Immune profiles in sheep following experimental infection with Mycobacterium paratuberculosis
Johne's disease in ruminants is caused by the pathogenic bacterium Mycobacterium avium subspecies paratuberculosis. An experimental infection model in sheep was developed as a prelude to the testing of new vaccines and the development of improved diagnostic assays for Johne's disease. The final challenge model developed used four doses of 10⁹ viable organisms given at two to three day intervals. Gross and microscopic lesions were found in a high proportion of sheep (80%) at ten months post challenge. There was considerable variation in immune responses from animals challenged with different strains of M. paratuberculosis. Sheep challenged with a low passage laboratory culture of strain (W) M. paratuberculosis, produced strong lymphocyte transformation responses and Interferon gamma (IFN-γ) production at two months post challenge. Subsequent necropsy and culture from intestinal tissues showed only a low level of infection (25%). In comparison a primary tissue isolate of M. paratuberculosis (JD3) resulted in higher (60-90%) infection rates in orally challenged animals. The immune profile from these animals showed very little reactivity for the first three months post challenge, after which IFN-γ production could be detected. Antibody production and lymphocyte transformation response could not be measured until at least seven months post challenge. Sheep challenged with the primary tissue isolate instilled directly into the tonsil resulted in equivalent levels of Johne's disease to those obtained with oral challenge. However, intratonsillar challenge resulted in higher levels of immune reactivity than oral challenge. The proprietary Johne's vaccines; NeoparsecTM and GudairTM and an Aqueous vaccine were tested in sheep. The immunological reactions of the sheep to these vaccines showed some variations between the two separate studies, with the NeoparasecTM and GudairTM vaccines evoking high levels of CMI and humoral reactivity within two months of vaccination. Detailed immunological examination of gut associated lymphoid tissues were carried out on subgroups of animals that were either vaccinated or non-vaccinated and went on to develop disease or were immune to experimental challenge. The results showed that the diseased animals examined had multibacillary lesions and strong CMI and humoral responses. There were decreased proportions of CD4⁺, CD8⁺ and CD25⁺ T cells in peripheral blood and gut associated lymphatics of diseased animals compared with the immune or unchallenged subgroups. Profiles from the immune subgroups showed a stronger lymphocyte transformation response than case matched diseased animals. Tissues from immune animals showed increased proportions of B cells above those seen in diseased or unchallenged animals. This study has resulted in the development of a robust experimental sheep model in which Johne's disease occurs in a high proportion of challenged animals. Critical time points for the establishment of infection or disease have been determined. It can be used in the future to evaluate protective efficacy of vaccines or to critically chart immunological profiles that are associated with infection, disease or protective immunity. Considerable research is needed to develop improved diagnostic tests to identify patterns of immunity during the early stages of infection or while the animal has subclinical disease.
Advisor: Langley, John; Williams, Sheila; Cooke, Ken
Degree Name: Doctor of Philosophy
Degree Discipline: Department of Microbiology & Immunology
Publisher: University of Otago
Keywords: sheep; immunology; tuberculosis in animals; immunological aspects; Mycobacterium paratuberculosis
Research Type: Thesis