Knowledge of porcine astrovirus diversity and epidemiology remains limited. We used a broad range PCR approach to investigate the presence and diversity of astroviruses in healthy pigs of different ages on 20 farms and in 3 slaughterhouses situated in the province of Quebec, Canada between 2005 and 2007. Our study unexpectedly revealed remarkable levels of genetic diversity and high prevalence of astroviruses in pigs of this province. Astroviruses were detected on every farm investigated and in all age groups of pigs, from suckling piglets to adults. In addition, we found that nearly 80% of healthy finisher pigs harbour astroviruses in their intestine at slaughter. Phylogenetic evidence based on partial polymerase and complete capsid sequences, suggests that porcine astroviruses do not form a monophyletic group but are rather found on separate branches across the mamastrovirus tree. In addition to type species strains, we found highly divergent strains that form two additional lineages, one of which falls outside existing taxonomic groups. The presence of diverse astroviruses in a majority of healthy pigs likely represents a continuous source of infection to piglets and possibly to other animal species including humans. Porcine astrovirus strains appeared phylogenetically related not only to prototypical human astroviruses, as was already known, but also to novel human strains recently discovered suggesting multiple cross species transmission events between these hosts and other animal species. Overall, the findings reported in this study suggest an active role of pigs in the evolution and ecology of the Astroviridae.

f Irrigation water has been implicated as a likely source of produce contamination by Salmonella enterica. Therefore, the distribution of S. enterica was surveyed monthly in irrigation ponds (n ‫؍‬ 10) located within a prime agricultural region in southern Georgia and northern Florida. All ponds and 28.2% of all samples (n ‫؍‬ 635) were positive for Salmonella, with an overall geometric mean concentration (0.26 most probable number [MPN]/liter) that was relatively low compared to prior reports for rivers in this region. Salmonella peaks were seasonal; the levels correlated with increased temperature and rainfall (P < 0.05). The numbers and occurrence were significantly higher in water (0.32 MPN/liter and 37% of samples) than in sediment (0.22 MPN/ liter and 17% of samples) but did not vary with depth. Representative isolates (n ‫؍‬ 185) from different ponds, sample types, and seasons were examined for resistance to 15 different antibiotics; most strains were resistant to streptomycin (98.9%), while 20% were multidrug resistant (MDR) for 2 to 6 antibiotics. DiversiLab repetitive extragenic palindromic-element sequence-based PCR (rep-PCR) revealed genetic diversity and showed 43 genotypes among 191 isolates, as defined by >95% similarity. The genotypes did not partition by pond, season, or sample type. Genetic similarity to known serotypes indicated Hadar, Montevideo, and Newport as the most prevalent. All ponds achieved the current safety standards for generic Escherichia coli in agricultural water, and regression modeling showed that the E. coli level was a significant predictor for the probability of Salmonella occurrence. However, persistent populations of Salmonella were widely distributed in irrigation ponds, and the associated risks for produce contamination and subsequent human exposure are unknown, supporting continued surveillance of this pathogen in agricultural settings.

Outbreaks of enteritis caused by Escherichia coli O157 associated with fresh produce have resulted in questions about the safety of irrigation water; however, associated risks have not been systematically evaluated. In this study, the occurrence and distribution of the human pathogen E. coli O157 from vegetable irrigation ponds within the Suwannee River Watershed in Georgia were investigated, and the relationship to environmental factors was analyzed. Surface and subsurface water samples were collected monthly from 10 vegetable irrigation ponds from March 2011 to February 2012. Escherichia coli O157 was isolated from enriched filtrates on CHROMagar and sorbitol MacConkey agar media and confirmed by an agglutination test. Presence of virulence genes stx1, stx2 , and eae was tested by polymerase chain reaction. In addition, 27 environmental variables of the sampled ponds were measured. Denaturing gradient gel electrophoresis was conducted for the analysis of bacterial communities in the water samples. Biserial correlation coefficients were calculated to evaluate the log10 colony-forming unit per millilitre correlations between the environmental factors and the occurrence of E. coli O157. Stepwise and canonical discriminant analyses were used to determine the factors that were associated with the presence and absence of E. coli O157 in water samples. All 10 ponds were positive for E. coli O157 some of the time, mainly in summer and fall of 2011. The temporal distribution of this bacterium differed among the 10 ponds. Temperature, rainfall, populations of fecal coliform, and culturable bacteria were positively correlated with the occurrence of E. coli O157 (P < 0.05), while the total nitrogen concentration, oxidation-reduction potential, and dissolved oxygen concentration were negatively correlated with the occurrence of this pathogen (P < 0.05). Temperature and rainfall were the most important factors contributing to the discrimination between samples with and without E. coli O157, followed by bacterial diversity and culturable bacteria population density. Bacterial numbers and diversity, including fecal coliforms and E. coli O157, increased after rainfall (and possibly runoff from pond margins) in periods with relatively high temperatures, suggesting that prevention of runoff may be important to minimize the risk of enteric pathogens in irrigation ponds.

The dissemination of antibiotic resistant bacteria from anthropogenic sources into the environment poses an emerging public health threat. Antibiotic resistance genes (ARGs) and gene-capturing systems such as integron-associated integrase genes (intI) play a key role in alterations of microbial communities and the spread of antibiotic resistant bacteria into the environment. In order to assess the effect of anthropogenic activities on watersheds in southwestern British Columbia, the presence of putative antibiotic resistance and integrase genes was analyzed in the microbiome of agricultural, urban influenced, and protected watersheds. A metagenomics approach and high-throughput quantitative PCR (HT qPCR) were used to screen for elements of resistance including ARGs and intI. Metagenomic sequencing of bacterial genomic DNA was used to characterize the resistome of microbial communities present in watersheds over a 1-year period. There was a low prevalence of ARGs relative to the microbial population (<1%). Analysis of the metagenomic sequences detected a total of 60 elements of resistance including 46 ARGs, intI1, and groEL/intI1 genes and 12 quaternary ammonium compounds (qac) resistance genes across all watershed locations. The relative abundance and richness of ARGs was found to be highest in agriculture impacted watersheds compared to urban and protected watersheds. A downstream transport pattern was observed in the impacted watersheds (urban and agricultural) during dry months. Similar to other reports, this study found a strong association between intI1 and ARGs (e.g., sul1), an association which may be used as a proxy for anthropogenic activities. Chemical analysis of water samples for three major groups of antibiotics was below the detection limit. However, the high richness and gene copy numbers (GCNs) of ARGs in impacted sites suggest that the effects of effluents on microbial communities are occurring even at low concentrations of antimicrobials in the water column. Antibiotic resistance and integrase genes in a year-long metagenomic study showed that ARGs were driven mainly by environmental factors from anthropogenized sites in agriculture and urban watersheds. Environmental factors such as land-use and water quality parameters accounted for 45% of the variability observed in watershed locations.

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