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.

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.

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.

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).

Association of age and gender with Torque teno virus detection in stools from diarrheic and non-diarrheic people.

BACKGROUND: Torque teno virus (TTV) is a small virus belongs to Anelloviridea family. TTV is a disease orphan virus but it has often been associated with a variety of pathologies and co-infections. TTV was recently identified as an infectious agent that could potentially be involved in cases of acute enteritis. OBJECTIVES: To ascertain the presence of TTV in stools from diarrheic and not diarrheic people, and to investigate an association between infection, and patient age and gender. STUDY DESIGN: Stool samples from people exhibiting signs of enteritis (954) and from non-diarrheic individuals (76) were collected in the former Chinook Health Region (CHR) in Southwestern Alberta, Canada from May 2008 to April 2009. Viral genetic material was extracted, and detection and quantification of TTV were carried out by real-time PCR. The presence of other viral and bacterial enteric pathogens was also investigated. RESULTS: More (P<0.001) diarrheic people (38.8%) tested positive for TTV DNA than non-diarrheic individuals (18.4%). Furthermore, viral load was greater (P<0.001) in stools from diarrheic (2.0×10(7)copies/g) than non-diarrheic (2.0×10(3)copies/g) people. TTV DNA was detected most often in diarrheic individuals that were 0-5 (57.3%) and greater than 81 (59.0%) years of age. Combined across age, the prevalence of TTV was higher among men than women (P=0.003). Co-infections with other enteric pathogens were observed. CONCLUSIONS: This study revealed a significant association between TTV prevalence and viral load, and enteritis. Also, TTV prevalence was significantly higher in the very young and elderly suggesting that immunological status is important.

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.

A scoping review of the evidence for public health risks of three emerging potentially zoonotic viruses: hepatitis E virus, norovirus, and rotavirus.

Emerging zoonoses are defined as those newly recognized, or increasing in incidence or geographic range. Hepatitis E virus (HEV), norovirus (NoV), and rotavirus (RV), while well known to be transmitted person-person, have also been hypothesized to be emerging zoonoses. Our objective was to investigate their potential public health risks from animal reservoirs. Given the diversity of evidence sources, a scoping review incorporating a mixed methods synthesis approach was used. A broad search was conducted in five electronic databases. Each citation was appraised independently by two reviewers using screening tools designed and tested a priori. Level 1 relevance screening excluded irrelevant citations; level 2 confirmed relevance and categorized. At level 3 screening, data were extracted to support a risk profile. A stakeholder group provided input on study tools and knowledge translation and transfer. Level 1 screening captured 2471 citations, with 1270 advancing to level 2 screening, and 1094 to level 3. We defined criteria for case attribution to zoonosis for each virus. Using these criteria, we identified a small number of zoonotic cases (HEV n=3, NoV=0, RV=40 (zoonoses=3; human-animal re-assortants=37)) categorized as 'likely'. The available evidence suggests the following potential HEV human exposure sources: swine, other domestic animals, wildlife, surface waters, and asymptomatic human shedders. Possible at-risk groups include the immunocompromised and the elderly. Reports of NoV intergenogroup recombinants suggest potential for human-animal recombination. Greatest public health impact for RV zoonoses may be the potential effect of human-animal reassortants on vaccination efficacy.

Preliminary molecular epidemiological investigation of hepatitis E virus sequences from Québec, Canada.

Our study objective was to describe the Canadian Hepatitis E virus (HEV) sequences currently cataloged in GenBank from three populations: commercially raised pigs, retail pork, and locally acquired Hepatitis E cases, and to interpret the molecular evidence they provide. We searched the GenBank for any/all Canadian HEV sequences from these populations, and identified highly similar matches using the Basic Local Alignment Search Tool (BLAST) algorithm, studying sequences of the partial ORF2 gene. We validated the findings made using Multiple Sequence Comparison by Log-Expectation (MUSCLE) and Clustal 2 programs for multiple sequence alignments, as inputs to estimate dendrograms using both neighbour-joining and Unweighted Pair Group Method with Arithmetic Mean (UPGMA) methods. The GenBank search yielded 47 sequences collected from pigs: 32 sequences from two to four month old commercial pigs in Québec, one from three to four month old pigs at a research station in Ontario, one from two month old pigs in a commercial Saskatchewan herd, and 13 collected from finisher pigs in a national survey. Additionally, 14 sequences were collected from a national survey of Canadian retail pork livers, and seven sequences from two Canadian pediatric patients with locally acquired Hepatitis E, both from the province of Québec. All sequences belonged to genotype 3. Eight of the 14 sequences from retail pork livers had human-derived sequences in their top ten BLAST matches; six did not. Those eight sequences having close human BLAST matches clustered within a dendrogram, as did those with no close human BLAST matches. Human sequences with close matches to the eight retail sequences included both of the Québec Hepatitis E cases, as well as sequences from Japanese Hepatitis E cases, and Japanese blood donors. Seven of the eight HEV sequences from retail liver with close human BLAST matches originated in Québec. Kulldorff's spatial scan statistic showed a significant (P<0.05) spatial cluster of these sequences, but not of the overall dataset of 12 HEV sequences collected from Québec retail livers. All seven retail liver sequences with close human matches were processed in-store. We conclude that some Canadian sequences of HEV collected from pigs/pork are more closely related to human sequences than others, and hypothesize that detection of some HEV sequences recovered from Canadian retail pork livers may be associated with exposure to human shedding. More research needs to be conducted at the processing level to help understand the molecular epidemiology of HEV in Canadian retail pork.

Survey of Canadian retail pork chops and pork livers for detection of hepatitis E virus, norovirus, and rotavirus using real time RT-PCR.

Over the past 15 years, hepatitis E virus (HEV), norovirus (NoV), and rotavirus (RV) have been hypothesized to be potentially zoonotic; swine and pork have been suggested as possible human infection sources for all 3 viruses. Our objective was to estimate HEV, NoV, and RV prevalence and load on Canadian retail pork chops and livers. Using the Canadian Integrated Program for Antimicrobial Resistance Surveillance (CIPARS) sampling platform, pork livers (n=283) and chops (n=599) were collected, processed, and assayed for the 3 viruses by four collaborating federal laboratories using validated real time reverse transcriptase polymerase chain reactions (qRT-PCR). Follow-up qRT-PCR estimating viral load in genomic copies/g was followed by nested classical RT-PCR and isolate sequencing of a partial segment of the ORF2 gene. Local alignments were performed using MUSCLE (Multiple Sequence Comparison by Log-Expectation); a phylogenetic tree was created. Twenty-five livers and 6 chops were classified 'positive' (thresholds for viral RNA detected in both replicates of the assay) or 'suspect' (thresholds detected in one of two replicates) for HEV. Follow-up qRT-PCR detected HEV on 16 livers, 0 chops, and nested classical RT-PCR, on 14 livers and 0 chops. Initial qRT-PCR classified 12 chops 'suspect' for NoV. Follow-up qRT-PCR detected viral RNA on only one sample with thresholds greater than 40 in both replicates. No amplicon was yielded, and therefore no isolate was sequenced from this sample. Partial ORF2 genes from 14 HEV isolates were sequenced, and compared via sequence identity and phylogenetic analysis with selected human case isolates listed in NCBI-GenBank. Overall, HEV prevalence on retail pork was comparable with other published reports.

Presence, viral load and characterization of Torque teno sus viruses in liver and pork chop samples at retail.

Torque teno viruses (TTV) are widespread in humans, swine as well as in several other animal species. In market ready swine, the reported prevalence ranges between 11% and 100%. Through a national retail sampling plan from the Canadian Integrated Program for Antimicrobial Resistance Surveillance (CIPARS) program, 283 and 599 liver and pork chop samples, respectively, were collected over a 12-month period from commercial establishments in 5 selected geographical regions of Canada to assess the presence of Torque teno sus viruses (TTSuVs) in these products. TTSuVs were detected in 97.9% of pork chops with viral loads ranging between 1×10(4) and 9.9×10(5) genomic copies (gc)/g and 98.6% of liver samples with viral loads ranging from 1×10(5) to 9.9×10(6) gc/g. A selection of 20 positive samples (10 pork chop and 10 liver) from the 5 geographical regions were further tested for the production, of a 305bp fragment for TTSuV1 and a 253bp fragment for TTSuV2 in the non-coding region. TTSuV1 was present in all 10 liver and 10 pork chops samples while TTSuV2 was detected in 10 liver and 9 pork chop samples. Two different TTSuV1 sequences were simultaneously detected from 5 of 20 samples and 2 different TTSuV2 sequences were detected from 6 of 19 samples. The omnipresence of TTSuVs in commercial pork samples may allow its use as a viral indicator to monitor the effectiveness of cleaning and disinfecting process in slaughtering, cutting, slicing and packaging facilities.

Association of LIPA gene polymorphisms with obesity-related metabolic complications among severely obese patients.

The lipase A, lysosomal acid, cholesterol esterase enzyme (LIPA) is involved in the hydrolysis of triglycerides (TGs) and cholesteryl esters (CEs) delivered to lysosomes. LIPA deficiency in human causes two distinct phenotypes characterized by intracellular storage of CE and derangements in the control of cholesterol production, namely the Wolman disease (WD) and the CE storage disease (CESD). To test the potential association of LIPA gene polymorphisms with obesity-related metabolic complications, promoter, exons, and intronic flanking regions of the LIPA gene were first sequenced in 25 individuals. From the 14 common polymorphisms identified, 12 tagging single-nucleotide polymorphisms (tSNPs) were genotyped in a cohort of 1,751 obese individuals. After adjustments for the effect of age, sex, diabetes, and medication, the C allele of SNP rs1051338 was associated with lower blood pressure (BP; systolic (SBP) P = 0.004; diastolic (DBP) P = 0.006). Three of the tested SNPs were associated with modifications of the plasma lipid profile. The G/G genotype of rs2071509 was associated with higher high-density lipoprotein cholesterol (HDL-C) levels (P = 0.009) and minor allele of rs1131706 was also associated with higher HDL-C (P = 0.004) and an association between rs3802656 and total cholesterol (total-C)/HDL-C ratio was identified (P = 0.04). These results thus suggest that LIPA polymorphisms contribute to the interindividual variability observed in obesity-related metabolic complications.

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.

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.

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.

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.

Analytical performance of norovirus real-time RT-PCR detection protocols in Canadian laboratories.

BACKGROUND: Noroviruses (NoVs) are the leading cause of infectious gastroenteritis worldwide. Real-time reverse transcription PCR (real-time RT-PCR) is the preferred method of NoV detection for the majority of testing laboratories. Although the accepted target region for molecular detection assays is the conserved ORF1/ORF2 junction, multiple variations have been published with differences in primers, probes, reagents, multiplexing, etc. OBJECTIVES: We assessed the detection limit for GII.4 NoV real-time RT-PCR assays as well as the ability to detect the non-GII.4 NoV genotypes in each participating laboratory. STUDY DESIGN: A panel of 25 RNA samples was circulated to 18 testing laboratories for comparison of their real-time RT-PCR procedures for NoV detection. RESULTS: Multiple protocols with slight differences in reagents or conditions successfully detected 10 genome equivalents or fewer of NoV per reaction. Multiplex procedures were significantly associated (p=0.04) with false negative results, particularly for a GI.2 strain. Sensitive detection was associated with false positive results (p=0.03). CONCLUSIONS: Overall, the data indicate that comparable results are produced under slightly different assay conditions.

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.

Hepatitis E virus load in swine organs and tissues at slaughterhouse determined by real-time RT-PCR.

Although uncommon in North America, Hepatitis E virus (HEV) has been identified in some industrialized countries in patients without a history of travel to HEV-endemic countries. Its presence is ubiquitous worldwide in swine populations. Zoonotic transmission of swine HEV to non human primates has been achieved experimentally and transmission of HEV after ingestion of contaminated raw or undercooked meat is well documented. In Canada, so far, no HEV outbreak has been documented but HEV genotype 3 strains have been identified in sera and faecal samples of swine origin. The objective of the present study was to determine the viral load of HEV in liver, loin, bladder, hepatic lymph node, bile, tonsil, plasma and faeces samples of 43 pigs at slaughter. Feline calicivirus (FCV) was used as sample process control to validate the RNA extraction process, as a confirmation of the absence of sample inhibitors and as an amplification control. Using FCV/HEV multiplex TaqMan RT-qPCR system, HEV RNA was detected in 14 out of the 43 animals tested. HEV was detected in lymph nodes (11/43), bladder (10/43), liver (9/43), bile (8/43), faeces (6/43), tonsils (3/43), plasma (1/43) samples from infected animals. No HEV-positive loin samples were observed. Viral loads of 10(3) to 10(7) copies/g were estimated in positive liver and bile samples.

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.

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.