Tuberculosis in lions (Panthera leo) in South Africa : evaluation of the immune response towards Mycobacterium bovis
Maas, Miriam; University of Utrecht. Faculty of Veterinary Medicine. Dept. of Infectious Diseases and Immunology - Division of Immunology; University of Pretoria. Faculty of Veterinary Science. Dept. of Veterinary Tropical Diseases
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
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.