Trypanosoma (Trypanozoon) Evansi (Steel, 1885): immune responses and immunosuppression during experimental infection in sheep

This thesis describes studies on immune responses and immunosuppression during experimental Trypanosoma evansi infections in sheep. The studies were initiated by investigations into: 1) routine parasitology and haematology, 2) cell population dynamics in blood and. 3) assessment of parasite-specific antibody responses. Investigations into the role of T. evansi in suppression of immune responses to heterologous antigens were carried out using Pasteurella haemolytica vaccine as a model antigen and involved: 1) assessment of local inflammation, and measurement of skin thickness, at the site of vaccination. 2) cell phenotype dynamics in blood and in efferent lymph draining the site of vaccination, 3) assay of antiPasteurella antibodies, 4) cellular responses to T and B cell mitogens, Pasteurella and homologous tiypanosomal antigens in vitro and, 5) cell depletion experiments to try and identify the cell phenotype(s) responsible for immunosuppression.T. evansi infections in sheep produced scanty and frequently cryptic parasitaemia, ending in selfcure in some cases. There was marked lymphocytic leucocytosis from day 22 post infection (p.i.). Indirect immunofluorescence and flow cytometry revealed decreases in the proportions of CD5+, CD4+, CD8+ and SBU-T19"1" (yS) T cells, which were paralleled by increases in the slg+, CD45R+, CD 1+ cells and cells expressing the MHC Class II antigen. These changes were relatively minor in the selfcured sheep except that there was a greater decrease in CD8+ than CD4+ cells resulting in increased CD4:CD8 ratio. Trypanosome-specific IgG and IgM antibodies were detected in ail infected animals by enzyme immunoassays. However, the levels of these antibodies were greater in the selfcured animals.T. evansi infection actively suppressed the local inflammation and skin thickness normally induced in uninfected control sheep by subcutaneous inoculation of Pasteurella vaccine. Moreover, early systemic mobilisation of neutrophils as seen in uninfected controls was suppressed. Analysis of cell dynamics in the blood of infected, vaccinated sheep revealed that vaccination did not reverse the changes in the proportions of T cell subsets and B cells which was induced by T. evansi in the infected sheep. Two-colour flow cytometry also showed that T. evansi induced increases of up to 70 percent in B cells expressing the CD5 antigen (CD5+ B cells) which persisted after vaccination but was minimal in the selfcured animals. Vaccination caused little alteration in the normal expression of these lymphocyte subsets in the uninfected control sheep. In efferent lymph draining vaccination sites there were no major changes in the proportion of the T cell subsets in either group of sheep. However, two-colour flow cytometry revealed that infection was accompanied by the appearance of CD4+CD8+ (double positive, DP) cells. These increased in proportion upon vaccination, with up to 30 and 60 percent of CD4+ and CD8+ cells respectively being DP. Moreover, between 40 to 90 percent of B cells were CD5+. In contrast, in uninfected control sheep, the changes resulting in the co-expression of various T cell subsets and CD5 antigen on B cells were observed to a very limited extent. Anti-Pasteurella antibodies of the isotypes IgGl, IgG2 and IgM were produced in all immunised sheep, including those infected with T. evansi However, infection of sheep prior to vaccination resulted in the suppression of IgG antibody responses and production of non-specific IgM antibodies in that substantial level of this antibody was detected in the scrum collected from these animals prior to vaccine administrations. In contrast, the selfcurcd sheep responded to secondary vaccination and produced IgG antibodies at levels similar to those seen in vaccinated, uninfected control sheep.In vitro cell culture systems revealed that the proliferative responses of lymphocytes from infected sheep were depressed when stimulated with T or B cell mitogens, Concanavalin A (Con A) or bacterial lipopofysaccharide (LPS) respectively. Active infection also suppressed the responses of cells to specific stimulation with Pasteurella and homologous trypanosomal antigens. Treatment with trypanocidal drug restored these proliferative responses. These suppressive effects were not observed with cells collected from uninfected control sheep, even when 5 x 10^ trypanosomes ml" ^ or ultrasonicated trypanosomal antigens were added to the cultures. Cell depletion prior to culture showed that although CD8+ T cells and cells of the monocyte/macrophage lineage play a significant role in the suppression of lymphoproliferative responses, the latter cell type seems to play the more predominant role. Removal of either of these cell types restored the responsiveness of the cells to mitogcnic or specific antigenic stimulus to varying degrees. The responses were enhanced further by simultaneous removal of both cell types from the cell population