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.
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.
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.
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