Development Of Techniques For Detection Of Antibodies Against Mycoplasma Bovis Based On Recombinant Protein P48

Mycoplasma bovis is an important pathogen causing mastitis, pneumonia, arthritis, otitis media, keratitis and conjunctivitis in calves. It was generally estimated that M. bovis caused tremendous economic losses in the world cattle industry. Since the first report about infectious pneumonia caused by M. bovis in China was recorded in the year of 2008, M. bovis has been an increasinly important factor that threatens the Chinese cattle industry. However, there was no domestic comercialized reagents to diagnose M. bovis which hampers control of the disease so far. In this study, an indirect hemagglutination assay(IHA) and a colloidal gold-based immunochromatographic strip(ICS) based on recombinant protein P48 were developed for detection of antibodies against M. bovis and were evaluated for future filed application.The first part of this study was development of IHA and ICS to detect antibodies against M. bovis. According to a lot of foreign literature, the optimized p48 gene of M. bovis was synthesized and then cloned into expression vector pET32a(+). The recombinant plasmid pET32a(+)-p48 was transferred into Escherichia coli BL21(DE3) and a soluble recombinant protein P48 was then induced to express at 16℃. Western-blot showed that the purified recombinant protein P48 was well recognized by the hyperimmune serum against M. bovis.To establish an indirect hemagglutination assay(IHA), the purified recombinant protein P48 was injected into the sheep red blood cells(SRBC) treated with glutarldehyde and tannic acid. The optimum concentration of P48 antigen sensitizing SRBC was from 20 to 40 μg/mL, and the hyperimmune serum titer against M. bovis was up to 1:211. IHA was employed to detect the antisera to contagious bovine pleuropneumonia, bovine pasteurellosis, bovine viral diarrhea, brucellosis, bovine tuberculosis, foot-and-mouth disease, M.bovigenitalium infection, M.leachii infection and Escherichia coli infection, but all were negative. The analytical specificity sensitivity were 100% and 93.33%.The coincidence rate of IHA was 96.15% when compared with the commercialized M. bovis ELISA Kit(BioX). Detection of 833 field serum samples and 1084 milk samples with IHA revealed that the positive rates of antibodies to M. bovis were 15.37% and 19.56%, resepectively.The colloidal gold particles of 28.5nm was coated with 6 μg/mL purified recombinant protein P48 at pH7.4, and the conjugate pad was coated with the colloidal gold-labeled antigen solution. The test line and control line were coated with goat anti-bovine IgG(H+L) and rabbit anti-recombinant protein P48 IgG, respectively. Then the assembly of strip was established. The ICS possessed high specificity, because it did not cross-react with other positive serum to related pathogens as mentioned above. It had high sensitivity, the hyperimmune serum titer against M. bovis was up to 10-6. Compared with IHA, the positive coincidence rate was 93.57%, the negative coincidence rate was 93.02%, the average coincidence rate was 96.53%. Detection of 200 field serum samples and 108 milk samples with ICS revealed that the positive rates of antibodies to M. bovis were 21.30% and 17.59%, resepectively.In the second part of the study, according to guidelines of the 683 rd announcement from the Ministry of Agriculture, three batches of IHA reagents and three batches of ICS were produced in laboratory, and the sensitivity, specificity, reproductivity and stablity of IHA and ICS reagents were further evaluated for future commercialization development. The results showed that both reagents were good for detection of antibodies to M. bovis. It provided useful tools for seroprevalence investigation and helped to diagnose M. bovis infection.