Molecular and Immunological Characterization of Mycobacteria Associated with Bovine Farcy

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Victor Loku Kwajok, Kwajok
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The ability of delineate taxo-species has a profound influence in bacteriology, notably identification and classification of pathogenic bacteria. However, improved methods and automated data acquisition system can be expected to facilitate generation of high-quality data bases for variety of purposes. The aim of the study was to isolate and identify Mycobacterium farcinogenese from clinical samples (lymph nods and serum), to characterize these species including M. senegalense and the related taxa (M. chelonae, M. fortuitum, M. peregrimum and Nocardia farcinica) using molecular biology methods (DNA extraction, PCR amplification, restriction fragment length polymorphism determination using restriction enzymes and DNA sequencing) and immunological analysis of the species (animal pathogenicity tests, ELISA using serum samples from the clinical cases, protein antigen bands determination using SDS-PAGE method, and antigen-antibodies immunoassay using Western blotting and immunodiffusion tests. Seventeen clinical isolates identified as M. farcinogenese were obtained from 578 lymph nodes and 36 positive serum samples of the two 269 sera samples which were tested. Taxonomic criteria derived from the application of morphological, enzymatic and chemotaxonomic methods. DNA extraction method gave clearly resolved bands on agarose gel electrophoresis with clear common bands of 1500 base pairs. The extracted DNA was used as template for PCR amplification with universal primer 27f (5’AGAGTTTGATCCGGCTCAG-3’) and primer 1525r (5’AAGGAGGTGATCGAGCC-3’) with appended restriction sites being ideal primers for amplification. No significant difference in the DNA fingerprints was found, indicating that DNA fingerprints of the farcy agents were reproducible over successive generation and were in line with their placement in the genus Mycobacterium PCR-DNA fingerprinting using BamHI restriction enzymes for restriction fragment length polymorphism analysis a means for differentiating between M. farcinogenese and M. senegalense. The 16SrDNA sequencing of M. farcinogenese and M. senegalense the farcy sole agents, gave data of variable signals with 1482 nucleotides with 65 corresponding almost complete nucleotide sequences in 1404 positions. Manual alignment of the sequenced DNA computer, was clear and showed 16SrDNA similarity values of 99.8-99.9%, which corresponded to 7 and 12 nucleotide differences. Immunoassay of the protein antigens of the test strains of M. farcinogenese and the anti sera produced from the laboratory animals-guinea pigs gave positive results. M. farcinogenese isolates produced approximately similar profiles of protein bands.
Molecular and Immunological Characterization of Mycobacteria Associated with Bovine