Immune response to Coccidia
Hyun S. Lillehoj
Animal Parasitic Disease laboratory
Animal and Natural Resources Institute
Beltsville Agricultural Research Center
United States Department of Agriculture
Agricultural Research Service
Beltsville, MD 20705
SUMMARY
In view of rapid development of new biotechnologies
in veterinary science, novel control strategies using genomics, molecular
biology and immunology will offer an alternative way to prevent the spread of
coccidiosis in the future. Increasing understanding of the protective role of
local intestinal immune responses and the identification of various effector
molecules against coccidia provide optimism that novel means to control
coccidiosis will be feasible in the near future. The aim of this presentation is
to review the current progress in our understanding of the host immune response
to Eimeria and to discuss potential strategies which are currently being
developed for coccidiosis control. Due to complexities of the host immune system
and the parasite life cycle, comprehensive understanding of host-parasite
interactions that lead to protective immunity should precede before we can
develop successful prevention and disease control strategies. Recent progress in
Eimeria and poultry genome sequencing and rapidly developing functional genomics
technology is facilitating the identification and characterization of host and
parasite genes which are involved in immunoprotection and immunopathology in
avian coccidiosis. Recent studies have provided much evidence that molecular and
immunological-based strategies such as recombinant vaccines and dietary
immunomodulation enhance gut immunity. Thus, successful application of new
knowledge on host-parasite immunobiology, gut immunity and genomics in
commercial settings will lead to the development of novel disease prevention
strategies against coccidiosis in the near future.
INTRODUCTION
Avian coccidiosis is the major parasitic disease of
poultry with a substantial economic burden estimated to cost the industry
greater than $800 million in annual losses (Williams, 1998). In-feed medication
for prevention and treatment contributes a major portion of these losses in
addition to mortality, malabsorption, inefficient feed utilization and impaired
growth rate in broilers, and a temporary reduction of egg production in layers.
Eimeria spp. possess a complex life cycle comprising of both sexual and asexual
stages, and their pathogenicity varies in birds of different genetic backgrounds
(Lillehoj, 1988). In the natural host, immunity is species-specific, such that
chickens immune to one species of Eimeria are susceptible to others.
Additionally, Eimeria spp. exhibits different tissue and organ specificity in
the infected host. Understanding the interplay between the host and the
parasites in the intestine is crucial for the design of novel control approaches
against coccidiosis.
While natural infection with Eimeria spp. induces immunity, vaccination
procedures on a commercial scale have shown limited effectiveness and current
disease control remains largely dependent on routine use of anti-coccidial drugs
in most countries (Dalloul and Lillehoj, 2005). Recently several different live
vaccines have been commercially developed and these are mostly composed of
either virulent or attenuated parasite strains. Major disadvantages of live
parasite vaccines are labor-intensive production and high cost due to inclusion
of multiple parasite species in the vaccine. Although live oocyst vaccines
represent a limited but useful alternative to anticoccidial drugs, a recombinant
vaccine composed of parasite antigens/antigen-encoding genes that elicit
coccidia-specific immunity would be eminently preferable. While it would be
cost-effective to produce recombinant vaccines (proteins or DNA), the difficulty
remains to identify the antigens or genes which are responsible for eliciting
protective immunity and to devise the most efficient delivery method for these
recombinant vaccines to be delivered and presented to the bird’s immune system.
Also, such subunit vaccines would eliminate the danger of emerging resistant
strains encountered with live vaccines, but unfortunately until efficient
vaccines become commercially available, the poultry industry is forced to rely
upon prophylactic chemotherapy to control coccidiosis. Further, the introduction
of alternative prevention/treatment measures such as non-chemical feed
supplements that effectively enhance productivity and non-specific immunity may
help limit the use of anticoccidials. However, the lack of efficient vaccines
and the increasing incidence of drug resistant strains and escalating public
anxiety over chemical residues in meat and eggs mandate the development of
alternative control methods. For developing a successful recombinant vaccine
strategy, we need to better understand the chicken immune system and the means
to elicit effective immunity against coccidia.
PATHOGENESIS AND IMMUNOGENICITY OF Eimeria
The pathogenicity of coccidia depends largely on the
successful replication of developing parasites inside the host. Theoretical
estimates indicate that a single oocyst of a virulent species such as E. tenella
could yield 2,520,000 invasive parasites after the 2nd merogony stage (Levine,
1982). E. maxima is thought to undergo a minimum of four generations of
schizogony (McDonald et al., 1986). Most major enteric protozoa including
coccidia invade the intestinal mucosa and induce a certain degree of epithelial
cell damage, inflammation and villous atrophy (Pout, 1967). The signs of
coccidiosis depend on the degree of the damage and inflammation and include
watery, whitish diarrhea (E. acervulina) or hemorrhagic diarrhea (E. tenella),
petechial hemorrhages and the marked production of mucus (E. maxima),
dehydration, weight loss, rectal prolapse and dysentery. The profuse bleeding in
the ceca is a characteristic feature of E. tenella infection due to its
extensive destruction of the mucosa with histological lesions (Witlock et al.,
1975).
In general, young animals are more susceptible to coccidiosis and more readily
display signs of disease, whereas older chickens are relatively resistant to
infection (Lillehoj, 1998). Young animals which recover from coccidiosis may
later be able to partly compensate for the loss of body growth, but their growth
potential remains severely compromised. The magnitude of clinical signs
resulting from Eimeria infection is significantly influenced by host genetic
factors. In two genetically divergent strains of inbred chicken lines, SC (B2B2)
and TK (B15B21), the different degrees of disease pathogenesis depended upon the
genetics of host when they are infected with E. tenella or E. acervulina (Lillehoj,
1998). In general, SC chickens are more resistant than TK chickens to
coccidiosis. Because of their genetic differences in disease susceptibility to
coccidiosis, extensive research has been carried out regarding the underlying
immunological mechanisms controlling intestinal cell-mediated immune responses.
More detailed information concerning the genetic control of immune response to
Eimeria can be found in previous reviews (Lillehoj, 1991 and 1998; Lillehoj and
Lillehoj, 2000, Lillehoj et al., 2004).
Infection with Eimeria induces protective immunity that is long-lasting and
exquisitely specific to that particular Eimeria species. While a large number of
oocysts is generally required to generate a good immune response against
Eimeria, some exceptions have been noted, e.g. E. maxima is highly immunogenic
and requires only a small number of oocysts to induce almost complete immunity.
The early endogenous stages of Eimeria life cycle are considered to be more
immunogenic than the later sexual stages (Rose and Hesketh, 1976; Rose et al.,
1984) although gamete antigens of E. maxima were shown to be immunogenic and
induce protection against a challenge infection with the live parasites (Wallach
et al. 1990 and 1995). However, activation of immune T cells released IFN-g
which inhibited the intracellular development of coccidia (Lillehoj and Choi,
1998) indicating that host protective immunity is against the exponential growth
phase of the parasite life cycle. Because of the complexity of host-parasite
immunobiology which involve many different cell types and soluble factors,
further studies are necessary to obtain insights on host immunity eliciting
complete protection.
CHICKEN IMMUNE SYSTEM
Chickens have evolved sophisticated immune system
much like mammals. Major defense mechanisms include a non-specific immune
response which is activated immediately following exposure to potential
pathogens. Non-specific immunity is mediated by macrophages, granulocytes,
natural killer (NK) cells, serum proteins and soluble factors and precedes the
development of antigen-specific memory immune response mediated by lymphocytes.
Lymphocytes are generated in the primary lymphoid organs such as thymus and
bursa of Fabricius where they acquire functional identity whereas it is in the
secondary lymphoid organs such as lymph nodes, spleen and mucosal associated
lymphoid tissues where they differentiate into effector cells upon encounter
with antigens and potential pathogens. Lymphoid organs are organized into
different compartments where lymphocytes and non-lymphoid cells form a
microenvironment suitable for effective immune responses. In summary the major
cellular components of the avian immune system include thymus-derived T
lymphocytes, bursa-derived B lymphocytes, macrophages and NK cells.
B lymphocytes: B lymphocytes play an important role in host defense against many infectious diseases by producing antibodies which are specific for the eliciting antigen. Once produced, antibodies becomes effector molecules which can either block the invasion of host cells by pathogens, neutralize toxins or kill extracellular pathogens through antibody-dependent cell-mediated cytotoxicity (ADCC). Unlike other animals, chicken B cells develop in the bursa of Fabricius, a gut associated primary lymphoid tissue located near the cloaca. During embryonic development, pre-bursal stem cells enter the bursal rudiment in a single wave (Houssaint et al., 1976) where they undergo a maturation process which involves generation of antibody diversity by gene conversion and somatic diversification to generate different classes of immunoglobulins (IgM, IgG and IgA.) Bursal cells migrate out of bursa to the periphery a few days prior to hatch. Chickens generate strong antibody responses to both T cell-dependent and independent antigens. After an initial encounter with an antigen, B cells secrete the IgM isotype of antibody which later switches to IgG or IgA upon the secondary exposure. As in mammals, the secondary response is characterized not only by isotype switching but also an increase in magnitude compared with the initial response (Ratcliff, 1989). The activation of naďve B cells in vivo requires a direct interaction with helper T cells typically expressing CD4 and this interaction is restricted by antigen recognition in the context of class II genes of the major histocompatibility complex (MHC).
T lymphocytes: Thymus-derived lymphocytes in chickens are divided into 3 separate subpopulations on the basis of their cell surface antigen expression and biological function. Unlike mammals that possess two different types of antigen-recognizing receptors (TCRab and TCRgd), chicken T cells express 3 distinct T cell receptors, TCR1, TCR2 and TCR3. The TCR on a given T lymphocyte subset can be a heterodimer consisting either of a g and d chain (TCR1), an a and Vb1 chain (TCR2) or an a and Vb2 chain (TCR3) (Cooper et al., 1991; Gobel, 1996). As in mammals, immature T lymphocytes undergo differentiation in the thymus in chickens: CD4-CD8- thymocytes give rise to CD4+CD8+ which develop into CD4+CD8- or CD4-CD8+ T-cells. CD4 and CD8 T subsets express all 3 types of TCRs (Davidson and Boyd, 1992). Mature CD4 or CD8 single-positive T-cells leave the thymus to populate secondary immune organs, and also travel in the circulatory and lymphatic systems. In the blood, CD4-CD8-TCR1+ T cells, as well as CD4+CD8- and CD4-CD8+ T cells, expressing either TCR2 or TCR3 have been identified. The same T cell populations are found in the spleen where CD4-CD8+TCR1+ T cells also exist (Sowder et al., 1988; Gobel, 1996). With differentiation, functionally distinct T cell subsets express certain cell surface proteins. T cells expressing both CD4 and CD8 molecules are considered immature T cells and constitute the majority of cells in the thymus. Single-positive T cells, expressing either CD4 or CD8 are mature T cells. Most CD4+CD8- cells are helper or inflammatory T cells responding to exogenous antigen in association with MHC class II molecules, whereas CD4-CD8+ cells represent cytotoxic cells which respond to endogenous antigen in association with MHC class I molecules. The ab TCR (TCR2 and TCR3) are known to mediate MHC-restricted antigen recognition by single-positive T cells, whereas the physiological role of T cells expressing TCR1 is not well defined (Gobel, 1996).
Macrophages: Macrophages and dendritic cells represent components of the mononuclear phagocyte system (van Furth et al., 1972) and are involved in processing and presenting antigens to lymphocytes. Macrophages are highly heterogeneous cells present in primary and secondary lymphoid tissues and are important cells involved in host defense. Their functions are primarily phagocytosis, cytotoxic activity against tumors and production of chemokines and cytokines which mediate inflammatory responses. In chickens, interdigitating cells (IDC) which are found in situ in all T cell areas of all lymphoid tissues are characterized by dendritic extensions and express high level of MHC class II antigens and are probably the main antigen presenting cells to T helper cells in vivo during primary immune responses (Jeurissen et al., 1994). Macrophages express many cell surface antigens that have been detected by mouse monoclonal antibodies including K1 (Lillehoj et al., 1993) and KUL01 (Mast and Goddeeris,1995). Recent advances in our understanding of how these cells recognize diverse antigens led to the discovery of highly conserved pattern recognition receptors (PRRs) which are involved in recognition of highly conserved molecular structures on microbial components called pathogen-associated molecular patterns (PAMPs). The Toll-like receptor (TLR) family is membrane-bound PRRs that play critical roles in activating the innate immune response and phagocytosis (Underhill and Ozinsky, 2002). Activation of TLR by binding to a particular ligand leads to activation of the NF-kB signal transduction pathway inducing a wide variety of host genes involved in innate immunity, such as antimicrobial peptides, cytokines, chemokines and nitric oxide synthase (Barton and Medzhitov, 2002). Although in humans more than ten TLRs have been identified, only a limited number of homologues have been characterized in chickens. Thus, it appears that TLRs have been conserved through evolution and expressed in various immune related tissues and cell lines (Iqbal et al., 2005). Similar to the mammalian gene products, the secondary protein structure of chicken TLR1, 3, 5, 7, and 10 consist of several leucine rich domains, a transmembrane domain, and Toll/interleukin-1 receptor domains (Yilmaz et al., 2005). Understanding of how TLRs regulate immune response to pathogens in poultry will be important for future development of new strategies for disease control.
NK cells: NK cells are non-lymphoid, heterogeneous and nonphagocytic cells which mediate immediate response against infection. Along with macrophages, they are important in defense against pathogens and tumors in unimmunized hosts. NK cells in chickens have been identified from freshly obtained spleen of SPF chickens (Schat et al., 1986) and have been identified in many tissues including blood and the intestine (Lillehoj and Chai, 1988). In mammals, NK cells express the ? chain of CD3, the IL-2 receptor, CD2, and CD16. Chicken NK cells have been identified using different monoclonal antibodies such as K108 (Chung and Lillehoj, 1991) and 28-4 and they do not express CD3, CD4 or TCR (Gobel et al. 1996). Rather, NK cells have been classified as TCR0 cells since they do not express TCRab or TCRgd. NK cells have been implicated in resistance against MDV-induced tumors (Lam and Linna, 1979), and in intestinal defense against coccidia (Lillehoj, 1989) and rotavirus (Myers and Schat, 1990).
INNATE AND AQUIRED IMMUNE RESPONSE TO
Eimeria
Because the life cycle of Eimeria parasites is
complex and comprised of intracellular, extracellular, asexual, and sexual
stages, host immune responses are quite diverse and complex. After invasion of
the host intestine, Eimeria elicit both nonspecific and specific immune
responses which involve many facets of cellular and humoral immunity (Lillehoj,
1991; Lillehoj 1998; Lillehoj and Lillehoj, 2000; Dalloul and Lillehoj, 2005)
Nonspecific factors include physical barriers, phagocytes, leukocytes,
chemokines and complement components. Antigen-specific immunity is mediated by
antibodies, lymphocytes, and cytokines. Due to the specific invasion and
intracellular development of coccidia in the intestine, understanding of the
gut-associated lymphoid tissues (GALT) is important. The GALT serve three main
functions in host defense against enteric pathogens: processing and presentation
of antigens, production of intestinal antibodies, and activation of
cell-mediated immunity (CMI). In the naďve host, coccidia activate local
dendritic cells and macrophages eliciting various chemokines and cytokines
(Lillehoj, 1998). In immune hosts, parasites enter the gut early after
infection, but are prevented from further development, indicating that acquired
immunity to coccidiosis may involve mechanisms that inhibit the natural
progression of parasite development (Rose et al., 1984; Trout and Lillehoj,
1996; Lillehoj and Choi, 1998; Yun et al., 2000a). Recent studies demonstrated
the role of several cytokines produced locally during coccidiosis (Yun et al.,
2000b; Min et al., 2003; Lillehoj et al., 2004; Dalloul and Lillehoj, 2005)
which are responsible for enhancing protective immunity against Eimeria
(Lillehoj et al., 1998; Yun et al., 2000c).
Antibody responses: Following coccidiosis, both circulating and secretory
antibodies specific for coccidia parasites are detected in serum, bile and
intestine (Lillehoj and Ruff, 1987; Lillehoj, 1988; Yun et al., 2000c). However,
antibody titers in serum and intestine do not correlate with the level of
protection after oral infection with coccidia (Dalloul et al., 20003; Lillehoj
and Ruff, 1987). Convincing evidence on the minimum involvement of humoral
antibody to limit coccidian infection came from agammaglobulinemic chicken
models where it was observed that chickens Bursectomized by hormonal and
chemical means were resistant to reinfection with coccidia (Rose and Long, 1970;
Lillehoj, 1987). Three isotypes of antibodies are recognized in birds, IgM, IgA,
and IgY. IgY is considered the orthologue of the mammalian IgG (Leslie at al.,
1969), even though the cDNA encoding the IgY heavy chain shows similarity to
mammalian IgE (Parvari et al., 1998). The presence of other antibody classes
such as IgD or IgE in chickens has not yet been documented. The role of parasite
specific antibodies both in serum and mucosal secretions has been extensively
studied in coccidiosis (Girard et al., 1997; Lillehoj and Ruff, 1987). Maternal
IgY is concentrated in the yolk sac of the egg (Rose et al., 1974) where it is
transported to the embryo during late development by a mechanism similar to that
found in mammals (West el al., 2004), and is thus considered to be of some
relevance in maternal passive immunity (Wallach et al., 1992). When hens were
hyperimmunized with gametocyte surface antigens of E. maxima, passively
transferred antibodies in young birds protected against challenge with
sporulated E. maxima oocysts by reducing fecal oocyst production (Wallach et
al., 1992). The efficiency of maternally transferred antibodies in protection
against field infections needs to be verified using large scale trials.
Considering the short life span of maternal antibodies in young chicks, it may
not be feasible to maintain extremely high levels of antibodies in birds for a
long time.
Lately, immunotherapy using whole antibody molecules or single chain fragments
of the variable region (ScFv) with antigen binding activity has been gaining
interest as a potential immunotherapy against infectious agents. Currently
available immunological control strategies consist of sub-acute infection with
virulent or live attenuated parasites. The main obstacle to the development of
an antibody-based strategy against avian coccidiosis however, is the existence
of many different Eimeria species. With recent progress in molecular biology and
sequence information on chicken immunoglobulin genes, it is now possible to
generate recombinant chicken antibodies (Min et al., 2001; Park et al., 2004).
There are potentially two different approaches using antibodies against
coccidiosis. One is to produce hyperimmune serum against major immunogenic
proteins of coccidia and passively administer it to 18 day-old embryos or to
feed orally to young chicks at hatch. In a previous study, a surface protein
3-1E, which was identified from the merozoites of E. acervulina, was used as a
potential subunit vaccine for avian coccidiosis and found to be protective
against challenge infection with the homologous Eimeria (Lillehoj et al., 2000).
Moreover, a DNA vaccine prepared from the gene coding for the protein was
partially protective against challenge with E. acervulina (Lillehoj et al.,
2000). These results prompted us to investigate the potential use of chicken
antibodies against 3-1E in protection against coccidiosis. In a recent report
(Ngyen et al., 2003), we tested the protective effect of chicken egg antibody
(IgY) powder which was prepared from hens hyperimmunized with purified 3-1E
recombinant protein in a challenge model with E. acervulina and E. tenella.
Chickens which were fed standard diet with IgY powder containing antibodies
against 3-1E (3-1E/IgY) were better protected against oral challenge with E.
tenella or E. acervulina oocysts compared with those fed with standard diet
supplemented with IgY-containing powder only. These results clearly indicated
that 3-1E represents an important target antigen for coccidiosis prevention and
that passive immunization of chickens with antigen-specific IgY powder is a
promising method to confer protection against coccidiosis.
Another approach to generate therapeutic antibody is to develop recombinant
antibodies against protective epitopes. Using the chicken B cell line R27H4, we
previously developed several hybridomas producing coccidia-specific antibodies
(Lillehoj et al., 1994). One of them, 6D-12-G10, was reactive with an Eimeria
protein suggested to be involved in binding to a host cell receptor (Sasai et
al. 1996). Unfortunately, the amount of antibody secreted by this hybridoma into
culture medium was insufficient for further biochemical and physiological
characterization of the antigen. To circumvent the problems associated with low
yield, we produced an scFv fragment derived from the VH and VL genes encoding
the 6D-12-G10 antibody. The single chain Fv antibody was expressed in E. coli
and the recombinant gene product bound whole parasites (Min et al., 2002) by
immunoblot, immunofluorescence assay (IFA) and enzyme-linked immunosorbent assay
(ELISA). Chickens fed recombinant scFv antibodies showed reduced fecal oocysts
upon challenge infection with live coccidia (unpublished data). Using similar
approaches, we also generated other scFv antibodies detecting coccidia proteins
(Park et al., 2004). Like the native monoclonal antibodies from which they were
derived, these recombinant antibodies showed binding activity against Eimeria
antigens and were secreted at 5 mg/L into culture medium, indicating that
soluble, stable and functional chicken ScFv can be produced in large volume.
Thus, recombinant antibody technology has advantages over hybridomas, which
generally produce low quantities of antibodies (<0.5 mg/L), easily lose
antibody activity and are not able to make high titer ascites.
Although the role of antibodies produced during natural infection is debatable,
antibodies which are generated against specific epitope of coccidian parasites
can be used to reduce parasite invasion and have been shown to be beneficial
against coccidiosis infection (Walach et al., 1992; Ngyen et al., 2003).
Antibody-based therapies can be useful, for example, to prevent Eimeria
infections, where antibodies are known to play a role in protection against the
parasite. The results presented here demonstrate an example of recombinant
chicken antibodies useful to reduce parasites in the field. ScFv antibody
fragments may offer advantages for in vivo applications as diagnostic and
therapeutic reagents also. For example, because the scFv antibody is
approximately 33 kiloDalton in size, representing 20% of an intact IgG molecule,
it may penetrate tissues easily, an important consideration given the invasive
nature of Eimeria parasites. The ability to generate unlimited amount of soluble
and functional recombinant scFv antibodies will facilitate the investigation of
their potential therapeutic value in passive immunotherapy against avian
coccidiosis. Meanwhile evidence that antibodies in dietary supplements could
protect against oral coccidiosis infection opens a new door for novel
immunotherapy strategies against coccidiosis. Furthermore, given the limited
information concerning the nature of protective antigens of Eimeria, these
antibodies will be an important tool for affinity isolation of potential Eimeria
subunit vaccines.
Cell-mediated immunity: The evidence that the removal of the bursa by chemical or hormonal means (Rose and Long, 1970; Lillehoj, 1987) did not interfere with the development of protective immunity against Eimeria indicated the importance of cell-mediated immunity in coccidiosis. The role of T cells in the protection against coccidiosis has also been studied in immunosuppressed chickens using T cell-specific drugs that selectively abrogate or severely impair T cell function. These treatments included thymectomy (Rose and Long, 1970), cyclosporin A (Lillehoj, 1987), betamethasone, dexamethasone (Isobe and Lillehoj, 1993), and cell depletion using mouse monoclonal antibodies against CD8+ or abTCR-expressing cells (Trout and Lillehoj, 1996). In all of these studies, the abrogation of T cell function impaired host protective immunity against coccidiosis. Additional evidence for the protective role of T cells came from adoptive transfer studies where peripheral blood lymphocytes (PBL) and spleen cells from E. maxima-immune chickens protected syngeneic recipients against a live parasite challenge infection (Rose and Hesketh, 1982). Lillehoj and Choi (1998) and Miller et al. (1994), using an in vitro culture, showed that splenocytes from E. tenella-immune chickens inhibited the intracellular development of E. tenella in kidney cells. The nature of these cells was not determined, but may be NK cells since they did not show any MHC restriction in their action. Direct evidence for the presence of Eimeria-specific T cells was demonstrated by an in vitro antigen-driven lymphoproliferation assay (Rose and Hesketh, 1984; Lillehoj, 1986; Vervelde et al.,1996).
T lymphocytes: In the gut,
intraepithelial lymphocytes (IEL) represent an important component of the GALT
(Guy-Grand et al., 1974). A unique feature of IEL is that gd T cells are
predominant, whereas the vast majority of mature T lymphocytes in the peripheral
blood and lymphoid organs use the CD3-associated ab TCR heterodimer for antigen
recognition (Goodman and Lefrancois, 1988; Bonneville et al., 1988). Following
primary and secondary infections with E. acervulina, an increased percentage of
intraepithelial gd T cells was observed in the duodenum (Choi and Lillehoj,
2000). The percentage of gd T cells was significantly elevated by day 8
following primary infection with E. acervulina in SC chicken whereas a
significant increase was seen as early as day 4 in TK chickens (Choi et al.,
1999). Concurrent with the increase of gd T cells, a significant enhancement of
IL-2 mRNA transcripts was found (Choi and Lillehoj, 2000). The percentage of ab
T cells was elevated in IEL by day 4 after primary infection with E. acervulina
in SC chickens whereas a significant increase of ab T cells was not seen until 6
d post secondary infection in SC chickens (Choi et al., 1999).
The importance of CD8+ T cells has been shown in many intracellular parasitic
infections including toxoplasmosis (Hakim et al., 1991) and malaria (Weiss et
al., 1990). In avian coccidiosis, the selective elimination of CD8+ cells by
anti-CD8+ monoclonal antibody resulted in exacerbation of the disease, as
evidenced by increased oocyst shedding after infection with E. tenella or E.
acervulina (Trout and Lillehoj, 1996). Significant increase of T cells
expressing CD8+ molecules was noted in the intestinal IEL population following
challenge infections of chicken with E. acervulina (Lillehoj and Bacon, 1991).
Two-color immunofluorescence staining revealed that the majority of CD8+ cells
in the duodenum intraepithelium of immune chickens co-expressed the ab TCR. In
both SC and TK chickens, the ratio of CD8+ to CD4+ T lymphocytes in IEL was
elevated by day 4 following primary and secondary infections with E. acervulina.
These cells continued to increase in SC chickens but showed a marked decrease in
TK chickens following the secondary infection (Choi et al., 1999). When two MHC
congenic chickens with a different disease susceptibility to coccidiosis were
compared, the higher increase of ab TCR+CD8+ and gd TCR+CD8+ cells was
associated with B2B2 chickens which are less susceptible. Similarly, Bessay et
al. (1996) observed a significant increase in the proportion of CD4+, CD8+ and
TCR gd cells in duodenal IEL from day 4 to day 8 post-infection with E.
acervulina. In contrast, the proportion of CD8+ cells decreased significantly in
the blood and spleen on days 4 and 6 post-infection. After E. tenella infection,
the proportion of CD4+ cells increased on day 8 post-infection and CD8+ cells on
days 6 and 8 post-infection in cecal IEL. At the same time, the proportion of
CD4+ cells decreased in the spleen on day 8 post-infection and CD8+ cells
decreased in the blood on day 6. In chickens infected with E. mivati, the
percentages of splenic lymphocytes bearing CD8+, gd TCR, class II MHC, or
surface IgM antigens, were decreased in the dexamethasone-treated chickens when
compared to the normal chickens (Isobe and Lillehoj, 1993). Significantly higher
numbers of total oocyst output in the dexamethasone-treated chickens following
primary and secondary infections with E. mivati indicated the significance of
CD8+ cells in primary as well as secondary immune responses.
In the peripheral blood, a transient but sharp increase in the proportion of
CD8-expressing T cells was found in White Leghorn chickens at 8 days after a
primary infection with E. tenella (Breed et al., 1996; 1997a,b). This increase
was found to be concurrent with a marked increase in IFN-g as well as nitric
oxide (NO) production upon in vitro stimulation of PBL by T cell mitogen and E.
tenella sporozoite antigen (Breed et al., 1997a). In E. maxima infection, both
CD4+ and CD8+ cells were seen in the small intestine of Light Sussex chickens,
but the proportion of CD8+ cells was higher (Rothwell et al., 1995). CD4+ cells
represent a minor population of the IEL. During E. acervulina infection, CD4+
cells increased at day 7 after primary and day 14 after secondary infection
(Lillehoj, 1994). Bessay et al. (1996) examined the T-lymphocyte subsets in the
intestine following E. tenella and E. acervulina infections. Following E.
acervulina infection, a significant increase in the proportion of CD4+ was
observed in duodenal IEL from day 4 to day 8, and in the blood and spleen on day
8 post-infection. In E. tenella infection, CD4+ cells increased on day 8
post-infection in the cecal IEL but the proportion of CD4+ cells dropped in the
spleen on day 8 post-infection. In the ceca, the number of CD4+ cell increased
significantly at day 2 after E. tenella infection and in immune chickens, mainly
CD4+ and CD8+ T cells infiltrated the lamina propria (Vervelde et al., 1996). A
significantly higher number of sporozoites were found within or next to CD3+,
CD8+, and ab TCR+ cells in immune chickens. In a study aimed at elucidating the
immunologic differences between resistant SC (B2B2) and susceptible TK (B15B21)
chickens, duodenal CD4+ T lymphocytes increased significantly and rapidly at day
4 after primary and secondary infections with E. acervulina in SC as compared to
TK chickens (Choi et al., 1999). The role of CD4+ T cells in coccidiosis may
involve the production of soluble cytokines such as IFN-g (Yun et al.,
2000a,b,c). Using a quantitative RT-PCR, increased IFN-g mRNA expression was
observed in the cecal tonsil lymphocytes in E. tenella-infected SC chickens, and
the selective depletion of CD4+ cells, but not CD8+ cells, reduced IFN-g
production.
Non-T Cells:The role of NK-cells in parasitic diseases has been well documented
(Lillehoj et al., 2004). The chicken gut IEL are known to contain subpopulations
of cells that can mediate NK cell activities as demonstrated in 4 hr 51Cr
release assays using different avian tumor cell targets (Chai and Lillehoj,
1988). The NK cell activity was higher in the jejunum and ileum than in the
duodenum and cecum. Following infection with Eimeria parasites, the NK cell
activities of both splenic and intestinal IEL decreased to a subnormal level
during the early phase of infection (Lillehoj, 1989). NK cell activity returned
to normal or slightly higher than normal levels about 1 week after the primary
inoculation. Significant increases in the splenic and intestinal IEL NK cell
activities were seen during the early phase of secondary infection. This
increase in the IEL NK cell activity shortly after secondary infection was
accompanied by a substantial increase in the number of IEL expressing the
asialo-GM1 antigen, a NK marker (Lillehoj, 1989). In a recent study, we have
identified a major effector molecule from IEL which shows lytic activity against
sporozoites (unpublished observation). Chicken NK cells, defined phenotypically
as CD8+ cells lacking T- or B lineage specific markers, constitute approximately
30% of CD8+ intestinal IEL, but < 1% of splenocytes or PBL (Gobel et al.,
2001). Using the 28-4 monoclonal antibody, specific for CD8+CD3- IEL and an
antibody for CD3, IEL were separated into CD3+ IEL T cells and the 28-4+ cells,
both co-expressing the CD8 antigen. The 28-4+ IEL were able to lyse the
NK-sensitive target cells. These results define the two major phenotypically and
functionally distinct IEL subpopulations, and imply an important role of NK
cells in the mucosal immune system (Gobel et al., 2001). Using mouse antibodies
K-14 and K-108, Chung and Lillehoj (1991) identified NK cells which stain 6 to
17% of splenic lymphocytes, 11 to 14% of PBL, and fewer than 5% of thymic and
bursal lymphocytes.
Chicken macrophages identified using the monoclonal antibody K1, express MHC
class II antigens (Kaspers et al., 1993) and are involved in different phases of
the host immune response to coccidia (Lillehoj et al., 2004). In E.
tenella-immune chickens, more leukocytes were present in the lamina propria and
leukocytes infiltrated the ceca more rapidly than in the naive chickens
(Vervelde et al., 1996). By immunocytochemical staining, most infiltrating
leukocytes were macrophages and T cells. Macrophages pretreated with the culture
supernatants of Con A-stimulated spleen cells or T cells exerted cytostatic
effects on the growth of E. tenella sporozoites (Lillehoj et al., 1989; Dimier
et al., 1998). Pretreatment of macrophages with culture supernatants of Con
A-stimulated spleen cells induced NO synthesis, and the addition of NG
monomethyl-L-arginine, a NO synthase inhibitor, also overcame the inhibition of
E. tenella replication in macrophage cultures suggesting possible involvement of
NO or toxic oxygen intermediates in inhibiting E. tenella growth (Dimier-Poisson
et al., 1999).
Cytokine and chemokine responses: Extensive experimental evidence supports the
notion that immunity mediated by lymphocytes and their secreted products such as
cytokines mediate antigen specific protection against challenge infection with
Eimeria (Lillehoj and Lillehoj, 2000; Lillehoj et al., 2004). For example,
correlation of disease resistance and enhanced proliferation of T lymphocytes
and recruitment of intestinal T cells into the duodenum following primary and
secondary E. acervulina infections have been documented (Lillehoj, 1989 and
1998; Lillehoj et al., 2004). In the intestine, lymphocytes, macrophages,
dendritic cells and other effector cells act in harmony to secrete cytokines and
proinflammatory molecules which direct the appropriate immune responses to
eliminate the invading parasite and to induce the development of memory
responses. Recently, using T lymphocyte and macrophage cDNA microarrays, host
genes related to the immune response in the gut have been identified and their
role in protection against coccidiosis is being investigated (Min et al., 2003
and 2005; Dalloul et al., in preparation). Although the importance of cytokines
in mediating innate and acquired immunity against coccidiosis has been suggested
and documented, the nature of many chicken cytokines has not been well
characterized due to slow progress in characterizing chicken cytokine genes
(Lillehoj et al., 2003). In contrast to the plethora of mammalian cytokines,
only a few chicken homologues have been described, the main ones being IFN-g,
TGF, TNF, IL-1, IL-2, IL-6, IL-8 and IL-15 (Lillehoj, 2004). Of late, a series
of new chicken cytokines have been described including IL-17 (Min and Lillehoj,
2002), IL-18 (Gobel et al., 2003), IL-16 (Min and Lillehoj, 2003), IL-12(Degen
et al., 2004), and Th2 type cytokines such as IL-3, IL-4, IL-13 and GM-CSF
(Avery et al., 2004), IL-10 (Rothwell et al., 2004) and IL-5 (Koskela et al.,
2004).
Many different types of chemokines and cytokines are produced following primary
and secondary infection with Eimeria (Lillehoj et al., 2003). Interferons have
been shown to have various immunomodulating effects on a wide variety of
tissues. Interferons are classified into type I (IFN-a,-b, -w, and -t) and type
II (IFN-g). The chicken gene encoding IFN-g has been cloned and its biological
function studied by many laboratories (Digby and Lowenthal, 1995; Song et al.,
1997). IFN-g production during coccidiosis was examined using a quantitative
RT-PCR (Choi et al., 1999; Yun et al., 2000c), and recently using gene
expression profiling (Min et al., 2003). After E. acervulina infection, IFN-g
mRNA expression was detected in the cecal tonsils and spleen but not in the
duodenum of SC chickens (Choi et al., 1999). In E. tenella-infected chickens,
IFN-g transcripts were detected in the spleens, cecal tonsils, and IEL following
the primary and the secondary infections with E. tenella. The marked increase in
the transcripts of IFN-g was shown at day 6 after primary infection in the cecal
tonsils. Laurent et al. (2001) recently showed that IFN-g expression in the
cecum and jejunum of White Leghorn (PA12) chickens increased over 200-fold above
the control at day 7 after primary infection with E. tenella and E. maxima. The
effects of pretreatment of chicken macrophages or fibroblasts with crude culture
supernatants containing IFN-g on E. tenella sporozoites were examined in various
in vitro systems (Lillehoj et al., 1989; Lillehoj and Choi, 1998; Dimier et al.,
1998). Multiple intramuscular injections (three times) of the supernatant of
recombinant chicken IFN-g at one day prior to, and two and four days after
infection with E. acervulina,, conferred significant protection as measured by
body weight loss and oocyst shedding in both SC and TK strains (Lillehoj and
Choi, 1998). Furthermore, E. tenella sporozoites were inhibited to undergo
intracellular development in a chicken cell line stably transfected with the
chicken IFN-g gene. Treatment of chicken cells with recombinant IFN-g inhibited
the intracellular development of E. tenella without affecting sporozoite
invasion of host cells (Lillehoj and Choi, 1998). These results provide the
first direct evidence that IFN-g exerts an inhibitory effect against Eimeria and
provides a rational basis for the use of this cytokine as a vaccine adjuvant
against coccidiosis.
Interleukin-2 (IL-2) is a potent growth factor for a variety of cell types
including T cell differentiation, B cell development and NK cell activation
(Lillehoj et al., 1992; Farner et al., 1997). The chicken IL-2 gene has been
cloned (Sundick and Gill-Dixon, 1997) and its biological function characterized
(Choi and Lillehoj, 2000; Lillehoj et al., 2001). After primary and secondary
infections with E. acervulina, a significant enhancement of IL-2 mRNA
transcripts was observed in the spleen and intestine (Choi and Lillehoj, 2000).
The protective effect of IL-2 on vaccination of chickens with the recombinant
3-1E coccidia gene was recently demonstrated by DNA vaccination (Lillehoj et
al., 2000; Min et al., 2001). Co-injection of the IL-2 gene with the 3-1E or
MIC2 antigen or gene enhanced the host response to the vaccination procedure
(Ding et al., 2005a,b; Lillehoj et al., 2004, 2005).
IL-16 was originally described as a lymphocyte chemoattractant factor
synthesized by CD8+ and CD4+ T cells and released in response to antigens,
mitogens, histamine or serotonin (Cruikshank et al., 2000). Further analysis
indicated that IL-16 is generated by B cells, mast cells, epithelial cells,
macrophages, fibroblasts, and eosinophils (Cruikshank et al., 2000). Initially,
IL-16 is produced as a 67 kDa pro-IL-16 (Baier et al., 1997) that subsequently
is cleaved by caspase-3 producing a 17 kDa secreted form of the chemokine that
aggregates to form biogically active homotetramers (Zhang et al., 1998). IL-16
is chemoattractive for CD4+ T cells, eosinophils, and monocytes through a
mechanism involving binding to CD4 (Zhang et al., 1998), although recent data
suggest that CD4 is not the only receptor for IL-16 function (Mathy et al.,
2000). In addition to its chemotactic function, IL-16 induces the expression of
the IL-2 receptor alpha chain and MHC class II molecules (Cruikshank et al.,
1987). Recently, a cDNA from an expressed sequence tags (EST) cDNA library,
prepared from intestinal IEL of Eimeria-infected chickens and containing a
full-length open reading frame (ORF) of pro-IL-16, was characterized (Min and
Lillehoj, 2004). The encoded protein, predicted to consist of 607 amino acids,
showed 86% sequence homology to duck pro-IL-16 and 49-52% homology to various
mammalian homologues. By Northen blot analysis, IL-16 transcripts were
identified in chicken lymphoid tissues, but not in the non-lymphoid tissues
examined. A recombinant protein containing the COOH-terminal 149 amino acids of
pro-IL-16 when expressed in COS-7 cells showed chemoattractant activity for
splenic lymphocytes.
IL-17 was cloned originally from an activated T cell hybridoma produced by the
fusion of a mouse cytotoxic T cell clone with a rat T cell hybridoma, and
referred to as CTLA-8 (cytotoxic T lymphocyte-associated antigen 8)(Rouvier et
al., 1993). IL-17 was produced in a mixture of glycosylated (22 kDa) and
non-glycosylated (15 kDa) forms and secreted by activated CD4+ T cells as
covalently bound homodimers (Fossiez et al., 1996). Whereas IL-17 transcripts
were restricted to activated T cells, their receptors were found to be expressed
ubiquitously in a variety of mammalian tissues and cell lines (Yao et al.,
1995,1997). Functional studies indicated that IL-17 is involved in a broad range
of cellular activities. For example, IL-17 stimulated osteoclastogenesis (Kotake
et al., 1999), granulopoiesis (Schwarzenberger, et al., 1998), and T cell
proliferation by suboptimal concentrations of phytohemagglutinin (Yao et al.,
1995). Chicken IL-17 was cloned from an EST cDNA library prepared from
intestinal IEL of Eimeria-infected chickens (Min and Lillehoj, 2002). It
contained a 507 bp ORF predicted to encode a protein of 169 amino acids with a
molecular mass of 18.9 kDa, a 27 residue NH2-terminal signal peptide, a single
potential N-linked glycosylation site, and 6 cysteine residues conserved with
mammalian IL-17s. Chicken IL-17 shared 37-46% amino acid sequence identity with
the previously described mammalian homologues and also was homologous to ORF 13
of Herpes virus saimiri (HVS 13). By Northen blot analysis, IL-17 transcripts
were identified in a reticuloendotheliosis virus-transformed chicken lymphoblast
cell line (CU205) and Con A-stimulated splenic lymphocytes, but not other
chicken cell lines or normal tissues. Conditioned medium from COS-7 cells
transfected with the chicken IL-17 cDNA induced IL-6 production by chicken
embryonic fibroblasts suggesting a functional role for the cytokine in avian
immunity.
Tumor necrosis factor (TNF)-a or b have not been well characterized in poultry
at present. However, macrophages obtained during and immediately following an
infection with E. maxima or E. tenella produced a TNF-like activity in a
biphasic fashion, whereby the first peak was associated with the pathogenesis of
disease and the second peak with the development of a protective immunity
(Byrnes et al., 1993). The production of significantly greater amounts of TNF
during day 3-6 after inoculation correlated with the appearance of the most
characteristic local and systemic pathophysiological changes induced by coccidia
(Byrnes et al., 1993). Zhang et al. (1995a,b) investigated the effect of a
TNF-like activity on the pathogenesis of coccidiosis in inbred chickens. The
TNF-like factor was produced by peripheral blood macrophages in time- and
dose-dependent manners following primary, but not secondary, E. tenella
infection. Treatment of chickens with antibody against TNF resulted in a partial
abrogation of E. tenella induced body weight loss in SC chickens.
Transforming growth factor (TGF)-b is a pleiotropic anti-inflammatory cytokine
that stimulates the repair of damaged mucosal epithelial integrity following
injury (Robinson et al., 2000). Lymphocytes that secrete TGF-b downregulate host
immune and inflammatory responses, especially in the intestinal mucosa (Strober
et al., 1997). The expression of TGF-b2, 3, and 4 was investigated using cDNA
probes and antibodies specific for the different TGF-b isoforms in chickens
(Jakowlew et al., 1997). After infection with E. acervulina, expression of
TGF-b4 mRNA which is equivalent to TGF-b1 in mammals, increased 5- to 8-fold in
intestinal IEL and 2.5-fold in spleen cells, whereas the expression of mRNA for
TGF-b2 and TGF-b3 remained constant in these cells. Administration of TGF-b to
T. gondii-infected severe combined immunodeficiency (SCID) mice resulted in an
earlier mortality and shortening of the survival time of mice given exogenous
IL-12. Administration of anti-TGF-b to SCID mice beginning 4 hr prior to
infection and every 2 days thereafter prolonged the survival time significantly.
These data demonstrated the ability of TGF-b to antagonize IL-12-induced IFN-g
production by SCID mice and suggested a role for TGF-b in the regulation of T
cell-independent resistance mechanism to T. gondii (Hunter et al., 1995).
IL-6 is a pleotropic lymphokine originally described as a T cell-derived
lymphokine that induced the maturation of B cells into antibody-producing plasma
cells (Narazaki and Kishimoto, 1994). Chicken IL-6 shows about 35% sequence
identity to human IL-6 (Schneider et al., 2001). Bacterially expressed chicken
IL-6 carrying a histidine tag in place of the signal peptide was biologically
active and induced the proliferation of the IL-6-dependent murine hybridoma cell
line 7TD1 (Schneider et al., 2001). Production of chicken IL-6-like factor
activity was detected by a murine IL-6 7TD1 bioassay in serum taken from
chickens infected with E. tenella during the course of a primary infection
(Lynagh et al., 2000). IL-6 activity was detected during the first few hours
post-infection indicating a possible role of this cytokine in the development of
acquired immunity.
In vitro production of IL-1 by macrophages obtained from Eimeria-infected
chickens was observed during and immediately following infection with E. maxima
or E. tenella (Byrnes et al., 1993). Lymphocytes from Eimeria-infected chickens
produced a higher level of IL-1 following stimulation than cells from
non-infected birds. RT-PCR measurement of IL-1 production demonstrated a 27- to
80-fold increase in the IL-1b transcript levels at day 7 after infection with E.
tenella and E. maxima (Laurent et al., 2001). The precise role of IL-1 in the
development of resistance against coccidiosis needs to be better characterized
in view of its documented role in various infections. Chemokines are important
mediators of cell migration during inflammation and in normal leukocyte
trafficking. These proteins are generally active at the nanomolar concentration
and are produced by a wide variety of cell types in response to exogeneous
irritants and endogeneous mediators such as IL-1, TNF, PDGF, and IFN-g
(Oppenheim, 1991). Chemokines are grouped into four structural families
characterized by the position of their amino-terminal cysteine residues (Zlotnik
and Yoshie, 2000). The CXC class, which has one amino acid separated by two
cysteine residues, and the CC class, which possess two consecutive cysteine
residues, is the most common chemokines. IL-8 and K60 are CXC chemokines (Kaiser
et al., 1999; Sick et al., 2000) and K203 is a CC chemokine recently cloned from
chickens (Sick et al., 2000). The K203 cDNA cloned from the chicken macrophage
cell line HD-11 stimulated with LPS, revealed 50% sequence identity to the
mammalian macrophage inflammatory protein 1b (MIP-1b) (Sick et al., 2000).
Laurent et al. (2001) showed that mRNA levels of the CC chemokines K203 and
MIP-1b were upregulated 200- and 80-fold, respectively, in the cecum in response
to E. tenella infection, and 100- and 5-fold in the jejunum in response to E.
maxima infection. Interestingly, no discernible changes were observed in the
mRNA levels of the CXC chemokines IL-8, and K60.
The role of various cytokines and chemokines needs to be better studied to
understand how these different factors interact to eliminate parasites from the
host and to develop memory responses against later infections. To accomplish
this, we generated an EST cDNA library from IEL of Eimeria-infected chickens
(Min et al., 2005) and have identified two new chicken cytokines, IL-16 (Min and
Lillehoj, 2003) and IL-17 (Min and Lillehoj 2002). Both cytokines were elevated
in Eimeria-infected tissues, they may be involved in regulating local immune
response to coccidia. Analysis of 30 different cytokines and chemokines during
coccidia infection indicated that cytokines invloved in mediating Th1 responses
seem to be dominant during early times after coccidiosis (unpublished data) as
best manifested by the proven involvement of IFN-g (Lillehoj and Choi, 1998;
Lillehoj et al., 2000; Lillehoj et al., 2004; Lowenthal et al., 1997). The role
of Th2 type cytokines should also be investigated in coccidiosis in order to
obtain better insights on protective immunity. In toxoplasma infection, mice
defective in IL-10, an anti-inflammatory cytokine, showed enhanced
susceptibility to disease suggesting the role of IL-10 in downregulating
inflammatory response to prevent host immunopathology (Gazzinelli et al., 1996).
Recent evidence indicated that IL-10 is produced during coccidiosis (Rothwell et
al., 2004), but its role in disease pathogenesis has not been investigated.
Future studies to delineate cytokine regulation of local immunity to coccidia
will lead to better understanding of host-parasite immunobiology and novel
control strategies against coccidiosis.
FUTURE DIRECTIONS IN STUDYING HOST IMMUNE
RESPONSE GENES CONTROLLING COCCIDIOSIS RESISTANCE USING DNA MICROARRAY
With increased information on poultry genomics and
the availability of several tissue-specific cDNA EST libraries, high throughput
gene expression analysis is possible to study host immune response to Eimeria
(Min et al. 2005). DNA microarray is a revolutionary tool for genomic study of
interested traits in a high throughput manner. By immobilizing thousands of DNA
sequences in individual spots on a solid phase, DNA microarray allows
simultaneous analysis of a large number of genes in a single step, thereby
identifying genes whose expression levels are altered during natural biological
processes or experimental treatments, or vary due to genetic differences (Eisen
and Brown, 1999). In one approach, the sample of interest, such as mRNA isolated
from a certain tissue, is used to synthesize cDNA labeled with a fluorescent
dyes. The labeled cDNA probe is then hybridized to the array and a
post-hybridization image is developed. The color density of individual nucleic
acid species reflects the relative amount of labeled cDNA hybridized to the DNA
immobilized at the known position on the array. By comparing different samples
tested in well-controlled conditions, changes in expression levels of individual
genes can be detected. Once genes of interest are identified, Northern blotting
or RT-PCR can be used to confirm genes with differential expression. The genes
with significant differences can be used as potential candidate genes
influencing disease susceptibility traits.
Development of chicken intestinal cDNA
microarray to investigate host immunity against coccidia: We have established a
normalized chicken intestinal cDNA library using pooled intestinal tissues from
coccidia-infected chickens (Min et al., 2005). The library was prepared from
intestinal epithelial cells and lymphocytes at 0, 1, 2, 3, and 4 days
post-infection with Eimeria. According to the normalization control, the
redundancy in this library has been reduced by 37-fold. Individual clones
(n=34,078) were randomly picked and sequenced, generating 14,409
chicken-specific ESTs that could be grouped into 9,446 unique contigs. The
majority of contigs (7,595; 80.4%) consisted of single ESTs and the remaining
1,851 contigs were composed of clusters of 2 or more overlapping/identical ESTs
(average of 3.7 ESTs per cluster contig). Most cluster contigs (1,567; 84.7%)
contained 2-4 ESTs comprising 30.6% (4,418) of the total number of ESTs. The
contig average readable sequence length was 418 bp with almost half (45.8%)
falling within the range of 400-600 bp and the average sequence length per
contig was 712 bp and 71.2% (1,318) of all contigs ranged from 0.5-1.0 kb.
Using the Basic Local Alignment Search Tool (BLAST) program to perform
sequence-similarity searches against the GenBank nucleic acid sequence database,
we analyzed the 18 cluster contigs containing 15 or more ESTs, which could be
regarded as abundant transcripts and therefore were most likely to match
previously described genes. This group of contigs constituted 1.0% of the total
number of clusters and 3.6% of the total ESTs. Ten sequences were highly
homologous to previously described chicken genes. They included NK-lysin,
apolipoprotein AIV, fatty acid binding protein, acid ribosomal phosphoprotein,
a-tublin, GAPDH, and ferritin heavy chain. Comparison of our intestine cDNA
sequence data with chicken DNA sequences in GenBank identified 125 clones which
encoded novel genes. Of these, 110 genes were unknown and the remaining genes
showed weak homologies to CC chemokine receptor type 8, cell adhesion receptor
CD36, unc-51-like kinase 1, death-associated protein kinase 2, molybdenum
cofactor sulfurase, lysosomal alpha-glucosidase precursor, FYVE and coiled-coil
domain containing 1, NADH-ubiquinone oxidoreductase 23 kDa subunit, cytochrome
b, proline rich protein 2, XE7 protein, glypican-5 precursor, nuclear receptor
ROR gamma, and LPS-induced TNF-a factor. These EST sequences from
Eimeria-stimulated intestinal IEL transcripts will be used to study global gene
expression profiling and to identify novel immune-related genes during avian
coccidiosis and in other enteric diseases of poultry.
Analysis of cell-mediated immune
response to Eimeria using T lymphocyte cDNA microarray: In view of importance of
T cell-mediated immunity in coccidiosis resistance, we initially selected 450
clones encoding immune response associated genes from a cDNA library prepared
from mitogen-activated T lymphocytes (Min et al., 2003). To assess the changes
in intestinal gene expression of chickens infected with E. acervulina or E.
maxima, IEL were collected from the duodenum or ileum at 1, 2, 3, and 4 days
following primary or secondary infection. In general, E. acervulina primary and
secondary infection resulted in up- or down-regulation of more transcripts
compared with E. maxima infection and primary infection by either parasite
induced changes in a greater number of transcripts compared with secondary
infection (Min et al., 2003). Specifically, E. acervulina and E. maxima
infection affected the levels of 99 and 51 gene transcripts respectively
following primary infection and 46 and 25 transcripts following secondary
infection. Conversely, E. acervulina and E. maxima decreased the levels 88 and
56 gene transcripts respectively following primary infection and 22 and 37
transcripts following secondary infection. When considering all time points
examined following primary or secondary infection with E. acervulina or E.
maxima, the quantities of 5 gene transcripts were commonly induced (CMRF35
leukocyte immunoglobulin-like receptor, zinc finger gene, PmbA homolog,
granulysin precursor, cyclophilin A) and 8 were repressed (alpha-actinin,
hypothetical protein F39H12.5, spleen mitotic checkpoint BUB3,
interferon-induced granylate-binding protein 2, transcription factor NF-YC
subunit, transport associated protein 3, alpha-adaptin, homobox protein HOX-D8).
Since the changes in cytokine genes following Eimeria infection is an indication
of local cell-mediated immunity (Lillehoj et al., 2003), we included in our
microarray analysis 12 cytokine genes to monitor changes in their corresponding
transcripts subsequent to Eimeria infection. The transcript levels for IL-8,
IL-15, and lymphotactin genes were increased at all time points examined
following primary or secondary infection with E. acervulina or E. maxima,
whereas IL-18 and osteopontin gene transcripts were repressed (Min et al.,
2003). With the exception of TGF-b4, changes in the levels of all cytokine
transcripts examined were similar when comparing primary infection of E.
acervulina with E. maxima. Similarly, after secondary infection, all transcript
levels except those for IL-6 and TGF-b4 showed comparable changes when comparing
E. acervulina with E. maxima.
CONCLUDING REMARKS
In view of increasing consumer’s concern about drug
residues in the food supply and impending regulations on the use of growth
promoting drugs in poultry production, the industry will eventually look for
alternative method for coccidiosis control. Problems associated with antigenic
variation of field strains and the cost of producing multiple-species live
vaccines pose limits on the current vaccination approaches. Thus, novel
strategies to control coccidiosis are needed, but this will only be realized
after a systematic and detailed analysis of host-parasite interactions at the
molecular and cellular levels are completed. In particular, fundamental
knowledge on the basic immunobiology from initial parasite invasion to
intracellular development and ultimate elimination from the host is very
limited. Increasing evidence shows the magnitude of complexity involved in host
immune responses to Eimeria. Additional basic research is needed to ascertain
the detailed immunological and physiological processes mediating protective
immunity. The need to continue seeking more effective ways to minimize the
impact of poultry coccidiosis is undisputable, but critical resources are
severely lacking making it difficult to effect timely progress. Encouraging
results obtained from recent molecular and immunological studies that show the
ability of dietary modulation on intestinal immunity and enhanced disease
resistance against enteric pathogens of economic importance need to be further
explored. One encouraging finding is the feasibility to induce protective
immunity against live parasites using recombinant vaccines delivered in ovo. The
performance of these novel vaccines will have to be verified in field
evaluations in commercial settings.
REFERENCES
Avery, S., L. Rothwell, W.G.J.
Degen, V.E.J.C. Schijns, J.R. Young, J. Kaufman, and P. Kaiser. Characterization
of the first nonmammalian T2 cytokine gene cluster: The cluster contains
functional single-copy genes for IL-3, IL-4, IL-13, and GM-CSF, a gene for IL-5
that appears to be a pseudogene, and a gene encoding another cytokine -like
transcript, KK34. J. Interf. Cytok. Res. 24:600-610. 2004.
Baier, M., N. Bannert, A. Werner, K. Lang, and R. Kurth. Molecular cloning,
sequence, expression, and processing of the interleukin 16 precursor. Proc.
Natl. Acad. Sci. USA 94:5273-5277. 1997.
Barton G. M., and R. Medzhitov. Control of adaptive immune responses by
Toll-like receptors. Curr Opin Immunol 14:380–383. 2002.
Bessay, M., Y. Le Vern, D. Kerboeuf, P. Yvore, and P. Quere. Changes in
intestinal intra-epithelial and systemic T-cell subpopulations after an Eimeria
infection in chicken: comparative study between E. acervulina and E. tenella.
Vet. Res. 27:503-514. 1996.
Breed, D. G., J. Dorrestein, and A. N. Vermeulen. Immunity to Eimeria tenella in
chickens: phenotypical and functional changes in peripheral blood T-cell
subsets. Avian Dis. 40:37-48. 1996.
Breed, D. G. J., J. Dorrestein, T. P. M. Schetters, L. V. D. Waart, and E.
Rijke. Peripheral blood lymphocytes from Eimeria tenella infected chickens
produce g-interferon after stimulation in vitro. Parasit. Immunol. 19:127-135.
1997a.
Breed, D. G. J., T. P. M. Schetters, N. A. P. Verhoeven, and A. N. Vermeulen.
Characterization of phenotype related responsiveness of peripheral blood
lymphocytes from Eimeria tenella infected chicken. Parasit. Immunol. 19:
563-569. 1997b.
Byrnes, S., R. Eaton, and M. Kogut. In vitro interleukin-1 and tumor necrosis
factor-a production by macrophages from chicken infected with Eimeria maxima or
Eimeria tenella. Int. J. Parasitol. 23:639-645. 1993.
Chai, J.Y., and H. S. Lillehoj. Isolation and functional characterization of
chicken intestinal intraepithelial lymphocytes showing natural killer cell
activity against tumor target cells. Immunology 63:111?117. 1988.
Choi, K.D., and H. S. Lillehoj. Role of chicken IL-2 on gd T-cells and Eimeria
acervulina-induced changes in intestinal IL-2 mRNA expression and gd T-cells.
Vet. Immunol. Immunopathol. 73:309-321. 2000.
Choi, K. D., H. S. Lillehoj, and D. S. Zarlenga. Changes in local IFN-gamma and
TGF-beta4 mRNA expression and intraepithelial lymphocytes following Eimeria
acervulina infection. Vet. Immunol. Immunopathol. 71:263-275. 1999.
Chung, K. S., and H. S. Lillehoj. Development and functional characterization of
monoclonal antibodies recognizing chicken lymphocytes with natural killer cell
activity. Vet. Immunol. Immunopathol. 28:351-363. 1991.
Cooper, M.D., Chen, C.H., Bucy, R.P., and C. B. Thompson. Avian T-cell ontogeny.
Adv. Immunol. 50: 87–117. 1991.
Cooper, M.D. Characterization of avian killer cells and their intracellular CD3
protein complex. European J. Immunology. 24:1685—1691. 1994.
Cruikshank, W. W., K. Lim, A. C. Theodore, J. Cook, G. Fine, P. F. Weller, and
D. M. Center. IL-16 inhibition of CD3-dependent lymphocyte activation and
proliferation. J. Immunol. 157:5240-5248. 1996.
Cruikshank, W. W., H. Kornfeld, and D. M. Center. Interleukin-16. J. Leukoc.
Biol. 67:757-766. 2000.
Dalloul, R.A., H.S. Lillehoj, T.A. Shellem, and J.A. Doerr. Enhanced mucosal
immunity against Eimeria acervulina in broilers fed a Lactobacillus-based
probiotic. Poult. Sci. 82:62-66. 2003.
Dalloul, R.A., and H. S. Lillehoj. Recent advances in immunomodulation and
vaccination strategies against coccidiosis. Avian Diseases. 49:1-8. 2005.
Degen, W.G.J., N. van Daal, H.I. van Zuilekom, J. Burnside, and V.E.J.C.
Schijns. Identification and molecular cloning of functional chicken IL-12. J.
Immunol. 172:4371-4380. 2004.
Digby, M. R., and J. W. Lowenthal. Cloning and expression of the chicken
interferon-g gene. J. Interf. Cytok. Res. 15:939-945. 1995.
Dimier, I. H., P. Quere, M. Naciri, and D. T. Bout. Inhibition of Eimeria
tenella development in vitro mediated by chicken macrophages and fibroblasts
treated with chicken cell supernatants with IFN-g activity. Avian Dis.
42:239-247. 1998.
Dimier-Poisson, I. H., Z. Soundouss, M. Naciri, D. T. Bout, and P. Quere.
Mechanisms of the Eimeria tenella growth inhibitory activity induced by
concanavalin A and reticuloendotheliosis virus supernatants with interferon-g
activity in chicken macrophages and fibroblasts. Avian Dis. 43:65-74. 1999.
Ding, X., H.S. Lillehoj, M.A. Quiroz, E. Bevensee, and E.P. Lillehoj. Protective
immunity against Eimeria acervulina following in ovo immunization with a
recombinant subunit vaccine and cytokine genes. Infect. Immun. 72: 6939-6944.
2004.
Ding, X. C., H.S. Lillehoj, R. Dalloul, W. Min, Sato, T., and E. P. Lillehoj. In
ovo Vaccination with the Eimeria tenella EtMIC2 Gene Induces Immunity Against
Coccidiosis. Vaccine. 23:3733-3740. 2005.
Eisen, M. B., and P. O. Brown. DNA arrays for analysis of gene expression.
Methods Enzymol. 303:179-205. 1999.
Farner, N. L., J. A. Hank, and P. M. Sondel. Interleukin-2: molecular and
clinical aspects. Pages 29-40 in Cytokines in Health and Diseases. D. G. Remick
and J. S. Friedland, ed. Marcel Dekker, New York, NY. 1997.
Fossiez, F., O. Djossou, P. Chomarat, L. Flores-Romo, S. Ait-Yahia, C. Maat, J.
J. Pin, P. Garrone, E. Garcia, S. Saeland, D. Blanchard, C. Gaillard, B. Das
Mahapatra, E. Rouvier, P. Golstein, J. Banchereau, and S. Lebecque. T cell
interleukin-17 induces stromal cells to produce proinflammatory and
hematopoietic cytokines. J. Exp. Med. 183:2593-2603. 1996.
Gazzinelli, R. T., Wysocha, M., Hieny, S., Scharton-Kersten, T., Cheever, A.,
Kuhn, R., Muller, W., Trinchieri, G., and A. Sher. In the absence of endogenous
IL-10, mice acutely infected with Toxoplasma gondii succumb to a lethal immune
response dependent on CD4+ T cells and accompanied by overproduction of IL-12,
IFN-g and TNF-a. Journal of Immunology 157:798-805. 1996.
Girard, F., G. Fort, P. Yvore, and P. Quere. Kinetics
of specific immunoglobulin A, M and G production in the duodenal and caecal
mucosa of chickens infected with Eimeria acervulina or Eimeria tenella. Int. J.
Parasitol. 27:803-809. 1997
Gobel, T.W.F. The T-dependent immune system. in: Davison, T.F., Morris, T.R.,
Payne, L.N. Eds. , ¨Poultry Immunology, Poultry Science Symposium Series Vol.
24, Carfax Publishing Co. Abingdon, UK pp. 31–45. 1996.
Gobel, T. W., B. Kaspers, and M. Stangassinger. NK and T cells constitute two
major, functionally distinct intestinal epithelial lymphocyte subsets in the
chicken. Int. Immunol. 13:757-762. 2001.
Gobel, T.W., K. Schneider, B. Schaerer, I. Mejri, F. Puehler, S. Weigend, P.
Staeheli, and B. Kaspers. IL-18 stimulates the proliferation and IFN-gamma
release of CD4+ T cells in the chicken: Conservation of a Th1-like system in a
nonmammalian species. J. Immunol. 171:1809-1815. 2003.
Goodman, T., and L. Lefrancois. Expression of the gamma-delta T-cell receptor on
intestinal CD8+ intraepithelial lymphocytes. Nature 333:855-858. 1988.
Guy-Grand, D., C. Griscelli, and P. Vassalli. The gut-associated lymphoid
system: nature and properties of the large dividing cells. Eur. J. Immunol.
4:435-443. 1974.
Hakim, F. T., R. T. Gazzinelli, E. Denkers, S. Hieny, G. M. Shearer, and A.
Sher. CD8+ T cells from mice vaccinated against Toxoplasma gondii are cytotoxic
for parasite-infected or antigen-pulsed host cells. J. Immunol. 147:2310-2316.
1991.
Houssaint, E., Belo, M. and N. M. LeDouarin. Investigations of cell lineage
tissue interactions in the developing bursa of Fabricius through interspecies
chimeras. Developmental Biology, 53:250-264. 1976.
Hunter, C. A., L. Bermudez, H. Beernink, W. Waegell, and J. S. Remington.
Transforming growth factor-b inhibits interleukin-12-induced production of
interferon-g by natural killer cells: a role for transforming growth factor-b in
the regulation of T cell-independent resistance to Toxoplasma gondii. Eur. J.
Immunol. 25:994-1000. 1995.
Iqbal, M., Philbin, V.J. and A. L. Smith. Expression patterns of chicken
toll-like receptor mRNA in tissues, immune cell subsets and cell lines.
Veterinary Immunology and immunopathology 104: 117-127. 2005
Isobe, T., and H. S. Lillehoj. Dexamethasone suppresses T cell-mediated immunity
and enhances disease susceptibility to Eimeria mivati infection. Vet. Immunol.
Immunopathol. 39:431-446. 1993.
Jakowlew, S. B., A. Mathias, and H. S. Lillehoj. Transforming growth factor-b
isoforms in the developing chicken intestine and spleen: increase in
transforming growth factor b4 with coccidia infection. Vet. Immunol.
Immunopathol. 55:321-339. 1997.
Jeurissen S. H. M., Vervelde, L. and E. M.Janse. Structure and function of
lymphoid tissues of the chicken. Poultry Science Reviews. 5:183-207. 1994.
Kaiser, P., S. Hughes, and N. Bumstead. The chicken 9E3/CEF4 CXC chemokine is
the avian orthologue of IL8 and maps to chicken chromosome 4 syntenic with genes
flanking the mammalian chemokine cluster. Immunogenetics 49:673-684. 1999.
Kaspers, B., H. S. Lillehoj, and E. P. Lillehoj. Chicken macrophages and
thrombocytes share a common cell surface antigen defined by a monoclonal
antibody. Vet. Immun. Immunopathol. 36:333-346. 1993.
Kim, J. K., Min, W., Lillehoj, H. S., Kim, S. W., Sohn, E. J., Song, K. D. and
Han, J. Y. Generation and characterization of recombinant scFv antibodies
detecting Eimeria acervulina surface antigens. Hybridoma 20:175-181. 2001.
Koskela, K., P. Kohonen, H. Salminen, T. Uchida, J.M. Buerstedde, and O.
Lassila. Identification of a novel cytokine-like transcript differentially
expressed in avian gammadelta T cells. Immunogenetics 55:845-854. 2004.
Kotake, S., N. Udagawa, N. Takahashi, K. Matsuzaki, K. Itoh, S. Ishiyama, S.
Saito, K. Inoue, N. Kamatani, M. T. Gillespie, T. J. Martin, and T. Suda. IL-17
in synovial fluids from patients with rheumatoid arthritis is a potent
stimulator of osteoclastogenesis. J. Clin. Invest. 103:1345-1352. 1999.
Lam, KM, and T. J. Linna. Transfer of natural resistance to Marek’s disease
(JMV) with non-immune spleen cells. I. Studies of cell population transferring
resistance. International J. Cancer, 24:662-667. 1979.
Laurent, F., R. Mancassola, S. Lacroix, R. Menezes, and M. Naciri. Analysis of
Chicken Mucosal Immune Response to Eimeria tenella and Eimeria maxima Infection
by Quantitative Reverse Transcription-PCR. Infect. Immun. 69:2527-2534. 2001.
Leslie, G. A., and L. W. Clem. Phylogeny of immunoglobulin structure and
function. 3. Immunoglobulins of the chicken. J. Exp. Med. 130:1337-1352. 1969
Levine, N. D. Taxonomy and life cycles of coccidian, In: (edited by Long, P. L.)
The Biology of the Coccidia, Univeristy Park Press, Baltimore,pp 1-33. 1982.
Lillehoj, H. S. Effects of immunosuppression on avian coccidiosis: cyclosporin A
but not hormonal bursectomy abrogates host protective immunity. Infect. Immun.
55:1616-1621. 1987
Lillehoj, H.S., and M.D. Ruff. Comparison of disease susceptibility and
subclass-specific antibody response in SC and FP chickens experimentally
inoculated with Eimeria tenella, E. acervulina, or E. maxima. Avian Dis.
31:112-119. 1987.
Lillehoj, H.S. Influence of inoculation dose, inoculation schedule, chicken age,
and host genetics on disease susceptibility and development of resistance to
Eimeria tenella infection. Avian Dis. 32:437-444. 1988.
Lillehoj, H. S., and Chai, J. Y. Comparative natural killer cell activities of
thymic, bursal, splenic, and intestinal intraepithelial lymphocytes. Dev. Comp.
Immunol. 12:629?643. 1988.
Lillehoj, H.S. Intestinal intraepithelial and splenic natural killer cell
responses to Eimerian infections in inbred chickens. Infect. Immun.
57:1879-1884. 1989
Lillehoj, H. S., S. Y. Kang, L. Keller, and M. Sevoian. Eimeria tenella and E.
acervulina: lymphokines secreted by an avian T cell Lymphoma or by
sporozoite-stimulated immune T lymphocytes protect chickens against avian
coccidiosis. Exp. Parasitol. 69:54-64. 1989.
Lillehoj, H.S. Cell-mediated immunity in parasitic and bacterial diseases. In:
Avian Cellular Immunology. J. M. Sharma, ed. CRC Press, Boca Raton, FL. pp.
155-182. 1991.
Lillehoj, H. S., and L. D. Bacon. Increase of intestinal intraepithelial
lymphocytes expressing CD8+ antigen following challenge infection with Eimeria
acervulina. Avian Dis. 35:294-301. 1991.
Lillehoj, H. S., B. Kaspers, M. C. Jenkins, and E. P. Lillehoj. Avian interferon
and interleukin-2. A review by comparison with mammalian homologues. Poul. Sci.
Rev. 4:67-85. 1992.
Lillehoj, H. S., T. Isobe, and D. Weinstock. Tissue distribution and
cross-reactivities of new monoclonal antibody detecting chicken T lymphocytes
and macrophages: in: Coudert, F. (Ed) Avian Immunology in Progress, pp37-42
(Paris, INRA Editions). 1993.
Lillehoj, H. S. Analysis of Eimeria acervulina induced-changes in the intestinal
T lymphocyte subpopulations in two chicken strains showing different levels of
susceptibility to coccidiosis. Res. Vet. Sci. 56:1-7. 1994.
Lillehoj, H. S., K.S., Sasai, and H. Matsuda. Development and characterization
of chicken-chicken B-cell hybridomas secreting monoclonal antibodies that detect
sporozoite and merozoite antigens of Eimeria. Poultry Sci. 73:1685-1693. 1994.
Lillehoj, H.S., and J.M. Trout. Avian gut-associated lymphoid tissues and
intestinal immune responses to Eimeria parasites. Clin. Microbiol. Rev.
9:349-360. 1996.
Lillehoj, H.S. Role of T lymphocytes and cytokines in coccidiosis. Int. J.
Parasitol. 28:1071-1081. 1998.
Lillehoj, H.S., and K.D. Choi. Recombinant chicken interferon-gamma-mediated
inhibition of Eimeria tenella development in vitro and reduction of oocyst
production and body weight loss following Eimeria acervulina challenge
infection. Avian Dis. 42:307-314. 1998.
Lillehoj, H.S., and E.P. Lillehoj. Avian coccidiosis. A review of acquired
intestinal immunity and vaccination strategies. Avian Dis. 44:408-425. 2000.
Lillehoj, H.S., K.D. Choi, M.C. Jenkins, V.N. Vakharia, K.D. Song, J.Y. Han, and
E.P. Lillehoj. A recombinant Eimeria protein inducing interferon-gamma
production: comparison of different gene expression systems and immunization
strategies for vaccination against coccidiosis. Avian Dis. 44:379-389. 2000.
Lillehoj, H. S., W. Min, K. D. Choi, U. S. Babu, J. Burnside, T. Miyamoto, B. M.
Rosenthal, and E. P. Lillehoj. Molecular, cellular, and functional
characterization of chicken cytokines homologous to mammalian IL-15 and IL-2.
Vet. Immunol. Immunopathol. 82:229-244. 2001.
Lillehoj, H.S., R.A. Dalloul, and W. Min. Enhancing intestinal immunity to
coccidiosis. World Poult. 19 (Coccidiosis 4):18-21. 2003.
Lillehoj, H.S., W. Min, and R.A. Dalloul. Recent progress on the cytokine
regulation of intestinal immune responses to Eimeria. Poult. Sci. 83:611-623.
2004
Lillehoj, H.S., X. Ding, R.A. Dalloul, T. Sato, A. Yasuda, and E.P. Lillehoj.
Embryo vaccination against Eimeria tenella and E. acervulina infections using
recombinant proteins and cytokine adjuvants. J. Parasitology.91:666-673.2005a.
Lillehoj, H.S., X. Ding, M.A. Quiroz, E. Bevensee, and E.P. Lillehoj. Resistance
to intestinal coccidiosis following DNA immunization with the cloned 3-1E
Eimeria gene plus IL-2, IL-15, and IFN-g. Avian Diseases.49:112-117. 2005b.
Lowenthal, J.W., J.J. York, T.E. O'Neil, S. Rhodes, S.J. Prowse, D.G. Strom, and
M.R. Digby. In vivo effects of chicken interferon-gamma during infection with
Eimeria. J. Interf. Cytok. Res. 17:551-558. 1997.
Lynagh, G. R., M. Bailey, and P. Kaiser. Interleukin-6 is produced during both
murine and avian Eimeria infections. Vet. Immunol. Immunopathol. 76:89-102.
2000.
Mast, J. and B. M., Goddeeris, B. M. Monoclonal antibodies reactive with the
chicken monocytes/macrophage lineage. In:Davison, TF., Bumstead, N. and Kaiser,
P. (Eds) Advances in Avian Immunology Research. Pp39-48 (Abingdon, Oxford,
Carfax).1995.
Mathy, N. L., N. Bannert, S. G. Norley, and R. Kurth. Cutting edge: CD4 is not
required for the functional activity of IL-16. J. Immunol. 164:4429-4432. 2000.
McDonald, V.; Shirley, M. W. and Bellatti, M. A. Eimeria maxima: characteristics
of attenuated lines obtained by selection for precocious development in the
chicken. Exp. Parasitol. 61:192-200. 1986
Miller, T. K., D. D. Bowman, and K. A. Schat. Inhibition of the in vitro
development of Eimeria tenella in chick kidney cells by immune chicken
splenocytes. Avian Dis. 38:418-427. 1994.
Min, W., J. K. Kim, H. S. Lillehoj, E. Sohn, J. Y. Han, K. D. Song, and E.
Lillehoj. Characterization of recombinant scFv antibody reactive with an apical
antigen of Eimeria acervulina. Biotechnology Letters 23:949-955. 2001.
Min, W., H.S. Lillehoj, J. Burnside, K.C. Weining, P. Staeheli, and J.J. Zhu.
Adjuvant effects of IL-1beta, IL-2, IL-8, IL-15, IFN-alpha, IFN-gamma TGF-beta4
and lymphotactin on DNA vaccination against Eimeria acervulina. Vaccine
20:267-274. 2001.
Min, W., and H.S. Lillehoj. Isolation and characterization of chicken
interleukin-17 cDNA. J. Inter. Cyto. Res. 22:1123-1128. 2002.
Min, W., H. S. Lillehoj, S. Kim, J. J. Zhu, H. Beard, N. Alkharouf, and B.F.
Matthews. Profiling local gene expression changes associated with Eimeria maxima
and Eimeria acervulina using cDNA microarray. Appl. Microbiol. Biotechnol.
62:392-399. 2003
Min, W., and H.S. Lillehoj. Identification and characterization of chicken
interleukin-16 cDNA. Dev. Comp. Immunol. 28:153-162. 2004.
Min, W., R.A. Dalloul, and H.S. Lillehoj. Application of biotechnological tools
for coccidia vaccine development. J. Vet Science. 5:279-288. 2004.
Min, W., H. S. Lillehoj, C. M. Ashwell, C. van Tassell, L. L. Matukumalli, J.
Han, and E. P. Lillehoj. EST analysis of Eimeria-activated intestinal
intra-epithelial lymphocytes in chickens. Molecular Biotechnology. 30:143-150.
2005.
Myers, T. J. and K. A. Schat. Natural killer cell activity of chicken
intraepithelial leukocytes against rota-virus infected target cells. Veterinary
Immunology and Immunopathology 26:157-170. 1990
Nguyen, S., H. S. Lillehoj, J. Donohue, A. Yokohama, and Y. Kodama, Y. Passive
Protection against Two Eimeria Species in Chickens by Orally Administered
Antibodies Specific for a Single Eimeria Protein. American Association of Avian
Pathologists, American Veterinary Medical Association, p21. 2004.
Narazaki, M., and T. Kishimoto. Interleukin-6 (IL-6). Pages 56-61: in Guidebook
to Cytokines and their Receptors. N. Nicola, ed. A Sambrook and Tooze Pub.,
Oxford University Press, Oxford, UK. 1994.
Oppenheim, J. J., C. O. Zachariae, N. Mukaida, and K. Matsushima. Properties of
the novel proinflammatory supergene "intercrine" cytokine family. Ann.
Rev. Immunol. 9:617-648. 1991.
Park, K., D. C. Park, H. Kim, B. K. Han, J. Y. Han, H. S. Lillehoj and J. K.
Kim. Development and Characterization of a Recombinant Chicken Single-Chain Fv
Detecting Eimeria acervulina sporozoite antigen. Biotechnology Letter,
Biotechnology Letter, 27:289-295. 2005.
Parvari, R., A. Avivi, F. Lentner, E. Ziv, S. Tel-Or, Y. Burstein, and I.
Schechter. Chicken immunoglobulin gamma-heavy chains: limited VH gene
repertoire, combinatorial diversification by D gene segments and evolution of
the heavy chain locus. EMBO J. 7:739-744. 1988
Pout, D. D. Villous atrophy and coccidiosis. Nature 213:306-307. 1967.
Ratcliffe, M. J. H. Development of avian B lymphocyte lineage. CRC critical
Reviews in Poultry Biology, 2: 207-234. 1989.
Robinson, P., P. C. Okhuysen, C. L. Chappell, D. E. Lewis, I. Shahab, S. Lahoti,
and A. C. White Jr. Transforming growth factor b1 is expressed in the jejunum
after experimental Cryptosporidium parvum infection in humans. Infect. Immun.
68:5405-5407. 2000.
Rose, M. E., and P. L. Long. Resistance to Eimeria infections in the chicken:
the effects of thymectomy, bursectomy, whole body irradiation and cortisone
treatment. Parasitol. 60:291-299. 1970.
Rothwell, L., R. A. Gramzinski, M. E. Rose, and P. Kaiser. Avian coccidiosis:
changes in intestinal lymphocyte populations associated with the development of
immunity to Eimeria maxima. Parasit. Immunol. 17:525-533. 1995.
Rouvier, E., M. F. Luciani, M.G. Mattei, F. Denizot, and P. Golstein. CTLA-8,
cloned from an activated T cell, bearing AU-rich messenger RNA instability
sequences, and homologous to a herpesvirus saimiri gene. J. Immunol.
150:5445-5456. 1993.
Rose, M. E. and P. Hesketh. Immunity to coccidiosis: stages of the life-cycle of
Eimeria maxima which induce, and are affected by, the response of the host.
Parasitology 73:25-57. 1976.
Rose, M. E.; A. M. Lawn, and B. J. Millard. The effect of immunity on the early
events in the life-cycle of Eimeria tenella in the caecal mucosa of the chicken.
Parasitol. 88:199-210. 1984.
Rothwell, L., J. R. Young, R. Zoorob, C.A. Whittaker, P. Hesketh, A. Archer,
A.L. Smith, and P. Kaiser. Cloning and characterization of chicken IL-10 and its
role in the immune response to Eimeria maxima. J. Immunol. 173:2675-2682. 2004.
Schneider, K., R. Klaas, B. Kaspers, and P. Staeheli. Chicken interleukin-6.
cDNA structure and biological properties. Eur. J. Biochem. 268:4200-4206. 2001.
Sasai, K., H. S. Lillehoj, Matsuda, H., and W. P. Wergin. Characterization of a
chicken monoclonal antibody that recognizes the apical complex of Eimeria
acervulina sporozoites and partially inhibits sporozoite invasion of CD8+ T
lymphocytes in vitro. J. Parasitol. 82:82-87. 1996.
Schat, K. A, B. W. Calnek, and D. Weinstock. Cultivation and characterization of
avian lymphocytes with natural killer cell activity. Avian Pathology 15:539-556.
1986.
Schwarzenberger, P., V. La Russa, A. Miller, P. Ye, W. Huang, A. Zieske, S.
Nelson, G. J. Bagby, D. Stoltz, R. L. Mynatt, M. Spriggs, and J. K. Kolls. IL-17
stimulates granulopoiesis in mice: use of an alternate, novel gene
therapy-derived method for in vivo evaluation of cytokines. J. Immunol.
161:6383-6389. 1998.
Shirley, M.W., A. Ivens, A. Gruber, A. M. B. N. Madeira, K.-L. Wan, P. H. Dear,
and F. M. Tomley. The Eimeria genome projects: a sequence of events. Trends
Parasitol. 20:199-201. 2004.
Sick, C., K. Schneider, P. Staeheli, and K. C. Weining. Novel chicken CXC and CC
chemokines. Cytokine 12:181-186. 2000.
Song, K. D., H. S. Lillehoj, K. D. Choi, D. Zarlenga, and J. Y. Han. Expression
and functional characterization of recombinant chicken interferon-g. Vet.
Immunol. Immunopathol. 58:321-333. 1997.
Sowder, J.T., C.-L.H. Chen, L. L. Ager, M. M. Chan, and M. D. Cooper. A large
subpopulation of avian T-cells express a homologue of the mammalian Tgd
receptor. J. Exp. Med. 167: 315–322. 1988.
Strober, W., B. Kelsall, I. Fuss, T. Marth, B. Ludviksson, R. Ehrhardt, and M.
Neurath. Reciprocal IFN-g and TGF-b responses regulate the occurrence of mucosal
inflammation. Immunol. Today 18:61-64. 1997.
Sundick, R. S., and C. Gill-Dixon. A cloned chicken lymphokine homologous to
both mammalian IL-2 and IL-15. J. Immunol. 159:720-725. 1997.
Trout, J. M., and H. S. Lillehoj. T lymphocyte roles during Eimeria acervulina
and Eimeria tenella infections. Vet. Immunol. Immunopathol. 53:163-172. 1996
Underhill DM, and A. Ozinsky. Phagocytosis of microbes:complexity in action.
Annu Rev Immunol 20:825–852. 2002.
van Furth, R., Z. A. Cohn, J. G. Hirsch, J. H. Humphrey, W. G. Spector, and H.
L. Langevoort. The mononuclear phagocyte system: a new classification of
macrophages, monocytes, and their precursor cells. Bulletin World Health
Organization, 46:845-852. 1972.
Vervelde, L., A. N. Verleulen, and S. H. M. Jeurissen. In situ characterization
of leucocyte subpopulations after infection with Eimeria tenella in chickens.
Parasit. Immunol. 18:247-256. 1996.
Wallach, M.; G. Pillemer, S. Yarus, A. Halabi, T. Pugatsch, and D. Mencher.
Passive immunization of chickens against Eimeria maxima infection with a
monoclonal antibody developed against a gametocyte antigen. Infect. Immun.
58:557-562. 1990.
Weining, K. C., C. Sick, B. Kaspers, and P. Staeheli. A chicken homolog of
mammalian interleukin-1 b: cDNA cloning and purification of active recombinant
protein. Eur. J. Biochem. 258:994-1000. 1998.
Weiss, W. R., S. Mellouk, R. A. Houghten, M. Sedegah, S. Kumar, M. F. Good, J.
A. Berzofsky, L. H. Miller, and S. L. Hoffman. Cytotoxic T cells recognize a
peptide from the circumsporozoite protein on malaria-infected hepatocytes. J.
Exp. Med. 171:763-773. 1990.
Wallach, M., A. Halabi, G. Pillemer, O. Sar-Shalom, D. Mencher, M. Gilad, U.
Bendheim, H. D. Danforth, and P.C. Augustine. Maternal immunization with
gametocyte antigens as a means of providing protective immunity against Eimeria
maxima in chickens. Infect. Immun. 60:2036-2039. 1992.
Weber, F. H., K.C. Genteman, M. A. LeMay, D.O. Lewis, Sr., and N.A. Evans.
Immunization of broiler chicks by in ovo injection of infective stages of
Eimeria. Poult. Sci. 83:392-399. 2004.
West, J., Anthony P., A.B. Herr, and P. J. Bjorkman. The chicken yolk sac IgY
receptor, a functional equivalent of the mammalian MHC-related Fc receptor, is a
phospholipase A2 receptor homolog. Immunity 20:601-610. 2004.
Williams, R.B. Epidemiological aspects of the use of live anticoccidial vaccines
for chickens. Int. J. Parasitol. 28:1089-1098. 1998.
Witlock, D. R., W. B. Lushbaugh, H. D. Danforth, and M. D. Ruff, Scanning
electron microscopy of the cecal mucosa in Eimeria-tenella-infected and
uninfected chickens. Avian Dis. 19:293-304. 1975.
Yao, Z., W. C. Fanslow, M. F. Seldin, A. M. Rousseau, S. L. Painter, M. R.
Comeau, J. I. Cohen, and M. K. Spriggs. Herpesvirus Saimiri encodes a new
cytokine, IL-17, which binds to a novel cytokine receptor. Immunity 3:811-821.
1995.
Yao, Z., M. K. Spriggs, J. M. Derry, L. Strockbine, L. S. Park, T. VandenBos, J.
D. Zappone, S. L. Painter, and R. J. Armitage. Molecular characterization of the
human interleukin (IL)-17 receptor. Cytokine 9:794-800. 1997.
Yun, C. H., H. S. Lillehoj, and K.D. Choi. Eimeria tenella infection induces
local gamma interferon production and intestinal lymphocyte subpopulation
changes. Infect. Immun. 68:1282-1288. 2000a.
Yun, C.H., H. S. Lillehoj, and E.P. Lillehoj. Intestinal immune responses to
coccidiosis. Dev. Comp. Immunol. 24:303-324. 2000b.
Yun, C.H., H. S. Lillehoj, J. Zhu, and W. Min. Kinetic differences in intestinal
and systemic interferon-gamma and antigen-specific antibodies in chickens
experimentally infected with Eimeria maxima. Avian Dis. 44:305-312. 2000c.
Zhang, S., H. S. Lillehoj, and M. D. Ruff. Chicken tumor necrosis-like factor.
I. In vitro production by macrophages stimulated with Eimeria tenella or
bacterial lipopolysaccharide. Poult. Sci. 74:1304-1310. 1995a.
Zhang, S., H. S. Lillehoj, and M. D. Ruff. In vivo role of tumor necrosis-like
factor in Eimeria tenella infection. Avian Dis. 39:859-866. 1995b
Zhang, Y., D. M. Center, D. M. Wu, W. W. Cruikshank, J. Yuan, D. W. Andrews, and
H. Kornfeld. Processing and activation of pro-interleukin-16 by caspase-3. J.
Biol. Chem. 273:1144-1149. 1998.
Zhao, C., T. Nguyen, L. Liu, R. E. Sacco, K. A. Brogden, and R. I. Lehrer.
Gallinacin-3, an inducible epithelial b-defensin in the chicken. Infect. Immun.
69:2684-2691.2001.
Zhu, J.J., H.S. Lillehoj, P.C. Allen, C.P. Van Tassell, T.S. Sonstegard, H.H.
Cheng, D. Pollock, M. Sadjadi, W. Min, and M.G. Emara. Mapping quantitative
trait loci associated with resistance to coccidiosis and growth. Poult. Sci.
82:9-16. 2003
Zlotnik, A., and O. Yoshie. Chemokines: a new classification system and their
role in immunity. Immunity 12:121-127. 2000.
Yilmaz, A., S. Shen, D. L. Adeison, S. Xavier, and J. Zhu. Identification and
sequence analysis of chicken Toll-like receptors. Immunogenetics 56:743-753.
2005.