Analysis of the Axial Filaments of Treponema hyodysenteriae by SDS-PAGE and Immunoblotting

Kent, Karen; Sellwood, R.; Lemcke, Ruth M.; Burrows, M. R.; Lysons, R. J.

doi:10.1099/00221287-135-6-1625

Cited by 31 publications

(50 citation statements)

References 29 publications

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“…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.)…”

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confidence: 92%

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

Sakurai

Adachi

1998

The Journal of Veterinary Medical Science

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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...

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confidence: 92%

“…The supernatant was taken after centrifugation at 15,000 × g for 30 min and was used for SDS-PAGE. Immunoblotting technique was carried out essentially as previously described [10,13] using semidry type blotter (Sartblot II-S, Sartorius AG, Germany). Treatment with proteinase K was carried out as previously described [18].…”

mentioning

confidence: 99%

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

Sakurai

Adachi

1998

The Journal of Veterinary Medical Science

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“…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.…”

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confidence: 67%

“…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].…”

Section: Brachyspira (Bmentioning

confidence: 99%

“…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].…”

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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

Abe

Adachi

2004

The Journal of Veterinary Medical Science

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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...

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Cloning and Characteristics of a Gene Encoding NADH Oxidase, a Major Mechanism for Oxygen Metabolism by the Anaerobic Spirochete, Brachyspira (Serpulina) hyodysenteriae

Stanton

Sellwood

1999

Anaerobe

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Analysis of the Axial Filaments of Treponema hyodysenteriae by SDS-PAGE and Immunoblotting

Cited by 31 publications

References 29 publications

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

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

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

Cloning and Characteristics of a Gene Encoding NADH Oxidase, a Major Mechanism for Oxygen Metabolism by the Anaerobic Spirochete, Brachyspira (Serpulina) hyodysenteriae

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