Pathogenicity assessment of Arcobacter butzleri isolated from Canadian agricultural surface water.

BACKGROUND: Water is considered a source for the transmission of Arcobacter species to both humans and animals. This study was conducted to assess the prevalence, distribution, and pathogenicity of A. butzleri strains, which can potentially pose health risks to humans and animals. Cultures were isolated from surface waters of a mixed-use but predominately agricultural watershed in eastern Ontario, Canada. The detection of antimicrobial resistance (AMR) and virulence-associated genes (VAGs), as well as enterobacterial repetitive intergenic consensus-polymerase chain reaction (ERIC-PCR) assays were performed on 913 A. butzleri strains isolated from 11 agricultural sampling sites. RESULTS: All strains were resistant to one or more antimicrobial agents, with a high rate of resistance to clindamycin (99%) and chloramphenicol (77%), followed by azithromycin (48%) and nalidixic acid (49%). However, isolates showed a significantly (p < 0.05) high rate of susceptibility to tetracycline (1%), gentamycin (2%), ciprofloxacin (4%), and erythromycin (5%). Of the eight VAGs tested, ciaB, mviN, tlyA, and pldA were detected at high frequency (> 85%) compared to irgA (25%), hecB (19%), hecA (15%), and cj1349 (12%) genes. Co-occurrence analysis showed A. butzleri strains resistant to clindamycin, chloramphenicol, nalidixic acid, and azithromycin were positive for ciaB, tlyA, mviN and pldA VAGs. ERIC-PCR fingerprint analysis revealed high genetic similarity among strains isolated from three sites, and the genotypes were significantly associated with AMR and VAGs results, which highlight their potential environmental ubiquity and potential as pathogenic. CONCLUSIONS: The study results show that agricultural activities likely contribute to the contamination of A. butzleri in surface water. The findings underscore the importance of farm management practices in controlling the potential spread of A. butzleri and its associated health risks to humans and animals through contaminated water.

Real-time quantitative PCR assay development and application for assessment of agricultural surface water and various fecal matter for prevalence of Aliarcobacter faecis and Aliarcobacter lanthieri.

BACKGROUND: Aliarcobacter faecis and Aliarcobacter lanthieri are recently identified as emerging human and animal pathogens. In this paper, we demonstrate the development and optimization of two direct DNA-based quantitative real-time PCR assays using species-specific oligonucleotide primer pairs derived from rpoB and gyrA genes for A. faecis and A. lanthieri, respectively. Initially, the specificity of primers and amplicon size of each target reference strain was verified and confirmed by melt curve analysis. Standard curves were developed with a minimum quantification limit of 100 cells mL- 1 or g- 1 obtained using known quantities of spiked A. faecis and A. lanthieri reference strains in autoclaved agricultural surface water and dairy cow manure samples. RESULTS: Each species-specific qPCR assay was validated and applied to determine the rate of prevalence and quantify the total number of cells of each target species in natural surface waters of an agriculturally-dominant and non-agricultural reference watershed. In addition, the prevalence and densities were determined for human and various animal (e.g., dogs, cats, dairy cow, and poultry) fecal samples. Overall, the prevalence of A. faecis for surface water and feces was 21 and 28%, respectively. The maximum A. faecis concentration for water and feces was 2.3 × 107 cells 100 mL- 1 and 1.2 × 107 cells g- 1, respectively. A. lanthieri was detected at a lower frequency (2%) with a maximum concentration in surface water of 4.2 × 105 cells 100 mL- 1; fecal samples had a prevalence and maximum density of 10% and 2.0 × 106 cells g- 1, respectively. CONCLUSIONS: The results indicate that the occurrence of these species in agricultural surface water is potentially due to fecal contamination of water from livestock, human, or wildlife as both species were detected in fecal samples. The new real-time qPCR assays can facilitate rapid and accurate detection in < 3 h to quantify total numbers of A. faecis and A. lanthieri cells present in various complex environmental samples.

Novel virulence, antibiotic resistance and toxin gene-specific PCR-based assays for rapid pathogenicity assessment of Arcobacter faecis and Arcobacter lanthieri.

BACKGROUND: Arcobacter faecis and A. lanthieri are two newly classified species of genus Arcobacter. The prevalence and distribution of virulence, antibiotic resistance and toxin (VAT) genes in these species are required to assess their potential pathogenic health impacts to humans and animals. This study (i) developed species- and gene-specific primer pairs for the detection of six virulence, two antibiotic resistance, and three toxin genes in two target species; (ii) optimized eight single-tube multiplex and three monoplex PCR protocols using the newly developed species- and gene-specific primers; and (iii) conducted specificity and sensitivity evaluations as well as validation of eleven mono- and multiplex PCR assays by testing A. faecis (n= 29) and A. lanthieri (n= 10) strains isolated from various fecal and agricultural water sources to determine the prevalence and distribution of VAT genes and assess the degree of pathogenicity within the two species. RESULTS: Detection of all ten and eleven target VAT genes, and expression of cytolethal distending toxin (cdtA, cdtB and cdtC) genes in A. faecis and A. lanthieri reference strains with high frequency in field isolates suggest that they are potentially pathogenic strains. These findings indicate that these two species can pose a health risk to humans and animals. CONCLUSIONS: The study results show that the developed mono- and multiplex PCR (mPCR) assays are simple, rapid, reliable and sensitive for the simultaneous assessment of the potential pathogenicity and antibiotic resistance profiling of tet(O) and tet(W) genes in these two newly discovered species. Also, these assays can be useful in diagnostic and analytical laboratories to determine the pathotypes and assessment of the virulence and toxin factors associated to human and animal infections.

Aquatic Bacterial Communities Associated With Land Use and Environmental Factors in Agricultural Landscapes Using a Metabarcoding Approach.

This study applied a 16S rRNA gene metabarcoding approach to characterize bacterial community compositional and functional attributes for surface water samples collected within, primarily, agriculturally dominated watersheds in Ontario and Québec, Canada. Compositional heterogeneity was best explained by stream order, season, and watercourse discharge. Generally, community diversity was higher at agriculturally dominated lower order streams, compared to larger stream order systems such as small to large rivers. However, during times of lower relative water flow and cumulative 2-day rainfall, modestly higher relative diversity was found in the larger watercourses. Bacterial community assemblages were more sensitive to environmental/land use changes in the smaller watercourses, relative to small-to-large river systems, where the proximity of the sampled water column to bacteria reservoirs in the sediments and adjacent terrestrial environment was greater. Stream discharge was the environmental variable most significantly correlated (all positive) with bacterial functional groups, such as C/N cycling and plant pathogens. Comparison of the community structural similarity via network analyses helped to discriminate sources of bacteria in freshwater derived from, for example, wastewater treatment plant effluent and intensity and type of agricultural land uses (e.g., intensive swine production vs. dairy dominated cash/livestock cropping systems). When using metabarcoding approaches, bacterial community composition and coexisting pattern rather than individual taxonomic lineages, were better indicators of environmental/land use conditions (e.g., upstream land use) and bacterial sources in watershed settings. Overall, monitoring changes and differences in aquatic microbial communities at regional and local watershed scales has promise for enhancing environmental footprinting and for better understanding nutrient cycling and ecological function of aquatic systems impacted by a multitude of stressors and land uses.

Waterborne Viruses and F-Specific Coliphages in Mixed-Use Watersheds: Microbial Associations, Host Specificities, and Affinities with Environmental/Land Use Factors.

From the years 2008 to 2014, a total of 1,155 water samples were collected (spring to fall) from 24 surface water sampling sites located in a mixed-used but predominantly agricultural (i.e., dairy livestock production) river basin in eastern Ontario, Canada. Water was analyzed for viable F-specific DNA (F-DNA) and F-specific RNA (F-RNA) (genogroup I [GI] to GIV) coliphage and a suite of molecularly detected viruses (norovirus [GI to GIV], torque teno virus [TTV], rotavirus, kobuvirus, adenovirus, astrovirus, hepatitis A, and hepatitis E). F-DNA and F-RNA coliphage were detected in 33 and 28% of the samples at maximum concentrations of 2,000 and 16,300 PFU · 100 ml-1, respectively. Animal TTV, human TTV, kobuvirus, astrovirus, and norovirus GIII were the most prevalent viruses, found in 23, 20, 13, 12, and 11% of samples, respectively. Viable F-DNA coliphage was found to be a modest positive indicator of molecularly detected TTV. F-RNA coliphage, unlike F-DNA coliphage, was a modest positive predictor of norovirus and rotavirus. There were, however, a number of significant negative associations among F-specific coliphage and viruses. F-DNA coliphage densities of >142 PFU · 100 ml-1 delineated conditions when ∼95% of water samples contained some type of virus. Kobuvirus was the virus most strongly related to detection of any other virus. Land use had some associations with virus/F-specific coliphage detection, but season and surface water flow were the variables that were most important for broadly delineating detection. Higher relative levels of detection of human viruses and human F-RNA coliphage were associated with higher relative degrees of upstream human land development in a catchment. IMPORTANCE: This study is one of the first, to our knowledge, to evaluate relationships among F-specific coliphages and a large suite of enteric viruses in mixed-use but agriculturally dominated surface waters in Canada. This study suggested that relationships between viable F-specific coliphages and molecularly detected viruses do exist, but they are not always positive. Caution should be employed if viable F-specific coliphages are to be used as indicators of virus presence in surface waters. This study elucidates relative effects of agriculture, wildlife, and human activity on virus and F-specific coliphage detection. Seasonal and meteorological attributes play a strong role in the detection of most virus and F-specific coliphage targets.

Biosolids applied to agricultural land: Influence on structural and functional endpoints of soil fauna on a short- and long-term scale.

Biosolids have well-documented crop and soil benefits similar to other sources of organic amendment, but there is environmental concern due to biosolids-associated pollutants. The present study investigated two field sites that had received biosolids at commercial-scale rates in parallel to associated field sections which were managed similarly but without receiving biosolids (controls). The investigated endpoints were abundance and diversity of soil organisms (nematodes, enchytraeids and earthworms) and soil fauna feeding activity as measured by the bait lamina assay. Repeated sampling of one of the field sites following the only biosolids application demonstrated an enrichment effect typical for organic amendments, which was mostly exhausted after 44months. After an initial suppression, the proportion of free-living plant-parasitic nematodes tended to increase in the biosolids-amended soil over time. Yet, none of the endpoints at this site indicated significant negative effects resulting from the biosolids until 44months post application. In contrast to the repeatedly tilled first field site, the second one was left fallow after three biosolids applications, and was sampled 96months post last application. It was only at this field site that potential evidence for a long-term impact of biosolids was detected with regard to two endpoints: earthworm abundance and structure of the nematode assemblage. Agricultural management and correlation with abiotic soil parameters explained the observed difference in earthworm abundance. Yet, the development of a highly structured and mature nematode assemblage at the control but not at the biosolids-amended section of this fallow field could not be explained by such correlations nor by soil metal concentrations. Overall, the present study found only weak evidence for negative long-term impacts of biosolids applied at commercial rates on soil fauna. High-level community parameters such as the nematode structure index (SI) appeared more suitable to detect deleterious effects on soil fauna than simple abundance measurements.

Detection of virulence, antibiotic resistance and toxin (VAT) genes in Campylobacter species using newly developed multiplex PCR assays.

Campylobacter species are one of the leading causes of bacterial gastroenteritis in humans worldwide. This twofold study was sought to: i) develop and optimize four single-tube multiplex PCR (mPCR) assays for the detection of six virulence (ciaB, dnaJ, flaA, flaB, pldA and racR), three toxin (cdtA, cdtB and cdtC) and one antibiotic resistance tet(O) genes in thermophilic Campylobacter spp. and ii) apply and evaluate the developed mPCR assays by testing 470 previously identified C. jejuni, C. coli and C. lari isolates from agricultural water. In each mPCR assay, a combination of two or three sets of primer pairs for virulence, antibiotic resistance and toxin (VAT) genes was used and optimized. Assay 1 was developed for the detection of dnaJ, racR and cdtC genes with expected amplification sizes of 720, 584 and 182bp. Assay 2 generated PCR amplicons for tet(O) and cdtA genes of 559 and 370bp. Assay 3 amplified cdtB ciaB, and pldA genes with PCR amplicon sizes of 620, 527 and 385bp. Assay 4 was optimized for flaA and flaB genes that generated PCR amplicons of 855 and 260bp. The primer pairs and optimized PCR protocols did not show interference and/or cross-amplification with each other and generated the expected size of amplification products for each target VAT gene for the C. jejuni ATCC 33291 reference strain. Overall, all ten target VAT genes were detected at a variable frequency in tested isolates of thermophilic Campylobacter spp. where cdtC, flaB, ciaB, cdtB, cdtA and pldA were commonly detected compared to the flaA, racR, dnaJ and tet(O) genes which were detected with less frequency. The developed mPCR assays are simple, rapid, reliable and sensitive tools for simultaneously assessing potential pathogenicity and antibiotic resistance profiling in thermophilic Campylobacter spp. The mPCR assays will be useful in diagnostic and analytical settings for routine screening of VAT characteristics of Campylobacter spp. as well as being applicable in epidemiological studies by providing information that could be related to the risk of human infection.

A national investigation of the prevalence and diversity of thermophilic Campylobacter species in agricultural watersheds in Canada.

The occurrence and diversity of thermophilic Campylobacter species (C. jejuni, coli, and lari) were studied in water samples from four river basins located across Canada. These basins located in Quebec (Bras d'Henri), Alberta (Oldman), Ontario (South Nation), and British Columbia (Sumas) represented some of the most intensive farming areas in Canada for hog, beef cattle, dairy cattle, and poultry, respectively. This study analyzed 769 water samples collected from 23 monitoring sites with agricultural influence, and four reference sites with limited or no agricultural influence. Water samples were collected bi-weekly over two years and analyzed for Campylobacter using a semi-quantitative minimum probable number (MPN) enrichment protocol. Putative isolates were confirmed by genus- and species-specific multiplex polymerase chain reaction (PCR) assays. A total of 377 (49%) water samples were positive for campylobacters with 355 samples having a cell density ranging from 4 to 4000 MPN L(-1). Campylobacters were more common at agricultural than reference sites in each river basin, although this difference was not significant in the Oldman and South Nation (p > 0.05). Campylobacter was significantly more common in the Bras d'Henri and Sumas (63%) compared to the South Nation (45%) and Oldman (33%) River basins (p < 0.05). C. jejuni, C. coli and C. lari were detected in each river basin, and these species occurred in 45% (n = 168), 34% (n = 128) and 19% (n = 73), of all Campylobacter positive samples, respectively. The remaining Campylobacter positive water samples without these three species (n = 67; 18%) were identified as other Campylobacter species. C. jejuni was the predominant species occurring in the Sumas, Oldman and South Nation River basins. However, in the Bras d'Henri River basin with intensive hog production, C. coli was the predominant species. This study found campylobacters to be common in some agricultural systems with intensive livestock farming activities, and different river basins could have strikingly different profiles of either C. jejuni or C. coli as the predominant waterborne thermophilic Campylobacter species.

Long-term monitoring of waterborne pathogens and microbial source tracking markers in paired agricultural watersheds under controlled and conventional tile drainage management.

Surface waters from paired agricultural watersheds under controlled tile drainage (CTD) and uncontrolled tile drainage (UCTD) were monitored over 7 years in order to determine if there was an effect of CTD (imposed during the growing season) on occurrences and loadings of bacterial and viral pathogens, coliphages, and microbial source tracking markers. There were significantly lower occurrences of human, ruminant, and livestock (ruminant plus pig) Bacteroidales markers in the CTD watershed in relation to the UCTD watershed. As for pathogens, there were significantly lower occurrences of Salmonella spp. and Arcobacter spp. in the CTD watershed. There were no instances where there were significantly higher quantitative loadings of any microbial target in the CTD watershed, except for F-specific DNA (F-DNA) and F-RNA coliphages, perhaps as a result of fecal inputs from a hobby farm independent of the drainage practice treatments. There was lower loading of the ruminant marker in the CTD watershed in relation to the UCTD system, and results were significant at the level P = 0.06. The odds of Salmonella spp. occurring increased when a ruminant marker was present relative to when the ruminant marker was absent, yet for Arcobacter spp., the odds of this pathogen occurring significantly decreased when a ruminant marker was present relative to when the ruminant marker was absent (but increased when a wildlife marker was present relative to when the wildlife marker was absent). Interestingly, the odds of norovirus GII (associated with human and swine) occurring in water increased significantly when a ruminant marker was present relative to when a ruminant marker was absent. Overall, this study suggests that fecal pollution from tile-drained fields to stream could be reduced by CTD utilization.

Using SWAT, Bacteroidales microbial source tracking markers, and fecal indicator bacteria to predict waterborne pathogen occurrence in an agricultural watershed.

Developing the capability to predict pathogens in surface water is important for reducing the risk that such organisms pose to human health. In this study, three primary data source scenarios (measured stream flow and water quality, modelled stream flow and water quality, and host-associated Bacteroidales) are investigated within a Classification and Regression Tree Analysis (CART) framework for classifying pathogen (Escherichia coli 0157:H7, Salmonella, Campylobacter, Cryptosporidium, and Giardia) presence and absence (P/A) for a 178 km(2) agricultural watershed. To provide modelled data, a Soil Water Assessment Tool (SWAT) model was developed to predict stream flow, total suspended solids (TSS), total N and total P, and fecal indicator bacteria loads; however, the model was only successful for flow and total N and total P simulations, and did not accurately simulate TSS and indicator bacteria transport. Also, the SWAT model was not sensitive to an observed reduction in the cattle population within the watershed that may have resulted in significant reduction in E. coli concentrations and Salmonella detections. Results show that when combined with air temperature and precipitation, SWAT modelled stream flow and total P concentrations were useful for classifying pathogen P/A using CART methodology. From a suite of host-associated Bacteroidales markers used as independent variables in CART analysis, the ruminant marker was found to be the best initial classifier of pathogen P/A. Of the measured sources of independent variables, air temperature, precipitation, stream flow, and total P were found to be the most important variables for classifying pathogen P/A. Results indicate a close relationship between cattle pollution and pathogen occurrence in this watershed, and an especially strong link between the cattle population and Salmonella detections.

Coherence among different microbial source tracking markers in a small agricultural stream with or without livestock exclusion practices.

Over 1,400 water samples were collected biweekly over 6 years from an intermittent stream protected and unprotected from pasturing cattle. The samples were monitored for host-specific Bacteroidales markers, Cryptosporidium species/genotypes, viruses and coliphages associated with humans or animals, and bacterial zoonotic pathogens. Ruminant Bacteroidales markers did not increase within the restricted cattle access reach of the stream, whereas the ruminant Bacteroidales marker increased significantly in the unrestricted cattle access reach. Human Bacteroidales markers significantly increased downstream of homes where septic issues were documented. Wildlife Bacteroidales markers were detected downstream of the cattle exclusion practice where stream and riparian habitat was protected, but detections decreased after the unrestricted pasture, where the stream and riparian zone was unprotected from livestock. Detection of a large number of human viruses was shown to increase downstream of homes, and similar trends were observed for the human Bacteroidales marker. There was considerable interplay among biomarkers with stream flow, season, and the cattle exclusion practices. There were no to very weak associations with Bacteroidales markers and bacterial, viral, and parasitic pathogens. Overall, discrete sample-by-sample coherence among the different microbial source tracking markers that expressed a similar microbial source was minimal, but spatial trends were physically meaningful in terms of land use (e.g., beneficial management practice) effects on sources of fecal pollution.

Quantitative multi-year elucidation of fecal sources of waterborne pathogen contamination in the South Nation River basin using bacteroidales microbial source tracking markers.

Over a seven-year period (2004-2010) 1095 water samples were obtained from the South Nation River basin at multiple watershed monitoring sites (Ontario, Canada). Real-time PCR using Bacteroidales specific markers was used to identify the origin (human (10% prevalence), ruminant (22%), pig (~2%), Canada goose (4%) and muskrat (7%)) of fecal pollution. In parallel, the distribution of fecal indicator bacteria and waterborne pathogens (Cryptosporidium oocysts, Giardia cysts, Escherichia coli O157:H7, Salmonella enterica and Campylobacter spp.) was evaluated. Associations between the detection of specific Bacteroidales markers and the presence of fecal indicator bacteria, pathogens, and distinct land use or environmental variables were evaluated. Linear correlations between Bacteroidales markers and fecal indicator bacteria were weak. However, mean marker densities, and the presence and absence of markers could be discriminated on the basis of threshold fecal indicator densities. The ruminant-specific Bacteroidales marker was the most frequently detected marker in water, consistent with the large number of dairy farms in the study area. Detection of the human or the ruminant markers were associated with a slightly higher risk of detecting S. enterica. Detection of the muskrat marker was related to more frequent Campylobacter spp. detections. Important positive associations between markers and pathogens were found among: i) total Bacteroidales and Cryptosporidium and Giardia, ii) ruminant marker and S. enterica, and iii) muskrat and Campylobacter spp.

Spatiotemporal analysis of Cryptosporidium species/genotypes and relationships with other zoonotic pathogens in surface water from mixed-use watersheds.

Nearly 690 raw surface water samples were collected during a 6-year period from multiple watersheds in the South Nation River basin, Ontario, Canada. Cryptosporidium oocysts in water samples were enumerated, sequenced, and genotyped by detailed phylogenetic analysis. The resulting species and genotypes were assigned to broad, known host and human infection risk classes. Wildlife/unknown, livestock, avian, and human host classes occurred in 21, 13, 3, and <1% of sampled surface waters, respectively. Cryptosporidium andersoni was the most commonly detected livestock species, while muskrat I and II genotypes were the most dominant wildlife genotypes. The presence of Giardia spp., Salmonella spp., Campylobacter spp., and Escherichia coli O157:H7 was evaluated in all water samples. The greatest significant odds ratios (odds of pathogen presence when host class is present/odds of pathogen presence when host class is absent) for Giardia spp., Campylobacter spp., and Salmonella spp. in water were associated, respectively, with livestock (odds ratio of 3.1), avian (4.3), and livestock (9.3) host classes. Classification and regression tree analyses (CART) were used to group generalized host and human infection risk classes on the basis of a broad range of environmental and land use variables while tracking cooccurrence of zoonotic pathogens in these groupings. The occurrence of livestock-associated Cryptosporidium was most strongly related to agricultural water pollution in the fall (conditions also associated with elevated odds ratios of other zoonotic pathogens occurring in water in relation to all sampling conditions), whereas wildlife/unknown sources of Cryptosporidium were geospatially associated with smaller watercourses where urban/rural development was relatively lower. Conditions that support wildlife may not necessarily increase overall human infection risks associated with Cryptosporidium since most Cryptosporidium genotypes classed as wildlife in this study (e.g., muskrat I and II genotype) do not pose significant infection risks to humans. Consequently, from a human health perspective, land use practices in agricultural watersheds that create opportunities for wildlife to flourish should not be rejected solely on the basis of their potential to increase relative proportions of wildlife fecal contamination in surface water. The present study suggests that mitigating livestock fecal pollution in surface water in this region would likely reduce human infection risks associated with Cryptosporidium and other zoonotic pathogens.

Molecular and phylogenetic approaches for assessing sources of Cryptosporidium contamination in water.

The high sequence diversity and heterogeneity observed within species or genotypes of Cryptosporidium requires phylogenetic approaches for the identification of novel sequences obtained from the environment. A long-term study on Cryptosporidium in the agriculturally-intensive South Nation River watershed in Ontario, Canada was undertaken, in which 60 sequence types were detected. Of these sequence types 33 were considered novel with no identical matches in GenBank. Detailed phylogenetic analysis identified that most sequences belonged to 17 previously described species: Cryptosporidium andersoni, Cryptosporidium baileyi, Cryptosporidium hominis, Cryptosporidium parvum, Cryptosporidium ubiquitum, Cryptosporidium meleagridis, muskrat I, muskrat II, deer mouse II, fox, vole, skunk, shrew, W12, W18, W19 and W25 genotypes. In addition, two new genotypes were identified, W27 and W28. C. andersoni and the muskrat II genotype were most frequently detected in the water samples. Species associated with livestock made up 39% of the total molecular detections, while wildlife associated species and genotypes accounted for 55% of the Cryptosporidium identified. The human pathogenic species C. hominis and C. parvum had an overall prevalence of 1.6% in the environment, indicating a small risk to humans from the Cryptosporidium present in the watershed. Phylogenetic analysis and knowledge of host-parasite relationships are fundamental in using Cryptosporidium as a source-tracking or human health risk assessment tool.

Spatial and temporal drivers of zoonotic pathogen contamination of an agricultural watershed.

In regions where animal agriculture is prominent, such as southern Alberta, higher rates of gastrointestinal illness have been reported when compared with nonagricultural regions. This difference in the rate of illness is thought to be a result of increased zoonotic pathogen exposure through environmental sources such as water. In this study, temporal and spatial factors associated with bacterial pathogen contamination of the Oldman River, which transverses this region, were analyzed using classification and regression tree analysis. Significantly higher levels of fecal indicators; more frequent isolations of Campylobacter spp., Escherichia coli O157:H7, and Salmonella enterica spp.; and higher rates of detection of pig-specific Bacteroides markers occurred at downstream sites than at upstream sites, suggesting additive stream inputs. Fecal indicator densities were also significantly higher when any one of these three bacterial pathogens was present and where there were higher total animal manure units; however, occasionally pathogens were present when fecal indicator levels were low or undetectable. Overall, Salmonella spp., Campylobacter spp., and E. coli O157:H7 presence was associated with season, animal manure units, and total rainfall on the day of sampling and 3 d in advance of sampling. Several of the environmental variables analyzed in this study appear to influence pathogen prevalence and therefore may be useful in predicting water quality and safety and in the improvement of watershed management practices in this and other agricultural regions.

Distribution and diversity of Escherichia coli populations in the South Nation River drainage basin, eastern Ontario, Canada.

We investigated the prevalence and diversity of Escherichia coli strains isolated from surface waters from multiple watersheds within the South Nation River basin in eastern Ontario, Canada. The basin is composed of mixed but primarily agricultural land uses. From March 2004 to November 2007, a total of 2,004 surface water samples were collected from 24 sampling sites. E. coli densities ranged from undetectable to 1.64 x 10(5) CFU 100 ml(-1) and were correlated with stream order and proximity to livestock production systems. The diversity of 21,307 E. coli isolates was characterized using repetitive extragenic palindromic PCR (rep-PCR), allowing for the identification of as many as 7,325 distinct genotypes, without capturing all of the diversity. The community was temporally and spatially dominated by a few dominant genotypes (clusters of more than 500 isolates) and several genotypes of intermediary abundance (clustering between 10 and 499 isolates). Simpson diversity indices, assessed on a normalized number of isolates per sample, ranged from 0.050 to 0.668. Simpson indices could be statistically discriminated on the basis of year and stream order, but land use, discharge, weather, and water physical-chemical properties were not statistically important discriminators. The detection of Campylobacter species was associated with statistically lower Simpson indices (greater diversity; P < 0.05). Waterborne E. coli isolates from genotypes of dominant and intermediary abundance were clustered with isolates obtained from fecal samples collected in the study area over the same period, and 90% of the isolates tested proved to share genotypes with fecal isolates. Overall, our data indicated that the densities and distribution of E. coli in these mixed-use watersheds were linked to stream order and livestock-based land uses. Waterborne E. coli populations that were distinct from fecal isolates were detected and, on this basis, were possibly naturalized E. coli strains.

Seasonal relationships among indicator bacteria, pathogenic bacteria, Cryptosporidium oocysts, Giardia cysts, and hydrological indices for surface waters within an agricultural landscape.

The South Nation River basin in eastern Ontario, Canada is characterized by mixed agriculture. Over 1600 water samples were collected on a bi-weekly basis from up to 24 discrete sampling sites on river tributaries of varying stream order within the river basin between 2004 and 2006. Water samples were analyzed for: densities of indicator bacteria (Escherichia coli, Clostridium perfringens, enterococci, total and fecal coliforms), the presence of pathogenic bacteria (Listeria monocytogenes, E. coli O157:H7, Salmonella spp., Campylobacter spp.), and densities of parasite Giardia cysts and Cryptosporidium oocysts. Relationships between indicator bacteria, pathogens, and parasite oocysts/cysts were overall weak, seasonally dependent, site specific, but primarily positive. However, L. monocytogenes was inversely related with indicator bacteria densities. Campylobacter, Salmonella, Giardia cysts and Cryptosporidium oocysts were most frequently detected in the fall. E. coli O157:H7 was detected at a very low frequency. Exploratory decision tree analyses found overall that E. coli densities were the most utilitarian classifiers of parasite/pathogen presence and absence, followed closely by fecal coliforms, and to a lesser extent enterococci and total coliforms. Indicator bacteria densities that classified pathogen presence and absence groupings, were all below 100 CFU per 100 mL(-1). Microorganism relationships with rainfall indices and tributary discharge variables were globally weak to modest, and generally inconsistent among season, site and microorganism. But, overall rainfall and discharge were primarily positively associated with indicator bacteria densities and pathogen detection. Instances where a pathogen was detected in the absence of a detectable bacterial indicator were extremely infrequent; thus, the fecal indicators were conservative surrogates for a variety of pathogenic microorganisms in this agricultural setting. The results from this study indicate that no one indicator or simple hydrological index is entirely suitable for all environmental systems and pathogens/parasites, even within a common geographic setting. These results place more firmly into context that robust prediction and/or indicator utility will require a more firm understanding of microorganism distribution in the landscape, the nature of host sources, and transport/environmental fate affinities among pathogens and indicators.

Characteristics and frequency of detection of fecal Listeria monocytogenes shed by livestock, wildlife, and humans.

Listeria monocytogenes is a facultative intracellular pathogen that can be carried asymptomatically in various animals and can be shed in feces. We investigated the prevalence and characteristics of L. monocytogenes isolated from livestock, wildlife, and human potential sources of contamination in 2 areas in Ontario, Canada. From February 2003 to November 2005, a total of 268 fecal samples were collected from different animals. Listeria monocytogenes was isolated using selective enrichment, isolation, and confirmation procedures, and 15 samples (6%) yielded to the isolation of 84 confirmed strains. Listeria monocytogenes was isolated from livestock (beef and dairy), wildlife (deer, moose, otter, and raccoon), and human (biosolids and septic) fecal sources. Thirty-two isolates were from serovar 1/2a, 34 from serovar 1/2b, 1 from serovar 3a, and 17 from serovar 4b. Listeria monocytogenes populations were resolved into 13 EcoRI ribotypes, and 18 ApaI and 18 AscI pulsotypes, with Simpson indexes of discrimination of 0.878 and 0.907, respectively. A majority (59%) of L. monocytogenes isolates exhibited potential virulence linked to the production of a functional internalin A, which was supported by higher entry into Caco-2 cells (9.3%) than isolates producing truncated and secreted internalin A (1.3% of entry). Listeria monocytogenes fecal isolates were on average resistant to 6.4 +/- 2.5 antibiotics out of 17 tested, and potentially virulent isolates exhibited an enhanced resistance to kanamycin, gentamicin, streptomycin, and rifampicin. Livestock, wildlife, and human L. monocytogenes fecal communities exhibited overlapping but distinct populations, and some genotypes and phenotypes were similar to those previously described for surface water isolates in the same area.

Distribution and characteristics of Listeria monocytogenes isolates from surface waters of the South Nation River watershed, Ontario, Canada.

Listeria monocytogenes is a facultative intracellular pathogen thought to be widely distributed in the environment. We investigated the prevalence and characteristics of L. monocytogenes isolates from surface waters derived from catchments within the South Nation River watershed (Ontario, Canada). This watershed is dominated by urban and rural development, livestock and crop production, and wildlife habitats. From June to November 2005, a total of 314 surface water samples were collected biweekly from 22 discrete sampling sites characterized by various upstream land uses. Presumptive Listeria spp. were isolated using a selective enrichment and isolation procedure, and 75 L. monocytogenes isolates were identified based on colony morphology, hemolytic activity, and amplification of three pathogenicity genes: iap, inlA, and hlyA. Thirty-two of 314 (10%) surface water samples were positive for the presence of L. monocytogenes, but detection ranged between 0 and 27% depending on the sampling date. Isolates belonging to serovar group 1/2a, 3a (50%) and group 4b, 4d, 4e (32%) were dominant. L. monocytogenes populations were resolved into 13 EcoRI ribotypes and 21 ApaI and 21 AscI pulsotypes. These had Simpson indexes of discrimination of up to 0.885. Lineage I-related isolates were dominant (61%) during the summer, whereas lineage II isolates were dominant (77%) in the fall. Isolates were, on average, resistant to 6.1 +/- 2.1 antibiotics out of 17 tested. Half of the L. monocytogenes isolates exhibited potential virulence linked to the production of a functional internalin A, and some isolates were found to be moderately to highly virulent by in vitro Caco-2 plaque formation assay (up to 28% of entry). There was a statistically significant link between the occurrence of L. monocytogenes and proximity to an upstream dairy farm and degree of cropped land. Our data indicate that L. monocytogenes is widespread in the studied catchments, where it could represent a public health issue related to agricultural land use.

Tracking host sources of Cryptosporidium spp. in raw water for improved health risk assessment.

Recent molecular evidence suggests that different species and/or genotypes of Cryptosporidium display strong host specificity, altering our perceptions regarding the zoonotic potential of this parasite. Molecular forensic profiling of the small-subunit rRNA gene from oocysts enumerated on microscope slides by U.S. Environmental Protection Agency method 1623 was used to identify the range and prevalence of Cryptosporidium species and genotypes in the South Nation watershed in Ontario, Canada. Fourteen sites within the watershed were monitored weekly for 10 weeks to assess the occurrence, molecular composition, and host sources of Cryptosporidium parasites impacting water within the region. Cryptosporidium andersoni, Cryptosporidium muskrat genotype II, Cryptosporidium cervine genotype, C. baileyi, C. parvum, Cryptosporidium muskrat genotype I, the Cryptosporidium fox genotype, genotype W1, and genotype W12 were detected in the watershed. The molecular composition of the Cryptosporidium parasites, supported by general land use analysis, indicated that mature cattle were likely the main source of contamination of the watershed. Deer, muskrats, voles, birds, and other wildlife species, in addition to sewage (human or agricultural) may also potentially impact water quality within the study area. Source water protection studies that use land use analysis with molecular genotyping of Cryptosporidium parasites may provide a more robust source-tracking tool to characterize fecal impacts in a watershed. Moreover, the information is vital for assessing environmental and human health risks posed by water contaminated with zoonotic and/or anthroponotic forms of Cryptosporidium.