Tuberculosis in lions (Panthera leo) in South Africa : evaluation of the immune response towards Mycobacterium bovis

Abstract

Bovine tuberculosis (BTB), caused by Mycobacterium bovis (M. bovis), was most likely introduced in South Africa by the first imported European cattle breeds during the 18th and 19th century. The rapid spread of BTB amongst the lion population in the Kruger National Park (KNP) raises concerns about the future of these animals, one of the main tourist attractions of the park. The main goal of the presented study was to develop better insight in the development of the immune response of lions infected with M. bovis. During a field survey in the Kruger National Park, the tuberculin skin test, based on responsiveness of cell-mediated immunity in the early stages of infection, was performed to determine disease status of the lions. However, the reading of the skin test during recapture could only be performed for 29 lions, confirming the need of a test that does not need recapture of the animals. Of these 29 animals, 23 animals were classified as positive animals. To find proof of lions shedding M. bovis six tracheal flushings were performed, smears of the flushings were coloured with Ziehl- Neelsen, checked for mycobacteria with microscopy and subsequently cultured. In three smears, acid-fast bacteria were found, an indication of presence of M. bovis, but this could not be confirmed by culture. A multi-species IFN-γ ELISA was optimized for lions, but the ELISA could not be completed due to difficulties that were experienced in the process of optimization of the test. Sequencing of the lion IFN-γ gene has been performed, which may be a first step in development of a lion-specific IFN-γ ELISA. The similarity between the sequences of the lion, cheetah and domestic cat suggests that this IFN-γ ELISA may also be used as a feline-specific IFN-γ ELISA. To determine BTB infection status in lions in later stages of the disease, indirect ELISA’s were developed for the recombinant antigens CFP10 and DiaSer3. Test characteristics of these two iELISA’s and the MPB70 and MPB83 iELISA’s, showed that, with a high specificity, the iELISA’s for these four antigens have low sensitivities –a difficulty of serology-based tests that is generally acknowledged and that might be avoided by using multiple recombinant antigens together. The four iELISA’s where subsequently used to test the KNP lion serum samples collected during the field surveys, classifying a low number of samples as positive. With the development of a Real-Time reverse transcriptase PCR for lion cytokines IFN-γ, TNF-α, IL-4, and IL-10 we introduced new methods for BTB diagnostics in Abstract Excellent Track research report - Tuberculosis in lions in South Africa 112 lions. This RT qPCR has been used on 29 lions of the KNP with confirmed skin test results. For IFN-γ, TNF-α, and IL-10 increases of gene expression were seen in skin positive animals, of which the most pronounced was the increase of gene expression for the IFN-γ gene. IL-4 was less expressed in skin test positive animals than in skin test negative animals. In four out of six negative skin test lions the Ct for IL-4 was beyond detection level however. If more samples and data become available in the future, the test can be validated and the technique could be used as well to classify stage of disease, i.e. Th1 or Th2 stage animals. With the data of this study, no correlation can be shown between LLV and BTB infection. An average of almost 50% of the KNP lions is infected with LLV, but the prevalence of LLV infection in lions in the north and the south of the KNP is almost equal, and the prevalence of BTB in lentivirus-infected lions is also similar to the prevalence of BTB in non-lentivirus-infected lions. Low sample sizes and to a certain extend variable sample quality due to field conditions, of the samples used to perform the various tests –some of which need further development still or have low sensitivity– complicates analysis of test outcomes and interpretation of results in view of progression of BTB in lions. Without an easy, sensitive and specific test it is difficult to determine M. bovis infection status in lions, which makes it difficult to validate new diagnostic tests, and the preliminary results of the various diagnostic tests in this study could change if larger data sets become available. This emphasizes the need for development of a useful diagnostic test for M. bovis infection in lions and with this study, progress to achieve that goal has been made.