Germ-free mice exhibit profound gut microbiota-dependent alterations of intestinal endocannabinoidome signaling.

The gut microbiota is a unique ecosystem of microorganisms interacting with the host through several biochemical mechanisms. The endocannabinoidome (eCBome), a complex signaling system including the endocannabinoid system, approximately 50 receptors and metabolic enzymes, and more than 20 lipid mediators with important physiopathologic functions, modulates gastrointestinal tract function and may mediate host cell-microbe communications there. Germ-free (GF) mice, which lack an intestinal microbiome and so differ drastically from conventionally raised (CR) mice, offer a unique opportunity to explore the eCBome in a microbe-free model and in the presence of a reintroduced functional gut microbiome through fecal microbiota transplant (FMT). We aimed to gain direct evidence for a link between the microbiome and eCBome systems by investigating eCBome alterations in the gut in GF mice before and after FMT. Basal eCBome gene expression and lipid profiles were measured in various segments of the intestine of GF and CR mice at juvenile and adult ages using targeted quantitative PCR transcriptomics and LC-MS/MS lipidomics. GF mice exhibited age-dependent modifications in intestinal eCBome gene expression and lipid mediator levels. FMT from CR donor mice to age-matched GF male mice reversed several of these alterations, particularly in the ileum and jejunum, after only 1 week, demonstrating that the gut microbiome directly impacts the host eCBome and providing a cause-effect relationship between the presence or absence of intestinal microbes and eCBome signaling. These results open the way to new studies investigating the mechanisms through which intestinal microorganisms exploit eCBome signaling to exert some of their physiopathologic functions.

The prevalence of enteric RNA viruses in stools from diarrheic and non-diarrheic people in southwestern Alberta, Canada.

Southwestern Alberta is a region of Canada that has high rates of enteritis as well as high densities of livestock. The presence of enteric RNA viruses, specifically norovirus (NoV) GI, GII, GIII, GIV; sapovirus (SaV); rotavirus (RV); and astrovirus (AstV), was evaluated in stools from diarrheic (n = 2281) and non-diarrheic (n = 173) people over a 1-year period in 2008 and 2009. Diarrheic individuals lived in rural (46.6 %) and urban (53.4 %) settings and ranged in age from less than 1 month to 102 years, and the highest prevalence of infection in these individuals was in November. In all, viruses were detected in diarrheic stools from 388 individuals (17.0 %). NoV GII was the most frequently detected virus (8.0 %; n = 182) followed by SaV (4.3 %; n = 97), RV (2.0 %; n = 46), AstV (1.8 %; n = 42), NoV GI (0.9 %; n = 20), and NoV GIV (0.1 %; n = 1). Animal NoV GIII was never detected. The prevalence of mixed viral infections in diarrheic individuals was 2.8 % (n = 11). Children from 1 to 5 years of age accounted for the highest prevalence of positive stools, followed by the elderly individuals (≥70 years). Only NoV GII (1.2 %; n = 2) and SaV (1.2 %; n = 2) were detected in stools from non-diarrheic people. Sequence analysis of a subset of stools revealed homology to NoV, SaV and RV sequences from humans but not to strains from non-human animals. The results of this study do not support the hypothesis that viruses of animal origin have a significant impact on the occurrence of acute gastroenteritis caused by RNA enteric viruses in people living in southwestern Alberta.

Analysis of complete genome sequences and a V239A substitution in the helicase domain of swine hepatitis E virus strains isolated in Canada.

Among Canadian swine HEV strains, only one complete genome sequence has been published so far, and there are no data on the virulence of these strains. A collection of 28 Canadian swine HEV strains was used in this study. After RNA extraction, a portion of ORF2, the 3' end of the helicase domain, and two complete genomes were amplified and sequenced. These two new Canadian complete genomes belonged to two different subtypes and showed 87.5 and 87.7% sequence identity to the Canadian swine HEV strain Arkell. The V239A substitution within the helicase domain, which is associated with increased virulence of genotype 3 HEV, was detected in one Canadian swine HEV strain. However, no human hepatitis E infections have been associated with this strain.

Chronic hepatitis E infection in children with liver transplantation.

OBJECTIVE: Chronic hepatitis E virus (HEV) infection has been described in immunosuppressed adult patients. A study was undertaken to establish the presence of HEV infection in children after orthotopic liver transplantation (OLT). METHODS: Children undergoing liver transplantation between 1992 and 2010 with available serum were classified into two groups: group 1 (control group, n=66) with normal serum aminotransferases and group 2 (n=14) with persistently increased serum aminotransferases and histological features of chronic hepatitis. Patients were tested for HEV RNA by reverse transcription-polymerase chain reaction (RT-PCR). HEV amplicons were sequenced and compared with published sequences. Antibody titres (IgG and IgM) to 12 HEV immunodominant regions were measured by enzyme-linked immunosorbent assays. RESULTS: In group 1 (control group), 15% of children were anti-HEV IgG-positive during follow-up. No anti-HEV IgM antibodies were detected in any of these children. After OLT, 86% of patients in group 2 had anti-HEV IgG compared with 36% pre-OLT. Thus, two-thirds of children acquired anti-HEV IgG after OLT. Seven anti-HEV IgG-positive patients (58%) were also anti-HEV IgM-positive more than once during follow-up after OLT. Eight years post-OLT, one girl presented with anti-HEV IgG and IgM that remained positive afterwards. In this patient, HEV RNA was found in five different annual samples from 10 years post-OLT, concomitantly with increased serum aminotransferases and cirrhosis development during that period. Phylogenetic analysis revealed two different HEV strains (detected 3 years apart) that were highly similar to swine genotype 3, suggesting a possible case of zoonotic re-infection. CONCLUSION: The diagnosis of HEV infection is technically challenging and should be made simultaneously with RT-PCR methods, viral load quantification and serological markers. In immunosuppressed children who develop chronic hepatitis, the prevalence of HEV is high and could explain the chronic liver inflammation potentially leading to cirrhosis. Re-infection by different HEV strains from zoonotic transmission can result in progressive liver disease in immunocompromised children.

Genetic contribution to C-reactive protein levels in severe obesity.

Obese individuals are characterized by a chronic, low-grade inflammatory state. Increased levels of C-reactive protein (CRP), a marker of inflammation, have been observed in subjects with the metabolic syndrome. We have previously reported that genes encoding proteins involved in the anti-inflammatory and immune response are differentially expressed in visceral adipose tissue of obese men with or without the metabolic syndrome. Among these genes, the interferon-gamma-inducible protein 30 (IFI30), CD163 molecule (CD163), chemokine (C-X-C motif) ligand 9 (CXCL9) and thymic stromal lymphopoietin (TSLP), were selected for further genetic analyses. The aim of the study was to verify whether IFI30, CD163, CXCL9 and TSLP gene polymorphisms contribute to explain the inter-individual variability of the inflammatory profile of obesity assessed by plasma high-sensitivity CRP concentrations. A total of 1185 severely obese individuals were genotyped for single nucleotide polymorphisms (SNPs) covering most of the sequence-derived genetic variability at the IFI30, CD163, CXCL9 and TSLP gene loci (total of 27 SNPs). Following measurement of plasma CRP levels, subjects were divided into two groups, low vs. high using the median value of plasma CRP levels (8.31 mg/L) as a cutoff point. Genotype frequencies were compared between groups. Associations between genotypes and plasma CRP levels (continuous variable) were also tested after adjustments for age, sex, smoking and BMI. The rs11554159 and rs7125 IFI30 SNPs showed a significant difference in genotype frequencies (p<0.05) between subgroups of low vs. high plasma CRP levels (wild type homozygotes: rs11554159=47% vs. 55%, rs7125=31% vs. 24%, for low vs. high CRP groups, respectively). The association between rs11554159 and CRP levels as a continuous variable remained significant (p=0.004). Both carriers of the GA and AA genotypes demonstrated, on average, a 13% lower CRP levels in comparison with GG homozygotes. No association was observed between SNPs in the CD163, CXCL9 and TSLP genes and CRP levels. The IFI30 rs11554159 polymorphism could partially explain the inter-individual variability observed in the inflammatory profile associated with obesity.

Cirrhosis due to chronic hepatitis E infection in a child post-bone marrow transplant.

Chronic hepatitis E virus (HEV) infection occurs in immunosuppressed adults. We detected HEV ribonucleic acid in serum of an adolescent patient who had undergone bone marrow transplantation and subsequently presented with persistently increased aminotransferases and histologic chronic hepatitis, and eventually developed cirrhosis. Phylogenetic analysis revealed these HEV strains were similar to swine genotype 3a, suggesting a possible zoonosis.

Enteric campylobacteria and RNA viruses associated with healthy and diarrheic humans in the Chinook health region of southwestern Alberta, Canada.

The presence of Campylobacter species and enteric RNA viruses in stools from diarrheic (n = 442) and healthy (n = 58) humans living in southwestern Alberta was examined (May to October 2005). A large number of diarrheic individuals who were culture negative for C. jejuni (n = 54) or C. coli (n = 19) were PCR positive for these taxa. Overall detection rates for C. jejuni and C. coli in diarrheic stools were 29% and 5%, respectively. In contrast, 3% and 0% of stools from healthy humans were positive for these taxa, respectively. Infection with C. jejuni was endemic over the study period. However, there was no difference in infection rates between individuals living in urban or rural locations. Stools from a large number of diarrheic (74%) and healthy (88%) individuals were positive for Campylobacter DNA. The prevalence rates of C. concisus, C. curvus, C. fetus, C. gracilis, C. helveticus, C. hominis, C. hyointestinalis, C. mucosalis, C. showae, C. sputorum, and C. upsaliensis DNA were either not significantly different or were significantly lower in stools from diarrheic than from healthy individuals. No C. lanienae or C. lari DNA was detected. Stools from 4% and 0% of diarrheic and healthy humans, respectively, were positive for rotavirus, sapovirus, or norovirus (GI/GII). Our results showed a high prevalence of diarrheic individuals living in southwestern Alberta who were infected by C. jejuni and, to a lesser extent, by C. coli. However, other Campylobacter species, norovirus, rotavirus, sapovirus, and bovine enteric calicivirus were either inconsequential pathogens during the study period or are not pathogens at all.

Unusual central nervous system lesions in slaughter-weight pigs with porcine circovirus type 2 systemic infection.

Porcine circovirus type 2 systemic infection was diagnosed in 2 slaughter-weight pigs based on postmortem examination. The infection was associated with unusual central nervous system lesions characterized by a multifocal lymphohistiocytic to granulomatous meningoencephalomyelitis with giant cell formation. The role of these nervous system lesions in the development of the clinical signs in these pigs remains uncertain.

Multicenter comparison of two norovirus ORF2-based genotyping protocols.

Point source norovirus outbreaks can be difficult to track due to high background levels of the virus in the environment and the limited strain variation in some genotyping regions. However, rapid and accurate source identification can limit the spread of a foodborne outbreak and reduce the number of cases. Harmonization of genotyping assays is critical for enabling the rapid exchange of sequence data nationally and internationally. Several regions of the genome have been proposed for this purpose, but no consensus has been reached. In the present study, two standardized genotyping protocols (region C and region D) were evaluated by nine laboratories in Canada and the United States, using a coded panel of 96 fecal specimens representing 22 different norovirus genotypes. Overall, region C typing had a success rate of 78% compared to 52% for region D; however, region D provides greater nucleotide sequence diversity for identifying new GII.4 variant strains. Significant differences in the genotyping success rate were observed among the nine participating laboratories (10% to 100%) and among the different genotypes (6% to 100%). For several genogroup II strains, reduced region D amplification correlated directly with mismatches between primer sequences and the template. Based on overall performance, we recommend the region C protocol for routine genotyping of noroviruses, while the region D protocol may be useful for identifying new GII.4 variants. Standardized genotyping protocols will enable rapid exchange of outbreak and sequence data through electronic norovirus surveillance networks.

Comparison of commercial viral genomic extraction kits for the molecular detection of foodborne viruses.

When genetic material is extracted from viruses responsible for food illnesses, two broad types of possibilities are offered: conventional methods, which are well established but usually long and exacting to perform, or commercial kits, which are faster and easy to use but much more expensive. Thus, it is important to evaluate some performance parameters such as the analytical sensitivity to be able to select the optimal technique for each situation. The principal objective of this study was to establish and compare the analytical sensitivities of three commercial genetic material extraction methods (TRIzol reagent, FTA cards, and QIAGEN kits) along with three selected viruses, adenovirus, hepatitis A virus, and rotavirus. Viral detection was carried out using a standard PCR technique for adenovirus and reverse transcription PCR for rotavirus and hepatitis A virus. The results obtained showed that with the QIAGEN kit, the sensitivity was 2 logs lower than with the two other methods for all three viruses studied. Nevertheless, despite their lower analytical sensitivities, the other two extraction methods should not be overlooked and ought to be considered when evaluating the most efficient approach suitable for a specific commodity, since food-related outbreaks may be traced to a wide variety of food types.

Rapid and Concomitant Gut Microbiota and Endocannabinoidome Response to Diet-Induced Obesity in Mice.

The intestinal microbiota and the expanded endocannabinoid (eCB) system, or endocannabinoidome (eCBome), have both been implicated in diet-induced obesity and dysmetabolism. These systems were recently suggested to interact during the development of obesity. We aimed at identifying the potential interactions between gut microbiota composition and the eCBome during the establishment of diet-induced obesity and metabolic complications. Male mice were fed a high-fat, high-sucrose (HFHS) diet for 56 days to assess jejunum, ileum, and cecum microbiomes by 16S rRNA gene metataxonomics as well as ileum and plasma eCBome by targeted liquid chromatography-tandem mass spectrometry (LC-MS/MS). The HFHS diet induced early (3 days) and persistent glucose intolerance followed by weight gain and hyperinsulinemia. Concomitantly, it induced the elevation of the two eCBs, anandamide, in both ileum and plasma, and 2-arachidonoyl-glycerol, in plasma, as well as alterations in several other N-acylethanolamines and 2-acylglycerols. It also promoted segment-specific changes in the relative abundance of several genera in intestinal microbiota, some of which were observed as early as 3 days following HFHS diet. Weight-independent correlations were found between the relative abundances of, among others, Barnesiella, Eubacterium, Adlercreutzia, Parasutterella, Propionibacterium, Enterococcus, and Methylobacterium and the concentrations of anandamide and the anti-inflammatory eCBome mediator N-docosahexaenoyl-ethanolamine. This study highlights for the first time the existence of potential interactions between the eCBome, an endogenous system of multifunctional signaling lipids, and several intestinal genera during early and late HFHS-induced dysmetabolic events, with potential impact on the host capability of adapting to increased intake of fat and sucrose.IMPORTANCE The intestinal microbiota and the expanded endocannabinoid system, or endocannabinoidome, have both been implicated in diet-induced obesity and dysmetabolism. This study aims at identifying the potential interactions between these two fundamental systems-which form the gut microbiota-endocannabinoidome axis-and their involvement in the establishment of diet-induced obesity and related metabolic complications. We report here time- and segment-specific microbiome disturbances as well as modifications of intestinal and circulating endocannabinoidome mediators during high-fat, high-sucrose diet-induced glucose intolerance and subsequent obesity and hyperinsulinemia. This highlights the involvement of, and the interaction between, the gut microbiota and the endocannabinoidome during metabolic adaptation to high-fat and high-sucrose feeding. These results will help identifying actionable gut microbiome members and/or endocannabinoidome mediators to improve metabolic health.

Variants within the muscle and liver isoforms of the carnitine palmitoyltransferase I (CPT1) gene interact with fat intake to modulate indices of obesity in French-Canadians.

Obesity is under the influence of genetic and nutritional factors. The objective was to verify whether variants in the gene encoding the carnitine palmitoyltransferase I (CPT1), a key enzyme in beta-oxidation of fatty acids, are associated with obesity phenotypes, alone or in interaction with fat intake. Sequencing of CPT1 was performed in 40 overweight subjects and 4 controls. Genotypes were determined in 351 French-Canadians. Fat intake was evaluated by a food frequency questionnaire. We identified 14 genetic variations in CPT1A and 26 in CPT1B. Nine variants within CPT1B and one variant within CPT1A were selected for further analyses based on the minor allele frequency (>10%) or on potential functional impact. A significant association between obesity phenotypes (BMI, weight, and waist girth) and CPT1B c.282-18C > T and p.E531K variants was observed (p < 0.05) No other association was found with variants in CPT1A and CPT1B. When subjects were divided into six groups according to p.E531K genotypes and further on the basis of fat using the median value as a cutoff point (34.4% of energy), BMI, weight, and waist girth were higher in E531/K531 on a high-fat diet compared to E531/K531 subjects under a low-fat diet (p = 0.004, p = 0.006, p = 0.003, respectively). There was no difference among E531/E531 and K531/K531. Similar results were obtained with the CPT1A p.A275T variant as BMI and waist girth were higher in A275/A275 on a high fat compared to A275/A275 subjects on a low-fat diet. Among carriers of the T275 allele, obesity indices were not affected by fat intake (p = 0.05 and p = 0.008 for BMI and waist girth, respectively). Among variants within the CPT1B gene, 13 haplotypes were inferred and the two most frequent haplotypes, H7 (38.0%) and H5 (27.7%), were kept for diplotype analysis. These haplotypes were not associated with indices of obesity, but as observed with the CPT1B E531K variant fat intake modulated this association. In conclusion, this finding suggests that indices of obesity might be modulated by an interaction between CPT1 variants and fat intake.

Molecular characterization of hepatitis E virus detected in swine farms in the province of Quebec.

Swine Hepatitis E virus (HEV) could be a zoonotic agent for HEV infection in humans. In Canada, approximately 60% of 6-mo-old commercial pigs are seropositive for HEV; the prevalence is higher in the provinces of Quebec and Ontario. A study was set up to evaluate the presence of swine HEV in Quebec farms and to compare the strains detected in fecal samples with human and swine HEV strains reported worldwide. Fecal samples were collected randomly from May 2003 to January 2004 from 70 swine farms in Quebec. In 24 specimens, representing 34% of the visited farms, HEV RNA was extracted and detected by nested reverse-transcription polymerase chain reaction (RT-PCR). All amplified nested RT-PCR products were purified, cloned, and sequenced. The nucleotide sequences of a 304 base pair fragment at the 5' end of the open reading frame 2 gene were determined. Phylogenetic analysis revealed that the 24 amplified fragments clustered in genotype 3 and had 85% to 99% nucleotide-sequence similarity with HEV strains identified in Japan, the United States, and Canada. Three strains identified in the study (swSTHY1, swSTHY31, and swSTHY47) showed 95% homology with 2 Japanese (swJ1-1 and HE-JA10) and 1 American (US1) HEV human strains.

Antibiotic resistance in Escherichia coll and Enterococcus spp. isolates from commercial broiler chickens receiving growth-promoting doses of bacitracin or virginiamycin.

Antibacterial agents such as zinc bacitracin (ZB) and virginiamycin (VG) are used as growth promoting agents (GP) in broiler chicken production. The objective of this study was to evaluate the effect of the use of ZB and VG on the emergence of antibacterial resistance in a commercial broiler chicken farm. Three trials were conducted using 3 different diets: one without antibacterial agents, one containing VG, and one with ZB. Escherichia coli and Enterococcus spp. strains were isolated and tested for their susceptibility to various antibacterial agents. The occurrence of the resistance genes vatD, ermB, and bcrR in Enterococcus spp. isolates was determined by polymerase chain reaction (PCR). Comparative quantification of vatD and bcrR genes in total deoxyribonucleic acid (DNA) extracts from litter was done by SYBR Green Real-Time PCR (QPCR). Escherichia coli and Enterococcus spp. isolates from diet groups had different levels of resistance to various antibacterial agents over time. These GPs did not select for specific antibacterial agent resistance (AAR) in Enterococcus spp. The use of GPs seemed to lower the percentage of E. coli isolates resistant to some antibacterial agents. The presence of the bcrR gene could not explain all resistant phenotypes to ZB. Genes other than vatD and ermB might be involved in the resistance to VG in Enterococcus spp. Use of GPs was not associated with presence of the bcrR gene in DNA extracts from litter, but use of VG was associated with vatD presence.

Comparative evaluation of new TaqMan real-time assays for the detection of hepatitis A virus.

Three novel real-time TaqMan RT-PCR assays targeting the 5'-UTR, the viral protease and the viral polymerase regions of the hepatitis A virus (HAV) were developed, evaluated and compared against a new published 5'-UTR TaqMan assay (JN) and a widely used conventional RT-PCR assay (HAVc). All conventional RT-PCR (HAV, SH-Prot and SH-Poly systems) and TaqMan (SH-Prot, SH-Poly, JN and SH-5U systems) assays evaluated were consistent for the detection of the three different HAV strains (HM-175, HAS-15 and LSH/S) used and reproducible for both RNA duplicates with the exception of two reproducibility discrepancies observed with both 5'-UTR real-time systems (JN and SH-5U). Limits of detection for conventional HAV, SH-Prot and SH-Poly RT-PCR systems were found to be equivalent when tested with serially diluted suspensions of the HM-175 strain. Although the four real-time RT-PCR TaqMan assays evaluated herein produced similar and consistent quantification data down to the level of one genomic equivalent copy with their respectively cloned amplicons, significant and important differences were observed for the detection of HAV genomic RNA. Results showed that the new real-time TaqMan SH-Poly and SH-Prot primer and probe systems were more consistent and sensitive by 5 logs as compared to both 5'-UTR designs (JN and SH-5U) used for the detection of HAV genomic RNA as well as for the detection in cell culture by cytopathic effect. Considering their higher analytical sensitivity, the proposed SH-Poly and SH-Prot amplification systems could therefore represent valuable tools for the detection of HAV in clinical, environmental and food samples.

Presence of hepatitis E virus in a naturally infected swine herd from nursery to slaughter.

Hepatitis E virus has recently been recognized as having zoonotic potential and could be transmitted from pig to human. Pigs are identified as a potential animal reservoir and HEV is highly prevalent in the swine population around the world. In this study, the presence of HEV was investigated in 51 subjects reared on a simulated commercial farm setting from the age of 2 weeks up to slaughter. Samples were collected on four occasions: at 2, 8, and 18 weeks and between 22-29 weeks of age. Anti-HEV IgG in plasma samples, presence of HEV RNA in plasma samples and feces were monitored. At 2 weeks of age, HEV RNA was detected in feces of 6 subjects (11.8%) but not in their plasma. At 8 weeks, HEV was detected in feces of 27 subjects (52.9%) and in plasma of one subject. At 18 weeks, HEV was detected in feces of 44 subjects (86.2%) and in plasma of 24 subjects (47.1%). At slaughter time (22-29 weeks), HEV was present in plasma of 6 subjects (11.8%) and in stools of 21 subjects (41.2%). Spread of the virus inside the population was evaluated by comparison of means (paired t-test, P<0.05) of anti-HEV IgG ELISA results from the 4 bleedings. Significant differences were noted between the results of populations at 8 and 18 weeks and also between 18 and 22 to 29 weeks indicating an immune response to the virus. Based on the comparison of a 304 nucleotides sequence of the 5' ORF 2 gene, all amplified fragments clustered in genotype 3a.

The emergence of porcine circovirus 2b genotype (PCV-2b) in swine in Canada.

Since late 2004, the swine industry in the province of Quebec has experienced a significant increase in death rate related to postweaning multisystemic wasting syndrome (PMWS). To explain this phenomenon, 2 hypotheses were formulated: 1) the presence of a 2nd pathogen could be exacerbating the porcine circovirus 2 (PCV-2) infection, or 2) a new and more virulent PCV-2 strain could be infecting swine. In 2005, 13 PMWS cases were submitted to the Quebec provincial diagnostic laboratory and PCV-2 was the only virus that could be found consistently by PCR in all 13 samples. The PCR detection results obtained for other viruses revealed the following: 61.5% were positive for porcine reproductive and respiratory syndrome virus, 30.8% for swine influenza virus, 15.4% for porcine parvovirus, 69.2% for swine torque teno virus (swTTV), 38.5% for swine hepatitis E virus (swHEV) and 84.6% for Mycoplasma hyorhinis; transmissible gastroenteritis virus and porcine respiratory coronavirus (TGEV/PRCV) was not detected. Sequences of the entire genome revealed that these PCV-2 strains belonged to a genotype (named PCV-2b) that has never been reported in Canada. Further sequence analyses on 83 other Canadian PCV-2 positive cases submitted to the provincial diagnostic laboratory during years 2005 and 2006 showed that 79.5% of the viral sequences obtained clustered in the PCV-2b genotype. The appearance of the PCV-2b genotype in Canada may explain the death rate increase related to PMWS, but this relationship has to be confirmed.

Development of an extraction and concentration procedure and comparison of RT-PCR primer systems for the detection of hepatitis A virus and norovirus GII in green onions.

Vegetables can be considered as a vector of transmission for human hepatic and enteric viruses such as hepatitis A virus (HAV) and noroviruses when contaminated by spoiled irrigation water or when prepared by infected food handlers. Recently, outbreaks of HAV have been reported in the USA involving fresh green onions. A viral elution-concentration method was developed for the detection of HAV and norovirus contaminated green onions by RT-PCR. Repeated pipetting/washings of the surface with a pH 9.5 glycine-buffered solution allowed the elution of viruses from the vegetables. Concentration of the viral load was performed by a polyethylene glycol (PEG) precipitation procedure. Viral RNAs were extracted and purified using a combination of Trizol-chloroform and poly(dT) magnetic beads methods. Different sets of primers, including two newly designed primers sets for HAV RT-PCR, were tested in order to achieve the best analytical sensitivity. Using the new primer design, it was possible to detect 10(0) TCID(50%)/25 g of HAV in fresh green onions, while 1 RT-PCRU/25 g was detected for noroviruses GII using previously described primers. This method, based on molecular tools, would be useful for diagnostic laboratories in order to perform viral analyses of such commodities as fresh vegetables in cases of foodborne infections.

Comparative evaluation of RT-PCR, nucleic acid sequence-based amplification (NASBA) and real-time RT-PCR for detection of noroviruses in faecal material.

Using different primer and probe sets, RT-PCR, NASBA and TaqMan RT-PCR molecular methods were compared to detect NoV GII in 13 clinical stool samples. The RT-PCR results observed by gel electrophoresis (Ando, Kageyama and Anderson amplification and probe systems), dot blot hybridization (Ando and Kageyama) and real-time TaqMan assay (Ando and Kageyama) were shown to be consistent and reproducible for the detection of NoV GII. Whereas, the NASBA assay using Ando primers showed some reproducibility discrepancies. Detection limits of the NoV GII/Kageyama system were found to be equal or significantly higher than the Ando system. Real-time TaqMan RT-PCR assay showed similar detection limits to that of NASBA with the Kageyama amplification and detection system, while it was 1log less sensitive than the Ando system. In a clinical context, RT-PCR, NASBA and real-time TaqMan RT-PCR methods using undiluted samples were all suitable for the detection of NoV GII, however the NASBA assay provided less consistent signals. The NoV GII Kageyama real-time TaqMan RT-PCR assay was reliable with a high analytical sensitivity and has shown the capability of detecting one genomic equivalent copy.

Farm-level prevalence and risk factors for detection of hepatitis E virus, porcine enteric calicivirus, and rotavirus in Canadian finisher pigs.

Hepatitis E virus (HEV), norovirus (NoV), and rotavirus (RV) are all hypothesized to infect humans zoonotically via exposure through swine and pork. Our study objectives were to estimate Canadian farm-level prevalence of HEV, NoV [specifically porcine enteric calicivirus (PEC)], and RV in finisher pigs, and to study risk factors for farm level viral detection. Farms were recruited using the Canadian Integrated Program for Antimicrobial Resistance Surveillance (CIPARS) and FoodNet Canada on-farm sampling platforms. Six pooled groups of fecal samples were collected from participating farms, and a questionnaire capturing farm management and biosecurity practices was completed. Samples were assayed using validated real-time polymerase chain reaction (RT-PCR). We modeled predictors for farm level viral RNA detection using logistic and exact logistic regression. Seventy-two herds were sampled: 51 CIPARS herds (15 sampled twice) and 21 FoodNet Canada herds (one sampled twice). Hepatitis E virus was detected in 30/88 farms [34.1% (95% CI 25.0%, 44.5%)]; PEC in 18 [20.5% (95% CI: 13.4%, 30.0%)], and RV in 6 farms [6.8% (95% CI: 3.2%, 14.1%)]. Farm-level prevalence of viruses varied with province and sampling platform. Requiring shower-in and providing boots for visitors were significant predictors (P < 0.05) in single fixed effect mixed logistic regression analysis for detection of HEV and PEC, respectively. In contrast, all RV positive farms provided boots and coveralls, and 5 of 6 farms required shower-in. We hypothesized that these biosecurity measures delayed the mean age of RV infection, resulting in an association with RV detection in finishers. Obtaining feeder pigs from multiple sources was consistently associated with greater odds of detecting each virus.

Le virus de l'hépatite E (VHE), le norovirus (NoV), et le rotavirus (RV) sont tous suspectés être des agents zoonotiques associés à une exposition aux porcs ou à la viande de porc. Les objectifs de la présente étude étaient d'estimer, dans des fermes canadiennes, la prévalence de VHE, NoV [spécifiquement le calicivirus entérique porcin (CEP)], et le RV chez des porcs en finition, et d'étudier les facteurs de risque pour la détection virale à la ferme. Les fermes ont été recrutées à l'aide des plateformes d'échantillonnage à la ferme du Programme intégré canadien de surveillance de la résistance aux antimicrobiens (PICRA) et de FoodNet Canada. Six groupes d'échantillons amalgamés de matières fécales ont été récoltés dans les fermes participantes, et un questionnaire relevant les pratiques de gestion à la ferme et les mesures de biosécurité a été complété. Les échantillons ont été analysés au moyen d'une méthode validée de réaction d'amplification en chaîne par la polymérase en temps réel (RT-PCR). Des prédicteurs de détection de l'ARN viral sur la ferme ont été modélisés à l'aide de régressions logistiques et de régressions logistiques exactes. Soixante-douze troupeaux ont été échantillonnés : 51 troupeaux du programme CRIPA (15 troupeaux échantillonnés deux fois) et 21 troupeau du programme FoodNet Canada (un troupeau échantillonné deux fois). Le VHE a été détecté dans 30/88 fermes [34,1 % (IC 95 % : 25,0 %, 44,5 %)], CEP dans 18 [20,5 % (IC 95 % : 13,4 %, 30,0 %)], et RV dans 6 fermes [6,8 % (IC 95 % : 3,2 %, 14,1 %)]. La prévalence des virus dans les fermes variait selon la province et la plate-forme d'échantillonnage. Une douche obligatoire avant l'entrée dans la porcherie et le fait de fournir des bottes aux visiteurs s'avéraient des prédicteurs significatifs (P < 0,05) pour la détection du VHE et du CEP, respectivement, dans une analyse par régression logistique mixte à effet fixe unique. Ceci contrastait avec le fait que toutes les fermes positives pour RV fournissaient des bottes et des couvre-tout, et 5 des 6 fermes exigeaient une douche à l'entrée. Nous émettons l'hypothèse que ces mesures de biosécurité ont retardé l'âge moyen d'une infection par le RV, ce qui résultait en une association entre la détection de RV et les animaux en finition. L'acquisition de porcs en croissance de sources multiples était constamment associée avec une probabilité plus grande de détecter chaque virus.(Traduit par Docteur Serge Messier).