All samples with high absorbance values but low blocking percentages were taken as positive in repeated analyses under different conditions of sample or SwSneg

All samples with high absorbance values but low blocking percentages were taken as positive in repeated analyses under different conditions of sample or SwSneg./pos. pigs of AMG-333 all ages with high morbidity but low mortality (Pensaert and DeBouck, 1978, Pensaert et al., 1981, Pritchard et al., 1999), whereas in Asia (Japan, Korea and China) mortality reaches high percentages (30C80%) in suckling piglets (Takahashi et al., AMG-333 1983, Shibata et al., 2001). Three major structural proteins occur in coronaviruses: the glycoprotein S (Mr 150C220?K) found in the viral protrusions (corona), the nucleocapsid phosphoprotein N AMG-333 (Mr 45C57?K), and the membrane protein M (Mr 20C30?K) (Saif, 1993, Utiger et al., 1995). Conventional diagnostic methods of porcine viral gastroenteritis are based on the detection of either antibodies or the virus. Before local immunity is usually actively established, the intestine of the piglet is usually AMG-333 protected against contamination by maternal antibodies obtained through colostrum and, later on, mainly through milk. Serological examination allows assessing earlier contacts of sows with the virus, but even litters from seropositive sows are not protected against contamination if the milk contains non-protective antibody levels. Therefore, information on a current epizootologic situation in a herd is best obtained by virus detection. Besides virus isolation, immunohistochemical and electron microscopic detection methods are often used (Guscetti et al., 1998, Kim et al., 1999, Rodk et al., 1999). As these techniques are labourious and time demanding, only few samples are usually examined. This holds also for highly sensitive and specific methods of molecular virology (PCR, RT-PCR) (Ishikawa et al., 1997, Kweon et al., 1997, Kim et al., 2000), although miniaturization and automation of e.g. real-time PCR methods are gradually reaching the veterinary diagnostic laboratory. Of the ELISA variants the double antibody sandwich form of the assay (DAS), with specificity checked by blocking, has been described for PEDV detection (Callebaut et al., 1982; van Nieuwstadt and Zetstra, 1991, Carvajal et al., 1995). The objective of the present study was to verify modifications of the ELISA blocking method for the detection of PEDV in culture media and faecal samples, and to use the optimal one in routine examination of field samples. 2.?Material and methods 2.1. Viral and control antigens The reference strain of PEDV (CV-777) was propagated in Vero cells grown in minimum essential medium Eagle (MEM) in the presence of trypsin (Hofmann and Wyler, 1988). As soon as the cytopathic effect was fully developed, the virus was PAPA1 released from the cells by repeated freezing/thawing. After centrifugation (2000?? for 15?min) of the culture medium, the pellet was resuspended in 1/100 of the original medium volume in phosphate buffer saline (PBS) as crude viral antigen (V-Ag). Purified V-Ag was prepared (Hofmann and Wyler, 1990) by ultracentrifugation in saccharose gradients (Beckman, Optima LE-80K Ultracentrifuge, USA) from the supernatant of the infectious culture medium (without cell debris). Similarly, crude control antigens (C-Ag) were prepared from non-infected cell lines. All antigens were kept at ?80?C before use. Culture media with defined virus concentrations (decided as plaque-forming units-PFU/ml medium) were used for determination of the sensitivity of the ELISA method by box titration. V-Ag and C-Ag were prepared similarly by propagation of TGEV (collection of animal pathogenic microorganisms, Brno; strain CAPM V-344), and group A rotavirus (strain OSU and our own isolates) in the porcine kidney (PK-15) and monkey kidney (MA-104) cell lines, respectively. To exclude potential nonspecificity due to cross-reactivity with other brokers, crude V-Ag (PED, TGE, rota) and C-Ag were tested for specificity in all ELISA methods used. Purified PED V-Ag was used for immunisation of mice and for Western blot analysis. 2.2. Experimental contamination Two hysterectomy-derived, colostrum-deprived piglets kept in sterile box were fed sterile condensed milk made up of 7% of fat. On the day 19 they were orally infected with 2.2??104 PFU PEDV strain CV-777. In one of the piglets, diarrhoea appeared 24?hpi and the animal died 48?hpi. The intestinal contents were collected for EM and ELISA virus detection. Small intestine samples were frozen in liquid nitrogen and kept at ?80?C before immunohistochemical examination. In the second piglet, diarrhoea occured 48?hpi, and serial faecal samples were collected during one week. Seven weeks post contamination, the piglet was orally challenged with 1.1??105 PFU PEDV and sacrificed 12 days later by exsanguination under total anaesthesia. The titre of PEDV antibodies in the obtained serum (SwSpos.) was determined by indirect ELISA (51,200). Good results were obtained by comparative estimation of PEDV blocking activities of this serum and the swine AMG-333 PEDV sera kindly provided by Dr. Pensaert (Belgium) and Dr. M?stl.