The detection of Mycobacterium avium subsp. paratuberculosis in sheep in the Western Cape, South Africa

Howell, Shannon Kimlynn (2018-12)

Thesis (MScFoodSc)--Stellenbosch University, 2018.

Thesis

ENGLISH ABSTRACT: Mycobacterium avium subsp. paratuberculosis (MAP) is the cause of a gastroenteritis ruminant (cattle, sheep, and goat) disease known as Johne’s disease (JD). Subsequently MAP has been speculated as the cause of Crohn’s disease (CD) in humans, an infection with similar symptoms, as MAP has been isolated from CD patients. Research has detected MAP in animal derived products such as pasteurised milk, soft cheeses and liver products. It is thought that through these routes, the transmission of MAP to humans has occurred. Statistics in South Africa depict that the Western Cape has the highest ovine JD outbreak, but the true prevalence is unknown. Currently used detection methods in South Africa lack sensitivity and are unreliable, therefore investigation of other more sensitive and reliable methods is required. The aim of this study was to detect MAP in sheep in the Western Cape, South Africa. The first objective was to optimise a phage assay to specifically detect viable MAP in blood. This followed optimising a polymerase chain reaction (PCR) method. The last objective was to investigate the prevalence of MAP in various sheep sample matrices (blood, faecal and milk), and further compare the results to an enzyme-linked immunosorbent assay (ELISA) test. Optimisation of the phage assay included establishing a Mycobacterium smegmatis (sensor cells) stocks, determine the D29 phage titre and apply these stocks to produce a positive and negative control. However, the optimisation proved to be problematic as the Mycobacterium smegmatis (sensor cells) stocks, used to create the lawn for the phage, were contaminated during storage. Therefore the positive and negative controls produced incorrect plaque results and the assay could not be used as a detection method. As there is currently no published work in South Africa on the investigation of other detection methods besides ELISA, culturing and Ziehl-Neelsen stain; it was decided to investigate PCR as a possible detection method. Optimisation of DNA extraction was conducted on whole blood and buffy coat using two different DNA extraction kits. The DNA extracted from the buffy coat using the QuickDNA Mini Prep kit produced the most and purest DNA compared to the whole blood. Various MAP specific primer sets that target different regions of the MAP genome were tested. The F57 primer sequence was the only primer sequence to detect the presence of MAP. Positive PCR samples were sequenced and aligned using the BLAST website. Results indicated that the PCR samples was 100% identical to MAP ATCC 19698, a bovine strain. The optimised PCR protocol was used to determine the prevalence of MAP in various sheep sample matrices (faecal, blood and milk). Samples were taken from pregnant Merino and Dohne Merino ewes. Faecal samples did not produce any positive results due to lack of MAP shedding or faecal PCR inhibitors. Blood samples produced the highest positive prevalence at 47.6% from nonvaccinated ewes. The ELISA results indicated positive results for the vaccinated ewes, and also produced three false-positive results between 2017 and 2018, demonstrating the unreliability of the ELISA test. From the 47.6% positive blood PCR results, only two of the ewes produced positive ELISA results. This demonstrated that the PCR method is more sensitive than the ELISA method. The milk samples produced a prevalence of 26.3%. The low positive prevalence was thought to be as a result of low MAP shedding during sampling. The live weight and body condition score was also taken into consideration, and showed no significant difference between 2017 and 2018. To conclude, the PCR demonstrated to be more sensitive than the ELISA test, and the blood samples produced the highest prevalence of MAP.

AFRIKAANSE OPSOMMING: Micobacterium avium subsp. paratuberculosis (MAP) is die oorsaak van 'n gastro-enteritis herkouer (bees, skape, bok) siekte bekend as Johne se siekte (JD). Daar word gespekuleer dat MAP moontlik die oorsaak is of ʼn rol speel by Chrohn siekte (CD) in mense, 'n infeksie met soortgelyke simptome as Johne se siekte, aangesien MAP ook al geisoleer is van CD-pasiente. Navorsing het MAP bespeur in diere afkomstige produkte soos gepasteuriseerde melk, sagte kaas en lewerprodukte. Daar word vermoed dat die oordrag van MAP aan mense deur hierdie roetes plaasgevind het. Statistiek in Suid-Afrika toon dat die Wes-Kaap die hoogste skaap-JD-uitbraak het, maar dat die ware voorkoms daarvan onbekend is. Huidige toetsmetodes in Suid-Afrika is nie sensitief genoeg nie en is onbetroubaar, daarom is die ondersoek na ander meer sensitiewe en betroubare metodes nodig. Die doel van hierdie studie was dus om die opsporing van MAP in skape in die Wes-Kaap, Suid-Afrika na te vors. Die eerste doel was om 'n fage-toets te optimaliseer, om sodoende lewensvatbare MAP in bloed te kan bepaal. Tweedens, die optimalisering van 'n polimerase ketting reaksie (PKR) metode. Die laaste doelwit was om die voorkoms/teenwoordigheid van MAP in verskillende skaapmonster matrikse (bloed, fekale en melk) te ondersoek en die resultate verder te vergelyk met 'n ensiem-gekoppelde immunosorbens-toets (ELISA). Optimisering van die fage-toets behels die groei van 'n Mycobacterium smegmatis (sensor selle) laag, bepaling van die D29 fage titer, en die gebruik hiervan as ʼn positiewe en negatiewe kontrole. Die optimisering was problematies, aangesien die Mycobacterium smegmatis (sensor selle) wat gebruik is om die groei-laag vir die fage te skep, tydens die berging daarvan besmet geraak het. Die positiewe en negatiewe kontrole het dus foutiewe plaakresultate opgelewer, en die toets kon nie as 'n opsporingmetode gebruik word nie. Aangesien daar tans geen gepubliseerde werk in Suid-Afrika is nie oor die ondersoek van ander opsporingsmetodes behalwe ELISA, kultivering en die Ziehl-Neelsen kleuring; is daar besluit om PKR as moontlike opsporingmetode te ondersoek. Optimalisering van DNA-ekstraksie is uitgevoer op die bloed en witbloedselle laag (buffy coat layer) met behulp van twee verskillende DNA-ekstraksie toets-stelle. Die DNA wat uit die witbloedselle geekstraheer is met behulp van die Quick-DNA Mini Prep toets-stel, het die meeste en suiwerste DNA in vergelyking met die bloed geproduseer. Verskeie MAP spesifieke aanvangskodon stelle wat verskillende streke van die MAP genoom teiken, is getoets. Die F57-primerreeks was die enigste aanvangskodon volgorde om die teenwoordigheid van MAP op te spoor. Positiewe PKR monsters is op volgorde van die BLASTwebblad geordend en in lyn gebring. Resultate het aangedui dat die PKR monsters 100% identies was aan MAP ATCC 19698, 'n bees stam. Die geoptimaliseerde PKR protokol is gebruik om die voorkoms van MAP in verskillende skaapmonster matrikse (bloed, fekale en melk) te bepaal. Monsters is van dragtige Merino- en Dohne Merino-ooie geneem. Fekale monsters het nie enige positiewe resultate opgelewer nie as gevolg van 'n gebrek aan MAP-skuur of fekale PKR-inhibeerders. Bloedmonsters het die hoogste positiewe voorkoms van 46.7% opgelewer. Die melkmonsters het 'n voorkoms van 26.3% opgelewer. Die lae positiewe voorkoms was vermoedelik die gevolg van lae MAP-vergieting tydens steekproefneming. Die ELISA-resultate het positiewe resultate vir die ingeente ooie aangedui, en het ook drie vals positiewe resultate tussen 2017 en 2018 opgelewer, wat die onbetroubaarheid van die ELISA-toets getoon het. Die lewendige gewig en liggaamskondisie telling is ook in ag geneem en het geen beduidende verskil tussen 2017 en 2018 getoon nie. Wat dus hieruit afgelei kan word, is dat die PKR blyk om meer sensitief te wees as die ELISAtoets, en dat die bloedmonsters die hoogste voorkoms van MAP opgelewer het.

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