Absence of Tuberculosis-Causing Mycobacteria from Slaughtered Livestock Tissues and Environmental Samples, Gauteng Province, South Africa

Mycobacterium tuberculosis complex (MTBC) is a group of bacteria responsible for causing tuberculosis in animals and humans. In South Africa (S.A), slaughterhouses are registered by the government and closely inspected and audited for hygienic slaughter practices. Meat inspection to detect lesions has been used for passive surveillance, monitoring, and diagnosis of the disease status. Information on the current status of bovine tuberculosis (bTB) in livestock in the country is limited. Hence, we investigated the occurrence of Mycobacterium spp. in the tissues of slaughtered livestock and environmental samples in abattoirs in Gauteng province of South Africa (S.A). The cross-sectional study employing random sampling from cattle, pigs, and sheep (with the collection of liver, lung, spleen, and different lymph nodes) irrespective of lesions was carried out in 19 red meat abattoirs. Five hundred animals were sampled, comprising cattle (n = 369), pigs (n = 90), and sheep (n = 41). Additionally, 19 environmental samples were collected from feedlots, or where animals drink water while awaiting slaughter, to identify mycobacterial species using culture, acid-fast bacteria staining, and polymerase chain reaction (PCR). The Chi-square and Fisher's Exact tests were used to detect statistically significant differences in the frequency of detection of Mycobacterium spp. according to the variables investigated (types of tissues, livestock, abattoirs, etc.). The PCR assays detected no MTBC complex species DNA in the bacterial isolates from cattle (n = 32). Sequence analysis (16S rDNA) of the isolates from eight cattle confirmed only two species, namely Mycobacterium colombiense (99.81% identity) and Mycobacterium simiae (99.42% identity). The remaining isolates were identified as members of the Actinomadura species. From the environmental samples, bacterial isolation was made from three samples, and two could only be identified up to the genus level (Mycobacterium species) while the remaining isolate was identified as Mycobacterium senuense (99.22% identity). The study revealed the absence of bovine tuberculosis-causing pathogens in red meat abattoirs of the Gauteng province. Although non-tuberculous Mycobacteria have been implicated as potentially causing tuberculosis-like diseases in livestock, their occurrence in the current study was found to be low, but the potential to cause disease cannot be ignored.


Introduction
Bovine tuberculosis (bTB), caused mainly by Mycobacterium bovis, is a zoonotic disease with economic losses estimated at billions of dollars annually worldwide, yet the economic impact of the disease in African countries remains unquantifed [1].In South Africa, TB in cattle is mostly known to be caused by M. bovis and it is classifed as a controlled disease according to the national Animal Diseases Act, Act 35 of 1984.Control strategies have been put in place since 1969, and reduced the disease prevalence in communal cattle herds to less than 1%, although sporadic outbreaks still occur [2,3].Te true disease prevalence in diferent provinces of the country including Gauteng province is however, currently unknown, more especially in communal cattle populations.Tis is mostly due to the fact that control of the disease has become less prioritised by the government over the years due to lack of funding [4].
In S.A, slaughtering of livestock is ofcially conducted at abattoirs, although some back yard slaughtering also occur.Ofcial abattoirs are registered by government and closely inspected and audited for hygienic slaughter practices [4].Meat inspection in slaughterhouses to detect lesions has been used for passive surveillance, monitoring, and diagnosis of the disease [5].However, this method only detects lesions in advanced stages of infection when visible lesions are observed [6].Furthermore, it should be noted that lesions observed are not only due to M. bovis or other members of the Mycobacterium tuberculosis complex (MTBC), but some members of the Mycobacterium avium complex (MAC) and other nontuberculous mycobacteria (NTM) are also both pathogenic and opportunistic and may produce lesions in the infected animals [7].Hence, confrmation of the causative agent is of outmost importance, as NTM were previously also found to interfere with cell mediated immunity (CMI) response diagnostic tests for bTB [8,9].Studies have suggested that meat inspection should be done together with routine culturing and followed by molecular methods for characterizing different mycobacterial species [10] especially in regions with a high TB burden and where NTMs remain undiagnosed [11].It has been reported that approximately one-third of the NTMs are responsible for human disease [12].In the last three decades, there has been a recorded increase in NTM laboratory isolation [13].In a study by Oloya [14] in Uganda, 19 M. bovis and 11 NTM were isolated from 61 tissue samples from slaughter livestock, confrming the widespread prevalence of bTB in this country.In another study, Awah-Ndukum et al. [15] confrmed the presence of bTB by isolating M. bovis from samples collected at abattoirs.In a recent study, from Rwanda, both M. bovis and Mycobacterium tuberculosis were isolated from slaughtered cattle, indicating that bTB is present in this country, although at low prevalence [16].Molecular epidemiology of bTB is useful in the determination of risk factors of bTB transmissions, identifcation of the sources of contamination, tracking of the geographic distribution, and the spread of Mycobacterial species.
Information on the current status of bTB in livestock in the country is limited.Hence, we investigated the occurrence of Mycobacterium spp. in the tissues of slaughtered livestock and environmental samples in abattoirs in Gauteng province of S.A and determined the possible food safety risks posed to meat consumers.

Description of the Study Area, Abattoirs, Sample Type, and
Size Determination.Tis study was conducted in 19 red meat abattoirs across the Gauteng province of South Africa and was done in parallel with our previous study [17].Briefy, the investigators obtained a list of functional red meat abattoirs (mono-and multi-species) located across Gauteng province from the Gauteng Department of Agriculture and Rural Development (GDARD).Based on the information provided for each abattoir which included the type and number of livestock slaughtered daily, location, and their facilities, a total of 19 abattoirs were randomly selected, comprising 16 high-throughput (HT) and three low-throughput (LT) abattoirs for the study.Te study was conducted in all six districts of Gauteng province, namely, the City of Johannesburg, the City of Tshwane, Ekurhuleni, Metsweding, Sedibeng, and West Rand (Figure 1).At each selected abattoir, slaughtered cattle, pigs, and sheep were sampled on a single day using a systematic random sampling method, and the abattoir setting determined this.Approximately 30 animals were sampled during each sampling visit to the abattoir, and the number sampled per livestock species was proportional to the expected number to be slaughtered on that day, as provided by the abattoir workers.Te prevalence of Mycobacterium spp. in tissues collected from slaughtered livestock in South Africa is currently unknown.Te prevalence was therefore estimated to be 50% based on the standard practice used for unknown prevalence, and sample size determination was calculated using the formula n o = 1.96 2 × P exp × (1 − P exp )/d 2 recommended by Trusfeld [18], where n o represented the minimum sample size, P exp represented the expected prevalence and d 2 represented the desired precision value of 5%.Although the minimum estimated sample size was 384, a total of 500 animals were sampled, and the distribution per livestock species (cattle, sheep, and pigs) was proportional to the number of animals slaughtered per species during abattoir visits.

Pre-and Post-Slaughter Inspection.
Te professional meat inspectors assigned to each abattoir conducted preand post-slaughter inspections according to a standard procedure, which included detecting clinical TB lesions as previously described [17].Biographical information about the slaughtered livestock, including gender, breed, district, and municipality origin of animals, was recorded [17].

Sample Collection.
While the inspectors were conducting physical examinations of the carcasses, the following organs were collected, i.e., lymph nodes (retropharyngeal, abdominal, mesenteric), liver, lung, and spleen for the Mycobacterium species culture test.A total of 500 animals were sampled, i.e., cattle (n � 369), pigs (n � 90), and sheep (n � 41).Overall, 2000 tissue samples were collected from diferent organs of each animal.Tissue samples were labelled and placed in sterile Ziploc bags or specimen containers.All the tissues were transported to the Tuberculosis laboratory at the Agricultural Research Council-Onderstepoort Veterinary Research (ARC-OVR) in ice-cooled boxes.Environmental samples (water; n � 19), one from each abattoir, were collected from feedlots or where animals drink water while waiting for slaughter.All the collected environmental and 2 International Journal of Microbiology tissue samples were cultured according to established standard laboratory protocols and monitored weekly for bacterial growth.

Isolation of Mycobacterium Species from Tissue Samples.
To culture tissue samples for Mycobacterium spp., standard methods available in the ARC-OVR Tuberculosis laboratory were used [8].Approximately 944 samples from 236 animals were individually processed.Representatives from the same animals were processed as a pool for the remaining 1056 samples (264).Briefy, the collected tissue samples were cut into approximately 5 g pieces, and all the fat was removed.Using the Ultra-Turrax ® homogenizer (Separation Scientifc, SA), the samples were then homogenized.Te homogenates were poured into two 50 ml tubes in preparation for the decontamination.Approximately 7 ml of each homogenized sample was transferred into two 15 ml centrifuge tubes each and then decontaminated in the frst tube with HCL to a fnal concentration of 1.0% and in the second tube with NaOH to a fnal concentration of 2.0% for 10 minutes, followed by centrifugation (3500 rpm).Te Lowenstein-Jensen (LJ) media supplemented with pyruvate (4 slopes) and glycerol (2 slopes) was inoculated with the sample pellets following neutralization with sterile distilled water and centrifugation (3500 rpm).For each batch of samples tested, positive and negative controls were included.Te positive control was a sample collected from either skin test positive/suspect reactor or slaughter cattle with suspect tuberculous lesions and confrmed by culture and PCR, while the negative control sample was collected from a carcass declared ft for human consumption and underwent microbiological testing for confrmation.Te inoculated media slopes were incubated at 37 °C for 10 weeks and examined weekly for bacterial growth (colonies).

Isolation of Mycobacterium Species from Water Samples.
For the isolation of Mycobacterium species, water samples were collected from the selected abattoirs and cultured according to the standard laboratory protocol at the Tuberculosis laboratory.Positive (water sample spiked with a colony of an in-house Mycobacterium bovis, TB 9854A) and negative (unspiked sterile distilled water) controls were included for each batch of samples tested.Briefy, 2% NaOH was added to the samples and centrifuged, thereafter 5.0% oxalic acid was added.After centrifugation, Lowenstein-Jensen (LJ) slopes (3x) were inoculated with the sample and incubated at 27 °C.Also, another set was inoculated and incubated at 37 °C and monitored for up to 10 weeks for colonies typical of Mycobacterium species.Tereafter, colonies were selected for Ziehl-Neelsen staining and PCR identifcation.International Journal of Microbiology 2.6.Ziehl-Neelsen Staining and Microscopy.Te presence of colonies following the detection of growth on the selective media after incubation was suggestive of the presence of Mycobacteria, and bacterial smears were prepared on microscopic slides for Ziehl-Neelsen (ZN) staining to confrm acidfastness.Te ZN-stained smears were then observed under a microscope.

DNA Extraction in Preparation for Polymerase Chain
Reaction.DNA extraction was conducted as previously described [19].DNA templates were prepared from colonies typical of Mycobacterium species, and individual colonies were picked up from the L-J media and mixed with 100 μl ultra-pure water.DNA was extracted from isolates by heattreating the suspension at 100 °C for 25 minutes and allowed to cool down at room temperature.DNA templates were stored at −20 °C until PCR analysis [19].
2.9.Gel Electrophoresis.Te PCR products were visualized on a 1.5% agarose gel stained with 20 μl ethidium bromide (10 μg/ml) and run at 80 V for 3 h.A 100 bp ladder (Inqaba Biotechnical Industries) was included and used to estimate the size of the resulting PCR products.Positive and negative controls (previously tested DNA templates) were included for quality control.In addition, distilled water was included as a blank control.For the study, the dependent variable was the isolation of Mycobacterium spp., and the independent variables were the type, gender, and breed of animal species, livestock origin, district, municipality, and abattoirs (HT and LT).Chi-square and Fisher's Exact analysis were conducted to determine whether there were statistically signifcant diferences in the frequency of isolation of Mycobacterium spp.among the livestock tissues tested according to the independent variables mentioned above.Te level of signifcance was determined at an alpha level of 0.05.

Bacterial Isolation from Tissue Samples and Ziehl Neelsen
Staining.Tissue samples originating from 19 diferent red meat abattoirs located in the Gauteng province (Figure 1) were collected and tested at the Tuberculosis Laboratory at the ARC-OVR for Mycobacterium spp.isolation.Colonies of bacterial growth typical of mycobacteria were observed.Bacterial isolation was made from 32 samples representing individual cattle from 12 diferent red meat abattoirs.Because of the single colony growth from most of the samples, only a few could be subjected to Ziehl Neelsen staining to confrm acid fastness.No isolation was made from pig and sheep samples.

Identifcation of Mycobacterium tuberculosis Complex
Species by PCR.Out of the 32 isolates, no MTBC complex species was detected.

Characterization of Mycobacterium Isolates from Tissue Samples by 16S rRNA PCR and Gene Sequence Analysis.
Out of the 32 isolates, 8 (25%) from individual animals originating from 4 abattoirs displayed a 577 bp PCR product by 16S rRNA PCR analysis (Figure 2).Te 16S   International Journal of Microbiology PCR product was however observed for an isolate from abattoir P (results not shown), hence it could not be submitted for sequencing.Sequence data analysis following NCBI blasting identifed one of the isolates up to genus level (Mycobacterium species at 98.89% identity).Tis isolate was from a sample collected at the abattoir G. Mycobacterium avium (99.61% identity) was isolated from a sample collected at abattoir L, and Mycobacterium senuense (99.02% identity) from abattoir D. Te overall prevalence of Mycobacteria isolated from environmental samples was 15.8%.

Discussion
In this study, no MTBC species were isolated from tissues of slaughtered livestock and environmental samples in abattoirs in the Gauteng province of S.A, and no possible food safety risks were posed to meat consumers.Tis is not a surprise because no lesions resembling M. bovis infection were visible on any of the samples processed.Tuberculosislike lesions are an indicator of the presence of tuberculosis [21,22].According to Botha et al. [23], tuberculosis is a slow and progressive disease that remains asymptomatic for an extended period until the advanced stages of infection when lesions appear.Tis may have been the case in this study that when livestock are taken to the abattoirs for slaughter, the animals could still be in the early stages of infection and the bacterial concentrations are too low to be detected by culture.In South Africa, implementing the national bovine tuberculosis programme, especially in commercial cattle, decreased the national prevalence of bovine tuberculosis to 0.4% over 25 years ago, from 11.85% over half-century ago [3] with sporadic outbreaks still occurring [2].In communal cattle farming, the disease prevalence is largely still unknown, with a few isolated studies reporting varying prevalence ranging from less than 0.5% to more than 15% [24,25].It should be noted that animals sampled in the current study were from auctions and feedlots originating from diferent provinces of the country.Te prevalence of bovine tuberculosis in SA is however, still low contrary to other African countries such as Zambia, Uganda, and Ethiopia, where herd prevalence of up to 50% have been reported in cattle [14,26,27].No isolation was made from pigs and sheep sampled in the current study.Tuberculosis in sheep is generally rare and there are conficting opinions regarding the susceptibility of sheep to MTBC species.
Considering the chronic nature of the disease, potentially infected sheep get slaughtered before the disease develops [28,29].With that said, a few studies have however, reported cases of M. bovis infection in sheep [29][30][31].Although bovine tuberculosis in sheep has never been reported in South Africa [17], known zoonotic mycobacteria, including Mycobacterium avium subsp.hominisuis, Mycobacterium avium subsp.avium, Mycobacterium intracellulare, Mycobacterium bovis and Mycobacterium tuberculosis were previously isolated from slaughtered pigs during the years 1991-2002, hence a potential public health concern [32].
In our previous study, conducted in parallel with the current research, Mareledwane [17] reported an estimated bTB seroprevalence of 4.4% in cattle, indicating past exposure or detection of early infection, an advantage of the cell-mediated immune response-based tests.Although a 0.0% prevalence (absence) for MTBC species was recorded in the current study, NTM were isolated, with the overall prevalence of Mycobacterium spp.estimated to be 0.5% for cattle and 15.8% for environmental sample.Non-tuberculous mycobacteria isolated include Mycobacterium colombiense, Mycobacterium simiae, Mycobacterium avium, and Mycobacterium senuense.It is well known that non-tuberculous mycobacteria interfere with the most widely used tests for the diagnosis of bovine tuberculosis such as the intradermal tuberculin test as well as the gamma interferon test, hence of outmost importance to identify such interferences [9,33].Te NTMs identifed in the current study were previously found to occur in the environment and animals during a country wide study conducted [9].
One of the NTM isolated in our study, M. simiae, has been shown to cause pathological conditions, especially in humans, and has been isolated more especially in immunocompromised patients [34].It has been reported that infection by this NTM leads to extrapulmonary infections [35].Tis is the frst study to investigate the occurrence of Mycobacteria and to isolate NTMs from slaughtered cattle in red meat abattoirs in the Gauteng province of SA.In a previous report, Hlokwe and co-workers isolated Mycobacterium nonchromogenicum and an NTM species closely related to Mycobacterium moriokaense from cattle on two farms in the Eastern Cape province of South Africa.Similar to the outcome of the current study, these NTM species were detected from samples without visible lesions, hence their clinical signifcance could not be determined but the potential to cause disease cannot be ignored [8].

. Conclusion
Te current study demonstrated a very low prevalence of Mycobacterium species in Gauteng red meat abattoirs as confrmed by culture and molecular assays, hence posing a low risk of infection to meat consumers.Importantly, the study demonstrated the absence (0.0% prevalence) of MTBC species in all the abattoir samples tested.However, future studies should pay attention to NTM isolated from abattoirs, as their role in causing mycobacteriosis both in humans and animals still needs to be better understood.

Figure 1 :
Figure 1: Map showing the six districts where red meat abattoirs sampled are located within the Gauteng province of South Africa.

Figure 2 :
Figure 2: Gel electrophoresis results obtained from 16S rRNA PCR analysis.Lane M is a 100 bp DNA marker; lane 1 represents an isolate from abattoir N, lanes 2-4 represent isolates from abattoir P, lanes 5-7 represent isolates from abattoir B while lane 8 represents an isolate from abattoir K, lane + represent positive control (TB 9845A), lane MOTT represents a Mycobacteria positive control (TB9801B) and lanerepresents the negative control which was distilled water.
μl of each forward and reverse primers (50 pmol) and 1-2 μl DNA template.Te PCR cycling parameters were as follows: initial denaturation at 95 °C for 15 minutes, followed by 35 cycles of denaturation at 95 °C for 30 seconds, annealing at 60 °C for 30 seconds, and elongation at 72 °C for 30 seconds and a fnal extension at 72 °C for 10 minutes.

Table 1 :
Characteristics of cattle sampled and the estimated prevalence of Mycobacterium species in Gauteng abattoirs.
3.5.Isolation of Mycobacteria from Environmental Samples.Bacterial isolation was made from water samples from fve abattoirs: L, P, O, G, and D. All isolates were confrmed as acid-fast by Ziehl-Neelsen staining.3.6.Characterization of Mycobacterium spp.fromEnvironmental Samples by 16S rRNA PCR and Gene SequenceAnalysis.Bacterial isolation was made from fve of the 19 (26.3%) environmental samples.Specifc PCR amplifcation was obtained from four of the fve (15.8%) acid-fast isolates, and the remaining isolate was negative.Te expected size of the PCR product was approximately 577 bp indicative of a mycobacterium species or closely related species.A weak N: indicates number of cattle sampled.

Table 2 :
Characteristics of sheep sampled in the study and the estimated prevalence of Mycobacterium species in Gauteng abattoirs.
N: indicates the number of sheep sampled.

Table 3 :
Characteristics of pigs sampled in the study and the estimated prevalence of Mycobacterium species in Gauteng abattoirs.