A Possibility of Application of the 105-Kilodaltons Protein of <i>Brachyspira alvinipulli</i> Cross-Reacting with Antisera to Five Species in the Genus <i>Brachyspira</i> to Diagnosis

Abstract

ABSTRACT. The antigenic properties of Brachyspira (B.) Brachyspira (B.) alvinipulli is an anaerobic and weakly beta-hemolytic spirochete [14], and a causal agent of the deterioration of chicks and reduction in egg production [17]. The spirochete colonized the mucosal surface of ceca in one-day-old chicks and 14-month-old hens, and the ceca affected with the spirochetes were dilated and contained pale-yellow watery contents [17]. Lymphocytic typhlitis and proctitis in the infected chicks were observed as histological changes [17].Many studies on the antigens of B. hyodysenteriae have been published [7,9,11,13,19]. Amongst the reports, a 45-kilodaltons (kDa) polypeptide located in the outer envelope of B. hyodysenteriae P18A is more interesting, because it has the ability to produce antibody in gnotobiotic pig serum [13]. A 45-kDa protein in the B. hyodysenteriae B204 was related to endotoxin [19]. A 44-kDa periplasmic flagellar sheath protein of B. hyodysenteriae was confirmed as a species-specific antigen consisting of glycoprotein, and the protein could produce antibody in adult New Zealand White rabbits [9]. Ochiai et al. [11] reported that 22-and 17-kDa proteins present in eight strains of B. hyodysenteriae reacted strongly with convalescent pig sera and were intimately related to the antigens responsible for the strong stimulation in pigs [11]. Amongst the protein antigens, the periplasmic flagella have been studied intensively. The hyperimmune pig serum to periplasmic flagella derived from B. hyodysenteriae P18A cross-reacted with strains B78, S75/1, B169, P18A, KF9, VS1, MC52/80 and P35/2 of B. hyodysenteriae. Convalescent serum of a pig affected with dysentery also reacted strongly with the periplasmic flagella of B. hyodysenteriae P18A. These results mentioned above demonstrated that the periplasmic flagella of B. hyodysenteriae were major antigens in the spirochete [7], but there are no reports on the immunochemical characteristics of proteins of B. alvinipulli. In this study we analyzed the proteins immunochemically and compared the antigens with those of the other Brachyspira species. MATERIALS AND METHODSStrains used: B. alvinipulli ATCC 51933 and strain C2, B. hyodysenteriae ATCC 27164 and ATCC 31212, B. pilosicoli ATCC 51139, B. innocens ATCC 29796 and B. aalborgi NCTC 11492 were used in this study. All strains were grown at 37°C for 72 hr on trypticase soy agar (BBL, U.S.A.) containing 5% (volume/volume) sheep blood under anaerobic conditions with the GasPak System (BBL, U.S.A.). The cells were harvested and washed twice with physiological saline by centrifugation at 15,000 rpm for 10 min at 4°C, and the pellets were stored at -80°C before use.Hyperimmune serum: Hyperimmune sera were prepared as previously described [1]. The cells in 0.01 M phosphate buffer (pH7.2) were adjusted to approximately 1 × 10 9 cells ml -1 , and were inoculated intravenously into rabbits at intervals of four days. When the antibody titer to the inoculum, reached the maximum titer in agglutination and immunodiffusion t...

A Spirochaete is suggested as the causative agent of Akoya oyster disease by metagenomic analysis

“…Rabbit polyclonal antibodies against Brachyspira aalborgi NCTC11492, B . pilosicoli ATCC51139 [ 29 ], and Treponema pallidum (Abcam, Cambridge, UK) were labeled with Alexa555, and naïve rabbit antibody was labeled with Alexa488 by Zenon labeling kit (ThermoFisher). Hemolymph was drawn from three diseased pearl oysters collected at Shima (a-value 3.03, 6.21, 6.50) and three healthy pearl oysters collected at Noto (a-value 0.21, 1.22, 1.04), and smears were prepared as described above.…”