Angela Cook

Human noroviruses in swine and cattle.

Detection of GII.4 norovirus sequences in animal fecal samples and retail meats demonstrates that noroviruses may be transmitted zoonotically.

The potential for zoonotic transmission of Giardia duodenalis and Cryptosporidium spp. from beef and dairy cattle in Ontario, Canada.

The objective of this study was to compare the occurrence and the genotypes and species of Giardia duodenalis and Cryptosporidium spp. in beef and dairy cattle from farms in the Regional Municipality of Waterloo, Ontario, in an effort to determine the potential for zoonotic transmission from these animals. Pooled manure samples were collected from 45 dairy cattle farms and 30 beef cattle farms. The presence of Giardia cysts and Cryptosporidium oocysts was determined by immunofluorescence microscopy, while nested-PCR and DNA sequencing were used to determine genotypes and species. The overall farm prevalence was very high for both Giardia and Cryptosporidium, and was similar for dairy cattle farms (96 and 64%, respectively) and beef cattle farms (97 and 63%, respectively). However, on dairy cattle farms, G. duodenalis and Cryptosporidium spp. were detected in 44% and 6% of total pooled pen manure samples, respectively, with the occurrence of both parasites being generally higher in calves than in older animals. Most Giardia isolates were identified as either the host-adapted genotype G. duodenalis Assemblage E or the zoonotic Assemblage B. Cryptosporidium parvum and Cryptosporidium andersoni were the most frequently identified species in dairy cattle, while the non-zoonotic species Cryptosporidium ryanae and Cryptosporidium bovis were also found. On beef cattle farms, 72% and 27% of the total pooled pen manure samples were positive for Giardia and Cryptosporidium, respectively, with no obvious correlation with age. All Giardia isolates in beef cattle were identified as G. duodenalis Assemblage E, while all Cryptosporidium isolates were identified by sequence analysis as C. andersoni, although microscopic analyses, and subsequent restriction fragment length polymorphism analyses, indicated that other Cryptosporidium species were also present. The results of this study indicate that although Giardia and Cryptosporidium were identified in a higher overall percentage of the pooled beef cattle manure samples than in dairy cattle, firmly established zoonotic genotypes and species were much more common in dairy cattle than in beef cattle in this region. Dairy cattle, and especially dairy calves, may, therefore, pose a greater risk of infection to humans than beef cattle. However, these results may also provide evidence of potential zooanthroponotic transmission (human to animal).

Detection of Cyclospora, Cryptosporidium, and Giardia in ready-to-eat packaged leafy greens in Ontario, Canada.

Numerous foodborne outbreaks of diarrheal illness associated with the consumption of produce contaminated with protozoan parasites have been reported in North America in recent years. The present study reports on the presence of Cyclospora, Cryptosporidium, and Giardia in precut salads and leafy greens purchased at retail in Ontario, Canada. A total of 544 retail samples were collected between April 2009 and March 2010 and included a variety of salad blends and individual leafy greens. Most of these products were grown in the United States, with some from Canada and Mexico. Parasites were eluted and concentrated before detection by PCR and immunofluorescence microscopy. DNA sequences were aligned with reference sequences in GenBank. Cyclospora spp. were identified by PCR-restriction fragment length polymorphism in nine (1.7 % ) samples and by DNA sequence analysis. Cryptosporidium spp. were identified in 32 (5.9%) samples; 29 were sequenced and aligned with the zoonotic species Cryptosporidium parvum. Giardia duodenalis was identified in 10 (1.8%) samples, and of the 9 samples successfully sequenced, 7 aligned with G. duodenalis assemblage B and 2 with assemblage A, both of which are also zoonotic. The presence of Cryptosporidium oocysts and Giardia cysts was confirmed in some of the PCR-positive samples using microscopy, while Cyclospora -like oocysts were observed in most of the Cyclospora PCR-positive samples. The relatively high prevalence of these parasites in packaged salads and leafy greens establishes a baseline for further studies and suggests a need for additional research with respect to the possible sources of contamination of these foods, the determination of parasite viability and virulence, and means to reduce foodborne transmission to humans.

Enteric Viruses in Ready-to-Eat Packaged Leafy Greens

To the Editor: Fresh produce increasingly has been implicated in viral disease outbreaks (1). In some instances, lettuce was contaminated before wholesale distribution (1). Enteric viruses can be introduced in the field if produce is exposed to human waste. Processed and packaged produce can be contaminated if equipment or wash water is not effectively sanitized. Fewer than 10 infectious viral particles are sufficient to cause disease (2), and these organisms are resistant to disinfectants at concentrations that reduce bacterial levels (3). Contamination of fresh produce could pose a health risk to humans because fresh produce is eaten raw. High levels of viral contamination can result in large outbreaks, but intermittent contamination of fresh produce accounts for some sporadic cases of norovirus and rotavirus gastroenteritis. During April 27–November 23, 2009, we performed viral testing on 328 samples of packaged leafy greens (representing 12–14 different lots from 3–6 companies per week; no samples were taken on weeks with a statutory holiday) for norovirus or rotavirus RNA. Packaged leafy greens were purchased from retail stores in southern Ontario, Canada. Shipments maintained an average temperature of 3.8°C during transit to the testing laboratory. Each 25-g sample was spiked with 106 PFU of feline calicivirus (FCV) as a sample process control (4). Virus was concentrated by using an adsorption-elution-ultrafiltration filtration protocol (4). Recovery of FCV was quantified from an RNA standard curve. FCV process control recovery was 0.01% of the FCV was observed for the remaining 273 (83%) samples. Two samples from which FCV was not recovered were positive for norovirus (CE-V-09–0138) and rotavirus (CE-V-09–0129); they were considered true positive results. Of these 275 samples, 148 (54%) were positive for norovirus by real-time reverse transcription–PCR (RT-PCR) (5), and 1 (0.4%) was positive for rotavirus group A by RT-PCR (6). To confirm detection of norovirus RNA, we amplified a second norovirus target by RT-PCR of region C (5). Only 40 samples (15% of total) produced a band of the expected size for this second norovirus amplicon. Of these 40 amplicons, only 16 (6% of total) could be sequenced to confirm norovirus RNA. The rotavirus-positive sample was confirmed by sequencing. For some sample dates, multiple lots were positive; for others, no positive samples were identified (Figure). Multiple detections on the same date were not caused by cross-contamination; partial capsid sequencing showed different genetic types on dates when multiple samples were positive (Figure). Results were positive from 5 different brands, and no organic samples were confirmed positive for enteric virus contamination. Of the 16 norovirus strains confirmed, 13 belonged to genogroup I (GI) and 3 to genogroup II (GII) (Figure). All were strain types known to be human pathogens. The group A rotavirus was not subtyped; group A rotaviruses can be human or animal pathogens. Figure Phylogenetic analysis of the partial capsid sequence from genogroup I (A) and genogroup II (B) norovirus strains detected on leafy greens samples, Ontario, Canada, 2009, compared with the ViroNet Canada reference set for this region. Dates in parentheses ... Most noroviruses detected belonged to GI. Previous reports indicate that GI norovirus are more frequently identified in foodborne or waterborne outbreaks; GII.4 noroviruses are more common in large outbreaks spread person to person (7). Identification of GI norovirus is consistent with occasional contamination of produce or wash water. Disinfectants and sanitation agents are used in wash water at low concentrations, at which they have limited efficacy against norovirus (3). Washing and disinfecting produce before eating it can reduce the risk for infection by reducing the viral load by 10- to 1,000-fold (8). The median level of confirmed contamination in this study was ≈500 RNA copies for norovirus (range 1.4 copies to 9 × 106 copies). A limitation of our findings is the inability to determine the association between molecular detection results and infectious virus. No outbreaks were related to the sequences detected here. There is no routine cell culture system for the laboratory growth of human norovirus. Genomic RNA can persist after the virus has been inactivated (9). The new ViroNet Canada network, which went online in April 2010, will monitor strains detected in leafy greens and other food products together with strains from community outbreaks to identify outbreaks linked to contaminated foods. Our comprehensive surveillance study identified norovirus and rotavirus contamination of packaged leafy greens. We detected noroviruses on 6% and rotavirus on 0.4% of lots tested from retail markets in southern Ontario. Packages with confirmed positive samples were both imported into Canada and had been conventionally grown. Noroviruses have a low infectious dose (2), and detection of viral RNA is associated with human health risk in oysters, another commodity that is eaten raw (10). Our results suggest a possible risk for foodborne transmission of norovirus and rotavirus from packaged leafy greens.

Antimicrobial resistance in Campylobacter, Salmonella, and Escherichia coli isolated from retail turkey meat from southern Ontario, Canada.

This study estimated the prevalence of Salmonella, Campylobacter, and Escherichia coli isolates in fresh retail grain-fed veal obtained in Ontario, Canada. The prevalence and antimicrobial resistance patterns were examined for points of public health significance. Veal samples (n = 528) were collected from February 2003 through May 2004. Twenty-one Salmonella isolates were recovered from 18 (4%) of 438 samples and underwent antimicrobial susceptibility testing. Resistance to one or more antimicrobials was found in 6 (29%) of 21 Salmonella isolates; 5 (24%) of 21 isolates were resistant to five or more antimicrobials. No resistance to antimicrobials of very high human health importance was observed. Ampicillin-chloramphenicolstreptomycin-sulfamethoxazole-tetracycline resistance was found in 5 (3%) of 21 Salmonella isolates. Campylobacter isolates were recovered from 5 (1%) of 438 samples; 6 isolates underwent antimicrobial susceptibility testing. Resistance to one or more antimicrobials was documented in 3 (50%) of 6 Campylobacter isolates. No Campylobacter isolates were resistant to five or more antimicrobials or category I antimicrobials. E. coli isolates were recovered from 387 (88%) of 438 samples; 1,258 isolates underwent antimicrobial susceptibility testing. Resistance to one or more antimicrobials was found in 678 (54%) of 1,258 E. coli isolates; 128 (10%) of 1,258 were resistant to five or more antimicrobials. Five (0.4%) and 7 (0.6%) of 1,258 E. coli isolates were resistant to ceftiofur and ceftriaxone, respectively, while 34 (3%) of 1,258 were resistant to nalidixic acid. Ciprofloxacin resistance was not detected. There were 101 different resistance patterns observed among E. coli isolates; resistance to tetracycline alone (12.7%, 161 of 1,258) was most frequently observed. This study provides baseline prevalence and antimicrobial resistance data and highlights potential public health concerns.

Campylobacter, Salmonella, Listeria monocytogenes, verotoxigenic Escherichia coli, and Escherichia coli prevalence, enumeration, and subtypes on retail chicken breasts with and without skin.

This study examined the prevalence, counts, and subtypes of Campylobacter, Salmonella, Listeria monocytogenes, verotoxigenic Escherichia coli (VTEC), and E. coli on raw retail chicken breast with the skin on versus the skin off. From January to December 2007, 187 raw skin-on chicken breasts and 131 skin-off chicken breasts were collected from randomly selected retail grocery stores in the Region of Waterloo, Ontario, Canada. Campylobacter isolates were recovered from a higher proportion of the skin-off chicken breasts, 55 (42%) of 131, than of the skin-on chicken breasts tested, 55 (29%) of 187 (P = 0.023). There was no difference in the proportion of Salmonella isolates recovered from the two meat types (P = 0.715): 40 (31%) of 131 skin-off chicken breasts versus 61 (33%) of 187 skin-on chicken breasts. L. monocytogenes isolates were recovered from a statistically lower proportion of the skin-off chicken breasts, 15 (15%) of 99, than of the skin-on chicken breasts, 64 (34%) of 187 (P = 0.001). There was no difference in the proportion of E. coli isolates recovered from the skin-off chicken breasts, 33 (33%) of 99, than from the skin-on chicken breasts, 77 (41%) of 187 (P = 0.204). VTEC was detected on a single skin-off chicken breast. Campylobacter jejuni was the most frequent species isolated on both types of chicken meat: skin-on, 48 (87%) of 55, and skin-off, 51 (94%) of 54. Salmonella serotypes Kentucky and Heidelberg and L. monocytogenes serotype 1/2a were the most frequently detected serotypes from both skin-off and skin-on chicken breasts. Although there appeared to be a trend toward higher enumeration values of these pathogens and E. coli on the skin-on chicken, the differences did not exceed 1 log. This study suggested that skin-off chicken breast may represent a higher risk of consumer exposure to Campylobacter, a similar risk for Salmonella, VTEC, and E. coli, and a lower risk for L. monocytogenes than skin-on chicken breast.

Occurrence of Salmonella, Campylobacter, Yersinia enterocolitica, Escherichia coli O157 and Listeria monocytogenes in Swine

The objective of this study was to investigate the occurrence of major bacterial foodborne pathogens in swine. In total, 359 samples from manure storage tanks (91) and fresh pooled faeces (268) obtained from finisher (110), sows (78) and weanlings (80) were collected and tested. Campylobacter, Salmonella, Yersinia enterocolitica, Escherichia coli O157 and Listeria monocytogenes were isolated from 36.5%, 31.5%, 5.8%, 3.3% and 3.3% of samples respectively. All E. coli O157 isolates found on 10 farms were tested but none was determined to be E. coli O157:H7. Salmonella and Campylobacter were more likely to be detected from stored manure rather than from fresh faecal samples. Yersinia enterocolitica tended to be detected more commonly from fresh samples than from manure pits. Listeria monocytogenes was not recovered from manure pits or from sow faecal samples and only infrequently found in the faeces of weanling pigs and finisher pigs. The proportion of positive samples showed a seasonal change. Salmonella was twice as likely not be recovered in winter, whereas the chance of culturing Campylobacter was higher in winter. The 113 Salmonella isolates recovered on 24 farms and the four most common serovars were Salmonella Typhimurium var. Copenhagen (31.0%), Salmonella Derby (12.4%), S. Typhimurium (10.6%) and Salmonella Agona (10.6%). Of 131 Campylobacter isolates recovered on 21 farms, 118 isolates were Campylobacter coli and 13 isolates could not be speciated. Fifteen of 21 Y. enterocolitica isolates found on 15 farms were detected in finisher pigs. The sero/biogroups of Y. enterocolitica were O3/biotype 4 (16 isolates), O6,30/biotype 1A (three isolates), O5/biotype 1A (one isolate) and O8/biotype 1B (one isolate). These findings provide baseline information on the distribution of important zoonotic pathogens in swine and indicate that pigs should be considered as a possible source of foodborne diseases in humans.

Seasonality in Human Salmonellosis: Assessment of Human Activities and Chicken Contamination as Driving Factors

This study used integrated surveillance data to assess the seasonality in retail chicken contamination and of human activities and their role on the seasonality of human endemic salmonellosis. From June 2005 to May 2008, reported cases of salmonellosis were followed-up comprehensively using a standardized questionnaire, and 616 retail chicken breasts were systematically tested for Salmonella, in one Canadian community. Poisson regression was used to model seasonality of human cases, Salmonella in retail chicken, and to assess the relationship between these and selected meteorological variables. The case-case approach was used to compare the activities of salmonellosis cases that occurred during the summer peak to the other cases. There were 216 human endemic salmonellosis cases (incidence rate: 14.7 cases/100,000 person-years), predominantly of Typhimurium and Enteritidis serotypes (28.4% and 20.8%, respectively). The monthly distribution of cases was associated with ambient temperature (p < 0.001) with a significant seasonal peak in June (p = 0.03) and July (p = 0.0005), but it was not associated with precipitation (p = 0.38). Several activities reported by cases tended to be more frequent during summer. Particularly, attending a barbeque and gardening within the 3 days before the disease onset were two significant risk factors for salmonellosis in June or July compared with the salmonellosis cases that occurred in the other months. Out of all chicken samples, 185 (30%) tested positive for Salmonella spp., Kentucky being the dominant serotype (44.3% of positive samples). The monthly proportion of positive chicken samples showed no seasonal variations (p = 0.30) and was not associated with the monthly count of human cases (p = 0.99). In conclusion, even though evidence generally supports chicken as a primary vehicle of Salmonella to humans, the contamination of retail chicken was not driving the seasonality in human salmonellosis. Attending a barbeque or gardening during the hotter months of the year should be further assessed for their risk.

Rotaviruses from Canadian farm samples

Animal rotavirus (RoV) strains detected in Canadian swine and dairy cattle farms were characterized by sequence analysis of viral protein 4 (VP4), VP6, VP7 and non-structural protein 4 segments from 15 RoV strains. Some porcine strains were found to contain a mixture of segments typical of human and animal viruses. One strain represented a novel VP6 genotype “I14”, G2-P[27]-I14. Other strains detected in porcine samples represented multiple different segment types. These results illustrate the active evolution of animal RoV strains and underline the need for surveillance of both animal and human strains in public health-monitoring programs.

Food consumption patterns in the Waterloo Region, Ontario, Canada: a cross-sectional telephone survey

BackgroundThe demographics and lifestyles of Canadians are changing, thereby influencing food choices and food preparation in the home. Although different dietary practices are associated with increased risk of foodborne illness, our ability to evaluate food consumption trends and assess risks associated with foodborne illness is limited by lack of data on current eating habits and consumer food safety practices. The objective of this study was to describe, for the first time, the food consumption patterns in a Canadian-based population from a food safety perspective, in order to establish baseline data on actual food intake of individuals.MethodA cross-sectional telephone survey of 2,332 randomly selected residents of Waterloo Region, Ontario, Canada (C-EnterNet pilot site) was conducted between November 2005 and March 2006. Food intake was assessed using a 7-day dietary recall method.ResultsCertain food items were consumed more than others among the same food groups, and consumption of many food items varied by gender and age. Specific foods considered high-risk for the transmission of certain enteric pathogens were significantly more likely to be consumed by males (i.e. unpasteurized juice, bean sprouts, and undercooked meat) and elderly individuals (i.e. undercooked eggs). The majority of households prepared and consumed most meals at home, allocating an average of 44 minutes to prepare a meal.ConclusionBaseline data on actual food intake is useful to public health professionals and food safety risk assessors for developing communication messages to consumers and in foodborne outbreak investigations.