Development of a qPCR assay for the quantification of porcine adenoviruses as an MST tool for swine fecal contamination in the environment.

The Adenoviridae family comprises a wide diversity of viruses that may be excreted for long periods in feces or urine. Previous studies have suggested that the detection of human and animal adenoviruses as well as human and animal polyomaviruses by PCR could be used as an index of fecal contamination of human and animal origin. In this study, quantitative PCR assays targeting specifically porcine adenoviruses have been developed and applied to fecal and environmental samples, including pig slurries, urban sewage, slaughterhouse sewage and river water samples. To develop real-time quantitative PCR for the detection and quantitation of porcine adenoviruses, primers and a TaqMan probe targeting a 68-bp region of the porcine adenovirus hexon gene were designed to amplify specifically porcine adenovirus, and the conditions of the reaction were optimized. The assay detected 1-10 genome copies per test tube and was specific in showing no positive results when samples containing human or bovine adenoviruses were analyzed. Fecal samples contained mean concentrations of porcine adenoviruses of 10(5) GC/g while slaughterhouse wastewater samples showed mean values of 10(3) GC/ml. The assay detected porcine fecal pollution in samples that were highly diluted and had been collected at a considerable distance from the input source, such as river water. In general, the data presented here provide a quantitative tool for the analysis of porcine adenoviruses as indicators of the presence of porcine contamination in the environment, and support the detection of porcine adenoviruses by real-time quantitative PCR as a promising and valuable tool for source-tracking studies.

Detection and survival of prion agents in aquatic environments.

Environmental contamination is considered a potential mechanism of transmission of prion diseases. Sheep scrapie and cervid chronic wasting diseases (CWD) epizootics are thought to be maintained by natural horizontal transmission through the environment. Here, we describe a method for the detection of prion proteins (PrPres) in aquatic environments. The procedure is based on a glycine buffer-mediated extraction, sonication, and an ultracentrifugation step. The detection limit of the method was estimated to be over 5-10 microg of infected tissue. In order to determine the inactivation of these agents, we spiked infected brain tissue in urban sewage, seawater and a buffered solution (final concentrations of 0.1-0.2% brain in matrix), and studied the decay of BSE- and scrapie-associated PrPres over time (up to 265 days). Densitometric data from Western blots were plotted in logarithmic scale against time. Reduction of PrPres titer in sewage was quantified in one logarithm after 13.5 days for BSE, 27.9 days for mouse-passaged scrapie and 32.6 days for sheep scrapie. In the buffered solution, a logarithm of BSE-associated PrPres also disappeared earlier than that of scrapie (113.9 and 214.3 days, respectively). By means of the covariance analysis, these differences in the inactivation patterns were shown to be statistically significant. According to the data, prions may be stable for extended periods of time in buffered solutions like PBS, but would show limited survival in aquatic environmental matrices.

Assessing the presence of BSE and scrapie in slaughterhouse wastewater.

AIMS: This paper describes a procedure for evaluating the presence and the stability of the proteinase K-resistant form of the prion protein (PrP(res)) in slaughterhouse wastewater. METHODS AND RESULTS: Wastewater samples were spiked with either scrapie or bovine spongiform encephalopathy agents and PrP(res) was concentrated and detected by western blotting. The detection limit was estimated to be 2-4 microg of either scrapie or BSE-infected brain tissue in 15 ml of sewage. Wastewater samples from three abattoirs were analysed, two of which had processed BSE-infected animals. No PrP(res) was detected. The effect of sewage on the inoculum and the persistence of transmissible spongiform encephalopathy agents in wastewater were also considered. CONCLUSIONS: The results of the assay suggest that wastewaters from abattoirs where one positive BSE case has been identified would contain titres lower than 0.6-26 x 10(-4) cattle oral ID(50) per litre resulting from specified risk material tissue contamination. Moreover, the effect of abattoir wastewaters is to reduce the persistence of PrP(res). SIGNIFICANCE AND IMPACT OF THE STUDY: The assay may be a useful tool for risk assessment studies and for reducing the potential risk of contamination with BSE via sewage sludge fertilizer procedures.