance to challenge infection has yet to be deter-
mined. Immunogenic Eimeria antigens have been administered
as isolated proteins with adjuvants (18, 100), as recombinant
antigens in live vectors such as nonpathogenic strains of Esch-
erichia coli, Salmonella enterica serovar Typhimurium, poxvi-
ruses, fowlpox virus, and turkey herpesvirus (93), and by direct
plasmid DNA injection (43, 48, 54), with various degrees of
success. More research is needed to determine how to target
vaccines to the cellular immune effector systems that operate
during natural primary and challenge infections and to deter-
mine the unique host immune responses to each parasite.
Because T-cell-stimulatory antigens appear to be the most
relevant to protective immunity, T-cell proliferation assays (us-
ing T cells from recovering chickens) and IFN-␥ production
have been used to screen for protective antigens against E.
tenella infections (20). The fraction of antigens that produced
BIOLOGY AND IMMUNOBIOLOGY OF EIMERIA 61
the highest T-cell proliferation and macrophage-activating ac-
tivity, when administered as a vaccine, also yielded chickens
with the lowest cecal lesion scores after challenge (20).
Live vaccines. Live vaccines for coccidiosis control have
been used to a limited degree by the poultry industry for about
50 years. Their effectiveness hinges on the recycling of initially
very low doses of oocyst, and the gradual buildup of solid
immunity. They have been used primarily to protect breeder
and layer flocks (85). However, their use, particularly in broiler
flocks, is increasing.
Live vaccines contain either attenuated or virulent coccidial
strains. Paracox (consisting of precocious strains of all seven
species of chicken coccidia) and Livacox (consisting of preco-
cious and egg-passaged lines) are attenuated vaccines. All coc-
cidial strains in these vaccines are drug sensitive. Coccivac and
Immucox are each composed of several virulent species. All
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four vaccines are commercially available. An in-depth compar-
ison of these vaccines is provided by Bedrnik et al. (17). All of
these vaccines are administered during the first week after
hatch and will produce solid immunity when they are used
carefully under good rearing conditions (30). Because live vac-
cines are multivalent (containing more than one species), tests
of their efficacies require a different approach from tests of the
efficacies of anticoccidial compounds. A standard protocol that
is based on growth rates after challenge and that avoids lesion
scoring and oocyst output enumeration has been published
recently (109).
An advantage of attenuated vaccines is that they have low
reproductive potentials, thus avoiding crowding in the specific
mucosal areas of infection and resulting in the development of
optimal immunity with minimal tissue damage (105). It is be-
lieved that the drug-sensitive, attenuated strains and wild, na-
tive strains interbreed, reducing both virulence and drug re-
sistance in local populations. Thus, the useful period of
anticoccidial drugs could be extended by rotating their appli-
cations with live vaccines (107).
Coccivac and Immucox have also been used to treat broiler
breeders, particularly in the United States and Canada. There
has been a general reluctance to try them in broilers and heavy
roaster birds, because of reduced weight gain and feed con-
version compared to those in prophylactically medicated birds
(30). Additionally, there is a risk of introducing unwanted
Eimeria species into the environment. However, recent battery,
floor pen, and broiler house trials (30) have shown that this
type of vaccine (e.g., Immucox) can provide equal or superior
performance, compared with prophylactic medication, when
given in gel form at 1 day of age, because this method ensures
the synchronous exposure of all birds to small uniform num-
bers of oocysts. (32, 33).
Antigenicity of coccidial strains can vary geographically (39,
66). Therefore, it is important to screen live vaccines against
local populations before administering them to large flocks.
Immunovariant strains can be isolated and substituted to pro-
vide region-specific, autogenous vaccines (30). Live vaccines
can also be modified by incorporating drug-resistant strains. In
floor pen trials, anticoccidially medicated broiler birds that had
been immunized at 1 day of age with a drugresistant strain of
coccidia were protected almost completely against homologous
challenge (30). When this vaccination regimen was applied to
large flocks in two sets of paired broiler houses, vaccinated-
62 ALLEN AND FETTERER
medicated birds had consistently lower (more efficient) feed
conversions but slightly more variable final weights than did
the medicated controls (30). Thus, this type of vaccine treat-
ment has the potential to extend the life of anticoccidials
cleared for use in geographical areas where drug resistance and
lack of coccidial control is a problem (30).
Use of cytokines as adjuvants. As mentioned above, cyto-
kines are major modulators of immune responses to infection,
and therefore they represent natural sources of immunostimu-
lation that could be used as adjuvants in vaccines. IFN-␥ has
received the most attention as a possible adjuvant for an an-
ticoccidial vaccine. Chicken IFN-␥ has been cloned (35, 62,
89). Treatment with recombinant IFN-␥ alone has been shown
to moderate reductions in weight gain (55, 64) during E. acer-
vulina and E. tenella infections and to moderate oocyst output
(55) during E. acervulina infection. One problem with the in
vivo use of cytokines is their rapid degradation and clearance
(63). Administering them in DNA form or in a viral or bacte-
rial vector could overcome this problem and provide a more
practical method for treating large flocks. For example, a re-
combinant protein (3-1E) from E. acervulina merozoites,
which is also found in E. tenella sporozoites and merozoites,
stimulated IFN-␥ production in chicken spleen lymphocytes.
Direct injection of 3-1E cDNA induced protective immunity
against E. acervulina challenge. Simultaneous injection of
cDNA encoding chicken IFN-␥ further enhanced immunity
(56). Chickens treated with a fowl adenovurus chicken IFN-␥
construct showed increased weight gains compared to un-
treated controls and exhibited smaller weight gain reductions
on challenge with E. acervulina (47).
Alternative Controls Including Natural-Product
Feed Additives
Many different types of substances have been investigated in
the search for alternative controls of coccidiosis. Peptidyl
membrane-interactive molecules are known to have antibacte-
rial activities. Three of seven tested produced ultrastructural
damage to sporozoite pellicles after a 5- to 10-min exposure to
5 ␮M concentrations in vitro (67). Oral doses (10, 50, or 75
␮M) of two of them, which were methylated to prevent pro-
teolysis, reduced lesion scores from challenge infections with
E. acervulina, E. tenella, and E. maxima, suggesting that they
have potential for coccidiosis control.
A number of natural products or feedstuffs have been tested
as anticoccidial dietary additives. Some of this work has been
recently reviewed (5). Sources of fats containing high concen-
trations of n-3 fatty acids (n-3 FA) (docosahexaenoic acid,
eicosapentaenoic acid, and linolenic acid), such as fish oils,
flaxseed oil, and whole flaxseed, when added to starter rations
and fed to chicks from 1 day of age, effectively reduced lesions
resulting from challenge infections with E. tenella (6, 7) but not
E. maxima (7). The fish oil and flaxseed oil diets significantly
reduced the degree of parasitization by and development of E.
tenella (6) and caused ultrastructural degradation of both asex-
ual and sexual stages, characterized by cytoplasmic vacuoliza-
tion, chromatin condensation within the nucleus, and lack of
parasitophorous vacuole delineation (31). These results are
consistent with reports of the effects of high n-3 FA diets on
other parasites (5) and suggest that these diets induce a state
CLIN. MICROBIOL. REV.
of oxidative stress (due to the high concentration of easily
oxidized double bonds) that is detrimental to parasite devel-
opment. Further support for the oxidative-stress hypothesis
include the observations that (i) n-3 FA diets effective in con-
trolling E. tenella reduce plasma carotenoid levels even in
uninfected chickens (8), (ii) the effect on E. tenella appears
related to the concentrations of double bonds in diets supple-
mented with n-3 FA ethyl esters (4), (iii) antioxidant-stabilized
diets supplemented with up to 10% flaxseed do not protect
against E. tenella (11) and (iv) diets supplemented with 15 and
20% chia seed (another seed with high linolenic acid content)
and containing no additional antioxidants reduce E. tenella
lesion scores (P. C. Allen and J. DeGroot, unpublished obser-
vations). Additionally, Michalski and Prowse (69) have shown
sporulated oocysts and sporozoites of E. tenella to be deficient
in superoxide dismutase, an enzyme that would protect them
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from damage by reactive oxygen species.
Artemisinin is a Chinese herb isolated from Artemisia an-
nua; it is a naturally occurring endoperoxide with antimalarial
properties. It has been found effective in reducing oocyst out-
put from both E. acervulina and E. tenella infections when fed
at levels of 8.5 and 17 ppm in starter diets (10). The mode of
action is also thought to involve oxidative stress.
Most recently, extracts from 15 Asian herbs were tested for
anticoccidial activity against E. tenella. Of the species tested,
extracts from Sophora flavescens Aiton was the most effective in
reducing lesion scores, maintaining body weight gain, and re-
ducing oocyst production. (111)
Feed supplementation with antioxidants such as ␥-tocoph-
erol (8 ppm), found plentifully in seed oils such as wheat, corn,
and soybean, and the spice tumeric (1%), as well as its main
medicinal component, curcumin (0.05%), appear effective in
reducing upper- and mid-small-intestinal infections caused by
E. acervulina and E. maxima (5). They are not beneficial for E.
tenella infections, however. The osmoprotectant betaine (from
sugar beets) has long been known to have beneficial effects on
livestock growth and performance. When fed at 0.15% along
with 66 ppm salinomycin in floor pen studies, betaine had
beneficial effects on weight gains and feed conversion and
resulted in reduced mortality. In tissue culture, betaine and
salinomycin significantly reduced cell invasion by E. acervulina.
It is concluded that this combination may improve perfor-
mance in coccidially infected chickens directly by its action on
E. acervulina development and indirectly by its action as an
osmoprotectant, supporting intestinal structure and function.
(13, 15, 16).
These studies emphasize the unique biology of the individual
Eimeria species that parasitize chickens, and a pattern of ef-
fectiveness of various dietary treatments seems apparent.
Products that can generate a state of oxidative stress, such as
n-3 FA and artemisinin, are particularly effective against the
cecal parasite E. tenella. Products that have antioxidant prop-
erties, such as ␥-tocopherol and curcumin, seem to be more
effective against the mid- and upper-small intestinal species E.
maxima and E. acervulina. The osmoprotectant betaine ap-
pears to be most active against E. acervulina. Practical appli-
cations of these findings, such as the use of the products in
starter rations or combinations of them with current anticoc-
cidials or vaccines, appear possible and need to be investigated.
VOL. 15, 2002
DIRECTION OF FUTURE RESEARCH
Over the past several decades, knowledge about the genetic
makeup of avian Eimeria spp. and the immunobiological inter-
action of the avian host with the coccidian parasites has in-
creased dramatically. The impetus for this expansion has come
from efforts to find new, effective ways to control coccidiosis in
the face of increased resistance of the parasites to traditional
anticoccidal pharmaceuticals. Much of the work on both the
parasites and the host has been aided by the development of
techniques in molecular biology and driven by the search for
effective vaccines.
The ultimate key to coccidiosis control may well come in
part from research directed toward a better understanding of
parasite biology and biochemistry. Some of the questions that
need to be answered include the following. Why are avian
coccidia host and site specific? What are the important criteria
that allow parasites to invade a host cell? What are the bio-
chemical defense mechanisms that promote survival within the
host? What genes control the development of coccidia within
the host? New developments or improvements in in vitro cul-
tivation methods, the current worldwide push to unravel the
parasite genome, and the availability of genetic sequence in-
formation in publicly accessible databases, along with new
techniques in proteomic research, should help to answer these
questions.
Further insight into the control of coccidiosis will also come
from research on the avian host that is directed to the following
questions. What are the host genetic components associated
with innate and acquired resistance to coccidial infections (i.e.,
identification of qualitative trait loci) (115)? What are the
immunological restrictions that limit cross-protection among
species? What are the differences in host cellular responses
between live infection and vaccination? Current and future
research using microarray and proteomic technologies will be-
gin to answer some of these questions. It is anticipated that
large advances will be made in the understanding and control
of avian coccidiosis within the next decade.
REFERENCES
1. Al-Attar, M. A., and M. A Fernando. 1987. Transport of Eimeria necatrix
sporozoites in the chicken: effects of irritants injected intraperitonally. J.
Parasitol. 73:494–502.
2. Allen, P. C. 1996. Production of free radical species during Eimeria maxima
infections in chickens. Poult. Sci. 76:814–821.
3. Allen, P. C. 1997. Nitric oxide production during Eimeria tenella infections
in chickens. Poult. Sci. 76:810–813.
4. Allen, P. C., and H. D. Danforth. 1998. Effects of dietary supplementation
with n-3 fatty acid ethyl esters on coccidiosis in chickens. Poult. Sci. 77:
1631–1635.
5. Allen, P. C., H. D. Danforth, and P. C. Augustine. 1998. Dietary modulation
of avian coccidiosis. Int. J. Parasitol. 28:1131–1140.
6. Allen, P. C., H. D. Danforth, and O. A. Levander. 1996. Diets high in n-3
fatty acids reduce cecal lesion scores in broiler chickens infected with
Eimeria tenella. Poult. Sci. 75:179–185.
7. Allen, P. C., H. D. Danforth, and O. A. Levander. 1997. Interaction of
dietary flaxseed with coccidia infections in chickens. Poult. Sci. 76:822–827.
8. Allen, P. C., H. D. Danforth, V. L. Morris, and O. A. Levander. 1996.
Association of lowered plasma carotenoids with protection against cecal
coccidiosis by diets high in n-3 fatty acids. Poult. Sci. 75:966–973.
9. Allen, P. C., and H. S. Lillehoj. 1998. Genetic influence of nitric oxide
production during Eimeria tenella infections in chickens. Avian Dis. 42:397–
403.
10. Allen, P. C., J. Lydon, and H. D. Danforth. 1997. Effects of components of
Artemisia annua on coccidia infections in chickens. Poult. Sci. 76:1156–
1163.
11. Allen, P. C., H. D. Danforth, and P. A. Stitt. 2000. Effects of nutritionally
BIOLOGY AND IMMUNOBIOLOGY OF EIMERIA 63
balanced and stabilized flaxmeal-based diets on Eimeria tenella infections in
chickens. Poult. Sci. 79:489–492.
12. Allocco, J. J., H. Profous-Juchelka, R. W. Myers, B. Nare, and D. M.
Schmatz. 1999. Biosynthesis and catabolism of mannitol is developmentally
regulated in the protozoan parasite Eimeria tenella. J. Parasitol. 85:167–173.
13. Augustine, P. C. 1997. Effect of betaine on invasion and development of
avian coccidia and growth performance in coccidia-infected chickens. Aust.
Poult. Sci. Symp. 9:39–45.
14. Augustine, P. C., and H. D. Danforth. 1986. A study in the dynamics of the
invasion of immunized birds by Eimeria sporozoites. Avian Dis. 30:347–351.
15. Augustine, P. C., and H. D. Danforth. 1999. Influence of betaine and
salinomycin on intestinal absorption of methionine and glucose and on the
ultrastructure of intestinal cells and parasite development stages in chicks
infected with Eimeria acervulina. Avian Dis. 43:89–97.
16. Augustine, P. C., J. L. McNaughton, E. Virtanen, and L. Rosi. 1997. Effect
of betaine on the growth performance of chicks inoculated with mixed
cultures of avian Eimeria species and on invasion and development of
Eimeria tenella and Eimeria acervulina in vivo and in vitro. Poult. Sci.
76:802–809.
17. Bedrnik, P., T. Hiepe, D. Mielke, and U. Drossigk. 1995. Antigens and
immunization procedures in the development of vaccines against poultry
coccidiosis, p. 176–189, In J. Eckert, R. Braun, M. W. Shirley, and F.
Downloaded from cmr.asm.org at MAHIDOL UNIV FAC OF MED on June 25, 2007
Coudert (ed.), COST 89/820. Biotechnology: guidelines on techniques in
coccidiosis research. European Commission, Luxembourg; Luxembourg.
18. Brake, D. A., G. Strang, and J. E. Lineberger. 1997. Immunogenic charac-
terization of a tissue culture-derived vaccine that affords partial protection
against avian coccidiosis. Poult. Sci. 76:974–983.
19. Breed, D. G., J. Dorrestein, and A. N. Vermeulen. 1996. Immunity to
Eimeria tenella in chickens: phenotypical and functional changes in periph-
eral blood T-cell subsets. Avian Dis. 40:37–48.
20. Breed, D. G., T. M. P. Schetters, N. A. P. Verhoeven, and A. N. Vermeulen.
1997. Characterization of phenotype related responsiveness of peripheral
blood lymphocytes from Eimeria tenella infected chickens. Parasite Immu-
nol. 19:563–569.
21. Bucy, R. P., C. L. Chen, J. Cihak, U. Lasch, and M. D. Cooper. 1988. Avian
T cells expressing receptors localize in the splenic sinusoids and the intes-
tinal epithelium. J. Immunol. 141:2200–2205.
22. Byrnes, S., R. Eaton, and M. Kogut. 1993. In vitro interleukin-1 and tumor
necrosis factor alpha production by macrophages from chickens infected
with either Eimeria maxima or Eimeria tenella. Int. J. Parasitol. 23:639–645.
23. Chai, J. Y., and H. S. Lillehoj. 1988. Isolation and functional characteriza-
tion of chicken intestinal intraepithelial lymphocytes showing natural killer
cell activity against tumor target cells. Immunology 63:111–117.
24. Chan, M. M., C. L. Chen, L. L. Ager, and M. D. Cooper. 1988. Identification
of the avian homologues of mammalian CD4 and CD8 antigens. J. Immu-
nol. 140:2133–2138.
25. Chapman, H. D. 1994. Sensitivity of field isolates of Eimeria to monensin
following the use of a coccidiosis vaccine in broiler chickens. Poult. Sci.
73:476–478.
26. Chapman, H. D. 1998. Evaluation of the efficacy of anticoccidial drugs
against Eimeria species in the fowl. Int. J. Parasitol. 28:1141–1144.
27. Chen, C. H., J. Cihak, U. Losch, and M. D. Cooper. 1988. Differential
expression of two T cell receptors, TCR1 and TCR2 on chicken lympho-
cytes. Eur. J. Immunol. 18:539–543.
28. Cheung, K., and H. S. Lillehoj. 1991. Characterization of monoclonal an-
tibodies detecting avian macrophages and NK cells. Vet. Immunol. Immu-
nopath. 28:351–363.
29. Conway, D. P., and M. E. McKenzie. 1991. Poultry coccidiosis diagnosis and
testing procedures, 2nd ed. Pfizer, Inc., New York, N.Y.
30. Danforth, H. D. 1998. Use of live oocyst vaccines in the control of avian
coccidiosis: experimental studies and field trials. Int. J. Parasitol. 28:1099–
1109.
31. Danforth, H. D., P. C. Allen, and O. A. Levander. 1997. The effect of high
n-3 fatty acid diets on the ultrastructural development of E. tenella. Para-
sitol. Res. 83:440–444.
32. Danforth, H. D., E.-H. Lee, A. Martin, and M. Dekich. 1997. Evaluation of
a gel-immunization technique used with two different Immucox vaccine
formulations in battery and floor-pen trials with broiler chickens. Parasitol.
Res. 83:445–451.
33. Danforth, H. D., K. Watkins, A. Martin, and M. Dekich. 1997. Evaluation
of the efficacy of Eimeria maxima oocysts immunization with different
strains of day-old broiler and roaster chickens. Avian Dis. 41:792–801.
34. Daszak, P., S. J. Ball, R. M. Pittilo, and C. C. Norton. 1993. Ultrastructural
observations on cecal epithelial cells invaded by first generation merozoites
of Eimeria tenella in vivo. Ann. Trop. Med. Parasitol. 87:259–364.
35. Digby, M. R., and J. W. Lowenthal. 1995. Cloning and expression of the
chicken interferon-gamma gene. J. Interferon Cytokine Res. 15:939–945.
36. Dimier-Poisson, I. H., Z. Soundous, M. Naciri, D. T. Bout, and P. Quere.
1999. Mechanisms of the Eimeria tenella growth inhibitory activity induced
by concanavalin A and reticuloendotheliosis virus supernatants with inter-
feron gamma activity in chicken macrophages and fibroblasts. Avian Dis.
43:65–74.
64 ALLEN AND FETTERER
37. Ellis, J., and J. Bumstead. 1990. Eimeria species: studies using rRNA and
rDNA probes. Parasitology 101:1–6.
38. Fernando, A. M. 1990. Eimeria: Infections of the intestine, p. 63–75. in P. L.
Long (ed.), Coccidiosis of man and domestic animals. CRC Press, Inc.,
Boca Raton, Fla.
39. Fitz-Coy, S. H. 1992. Antigenic variation among strains of Eimeria maxima
and E. tenella of the chicken. Avian Dis. 36:40–43.
40. Fowler, N. G. 1995. Anticoccidial compendium. Janssen Pharmaceutica,
Animal Health, Beerse, Belgium.
41. Jeffers, T. 1997. Tyzzer to tomorrow: control of avian coccidiosis into the
next millenium, p 16. In M. W. Shirley, F. M. Tomley, and B. M. Freeman
(ed.), Control of coccidiosis into the next millennium. Proc. VII Int. Coc-
cidiosis Conf.
42. Jenkins, M. C. 1998. Progress on developing a recombinant coccidiosis
vaccine. Int J. Parasitol. 28:1111–1119
43. Jenkins, M. C., P. Allen, H. Danforth, and P. Augustine. 1998. Immuniza-
tion of chickens with plasmid DNA encoding recombinant Eimeria acervu-
lina antigen via jet-gun injection elicits immunity against coccidiosis, p. 47.
Proc. 43rd Annu. Meet. AAVP.
44. Jenkins, M. C., P. C. Augustine, J. R. Barta, and M. D. Castle. 1991.
Development of resistance to coccidiosis in the absence of merogonic
development using X-irradiated Eimeria acervulina oocysts. Exp. Parasitol.
72:285–293.
45. Jenkins, M. C., P. C. Augustine, H. D. Danforth, and J. R. Barta. 1991.
X-irradiation of Eimeria tenella oocysts provides direct evidence that sporo-
zoite invasion and early schizont development induce protective immune
response(s). Infect. Immun. 59:4042–4048.
46. Jenkins, M. C., P. G. Sefarian, P. C. Augustine, and H. D. Danforth. 1993.
Protective immunity against coccidiosis elicited by radiation-attenuated Ei-
meria maxima sporozoites that are incapable of asexual development. Avian
Dis. 37:74–82.
47. Johnson, M. A., C. Pooley, and J. W. Lowenthal. 2000. Delivery of avian
cytokines by adenovirus vectors. Dev. Comp. Immunol. 24:343–354.
48. Kopko, S. H., D. S. Martin, and J. R. Barta. 2000. Responses of chickens to
a recombinant refractile body antigen of Eimeria tenella administered using
various immunizing strategies. Poult. Sci. 79:336–342.
49. Lawn, A. M., and M. E. Rose. 1982. Mucosal transport of Eimeria tenella in
the cecum of the chicken. J. Parasitol. 68:1117–1123.
50. Lillehoj, H. S. 1986. Immune response during coccidiosis in SC and FP
chickens. I. In vitro assessment of T cell proliferation response to stage
specific parasite antigens. Vet. Immunol. Immunopathol. 13:321–330.
51. Lillehoj, H. S. 1987. Effects of immunosuppression of avian coccidiosis:
cyclosporin A but not hormonal bursectomy abrogates host protective im-
munity. Infect. Immun. 55:1616–1621.
52. Lillehoj, H. S. 1989. Intestinal intraepithelial and splenic natural killer cell
responses to Eimeria infections in inbred chickens. Infect. Immun. 57:1879–
1884.
53. Lillehoj, H. S. 1994. Analysis of Eimeria acervulina induced changes in
intestinal T lymphocyte subpopulations in two inbred chickens showing
different levels of disease susceptibility to coccidia. Res. Vet. Sci. 56:1–7.
54. Lillehoj, H. S., K. D. Chai., D. Heller, M. C. Jenkins, and V. N. Vakharia.
1997. Naked DNA immunization with a plasmid encoding an Eimeria
surface sporozoite protein and oral feeding of baculovirus-expressed Eime-
ria protein elicit serum antibodies and reduced oocyst output, p. 105. Proc.
VII Int. Coccidiosis Conf.
55. Lillehoj, H. S., and K. D. Choi. 1998. Recombinant chicken interferon-
gamma inhibits in vitro Eimeria tenella development, and reduces in vivo
oocyst production and body weight loss following challenge infection with
Eimeria acervulina. Avian Dis. 42:307–314.
56. Lillehoj, H. S., K. D. Choi, M. C. Jenkins, V. N. Vakharia, K. D. Song, J. Y.
Han, and E. P. Lillehoj. 2000. A recombinant Eimeria protein inducing
interferon-gamma production: comparison of different gene expression sys-
tems and immunization strategies for vaccination against coccidiosis. Avian
Dis. 44:379–389.
57. Lillehoj, H. S., and E. P. Lillehoj. 2000. Avian coccidiosis. A review of
acquired intestinal immunity and vaccination strategies. Avian Dis. 44:408–
425.
58. Lillehoj, H. S., and J. M. Trout. 1993. Coccidia: a review of recent advances
on immunity and vaccine development. Avian Pathol. 22:3–21.
59. Lillehoj, H. S., and J. M. Trout. 1994. CD8⫹ T cell-coccidia interactions.
Parasitol. Today 10:10–14.
60. Lillehoj, H. S., and J. M. Trout. 1996. Avian gut-associated lymphoid tissues
and intestinal immune responses to Eimeria parasites. Clin. Microbiol. Rev.
9:349–360.
61. Lillehoj, E. P., C. H. Yun, and H. S. Lillehoj. 2000. Vaccines against the
avian enteropathogens Eimeria, Cryptosporidium and Salmonella. Anim.
Health Res. Rev. 1:47–65.
62. Lowenthal, J. W., M. R. Digby, and J. J. York. 1995. Production of inter-
feron-gamma by chicken T-cells. J. Interferon Cytokine Res. 15:933–938.
63. Lowenthal, J. W., T. E. O’Neil, A. David, G. Strom, and M. E. Andrew. 1999.
Cytokine therapy: a natural alternative for disease control. Vet Immunol.
Immunopathol. 72:183–188.
CLIN. MICROBIOL. REV.
64. Lowenthal, J. W., J. J. York, T. E. O’Neil, S. Rhodes, S. J. Prowse, D. G.
Strom, and M. R. Digby. 1997. In vivo effects of chicken interferon-gamma
during infection with Eimeria. J. Interferon Cytokine Res. 17:551–558.
65. MacMicking, J., Q.-W. Xie, and K. Nathan. 1997. Nitric oxide and macro-
phage function. Annu. Rev. Immunol. 15:323–350.
66. Martin, A. G., H. D. Danforth, J. R. Barta, and M. A. Fernando. 1997.
Analysis of immunological cross protection and sensitivities to anticoccidial
drugs among five geographical and temporal strains of Eimeria maxima. Int.
J. Parasitol. 5:527–533.
67. Martin, A. G., H. D. Danforth, J. M. Jaynes, and J. E. Thornton. 1999.
Evaluation of the effect of peptidyl membrane-interactive molecules on
avian coccidia. Parasitol. Res. 85:331–336.
68. Martin, A. G., H. S. Lillehoj, B. Kaspers, and L. Bacon. 1994. Mitogen-
induced lymphocyte proliferation and interferon production following coc-
cidia infection. Avian Dis. 38:262–268.
69. Michalski, W. P., and S. J. Prowse. 1991. Superoxide dismutases in Eimeria
tenella. Mol. Biochem. Parasitol. 47:189–195.
70. Ouarzane, M., M. Labbe, and P. Pery. 1998. Eimeria tenella: cloning and
characterization of cDNA encoding a s3a ribosomal protein. Gene 28:125–
130.
71. Ovington, K. S., and N. C. Smith. 1992. Cytokines, Free radicals and
resistance to Eimeria. Parasitol. Today 8:422–426.
Downloaded from cmr.asm.org at MAHIDOL UNIV FAC OF MED on June 25, 2007
72. Procunier, J. D., M. A. Fernando, and J. R. Barta. 1993. Species and strain
differentiation of Eimeria spp. of the domestic fowl using DNA polymor-
phisms amplified by arbitrary primers. Parasitol. Res. 79:98–102.
73. Prowse, S. J., and J. Pallister. 1989. Interferon release as a measure of the
T-cell response to coccidial antigens in chickens. Avian Pathol. 18:619–630.
74. Rose, M. E. 1987. Eimeria, Isospora, and Cryptosporidium, p. 275–312. In
E. J. L. Soulsby (ed.), Immune responses in parasitic infections: immunol-
ogy, immunopathology and immunoprophylaxis. CRC Press, Inc., Boca
Raton, Fla.
75. Rose, M. E., and D. L. Lee. 1977. Interactions in vitro between sporozoites
of Eimeria tenella and host peritoneal exudate cells: electron microscopal
observations. Z. Parasitenkd. 54:1–7.
76. Rose, M. E., B. M. Ogilvie, and J. W. Bradley. 1980. Intestinal mast cell
response in rats and chickens to coccidiosis, with some properties of
chicken mast cells. Int. Arch. Allergy Appl. Immunol. 63:21–29.
77. Rosen, G. M., S. Pou, C. L. Ramos, M. Cohen, and B. E. Britigan. 1995.
Free radicals and phagocytic cells. FASEB J. 9:200–209.
78. Rothwell, L., W. Muir, and P. Kaiser. 2000. Interferon-gamma is expressed
in both gut and spleen during Eimeria tenella infection. Avian Pathol.
29:333–342.
79. Ruff, M. D., and H. D. Danforth. 1966. Resistance of coccidia to medica-
tions, p. 427–430. In Proc. XX World’s Poult. Congr., vol. II.
80. Schmatz, D. M. 1997. Anticoccidial drug discovery and design, p. 20–21, In
M. W. Shirley, F. M. Tomley, and B. M. Freeman (ed.), Control of coccid-
iosis into the next millennium. Proc. VII Int. Coccidiosis Conf.
81. Schnitzler, B. E., P. Thebo, F. M. Tomley, M. W. Shirley, and A. Uggla.
1997. Identification of Eimeria sp in poultry by in vitro amplification of the
internal transcribed spacer 1, p. 64. In M. W. Shirley, F. M. Tomley, and
B. M. Freeman (ed.), Control of coccidiosis into the next millennium. Proc.
VII Int. Coccidiosis Conf.
82. Shirley, M. W. 1975. Enzyme variation in Eimeria species of the chicken.
Parasitology 71:369– 376.
83. Shirley, M. W. 1986. New methods for the identification of species and
strains of Eimeria, p. 13–35. In L. R. McDougald, P. L. Long, and L. P.
Joyner (ed.), Research in avian coccidiosis. University of Georgia, Athens.
84. Shirley, M. W. 1994. Coccidial parasites from the chicken: discrimination of
different populations of Eimeria tenella by DNA hybridisation. Res. Vet.
Sci. 57:10–14.
85. Shirley, M. W., A. C. Bushell, J. E. Bushell, V. McDonald, and B. Roberts.
1995. A live attenuated vaccine for control of avian coccidiosis: trials in
broiler breeders and replacement layer flocks in the United Kingdom. Vet.
Rec. 137:453–457.
86. Shirley, M. W., and D. A. Harvey. 1996. Eimeria tenella: genetic recombi-
nation of markers for precoccious development and arprinocid resistance.
Appl. Parasitol. 37:293–299.
87. Shirley, M. W., and D. A. Harvey. 2000. A genetic linkage map of the
apicomplexan protozoan parasite Eimeria tenella. Genome Res. 10:1587–
1593.
88. Smith, A. L., and A. C. Hayday. 1998. genetic analysis of the essential
components of the immunoprotective response to infection with Eimeria
vermiformis. Int. J. Parasitol. 28:1060– 1069.
89. Song, K. D., H. S. Lillehoj, K. D. Choi, D. Zarlenga, and J. Y. Han. 1997.
Expression and functional characterization of recombinant chicken inter-
feron-gamma. Vet. Immunol. Immunopathol. 58:321–333.
90. Tennyson, S. A., and J. R. Barta. 2000. Localization and immunogenicity of
a low molecular weight antigen of Eimeria tenella. Parasitol. Res. 86:453–
460.
91. Tomley, F. M. 1994. Characterization of rhoptry proteins of Eimeria tenella
sporozoites: antigenic diversity of rhoptry epitopes within species of the
genus Eimeria and among three asexual generations of a single species, E.
VOL. 15, 2002
tenella. Infect. Immun. 62:4656–4658.
92. Tomley, F. M., J. M. Bumstead, K. J. Billington, and P. P. J. Dunn. 1996.
Molecular cloning and characterization of a novel acidic microneme protein
(Etmic-2) from the apicomplexan protozoan parasite, E. tenella. Mol. Bio-
chem. Parasitol. 79:195–206.
93. Tomley, F. M., L. E. Clark, U. Kawazoe, R. Dijkema, and J. J. Kok. 1991.
Sequence of gene encoding an immunodominant microneme protein of E.
tenella. Mol. Biochem. Parasitol. 49:277–288.
94. Tomley, F., A. N. Vermeulen, and P. van den Boogart. 1993. The use of live
vectors for presentation of recombinant antigens to chickens, p. 124–126. In
Proc. VI Int. Coccidiosis Conf.
95. Trout, J. M., and H. S. Lillehoj. 1993. Transport of Eimeria acervulina
sporozoites, evidence of a role for intestinal CD8⫹ lymphocytes and mac-
rophages. J. Parasitol. 79:790–792.
96. Trout, J. M., and H. S. Lillehoj. 1996. T lymphocyte roles during Eimeria
acervulina and Eimeria acervulina infections. Veterinary immnuology and
immunopathology 53:163–172.
97. Trout, J. M., and H. S. Lillehoj. 1995. Eimeria acervulina infection: evidence
for the involvement of CD8⫹ T lymphocytes in sporozoite transport and
host protection. Poult. Sci. 74:1117–1125
98. Tsuji, N., S. Kawazu, M. Ohta, T. Kamio, T. Isobe, K. Shimura, and K.
Fujisaki. 1997. Discrimination of eight chicken Eimeria species using the
two-step polymerase chain reaction. J. Parasitol. 83:966–970.
99. Van Doornick, W. M., and E. R. Becker. 1957. Transport of Eimeria necatrix
in macrophages. J. Parasitol. 43:40–44.
100. Vermeulen, A. N. 1998. Progress in recombinant vaccine development
against coccidiosis. A review and prospects into the next millenium. Int J.
Parasitol. 28:1121–1130.
101. Vermeulen, A., R. Dijkema, and J. J. Kok. Mar. 1994. Coccidiosis vaccine.
European patent 0349071B1.
102. Vermeulen, A. N., J. J. Kok, P. van den Boogart, R. Dijkema, and J. A. J.
Claessens. 1993. Eimeria refractile body proteins contain 2 potentially func-
tional characteristice-transhydrogenase and carbohydrate transport. FEMS
Microbiol. Lett. 1993:223–230.
103. Wallach, M., A. Halabi, G. Pillemer, O. Sar Shalom, D. Mencher, M. Gilad,
U. Bendheim, H. D. Danforth, and P. C. Augustine. 1992. Maternal immu-
nization with gametocyte antigens as a means of providing immunity against
Eimeria maxima in chickens. Infect. Immun. 60:2036– 2039.
BIOLOGY AND IMMUNOBIOLOGY OF EIMERIA 65
104. Wallach, M., N. C. Smith, M. Petracca, C. M. D. Miller, J. Eckert, and R.
Braun. 1995. Eimeria maxima gametocyte antigens: potential use as a sub-
unit maternal vaccine against coccidiosis in chickens. Vaccine 13:347–354.
105. Williams, R. B. 1994. Safety of the attenuated anticoccidial vaccine “Para-
cox” in broiler chickens isolated from extraneous coccidial infection. Vet.
Res. Commun. 18:189–198.
106. Williams, R. B. 1995. Epidemiological studies of coccidiosis in the domestic
fowl (Gallus gallus). II. Physical condition and survival of Eimeria acervulina
oocysts in poultry house liter. Appl. Parasitol. 36:90–96.
107. Williams, R. B. 1998. Epidemiological aspects of the use of live anticoc-
cidial vaccines for chickens. Int. J. Parasitol. 28:1089–1098
108. Williams, R. B. 1999. Three enzymes newly identified from the genus
Eimeria and two more newly identified from E. maxima, leading to the
discovery of some aliphatic acids with activity against coccidia of the do-
mestic fowl. Vet. Res. Commun. 23:151–163.
109. Williams, R. B., and J. Catchpole. 2000. A new protocol for a challenge test
to assess the efficacy of live anticoccidial vaccines for chickens. Vaccine
18:1178–1185.
110. Woods, W. G., K. G. Whithear, D. G. Richards, G. R. Anderson, W. K.
Jorgensen, and R. B. Gasser. 2000. Single-strand restriction fragment
length polymorphism analysis of the second internal transcribed spacer
(ribosomal DNA) for six species of Eimeria from chickens in Australia. Int.
Downloaded from cmr.asm.org at MAHIDOL UNIV FAC OF MED on June 25, 2007
J. Parasitol. 30:1019–1023.
111. Youn, H. J., and J. W. Noh. 2001. Screening of the anticoccidial effects of
herb extracts against Eimeria tenella. Vet. Parasitol. 96:257–263.
112. Yun, C. H., H. S. Lillehoj, J. Zhu, and W. Min. 2000. Kinetic differences in
intestinal and systemic interferon-gamma and antigen-specific antibodies in
chickens experimentally infected with Eimeria maxima. Avian Dis. 44:305–
312.
113. Zhang, S., H. S. Lillehoj, and M. D. Ruff. 1995. Chicken tumor necrosis-like
factor. I. In vitro production by macrophages stimulated with Eimeria
tenella or bacterial lipopolysaccharide. Poult. Sci. 74:1304–1310.
114. Zhang, S., H. S. Lillehoj, and M. D. Ruff. 1995. In vivo role of tumor
necrosis-like factor in Eimeria tenella infection. Avian Dis. 39:859–866.
115. Zhu, J., H. S. Lillehoj, P. Allen, C. H. Yun, D. Pollock, and E. Emara. 2000.
Analysis of disease resistance associated parameters in broiler chickens
challenged with Eimeria maxima. Poult. Sci. 79:619–625.