After incubation for 1 h the plates were washed three times with 0
After incubation for 1 h the plates were washed three times with 0.85% saline containing 0.05% Tween 20. erysipelas on farms each year, and each year meat inspection government bodies condemn about 2, 000 pigs because they have the subacute or chronic form of the disease. Various serological methods for the analysis of chronic swine erysipelas or for assay of maternal antibody and acquired antibody before and after vaccination have been reported, e.g., growth agglutination checks (12, 20, 25), the latex agglutination test (19), and enzyme-linked immunosorbent assay (ELISA) (1, 2, 4, 9, 10, 11, 16, 17, 19). Unlike in other countries, the attenuated live vaccine is the most commonly used type of vaccine in Japan, and the growth agglutination test is used for the detection of maternal antibody and acquired antibody before and after vaccination. This double test is carried out since the production of antibody against the live vaccine is definitely affected by the presence of maternal antibody (26). However, the growth agglutination test requires MPL tradition of live pathogenic bacteria, which can be dangerous to laboratory workers. For this reason, recently developed latex agglutination packages are progressively being utilized. On the other hand, ELISA is the test of choice among existing serological methods because it is simple, permits the screening of large numbers of samples in a short time, and gives precise, objective results. The major protecting antigen of is the so-called 64- to 66-kDa antigen (1, 6, 7, 13, 19). Makino et al. (14) cloned the gene encoding the 69.9-kDa protecting antigen of strain Tama of serotype 2 and named it the protein surface protecting antigen (SpaA). The gene, which encodes a 69.0-kDa protecting antigen, of the virulent Fujisawa strain of serotype 1a was also cloned, and it was shown for the first time that purified truncated recombinant SpaA (amino acids 61 to 408; SpaA348) of serotype 1a can elicit total safety in pigs challenged with serotypes 1a PF-4840154 and 2b (9). The antibody production of these immunized pigs was PF-4840154 sensitively recognized by an indirect ELISA with SpaA348 as the antigen and by a double-antibody sandwich ELISA with alkaline components of as the antigen (9). However, the sensitivity of the indirect SpaA348 ELISA was insufficient for the detection of antibody in pigs immunized with the live vaccine. In this study, we constructed five regions of SpaA and compared their sensitivities and specificities in an indirect ELISA. We also evaluated the applicability of the SpaA ELISA using sera collected from experimentally immunized pigs, nonimmunized control pigs, experimentally challenged pigs, and pigs reared on farms. MATERIALS AND METHODS Bacterial strains and viral strain. The Fujisawa strain (a virulent strain of serotype 1a) and a Japanese established challenge strain for the assay of vaccine effectiveness were used in most of the experiments for PF-4840154 intradermal challenge of pigs. 82-875, a virulent strain of serotype 2b, was from the National Veterinary Assay Laboratory and was used in the cross-protection PF-4840154 checks with truncated recombinant SpaA348. strain C42 of serotype 1a was isolated from a pig that experienced died from a dual illness with porcine reproductive and respiratory syndrome disease (PRRSV) and in 1995. PRRSV strain E4 was isolated from a seriously affected pig in 1993. strain C42 and PRRSV strain E4 were utilized for the challenge exposure of pigs to study whether PRRSV illness in pigs inhibits the effect of the attenuated vaccine (18). strain SE-9, an official strain utilized for the production of bacterin in the United States, was used to prepare alkaline components for the double-antibody sandwich ELISA. Manifestation of SpaA in like a fusion.