2018-12-11 12:00 - Maintenance complete.

The occurrence of shiga-toxin producing Escherichia coli in South African game species

Germishuys, Zandre (2017-03)

Thesis (MScFoodSc)--Stellenbosch University, 2017.

Thesis

ENGLISH ABSTRACT: The consumption of game meat on a global scale is increasing every year and South Africa is no exception. Consumers have started incorporating low kilojoule and low cholesterol products into their lifestyles, of which game meat is one such product. These increases give rise to a range of challenges for the game meat industry in terms of the microbial safety of the meat. The aims of this study were to optimise a DNA extraction protocol for the isolation of the Shiga-toxin producing Escherichia coli (STEC) positive control and PCR conditions for the amplification of the stx1, stx2 and eaeA virulence genes; to determine the prevalence of STEC contamination in South African game species; to determine the microbial population present on South African game carcasses after dressing; and to develop and implement an organic acid spray in order to reduce microbial growth on game carcasses. Optimisation of a DNA extraction protocol for STEC positive control included an ethanol wash and re-suspension in sterile distilled water to purify the DNA from any PCR inhibitors. Primer concentrations were also optimised to prevent non-specific binding, which causes primer-dimer formation. PCR amplification conditions were compared to determine the optimum annealing temperature, as a too low or too high temperature led to unsatisfactory amplification results. The optimised protocol and conditions were used to determine the prevalence of STEC in South African game species. Animals from two different farms were used in this study. Farm 1 consisted of four species, of which two species (Zebra and Black Wildebeest) had STEC present in the faecal matter. The meat of all four species (Zebra, Black Wildebeest, Impala and Eland) contained STEC suggesting possible cross contamination from the hide to the carcass. Faecal samples from Farm 2 (Blesbok and Springbok) tested negative for STEC. During the microbial population study it was found that aerobic bacteria prevalence on the meat ranged from 1.60 to 4.97 log cfu·cm-2 whereas total coliforms varied from 5.04 to 5.59 log cfu·cm-2. E. coli prevalence ranged from 0.00 to 1.71 log cfu·cm-2 while Staphylococcus aureus varied from 0.00 to 2.97 log cfu·cm-2. The results from the faecal matter displayed aerobic bacteria prevalence in the range of 5.78 to 6.44 log cfu·g-1 while total coliforms ranged from 6.53 to 7.04 log cfu·g-1. E. coli prevalence varied from 3.00 – 4.54 log cfu·g-1 while Staphylococcus aureus ranged from 3.63 to 4.40 log cfu·g-1. None of the faecal samples tested positive for Salmonella or Listeria. Throughout the organic acid spray development, different organic acids (lactic acid, citric acid, acetic acid and octanoic acid) and sodium benzoate were tested singly and in combination. Development started with enrichment mediums and progressed to meat samples. Based on the results of the meat samples a final combination of 5% lactic acid, 0.5% octanoic acid and 0.1% sodium benzoate was chosen to use on game carcasses. Although the combination provided promising results in laboratory trials, its efficacy on the carcasses was unsatisfactory with low overall log reductions achieved. To conclude, the high prevalence of STEC (26.9%) detected in the game species was alarming and requires further investigation.

AFRIKAANSE OPSOMMING: Die verbruik van wildsvleis op ‘n globale skaal neem jaarliks toe en Suid-Afrika is geen uitsondering nie. Verbruikers het begin om lae kilojoule en lae cholesterol produkte in hul lewenswyse te inkorporeer, waarvan wildsvleis een so 'n produk is. Hierdie verbruiksverhoging gee aanleiding tot 'n reeks uitdagings vir die vleis industrie in terme van die mikrobiologiese veiligheid van die vleis. Die doelwitte van hierdie studie was om 'n DNA ekstraksie protokol vir die isolasie van die Shiga-toksien produserende Escherichia coli (STEC) positiewe kontrole asook die Polimerase Ketting Reaksie (PKR) kondisies vir die amplifisering van die stx1, stx2 en eaeA virulensie gene te optimiseer; om die voorkoms van STEC kontaminasie in Suid-Afrikaanse wildspesies te bepaal; om die mikrobiologiese populasie teenwoordig op Suid-Afrikaanse wildsvleis karkasse na afslag te bepaal; en om 'n organiese suur spuitmiddel te ontwikkel en te implementeer om mikrobiese groei op wildsvleis karkasse te verminder. Optimisering van 'n DNA ekstraksie protokol vir die STEC positiewe kontrole het 'n etanolwas en heroplossing in steriele gedistilleerde water ingesluit om te verseker dat die DNA vry is van PKR inhibeerders. Inleier konsentrasies is ook geoptimiseer om nie-spesifieke binding wat inleier dimere tot gevolg het, te verhoed. PKR amplifiseringskondisies is met mekaar vergelyk om die optimale inleier aanhegtings temperatuur te bepaal, aangesien ‘n te hoë of te lae temperatuur tot onbevredigende amplifikasie resultate sou lei. Die geoptimiseerde protokol en kondisies is gebruik om die voorkoms van STEC in Suid- Afrikaanse wildspesies te bepaal. Diere vanaf twee verskillende plase is tydens hierdie studie gebruik. Plaas 1 het bestaan uit vier spesies, waarvan twee spesies (Zebra en Swart Wildebees) STEC in die ontlasting teenwoordig gehad het. Die vleis van al vier spesies (Zebra, Swart Wildebees, Impala en Eland) het STEC bevat wat moontlike kruis-kontaminasie vanaf die vel na die karkas aandui. Fekale monsters vanaf Plaas 2 (Blesbok en Springbok) het negatief getoets vir STEC. Gedurende die mikrobiologiese populasie studie is gevind dat die voorkoms van aerobiese bakterieë op die vleis gewissel het tussen 1.60 en 4.97 log kve·sm-2 terwyl totale kolivorme gewissel het tussen 5.04 – 5.59 log kve·sm-2. Die voorkoms van E. coli het gewissel vanaf 0.00 tot 1.71 log kve·sm-2 terwyl Staphylococcus aureus gewissel het tussen 0.00 en 2.97 log kve·sm-2. Die resultate van die fekale monsters het getoon dat die voorkoms van aerobiese bakterieë gevarieer het tussen 5.78 en 6.44 log kve·g-1, terwyl totale kolivorme gewissel het tussen 6.53 en 7.04 log kve·g-1. Die voorkoms van E. coli het gevarieer tussen 3.00 en 4.54 log kve·g-1, terwyl Staphylococcus aureus gewissel het tussen 3.63 en 4.40 log kve·g-1. Geen fekale monsters het positief getoets vir Salmonella of Listeria nie. Gedurende die organiese suur spuitstof ontwikkeling, is verskillende organiese sure (melksuur, sitroensuur, asynsuur en oktanoësuur) en natriumbensoaat afsonderlik asook in kombinasie getoets. Ontwikkeling het begin met verrykingsmediums en daarna voortgegaan met vleismonsters. Op grond van die resultate behaal vir die vleismonsters was 'n finale kombinasie van 5% melksuur, 0.5% oktanoësuur en 0.1% natriumbensoaat gekies om op die wildsvleis karkasses te gebruik. Alhoewel dié kombinasie belowende resultate in laboratoriumtoetse getoon het, was die doeltreffendheid daarvan op die karkasse onbevredigend in terme van die algehele log reduksies wat bereik is. Om af te sluit, die hoë voorkoms van STEC (26.9%) wat waargeneem is in die wildspesies, is kommerwekkend en vereis verdere ondersoek.

Please refer to this item in SUNScholar by using the following persistent URL: http://hdl.handle.net/10019.1/101283
This item appears in the following collections: