Expert Elicitation To Estimate the Feed Safety Impact of Criteria Included in the Canadian Food Inspection Agency Risk Assessment Model for Feed Mills.

ABSTRACT: The Canadian Food Inspection Agency is developing an Establishment-based Risk Assessment (ERA) model for commercial and on-farm mills involved in the manufacture, storage, packaging, labeling, or distribution of livestock feed (ERA-Feed Mill model). This model will help inform the allocation of inspection resources on the basis of feed safety risk, including animal health and food safety risk. In a previous study, 34 risk factors, grouped into inherent, mitigation, and compliance clusters, along with assessment criteria were selected. The objective of this current study was to estimate the relative risk (RR) of the 203 assessment criteria on the basis of the impact on feed safety to design an ERA-Feed Mill model algorithm. Furthermore, the intent of this study was to assess the maximum increase or decrease of risk obtained when multiple criteria belonging to a same cluster were identified in a specific feed mill. To do so, a two-round face-to-face expert elicitation was conducted with 28 Canadian feed experts. Results showed no significant association between respondent profiles (years of experience and work sector) and estimated RR. Uniformity of answers between experts improved between rounds. Criteria having the highest increase in risk (median RR ≥ 4) included the presence of materials prohibited to be fed to ruminants in a facility that produces ruminant feed, the presence of multiple livestock species on-site, and historical noncompliances related to the inspection of the feed mill's process control and end-product control programs. Risk mitigation criteria having the highest impact on decreasing the risk were the implementation of feed safety certifications, the use of dedicated manufacturing lines (prohibited materials or medications), and having a hazard sampling plan in place for finished feed. The median RR assigned to each criterion and cluster will be used to build an algorithm of the Canadian Food Inspection Agency's ERA-Feed Mill model.

Expert Elicitation to Estimate the Relative Risk of Food Safety Criteria Included in the Establishment-Based Risk Assessment Model for Canadian Hatcheries.

The Canadian Food Inspection Agency is developing an Establishment-based Risk Assessment model for Hatcheries to allocate inspection resources according to the food safety risk associated with each hatchery falling under its jurisdiction. In a previous study, 29 factors contributing to the food safety risk of hatcheries were identified and grouped into three clusters (inherent risk, risk mitigation, and compliance) and assessment criteria were defined. The objective of the current study was to estimate the relative risk (RR) of these criteria. Two rounds of expert elicitations were conducted to allow 13 Canadian experts to estimate the RR of each criterion (n = 96) based on its potential impact on human health, with a specific focus on Salmonella spp. This process also aimed to estimate the maximum increase or decrease in the overall food safety risk of a hatchery when considering multiple criteria belonging to a specific cluster and to assess the risk attribution of Salmonella spp. at the hatchery and bird-type levels. Results showed that the respondent profile had no influence on the importance given to a majority of criteria. Uniformity of answers among experts improved from the first to the second round. Overall, 62.5%, 32.3%, and 5.2% of the criteria were attributed to an RR that was less than 2, between 2 and 3, and greater than 3, respectively. Mixing eggs from different supply flocks when placed into the same hatching unit, hatching multiple species, and importing eggs with unknown quality status were identified as having the highest contribution to a hatchery's inherent risk. Requiring information on the foodborne pathogen status of supplying flocks and the occurrence of regulatory enforcement actions were the most impactful risk mitigation and compliance factors, respectively. The median RR value assigned to each criterion and cluster will be used to build this new model.

Source attribution at the food sub-product level for the development of the Canadian Food Inspection Agency risk assessment model.

Decreasing the health burden caused by foodborne pathogens is challenging and it depends on the identification of the most significant hazards and food sources causing illnesses, so adequate mitigation strategies can be implemented. In this regard, the Canadian Food Inspection Agency (CFIA) has developed the Establishment-based Risk Assessment (ERA) model, so that a more effective and efficient allocation of resources can be assigned to the highest food safety risk areas. To assess risk, the model considers the type of food sub-products being manufactured by establishments and its scope is limited to the 17 most important foodborne pathogens representing the highest level of food safety risk. However, the information on source attribution at the sub-product level based on a structured approach is limited. To overcome this challenge, an expert elicitation was conducted in 2016 to estimate the relative contribution and associated certainty of each sub-product for 31 pathogen-commodity combinations to the total Canadian health burden associated with foodborne illnesses (expressed in DALYs). These DALYs represent 78% of the total Canadian health burden associated with federally-regulated food commodities considered within the model. A total of 49 Canadian experts recruited using a "snow ball" sampling strategy participated in the study by completing an electronic survey. Results of the elicitation displayed variable levels of health burden allocation between the pathogens and the different commodity sub-products. Assessment of the certainty levels showed some combinations being evaluated with more confidence (e.g., Campylobacter and eggs/poultry sub-products) than others, where a bimodal distribution of certainty was observed (e.g., Toxoplasma in pork sub-products). Furthermore, no participant raised concerns on the food classification scheme, suggesting their agreement with the proposed sub-products categorization of the elicitation. Relative contribution estimates will be included in the CFIA ERA model and used to enhance its applicability for risk prioritization and effective resource allocation during food establishment inspections. While substantial uncertainty around the central tendency estimates was found, these estimates provide a good basis for regulatory oversight and public health policy.

Magnetic resonance imaging in foals with infectious arthritis.

The magnetic resonance (MR) imaging findings of foals with infectious and noninfectious arthritis are described. Six foals with infectious arthritis and three foals with noninfectious arthritis were grouped based on synovial fluid analysis results and examined with radiography and MR imaging. Four out of six foals with infectious arthritis had osseous lesions in MR images indicative of osteomyelitis and only 4/19 lesions were detected on digital radiographs. The three foals with noninfectious arthritis had no osseous lesions in MR images or radiographically. Of the six joints that had osseous lesions detected with MR imaging, three had at least one lytic lesion detected radiographically. Osseous lesions in the epiphysis, metaphysis, and physis appeared in MR images as T2W, short tau inversion recovery, and proton density hyperintense foci with a hypointense halo. The same lesions appeared hyperintense in the 3D RSSG water excitation pulse sequence but lacked a surrounding hypointense halo. Most joints of foals with infectious arthritis had heterogenous signals within the synovial fluid whereas all of the nonseptic joints had homogenous synovial fluid signals. MR imaging appears to be better than radiography in the detection of osseous lesions in foals diagnosed with infectious arthritis and may be a valuable screening test for the presence of osteomyelitis.

The Schizosaccharomyces pombe Hsp104 disaggregase is unable to propagate the [PSI] prion.

The molecular chaperone Hsp104 is a crucial factor in the acquisition of thermotolerance in yeast. Under stress conditions, the disaggregase activity of Hsp104 facilitates the reactivation of misfolded proteins. Hsp104 is also involved in the propagation of fungal prions. For instance, the well-characterized [PSI(+)] prion of Saccharomyces cerevisiae does not propagate in Deltahsp104 cells or in cells overexpressing Hsp104. In this study, we characterized the functional homolog of Hsp104 from Schizosaccharomyces pombe (Sp_Hsp104). As its S. cerevisiae counterpart, Sp_hsp104(+) is heat-inducible and required for thermotolerance in S. pombe. Sp_Hsp104 displays low disaggregase activity and cannot propagate the [PSI(+)] prion in S. cerevisiae. When overexpressed in S. cerevisiae, Sp_Hsp104 confers thermotolerance to Deltahsp104 cells and reactivates heat-aggregated proteins. However, overexpression of Sp_Hsp104 does not propagate nor eliminate [PSI(+)]. Strikingly, [PSI(+)] was cured by overexpression of a chimeric chaperone bearing the C-terminal domain (CTD) of the S. cerevisiae Hsp104 protein. Our study demonstrates that the ability to untangle aggregated proteins is conserved between the S. pombe and S. cerevisiae Hsp104 homologs, and points to a role of the CTD in the propagation of the S. cerevisiae [PSI(+)] prion.

The epigenetic calnexin-independent state is induced in response to environmental changes.

Yeasts have evolved numerous responsive pathways to survive in fluctuating and stressful environments. The endoplasmic reticulum (ER) is sensitive to adverse conditions, which are detected by response pathways to ensure correct protein folding. Calnexin is an ER transmembrane chaperone acting in both quality control of folding and response to persistent stress. Calnexin is a key protein required for viability in certain organisms such as mammals and the fission yeast Schizosaccharomyces pombe. Nevertheless, S. pombe calnexin-independent (Cin) cells were obtained after transient expression of a particular calnexin mutant. The Cin state is dominant, is stably propagated by an epigenetic mechanism and segregates in a non-Mendelian fashion to the meiotic progeny. The nucleolar protein Cif1p was identified as an inducer of the Cin state in a previous genetic screen. Here, we report the identification of novel inducers isolated in an overexpression genetic screen: pyruvate kinase (Pyk1p) and phosphoglycerate kinase (Pgk1p). Addition of pyruvate, the end product of pyruvate kinase and glycolysis, also induced calnexin independence in a dose-dependent manner. Remarkably, growth in respiration media or cold temperatures induced the appearance of Cin cells at high frequencies. Taken together, our results indicate that the Cin state can be triggered by extracellular changes, suggesting that this state represents an epigenetic adaptative response to environmental modifications.

Calnexin regulates apoptosis induced by inositol starvation in fission yeast.

Inositol is a precursor of numerous phospholipids and signalling molecules essential for the cell. Schizosaccharomyces pombe is naturally auxotroph for inositol as its genome does not have a homologue of the INO1 gene encoding inositol-1-phosphate synthase, the enzyme responsible for inositol biosynthesis. In this work, we demonstrate that inositol starvation in S. pombe causes cell death with apoptotic features. This apoptotic death is dependent on the metacaspase Pca1p and is affected by the UPR transducer Ire1p. Previously, we demonstrated that calnexin is involved in apoptosis induced by ER stress. Here, we show that cells expressing a lumenal version of calnexin exhibit a 2-fold increase in the levels of apoptosis provoked by inositol starvation. This increase is reversed by co-expression of a calnexin mutant spanning the transmembrane domain and C-terminal cytosolic tail. Coherently, calnexin is physiologically cleaved at the end of its lumenal domain, under normal growth conditions when cells approach stationary phase. This cleavage suggests that the two naturally produced calnexin fragments are needed to continue growth into stationary phase and to prevent cell death. Collectively, our observations indicate that calnexin takes part in at least two apoptotic pathways in S. pombe, and suggest that the cleavage of calnexin has regulatory roles in apoptotic processes involving calnexin.

Pro-aging effects of glucose signaling through a G protein-coupled glucose receptor in fission yeast.

Glucose is the preferred carbon and energy source in prokaryotes, unicellular eukaryotes, and metazoans. However, excess of glucose has been associated with several diseases, including diabetes and the less understood process of aging. On the contrary, limiting glucose (i.e., calorie restriction) slows aging and age-related diseases in most species. Understanding the mechanism by which glucose limits life span is therefore important for any attempt to control aging and age-related diseases. Here, we use the yeast Schizosaccharomyces pombe as a model to study the regulation of chronological life span by glucose. Growth of S. pombe at a reduced concentration of glucose increased life span and oxidative stress resistance as reported before for many other organisms. Surprisingly, loss of the Git3 glucose receptor, a G protein-coupled receptor, also increased life span in conditions where glucose consumption was not affected. These results suggest a role for glucose-signaling pathways in life span regulation. In agreement, constitutive activation of the Galpha subunit acting downstream of Git3 accelerated aging in S. pombe and inhibited the effects of calorie restriction. A similar pro-aging effect of glucose was documented in mutants of hexokinase, which cannot metabolize glucose and, therefore, are exposed to constitutive glucose signaling. The pro-aging effect of glucose signaling on life span correlated with an increase in reactive oxygen species and a decrease in oxidative stress resistance and respiration rate. Likewise, the anti-aging effect of both calorie restriction and the Deltagit3 mutation was accompanied by increased respiration and lower reactive oxygen species production. Altogether, our data suggest an important role for glucose signaling through the Git3/PKA pathway to regulate S. pombe life span.

Inter-species complementation of the translocon beta subunit requires only its transmembrane domain.

In eukaryotes, proteins enter the secretory pathway through the translocon pore of the endoplasmic reticulum. This protein translocation channel is composed of three major subunits, called Sec61alpha, beta and gamma in mammals. Unlike the other subunits, the beta subunit is dispensable for translocation and cell viability in all organisms studied. Intriguingly, the knockout of the Sec61beta encoding genes results in different phenotypes in different species. Nevertheless, the beta subunit shows a high level of sequence homology across species, suggesting the conservation of a biological function that remains ill-defined. To address its cellular roles, we characterized the homolog of Sec61beta in the fission yeast Schizosaccharomyces pombe (Sbh1p). Here, we show that the knockout of sbh1(+) results in severe cold sensitivity, increased sensitivity to cell-wall stress, and reduced protein secretion at 23 degrees C. Sec61beta homologs from Saccharomyces cerevisiae and human complement the knockout of sbh1(+) in S. pombe. As in S. cerevisiae, the transmembrane domain (TMD) of S. pombe Sec61beta is sufficient to complement the phenotypes resulting from the knockout of the entire encoding gene. Remarkably, the TMD of Sec61beta from S. cerevisiae and human also complement the gene knockouts in both yeasts. Together, these observations indicate that the TMD of Sec61beta exerts a cellular function that is conserved across species.