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.

Prevalence of methicillin-resistant Staphylococcus aureus (MRSA) in broilers and workers at 'pluck shops' in Trinidad.

Livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA) is a cause of zoonotic infections in many countries. People with occupational contact with food animal production are at risk of colonization. The aim of this study was to determine the prevalence of MRSA and their frequency of resistance to other antimicrobial agents from broilers and workers at the 'pluck shops' in Trinidad. For isolation of MRSA, choanal, cloacal and pharyngeal swabs taken from broilers and nasal swabs from humans were enriched then plated on CHROMagar MRSA and Brilliance MRSA. MRSA was confirmed using the PBP2a test kit, resistance to oxacillin and cefoxitin and polymerase chain reaction (PCR) for the mecA gene. Antimicrobial resistance of the MRSA isolates to 16 antimicrobial agents was determined using the disc diffusion method. Of the 287 broilers and 47 humans sampled, MRSA was isolated at a frequency of 2 (0.7%) and 0 (0.0%) respectively. All the MRSA isolates exhibited resistance to one or more of the 16 antimicrobial agents. The study demonstrated that broilers at 'pluck shops' in Trinidad harbor MRSA. This is the first isolation of MRSA from poultry in Trinidad, West Indies, and this finding is of public health significance since occupational exposure of humans can lead to increased risk of acquiring MRSA infections.

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.

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.

Evidence for Horizontal Transmission and Recirculation of Shiga Toxin-Producing Escherichia coli in the Beef Production Chain in South Africa Using Whole Genome Sequencing.

We used whole genome sequencing (WGS) as an epidemiologic surveillance tool to elucidate the transmission dynamics of Shiga toxin-producing Escherichia coli (STEC) strains along the beef production chain in South Africa. Isolates were obtained from a cattle farm, abattoirs and retail outlets. Isolates were analysed using WGS on a MiSeq platform (Illumina, San Diego, CA, USA) and phylogenetic analysis was carried out. Of the 85 isolates, 39% (33) carried the stx gene and 61% (52) had lost the stx gene. The prevalence of stx subtypes was as follows; stx1a 55% (18/33), stx1b 52% (17/33), stx2a 55% (18/33), stx2b 27% (9/33), stx2dB 30% (10/33) and stx2d1A 15% (5/33). Thirty-five different serogenotypes were detected, of which 65% (56) were flagellar H-antigens and 34% (29) were both O-antigens and flagellar H-antigens. We identified 50 different sequence types (STs), and only nine of the isolates were assigned to three different clonal complexes. Core genome phylogenetic analysis revealed genetic relatedness, and isolates clustered mainly according to their STs and serogenotypes regardless of stx subtypes. This study provides evidence of horizontal transmission and recirculation of STEC strains in Gauteng province and demonstrates that every stage of the beef production chain plays a significant role in STEC entry into the food chain.

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.

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.

Brucellosis Seropositivity Using Three Serological Tests and Associated Risk Factors in Abattoir Workers in Gauteng Province, South Africa.

Abattoir workers are liable to zoonotic infections from animals and animal products, primarily to diseases with asymptomatic and chronic clinical manifestations in animals, such as brucellosis. No published reports exist on the seroprevalence of brucellosis in abattoir workers in South Africa. Therefore, this cross-sectional study was conducted to estimate the occurrence and risk factors for Brucella exposure in abattoir workers in Gauteng Province. A total of 103 abattoir workers and managers from 6 abattoirs, where brucellosis-positive slaughtered cattle and sheep were previously detected, were interviewed and tested with serological assays using the Rose Bengal test (RBT), BrucellaCapt, and IgG-ELISA. A pre-tested questionnaire was administered to consenting respondents to obtain information on risk factors for brucellosis. Of the 103 respondents tested, the distribution of female and male workers was 16 (15.5%) and 87 (84.5%), respectively. The seroprevalence for exposure to brucellosis was 21/103 (20.4%, 95%CI: 13.1-29.5) using a combination of RBT, BrucellaCapt, or IgG-ELISA. For test-specific results, seroprevalences by RBT, BrucellaCapt, and IgG-ELISA were 13/103 (12.6%, 95%CI: 6.9-20.6), 9/103 (8.74%, 95%CI: 4.1-15.9), and 18/103 (17.5%, 95%CI: 10.7-26.2), respectively. Low-throughput abattoirs were identified as associated risks, as 29.3% of workers were seropositive compared with 12.7% of workers in high-throughput abattoirs, which highlights that direct contact at abattoirs poses higher risk to workers than indirect and direct contact outside abattoirs. This study confirms the occurrence of Brucella spp. antibodies among abattoir workers in South Africa, possibly due to occupational exposure to Brucella spp., and highlights the occupational hazard to workers. Furthermore, findings underscore that abattoir facilities can serve as points for active and passive surveillance for indicators of diseases of public health importance. We recommend periodic implementation of brucellosis testing of abattoir workers country-wide to establish baseline data for informing appropriate preventive practices and reducing the potential burden of infection rates among these high-risk workers.

Immune response at a vaccine-challenge study using beagle dogs and locally isolated Leptospira spp.

Determination of the immune response of dogs by measuring the antibody levels (utilizing MAT) and levels of cytokines (TNF-α, IL-4 and IFN-γ) post-vaccination with locally produced killed whole-celled Leptospiral vaccine and post-challenge with a locally isolated Leptospira icterohaemorrhagiae Copenhageni strain. For assessment of immunity of the vaccine serum antibodies were detected before and after vaccination and challenge in three studies. The effects of the challenge were determined by a variety of parameters including reisolation of the challenge Leptospira spp. via blood, urine, and kidney samples. The challenge strain did not produce generalised infection but elevated circulating antibody levels in both the control and vaccinated dogs in any of the three studies, however leptospires were reisolated from the urine of the control dogs but not the vaccinated dogs. Cytokine levels (TNF-α, IFN-γ and IL-4) were detected post-challenge in the vaccinated dogs to determine the immune profile response. The whole-killed cell vaccine in this study did not prevent leptospireamia but prevented leptospiruria in vaccinated dogs after a challenge with a live Leptospira icterohaemorrhagiea Copenhageni. The vaccine-challenge showed increased antibody (MAT) levels due to vaccination and infection (through challenge). Cytokine production (TNF-α, IFN-γ and IL-4) by the host immune system was observed post-challenge with live leptospires.

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.

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.

Phylogenetic analyses of Salmonella detected along the broiler production chain in Trinidad and Tobago.

This study was conducted to determine the phylogenies of Salmonella strains isolated from cross-sectional studies conducted at hatcheries, broiler farms, processing plants, and retail outlets (broiler production chain) in Trinidad and Tobago over 4 yr (2016-2019). Whole-genome sequencing (WGS) was used to characterize Salmonella isolates. Core genome phylogenies of 8 serovars of public health significance were analyzed for similarities in origin and relatedness. In addition, Salmonella strains isolated from human salmonellosis cases in Trinidad were analyzed for their relatedness to the isolates detected along the broiler production chain. The common source of these isolates of diverse serovars within farms, within processing plants, between processing plants and retail outlets, and among farm-processing plant-retail outlet continuum was well-supported (bootstrap value >70%) by the core genome phylogenies for the respective serovars. Also, genome analyses revealed clustering of Salmonella serovars of regional (intra-Caribbean) and international (extra-Caribbean) origin. Similarly, strains of S. Enteritidis and S. Infantis isolated from human clinical salmonellosis in 2019 from Trinidad and Tobago clustered with our processing plant isolates recovered in 2018. This study is the first phylogenetic analysis of Salmonella isolates using WGS from the broiler industry in the Caribbean region. The use of WGS confirmed the genetic relatedness and transmission of Salmonella serovars contaminating chickens in broiler processing, and retailing in the country, with zoonotic and food safety implications for humans.

Molecular Characterization of Leptospira Species Detected in the Kidneys of Slaughtered Livestock in Abattoirs in Gauteng Province, South Africa.

Leptospira was investigated in kidneys (n = 305) from slaughtered livestock in the Gauteng Province abattoirs, South Africa, using a culture medium to isolate Leptospira, followed by the LipL32 qPCR to detect Leptospira DNA. The SecY gene region was amplified, sequenced, and analyzed for LipL32 qPCR-positive samples or Leptospira isolates. The overall frequency of isolation of Leptospira spp. was 3.9% (12/305), comprising 4.8% (9/186), 4.1% (3/74), and 0% (0/45) from cattle, pigs, and sheep, respectively (p > 0.05). However, with LipL32 qPCR, the overall frequency of Leptospira DNA was 27.5%, consisting of 26.9%, 20.3%, and 42.2% for cattle, pigs, and sheep, respectively (p = 0.03). Based on 22 SecY sequences, the phylogenetic tree identified the L. interrogans cluster with serovar Icterohaemorrhagiae and the L. borgpetersenii cluster with serovar Hardjo bovis strain Lely 607. This study is the first molecular characterization of Leptospira spp. from livestock in South Africa. The reference laboratory uses an eight-serovar microscopic agglutination test panel for leptospirosis diagnosis, of which L. borgpetersenii serovar Hardjo bovis is not part. Our data show that pathogenic L. interrogans and L. borgpetersenii are circulating in the livestock population. Diagnostic use of molecular methods will eliminate or reduce the under-reporting of leptospirosis in livestock, particularly sheep, in South Africa.

Serological and bacteriological responses of water buffalo (Bubalus bubalis) vaccinated with two doses of Brucella abortus strain RB51 vaccine.

Thirty-two water buffalo (Bubalus bubalis) calves aged 6­10 months were used to evaluate serological responses to Brucella abortus strain RB51 (RB51) vaccination in a dose-response study and to compare the use of two selective media for the isolation of RB51. The animals were randomly divided into three treatment groups. Groups I-III received the recommended vaccine dose (RD) twice 4 weeks apart, RD twice 18 weeks apart and saline once, respectively. Lymph nodes were excised from the three groups and subjected to bacteriological examination to determine the frequency of detection of RB51. Pre- and post-vaccination blood samples were collected and tested for B. abortus antibodies using the buffered plate agglutination test (BPAT), complement fixation test (CFT), and dot-blot assay. Sera taken at all post-inoculation weeks (PIW) were negative for field strain B. abortus using the BPAT. Antibody responses to RB51 were demonstrated in all vaccinates but not in controls by CFT and dot-blot assay from 1 PIW up to 16 weeks following booster vaccination. The agreement for both assays was 80.7% and there was a linear interdependence with a Pearson's correlation coefficient value of 0.578. The frequency of isolation of RB51 from the two selective media used was not significantly different (P > 0.05).

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.

Prevalence and Characterization of Campylobacter Species from Chickens Sold at Informal Chicken Markets in Gauteng, South Africa.

ABSTRACT: This study determined the prevalence, characteristics, and risk factors of Campylobacter species contamination of chicken carcasses sold at informal poultry outlets in Gauteng province, South Africa. Within six townships, 151 chicken carcasses were collected from 47 outlets. Carcass swab, cloacal swab, and carcass drip samples were collected from each chicken, along with a matched questionnaire on risk factors regarding Campylobacter contamination. Sample-inoculated Bolton broth (BB) was cultured to isolate Campylobacter species by bacteriological methods. Subsequent confirmation and characterization of Campylobacter were conducted using polymerase chain reaction (PCR). Isolated Campylobacter strains were evaluated for the presence of six virulence genes (ciaB, dnaj, pldA, racR, flaA, and flaB), three toxin genes (cdtA, cdtB, and cdtC), and one antimicrobial resistance gene (tetO). The overall prevalence of Campylobacter was 23.4% (106 of 453), with sample type-specific prevalence being 17.2% (26 of 151), 25.8% (39 of 151), and 27.2% (41 of 151) for the carcass swabs, cloacal swabs, and carcass drip, respectively, following bacteriological isolation and confirmation by PCR. The overall prevalence of Campylobacter species was 93.5% by PCR, which varied significantly (P = 0.000) by sample: 99.2, 98.4, and 82.8% for carcass swabs, cloacal swabs, and carcass drip, respectively, by using PCR to detect Campylobacter in BB. Important risk factors for carcass contamination by Campylobacter included the slaughter of culled breeders and spent chickens, the use of stagnant water, and poor sanitation. Virulence and toxin gene frequencies were higher in C. jejuni-positive (82.5%) than in C. coli-positive (71.4%) BB cultures, but tetracycline resistance gene (tetO) frequency was higher in C. coli (75.9%) than in C. jejuni (48.10%). The observed high frequencies in C. jejuni recovered from street-vended chickens may pose food safety and therapeutic concerns to consumers.

Genomic Comparison of Eight Closed Genomes of Multidrug-Resistant Salmonella enterica Strains Isolated From Broiler Farms and Processing Plants in Trinidad and Tobago.

Salmonella enterica is an important foodborne pathogen worldwide. We used long and short-read sequencing to close genomes of eight multidrug-resistant (MDR) S. enterica strains, belonging to serovars Infantis (2), Albany, Oranienburg, I 4,[5],12:i:-, Javiana, Schwarzengrund, and Kentucky from broiler chicken farms and processing plants in Trinidad and Tobago. They also belonged to seven different sequence types (STs- 32, 292, 1510, 19, 24, 152, and 96). Among the strains, seven had demonstrated multi-drug resistance with the presence of at least three AMR genes, whereas three isolates contained the quinolone resistance gene qnr B19 in plasmids (CFSAN103840, CFSAN103854, and CFSAN103872). The extended-spectrum ß-lactamase genes bla CTX-M-65 (CFSAN103796) and bla TEM-1 (CFSAN103852) were detected in this study. The genomes closed in this study will be useful for future source tracking and outbreak investigations in Trinidad and Tobago and worldwide.

Occurrence, Risk Factors, Serotypes, and Antimicrobial Resistance of Salmonella Strains Isolated from Imported Fertile Hatching Eggs, Hatcheries, and Broiler Farms in Trinidad and Tobago.

ABSTRACT: This cross-sectional study was conducted to determine the occurrence, risk factors, and characteristics of Salmonella isolates recovered from imported fertile broiler hatching eggs, hatcheries, and broiler farms in Trinidad and Tobago. Standard methods were used to isolate and characterize Salmonella isolates from two broiler hatcheries and 27 broiler farms in the country. The frequency of isolation of Salmonella was 0.0% for imported fertile hatching eggs (0 of 45 pools of 10 eggs each, i.e., 450 eggs), 7.6% for hatcheries (12 of 158 samples), and 2.8% for broiler farms (24 of 866 samples) (P = 0.006). Stillborn chicks at hatcheries had the highest prevalence of Salmonella (7 of 28 samples, 28.0%), whereas on broiler farms the cloacal swabs had the highest prevalence of Salmonella (15 of 675 samples, 2.2%). None of the 15 farm management and production practices investigated were significantly associated (P > 0.05) with the isolation of Salmonella. The predominant Salmonella serotypes were Kentucky (83.3%) and Infantis (62.5%) among hatchery and farm isolates, respectively. The disk diffusion method revealed frequencies of antimicrobial resistance (i.e., resistance to one or more agents) of 44.0% (11 of 25 isolates) and 87.5% (35 of 40 isolates) at hatcheries and broiler farms, respectively (P = 0.0002). Antimicrobial resistance among hatchery isolates was highest (28.0%) to doxycycline and kanamycin and was very high (>65%) among farm isolates to sulfamethoxazole-trimethoprim, gentamicin, ceftriaxone, kanamycin, and doxycycline. Multidrug resistance (MDR; i.e., resistance to antimicrobial agents from three or more classes) was exhibited by 4.0 and 85.7% of Salmonella isolates recovered from several environmental and animal sources at the hatcheries and farms, respectively (P < 0.0001). The high level of antimicrobial resistance and the presence of MDR among Salmonella isolates from broiler farms highlight the therapeutic implications and the potential for MDR strains to enter the food chain.