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Wieland B.,Lane College | Dhollander S.,Animal Health and Welfare Unit | Salman M.,Colorado State University | Koenen F.,Veterinary and Agrochemical Research Center
Preventive Veterinary Medicine | Year: 2011

In the absence of data, qualitative risk assessment frameworks have proved useful to assess risks associated with animal health diseases. As part of a scientific opinion for the European Commission (EC) on African Swine Fever (ASF), a working group of the European Food Safety Authority (EFSA) assessed the risk of ASF remaining endemic in Trans Caucasus Countries (TCC) and the Russian Federation (RF) and the risk of ASF becoming endemic in the EU if disease were introduced. The aim was to develop a tool to evaluate how current control or preventive measures mitigate the risk of spread and giving decision makers the means to review how strengthening of surveillance and control measures would mitigate the risk of disease spread. Based on a generic model outlining disease introduction, spread and endemicity in a region, the impact of risk mitigation measures on spread of disease was assessed for specific risk questions. The resulting hierarchical models consisted of key steps containing several sub-steps. For each step of the risk pathways risk estimates were determined by the expert group based on existing data or through expert opinion elicitation. Risk estimates were combined using two different combination matrices, one to combine estimates of independent steps and one to combine conditional probabilities. The qualitative risk assessment indicated a moderate risk that ASF will remain endemic in current affected areas in the TCC and RF and a high risk of spread to currently unaffected areas. If introduced into the EU, ASF is likely to be controlled effectively in the production sector with high or limited biosecurity. In the free range production sector, however, there is a moderate risk of ASF becoming endemic due to wild boar contact, non-compliance with animal movement bans, and difficult access to all individual pigs upon implementation of control measures. This study demonstrated the advantages of a systematic framework to assist an expert panel to carry out a risk assessment as it helped experts to disassociate steps in the risk pathway and to overcome preconceived notions of final risk estimates. The approach presented here shows how a qualitative risk assessment framework can address animal diseases with complexity in their spread and control measures and how transparency of the resulting estimates was achieved. © 2011 Elsevier B.V. Source


Siah A.,British Columbia Center for Aquatic Health science | Siah A.,University of Prince Edward Island | McKenna P.,University of Prince Edward Island | Johnson G.,University of Prince Edward Island | Berthe F.C.J.,Animal Health and Welfare Unit
Journal of Shellfish Research | Year: 2012

Previous studies have shown that hemic neoplasia in softshell clams is related to the level of P53 -like mRNA in hemocytes. Traditionally, the p53-like mRNA level has been quantified using quantitative real-time RT-PCR (Q RT-PCR). However, this technique requires several steps that are sources of contamination and may result in a low accuracy of the analysis. The novel aspect of this study is that the p53-like mRNA level was quantified directly from a lysate of hemocytes without any RNA extraction or reverse transcription steps. This assay is based on branched DNA (bDNA) signal amplification technology and enables quantification of p53-like mRNA levels in as few as 2,500 hemocytes, with a coefficient of variation close to 6% (range, 212%). A significant correlation (R2= 0.99, P ≤0.01, n= 5) was found between the p53-like mRNA quantified directly from hemocytes without RNA extraction and from 1 g total RNA extracted from hemocytes using the classic TRIzol protocol. To compare p53-like mRNA levels in hemocytes from diseased clams collected in North River (Prince Edward Island, Canada) and from healthy clams found in Havre aux Maisons at Magdalene Island (Quebec, Canada), the quantification of relative p53-like mRNA levels was performed using our single plex assay. Data showed a significantly (P ≤0.01, n= 5) high expression of p53-like in the hemocytes of clams collected from North River. Therefore, although Q RT-PCR remains the most widely used technique for the quantification of gene expression level, we believe that single plex using bDNA technology could represent a new generation of mRNA quantification tool, enabling a more efficient mollusc health management. Source


Siah A.,University of Prince Edward Island | McKenna P.,University of Prince Edward Island | Danger J.-M.,University of Le Havre | Johnson G.R.,University of Prince Edward Island | Berthe F.C.J.,Animal Health and Welfare Unit
Developmental and Comparative Immunology | Year: 2011

In Prince Edward Island, a high mortality of soft-shell clams Mya arenaria was found to be related to the disease known as disseminated neoplasia (DN). However, the molecular mechanisms by which hemocytes of clams are transformed in the course of DN remain by far unknown. This study aims at identifying the transcripts involved in the development of the disease. Four subtractive cDNA sequence libraries were generated and more than 200,000 reads were obtained. Following similarity searches in genome databases, the transcripts were assigned to cellular functions including mitochondrial respiration, structural proteins, cytoskeleton, nucleic acid regulation, general metabolism, signal transduction, apoptosis, cell cycle regulation, as well as virus transcripts. The expression levels of transposase and polyprotein genes were evaluated in clams with various percentages of tetraploid hemocytes. Data have shown that expression levels were significantly higher in clams with a high percentage of tetraploid hemocytes. These results reinforce the hypothesis of endogenous retrotransposon involvement in the etiology of the disease. Further investigations are needed, however, to elucidate the role of transposase and polyprotein in the disease development. © 2010 Elsevier Ltd. Source


Araya M.T.,University of Prince Edward Island | Markham F.,University of Prince Edward Island | Mateo D.R.,University of Prince Edward Island | McKenna P.,University of Prince Edward Island | And 3 more authors.
Fish and Shellfish Immunology | Year: 2010

Although the mollusc immune system has been studied at the cellular level, the response to pathogens at gene expression level has not been thoroughly investigated. This study aimed to investigate the early molecular response of hemocytes of soft-shell clams, Mya arenaria, to Vibrio splendidus strain LGP32 by identification of transcripts involved in immune defense. The Suppression Subtractive Hybridization (SSH) was used to selectively identify differentially expressed genes in hemocytes exposed to V. splendidus at a ratio 1:1 for 2 h. Both forward and reverse subtracted cDNA were constructed and a total of 16,000 reads were obtained and analyzed. Identity searches in genome databases were performed using BlastX program and transcripts were clustered to cellular functions including structural proteins, immunity, stress proteins, apoptosis, cell process, metabolism and signal transduction. Among the differentially expressed immune associated genes were ficolin, killer cell lectin-like receptor, natural resistance-associated macrophage protein 1 (Nramp-1), mitogen-activated protein kinases (MAPK), ferritin, heat shock proteins 90 (HSP90) and cathepsin and their expressions were quantified using Reverse Transcription quantitative Polymerase Chain Reaction (RT-qPCR) at 1, 2 and 3 h post- Vibrio challenge. These genes showed similar expression patterns, up-regulation at 1 h, followed by a down-regulation at 2 and 3 h. These data corroborates our previous observations of cell rounding, reduced phagocytosis and respiratory burst activity. To our knowledge, this is the first study to demonstrate an effect of V. splendidus on expression of genes related to immune system in soft-shell clams M. arenaria. However, further investigations are needed to unravel the molecular mechanisms of hemocytes subjected to V. splendidus. © 2010. Source


Mateo D.R.,University of Prince Edward Island | Greenwood S.J.,University of Prince Edward Island | Araya M.T.,University of Prince Edward Island | Berthe F.C.J.,Animal Health and Welfare Unit | And 2 more authors.
Developmental and Comparative Immunology | Year: 2010

Immune function gene expression in Mya arenaria haemocytes was evaluated following in vivo infection with Vibrio splendidus LGP32-GFP and 7SHRW. Elongation factor 1α (EF-1α) with 2 (EF-2), after challenge with LGP32-GFP, and EF-1α with the ribosomal protein S-18, after challenge with 7SHRW, were found to be the most stable housekeeping genes. Using these internal controls and comparing the regulation induced by both strains, up-regulation of γ-actin, down-regulation of TLR-2 and up-regulation of IRAK-4 was significantly higher after challenge with LGP32-GFP (p<0.001, p=0.001 and p<0.05, respectively). These results suggest specific responses at a molecular level modulated by the bacterial strains. LGP32-GFP induced marked responses which coincide with a similar trend previously found on phenotypic responses under our experimental model. © 2010 Elsevier Ltd. Source

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