Studies on cytokines and nitric oxide in relation to Cowdria Ruminantium infection
This thesis describes studies on the pathogenesis of heartwater by investigating the effects of cytokines and nitric oxide (NO) in experimental Cowdria ruminantium infections in in vitro and in vivo models. The latter were carried out in sheep and mice. In sheep infections, there was increased levels of antioxidation enzymes, glutathione peroxidase and superoxide dismutase with the highest levels coinciding with the period of the clinical disease. C. ruminantium was also found to induce increased levels of nitrite, indicative of NO, in plasma where the nitrite levels were found to have an earlier and higher increase in sheep with prior exposure to C. ruminantium antigens as compared to naive sheep infections. Mean interferon gamma (IFN-y) levels in plasma were found to be increased after infection just prior to or at time of the febrile reaction, with higher levels being recorded in the sheep with prior exposure to C. ruminantium antigens than in the naive animals. There was a gradual increase in mean interleukin 8 (IL-8) and tumor necrosis factor alpha (TNF-a.) levels after infection and the highest levels coincided with the febrile reaction. Higher levels of IL-8 were recorded in the primary infections than in the sheep with prior exposure to C. ruminantium antigens. To determine the source of the NO detected in in vivo studies, in vitro studies of C. ruminantium infections of bovine pulmonary endothelial cells (BPEC) were conducted and NO was found to be induced by C. ruminantium infections. The NO production was found to be enhanced by bovine recombinant IFN-y and was inhibited by nitric oxide inducible synthase inhibitor, LNMMA. Induction of apoptotic cell death was seen in IFN- y treated cells with the infected cells showing higher apoptotic cell death than the uninfected cells. Treatment of elementary bodies with NO was found to reduce both infectivity and viability of C. ruminantium. This indicates that NO is released during endothelial infection and has an effect upon infectivity and viability of the organism. The importance of interferons in the control of C. ruminantium infections was further investigated in an in vivo model using mice deficient in IFN- Y and IFN-a/b receptors. Infection of these mice resulted in a more severe disease characterised by a shorter incubation period, higher morbidity and mortality rates in relation to time, with more pronounced pathology characterised by increased amount of oedema fluid, infiltration of inflammatory cells and higher lung and brain infection rate as compared to control mice. Electron micrographs from infected mice 7 days post-infection indicated that organisms had developed to later developmental stages in the mice deficient in IFN- Y receptors, where as earlier developmental forms were seen in controls, with mixed stages being seen in the IFN-a/13 receptor deficient mice. Studies then looked at the time course of cytokine production in relation to C. ruminantium infection in mice. Infection of mice with C. ruminantium induced transient productions of IFN-y and the biologically active 70 kDa heterodimeric form ofIL-l2, (IL-l2p70) and a corresponding initial reduction in IL-4 and IL-lO levels. ILl2p70 levels were highest on days 2-4 while IFN-y levels were highest on day 6, just prior to disease onset followed by a dramatic reduction which also coincided with the clinical onset on day 8. Levels of IL-4 and IL-lO were reduced immediately after C. ruminantium infection with the lowest levels coinciding with the highest IFN-y levels on day 6 post-infection. This study showed induction of a transient Th 1 immune response which was not sustained, possibly resulting in the fatal outcome of infection of nnce. In contrast, inoculations of mice with murine recombinant interleukin 12 (MrIL- 12) were found to have immunomodulatory effects on C. ruminantium infections in mice and to protect a significant number of mice. The highest protection was afforded by mice inoculated with MrIL-12 from beginning of infection with booster doses every 2 days; 20% of these did not show any clinical signs and were immune to challenge infection with an overall recovery rate of 45% as compared to the control. There was reduction of mortality and morbidity rates in mice inoculated with MrIL-12 before or at time of infection but giving IL-12 after disease onset was found not to offer any protection to the animals against C. ruminantium infection. The increased resistance or reduced severity of infection coincided with sustained increase in IFN- y, nitrite levels and in IgG2a immunoglobulins and a reduction in IL-4 and IL-l O. This study showed that the course taken by C. ruminantium infection in both in vivo and in vitro models is dependent on the cytokine and antibody milieu in plasma and possibly tissues prior to or during infection. Involvement of NO in C. ruminantium infections was suggested in this study. Infection in mice was characterised by a transient high IFN-y response at about day 6 which was not sustained. It is likely that this response controls C. ruminantium to some extent because mice deficient in IFN-y receptors died earlier in the disease with higher infection levels as compared to the control. Sustained release oflFN-y occurred in mice treated with MrIL-12 and provided protection in a significant proportion. The failure to sustain IFN -y in mice may be related to a lack of sustainable IL-12 production. The importance of a Th 1 type immune response was, therefore, underlined and the possible roles for IL-12 and IFN- y with involvement of nitric oxide in the killing of the infectious agent suggested. The study showed for the first time the possible roles of nitric oxide in the killing immune response and pathology of the disease and the importance of availability of IL-12 early in infection in the development of protective . . . nnmune response m mice.