ABSTRACT
Objective To observe clinical manifestations and immune responses in rabbit models of syphilis established by inoculation with Treponema pallidum through different routes. Methods A total of 20 rabbits were randomly and equally divided into two groups: testis-inoculated group injected with Treponema pallidum (Tp) suspensions into the testes, back-inoculated group intracutaneously injected with Tp suspensions at six sites on the shaved back. All the rabbits received a single injection with the total injection amount of Tp (Nichols strains)being 2 × 107 in both groups. Clinical symptoms and immune responses were evaluated in both groups every other day. Statistical analysis was carried out by a two-sample t-test and rank sum test with the SPSS 20.0 software. Results On day 8 after the inoculation, the testes of rabbits in the testis-inoculated group started to swell with induration, and the swelling was most severe during days 13 - 16. Afterwards, the testes gradually diminished, and returned to normal in size without swelling or hardening on day 28. No skin lesions occurred in the other sites in the testis-inoculated group within 2 months after the inoculation. Erythematous swelling occurred at inoculation sites on day 7 after inoculation in the back-inoculated group, which was most obvious between days 15 and 45. Moreover, cartilage-like indurated ulcers were observed at 12 inoculation sites in 3 rabbits in the back-inoculated group, and dark-field examination of the ulcers showed the presence of Tp. There was a significant difference in the time required for the severity of lesions to peak between the testis-inoculated group and back-inoculated group (14.50 ± 1.08 days vs. 29.00 ± 10.30 days, t=5.02, P<0.05). Rank sum test showed significant differences in the distribution of highest RPR titers between the two groups(P<0.05), and RPR titers were higher in the testis-inoculated group than in the back-inoculated group. Conclusions The injection of Tp suspensions at different sites can cause different clinical symptoms and immune responses in rabbits. RPR turned positive earlier with a higher titer in the testis-inoculated group compared with the back-inoculated group. The clinical manifestations in the back-inoculated group were similar to those of chancre in primary syphilis in human.
ABSTRACT
ObjectiveTo clone and express the Tp0136 gene of Treponema pallidum,to purify the recombinant protein and to evaluate its immunocompetence.MethodsThe full-length Tp0136 gene was synthesized,then subcloned into the expression vector pET28a to construct a recombinant plasmid,pET28a-Tp0136,which was subsequently transfected into E.coli Rosetta for protein expression.The recombinant protein was purified with nickel-nitrilotriacetic acid(Ni-NTA) affinity chromatography,and identified by using sodium dodecyl sulfate polyacrylamide gel electropheresis (SDS-PAGE) and Western blot.New Zealand rabbits were immunized with the recombinant Tp0136 (rTp0136)protein,and anti-Tp0136 polyclonal antibodies in sera of the rabbits were examined by indirect enzyme linked immunosorbent assay(ELISA) with rTp0136 protein as the coating antigen.Also,positive sera were obtained from patients with syphilis and examined by Western blot to identify the immunoreactivity of the rTp0136 protein.ResultsThe E.coli expression vector pET28a-Tp0136 was constructed successfully,and rTp0136 protein was also successfully expressed with a molecular weight of about 50 kD and a purity above 95％.High titres of anti-rTp0136 antibodies were detected in sera of rabbits immunized with the rTp0136 protein,and Western blot showed that the rTp0136 could specifically react with the sera from syphilitic patients,which proved the high immunogenicity and immunoreactivity of the recombinant protein.ConclusionsThe full-length Tp0136 membrane protein is successfully expressed with a high immunocompetence,and Tp0136 membrane protein may play an important role in the pathogenic mechanisms of Treponema pallidum.