The following technical article is related to the event:
XXII Latin American Poultry Congress 2011

Immune mechanisms in avian Coccidiosis

Published on: 10/20/2011
Author/s : G Pascual*, M De Marzi, H Barrios, M De Franceschi - Universidad Nacional de Luján
Summary

This paper is a review that summarizes current knowledge about the immunological mechanisms against avian coccidiosis. Coccidiosis is an intestinal infection caused by intracellular parasites, belonging to the genus Eimeria, considered one of the most economically important diseases of poultry. Chickens infected with Eimeria produce specific antibodies; both in circulation and in the mucous secretions, but the humoral immunity plays a minor role in protection against the disease. Sporozoites within the parasite''''s life cycle, are critical for the development of the immune response. Eimeria sp induces protective immunity against a subsequent challenge, the protection is specific to the species that produced it, but has no cross-protection among different species. During an infection in immune chicken, leukocytes infiltrate the lamina propria; sporozoites are found in or near LT CD8+, CD4+ and macrophages. This indicates that more than one cell type is involved in inhibiting the development of sporozoites in the lamina propria of immune chicken. The activation of macrophages is one of the first events induced by different species of Eimeria, and those would be the key effector inflammatory cells at the site of the infection. This knowledge has led researchers to search for antigens that can stimulate cross-protection. Today, subunit vaccines (parasite proteins) or DNA vaccines inoculated simultaneously with cytokine-expressing plasmids or co-stimulating molecules are being studied, in order to obtain the desired protection.
Keywords: Birds, Eimeria, Coccidiosis, Immunity, Vaccines.

Introduction

Coccidiosis is an intestinal infection caused by protozoa, intracellular parasites of the Eimeria genus present in the whole world, that affects all productive values of commercial birds and it is considered as a disease of great economic importance.

For many years, the preventive use of anticoccidians in chicken has been the principal means of control.  However, the presence of resistant strains, plus the regulations by the European Community over the use of additives in animal feeds, creates the need to develop new strategies for the prevention of avian coccidiosis.

Research efforts focus on finding alternate control methods, as greater knowledge of the parasite biology and the host response is gained.   

In spite of the acquired immunity of birds after a natural infection with Eimeria, the complex life cycle and the complicated immune response to the parasite, have made the development of efficacious vaccines a difficult task.  

Important progress has been made in the definition of the parasite antigens with a potential use for vaccines, in the definition of the Eimeria genome, in the understanding of the immunology of coccidian infections and in the practical applications of live vaccines.

Cell-mediated immunity

Since the Eimeria invasion takes place through the intestinal mucosa, the mucosa-associated lymphoid tissue (MALT) plays a decisive role as defense barrier.  

Cell-mediated immunity, mainly by intraepithelial lymphocytes (IEL) and lymphocytes of the lamina propria, represents the main component of protective immunity against avian coccidiosis (Lillehoj et al., 2004).

The CD4+ TC and IEL are involved in the response against a primary Eimeria infection (Lillehoj, 1998) and CD8+ TC and IFN-γ were identified as components of the protective immune response against the infection (Lillehoj et al., 2007).

By analyzing the immune response induced by E. acervulina, E. maxima and E. tenella, main species that affect the production, similarities and differences were observed.

Sporozoites seem to be, within the parasite´s life cycle, the most important elements for the development of the immune response. The immune system can inhibit parasite development during the time in which parasites look for an entry site through the epithelium, or when they lodge in the epithelium of the intestinal villi among the IEL or during its passage through the lamina propria towards the crypts, where the asexual phase is developed (Jeurissen, S., et al., 1996). Sporozoites are carried by the IEL (Laurent et al., 2001; Lawn et al., 1988; Rose et al., 1984).

Once sporozoites get to the lamina propria, their distribution within the villi varies depending on the immunization status of the chicken.  In non-immunized birds sporozoites may get to the crypt epithelium and develop. In immunized birds, fewer sporozoites get to the crypts and the creation of schizonts is inhibited by the lamina propria leukocytes (Jeurissen et al., 1996).

The reduction in the number of developing parasites is higher due to the failure of sporozoite transfer from IEL to the crypt''''s enterocytes (Rose et al., 1984).

During the first day of a primary infection with E. tenella, macrophages, granulocytes and lymphocytes massively infiltrate the lamina propria, but in immunized birds the infiltration is faster than in non-immunized birds. (Dalloul, R., et al, 2007).

Eimeria induces immune protection against a subsequent challenge; such protection is species-specific with no cross protection (Dalloul, R., et al, 2007).

E.maxima is characterized by immunogenicity. Some few oocysts induce complete immune protection upon homologous challenges. In contrast, many more E. acervulina and E. tenella oocysts are needed to induce similar protection levels (Dalloul et al., 2007; Lillehoj et al., 2007).

In the cecum of non-immunized birds, the number of LCD4+ TC increases significantly two days after the first infection with E. tenella, while in immune birds, CD4+ and CD8+ infiltrate the lamina propria (Lillehoj et al., 2004). Some sporozoites were detected inside or near the macrophages in the lamina propria of immunized birds, but many more were found inside or near the T-cell, especially CD8+. These results indicate that there is more than one cell type involved in the inhibition of the sporozoites development in the lamina propria of immune birds. CD8+ TC could inhibit sporozoite development directly or indirectly through the production of cytokines (Jeurissen et al., 1996).

Also, upon an infection with E. maxima, CD4+ and CD8+ participate in the immune response, yet CD8+ TC plays the greatest role. By the same token, after an E. acervulina challenge, a significant increase in the CD4+ and CD8+ ratio was observed; with a sharp increase of CD8+ in the duodenum after a second infection (Girard et al., 1997). The role of CD4+ in coccidiosis could involve the production of soluble cytokines such as IFN-γ.

Macrophages activation is one of the first events induced by Eimeria sp.

Cytokine and chemokine expressions

IL-1 proinflammatory cytokine was highly induced by the three species. It regulates the production of other chemokines and cytokines such as osteopontin (OPN), by magnifying the immune response. Osteopontin enhances the Th1 response and inhibits Th2. An early expression of Th1 cytokines is critical in a protective response against an intracellular infection.  Therefore, the factors that stimulate Th1 cytokines and inhibit Th2 would act as modulators of cell-mediated immunity.

However, Lillehoj et al. (2007) by using quantitative RT-PCR (reverse transcription-PCR), observed that many cytokines involved in Th1 and Th2 were induced simultaneously after the infection, reflecting the complexity of the immune response induced by Eimeria in the intestine.

IFN-γ rapidly appears upon an E. tenella infection, but not so upon infections of other species. Levels of IFN-γ increase in response to E. tenella infections and inhibit its development in vitro (Dalloul et al., 2007; Lillehoj et al., 2007).

Other cytokines and chemokines show different expressions to the Eimeria species. Thus IL-18, a type of Th1 cytokine, was induced after 18 hours with E. acervulina and E. tenella and after 48 hours in response to E. maxima (Dalloul et al., 2007).

The CC-chemokines K203 and MIP-1β, involved in the macrophage recruitment (Dalloul et al., 2007; Lillehoj et al., 2007), expressed during an E. tenella infection, which suggests that these molecules play a role in the immune response of the intestinal mucosa  (Laurent et al., 2001).

Also, in the immune response to coccidiosis, a peptide released by T-cells and NK cells with anti-parasitic activity was identified as NK-lysin. High levels of NK-lysin transcription were identified in IEL and in splenic cells and low levels in thyme lymphocytes (Lillehoj, 2007).

IFN-γ induces the expression of NOS (nitric oxide synthetase) in various types of cells, being more important during an E. tenella infection. This could contribute to the bleeding observed frequently after an E. tenella infection (Laurent et al., 2001).

With the use of RT-PCR, an increase in the expression of IFN-γ mRNA was observed in lymphocytes of cecal tonsils of E. tenella infected birds (Lillehoj et al., 2004).

Role of Ig in the protection against coccidiosis

Lee et al. (2009) demonstrated that feeding chicks with a hyper immune egg yolk, containing IgY (IgG) against E. tenella and E. maxima, provides them with a significant protection against coccidiosis.

The production of IgA increases significantly, more than IgM and IgG, after immunization or infection by E. tenella (Zigterman et al., 1993). It also inhibits the sporozoite invasion and development in cell cultures.  

A primary infection of E. acervulina triggers a significant production of antibodies, IgM appears in the first week post inoculation and IgA and IgG appear during the second week. The IgA response had a short duration and the specific IgM response ped in the following weeks. On the second week of the infection, specific IgG is produced in the duodenum and cecum of birds infected with E. acervulina or E. tenella, respectively; while the production of specific IgM is high during the first week of the infection and the IgA response is high during the second week (Girard et al., 1997).

Development of vaccines against avian coccidiosis

Today coccidiosis may be controlled by vaccines made with mixes of live attenuated oocysts from various Eimeria species.

  • There is a commercial vaccine made up of isolated and purified antigens which are subunits of the E. maxima gametocytes cellular wall. Immunized birds would be capable of producing antibodies against all Eimeria species on birds, including cross immunization. However, this could be arguable due to the importance of the cellular immune response in coccidiosis.

However, Wallach et al. (2008) confirm that the vaccine used to stimulate the production and transfer of antibodies among breeder hens and their chicks, would be safe and effective.  

Initially, the selection of a candidate antigen for the vaccine, was carried out by using antibodies from immune birds; today, the search is for an antigen capable of stimulating the T-cell specific response.  

Both for subunit or DNA vaccines, the effort is focused on finding an antigen capable of producing cross immunization.  

The new generation of recombinant DNA and subunit vaccines, especially when they include IL-2 and IFN-γ, are promising in the experimental control of the disease. 

Conclusions

The role of antibodies in the protection against coccidiosis is controversial.

More than one cellular type is involved in the inhibition of sporozoite development in the lamina propria of immune birds.  

Literature shows many discrepancies among research works. This may be due to the various techniques used, oocysts doses and Eimeria species utilized, types of chicken, genetics and age, that often times are not mentioned.

Nevertheless, new knowledge gave rise to a new generation of recombinant DNA and subunit vaccines in order to achieve cross immunization against all Eimeria species or, at least, the most prevalent ones.  

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Girard F, Fort G, Yvoré P, Quéré. 1997. Kinetics of Specific Immuoglobulin A, M and G Production in the Duodenal and Caecal Mucosa of Chickens Infected with Eimeria acervulina or Eimeria tenella. Internacional Journal for Parasitology 27(7):803-809.

Jeurissen S, Janse E, Vermeulen A, Vervelde L.1996. Eimeria tenella infections in chickens: spects of host-parasite: interaction. Veterinary Immunology and Immunopathology 54:231-238.

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Zigterman G, van de Ven W, van Geffen C, Loeffen A, Panhuijzen J, Rijke E, Vermeulen A. 1993. Detection of mucosal immune responses in chickens after immunization or infection. Veterinary Immunology and Immunopathology 36(3):281-291.

 

 

 
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