Seroprevalence and risk factors of Toxoplasma gondii in sheep and goats of North West Province, South Africa.

BACKGROUND: The protozoan parasite Toxoplasma gondii causes toxoplasmosis, one of the most prevalent parasitic zoonotic diseases with significant economic and public health implications worldwide. Infection with the parasite has a significant adverse effect on sheep and goat production and can frequently go undetected in the herd, resulting in abortions and weak or dead offspring. Although there are few studies on seroprevalence and risk factors associated with T. gondii infections in livestock in other provinces of South Africa, there is no data in the North West province. Therefore, a cross-sectional study was conducted to investigate the seroprevalence of T. gondii and risk factors associated with exposure in sheep and goats of the North West province of South Africa. Sera from 439 livestock (164 sheep and 285 goats) were collected and analysed for the presence of T. gondii IgG antibodies using indirect ELISA (Enzyme-linked immunosorbent assay). An assessment of potential risk factors in farms associated with seropositivity was also conducted using a structured questionnaire. RESULTS: Out of the 439 tested sheep and goats, 13.9% (61/439) were positive for IgG antibodies against T. gondii. Sheep and goats had seroprevalences of 19.5% (32/164) and 10.5% (29/275) respectively. In the multivariable logistic regression model, the risk of acquiring T. gondii was significantly higher in the mixed breed [Odds ratio (OR) = 71.07; 95% confidence interval (CI): 266.8-1893.1; p < 0.011)] animals than white dorper sheep and in farms that burn or bury aborted material (OR = 42.04; CI: 179.9-982.5; p = 0.020) compared to those that only burn aborted material. The risk was lower for the farms in Kagisano-Molopo (OR = 0.00; CI: 0.0-25.4; p = 0.015) and Mahikeng (OR = 0.00; CI: 0.0-4.9; p < 0.001) local municipalities than Greater Taung local municipality, and for the animals that drink water from dams (OR = 0.03; CI: 0.2-58.8; p = 0.021) than those that drink from boreholes. CONCLUSION: The seroprevalence and risk factors associated with transmission observed show that T. gondii infection is widespread in sheep and goats of the North West province.

Q fever and toxoplasmosis in South African livestock and wildlife: a retrospective study on seropositivity, sporadic abortion, and stillbirth cases in livestock caused by Coxiella burnetii.

BACKGROUND: Q fever and toxoplasmosis are economically important zoonoses as they cause considerable losses in livestock (cattle, sheep and goats) and wildlife (antelopes, giraffes, lions, and cheetahs) through reproductive disorders such as abortions and stillbirths. Q fever and toxoplasmosis testing in South Africa is conducted by the Agricultural Research Council-Onderstepoort Veterinary Research (ARC-OVR). However, both zoonoses are understudied and not monitored in South Africa as they are not considered controlled or notifiable diseases in the Animal Disease Act 35 of 1984. A retrospective study was conducted on Q fever (2007-2009) and toxoplasmosis (2007-2017) using diagnostic laboratory data at the ARC-OVR. Also, we report on sporadic abortion and stillbirth cases in livestock from diagnostic tissue samples submitted for Coxiella burnetii polymerase chain reaction (PCR) detection at the ARC-OVR. RESULTS: During 2007 to 2009, 766 animal samples were tested for C. burnetii antibodies and seropositivity was 0.9% (95%CI: 0.3-1.7) with sheep (1.9%; 95%CI: 0.6-4.4) having the highest seropositivity followed by cattle (0.7%; 95%CI: 0.09-2.6), while all goats (0.0%; 95%CI: 0.0-4.2) and wildlife (0.0%; 95%CI: 0.0-2.5) tested were negative. From 2007 to 2017, 567 sera were tested for T. gondii antibodies; overall seropositivity was 12.2% (95%CI: 9.6-15). Wildlife had highest seropositivity to T. gondii antibodies (13.9%; 95%CI: 9.0-19.7) followed by goats (12.9%; 95%CI: 9.2-17.4) and sheep (12.3%; 95%CI: 5.1-23.8) while seropositivity in cattle was 2.4% (95%CI: 0.06-12.9). Of 11 animals tested by C. burnetii PCR detection (2021-2022), 10 (91.0%) were positive. The amplicon sequences showed similarity to Coxiella burnetii strain 54T1 transposase gene partial coding sequence. CONCLUSIONS: We have confirmed the occurrence of the causative agents of Q fever and toxoplasmosis in livestock and wildlife in South Africa, with data limitations. These zoonoses remain of importance with limited information about them in South Africa. This study provides baseline information for future studies on Q fever and toxoplasmosis in South African livestock and wildlife, as well other African countries. Due to limited data collection experienced in this study, it is recommended that improvements in data collection samples tested should include associated factors such as sex, age, and breed of the animals.

Non-tuberculous Mycobacterium species causing mycobacteriosis in farmed aquatic animals of South Africa.

BACKGROUND: Mycobacteriosis caused by non-tuberculous mycobacteria (NTM), is among the most chronic diseases of aquatic animals. In addition, fish mycobacteriosis has substantial economic consequences especially in the aquaculture and fisheries industry as infections may significantly decrease production and trade. Some fish NTM pathogens are highly virulent and zoonotic; as such, infection of aquaria with these pathogens is a public health concern. In this study, we report isolation of nine different NTM species from sixteen aquatic animals including different fish species, frogs and a crocodile. Given the clinical significance of Mycobacterium marinum and its close relation to Mycobacterium tuberculosis, as well as the significance of ESAT 6 and CFP-10 secretion in mycobacterial virulence, we analysed the esxA and esxB nucleotide sequences of M. marinum isolates identified in this study as well as other mycobacteria in the public databases. RESULTS: Mycobacterium shimoidei, Mycobacterium marinum, Mycobacterium chelonae, Mycobacterium septicum /M. peregrinum and Mycobacterium porcinum were isolated from gold fish, Guppy, exotic fish species in South Africa, koi and undefined fish, Knysna seahorse, as well Natal ghost frogs respectively, presenting tuberculosis like granuloma. Other NTM species were isolated from the studied aquatic animals without any visible lesions, and these include Mycobacterium sp. N845 T, Mycobacterium fortuitum, a member of the Mycobacterium avium complex, and Mycobacterium szulgai. Phylogenetic analysis of mycobacteria, based on esxA and esxB genes, separated slow growing from rapidly growing mycobacteria as well as pathogenic from non-pathogenic mycobacteria in some cases. CONCLUSIONS: Isolation of the different NTM species from samples presenting granuloma suggests the significance of these NTM species in causing mycobacteriosis in these aquatic animals. The study also revealed the potential of esxA and esxB sequences as markers for phylogenetic classification of mycobacteria. Observations regarding use of esxA and esxB sequences for prediction of potential pathogenicity of mycobacteria warrants further investigation of these two genes in a study employing NTM species with well-defined pathogenicity.

Mycobacterium komaniense sp. nov., a rapidly growing non-tuberculous Mycobacterium species detected in South Africa.

Some species of non-tuberculous mycobacteria (NTM) have been reported to be opportunistic pathogens of animals and humans. Recently there has been an upsurge in the number of cases of NTM infections, such that some NTM species are now recognized as pathogens of humans and animals. From a veterinary point of view, the major significance of NTM is the cross-reactive immune response they elicit against Mycobacterium bovis antigens, leading to misdiagnosis of bovine tuberculosis. Four NTM isolates were detected from a bovine nasal swab, soil and water, during an NTM survey in South Africa. These were all found using 16S rRNA gene sequence analysis to be closely related to Mycobacterium moriokaense. The isolates were further characterised by sequence analysis of the partial fragments of hsp65, rpoB and sodA. The genome of the type strain was also elucidated. Gene (16S rRNA, hsp65, rpoB and sodA) and protein sequence data analysis of 6 kDa early secretory antigenic target (ESAT 6) and 10 kDa culture filtrate protein (CFP-10) revealed that these isolates belong to a unique Mycobacterium species. Differences in phenotypic and biochemical traits between the isolates and closely related species further supported that these isolates belong to novel Mycobacterium species. We proposed the name Mycobacterium komaniense sp. nov. for this new species. The type strain is GPK 1020T (=CIP 110823T=ATCC BAA-2758).

Mycobacterium malmesburyense sp. nov., a non-tuberculous species of the genus Mycobacterium revealed by multiple gene sequence characterization.

Non-tuberculous mycobacteria (NTM) are ubiquitous in the environment, and an increasing number of NTM species have been isolated and characterized from both humans and animals, highlighting the zoonotic potential of these bacteria. Host exposure to NTM may impact on cross-reactive immune responsiveness, which may affect diagnosis of bovine tuberculosis and may also play a role in the variability of the efficacy of Mycobacterium bovis BCG vaccination against tuberculosis. In this study we characterized 10 NTM isolates originating from water, soil, nasal swabs of cattle and African buffalo as well as bovine tissue samples. These isolates were previously identified during an NTM survey and were all found, using 16S rRNA gene sequence analysis to be closely related to Mycobacterium moriokaense. A polyphasic approach that included phenotypic characterization, antibiotic susceptibility profiling, mycolic acid profiling and phylogenetic analysis of four gene loci, 16S rRNA, hsp65, sodA and rpoB, was employed to characterize these isolates. Sequence data analysis of the four gene loci revealed that these isolates belong to a unique species of the genus Mycobacterium. This evidence was further supported by several differences in phenotypic characteristics between the isolates and the closely related species. We propose the name Mycobacterium malmesburyense sp. nov. for this novel species. The type strain is WCM 7299T (=ATCC BAA-2759T=CIP 110822T).

Mycobacterium tuberculosis infection in cattle from the Eastern Cape Province of South Africa.

BACKGROUND: Mycobacterium tuberculosis is the main causative agent of tuberculosis (TB) in human and Mycobacterium bovis commonly causes tuberculosis in animals. Transmission of tuberculosis caused by both pathogens can occur from human to animals and vice versa. RESULTS: In the current study, M. tuberculosis, as confirmed by polymerase chain reaction (PCR) using primers targeting 3 regions of difference (RD4, RD9 and RD12) on the genomes, was isolated from cattle originating from two epidemiologically unrelated farms in the Eastern Cape (E.C) Province of South Africa. Although the isolates were genotyped with variable number of tandem repeat (VNTR) typing, no detailed epidemiological investigation was carried out on the respective farms to unequivocally confirm or link humans as sources of TB transmission to cattle, a move that would have embraced the 'One Health' concept. In addition, strain comparison with human M. tuberculosis in the database from the E.C Province and other provinces in the country did not reveal any match. CONCLUSIONS: This is the first report of cases of M. tuberculosis infection in cattle in South Africa. The VNTR profiles of the M. tuberculosis strains identified in the current study will form the basis for creating M. tuberculosis VNTR database for animals including cattle for future epidemiological studies. Our findings however, call for urgent reinforcement of collaborative efforts between the veterinary and the public health services of the country.

Virulence Potential and Antimicrobial Resistance of Listeria monocytogenes Isolates Obtained from Beef and Beef-Based Products Deciphered Using Whole-Genome Sequencing.

Listeria monocytogenes is a ubiquitous bacterial pathogen that threatens the food chain and human health. In this study, whole-genome sequencing (WGS) was used for the genomic characterization of L. monocytogenes (n = 24) from beef and beef-based products. Multilocus Sequence Type (MLST) analysis revealed that ST204 of CC204 was the most common sequence type (ST). Other sequence types detected included ST1 and ST876 of CC1, ST5 of CC5, ST9 of CC9, ST88 of CC88, ST2 and ST1430 of CC2, and ST321 of CC321. Genes encoding for virulence factors included complete LIPI-1 (pfrA-hly-plcA-plcB-mpl-actA) from 54% (13/24) of the isolates of ST204, ST321, ST1430, and ST9 and internalin genes inlABC that were present in all the STs. All the L. monocytogenes STs carried four intrinsic/natural resistance genes, fosX, lin, norB, and mprF, conferring resistance to fosfomycin, lincosamide, quinolones, and cationic peptides, respectively. Plasmids pLGUG1 and J1776 were the most detected (54% each), followed by pLI100 (13%) and pLM5578 (7%). The prophage profile, vB_LmoS_188, was overrepresented amongst the isolates, followed by LP_101, LmoS_293_028989, LP_030_2_021539, A006, and LP_HM00113468. Listeria genomic island 2 (LGI-2) was found to be present in all the isolates, while Listeria genomic island 3 (LGI-3) was present in a subset of isolates (25%). The type VII secretion system was found in 42% of the isolates, and sortase A was present in all L. monocytogenes genomes. Mobile genetic elements and genomic islands did not harbor any virulence, resistance, or environmental adaptation genes that may benefit L. monocytogenes. All the STs did not carry genes that confer resistance to first-line antibiotics used for the treatment of listeriosis. The characterization of L. monocytogenes in our study highlighted the environmental resistance and virulence potential of L. monocytogenes and the risk posed to the public, as this bacterium is frequently found in food and food processing environments.

Coxiella burnetii infections from animals and ticks in South Africa: a systematic review.

Coxiella burnetii is a zoonotic intracellular bacterium that is widely distributed and affects domestic animals, wildlife, humans and non-mammalian species. This systematic review was aimed at synthesizing research findings on C. burnetii in both domestic and wild animals of South Africa. The systematic review protocol was registered with Open Society Foundations of systematic reviews ( https://doi.org/10.17605/OSF.IO/8WS ). PRISMA guidelines were followed to collect and evaluate relevant scientific articles published on C. burnetii infecting domestic and wild animals in South Africa. Published articles were sourced from five electronic databases, namely, Google Scholar, PubMed and ScienceDirect, EBSCO and Scopus. Results showed 11 eligible studies involving four domestic animals, three wild animals and one ectoparasite species from seven provinces across South Africa. The occurrence of C. burnetii infection was high in Ceratotherium simum (white rhinoceros) (53.9%), medium in sheep (29.0%) and low in pigs (0.9%). Limpopo province (26%) had the most recorded infections followed by KwaZulu-Natal (19%) and Free State (3%) had the least reported occurrence of C. burnetii. The current study discovered that there is scarcity of published research on prevalence and distribution of C. burnetii infecting domestic and wild animals in South Africa, and this is of concern as this bacterium is an important zoonotic pathogen of "One Health" importance.

Antimicrobial resistance profiles of Listeria species recovered from retail outlets in Gauteng Province, South Africa.

The study determined the antimicrobial resistance (AMR) profiles of Listeria spp. (L. monocytogenes, L. innocua and L. welshimeri) recovered from beef and beef products sold at retail outlets in Gauteng Province, South Africa. A total of 112 isolates of Listeria spp., including L. monocytogenes (37), L. innocua (65), and L. welshimeri (10), were recovered from beef and beef products collected from 48 retail outlets. Listeria spp. was recovered by direct selective plating following selective enrichment, and PCR was used to confirm and characterize recovered isolates. The disc diffusion method determined the resistance to 16 antimicrobial agents. All 112 isolates of Listeria spp. exhibited resistance to one or more antibiotics (P<0.05). The prevalence of AMR in Listeria isolates was high for nalidixic acid (99.1%) and cefotaxime (80.4%) but low for gentamycin (2.7%), sulfamethoxazole-trimethoprim (3.6%), azithromycin (5.4%), and doxycycline (6.3%). Overall, for the three species of Listeria, the prevalence of resistance varied significantly only for streptomycin (P=0.016) and tetracycline (P=0.034). Multidrug-resistant isolates were detected in 75.7% (28/37), 61.5% (40/65), and 80% (8/10) isolates of L. monocytogenes, L. innocua, and L. welshimeri, respectively. The prevalence of AMR was significantly affected by the location and size of retail outlets, type of beef and beef products, and serogroups of L. monocytogenes. The high prevalence of AMR, particularly among the L. monocytogenes isolates, poses potential therapeutic implications for human consumers of contaminated beef products. There is, therefore, a need to regulate and enforce the use of antimicrobial agents in humans and animals in South Africa.

The Presence of esat-6 and cfp10 and Other Gene Orthologs of the RD 1 Region in Non-Tuberculous Mycobacteria, Mycolicibacteria, Mycobacteroides and Mycolicibacter as Possible Impediments for the Diagnosis of (Animal) Tuberculosis.

The Esx-1 family proteins of the Type VII secretion systems of Mycobacterium bovis and Mycobacterium tuberculosis have been assessed and are frequently used as candidates for tuberculosis (TB) diagnosis in both humans and animals. The presence of ESAT-6 and CFP 10 proteins, which are the most immunogenic proteins of the Esx-1 system and have been widely investigated for the immunodiagnosis of tuberculosis, in some Mycobacteriaceae and in Mycobacterium leprae, poses limitations for their use in specific diagnoses of TB. As such, to improve the specificity of the ESAT-6/CFP 10-based cell-mediated immunity (CMI) assays, other proteins encoded by genes within and outside the RD 1 region of the esx-1 locus have been evaluated as candidate antigens for CMI, as well as to investigate humoral responses in combination with ESAT-6 and or CFP 10, with varying specificity and sensitivity results. Hence, in this study, we evaluated various non-tuberculous mycobacteria (NTM), Mycolicibacterium, Mycolicibacter and Mycobacteroides species genomes available on the NCBI database for the presence and composition of the RD1 region of the esx-1 locus. In addition, we also assayed by polymerase chain reaction (PCR) and sequencing of Mycobacteriaceae available in our culture collection for the presence and sequence diversity of esxA and esxB genes encoding ESAT-6 and CFP 10, respectively. Whole genome sequence (WGS) data analysis revealed the presence of RD 1 gene orthologs in 70 of the over 100 published genomes of pathogenic and non- pathogenic Mycobcteriaceae other than tuberculosis. Among species evaluated from our culture collection, in addition to earlier reports of the presence of esxA and esxB in certain Mycolicibacterium, Mycolicibacterium septicum/peregrinum, Mycolicibacterium porcinum and Mycobacterium sp. N845T were also found to harbour orthologs of both genes. Orthologs of esxA only were detected in Mycobacterium brasiliensis, Mycolicibacterium elephantis and Mycolicibacterium flouroantheinivorans, whereas in Mycolicibacter engbackii, Mycolicibacterium mageritense and Mycobacterium paraffinicum, only esxB orthologs were detected. A phylogenetic analysis based on esxA and esxB sequences separated slow-growing from rapidly growing bacteria. These findings strengthen previous suggestions that esxA and esxB may be encoded in the majority of Mycobacteriaceae. The role of the Esx-1 system in both pathogenic and non-pathogenic Mycobacteriaceae needs further investigation, as these species may pose limitations to immunological assays for TB.

Detection of Pathogenic Serogroups and Virulence Genes in Listeria monocytogenes Strains Isolated from Beef and Beef Products Retailed in Gauteng Province, South Africa, Using Phenotypic and Polymerase Chain Reaction (PCR)-Based Methods.

South Africa recently (2017-18) experienced the largest outbreak of human listeriosis in the world caused by L. monocytogenes following the consumption of "polony," a ready-to-eat meat product. Most (59%) cases originated from Gauteng province, South Africa. As a follow-up study to the outbreak, we used standard bacteriological and molecular methods to determine the prevalence of pathogenic and virulent serogroups of L. monocytogenes in various beef and beef products retailed in Gauteng province, South Africa. The overall prevalence of Listeria spp. was 28% (112/400), comprising Listeria monocytogenes (9.3%), Listeria innocua (16.3%), and Listeria welshimeri (2.5%) (p < 0.001). It is crucial to have detected that the region (p=0.036), type of product (p=0.032), and temperature at storage (p=0.011) significantly affected the occurrence of L. monocytogenes in beef products. It is alarming that pathogenic serogroups 4b-4d-4e (51.4%) and 1/2a-3a (43.2%) were detected among the isolates of L. monocytogenes. Importantly, they were all carriers of seven virulence-associated genes (hlyA, inlB, plcA, iap, inlA, inlC, and inlJ). Our study also demonstrated that 16.7% of "polony" samples investigated were contaminated with L. monocytogenes. Considering that pathogenic and virulent L. monocytogenes contaminated beef and beef products retailed in South Africa, the food safety risk posed to consumers remains and cannot be ignored. Therefore, it is imperative to reduce the contamination of these products with L. monocytogenes during beef production, processing, and retailing to avoid future outbreaks of human listeriosis in the country.

Whole Genome Sequence Analysis of Listeria monocytogenes Isolates Obtained from the Beef Production Chain in Gauteng Province, South Africa.

The study used whole-genome sequencing (WGS) and bioinformatics analysis for the genomic characterization of 60 isolates of Listeria monocytogenes obtained from the beef production chain (cattle farms, abattoirs, and retail outlets) in Gauteng province, South Africa. The sequence types (STs), clonal complexes (CCs), and the lineages of the isolates were determined using in silico multilocus sequence typing (MLST). We used BLAST-based analyses to identify virulence and antimicrobial genes, plasmids, proviruses/prophages, and the CRISPR-Cas system. The study investigated any association of the detected genes to the origin in the beef production chain of the L. monocytogenes isolates. Overall, in 60 isolates of Listeria monocytogenes, there were seven STs, six CCs, forty-four putative virulence factors, two resistance genes, one plasmid with AMR genes, and three with conjugative genes, one CRISPR gene, and all 60 isolates were positive for proviruses/prophages. Among the seven STs detected, ST204 (46.7%) and ST2 (21.7%) were the most prominent, with ST frequency varying significantly (p < 0.001). The predominant CC detected were CC2 (21.7%) and CC204 (46.7%) in lineages I and II, respectively. Of the 44 virulence factors detected, 26 (across Listeria Pathogenicity Islands, LIPIs) were present in all the isolates. The difference in the detection frequency varied significantly (p < 0.001). The two AMR genes (fosX and vga(G)) detected were present in all 60 (100%) isolates of L. monocytogenes. The only plasmid, NF033156, was present in three (5%) isolates. A CRISPR-Cas system was detected in six (10%), and all the isolates carried proviruses/prophages. The source and sample type significantly affected the frequencies of STs and virulence factors in the isolates of L. monocytogenes. The presence of fosX and vga(G) genes in all L. monocytogenes isolates obtained from the three industries of the beef production chain can potentially cause therapeutic implications. Our study, which characterized L. monocytogenes recovered from the three levels in the beef production chain, is the first time genomics was performed on this type of data set in the country, and this provides insights into the health implications of Listeria.

Identification of Listeria species and Multilocus Variable-Number Tandem Repeat Analysis (MLVA) Typing of Listeria innocua and Listeria monocytogenes Isolates from Cattle Farms and Beef and Beef-Based Products from Retail Outlets in Mpumalanga and North West Provinces, South Africa.

In this study, Listeria isolates (214) were characterized as follows: L. innocua (77.10%), L. monocytogenes (11.21%), L. welshimeri (5.61%), L. grayi (1.40%), L. seeligeri (0.93%), and L. species (3.73%) that were not identified at the species level, from beef and beef based products from retail and farms in Mpumalanga and North West provinces of South Africa. MLVA was further used to type Listeria innocua isolates (165) and Listeria monocytogenes isolates (24). The L. monocytogenes isolates were also serogrouped using PCR. The MLVA protocol for L. monocytogenes typing included six tandem repeat primer sets, and the MLVA protocol for L. innocua included the use of three tandem repeats primer sets. The L. monocytogenes serogroups were determined as follows: 4b-4d-4e (IVb) (37.50%), 1/2a-3a (IIa) (29.16%), 1/2b-3b (IIb) (12.50%), 1/2c-3c (IIc) (8.33%), and IVb-1 (4.16%). MLVA could cluster isolates belonging to each specie, L. monocytogenes, and L. innocua isolates, into MLVA-related strains. There were 34 and 10 MLVA types obtained from the MLVA typing of L. innocua and L. monocytogenes, respectively. MLVA clustered the L. monocytogenes isolates irrespective of sample category, serogroups, and geographical origin. Similarly, the L. innocua isolates clustered irrespective of meat category and geographical origin. MLVA was able to cluster isolates based on MLVA relatedness. The clustering of isolates from farms and retailers indicates transmission of Listeria spp. MLVA is an affordable, simple, and discriminatory method that can be used routinely to type L. monocytogenes and L. innocua isolates.

Mycobacteriosis in slaughter pigs from South Africa from 1991 to 2002: Mycobacterium spp. diversity and Mycobacterium avium complex genotypes.

Introduction: Mycobacterium avium complex (MAC) bacteria are the most prominent etiological agents of lymphadenitis in pigs. M. avium subspecies hominissuis (MAH) is a member of MAC and has been reported in many parts of the world to be the most prevalent non-tuberculous mycobacteria (NTM) to cause mycobacteriosis in humans, mainly in children. Thus, the economic and zoonotic impact of MAC species are increasingly being recognized. In South Africa, little is known about the distribution of NTM and the molecular epidemiology of M. avium in pigs. Materials and methods: In this study, lymph nodes including mandibular, mesenteric, submandibular, and retropharyngeal, with tuberculosis-like lesions were collected during routine meat inspection of slaughter pigs with no disease symptoms (n = 132), between 1991 and 2002. These pigs were slaughtered at 44 abattoirs distributed across seven of the nine South African provinces. Mycobacterial culture, polymerase chain reaction (PCR), and sequencing of the Mycobacterium specific 577 bp 16S rRNA gene fragment were performed for species and subspecies identification. Results: The majority of the isolates (each per sample); 114 (86.4%) were identified as MAH, 8 (6%) as MAA/M. avium subsp. silvaticum, 4 (3%) were Mycobacterium tuberculosis, 2 (1.5%) as Mycobacterium intracellulare, and 1 (0.75%) as Mycobacterium bovis. The other isolates were identified as Mycobacterium lentiflavum (0.75%), Mycobacterium novocastrense (0.75%), and a Micrococcus spp. (0.75%). Using an eight-marker MLVA typing tool, we deciphered at least nine MIRU VNTR INMV types of MAH and MAA. Discussion: Identification of known zoonotic mycobacteria, including MAH, MAA, M. intracellulare, M. bovis, and M. tuberculosis, from slaughter pigs has a potential public health impact and also strengthens recognition of the potential economic impact of MAC. This study has also for the first time in South Africa, revealed MAC MIRU VNTR INMV genotypes which will aid in the future epidemiological investigation of MAC in South Africa.

Genomic Characterization of Listeria innocua Isolates Recovered from Cattle Farms, Beef Abattoirs, and Retail Outlets in Gauteng Province, South Africa.

Whole-genome sequencing (WGS) was used for the genomic characterization of one hundred and ten strains of Listeria innocua (L. innocua) isolated from twenty-three cattle farms, eight beef abattoirs, and forty-eight retail outlets in Gauteng province, South Africa. In silico multilocus sequence typing (MLST) was used to identify the isolates' sequence types (STs). BLAST-based analyses were used to identify antimicrobial and virulence genes. The study also linked the detection of the genes to the origin (industries and types of samples) of the L. innocua isolates. The study detected 14 STs, 13 resistance genes, and 23 virulence genes. Of the 14 STs detected, ST637 (26.4%), ST448 (20%), 537 (13.6%), and 1085 (12.7%) were predominant, and the frequency varied significantly (p < 0.05). All 110 isolates of L. innocua were carriers of one or more antimicrobial resistance genes, with resistance genes lin (100%), fosX (100%), and tet(M) (30%) being the most frequently detected (p < 0.05). Of the 23 virulence genes recognized, 13 (clpC, clpE, clpP, hbp1, svpA, hbp2, iap/cwhA, lap, lpeA, lplA1, lspA, oatA, pdgA, and prsA2) were found in all 110 isolates of L. innocua. Overall, diversity and significant differences were detected in the frequencies of STs, resistance, and virulence genes according to the origins (source and sample type) of the L. innocua isolates. This, being the first genomic characterization of L. innocua recovered from the three levels/industries (farm, abattoir, and retail) of the beef production system in South Africa, provides data on the organism's distribution and potential food safety implications.

Prevalence and risk factors of Q fever (Coxiella burnetii) in cattle on farms of Limpopo province, South Africa.

Q fever in animals and humans and its economic and public health significance has been widely reported worldwide but in South Africa. There are few studies on the prevalence of this zoonosis and its associated risk factors in South African livestock. Therefore, a cross-sectional study was conducted to determine the seroprevalence, molecular prevalence, and risk factors associated with C. burnetii in cattle on farms in South Africa's Limpopo province. Out of 383 cattle tested for antibodies, the overall seroprevalence was 24.28%. Herd size of >150 (OR: 9.88; 95%CI: 3.92-24.89; p < 0.01) remained associated with C. burnetii seropositivity in cattle. For PCR detection, targeting IS1111 fragment, cattle with no abortion history (OR: 0.37; 95%CI: 0.18-0.77; p < 0.01) and herd size of >150 (OR: 3.52; 95%CI: 1.34-9.24; p < 0.01) remained associated with C. burnetii positivity. The molecular prevalence in sheath scrapings and vaginal swabs by IS1111 PCR was 15.67%. Cohen's kappa agreement test revealed a fair agreement between the PCR and ELISA results (k = 0.40). Sequence analysis revealed that the amplicons had similarities to the C. burnetii transposase gene fragment, confirming the presence of the pathogen. The higher seroprevalence than molecular prevalence indicated a past C. burnetii infection, no bacterial shedding through vaginal mucus in cows, or preputial discharge in bulls. Similarly, the detection of C. burnetii by PCR in the absence of antibodies could be partly explained by recent infections in which antibodies have not yet been produced against the bacteria, or the level of these antibodies was below the detectability threshold. The presence of the pathogen in cattle and the evidence of exposure, as shown by both PCR and ELISA suggests an active circulation of the pathogen. This study demonstrated that C. burnetii is widespread in the study area and that a herd size of >150 is associated with C. burnetii seroprevalence and molecular prevalence.

A novel multiplex qPCR­HRM assay for the simultaneous detection of four abortive zoonotic agents in cattle, sheep, and goats.

Abortifacient pathogens induce substantial economic losses in the livestock industry worldwide, and many of these pathogens are zoonotic, impacting human health. As Brucella spp., Coxiella burnetii, Leptospira spp., and Listeria monocytogenes cause abortion, rapid differential molecular diagnostic tests are needed to facilitate early and accurate detection of abortion to establish effective control measures. However, the available molecular methods are laborious, time-consuming, or costly. Therefore, we developed and validated a novel multiplex real-time polymerase chain reaction (qPCR) method based on high-resolution melting (HRM) curve analysis to simultaneously detect and differentiate four zoonotic abortifacient agents in cattle, goats, and sheep. Our HRM assay generated four well-separated melting peaks allowing the differentiation between the four zoonotic abortifacients. Out of 216 DNA samples tested, Brucella spp. was detected in 45 samples, Coxiella burnetii in 57 samples, Leptospira spp. in 12 samples, and Listeria monocytogenes in 19 samples, co-infection with Brucella spp. and Coxiella burnetii in 41 samples, and 42 samples were negative. This assay demonstrated good analytical sensitivity, specificity, and reproducibility. This is a valuable rapid, cost-saving, and reliable diagnostic tool for detecting individual and co-infections for zoonotic abortifacient agents in ruminants.

Multiple-Locus Variable-Number Tandem Repeat Analysis Genotypes of Listeria monocytogenes Isolated from Farms, Abattoirs, and Retail in Gauteng Province, South Africa.

ABSTRACT: The use of multiple-locus variable-number analysis (MLVA) of tandem repeats (TRs) for subtyping Listeria monocytogenes has proven to be reliable and fast. This study determined the MLVA genotypes of 60 isolates of L. monocytogenes recovered from cattle farms, abattoirs, and retail outlets in Gauteng province, South Africa. The distribution of the 60 L. monocytogenes isolates analyzed by type of sample was as follows: raw beef (28, 46.7%), ready-to-eat beef products (9, 15.0%), beef carcass swabs (9, 15.0%), cattle environment (6, 10.0%), and cattle feces (8, 13.3%). The serogroups of the isolates were determined using PCR and the MLVA genotypes based on six selected loci. The frequency of the 60 serogroups detected was as follows: 1/2a-3a (IIa) (27, 45.0%); 4b-4d-4e (1Vb) (24, 40.0%); 1/2c-3c (IIc) (8, 13.3%); and 1/2b-3b (IIb) (1, 1.7%). MLVA successfully clustered genetically related isolates and differentiated nonrelated isolates, irrespective of their sources, sample types, and serogroups, as demonstrated by 16 MLVA pattern types detected. For serogroup 4b-4d-4e (IVb), there was no variation in TRs LM-TR2, LM-TR4, and LM-TR6, which each contained only one allele (02, 00, and 93, respectively). However, across the sources and sample types of isolates, there was variation in serogroup 4b-4d-4e (IVb): LM-TR1 contained 00, 03, and 05; LM-TR3 contained 14, 20, and 22; and LM-TR5 contained 14, 21, and 25. Similar patterns of variation in the TRs were detected in the other serogroups (1/2a-3a, 1/2b-3b, and 1/2c-3c). BioNumeric data analysis identified at least five types in Gauteng province. MLVA epidemiologically clustered the related isolates and differentiated unrelated isolates.

Antimicrobial Resistance Profiles of Salmonella Isolates on Chickens Processed and Retailed at Outlets of the Informal Market in Gauteng Province, South Africa.

The study determined the antimicrobial resistance profiles of Salmonella on chickens processed and retailed at outlets of the informal markets in Gauteng province, South Africa. The study also investigated the relationship of antimicrobial resistant Salmonella to the source and type of samples and their serotypes. Carcass swabs, cloacal swabs and carcass drips were randomly collected from each of 151 slaughtered chickens from six townships. Isolation and identification were performed using standard and polymerase chain reaction (PCR) methods. The disc diffusion method was used to determine the resistance of Salmonella isolates to 16 antimicrobial agents and PCR to determine their serovars. Ninety-eight (64.9%) of the 151 chickens were contaminated with Salmonella of which 94.9% (93/98) were resistant serovars. The frequency of antimicrobial resistance of Salmonella isolates was high to erythromycin (94.9%) and spectinomycin (82.7%) but was low to ciprofloxacin (1.0%) and norfloxacin (1.0%) (p < 0.05). All 170 isolates of Salmonella tested exhibited resistance to one or more antimicrobial agents and the frequency varied significantly (p < 0.05) across the townships, the type of samples and the serovars. The prevalence of multidrug resistance (MDR) in Salmonella was 81.8% (139/170). Our findings pose zoonotic, food safety and therapeutic risks to workers and consumers of undercooked, contaminated chickens from these outlets.

Prevalence, Serovars, and Factors Associated with Salmonella Contamination of Chicken Carcasses Sold in Outlets of the Informal Market in Gauteng Province, South Africa.

ABSTRACT: Salmonella has been linked to many foodborne illnesses and epidemics in both humans and animals. This cross-sectional study determined the prevalence, serovars, and factors associated with Salmonella contamination of chickens slaughtered in informal market outlets in Gauteng Province, South Africa. A total of 151 chicken carcasses were randomly collected from 47 outlets. Standard bacteriological and molecular methods were used to isolate, identify, and determine the serovar of Salmonella isolates. The prevalence of Salmonella in carcass swabs, cloacal swabs, and carcass drips was 29.1% (44 of 151), 27.2% (41 of 151), and 43.7% (66 of 151), respectively, and the differences were statistically significant (P = 0.004). Only 5 (township locations of outlet, throughput, carcass evisceration, location of carcass for sale, and outlet sanitation) of 10 factors investigated for the contamination of carcasses by Salmonella were statistically significantly (P < 0.05) associated with the isolation of Salmonella. Of the 268 isolates of Salmonella, 157 (58.6%) were typeable using a limited molecular PCR technique, and nine serovars were identified. The predominant Salmonella enterica serovars were Bovismorbificans (31.0%), Enteritidis (7.5%), and Hadar (6.7%). The five important factors found to be significantly associated with the isolation of Salmonella at these outlets offer opportunities for the reduction of Salmonella contamination. There is a need for further investigation of the probable causes of the predominant isolation of Salmonella serovar Bovismorbificans in chickens and its potential implications for human infections in South Africa. It is concluded that chickens purchased from the informal market in Gauteng Province can be a source for salmonellosis in humans if improperly cooked before consumption.