Presence of 22 and 17-kDa Proteins Reacting with Sera in Mice Experimentally Infected with Brachyspira (Serpulina) hyodysenteriae.

Abstract

ABSTRACT. The antibodies to B. (S.)hyodysenteriae in experimentally infected mice were detected by microscopic agglutination test (MAT) and enzyme-linked immunosorbent assay (ELISA). The reactions in MAT were serotype specific while those in ELISA were common to both strains. A further investigation with immunoblotting technique demonstrated that 22-and 17-kDa proteins reacted strongly with the sera. The proteins in ATCC 27164 strain strongly reacted with the serum from ATCC 31212 strain-infected mouse and vice versa. These proteins were sensitive to proteinase K. A.). Each suspension of ATCC 27164 and 31212 strains was adjusted at 4.05 × 10 7 cells/ml and 7.17 × 10 7 cells/ml, respectively, and 0.5 ml of the suspension was inoculated intragastrically into the mice. In this experiment, SP-treated mice were allocated into ATCC 27164 (n=4) and 31212 (n=5) infected groups while SP-nontreated mice were allocated into ATCC 31212 infected group (n=4) and only TSB-infected group (n=5). Reisolation of the spirochetes from the feces on days 7 and 14 after inoculation and from the cecal contents on day 21 after the inoculation was carried out using blood agar containing 400 µg/ml of SP [14]. All mice were bled on day 21 after the inoculation. The microscopic agglutination test (MAT) was carried out as previously described [9]. The results are presented as the reciprocal of the maximum dilution of the serum giving a 50% microscopic agglutination of the organism. An enzyme-linked immunosorbent assay (ELISA) was carried out essentially as previously described [8]. The preparation of the antigen for ELISA was carried out as previously described [1]. After harvest, the cells were suspended in 0.01 M phosphate buffer (pH 7.2), washed three times by centrifugation at 10,000 × g for 20 min and adjusted to 0.65 at a wave length of 625 nm. The cell suspension was sonicated for 10 min (100 W, 10 kHz) by a sonicator UR-200P (TOMY Seiko Co., Ltd., Tokyo, Japan) and centrifuged at 2,000 × g for 30 min. The supernatant was used as the antigen for ELISA. Alkaline phosphatase Brachyspira (Serpulina) hyodysenteriae is the causative agent of swine dysentery (SD) [3,17]. We proposed the unification of the genera Serpulina [15] and Brachyspira [4], and the use of genus Brachyspira [12]. Mice have been used for the experimental infectious model of SD [5]. However, there have been no reports on the antibodies in mice infected with B.(S) hyodysenteriae. Boyden et al. [2] cloned genes that code for B.(S.) hyodysenteriae endoflagella antigens into E. coli with the purpose of identifying protective antigens for vaccine development and confirmed the protective activity of the cloned endoflagella antigen using CF1 mice. However, the antibacterial activity of hyperimmune pig serum raised against axial filaments consisting of 37-, 34-, and 32-kDa proteins could not be demonstrated [10]. On the other hand, pigs that have been infected with virulent strains of B.(S.) hyodysenteriae and recover from the disease have been shown to be immune to further inf...

Presence of 22-kDa protein reacting with sera in piglets experimentally infected withBrachyspira hyodysenteriae

“…These results demonstrated that the antibodies may induce an antibacterial mechanism in vivo which could lead to the protection of pigs from swine dysentery. On the other hand, we have carried out studies with experimental infection of mice with B. hyodysenteriae which had been treated with SP and reported the presence of 22‐ and 17‐kDa proteins which reacted strongly with the sera from mice infected with B. hyodysenteriae [11]. The proteins in strain ATCC 27164 strongly reacted with the serum from mice infected with strain ATCC 31212 and vice versa.…”