Mycoplasma bovis is associated with a large number of disease conditions viz. mastitis, arthritis, calf pneumonia, infectious kerato-conjunctivitis and genital disorders in bovines (Jasper, 1977; Bocklisch et al., 1986; Howard et al., 1987 and Gunning and Shephered, 1996). Due to lack of effective vaccines and antimicrobial therapy, M. bovis infections remain a significant problem resulting in extensive economic losses to cattle industry. The routine diagnostic procedures viz. isolation of the agent and serological tests are tedious and time consuming. Moreover, extreme serological cross-reactions are also observed with other mycoplasmas, particularly the M. agalactiae (Simecka et al., 1992). In recent years, the PCR has been employed as a suitable assay for specific detection of M. bovis, even from chronic and subclinical cases with an additional advantage of its differentiation from M. agalactiae, which is difficult by conventional tests (Johansson et al., 1996). The present study was undertaken to defferentiate M. bovis strains from other very closely related mycoplasmas by species specific PCR assay.

Ten strains of Mycoplasma spp.and one strain of Acholeplasma laidlawii used in this study were obtained from National Referra l Laboratory on Mycoplasma, Division of Bacteriology & Mycology, IVRI, Izatnagar (Table 1). The specially enriched B medium (Ernφ and Stipkovits, 1973) was used for the growth of M. bovis, M. bovirhinis, M. bovigenitalium and M. alkalescens strains, whereas, the strains of M. mycoides subsp . mycoides SC , M. sp. bovine group 7 , M. agalactiae and Acholeplasma laidlawii were grown in MBHS-L liquid medium (Carmichael et al., 1972). The genomic DNA from each of the strains was extracted as described earlier (Wilson, 1987), the concentration has been determined by taking O.D. at 260 nm and then diluted in autoclaved distilled water to have 10 ng of DNA/μl.

PCR was carried out using M. bovis specific primers MboF2[5′-GAAGAAAAGTAGCATAGGAAATGAT -3′] and MboR2[5′-CGTCGTCCCCACCTTCCTCCCG-3′] (Johansson et al., 1996). A 20 ng of genomic DNA of each strain was used as a template in 50 μl reaction mixture containing 10 mM Tris-HCl, pH 8.8; 50 mM KCl; 1.5 mM MgCl ₂ ; 0.08% nonidet P40; 200 μM dNTP mixture, 20 p mole of each primer (MboF2 and Mbo R2) and 1 unit of Taq DNA polymerase (MBI Fermentas). The reaction mixture was subjected to an initial denaturation at 94°C for 2 minutes followed by 30 cycles of amplification with 94°C (denaturation) for 45 seconds, 65°C (annealing) for 1 minute and 72°C (extension) for 1 minute. Final extension was carried out at 72°C for 5 minutes.

Another PCR was carried out using a different set of M. bovis specific primers viz. PpMB 920-1 [5′-GGCTCTCATTAAGAATGTC-3′] and PpMB 920-2[5′-TTTTAGCTCTTTTTGAACAAAT-3′] (Hotzel et al., 1993 & 1996). The reaction mixture similar to that of previous PCR was prepared along with the primers i.e. PpMB 920-1 and PpMB 920-2 while the programme was carried out with initial denaturation of 94°C for 2 minutes; 30 cycles of denaturation (94°C for 30 seconds), primer annealing (48°C for 45 seconds) and primer extension (72°C for 90 seconds) followed by final extension at 72°C for 5 minutes.

The PCR products were analysed by agarose gel electrophoresis on 1.4% agarose gel ethidium bromide (0.3 μg/ml). A 10 μl of each PCR product was run separately along with the suitable DNA marker at 7 volts/cm for 1 hour 30 minute in 0.5 X TBE buffer (45 mM Tris, 45 mM boric acid and 2 mM EDTA, pH 8.0). The gel was visualized and photographed under UV gel documentation system.

Both the strains of M. bovis viz. NC 317 and 23 K showed the amplified product (734 bp) when screened by MboF2 and MboR2 primers while the other nine strains of bovine mycoplasma and acholeplasma failed to (Fig. 1). Johansson et al. (1996) reported similar results using type strains of 14 different Mycoplasma species viz.M. agalactiae, M. bovis, M. arginini, M. bovigenitalium, M. bovoculi, M. californicum, M. canadense, M. capricolum subsp . capripneumoniae, M. fermentans, M. meleagridis, M. mycoides subsp.mycoides LC type, M. mycoides subsp.mycoides SC type, M. ovipneumoniae and M. putrefaciens. However, the other PCR assay using another set of primers PpMB 920-1 and PpMB 920-2 yielded an amplified product of 2kbp only in both the strains of M. bovis (NC 317 and 23 K) without any cross-amplification with other nine strains of mycoplasma and acholeplasma (Fig. 2). These results support the findings of Hotzel et al. (1993 and 1996) who detected similar amplified product in M. bovis strain which was isolated from nasal swab and cow milk.

The present findings of both the PCR assay also revealed no cross reactivity even with very closely related M. agalactiae, although, M. bovis and M.agalactiae had only 8 nucleotide difference in 16S rRNA gene (Mattsson et al., 1994). So, both the M. bovis-specific PCR assay have the potential for specific detection of M. bovis in clinical samples such as milk, meat, nasal swabs and even contaminated field materials and slaughter house samples in a very short period of time without any cross-amplification with other mycoplasmas as compared to lengthy and cumbersome procedures involved in serological and biochemical tests.

References