| Bordetella bronchiseptica (Bb) is an important pathogenic bacterium and results in acute and chronic respiration system disease. Bb is found to infect numerous mammals, such as swine, canine, rabbits, guinea, pig ferret, koala bear, et al. Human beings are also infected by Bb, especially children and immunocompromised individuals. With the development of rabbitry in our country, B.bronchiseptica infection and co-infections with Pasteurella multocida and Staphylococcus et.al are increased notably. It has been an important disease which caused significant mortality and morbidity in rabbits and led to vast economic losses in rabbit industry in China.This study includes three parts. The first is the establishment of ELISA method, the second is comparison of IHA, SAT and AGID for detection antibody against fimbrial crude extract of Bordetella bronchiseptica, the third is the establishment of PCR mehod. The results are:1.An indirect enzyme-linked immunosorbent assay (ELISA) method was developed to detect rabbit antibody against Bordetella bronchiseptica. The outer membrane protein (OMP) of B.bronchiseptica was collected by ultrasonic treatment and ultracentrifugation to remove cell bodies. The optimal antigen dilution and serum dilution were further examined as following, the final concentration of the antigen was 14.44μg/mL and the dilution of the serum sample was 1:400. It proved that the method possessed high specificity to Bordetella bronchiseptica (Bb). There was no detectable crossreactivity between OMP and antisera to other pathogenic microbes of rabbits. The established ELISA demonstrated a sensitivity 33-fold greater than MAT. The indirect ELISA was sensitive, specific, rapid, simple, accurate and reproducible, and is capable to identify animals infected with or exposed to Bordetella bronchiseptica. Therefore, it can be a valuable diagnostic tool in both clinical diagnosis and research.2.The fimbrial crude extract of Bordetella bronchiseptica was prepared by subjecting the bacterium to TritonX-100 and ultrasonic treatment. 458 rabbit serum samples were collected to detected antibody against fimbrial crude extract of Bordetella bronchiseptica by agar-gel immunodiffusion test (AGID), slide agglutination test (SAT) and indirect hemagglutination test (IHA). The results of the three assays were compared, and the positive ratio of IHA, SAT and AGID was 73.36%, 63.54% and 61.79% respectively. The correlation between IHA and AGID, IHA and SAT, AGID and SAT was 90.17%, 86.68% and 89.08% respectively. The results showed that IHA was specific and sensitive for the antibody detection of Bordetella bronchiseptica in rabbits.3.With a pair of primers designed to amplify fimbrial gene of Bordetella bronchiseptica according to the sequence of the gene, a fragment of 425bp in length on the target gene was amplified by PCR and a standard PCR assay was developed for quick detection of Bordetella bronchiseptica infection. Comparing the DNA sequence of the amplified fragment with the published sequence of gene fim2 showed that they were 100% homologous. Specificity and sensitivity assays revealed that the assay did not cross react with Escherichia coli, Pasteurella multocida, Clostridieum welchii and Staphylococcus aureus. 146 samples of nasal mucus from rabbits from Jiangsu and Shandong province were examined by the developed PCR assay. 92 samples were positive and the positive rates were 63.01%.In summary, this test extracted the outer membrane protein (OMP) of B. bronchiseptica to establish indirect enzyme-linked immunosorbent assay (ELISA) method and optimized the best working codition, which contributed to the research of indirect ELISA detection kit. The establishing of three kinds of method to detect rabbit antibody against fimbrial crude extract of Bordetella bronchiseptica paved the way for the antibody detection and epidemiological survey. The fim2 gene was amplified by PCR and a standard PCR assay was developed for quick detection of Boedetella bronchiseptica infection.
|
PDF Full Text Request