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.

Comparison of intestinal bacterial populations between two dairy cattle herds colonized or not by Campylobacter jejuni.

Campylobacter jejuni is an important foodborne pathogen. It can be isolated from bovine feces and its presence is influenced by farm characteristics and management practices. The impact of the bovine gut microbiota on the presence of C. jejuni is poorly documented. Two herds of lactating cows were selected: one where C. jejuni was not detected in 20 animals and the other where 55% of the sampled animals (11/20) were contaminated by C. jejuni. The bacterial diversity of bovine feces from these two herds was analyzed by terminal restriction fragment length polymorphism. The full-length 16S rRNA gene was amplified using fluorescently labeled primers and subsequently digested with HaeIII. Terminal restriction fragments profiles comparison showed a similarity level >79% between microbial populations from both herds. As profiles containing or not C. jejuni were clustered together, it is proposed that the presence of C. jejuni is not linked to a particular profile from the recovered intestinal bovine microbiota.

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.

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.

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.

The feline calicivirus as a sample process control for the detection of food and waterborne RNA viruses.

Many food and waterborne outbreaks of infectious disease are caused by viruses. While numerous methods exist and are being developed to test food and water for the presence of enteric viruses, there is no standard control for the comparison of different methods. Potential control viruses should be well characterized, share the physical characteristics of the enterically infecting viruses and not normally be associated with foods. Here, the feline calicivirus (FCV) is proposed as a sample process control for methods aimed at the extraction and detection of RNA viruses in food and water. FCV is shown to be useful as a control for the extraction of hepatitis A virus (HAV) from water using filtration technology and from strawberries using the Pathatrix system. The FCV standard provides a valuable quality control tool when testing potentially contaminated food samples.

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.

An outbreak of norovirus caused by consumption of oysters from geographically dispersed harvest sites, British Columbia, Canada, 2004.

OBJECTIVES: In January 2004, an increase in gastrointestinal illness following oyster consumption was reported in British Columbia. An investigation was initiated to explore the association between norovirus infection and consumption of British Columbia oysters and to identify the source of oyster contamination. METHODS: The outbreak investigation included active surveillance for human cases, two cohort studies, trace-back of oysters, and laboratory testing of oysters and human stools. RESULTS: Enhanced surveillance identified 26 confirmed and 53 clinical cases over 3 months. Oyster consumption was associated with illness in one cohort and suggestive in the other. Oysters were traced to 14 geographically dispersed harvest sites, 18 suppliers, and 45 points of purchase. Norovirus BCCDC03-028 (genotype I.2) was detected in 50% of human specimens. Experimental methods detected norovirus in 12 oyster samples. Sequencing identified mixed clonal patterns in the oysters with one direct sequence match between an oyster sample and the associated human specimen. CONCLUSIONS: The consumption of raw oysters led to norovirus infection. The source of oyster contamination remained unidentified. The geographical dispersion of implicated harvest sites was unusual. APPLICATIONS: This outbreak is unlike most shellfish outbreaks that can be traced back to a common source and challenges conventional thinking that all oyster-related norovirus outbreaks of are a result of point source contamination.

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.

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.

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.

Comparison of fructose-1,6-bisphosphatase gene (fbp) sequences for the identification of Lactobacillus rhamnosus.

Comparative analysis of fructose-1,6-bisphosphatase gene (fbp) sequences was evaluated for the differentiation of reference and clinical strains of Lactobacillus rhamnosus. The sequences of 1,971 nucleotides of the fbp gene were determined on both DNA strands for 21 L. rhamnosus strains, representing reference, probiotic, and clinical strains. No PCR amplification of the fbp gene was observed for other species of the Lactobacillus casei complex (L. casei and L. zeae) or strains of Lactobacillus acidophilus, Streptococcus thermophilus, and Escherichia coli. Phylogenetic analysis of the fbp putative amino acid sequences of L. rhamnosus strains by the neighbor-joining method showed clear distinct positions of this species. The phylogenetic tree, derived from fbp nucleotide sequences, showed four clear divisions between strains of L. rhamnosus. From a taxonomic point of view, our results confirm for the first time that fbp gene sequences have high discriminating power for strains of L. rhamnosus that are difficult to differentiate.

Rapid Detection of Bifidobacterium dentium by Enzymatic Hydrolysis of ß-Glucuronide Substrates.

Enzyme profiles and fermentation patterns of bifidobacteria were studied to determine phenotypic characteristics that allow the rapid detection of Bifidobacterium dentium and its differentiation from Bifidobacterium adolescentis , Bifidobacterium angulatum , Bifidobacterium catenulatum , and Bifidobacterium pseudocatenulatum . Among 43 bifidobacterial strains tested, the production of ß-glucuronidase was limited to six strains of B. dentium . The presence of B. dentium on a selective medium may be rapidly confirmed by the detection of ß-glucuronidase activity. Columbia agar containing propionic acid was chosen to enumerate bifidobacteria previously cultivated in MRS medium. After 48 h of incubation, ß-glucuronidase activity was determined by using a plate staining procedure. B. dentium strains gave positive results for ß-glucuronidase activity after application of the overlay solution of ß-glucuronide substrate. The ß-glucuronidase assay is a rapid screening method for B. dentium . This method might be useful for selection of nonpathogenic strains or detection of fecal contamination from human origin.