Prevalence and characterisation of Mycobacterium species in cattle and sheep at Gauteng abattoirs

Abstract

Bovine tuberculosis (bTB) is a zoonotic disease with a great economic impact estimated at billions of dollars annually worldwide. It is a highly infectious disease infecting mainly wildlife, domestic animals, and humans. The causative agent for the disease is a group of bacteria belonging to the Mycobacterium tuberculosis complex (MTBC). The current study aimed at investigating the prevalence and characterize Mycobacterium species in slaughter animals at Gauteng province abattoirs and to assess the risk of zoonotic tuberculosis posed to abattoir workers. In an attempt to fulfil the objective samples were collected from Gauteng province abattoirs. There is limited data available on abattoir-based studies on bovine tuberculosis (bTB) in South Africa. Abattoirs were chosen for this study as they provide important information of data and they play a key role in passive surveillance on the status of the prevalence of bTB in livestock. The study first reviewed the retrospective data for Mycobacterium spp. in the laboratory data in the Tuberculosis Laboratory at the Agricultural Research Council-Onderstepoort Veterinary Research (ARC-OVR) between 2007 and 2016. Samples that were submitted at the ARC-OVR from nine provinces of South Africa, were analysed for the prevalence of Mycobacterium spp. over these 10 years. The findings revealed that the total prevalence of Mycobacterium spp. was 18.47% [834/4516; 95% Confidence Interval: 17.37 – 19.63] with individual species prevalence at 11.47%, 5.20%, 1.53%, 0.24%, and 0.02%, for Mycobacterium bovis, Mycobacterium Other Than Tuberculosis (MOTT), Mycobacterium tuberculosis, Mycobacterium avium, and Mycobacterium orygis respectively. The findings revealed that M.bovis seems to be the most prevalent Mycobacterium species in both domestic animals at 62.26% and wildlife at 63.68%. The study found that factors for bTB such as the presence of wildlife reservoirs and contact with these reservoirs are a great influence on the transmission of the disease that could result in animals testing positive for Mycobacterium spp. Samples from Limpopo, Mpumalanga, and Gauteng provinces were most likely positive for Mycobacterium spp. The majority of samples from Gauteng province originated from captive wildlife. M. tuberculosis, which is mostly a human pathogen rather than M. bovis, was isolated. The study also highlights that wild carnivores and marine animals are more likely to test positive as these animals are likely to feed off infected prey. Study results showed that most of the organs were prone to infection by Mycobacterium spp. Improved data collection is required so that scientific research can target several aspects highlighted by the information obtained from the records. The laboratory data obtained in this study gave insight into the occurrence of Mycobacterium spp. in wildlife, livestock and their environment in South Africa and the factors that influence the transmission of mycobacteriosis such as bTB. Furthermore, we reviewed serological laboratory data in the Tuberculosis Laboratory at the Agricultural Research Council-Onderstepoort Veterinary Research (ARC-OVR) for the period 2011 - 2016. This was done to further highlight the importance that the combination of both bacteriological data and serology provide better insight into the epidermiology. Out of 10,369 fresh blood samples that were submitted from all of the provinces around South Africa and tested using the Bovigam® kit, the results showed that 1.54% (95% Confidence Interval: 1.30 – 1.78) were positive for bTB, 3.75% (95% Confidence Interval: 3.38 - 4.11) were avian reactors, and 2.29% (95% Confidence Interval: 3.38 - 4.11) were a combination of multiple reactors, equal reactors as well as animals that had a positive screening test. (A multiple reactor means an animal reacted positively to all tuberculin suggesting a possible infection with either Mycobacterium bovis or Mycobacterium forfuitum or both and in some instances even Mycobacterium avium. An equal reactor means an animal reacted positively to both avian or bovine tuberculin due to possible infection with either Mycobacterium bovis or Mycobacterium avium or both). Also, the results showed that the most number of samples tested were buffalo samples with 88.96% (95% Confidence Interval: 88.35-89.56), followed by domestic bovine (cattle) at 9.94% (95% Confidence Interval: 9.37 – 10.52). Our results showed that there is generally a low prevalence of bTB in South Africa. Furthermore, we collected fresh blood samples with corresponding tissue samples at the point of slaughter at abattoirs and samples were subjected to serological assays and bacterial cultureto detect the active interferon-gamma (IFN-γ) and isolate mycobacterial species respectively. The study was conducted at selected abattoirs in the Gauteng Province in South Africa where animals were also subjected to routine meat inspection. A total of 410 fresh blood samples were collected during the slaughter of animals (369 cattle and 41 sheep) from 15 abattoirs and analysed using a Bovigam® 1G-test kit (Prionics AG, Lelystad, The Netherlands) Bovigam test kit with bovine, avian, and fortuitum purified protein derivatives (PPD) as blood stimulating antigens. The estimated prevalence of bTB in cattle was 4.4% (95% Confidence Interval: 2.4%-7.3%) and the prevalence of avian reactors was 6.0% (95% Confidence Interval: 3.6%-9.2%). None of the sheep with valid test results (i.e. test samples that have passed quality control checks) were positive for bTB and none were avian reactors (95% Confidence Interval: 0% - 15%). Additionally, we collected a total of 2000 tissue samples comprising of lungs, liver, spleen, and lymph nodes were collected from 19 abattoirs. Additionally, 19 environmental samples were collected from feedlots, where animals usually drink as they await slaughter. These samples were cultured on Lowenstein-Jensen (LJ) media containing both pyruvate and glycerol. The cultures were monitored for growth over 10 weeks. Colonies that displayed morphology resembling that of Mycobacteria were first subjected to Ziehl-Neelsen (ZN) staining, then acid fast bacteria were subjected to a polymerase chain reaction (PCR) assay targeting Mycobacterium tuberculosis complex bacteria. No MTBC species were detected by PCR. The same isolates were subjected to the 16S rRNA PCR and gene sequence analysis to investigate and identify non-tuberculous mycobacteria (NTM) species. Isolations were made from eight animals originating from four abattoirs and were identified as Mycobacterium species by amplification of a product corresponding to 577 bp in size following gel electrophoresis. Sequence data analysis of the 8 isolates revealed only two of the isolates to be Mycobacterium colombiense (99.81% identity) and Mycobacterium simiae (99.42% identity). The remaining six isolates were identified as members of the Actinomadura species. From the environmental samples, isolation was made from three samples, and two were identified to genus level (Mycobacterium species). The remaining isolate was identified as Mycobacterium senuense (99.22% identity). For the slaughtered livestock, the results suggest that there was no risk of transmission of bTB to abattoir workers and the meat was probably safe to consume. Although NTM have been implicated to be potentially involved in causing tuberculosis-like diseases, their rate of occurrence in the current study was extremely low, hence insignificant. The study has,however, highlighted that the additional use of serological assays such as the interferon-gamma (IFN-γ) assay can help detect early signs of infection, thus establishing the status of infection. Additionally, interviews were conducted in the form of questionnaires to establish the knowledge, attitude, and practices (KAP) of abattoir workers regarding Tuberculosis (TB). The objective of the study was to obtain data from abattoir workers in Gauteng Province, South Africa and to assess the risk of zoonotic tuberculosis transmission among workers. This study revealed an overall knowledge score of the respondents to be 42% (95% Confidence Interval: 37.48 - 46.42) (P<0.001), with 88.35% of the respondents knowing of the disease. The overall knowledge of TB score was determined by calculating the overall average knowledge of the respondents. More than 45% of the participants were aware of zoonotic TB and how the disease is transmitted. Overall, the results of the study suggested that there are certain practices that abattoir workers are engaged in, such as the consumption of unpasteurized milk and undercooked or raw meat, slaughtering of animals at home as well as taking care of animals at home/work that may promote transmission of zoonotic tuberculosis among themselves and their communities at large. In conclusion, we report on the prevalence of Mycobacterium spp in slaughter animals in abattoirs in Gauteng Province, South Africa, the potential zoonotic risks posed to abattoir workers and on the 10-year retrospective data on samples submitted to the Tuberculosis laboratory (ARC-OVR) analysed for the prevalence of Mycobacterium spp. over this 10-year