Research Of Molecular Diagnosis And Immunity Of Clostridium Perfringens

Clostridium perfringens (C. perfringens) is a Gram positive, spore-forming and rod-shaped anaerobe. It can cause a variety of toxic-specific lesions and gastrointestinal disease in domestic and wild animals as well as in humans under certain conditions. The organism is grouped into five types (type A, B, C, D and E) on the basis of the production of major four toxins (a-toxin,β-toxin, e-toxin,ι-toxin). Since pathogenicity correlate with the major toxins elaborated by the organism, typing of the bacterium as one of five types (type A-E) according to major toxin production has diagnostic and epidemiological significance. In the study, a C. perfringens strain was isolated and identified from a livestock with acute death by microscope, serum neutralization test, biochemical tests and PCR. The multiplex PCR, real-time quantitative PCR (qPCR) and monoclonal antibody sandwich ELISA (McAb-S ELISA) to detect toxins of C. perfringens were established and applied, of which estsblishments of the qPCR and McAb-S ELISA were firstly reported in China. Detection of C. perfringens genotypes in feces of healthy dairy cattle using qPCR is the first time applied in China. In addition, a non-cytotoxic epsilon (e) toxin mutant of C. perfringens was firstly constructed by oligonucleotide-mediated mutagenesis, and immune response of mice to mutated toxin was observated. The major research results were presented below:(1) A bacterium strain was isolated from a acutely dead cow, the bacteria growing quickly under anaerobic conditions, and it can produce acid and gas. In blood agar medium seeded with the isolate, colonies showed round, smooth, jagged edges and formed double-hemolytic loops. Finally, the bacterium was identified as C. perfringens type A by biochemical tests, serum neutralization test and PCR.(2) A multiplex PCR assay was developed to detect the alpha, beta, epsilon, and iota toxin genes of C. perfringens. Both the specificity and sensitivity of the PCR is higher. Only C. perfringens showed positive but other Clostridium species, Escherichia coli, Staphylococcus sp all showed negative in the PCR tests. The average sensitivity of assay was 1.2×103 CFU/ml using the PCR to determine broth bacterium samples. The method can be used to detect and type C. perfringens from feces and intestinal contents of infected animal. It is a reliable method for the clinical diagnosis and epidemiological investigation of C. perfringens.(3) Three strain monoclonal antibodies (McAbs) against a toxin, entitled 5A4,3H4 and 5H4, respectely, were produced by fusing between SP2/0 myeloma cells and spleen cells of BALB/c mice immunized with a toxin. Among the three McAbs,5A4 and 3H4 belonged to IgG1 subtype, 5H4 belonged to IgG2a subtype and they all have good stability. A sandwich ELISA for detection of a toxin was developed by using McAb 5A4 as coating antibody and rabbit polyclonal antiserum as detector antibody.(4) A real-time quantitative PCR assay was established for detection of toxin genes of C. perfringens directly from livestock's feces. Fecal samples from 261 lactating dairy cattle in Ningxia of China were examined using the multiplex assay. The results show that 176 of the 261 samples were positive for C. perfringens, and only C. perfringens type A and type D were isolated from the feces of clinic healthy dairy cattle. Of the 176 PCR positive feces samples,142 (80.7%) were positive for cpb2 gene. It is the first study about molecular typing of C. perfringens using real-time multiplex PCR assay in dairy cattle herd in China.(5) e-toxin from C. perfringens was cloned and expressed, and a e-toxin mutant of C. perfringens was constructed by oligonucleotide-mediated mutagenesis. The mutated protein expressed in Escherichia coli were recognized in ELISA test by native e-toxin antibody. Cytotoxicity of the mutated toxin to MDCK cells was assayed in vitro, and the test result revealed that the expressed e-toxin mutant protein was non-cytotoxic for MDCK cells. Immunization of mice with the mutated e-toxin resulted in the induction of a specific antibody response and immunized mice were protected against 1000 LD5o doses of wild-type e-toxin which indicated that the expressed e-toxin mutant protein better preserved immunogenicity of the native e-toxin.