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3 result(s) for "sequential real‐time PCR"
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Evaluation of a non-invasive screening approach to determine hepatitis E virus status of pig farms
BackgroundIdentifying pig farms infected with hepatitis E virus (HEV) is a key aspect to implement surveillance programmes for this emerging zoonotic agent. Detection of HEV in blood has several drawbacks, including animal handling, economic costs and animal stress. The objective of this study was to evaluate the effectiveness of a non-invasive screening approach for determining the HEV status of pig farms under different management systems.MethodsForty stool samples randomly collected from the pen floor of 17 intensive pig farms and the yard of nine extensive ones were tested for HEV RNA. The invasive method used to confirm the HEV status of the farm was HEV RNA analysis of serum samples randomly collected from 40 animals on each farm.ResultsTwenty-one HEV-positive farms were detected by invasive and non-invasive methods. No positive serum or stool samples were detected on five intensive farms. A high intertest agreement (K=1; P<0.00001) was observed between both methodologies, showing the stool screening approach a 100 per cent of sensitivity and specificity with respect to the invasive method. Likewise, a significant negative relationship was observed between the HEV within-farm prevalence and the number of the first HEV-positive stool sample found (Spearman’s rho=−0.64; P=0.0004). This negative relationship was higher in intensively managed farms.ConclusionThis non-invasive screening approach could be reliably applied in a large-scale surveillance programme for determining the HEV status of pig farms under different management systems.
Transcriptional regulation of cholesterol and bile acid metabolism after dietary soyabean meal treatment in Atlantic salmon (Salmo salar L.)
Inclusion of plant protein sources such as soyabean meal (SBM) in aquafeeds is associated with decreased lipid digestibility, reduced bile acid levels and hypocholesterolaemia. The mechanism for these metabolic abnormalities is unknown. The present study aimed at gaining further insight into how cholesterol and bile acid metabolism is modulated by SBM feeding by quantifying a number of mRNA species corresponding to key proteins involved in cholesterol and bile acid metabolism using quantitative real-time PCR. A 21 d feeding trial with sequential sampling at ten time points following initiation of 20 % SBM exposure was conducted on Atlantic salmon. A histological evaluation confirmed distal intestinal enteritis after 5 d of dietary exposure to the SBM, whereas diminished glycogen/lipid deposition was the only relevant finding observed in the liver. SBM inclusion resulted in reduced body pools of cholesterol and bile acids. Hepatic gene expression profiles revealed up-regulation of genes encoding rate-limiting enzymes in cholesterol (3-hydroxy-3-methyl-glutaryl-CoA reductase; HMGCR) and bile acid (cytochrome P4507A1 (CYP7A1)) biosynthesis, as well as up-regulation of their associated transcription factors (sterol regulatory element binding proteins 1 and 2, liver X receptor, farnesoid X receptor and PPAR isoforms). Hepatic gene expressions of cholesterol (ATP binding cassette G5 (ABCG5)) and bile acid (ATP binding cassette B11 (ABCB11)) transporters were, by and large, not influenced by the SBM, but distal intestinal expression patterns of ABCG5 and apical Na-dependent bile acid transporter indicated impaired cholesterol and bile acid reabsorption. In conclusion, hepatic gene expression profiles indicated that the capacity for cholesterol and bile acid synthesis was up-regulated, whereas the indicated impaired cholesterol and bile acid reabsorption probably occurred as a direct result of distal intestinal inflammation.
Mode-Dependent Antiviral Activity of Medicinal Plant Extracts against the Mosquito-Borne Chikungunya Virus
The lack of specific treatment for chikungunya fever makes the need for anti-chikungunya virus agents more crucial. This study was conducted to evaluate 132 extracts obtained by sequential solvent extraction from 21 medicinal plants for cytopathic effect inhibitory activity using virus-infected Vero cells in two different sample introduction modes. Among the extracts, 42 extracts (31.8%) from 12 plants in the concurrent mode and three extracts (2.3%) from a plant in the non-concurrent mode displayed strong cytopathic effect inhibitory activity (cell viability ≥70%). Viral load quantification analysis unveiled that the extracts of Clinacanthus nutans (chloroform, ethyl acetate, and ethanol), Hydrocotyle sibthorpioides (ethanol), and Ocimum americanum (ethanol and methanol) hindered the release of viral progeny from the infected cells while the extracts of Ficus deltoidea (ethanol), Gynura bicolor (water), H. sibthorpioides (water), and O. americanum (chloroform and ethyl acetate) blocked the entry of virus into the cells. The extracts of Diodella sarmentosa (ethyl acetate), Diplazium esculentum (chloroform, ethyl acetate, and ethanol), and G. bicolor (ethanol) possessed virucidal effect and caused 5.41-log to 6.63-log reductions of viral load compared to the virus control. The results indicate that these medicinal plants are potential sources of anti-chikungunya virus agents that have varied modes of action.