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.

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.

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.

Molecular Characterization of Salmonella Detected along the Broiler Production Chain in Trinidad and Tobago.

This cross-sectional study determined the serovars, antimicrobial resistance genes, and virulence factors of Salmonella isolated from hatcheries, broiler farms, processing plants, and retail outlets in Trinidad and Tobago. Salmonella in silico serotyping detected 23 different serovars where Kentucky 20.5% (30/146), Javiana 19.2% (28/146), Infantis 13.7% (20/146), and Albany 8.9% (13/146) were the predominant serovars. There was a 76.0% (111/146) agreement between serotyping results using traditional conventional methods and whole-genome sequencing (WGS) in in silico analysis. In silico identification of antimicrobial resistance genes conferring resistance to aminoglycosides, cephalosporins, peptides, sulfonamides, and antiseptics were detected. Multidrug resistance (MDR) was detected in 6.8% (10/146) of the isolates of which 100% originated from broiler farms. Overall, virulence factors associated with secretion systems and fimbrial adherence determinants accounted for 69.3% (3091/4463), and 29.2% (1302/4463) counts, respectively. Ten of 20 isolates of serovar Infantis (50.0%) showed MDR and contained the blaCTX-M-65 gene. This is the first molecular characterization of Salmonella isolates detected along the entire broiler production continuum in the Caribbean region using WGS. The availability of these genomes will help future source tracking during epidemiological investigations associated with Salmonella foodborne outbreaks in the region and worldwide.

Characterization of Salmonella Isolates Recovered from Stages of the Processing Lines at Four Broiler Processing Plants in Trinidad and Tobago.

This cross-sectional study determined the prevalence, characteristics, and risk factors for contamination of chicken with Salmonella at four operating broiler processing plants in Trinidad. Standard methods were used to isolate and characterize the Salmonella isolates. The overall prevalence of Salmonella at the four processing plants was 27.0% (107/396). The whole carcass enrichment (WCE) method yielded a statistically significantly (p = 0.0014) higher frequency of isolation (53.9%; 97/180) than the whole carcass rinse (35.0%; 63/180) and neck skin methods (42.2%; 38/90). S. enterica serotypes Enteritidis, Javiana, and Infantis were the predominant serotypes isolated accounting for 20.8%, 16.7% and 12.5%, respectively, of the serotyped isolates. Risk factors included the use of over 100 contract farmers (OR 4.4), pre-chiller (OR 2.3), addition of chlorine to chiller (OR 3.2), slaughtering sick broilers (OR 4.4), and flocks with >50% mortality. Multi-drug resistance was detected in 12.3% (14/114) of the isolates of Salmonella. Resistance was high to kanamycin (85.7%) and doxycycline (74.6%) but low to amoxicillin-clavulanic acid (2.4%) and sulphamethoxazole-trimethoprim (0.8%). The occurrence of resistant Salmonella in chickens processed at commercial broiler processing plants has implications for salmonellosis and therapeutic failure in consumers of improperly cooked contaminated chickens from these plants in the country.

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).

Human leptospirosis in the Caribbean, 1997-2005: characteristics and serotyping of clinical samples from 14 countries.

OBJECTIVE: To determine the frequency of human leptospirosis in the sera of suspected clinical cases sent by 14 Caribbean countries for diagnosis to a regional laboratory in 1997-2005. METHODS: All serum samples were initially tested using the immunoglobulin M (IgM) enzyme-linked immunosorbent assay (ELISA) for leptospirosis. Demographic data (such as age and sex), month of the year and clinical manifestations that had been observed by the attending physician were related to seropositivity. The microscopic agglutination test (MAT) was used to serotype sera using a panel of 23 international serovars. RESULTS: Of 3 455 samples tested, 452 (13.1%) were seropositive for IgM antibodies to leptospirosis by the ELISA, with frequencies significantly (P < 0.05; χ2) different across countries and years. Among seropositive patients, the frequency of detection of leptospirosis (23.1%) was significantly higher in the age groups 1-20 years and 31-40 years combined compared with other age groups; and in male patients (72.1%) compared with female patients (19.7%) (P < 0.05; χ2). Chills, jaundice, vomiting, weakness, diarrhea, and kidney failure/problems were significantly (P < 0.05; χ2) exhibited at a higher frequency in seropositive, rather than seronegative patients. Using the MAT on 100 sera tested, 98 (98%) were seropositive, of which the serogroup Icterohaemorrhagiae was most prevalent with the detection of serovars Copenhageni (70%), Icterohaemorrhagiae (67%), and Mankarso (29%). CONCLUSIONS: Since only 13.1% of the suspected cases of leptospirosis were seropositive for IgM ELISA antibodies, other clinical conditions may have been responsible for the clinical manifestations observed, or the patient may have had chronic leptospirosis (IgG). In the Caribbean, serovars of the serogroup Icterohaemorrhagiae were responsible for most infections in the cases tested.

Human leptospirosis in the Caribbean, 1997-2005: characteristics and serotyping of clinical samples from 14 countries / Leptospirosis humana en el Caribe entre 1997 y 2005: características y serotipificación de muestras clínicas de 14 países

OBJECTIVE: To determine the frequency of human leptospirosis in the sera of suspected clinical cases sent by 14 Caribbean countries for diagnosis to a regional laboratory in 1997-2005. METHODS: All serum samples were initially tested using the immunoglobulin M (IgM) enzyme-linked immunosorbent assay (ELISA) for leptospirosis. Demographic data (such as age and sex), month of the year and clinical manifestations that had been observed by the attending physician were related to seropositivity. The microscopic agglutination test (MAT) was used to serotype sera using a panel of 23 international serovars. RESULTS: Of 3 455 samples tested, 452 (13.1 percent) were seropositive for IgM antibodies to leptospirosis by the ELISA, with frequencies significantly (P < 0.05; χ2) different across countries and years. Among seropositive patients, the frequency of detection of leptospirosis (23.1 percent) was significantly higher in the age groups 1-20 years and 31-40 years combined compared with other age groups; and in male patients (72.1 percent) compared with female patients (19.7 percent) (P < 0.05; χ2). Chills, jaundice, vomiting, weakness, diarrhea, and kidney failure/problems were significantly (P < 0.05; χ2) exhibited at a higher frequency in seropositive, rather than seronegative patients. Using the MAT on 100 sera tested, 98 (98 percent) were seropositive, of which the serogroup Icterohaemorrhagiae was most prevalent with the detection of serovars Copenhageni (70 percent), Icterohaemorrhagiae (67 percent), and Mankarso (29 percent). CONCLUSIONS: Since only 13.1 percent of the suspected cases of leptospirosis were seropositive for IgM ELISA antibodies, other clinical conditions may have been responsible for the clinical manifestations observed, or the patient may have had chronic leptospirosis (IgG). In the Caribbean, serovars of the serogroup Icterohaemorrhagiae were responsible for most infections in the cases tested.

OBJETIVO: Determinar la frecuencia de leptospirosis humana en el suero de presuntos casos clínicos enviados por 14 países del Caribe a un laboratorio regional para la confirmación del diagnóstico entre 1997 y 2005. MÉTODOS: Todas las muestras de suero se analizaron inicialmente mediante el ensayo inmunoenzimático de adsorción (ELISA) para detectar inmunoglobulina M (IgM) contra Leptospira. Se relacionó la seropositividad con datos demográficos (como la edad y el sexo), el mes del año y las manifestaciones clínicas observadas por el médico a cargo. Se usó la prueba de aglutinación microscópica para serotipificar los sueros con un grupo de 23 serovariedades internacionales. RESULTADOS: De las 3 455 muestras analizadas por ELISA, 452 (13,1 por ciento) fueron seropositivas para anticuerpos IgM contra Leptospira, con frecuencias significativamente diferentes (P < 0,05; χ2) según el país y el año. En los pacientes seropositivos, la frecuencia de detección de leptospirosis (23,1 por ciento) fue significativamente mayor en los grupos etarios de 1 a 20 años y de 31 a 40 años combinados, en comparación con otros grupos de edad; y mayor en los varones (72,1 por ciento) en comparación con las mujeres (19,7 por ciento) (P < 0,05; χ2). Los escalofríos, la ictericia, los vómitos, la debilidad, la diarrea y la insuficiencia o los trastornos renales fueron significativamente más frecuentes (P < 0,05; χ2) en los pacientes seropositivos que en los seronegativos. De los 100 sueros que se analizaron con la prueba de aglutinación microscópica, 98 (98 por ciento) fueron seropositivos, y entre estos el serogrupo Icterohaemorrhagiae fue el más frecuente, con detección de las serovariedades Copenhageni (70 por ciento), Icterohaemorrhagiae (67 por ciento) y Mankarso (29 por ciento). CONCLUSIONES: Ya que solo 13,1 por ciento de los presuntos casos de leptospirosis fueron seropositivos por ELISA para anticuerpos IgM, las manifestaciones clínicas observadas pueden haberse debido a otras enfermedades, o el paciente puede haber tenido leptospirosis crónica (con anticuerpos IgG). En los casos analizados en el Caribe, las serovariedades del serogrupo Icterohaemorrhagiae causaron la mayoría de las infecciones.

Brucellosis in domestic water buffalo (Bubalus bubalis) of Trinidad and Tobago with comparative epidemiology to cattle.

The water buffalo is an important domestic animal worldwide, and the local Buffalypso variety was developed in Trinidad to have improved beef qualities. Brucellosis was diagnosed in Trinidad and Tobago during 1998 in both cattle and domestic water buffalo (Bubalus bubalis) populations. Brucellosis in the latter species is caused by infection with Brucella abortus, similar to bovine brucellosis. Control of brucellosis is of paramount importance to preservation of the genetic diversity of these animals in Trinidad, and this has been complicated by differences in the epidemiology of water buffalo and bovine brucellosis. Some diagnostic tests do not have comparable accuracy between the two species, and the RB51 vaccine does not adequately protect against infection in water buffalo. The water buffalo in Trinidad may also be more resistant to infection than cattle. Development of effective vaccination protocols is key to brucellosis control in Buffalypso in Trinidad, and prohibitions on import of virulent B. abortus strains for vaccine efficacy studies has impeded progress in this area. These Trinidadian strains are of variable virulence; some might be effective for challenge in vaccine efficacy studies, while other, of lower virulence, may be vaccine candidates for use in water buffalo.

Seroepidemiology of leptospirosis in livestock in Trinidad.

A study was conducted to determine the seroprevalence of leptospirosis and infecting serovars across livestock (cattle, sheep, goats, and pigs) in Trinidad using the microscopic agglutination test with an international panel of 23 serovars. Of a total of 590 cattle tested, 21.5% were seropositive with agglutinations to 13 of the 23 antigens used in the panel. Icterohaemorrhagiae (9.3%), Sejroe (4.1%), Ballum (4.1%), and Autumnalis (1.9%) were the predominant serogroups detected in the cattle sampled (n = 590). Of 222 sheep tested, 5.0% were seropositive with agglutinations to five serovars belonging to two serogroups. These serogroups were Autumnalis at 2.7%, and Icterohaemorrhagiae at 2.3% of all sheep tested (n = 222). Of a total of 180 goats tested, 3.3% were seropositive, all agglutinating to the Icterohaemorrhagiae serogroup, 1.7% to serovar Copenhageni, 1.1% to serovar Mankarso, and 0.6% to serovar Icterohaemorrhagiae. Among pigs (n = 200), 5.0% were seropositive for five serovars belonging to three serogroups. These serogroups were Icterohaemorrhagiae at 2.5%, Australis at 2%, and Ballum at 0.5%. Overall, age and sex of animals were not significantly associated with leptospirosis with the exception of cattle where age was a significant factor for seropositivity. It was concluded that for livestock, leptospirosis may be an important zoonotic and economic disease, particularly in the case of cattle. It is imperative that the impact of leptospirosis on abortion, stillbirths, and decreased milk production in livestock in the country be assessed.

Leptospirosis in water buffalo (Bubalus bubalis) in Trinidad.

The seroprevalence of leptospirosis in water buffalo (Bubalus bubalis) reared for meat in semi-intensive and extensive managed farms in Trinidad was determined. All sera were tested for specific antibodies against 17 internationally recognized serovars of Leptospira using the microscopic agglutination test (MAT). Animals withtitres greater or equal to 100 were considered as seropositive indicating exposure to Leptospira and those withtitres greater or equal to 800 were interpreted as cases of acute leptospirosis. Of a total of 226 apparently healthy water buffalo from five major farms in Trinidad tested, 33 (14.6 per cent) were seropositive with titres ranging from 100 to 400. Three (60.0 per cent) of 5 farms had seropositive animals with seropositivity rates ranging from 2.0 per cent (1 of 50) on Farm A to 32.7 per cent (16 of 49) on Farm B. The difference was statistically signifi cant (P<0.05; X2). Age and sex of animals had no significant (P>0.05; X2) effect on infection rate. The prevalent antibodies to serovars of Leptospira were farm specific with specific antibodies to serovars Copenhageni and Georgia being predominant on Farm B having been detected in 10 (62.5 per cent) and 9 (56.3 per cent) respectively of 16 seropositive animals. On Farm D however, also with 16 seropositive animals, specifi c antibodies to serovars Patoc and Bratislava were most frequently detected, found in 11 (68.8 per cent) and 5 (31.3 per cent) respectively of seropositive animals. This is the first documentation of leptospirosis in water buffalo in the Caribbean region and the health risk posed to farm workers, abattoir workers and veterinarians cannot be ignored.