Detection and Characterization of Mycobacterial Infections Occurring in Phacochoerus africanus (Gmelin, 1788) (Common Warthog)

Roos, Eduard Otto (2018-12)

Thesis (PhD)--Stellenbosch University, 2018.

Thesis

ENGLISH ABSTRACT: Mycobacterium bovis, a member of the Mycobacterium tuberculosis complex and the cause of bovine tuberculosis (bTB), has an extensive host range that includes livestock and wildlife. While warthogs are considered spill-over hosts for bTB, they could potentially become reservoir hosts, if conditions are favourable, i.e. increased population densities. With limited knowledge on the infection status of warthogs in South Africa and their epidemiological significance for other species, it is imperative to have readily available diagnostic tests for warthogs. Therefore, this study aimed to (i) establish reference cohorts of M. bovis-infected and uninfected warthogs; (ii) utilize these for the development and evaluation of diagnostic tools that can distinguish between infected and uninfected individuals; (iii) determine the seroprevalence of bTB in warthogs using the newly developed diagnostic tools; and (iv) characterize the genetic diversity of M. bovis isolates from warthogs. Three serological assays, i.e. the indirect PPD ELISA, the TB ELISA-VK® and the DPP® VetTB Assay, could distinguish between M. bovis-infected and uninfected warthogs with high sensitivity (75-88%) and specificity (79-89%). The overall seroprevalence from four M. bovis-endemic locations was high, i.e. 38%. Furthermore, three tests measuring the cellmediated immune responses of warthogs were developed. A cytokine release assay measuring interferon gamma induced protein 10 was able to distinguish between M. bovisinfected and uninfected warthog with a sensitivity of 68% and a specificity of 84%. The comparative intradermal tuberculin test classified 100% of culture-negative warthogs as test negative and 81% of culture-positive warthogs as test positive. Lastly, CXCL9, 10, 11, IFNG and TNFA gene expression were significantly increased in whole blood from M. bovis-infected warthogs in response to antigen stimulation, with CXCL10 showing the greatest mean fold increase. High genetic diversity of M. bovis isolates from warthogs was confirmed through spoligotyping and whole genome sequencing. Two distinct clades of M. bovis were identified by WGS, even though they shared the same spoligotype patterns This study has demonstrated that warthogs develop measurable and specific immune responses to M. bovis infection, which can be used to identify infected individuals ante-mortem. Furthermore, these tests will facilitate epidemiological studies of bTB in warthogs. With a high culture prevalence in warthogs from bTB endemic areas such as uMhkuze Nature Reserve and the Greater Kruger National Park, and high seroprevalence, warthogs seem to be highly susceptible to M. bovis infection. This suggests that warthogs may be an ideal sentinel species and strengthens the case that, under certain circumstances, they could be maintenance hosts. The genetic diversity of M. bovis isolates and the identification of two distinct clades challenges the current hypothesis that a single dominant strain circulates within a specific geographical location. Warthogs as a species should receive greater attention as potential disease maintenance hosts or as sentinels for bTB disease surveillance.

AFRIKAANSE OPSOMMING: Mycobacterium bovis, 'n lid van die Mycobacterium tuberculosis kompleks en die oorsaak van bees-tuberkulose (bTB), het 'n uitgebreide gashere stelsel wat strek vanaf vee tot-en-met wild. Terwyl vlakvarke as oorspoel gashere vir bTB beskou word, kan hulle potensieël instandhoudingsgashere wees, indien toestande gunstig is, d.w.s. wanneer daar ‘n hoë bevolkingsdigtheid is. Met ‘n beperkte kennis, ten opsigte van vlakvarke se infeksiestatus, in Suid-Afrika asook hul epidemiologiese betekenis vir ander spesies, is dit noodsaaklik om algemeen beskikbare diagnostiese toetse vir vlakvarke te hê wat besmette individue kan identifiseer. Daarom het hierdie studie daarop gemik om (i) verwysingskohorte van M. bovis-besmette en onbesmette vlakvarke te bevestig; (ii) dié te gebruik vir die ontwikkeling en evaluering van diagnostiese instrumente wat tussen besmette en onbesmette individue kan onderskei; (iii) die seroprevalensie van bTB in vlakvarkke te bepaal, deur gebruik te maak van die nuut ontwikkelde diagnostiese instrumente; en (iv) die genetiese diversiteit van M. bovis-isolate van vlakvarke te karakteriseer. Drie serologiese toetse, naamlik die indirekte PPD ELISA, die TB ELISA-VK® en die DPP® VetTB-assay, kan onderskei tussen M. bovis-besmette en onbesmette vlakvarke met hoë sensitiwiteit (75-88%) en spesifisiteit (79-89%). Die algehele seroprevalensie van vier M. bovis-endemiese gebiede was hoog, d.w.s. 38%. Nog drie toetse was ontwikkel wat die selbemiddelde immuunreaksie van vlakvarke gemeet het. 'n Sitokien-vrystellingstoets wat interferon-gamma-geïnduseerde proteïen 10 meet was in staat om te onderskei tussen M. bovis-besmette en onbesmette vlakvarke met 'n sensitiwiteit van 68% en 'n spesifisiteit van 84%. Die vergelykende intradermale tuberkulintoets het 100% van kultuur-negatiewe vlakvarke as toets-negatief geklassifiseer en 81% van kultuur-positiewe vlakvarke as toets positief geklassifiseer. Laastens, was die geen-uitdrukking van CXCL9, 10, 11, IFNG en TNFA beduidend hoër in die bloed van M. bovis-besmette vlakvarke, in reaksie op antigeenstimulasie, met CXCL10 wat die grootste gemiddelde vouverhoging toon. Hoë genetiese diversiteit van M. bovis-isolate, vanuit vlakvarke, is bevestig deur middel van spoligotipering en heelgenoom volgorde bepaling. Twee verskillende stamme van M. bovis is deur heelgenoom volgorde bepaling geïdentifiseer, alhoewel hulle dieselfde spoeligotipepatrone gedeel het. Hierdie studie het getoon dat vlakvarke ‘n meetbare en spesifieke immuunreaksie op M. bovis-infeksie ontwikkel, wat gebruik kan word om besmette individue ante-mortem (voor die dood) te identifiseer. Verder sal hierdie toetse epidemiologiese studies van bTB in vlakvarke fasiliteer. Met 'n hoë kultuurvoorkoms in vlakvarke afkomstig vanuit bTB-endemiese gebiede soos uMhkuze Natuur Reservaat en die Grooter Kruger Nationale Park, as ook die hoë seroprevalensie, lyk vlakvarke hoogs vatbaar vir M. bovis-infeksie. Dit dui daarop dat vlakvarke 'n ideale brandwag spesie kan wees en versterk die aanname dat hulle onder sekere omstandighede instandhoudingsgashere kan wees. Die genetiese diversiteit van M. bovis-isolate en die identifisering van twee duidelike stamme daag die huidige hipotese uit, wat voorstel dat 'n enkele dominante stam versprei word binne 'n bepaalde geografiese gebied. Vlakvarke as 'n spesie moet groter aandag geniet as ‘n moontlike instandhoudingsgasheer of as brandwag vir bTB.

Please refer to this item in SUNScholar by using the following persistent URL: http://hdl.handle.net/10019.1/104836
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