Vaccination against Eimeria tenella infection using a fraction of E. tenella sporozoites selected by the capacity to activate T cells.

At 8 days after a primary Eimeria tenella infection, a subset of T cells, of which the protective role is as yet unclear, circulates in the peripheral blood. In order to investigate this, the in vitro cellular responsiveness of these peripheral blood lymphocytes has been used as selection criterion to identify potentially protective E. tenella sporozoite antigens. The hydrophilic protein phase of purified E. tenella sporozoite homogenates obtained by Triton X-114 extraction was fractionated using preparative gel electrophoresis. Nine fractions, separated according to different molecular weight, were tested for their ability to stimulate T-cell responses. Both the proliferation of peripheral blood lymphocytes and the macrophage activating activity released in the culture supernatants were measured. On the basis of this responsiveness, four fractions were selected and used to vaccinate chickens. All vaccine preparations induced strong T-cell responses. One fraction immunised chickens against subsequent challenge infection, in that the caecal lesion scores were significantly lower as compared with that of the unvaccinated controls. This fraction contained hydrophilic polypeptides with a molecular mass that ranged from 26 to 30 kDa.

Peripheral blood lymphocytes from Eimeria tenella infected chickens produce gamma-interferon after stimulation in vitro.

Protective immunity to infection by Eimeria parasites has been demonstrated to be dependent on T-cell mediated immune responses and may be associated with the release of cytokines. We have previously shown that the proportion of CD8-expressing T-cells in the peripheral blood of chicken increases transiently at 8 days after a primary infection with Eimeria tenella oocysts. The increase in the CD8+ population coincided with an increased proliferative lymphocyte response upon stimulation with E. tenella sporozoite antigen in vitro. In this study, we further investigated the functional activity of these peripheral blood leucocytes (PBL) by determining both the potential to proliferative and to produce IFN upon stimulation with E. tenella sporozoite antigens and mitogens. Enhanced proliferative responses to parasite antigen were accompanied by reduced responses to T-cell mitogens around 1 week of infection. The IFN activity in the supernatants of the stimulated PBL was measured by the ability to inhibit Semliki Forest Virus (SFV) replication in chicken embryo fibroblasts (CEF) and to activate macrophages, as measured by nitric oxide production. At eight days after infection the highest levels of virus inhibition and NO-production were detected upon stimulation with both E. tenella sporozoite antigen and mitogen. A strong correlation between the individual data of the two methods was found at this timepoint indicating that the produced cytokine was indeed IFN-gamma. These results suggest that around eight days after a primary E. tenella infection a parasite specific T-cell subset with the capacity of produce IFN(-gamma) is circulating which would be involved in the induction of protective immunity against Eimeria tenella.

Characterization of phenotype related responsiveness of peripheral blood lymphocytes from Eimeria tenella infected chickens.

We have previously shown that the proportion of CD8-expressing T cells (CD8bright+ and CD4+ CD8dim+ cells) in the peripheral blood of chickens increases around 8 days after a primary infection with Eimeria tenella oocysts. The increase in the CD8+ eight cells coincides with enhanced responses after in vitro stimulation with parasite antigen. In the study described here, the responsiveness of these day 8 PBL was further characterized by determining their capacity to proliferate and to produce cytokine (IFN-gamma) upon stimulation with E. tenella sporozoite antigen, or non-specific stimuli like T cell growth factor (TCGF) and anti-CD3 monoclonal antibody (MoAb). Comparing the responsiveness of infected responder (day 8) and control chickens, non-specific triggering induced cytokine production in cells from infected animals and proliferation in cells from control animals. When triggered with E. tenella sporozoite antigen, lymphocytes from infected chickens responded with proliferation and cytokine production, in contrast to lymphocytes from control animals that did not respond. The phenotype of the lymphocytes involved in the parasite-specific proliferation and cytokine production, was characterized in a blocking assay using MoAb directed against the CD4 or CD8 molecule. The results suggest that CD8bright+ as well as CD4+ (CD4+ CD8dim+ and possible CD4+, single positive) lymphocytes are responsible for the IFN-gamma production measured after stimulation with parasite antigen, whereas the specific proliferative response appears to be caused by CD4+ (CD4+ CD8dim+ and possibly CD4+ single positive) lymphocytes. We speculate that the CD8bright+ cells, present in the circulation around 8 days after a primary E. tenella infection, act as effector cells in protective immune responses, whereas CD4+ cells play an important helper function in these responses.

Differential binding of two monoclonal antibodies directed against the chicken CD8 alpha molecule.

Two murine monoclonal antibodies (mAb), CT8 and AV14, have been shown to recognise the avian homologue of the mammalian CD8 alpha molecule. In previous flow cytometry studies we could discriminate two subpopulations of CD8+ T cells, expressing either a high level (CD8Bright+) or a low level (CD8Dim+) of CD8 molecules. The staining patterns of mAb AV14 and mAb CT8 were not always identical for individual chickens. In this study the discrepancy in the reactivity of these mAb was examined, using outbred White Leghorn chickens as well as (B14B14)-MHC inbred Wellcome chickens. The results show that mAb AV14 and mAb CT8 recognise different epitopes on the chicken CD8 alpha molecule. The CD8Bright+ cells appeared to express the CD8 alpha beta heterodimer and the CD8Dim+ cells the CD8 alpha alpha homodimer. Conformational differences between the alpha beta heterodimer and the alpha alpha homodimer could account for the differences in binding characteristics found for the two mAb. The existence of a polymorphism of the CD8 alpha molecule in outbred White Leghorn chickens was suggested by the failure of peripheral blood leucocytes from some chickens to react with mAb AV14. This heterogeneity was not observed in the Wellcome line.

Immunity to eimeria tenella in chickens: phenotypical and functional changes in peripheral blood T-cell subsets.

The changes in peripheral blood leukocyte (PBL) T-cell subsets following Eimeria tenella infection in outbred white leghorn chickens were studied, using a panel of murine monoclonal antibodies specific for the chicken homologues of the mammalian CD3, CD8, and CD4 markers on day-to-day samples of PBLs. Both flow cytometric analysis (FCA) and immunofluorescence microscopy with fixed cells on slides were used as read-out systems. The changes in the composition of the T-cell subsets measured with both techniques were similar. At 8 days post primary infection, a sharp transitory increase in the proportion of CD8-expressing cells was found. With FCA, CD8-expressing cells could be discriminated in CD8(Dim+) and CD8(Bright+) populations, which have not been described before. The proportion of CD4-expressing cells was decreased at days 9-10 after primary infection, which coincided with a less marked decrease in CD3-expressing cells. Such effects were not seen after secondary infection. When PBLs collected at day 8 post primary infection were stimulated in vitro with E. tenella sporozoite antigen, the response was higher than that in uninfected control chickens. The effects we observed coincide with the onset of recovery from primary infection. We speculate that the increase in CD8-expressing PBLs is the result of stimulation and expansion of a specific subset involved in the induction of protective immunity against Eimeria tenella.