William Robertson

Distribution and Characteristics of Listeria monocytogenes Isolates from Surface Waters of the South Nation River Watershed, Ontario, Canada

ABSTRACT 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

ABSTRACT 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.

Molecular subtypes of Campylobacter spp., Salmonella enterica, and Escherichia coli O157:H7 isolated from faecal and surface water samples in the Oldman River watershed, Alberta, Canada

Campylobacter spp., Salmonella enterica, and Escherichia coli O157:H7 isolated from 898 faecal, 43 sewage, and 342 surface water samples from the Oldman River were characterized using bacterial subtyping methods in order to investigate potential sources of contamination of the watershed. Among these pathogens, Campylobacter spp. were the most frequently isolated from faecal, sewage, and surface water samples (266/895, 11/43, and 91/342, respectively), followed by Salmonella (67/898, 8/43, and 29/342, respectively), and E. coli O157:H7 (16/898, 2/43, and 8/342, respectively). Salmonella Rubislaw was the most common serovar isolated from water. This serovar was also isolated from two wild bird species. Most other serovars isolated from water were either not isolated from animals or were isolated from multiple species. E. coli O157:H7 was predominantly isolated from cattle. The most common phage-types of this pathogen from cattle were also the most common among water isolates, and there were exact pulsed field gel electrophoresis and comparative genomic fingerprint matches between cattle, sewage, and water isolates. Campylobacters were commonly isolated from surface waters and faeces from most animal species. Restriction fragment length polymorphism of the Campylobacter flaA gene identified several location and host species-specific (cattle, goose, pig) fingerprints. Molecular subtyping of these bacterial pathogens shows considerable promise as a tool for determining the sources of faecal pollution of water.

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.

A methods comparison for the isolation and detection of thermophilic Campylobacter in agricultural watersheds

Campylobacter species contribute to an enormous burden of enteric illnesses around the world. This study compared two different methods for detecting Campylobacter species in surface water samples from agricultural watersheds across Canada. One method was based on membrane filtration (MF) of 500 ml water samples followed by selective microaerophilic enrichment at 42 degrees C in Bolton broth, isolation of Campylobacter on CCDA, and subsequent identification confirmation by a PCR assay. The second method was based on centrifugation (CF) of 1000 ml water samples, followed by selective microaerophilic enrichment at 42 degrees C in Bolton broth, isolation of Campylobacter on Modified Karmali Agar, and subsequent identification confirmation by a different PCR assay. Overall comparison of the CF and MF methods indicated that both methods found Camylobacterjejuni to be the most commonly detected Campylobacter species in 699 water samples from four agricultural watersheds across Canada, and that C. jejuni frequency of occurrence was similar by both methods. However, the CF method detected significantly higher frequencies of Campylobactercoli (17%) and other Campylobacter species (13%) than the MF method (11% and 3%, respectively). It was frequently found that one method would detect Campylobacter in a water sample when the other method would not for a simultaneously collected, duplicate water sample. This study indicates that methods can have significantly different recovery efficiencies for Campylobacter species, and that caution is needed when comparing studies that report on the frequency of occurrence of waterborne Campylobacter at the genus level when different detection methods are used.