MOLECULAR CHARACTERIZATION OF CAPRINE MYCOPLASMA ISOLATES
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Date
2010
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AAU, Anand
Abstract
Mycoplasmas belongs to the class Mollicutes and are among the smallest freeliving microorganisms capable of auto replication. Mycoplasmas are highly fastidious bacteria, difficult to culture and slow growing. Many species are important veterinary pathogens causing respiratory infection, mastitis, conjunctivitis, arthritis, and occasionally abortion. M mycoides subsp. capri (Mmc), M. capricolum subsp. capricolum (Mcc) and M. capricolum subsp. capripneumoniae (Mccp) are some of the most virulent species that have been associated with caprine respiratory disease which all are belongs to a group of very closely related mycoplasmas known as the "M mycoides cluster," which also includes 2 other pathogens of ruminants: M. mycoides subsp. mycoides SC (MmmSC), and the bovine group 7. The present work was carried out with the objective of characterization of ninteen isolates of M. mycoides subsp. capri and eleven isolates of M. capricolum subsp. capricolum (which are being maintained at Department of Veterinary Microbiology, College of Veterinary Science and A. H., Anand were isolated from healthy and infected goats of Gujarat state). Preliminary confirmation of the mycoplasma isolates by slide agglutination test, molecular characterization of the mycoplasma isolates by PCR and PCR- RFLP profiling, in vitro detection of virulence associated genes and characterization of membrane protein by one-dimensional SDS-PAGE of mycoplasma isolates was carried out during the present work. Revival of the mycoplasma isolates was done on Modified Hank's Balanced salt solution liquid ( MBHS-L) medium & Modified Hank's Balanced salt solution agar ( MBHS-A) medium. For preliminary confirmation of mycoplasma isolates slide agglutination test were employed. Identification of Mycoplasma isolates by PCR was carried out using mycoplasma genus specific primer pairs viz., (GPO-l(F)/ MGSO(R)), which yielded an amplified products of 715 bp for all the (30) isolates of mycoplasma. Identification of mycoplasma isolates was also carried out by PCR using M. mycoides subsp. capri and M. capricolum subsp. capricolum specific primers (CAP-21(F)/ CAP-21(R)), which yielded an amplified products of 1225 bp for all the (30) isolates of mycoplasma and also further restriction enzyme Asel and Sspl were used for characterization of the amplified products.. Detection of virulence associated LppA gene in M. mycoides subsp. capri was carried out by PCR using LppA gene specific primers (MMMLC2(F) and MMMLCl(R)). No amplified product could be detected in all the isolates (19) of M. mycoides subsp. capri. Detection of virulence associated LppA gene in M. mycoides subsp. capricolum was carried out by PCR using LppA gene specific primers (MCCPLl(F) and MCCPLl(R)), which yielded as expected 1356 bp long sequence of the LppA gene for all the isolates (11) of M mycoides subsp. capricolum. Detection of virulence associated GlpO gene was carried out by PCR using GlpO gene specific primers (GlpO(F) and GlpO(R)) of the M. mycoides subsp. capri, yielded as expected 1189 bp long sequence of the GlpO gene for all the isolates (19) of M. mycoides subsp. capri. Further digestion of the 1189 bp amplicons with the enzyme HpaI resulted in 2 fragments, 652 bp and 537 bp, while with the enzyme EcoAll resulted in 2 fragments, 826 bp and 363 bp, and with the enzyme Bpil resulted in 2 fragments, 941 bp and 248 bp. GlpO enzyme may play a central role in the virulence of M mycoides subsp. capri by the fact that the H2O2 and reactive oxygen species (ROS) act as powerful mediators for cell injury directly by inflammatory processes or by inducing pro-inflammatory genes. Characterization of the membrane protein was carried out by one-dimensional SDS-PAGE, yielded 6 band pattern for M. mycoides subsp. capri and 5 band pattern for M. mycoides subsp. capricolum. A band with 62 kDa is considered to be the lipoprotein A for M. mycoides subsp. capri and band with 58 kDa is considered to be the lipoprotein A for M. capricolum subsp. capricolum. The lipoprotein is reported to have role in pathogenesis and immune response against the mycoplasmal agents.
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VERTINARY MICROBIOLOGY, CHARACTERIZATION