Title:Examination of Mycoplasma bovis infection in cattle
In Chapter 1 the usage of capture ELISA test and a selective differentiating medium in the diagnostics of the Mycoplasma bovis infection is presented. Out of 52 strains isolated from 510 various clinical specimens 43 were proven to be M. bovis by both culturing and capture ELISA. Out of 92 lung specimens 15 mycoplasma strains were isolated. All of them were identified as M. bovis by the above mentioned diagnostic methods. Samples from M. bovis challenged animals were examined by culturing, selective culturing and capture ELISA. The latter two methods have been proven to be rapid, highly specific and useful in the diagnostics of Mycoplasma bovis infection of cattle. Both capture ELISA and selective-differentiating medium give the same result as the time consuming conventional culturing of this organism. In Chapter 2 the production and testing of monoclonal antibodies against Mycoplasma bovis is discussed. To produce monoclonal antibodies, Balb/c AnN Crl BR mice were inoculated with the cell suspension of a Hungarian Mycoplasma bovis strain designated 26034. Three days after the last immunisation the spleen of the immunised mouse was removed aseptically. The fusion of spleen cells with Sp2/0-Ag14 murine myeloma cells was performed in the presence of polyethylene glycol. The obtained hybrid cells were selected with HAT medium. Two weeks after the fusion the supernatants of the cells grown were tested by an own-developed indirect ELISA. The results showed that 63 antibody-producing hybridomas had been obtained. For accurate determination of the molecular weight of antigen determinants, the supernatants giving positive reaction in the ELISA were tested by Western blotting. According to the results, the obtained monoclonal antibodies recognise the antigen determinants of the following molecular weights: 1B11: 63 kDa, 1C7: 63 kDa, 2C5: 22, 25 and 27 kDa, 2C9: 69 kDa, 3G12: 67, 69 and 72 kDa, 4H9: 63 kDa, 5B8: 22, 25 and 27 kDa, 5D3: 22, 25 and 27 kDa, 5C11: 69 kDa, 5E5: 22, 25 and 27 kDa, 6F11: 63 kDa, and 6H10: 22, 25 and 27 kDa. The 12 cell groups selected on the basis of the Western blotting were cloned twice by end-point dilution method. The cloned cells were propagated, and with 5 cell lines antibodies were produced in the CELLine bioreactor. Cell line 3G12 showed the highest productivity with an average daily output of 1.5 mg immunoglobulin. Cell line 5E5 produced 1.1 mg, 6H10 0.8 mg, 2C9 0.47 mg and 6F11 0.4 mg antibody per day. The isotype of the antibodies was determined by ELISA. The antibodies produced by the 12 cell lines tested were assigned to the IgG1 subclass according to the heavy chain. Ten cell lines produced κ and two produced λ light-chain antibody. Possible crossreactions of the produced monoclonal anti-M. bovis antibodies with certain Mycoplasma, Ureaplasma and Acholeplasma species were tested by an indirect ELISA procedure. All of the 12 antibodies tested gave a reaction with the antigen of M. bovis strain designated 26034. Monoclonal antibodies 3G12 (67, 69, 72 kDa) and 5B8 (22, 25, 27 kDa) gave no cross-reaction with antigens other than strains of the homologous Mycoplasma species. The other antibodies reacted with the M. bovigenitalium F7, M. sp. 8389, M. oculi and M. gallisepticum S6 antigens. Owing to its high specificity and affinity, primarily the antibody produced by cell line 3G12 is considered suitable for use in immunodiagnostic tests of M. bovis infections. The produced antibodies were also tested with immunohystochemical method. These examinations demonstrated that the antibodies 6H10, 6F11 and 4H9 are suitable for the in situ detection of the M. bovis antigen. In Chapter 3 the prevalence of Mycoplasma bovis and the evaluation of its pathogenic role is discussed by application of a mathematical model. Thirty four large cattle herds were screened for the presence of Mycoplasma bovis infection by examination of cattle slaughtered at slaughterhouse for pneumonic lesions in the lungs, culturing of M. bovis from lung lesions and testing sera for presence of antibodies against M. bovis. A statistical model was developed, which confirmed the relationship between these 3 parameters. Among 595 examined cattle, 33.9% had pneumonic lesions, Mycoplasmas were isolated from 59.9% of pneumonic lung samples, only 10.9% of sera from those animals contained M. bovis antibodies. In 25.2% of 8 cases mycoplasmas were isolated from lungs with no macroscopic lesions. The average seropositivity rate of individuals was 11.3%, however in certain herds it reached more than 50%. The proportion of seropositive herds was 64.7%. Comprehensive associations were found between serological responses against M. bovis and the observed lung lesions with the help of statistical calculations. In Chapter 4 the efficacy of valnemulin is presented in an experimental challenge trial. Mycoplasma bovis infection was experimentally induced in groups of 6 young calves. A further group was uninfected and served as a control. Ten days after infection, medication with either enrofloxacin (Baytril, Bayer) or valnemulin (Econor, Novartis) was instituted via the milk replacer for a further 10 days, after which all calves were killed. Infection resulted in depression, pyrexia, inappetance and prominent respiratory signs. Arthritis occurred in 2 animals, and 2 (unmedicated) animals died. At post mortem examination extensive lesions were present in the lungs and M. bovis was reisolated from infected unmedicated calves’ lungs. Medication with either enrofloxacin or valnemulin resulted in a rapid diminution of clinical signs, restoration of appetite and reversal of weight loss. Isolation of Pasteurella multocida from the calves’ lungs was suppressed by both medicaments. Valnemulin resulted in a more rapid reduction of clinical scores, and eliminated M. bovis from the lungs more effectively than enrofloxacin. In Chapter 5 development and application of an improved PCR system is presented. This system was developed using the forward primer described by Ghadersohi et al (1997) and a new reverse primer Mbr2 based on the Vsp gene region of Mycoplasma bovis, since both the original system and its further developed semi-nested variant (Hayman et al., 2003) do not work. The PCR did not amplify the pathogenic and ubiquitous mycoplasmas as well as bacteria commonly occurring in bovine respiratory and mammary tract. The assay detected as low as 150 CFU/ml of Mycoplasma bovis in broth culture enabling the diagnostic use of it with high sensitivity.