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

McFadyen, Ross (2016-03)

Thesis (MSc)--Stellenbosch University, 2016

Thesis

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 protein saver 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 after heat-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 a suitable reference gene for whole-blood stimulation in African buffaloes.

AFRIKAANSE OPSOMMING : Beestuberkulose (bTB) word veroorsaak deur die bakterie Mycobacterium bovis en is ‘n kroniese siekte in beeste en wild. M. bovis is deel van die M. tuberculosis kompleks, ‘n groep geneties verwante bakterie wat tuberkulose veroorsaak in die mens en verskeie ander spesies. Die bekeuring van bTB word bemoeilik deur die voorkoms van die siekte in wild spesies soos die Kaapse buffel (Syncerus caffer). Buffels in die Kruger Nasionale Park en Hluhluwe- iMfolozi Park is endemies geïnfekteer en die moontlikheid van oordrag na vee en ander wild spesies, asook na die mens, bestaan. Onlangse navorsing het bevind dat die huidige beskikbare diagnostiese toetse nie die nodige sensitiwiteit beskik om alle geïnfekteerde diere te identifiseer nie, asook oneffektief is in diere met n gevorderde siekte toestand. Vorige navorsing het bevind dat die mate van teenliggaam teenwoordigheid in geinfekteerde diere sterk korreleer met sy siektestoestand as gevolg van die groot hoeveelhede antigeen teenwoordig in letsels. Die doel van die studie was om die humorale immuun reaksie in bTB- positiewe buffels te karakteriseer deur gebruik te maak van verskeie molekulêre tegnieke. In lig van hierdie doelwit is ‘n PPD ELISA ontwikkel om die teenwoordigheid van teemliggaampies teen M. bovis te meet en die resultate te vergelyk met die van ‘n kommersiële beskikbare toets, die Chembio DPP toets. Albei toetse kon die M. bovis-spesifieke teenliggaampies in die bTB-positiewe groep optel en albei toetse se resultate het goed gekorreleer. Die humorale reaksie was verder gekarakteriseer deur die vrystelling van MCP-1 in M. bovis antigeen gestimuleerde bloed te meet. Hierdie sitokien is verantwoordelik vir B-sel proliferasie asook klas omskakeling gedurende gevorde infeksies. Daar was ‘n betekenisvolle verskil gevind in antigeen-spesifieke MCP-1 vrystelling tussen bTB-positiewe en bTB-negatiewe buffels. Hierdie resultate ondersteun die potensiele gebruik van MCP-1 as ‘n biologiese merker van infeksie, al was die sensitiwiteit laag. Verder was die stabiliteit van die MCP-1 sitokien geevalueer deur die meting daarvan na storing van bloed plasma op Whatman filtreer papier, asook na die hitte-behandeling daarvan by 65 ºC. Na 11 dae op filtreer papier was die konsentrasie van MCP-1 dieselfde as in plasma gestoor by -80 ºC. MCP-1 plasma konsentrasies was hoër na hitte behandeling as by -80 ºC storing en het goed gekorreleer met die verdunnings faktor. Laastens het ons die stabiliteit van 8 verskillende verwysings gene ge-evalueer om die mees stabiel gene te identifiseer vir die verdere gebruik daarvan in n kwantitatiewe ware tyd polimerase kettingreaksie toets vir die diagnose van bTB in buffels. Verder was die mees stabiele gene se boodskapper RNA nukleotied volgorde bepaal deur “primers” te ontwerp vanaf gepubliseerde beskikbare informasie beskikbaar oor elk een vir beeste. Die mees stabiel verwysings geen was PPIA, met GAPDH as die minder toepaslike kandidaat gedurende hierdie eksperimentele omstandighede. Ten slotte, serologiese toetse se gebruik vir die diagnose van M. bovis infeksie in buffels word benadeel deur hulle lae sensitiwiteit wel die gebruik van hierdie toetse in kombinasie met konvensionele toetse mag dalk nuttige informasie bied oor die immuun status van n dier. MCP- 1 toon potensiaal as n biologiese merker van infeksie in buffels en die stabiliteit onder verskeie kondisies ondersteun die moontlikheid om die vervoer van bloed monsters meer doeltreffend en veiliger te maak deur die geprosesseerde plasma by hoë temperature te skok en te stoor op filtreer papier. Alhoewel ons bewys het dat die PPIA die mees toepaslike verwysings geen is vir die gebruik in n diagnostiese geen uitdrukkings-toets vir bTB diagnose in Kaapse buffels, word verdere optimaliserings navorsing wel voorgestel.

Please refer to this item in SUNScholar by using the following persistent URL: http://hdl.handle.net/10019.1/98311
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