Duplex sequencing provides detailed characterization of mutation frequencies and spectra in the bone marrow of MutaMouse males exposed to procarbazine hydrochloride.

Mutagenicity testing is an essential component of health safety assessment. Duplex Sequencing (DS), an emerging high-accuracy DNA sequencing technology, may provide substantial advantages over conventional mutagenicity assays. DS could be used to eliminate reliance on standalone reporter assays and provide mechanistic information alongside mutation frequency (MF) data. However, the performance of DS must be thoroughly assessed before it can be routinely implemented for standard testing. We used DS to study spontaneous and procarbazine (PRC)-induced mutations in the bone marrow (BM) of MutaMouse males across a panel of 20 diverse genomic targets. Mice were exposed to 0, 6.25, 12.5, or 25 mg/kg-bw/day for 28 days by oral gavage and BM sampled 42 days post-exposure. Results were compared with those obtained using the conventional lacZ viral plaque assay on the same samples. DS detected significant increases in mutation frequencies and changes to mutation spectra at all PRC doses. Low intra-group variability within DS samples allowed for detection of increases at lower doses than the lacZ assay. While the lacZ assay initially yielded a higher fold-change in mutant frequency than DS, inclusion of clonal mutations in DS mutation frequencies reduced this discrepancy. Power analyses suggested that three animals per dose group and 500 million duplex base pairs per sample is sufficient to detect a 1.5-fold increase in mutations with > 80% power. Overall, we demonstrate several advantages of DS over classical mutagenicity assays and provide data to support efforts to identify optimal study designs for the application of DS as a regulatory test.

Characteristics of Interval Colorectal Cancer: A Canadian Retrospective Population-Level Analysis from Newfoundland and Labrador.

Interval colorectal cancers (I-CRCs) arise during the interval time period between scheduled colonoscopies. Predicting which patients are at risk of I-CRCs remains an elusive undertaking, but evidence would suggest that most I-CRCs arise from lesions missed on index endoscopy. The procedural factors that lead to missed lesions are numerous and lack consensus in the literature. In Canada, the province of Newfoundland and Labrador has the highest incidence of CRCs. In this study our aim was to examine I-CRCs (3-60 months after last colonoscopy) in NL through a population-level analysis covering 67% of the province from 2001-2018. We estimated the I-CRC rate to be up to 9.3%. Median age of I-CRC diagnosis was 67.1 years with an interval time of 2.9 years. About 57% of these tumors occurred proximal to the splenic flexure, with 53% presenting as local disease. No temporal differences were observed in interval time or tumor distribution. On univariate and multivariable logistical regression, risk of right-sided I-CRC did not correlate to the index colonoscopy indication, bowel preparation quality, size of largest polyp removed, colonoscopy completion rate, or stage at presentation. Improvements in synoptic reporting utilization and national registries are needed to identity risk factors and reduce I-CRC frequency.

Effect of probiotic bacteria on porcine rotavirus OSU infection of porcine intestinal epithelial IPEC-J2 cells.

Rotavirus infections in nursing or post-weaning piglets are known to cause diarrhea, which can lead to commercial losses. Probiotic supplementation is used as a prophylactic or therapeutic approach to dealing with microbial infections in humans and animals. To evaluate the effect of probiotic bacteria on porcine rotavirus infections, non-transformed porcine intestinal epithelial IPEC-J2 cells were used as an in vitro model, and three different procedures were tested. When cells were exposed to seven probiotics at concentrations of 105, 106, or 107 CFU/mL for 16 h and removed before rotavirus challenge, infection reduction rates determined by flow cytometry were as follows: 15% (106) and 18% (105) for Bifidobacterium longum R0175, 15% (107) and 16% (106) for B. animalis lactis A026, and 15% (105) for Lactobacillus plantarum 299V. When cells were exposed to three selected probiotic strains for 1 h at higher concentrations, that is, 108 and 5 × 108 CFU/mL, before infection with rotavirus, no significant reduction was observed. When the probiotic bacteria were incubated with the virus before cell infection, a significant 14% decrease in the infection rate was observed for B. longum R0175. The results obtained using a cell-probiotics-virus platform combined with flow cytometry analysis suggest that probiotic bacteria can have a protective effect on IPEC-J2 cells before infection and can also prevent rotavirus infection of the cells.

Clinical characteristics, multiorgan dysfunction and outcomes of patients with COVID-19: a prospective case series.

BACKGROUND: Characterizing the multiorgan manifestations and outcomes of patients hospitalized with COVID-19 will inform resource requirements to address the long-term burden of this disease. We conducted a descriptive analysis using prospectively collected data to describe the clinical characteristics and spectrum of organ dysfunction, and in-hospital and longer-term clinical outcomes of patients hospitalized with COVID-19 during the first wave of the pandemic at a Canadian centre. METHODS: We conducted a prospective case series involving adult patients (aged ≥ 18 yr) with COVID-19 admitted to 1 of 2 hospitals in London, Ontario, from Mar. 17 to June 18, 2020, during the first wave of the pandemic. We recorded patients' baseline characteristics, physiologic parameters, measures of organ function and therapies administered during hospitalization among patients in the intensive care unit (ICU) and in non-ICU settings, and compared the characteristics of hospital survivors and nonsurvivors. Finally, we recorded follow-up thoracic computed tomography (CT) and echocardiographic findings after hospital discharge. RESULTS: We enrolled 100 consecutive patients (47 women) hospitalized with COVID-19, including 32 patients who received ICU care and 68 who received treatment in non-ICU settings. Respiratory sequelae were common: 23.0% received high-flow oxygen by nasal cannula, 9.0% received noninvasive ventilation, 24.0% received invasive mechanical ventilation and 2.0% received venovenous extracorporeal membrane oxygenation. Overall, 9.0% of patients had cerebrovascular events (3.0% ischemic stroke, 6.0% intracranial hemorrhage), and 6.0% had pulmonary embolism. After discharge, 11 of 19 patients had persistent abnormalities on CT thorax, and 6 of 15 had persistent cardiac dysfunction on echocardiography. INTERPRETATION: This study provides further evidence that COVID-19 is a multisystem disease involving neurologic, cardiac and thrombotic dysfunction, without evidence of hepatic dysfunction. Patients have persistent organ dysfunction after hospital discharge, underscoring the need for research on long-term outcomes of COVID-19 survivors.

Duplex sequencing identifies genomic features that determine susceptibility to benzo(a)pyrene-induced in vivo mutations.

Exposure to environmental mutagens increases the risk of cancer and genetic disorders. We used Duplex Sequencing (DS), a high-accuracy error-corrected sequencing technology, to analyze mutation induction across twenty 2.4 kb intergenic and genic targets in the bone marrow of MutaMouse males exposed to benzo(a)pyrene (BaP), a widespread environmental pollutant. DS revealed a linear dose-related induction of mutations across all targets with low intra-group variability. Heterochromatic and intergenic regions exhibited the highest mutation frequencies (MF). C:G > A:T transversions at CCA, CCC and GCC trinucleotides were enriched in BaP-exposed mice consistent with the known etiology of BaP mutagenesis. However, GC-content had no effect on mutation susceptibility. A positive correlation was observed between DS and the "gold-standard" transgenic rodent gene mutation assay. Overall, we demonstrate that DS is a promising approach to study in vivo mutagenesis and yields critical insight into the genomic features governing mutation susceptibility, spectrum, and variability across the genome.

Personal, professional, and psychological impact of the COVID-19 pandemic on hospital workers: A cross-sectional survey.

OBJECTIVES: We aimed to evaluate the personal, professional, and psychological impact of the COVID-19 pandemic on hospital workers and their perceptions about mitigating strategies. DESIGN: Cross-sectional web-based survey consisting of (1) a survey of the personal and professional impact of the COVID-19 pandemic and potential mitigation strategies, and (2) two validated psychological instruments (Kessler Psychological Distress Scale [K10] and Impact of Events Scale Revised [IES-R]). Regression analyses were conducted to identify the predictors of workplace stress, psychological distress, and post-traumatic stress. SETTING AND PARTICIPANTS: Hospital workers employed at 4 teaching and 8 non-teaching hospitals in Ontario, Canada during the COVID-19 pandemic. RESULTS: Among 1875 respondents (84% female, 49% frontline workers), 72% feared falling ill, 64% felt their job placed them at great risk of COVID-19 exposure, and 48% felt little control over the risk of infection. Respondents perceived that others avoided them (61%), reported increased workplace stress (80%), workload (66%) and responsibilities (59%), and 44% considered leaving their job. The psychological questionnaires revealed that 25% had at least some psychological distress on the K10, 50% had IES-R scores suggesting clinical concern for post-traumatic stress, and 38% fulfilled criteria for at least one psychological diagnosis. Female gender and feeling at increased risk due to PPE predicted all adverse psychological outcomes. Respondents favoured clear hospital communication (59%), knowing their voice is heard (55%), expressions of appreciation from leadership (55%), having COVID-19 protocols (52%), and food and beverages provided by the hospital (50%). CONCLUSIONS: Hospital work during the COVID-19 pandemic has had important personal, professional, and psychological impacts. Respondents identified opportunities to better address information, training, and support needs.

The impact of the COVID-19 pandemic on intensive care unit workers: a nationwide survey.

PURPOSE: To evaluate the impact of the COVID-19 pandemic on Canadian intensive care unit (ICU) workers. METHODS: Between June and August 2020, we distributed a cross-sectional online survey of ICU workers evaluating the impact of the pandemic, coping strategies, symptoms of post-traumatic stress disorder (PTSD; Impact of Events Scale-Revised), and psychological distress, anxiety, and depression (Kessler Psychological Distress Scale). We performed regression analyses to determine the predictors of psychological symptoms. RESULTS: We analyzed responses from 455 ICU workers (80% women; 67% from Ontario; 279 nurses, 69 physicians, and 107 other healthcare professionals). Respondents felt that their job put them at great risk of exposure (60%), were concerned about transmitting COVID-19 to family members (76%), felt more stressed at work (67%), and considered leaving their job (37%). Overall, 25% had probable PTSD and 18% had minimal or greater psychological distress. Nurses were more likely to report PTSD symptoms (33%) and psychological distress (23%) than physicians (5% for both) and other health disciplines professionals (19% and 14%). Variables associated with PTSD and psychological distress included female sex (beta-coefficient [B], 1.59; 95% confidence interval [CI], 1.20 to 2.10 and B, 3.79; 95% CI, 1.79 to 5.78, respectively; P < 0.001 for differences in scores across groups) and perceived increased risk due to PPE shortage or inadequate PPE training (B, 1.87; 95% CI, 1.51 to 2.31 and B, 4.88; 95% CI, 3.34 to 6.43, respectively). Coping strategies included talking to friends/family/colleagues (80%), learning about COVID-19 (78%), and physical exercise (68%). Over half endorsed the following workplace strategies as valuable: hospital-provided scrubs, clear communication and protocols by hospitals, knowing their voice is heard, subsidized parking, and gestures of appreciation from leadership. CONCLUSIONS: This survey study shows that ICU workers have been impacted by the COVID-19 pandemic with high levels of stress and psychological burden. Respondents endorsed communication, protocols, and appreciation from leadership as helpful mitigating strategies.

RéSUMé: OBJECTIF: Évaluer l'impact de la pandémie de COVID-19 sur les travailleurs canadiens des unités de soins intensifs (USI). MéTHODE: Entre juin et août 2020, nous avons fait parvenir un sondage transversal en ligne aux travailleurs des soins intensifs pour évaluer l'impact de la pandémie, les stratégies d'adaptation et les symptômes de stress post-traumatique (SPT; Échelle révisée de l'impact de l'événement - IES-R), ainsi que la détresse psychologique, l'anxiété et la dépression (Échelle de détresse psychologique de Kessler). Nous avons réalisé des analyses de régression pour déterminer les prédicteurs de symptômes psychologiques. RéSULTATS: Nous avons analysé les réponses de 455 travailleurs des soins intensifs (80 % de femmes; 67 % de l'Ontario; 279 infirmières/infirmiers, 69 médecins et 107 autres professionnels de la santé). Les répondants ont estimé que leur emploi les plaçait face à un risque élevé d'exposition (60 %), craignaient de transmettre la COVID-19 aux membres de leur famille (76 %), se sentaient plus stressés au travail (67 %) et avaient envisagé de quitter leur emploi (37 %). Dans l'ensemble, 25 % souffraient probablement d'un SPT et 18 % présentaient une détresse psychologique minimale ou supérieure. Les infirmières et infirmiers étaient plus susceptibles de rapporter des symptômes de SPT (33 %) et de détresse psychologique (23 %) que les médecins (5 % pour les deux) et les professionnels de la santé des autres disciplines (19 % et 14 %). Les variables associées à un SPT et à la détresse psychologique comprenaient le sexe féminin (coefficient bêta [B], 1,59; intervalle de confiance [IC] à 95 %, 1,20 à 2,10 et B, 3,79; IC 95 %, 1,79 à 5,78, respectivement; P < 0,001 pour les différences de scores entre les groupes) et la perception d'un risque accru en raison des pénuries d'EPI ou d'une formation inadéquate en EPI (B, 1,87; IC 95 %, 1,51 à 2,31 et B, 4,88; IC 95 %, 3,34 à 6,43, respectivement). Les stratégies d'adaptation comprenaient le fait de parler aux amis, à la famille ou aux collègues (80 %), l'acquisition de connaissances concernant la COVID-19 (78 %) et l'exercice physique (68 %). Plus de la moitié ont estimé que les stratégies de travail suivantes étaient utiles : des uniformes fournis par les hôpitaux, une communication et des protocoles clairs de la part des hôpitaux, le fait de savoir que leur voix est entendue, un stationnement subventionné et des gestes d'appréciation de la part des dirigeants. CONCLUSION: Cette étude montre que les travailleurs des soins intensifs ont été touchés par la pandémie de COVID-19 avec des niveaux élevés de stress et de fardeau psychologique. Les répondants ont déclaré que la communication, les protocoles et les gestes d'appréciation de la direction constituaient des stratégies d'atténuation utiles.

Folate Intake Alters Mutation Frequency and Profiles in a Tissue- and Dose-Specific Manner in MutaMouse Male Mice.

BACKGROUND: While cancer is common, its incidence varies widely by tissue. These differences are attributable to variable risk factors, such as environmental exposure, genetic inheritance, and lifetime number of stem cell divisions in a tissue. Folate deficiency is generally associated with increased risk for colorectal cancer (CRC) and acute lymphocytic leukemia (ALL). Conversely, high folic acid (FA) intake has also been associated with higher CRC risk. OBJECTIVE: Our objective was to compare the effect of folate intake on mutant frequency (MF) and types of mutations in the colon and bone marrow of mice. METHODS: Five-week-old MutaMouse male mice were fed a deficient (0 mg FA/kg), control (2 mg FA/kg), or supplemented (8 mg FA/kg) diet for 20 wk. Tissue MF was assessed using the lacZ mutant assay and comparisons made by 2-factor ANOVA. LacZ mutant plaques were sequenced using next-generation sequencing, and diet-specific mutation profiles within each tissue were compared by Fisher's exact test. RESULTS: In the colon, the MF was 1.5-fold and 1.3-fold higher in mice fed the supplemented diet compared with mice fed the control (P = 0.001) and deficient (P = 0.008) diets, respectively. This contrasted with the bone marrow MF in the same mice where the MF was 1.7-fold and 1.6-fold higher in mice fed the deficient diet compared with mice fed the control (P = 0.02) and supplemented (P = 0.03) diets, respectively. Mutation profiles and signatures (mutation context) were tissue-specific. CONCLUSIONS: Our data indicate that dietary folate intake affects mutagenesis in a tissue- and dose-specific manner in mice. Mutation profiles were generally tissue- but not dose-specific, suggesting that altered cellular folate status appears to interact with endogenous mutagenic mechanisms in each tissue to create a permissive context in which specific mutation types accumulate. These data illuminate potential mechanisms underpinning differences in observed associations between folate intake/status and cancer.

The 28 + 28 day design is an effective sampling time for analyzing mutant frequencies in rapidly proliferating tissues of MutaMouse animals.

The Organisation for Economic Co-Operation and Development Test Guideline 488 (TG 488) uses transgenic rodent models to generate in vivo mutagenesis data for regulatory submission. The recommended design in TG 488, 28 consecutive daily exposures with tissue sampling three days later (28 + 3d), is optimized for rapidly proliferating tissues such as bone marrow (BM). A sampling time of 28 days (28 + 28d) is considered more appropriate for slowly proliferating tissues (e.g., liver) and male germ cells. We evaluated the impact of the sampling time on mutant frequencies (MF) in the BM of MutaMouse males exposed for 28 days to benzo[a]pyrene (BaP), procarbazine (PRC), isopropyl methanesulfonate (iPMS), or triethylenemelamine (TEM) in dose-response studies. BM samples were collected + 3d, + 28d, + 42d or + 70d post exposure and MF quantified using the lacZ assay. All chemicals significantly increased MF with maximum fold increases at 28 + 3d of 162.9, 6.6, 4.7 and 2.8 for BaP, PRC, iPMS and TEM, respectively. MF were relatively stable over the time period investigated, although they were significantly increased only at 28 + 3d and 28 + 28d for TEM. Benchmark dose (BMD) modelling generated overlapping BMD confidence intervals among the four sampling times for each chemical. These results demonstrate that the sampling time does not affect the detection of mutations for strong mutagens. However, for mutagens that produce small increases in MF, sampling times greater than 28 days may produce false-negative results. Thus, the 28 + 28d protocol represents a unifying protocol for simultaneously assessing mutations in rapidly and slowly proliferating somatic tissues and male germ cells.

Chemically induced mutations in a MutaMouse reporter gene inform mechanisms underlying human cancer mutational signatures.

Transgenic rodent (TGR) models use bacterial reporter genes to quantify in vivo mutagenesis. Pairing TGR assays with next-generation sequencing (NGS) enables comprehensive mutation pattern analysis to inform mutational mechanisms. We used this approach to identify 2751 independent lacZ mutations in the bone marrow of MutaMouse animals exposed to four chemical mutagens: benzo[a]pyrene, N-ethyl-N-nitrosourea, procarbazine, and triethylenemelamine. We also collected published data for 706 lacZ mutations from eight additional environmental mutagens. We report that lacZ gene sequencing generates chemical-specific mutation signatures observed in human cancers with established environmental causes. For example, the mutation signature of benzo[a]pyrene, a carcinogen present in tobacco smoke, matched the signature associated with tobacco-induced lung cancers. Our results suggest that the analysis of chemically induced mutations in the lacZ gene shortly after exposure provides an effective approach to characterize human-relevant mechanisms of carcinogenesis and propose novel environmental causes of mutation signatures observed in human cancers.

Structure, amphipathy, and topology of the membrane-proximal helix 8 influence apelin receptor plasma membrane localization.

G-protein coupled receptors (GPCRs) typically have an amphipathic helix ("helix 8") immediately C-terminal to the transmembrane helical bundle. To date, a number of functional roles have been associated with GPCR helix 8 segments, but structure-function analysis for this region remains limited. Here, we examine helix 8 of the apelin receptor (AR or APJ), a class A GPCR with wide physiological and pathophysiological relevance. The 71 residue C-terminal tail of the AR is primarily intrinsically disordered, with a detergent micelle-induced increase in helical character. This helicity was localized to the helix 8 region, in good agreement with the recent AR crystal structure. A series of helix 8 mutants were made to reduce helicity, remove amphipathy, or flip the hydrophobic and hydrophilic faces. Each mutant AR was tested both biophysically, in the isolated C-terminal tail, and functionally in HEK 293 T cells, for full-length AR. In all instances, micelle interactions were maintained, and steady-state AR expression was efficient. However, removal of amphipathy or helical character led to a significant decrease in cell surface localization. Flipping of helix 8 amphipathic topology restored cell surface localization to some degree, but still was significantly reduced relative to wild-type. Structural integrity, amphipathy to drive membrane association, and correct topology of helix 8 membrane association all thus appear important for cell surface localization of the AR. This behavior correlates well to GPCR C-terminal tail sequence motifs, implying that these serve to specify key topological features of helix 8 and its proximity to the transmembrane domain.

Persistence of murine norovirus, bovine rotavirus, and hepatitis A virus on stainless steel surfaces, in spring water, and on blueberries.

The viability of murine norovirus (MNV-1), bovine rotavirus (boRV), and hepatitis A virus (HAV) was evaluated at 21 °C, 4 °C, and -20 °C on stainless steel surfaces, in bottled water, and on blueberries for up to 21 days. After 14 days of incubation at 21 °C on stainless steel, a viability loss >4 log for MNV-1, >8 log for boRV, and >1 log for HAV was observed. Losses were observed for MNV-1 (>1 log) and HAV (>2 log) incubated in water at 21 °C for 21 days. No significant loss was detected for MNV-1 and HAV at 4 °C and -20 °C and for boRV at 21 °C, 4 °C, and -20 °C. On blueberries incubated at 4 °C and -20 °C, they all maintained their infectivity. After 7 days at 21 °C, a loss >2 log, a loss of 3 log, and no loss were observed for boRV, MNV-1, and HAV, respectively. After RNase pretreatment, the detection of extracted RNA from infectious and noninfectious samples suggested the protection of RNA inside the capsid. Even though they all are enteric viruses, their persistence varied with temperature and the nature of the commodity. It is therefore important to use more than one viral surrogate, during inactivation treatments or implementation of control measures.

Folate deficiency increases chromosomal damage and mutations in hematopoietic cells in the transgenic mutamouse model.

Folate deficiency causes megaloblastic anemia and neural tube defects, and is also associated with some cancers. In vitro, folate deficiency increases mutation frequency and genome instability, as well as exacerbates the mutagenic potential of known environmental mutagens. Conversely, it remains unclear whether or not elevated folic acid (FA) intakes are beneficial or detrimental to the induction of DNA mutations and by proxy human health. We used the MutaMouse transgenic model to examine the in vivo effects of FA deficient, control, and supplemented diets on somatic DNA mutant frequency (MF) and genome instability in hematopoietic cells. We also examined the interaction between FA intake and exposure to the known mutagen N-ethyl-N-nitrosourea (ENU) on MF. Male mice were fed the experimental diets for 20 weeks from weaning. Half of the mice from each diet group were gavaged with 50 mg/kg body weight ENU after 10 weeks on diet and remained on their respective diet for an additional 10 weeks. Mice fed a FA-deficient diet had a 1.3-fold increase in normochromatic erythrocyte micronucleus (MN) frequency (P = 0.034), and a doubling of bone marrow lacZ MF (P = 0.035), compared to control-fed mice. Mice exposed to ENU showed significantly higher bone marrow lacZ and Pig-a MF, but there was no effect of FA intake on ENU-induced MF. These data indicate that FA deficiency increases mutations and MN formation in highly proliferative somatic cells, but that FA intake does not mitigate ENU-induced mutations. Also, FA intake above adequacy had no beneficial or detrimental effect on mutations or MN formation. Environ. Mol. Mutagen. 59:366-374, 2018. © 2018 Her Majesty the Queen in Right of Canada 2018.

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.

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.

Pulmonary and cutaneous O2gas exchange: a student laboratory exercise in the frog.

Gas exchange in animals is ultimately diffusion based, generally occurring across dedicated respiratory organs. In many aquatic amphibians, however, multiple modes of gas exchange exist, allowing for the partitioning of O2 uptake and CO2 excretion between respiratory organs with different efficiencies. For example, due to the physical properties of O2 being vastly different between air and water phases, the lung and skin play disproportionately important roles in O2 uptake. Many aquatic frogs are renowned for their cutaneous gas exchange capacity, where often the majority of CO2 is excreted across the skin. Furthermore, the roles of these gas exchange organs change with the animal's behavior. Under diving conditions, most of the frog's gas exchange needs must be met by the skin. In this article, we describe an interactive undergraduate laboratory that allows a class of students to share equipment while assessing pulmonary and cutaneous respiration in frogs provided with an air/water choice and under enforced dive conditions. Concepts explored in this laboratory exercise include animal energetics, diving reflex, pulmonary and cutaneous gas exchange processes, diffusion-based gas flux, and O2 debt.