Immunological characterization of protective antigens prepared by alkaline treatment of whole cells and from the culture filtrate of Erysipelothrix rhusiopathiae

Kobayashi, Shigeki; Sato, Hisaaki; Hirose, Kazuhiko; Saito, Hiroshi

doi:10.1016/0378-1135(92)90095-b

Cited by 10 publications

(29 citation statements)

References 13 publications

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“…It has been shown that animals immunized with a culture filtrate from E. rhusiopathiae are protected against homologous and heterologous strains (23)(24)(25). As observed in other Firmicutes bacteria, anchorless housekeeping glycolytic enzymes, including TPI, FBA, and GAPDH, were present in the supernatant of the Fujisawa strain.…”

Section: Discussionmentioning

confidence: 99%

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Characterization and Identification of a Novel Candidate Vaccine Protein through Systematic Analysis of Extracellular Proteins of Erysipelothrix rhusiopathiae

et al. 2013

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dErysipelothrix rhusiopathiae, the causative agent of swine erysipelas, is a facultative intracellular Gram-positive bacterium. It has been shown that animals immunized with a filtrate from E. rhusiopathiae cultures are protected against lethal challenge. In this study, we identified and characterized the extracellular proteins of E. rhusiopathiae to search for novel vaccine antigens. A concentrated culture supernatant from the E. rhusiopathiae Fujisawa strain, which has been found to induce protection in mice, was analyzed using two-dimensional electrophoresis. From more than 40 confirmed protein spots, 16 major protein spots were selected and subjected to N-terminal amino acid sequence determination, and 14 protein spots were successfully identified. The identified proteins included housekeeping proteins and other metabolic enzymes. We searched for surface-localized proteins by analyzing the genomes of two E. rhusiopathiae strains: Fujisawa and ATCC 19414. Genome analysis revealed that the ATCC 19414 strain has three putative surface-exposed choline-binding proteins (CBPs): CbpA, CbpB, and CbpC. Each CBP contains a putative choline-binding domain. The CbpC gene is mutated in Fujisawa, becoming a nonfunctional pseudogene. Immunogold electron microscopy confirmed that CbpA and CbpB, as well as the majority of the metabolic enzymes examined, are associated with the cell surface of E. rhusiopathiae Fujisawa. Immunization with recombinant CbpB, but not with other recombinant CBPs or metabolic enzymes, protected mice against lethal challenge. A phagocytosis assay revealed that antiserum against CbpB promoted opsonin-mediated phagocytosis by murine macrophages in vitro. The protective capabilities of CbpB were confirmed in pigs, suggesting that CbpB could be used as a vaccine antigen.

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Section: Discussionmentioning

confidence: 99%

“…The E. rhusiopathiae strain Fujisawa possesses at least 24 surface proteins that contain either an LPXTG motif or a GW repeat (2). Protective antigens are present in E. rhusiopathiae culture supernatants (23)(24)(25)(26)(27); however, the nature of these protective antigens is entirely unknown. Thus far, only a few protective antigens have been identified.…”

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confidence: 99%

Characterization and Identification of a Novel Candidate Vaccine Protein through Systematic Analysis of Extracellular Proteins of Erysipelothrix rhusiopathiae

et al. 2013

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“…Tryptic soy broth supplemented with 0.3 % Tris-hydroxymethyl aminomethane and 0.1 % Tween 80 (TT-TS broth) and TT-TS broth supplemented with 1.5 % agar (TT-TS agar) (KOBAYASHI et al, 1992) were used for the routine culture of E. rhusiopathiae. A bacterial suspension of E. rhusiopathiae (0.1 ml; 10 10 colony forming units (CFU/ml) was added to 1000 ml of TT-TS broth and incubated without shaking at 37°C for 24 h for the isolation of the AE.…”

Section: Media and Culturementioning

confidence: 99%

“…The protein concentrations of the AE and P64 were measured using the Bicinchoninic acid protein assay (SMITH et al, 1985) as described by KOBAYASHI et al (1992).…”

Section: Quantitation Of the Protein Concentrationmentioning

confidence: 99%

“…SDS-PAGE was performed using the method of LAEMMLI (1970) as described by KOBAYASHI et al (1992) and SATO et al (1995). Electrophoresis was performed at 60 mA/gel for concentration determination and 120 mA/gel for protein separation with a constant current for 5 h. The molecular weight markers used were purchased from Pharmacia Fine Chemical Co. Ltd (Uppsala, Sweden) and included cytochrome C (12 kDa), myoglobin (17 kDa), chymotrypsinogen (26 kDa), ovalbumin (43 kDa), bovine serum albumin (66 kDa) and ovotransferrin (78 kDa).…”

Section: Sodium Dodecyl Sulphate-polyacrylamide Gel Electrophoresis (mentioning

confidence: 99%

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Protective Activity of the Purified Protein Antigen of Erysipelothrix rhusiopathiae in Pigs

Yamazaki

Sato

Sakakura

et al. 1999

Journal of Veterinary Medicine, Series B

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We purified the protein antigen (P64), which contains 66 and 64 kDa proteins, from the alkaline extract (AE) of whole cells of Erysipelothrix rhusiopathiae strain Agata (serovar 5) to determine the protective activity of the antigen against E. rhusiopathiae infection in pigs. The serum titre of antibody against P64 rapidly increased in pigs immunized with 500 and 100 mg of P64 and reached maximum values at 3 weeks after the first immunization (1 week after the second immunization). However, the serum antibody titres were not increased in pigs immunized with 20 mg of P64 and in nonimmunized pigs. In the pigs immunized with live cell vaccine (acriflavin-fast attenuated strain Koganei 65-0.15), the serum titres of antibody against P64 also increased at 1-2 weeks after immunization. In a pig challenge test performed on immunized and nonimmunized pigs, all nonimmunized pigs showed typical clinical signs of swine erysipelas (fever, erysipeloid, arthritis), while all pigs immunized with 500 and 100 mg of P64 and live cell vaccine showed no clinical signs of this disease. In Western blot analysis, sera from pigs immunized with P64 and live cell vaccine strongly reacted with the 64 kDa protein. In contrast, the serum from nonimmunized pigs did not react with any proteins. From these results, it was suggested that a specific antibody against the 64 kDa protein could be increased in pigs immunized with P64 or live cell vaccine and that this anti-P64 antibody has a strong protective effect against E. rhusiopathiae infection in pigs. U. S.

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Protective activity and antigenic analysis of fractions of culture filtrates of Erysipelothrix rhusiopathiae

Sato

Hirose

Saito

1995

Veterinary Microbiology

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Immunological characterization of protective antigens prepared by alkaline treatment of whole cells and from the culture filtrate of Erysipelothrix rhusiopathiae

Cited by 10 publications

References 13 publications

Characterization and Identification of a Novel Candidate Vaccine Protein through Systematic Analysis of Extracellular Proteins of Erysipelothrix rhusiopathiae

Characterization and Identification of a Novel Candidate Vaccine Protein through Systematic Analysis of Extracellular Proteins of Erysipelothrix rhusiopathiae

Protective Activity of the Purified Protein Antigen of Erysipelothrix rhusiopathiae in Pigs

Protective activity and antigenic analysis of fractions of culture filtrates of Erysipelothrix rhusiopathiae

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