Immunological tools for the characterization of the humoral immune response to Mycobacterium bovis infection in African buffaloes (Syncerus caffer)

[摘要] ENGLISH ABSTRACT : Bovine tuberculosis (bTB), caused by Mycobacterium bovis, is a chronic infectious disease known to occur in free-ranging mammals, captive wildlife, and livestock. M. bovis forms part of the Mycobacterium tuberculosis complex (MTC), a genetically related group of bacteria that cause tuberculosis in humans or other species. bTB eradication efforts are complicated by the occurrence of wildlife reservoirs of the disease such as the African buffalo (Syncerus caffer). Buffalo in the Kruger National Park and Hluhluwe-iMfolozi Game Reserve are affected by endemic bTB and there is the potential for M. bovis to spill-over into other domestic or wild animals, including zoonotic transmission to humans. Current standard diagnostic tests lack the required sensitivity to successfully detect all infected animals and are known to be ineffective in animals who have become anergic in the late stages of disease. Antibodies have been shown to correlate with disease pathology due to a high antigen burden and are able to be detected in anergic animals. The aim of this study was to characterize the humoral (antibody mediated) immune response in bTB-positive buffalo using different molecular techniques. Towards this aim, an in-house PPD ELISA was developed to detect antibodies against M. bovis and was compared to the Chembio® DPP VetTB, a commercial antibody assay. Both tests were able to detect M. bovis-specific antibodies in bTB-positive buffalo and there was good correlation between the two. We aimed to further assess the humoral response by investigating the diagnostic performance of monocyte chemotactic protein 1 (MCP-1), a cytokine involved in the activation of B-cell proliferation and class switching. There was a significant difference in antigen-specific MCP-1 release between bTB-positive and bTB-negative buffalos, suggesting there is promise for the use of MCP-1 as a biomarker, although sensitivity was low. Furthermore we evaluated the plasma stability of the MCP-1 protein during storage on proteinsaver cards (PSC) as well as after heat-treatment at 65°C. After 11 days at room temperature on the PSC, MCP-1 was still detected at similar levels. MCP-1 concentrations were higher afterheat-treatment and correlated with the dilution factor. Lastly, we aimed to determine the stability of 8 reference genes to be used in a whole-blood qPCR gene expression assay in buffaloes. Selected gene transcripts of the African buffalo were sequenced using primers from the domestic cow and it was shown that PPIA is the most stable reference genes whereas GAPDH is the most unsuitable for use under our experimental conditions. In conclusion, the use of serological tests for the detection of M. bovis infected buffalo are disadvantaged by their low sensitivity, although using them in combination with conventional tests may provide useful information on the immune status of the animal. MCP-1 shows promise as a biomarker of disease in buffalo and its stability means that transporting of plasma samples can be made safe and efficient by heat-treating samples and storing them on PSC. Further research is needed to fully optimize a diagnostic GEA for bTB in buffaloes, however, we have shown that PPIA is asuitable reference gene for whole-blood stimulation in African buffaloes.

[发布机构] Stellenbosch University