[Identifying immunogenic proteins of Erysipelothrix rhusiopathiae C43065 by MALDI-TOF mass spectrometry and cloning their encoding genes].

OBJECTIVE: To identify immunogenic proteins of Erysipelothrix rhusiopathiae C43065. METHODS: Antigens were extracted from E. rhusiopathiae C43065 by the alkaline extraction method. Proteins in the NaOH-extracted antigen were separated by SDS-PAGE and transferred to nitrocellulose membranes, and then Western blotting was performed with rabbit antiserum against the NaOH-extracted antigens. The immunogenic protein bands were identified by MALDI-TOF mass spectrometry. The genes encoding 5 major immunogenic proteins was amplified by PCR from the genomic DNA of E. rhusiopathiae C43065, and inserted into the pMD18-T vector and then sequenced. RESULTS: A total of 9 immunogenic surface proteins in the NaOH-extracted antigen from E. rhusiopathiae C43065 were successfully identified by MALDI-TOF mass spectrometry. Four of the proteins were putative virulence-associated proteins: enolase, ATP-binding cassette transporter, glyceraldehyde-3 -phosphate dehydrogenase and fructose-bisphosphate aldolase class-II. The genes encoding the chaperone protein GroEL, enolase, ATP-binding cassette transporter, glyceraldehyde-3-phosphate dehydrogenase and fructose-bisphosphate aldolase class-II were 1614, 1296, 1260, 1005 and 867 bp in length, and the nucleotide sequences homologies of the genes between the C43065 strain and the previously reported E. rhusiopathiae Fujisawa strain was more than 98%. CONCLUSION: Several putative virulence-associated proteins in the NaOH-extracted antigen of E. rhusiopathiae C43065 will be useful for elucidating the roles of these proteins in the pathogenesis of the organism.

Role of surface protective antigen A in the pathogenesis of Erysipelothrix rhusiopathiae strain C43065.

To clarify the role of surface protective antigen A (SpaA) in the pathogenesis of Erysipelothrix rhusiopathiae C43065 (serotype 2), the spaA deletion mutant of E. rhusiopathiae ΔspaA was constructed by homologous recombination. The virulence of the ΔspaA mutant decreased more than 76-fold compared with that of the wild-type strain C43065 in mice. The mutant strain was sensitive to the bactericidal action of swine serum, whereas the wild-type strain was resistant. The adhesion of wild-type strain to MEF cells was inhibited significantly by treatment with rabbit antiserum against recombinant SpaA (rSpaA) as compared with the treatment with normal rabbit serum, but the mutant strain was not affected. The mutant strain was readily taken up by mouse peritoneal macrophages in the normal rabbit serum, whereas the wild-type strain was resistant. Whereas the rabbit antiserum against rSpaA promoted the phagocytosis of wild-type strain by macrophages, the mutant strain was not affected. In addition, mice vaccinated with the formalin-killed mutant strain were provided 40% protection against challenge by the homologous virulent strain as compared with those with wild-type strain, NaOH-extracted antigen, or rSpaA, which provided more than 80% protection against the same infection. These suggested that SpaA has an important role in the pathogenesis of E. rhusiopathiae infection and could be a target for vaccination against swine erysipelas.

[Construction and characterization of ompH gene knockout mutant of avian Pasteurella multocida C48-3].

OBJECTIVE: To study the role of the outer membrane protein H (OmpH) in pathogenicity of avian Pasteurella multocida. METHODS: The ompH knock-out mutant of avian P. multocida C48-3 was constructed by homologous recombination. The DNA replacement was confirmed by PCR, RT-PCR and Western blot. We compared the differences of biological characteristics such as growth rate, capsular structure, adhesion ability and virulence between the ompH knockout mutant of C48-3 Delta ompH and parent strain C48-3, as well as the complemented strain C48-3C. RESULTS: C48-3 Delta ompH was successfully constructed. Electron microscopy examination of C48-3 Delta ompH shows the absence of capsular material compared to the parent strain C48-3 and complemented strain C48-3C. The adhesion assay shows that the number of C48-3 Delta ompH adhered to CEF cells was significantly lower than that of C48-3 and C48-3C. C48-3 Delta ompH was relatively attenuated in mice by intraperitoneal injection. CONCLUSION: The construction of C48-3 Delta ompH would facilitate further study on pathogenesis of avian Pasteurella multocida.

[Cross-protective effect of the Cp39 adhesive proteins against avian Pasteurella multicida in mice].

OBJECTIVE: We evaluated the cross-protective effect of a 39-kDa adhesive protein (Cp39), recombinant adhesive protein (rCp39), and capsular proteins from in vitro and in vivo grown avian P. multocida in mice. METHODS: The Cp39 was purified by electroelution from capsular proteins of in vivo grown strain C48-3. The rCp39 was expressed in E. coli B21 as a soluble protein by IPTG inducing, and purified with pMAL protein fusion and purification system. Mice of each group were subcutaneously immunized twice with 100 microg of rCp39, Cp39 or capsular proteins from in vivo or in vitro grown strain C48-3 with in complete or incomplete Freund adjuvant at 2-week intervals. Five mice of each group were challenged with 100 LD50 of avian P. multocida strain C48-3 or rabbit P. multocida strain C51-3 two weeks after the second immunization, while the antibody response specific for rCp39 was determined by ELISA. RESULTS: SDS-PAGE indicated that the Cp39 protein was expressed in vitro and in vivo grown P. multocida. Mice immunized with rCp39, native Cp39 or capsular proteins from in vivo and in vitro grown strain C48-3 were protected completely against the challenge with the homologous strain C48-3, while 60 - 80% protection was demonstrated in the mice against the challenge with the heterologous strain C51-3. CONCLUSION: In conclusion, rCp39 is a cross immunoprotective antigen of P. multocida capsular serogroup A strains, and might be a useful vaccine candidate against fowl cholera.

[Immunity of native SpaA and recombinant SpaA-N against Erysipelothrix rhusiopathiae infection in mice].

OBJECTIVE: We evaluated the effects of surface protective antigen A (SpaA) and its N-teminal protective domain (rSpaA-N) against Erysipelothrix rhusiopathiae infection in mice. METHODS: The SpaA was purified by electroelution from NaOH-extracted antigen of Erysipelothrix rhusiopathiae strain C43311. The rSpaA-N was expressed in E. coli BL21 as a soluble protein by IPTG inducing, and purified with GST affinity chromatography. Mice of each group were subcutaneously immunized three times with 50 microg or 100 microg of native SpaA, rSpaA-N or NaOH-extracted antigen with in complete or incomplete Freund adjuvant at 2-week intervals. Five mice of each group were challenged with 100 LD50 of Erysipelothrix rhusiopathiae virulent strain C43065 two weeks after the third immunization, and the specific antibody responses for SpaA was determined by indirect ELISA. RESULTS: Mice immunized with 50 microg or 100 microg of native SpaA, rSpaA-N, or NaOH-extracted antigen were protected completely against the challenge with strain C43065. There was no significant difference (P < 0.05) between the antibody responses observed for native SpaA, rSpaA-N and NaOH-extracted antigen at any dosages. Western blot results indicated that the native SpaA and rSpaA-N were recognized specifically by an antiserum against the native SpaA of strain C43311. CONCLUSION: These results demonstrated that the rSpaA-N is a protective antigen of Erysipelothrix rhusiopathiae serotype 2 strains, and might be a useful vaccine candidate against swine erysipelas.

[Expression and purification of an adhesive protein of rabbit Pasteurella multocida C51-3 and detection of its antigenicity].

The cp36 gene encoding an adhesive protein was amplified by PCR from genomic DNA of rabbit P. multocida C51-3 strain, and cloned into the pMD18-T vector and then sequenced. The mature adhesive protein without a signal peptide of cpm36 gene was amplified by PCR from the recombinant plasmid pMD18-cp36, then cloned into the prokaryotic expression vector pQE30 to provide a recombinant plasmid pQE30-cpm36. The recombinant protein of CPM36 was produced in Escherichia coli M15 harboring the recombinant plasmid pQE30-cpm36 by IPTG induction, and the recombinant protein purified by the affinity chromatography with Ni(2+)-NTA resin. The sequence analyses showed that the ORF of cp36 gene was 1032 bp in length, and DNA homology of the cp36 genes between the C51-3 strain and the previously reported different serotype strains of P. multocida in GenBank was 76.9 to 100%. The SDS-PAGE analyses revealed a single fusion protein band with a molecular weight of 37 kD, and the Western blotting analysis demonstrated that the recombinant protein CPM36 and native 36 kD protein of C51-3 were recognized specifically by an antiserum against the recombinant protein, suggesting that the recombinant protein is an antigenic protein.

[Cloning and expression of N-terminal protective domain of spaA gene from Erysipelothrix rhusiopathiae C43311].

The spaA gene was amplified by PCR from the genomic DNA of Erysipelothrix rhusiopathiae C43311 strain, and inserted into the pMD18-T vector and then sequenced. The N-terminal protective domain of the spaA gene was amplified by PCR from the recombinant plasmid pMD18-spaA, then cloned into the prokaryotic expression vector pGEX-6p-2 and expressed in E. coli BL21 (DE3) by IPTG induction. The expressed protein was identified by SDS-PAGE and Western blot. The sequence analyses showed that the coding region of the spaA gene of C43311 strain was 1881bp in length, and the nucleotide sequence homology of the spaA genes between the C43311 strain and the previously reported different serotype strains of E. rhusiopathiae was 93 to 99%. The SDS-PAGE analyses revealed a single fusion protein band with a molecular weight of 64kDa, and the Western blot results showed that the GST-SpaA-N fusion protein was recognized specifically by an antiserum against the SpaA protein of C43311 strain, suggesting that the fusion protein of GST-SpaA-N possessed high immunoreactivity.