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The current study was designed to determine the prevalence of antimicrobial resistance and virulence genes in Escherichia coli isolated from canine diarrheal cases. Fecal samples were collected from 77 dogs with clinical diarrhea and 10 non-diarrheic dogs. Breed, age and sex, and clinical manifestations were recorded. Samples were plated on MacConkey and EMB agars, followed by standard isolation procedures and identification of E. coli. An antimicrobial susceptibility test using the disc diffusion method was performed against Ampicillin, Tetracycline, Trimethoprim, Gentamicin, and Streptomycin. PCR was used to determine if the isolates carry virulence intimin adherence protein (eaeA) and antimicrobial resistance genes. Body temperature, respiratory, and heart rates in dogs with diarrhea were significantly higher than in non-diarrheic dogs. PCR detected the eaeA virulence gene in 44(69.8%) of 63 isolates from diarrheic dogs. All isolates were resistant to ampicillin, and 55(87.3%), 47(74.6%), 29(46.0%), and 19(30.2%) of these were resistant to tetracycline, trimethoprim, gentamicin, and streptomycin, respectively. The frequency of antimicrobial resistance genes in the 63 isolates was 81.0, 52.4, 41.3, 33.3, 23.8 and 9.5% for CITM, tet (B), dfrA1, aac (3)-IV, aadA1 and tet (A), respectively. Overall, 6(9.4%), 16(25.4%), and 41(65.1%) were positive for one, two, three, or four resistance genes, respectively. In conclusion, the high prevalence of virulence (69.8%) and resistance 9.5-81.0% genes in E. coli isolates could be responsible for the diarrhea episodes, which may have posed therapeutic implications in affected dogs.

Current diagnostic methods for detecting foodborne pathogens are time-consuming, require sophisticated equipment, and have a low specificity and sensitivity. Magnetic nanoparticles (MNPs) and plasmonic/colorimetric biosensors like gold nanoparticles (GNPs) are cost-effective, high-throughput, precise, and rapid. This study aimed to validate the use of MNPs and GNPs for the early detection of Escherichia coli O157:H7, Salmonella enterica spp., Campylobacter jejuni, and Listeria monocytogenes in bovine fecal samples. The capture efficiency (CE) of the MNPs was determined by using Salmonella Typhimurium (ATCC_13311) adjusted at an original concentration of 1.5 × 108 CFU/mL. One (1) mL of this bacterial suspension was spiked into bovine fecal suspension (1 g of fecal sample in 9 mL PBS) and serially diluted ten-fold. DNA was extracted from Salmonella Typhimurium to determine the analytical specificity and sensitivity/LOD of the GNPs. The results showed that the CE of the MNPs ranged from 99% to 100% and could capture as little as 1 CFU/mL. The LOD of the GNPs biosensor was 2.9 µg/µL. The GNPs biosensor was also tested on DNA from 38 naturally obtained bovine fecal samples. Out of the 38 fecal samples tested, 81.6% (31/38) were positive for Salmonella enterica spp., 65.8% (25/38) for C. jejuni, 55.3% (21/38) for L. monocytogenes, and 50% (19/38) for E. coli O157:H7. We have demonstrated that MNP and GNP biosensors can detect pathogens or their DNA at low concentrations. Ensuring food safety throughout the supply chain is paramount, given that these pathogens may be present in cattle feces and contaminate beef during slaughter.

Abstract Background Coronaviruses have the potential to cross species barriers. To learn the molecular intersections among the most common coronaviruses of domestic and close-contact animals, we analyzed representative coronavirus genera infecting mouse, rat, rabbit, dog, cat, cattle, white-tailed deer, swine, ferret, mink, alpaca, Rhinolophus bat, dolphin, whale, chicken, duck and turkey hosts; reference or complete genome sequences were available for most of these coronavirus genera. Protein sequence alignments and phylogenetic trees were built for the spike (S), envelope (E), membrane (M) and nucleocapsid (N) proteins. The host receptors and enzymes aminopeptidase N (APN), angiotensin converting enzyme 2 (ACE2), sialic acid synthase (SAS), transmembrane serine protease 2 (TMPRSS2), dipeptidyl peptidase 4 (DPP4), cathepsin L (and its analogs) and furin were also compared. Results Overall, the S, E, M, and N proteins segregated according to their viral genera (α, β, or γ), but the S proteins of alphacoronaviruses lacked conservation of phylogeny. Interestingly, the unique polybasic furin cleavage motif found in severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) but not in severe acute respiratory syndrome coronavirus (SARS-CoV) or Middle East respiratory syndrome coronavirus (MERS-CoV) exists in several β-coronaviruses and a few α- or γ-coronaviruses. Receptors and enzymes retained host species-dependent relationships with one another. Among the hosts, critical ACE2 residues essential for SARS-CoV-2 spike protein binding were most conserved in white-tailed deer and cattle. Conclusion The polybasic furin cleavage motif found in several β- and other coronaviruses of animals points to the existence of an intermediate host for SARS-CoV-2, and it also offers a counternarrative to the theory of a laboratory-engineered virus. Generally, the S proteins of coronaviruses show crossovers of phylogenies indicative of recombination events. Additionally, the consistency in the segregation of viral proteins of the MERS-like coronavirus (NC_034440.1) from pipistrelle bat supports its classification as a β-coronavirus. Finally, similarities in host enzymes and receptors did not always explain natural cross-infections. More studies are therefore needed to identify factors that determine the cross-species infectivity of coronaviruses.

ABSTRACT: 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.

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.

The objective of this study was to evaluate the effect of varying levels of hempseed meal supplementation on antibody and cell-mediated immune responses, as well as the expression of some of the important immunoregulatory cytokines. Treatments consisted of hempseed meal supplementation at 0 (control), 10, 20, and 30% of the total diet. Goats were randomly assigned to one of the four treatments n = 10. Cell-mediated immune response was evaluated on day 59 of the feeding period by measuring skinfold thickness at 24 h following intradermal injection of phytohemagglutinin. A significant increase in skinfold thickness was observed with increasing levels of supplementation as compared to that of the control group. Serum antibody titers to chicken ovalbumin were not significantly different between treatment groups. Cytokine concentrations of IL-6 increased linearly with increasing level of supplementation (p < 0.05), contrarily to the linear decrease that was observed for TNF-α (p < 0.05). Although IL-2 tended to increase with the 10 and 30% levels of supplementation (p < 0.07), the result was not significant, and no significant differences were obtained with respect to IL-4 concentrations. Cytokine gene expression values measured by RT-PCR, however, demonstrated some significant differences. HSM supplementation had no significant effect on the expression of IL-2 or IL-6. However, significant differences were observed with the 30% supplementation for IL-4 and TNF-α as compared to that of the control group (p < 0.05). IL-4 was down regulated for the 10 and 20% treatment groups but was upregulated for the 30% treatment group. TNF-α was downregulated in the 10% but upregulated for the 20 and 30% treatment groups. No significant differences were observed for the serum cortisol concentration or white blood cell counts. These results suggested that hempseed meal supplementation may improve cell-mediated immune response while having no effect on antibody-mediated immune response. However, more research needs to be conducted to determine the most efficacious inclusion rate.

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.