Isolation and molecular characterization of mycoplasma gallisepticum from domestic poultry in Şanliurfa region

This study was conducted to isolate Mycoplasma gallisepticum (M. gallisepticum) (MG), which is the aetiologic agent of Chronic Respiratory Disease) (CRD) in domestic fowls, molecularly confirm of this agent with polymerase chain reaction (PCR), perform phylogenetic analysis by sequencing and restriction fragment length polymorphism (RFLP) of amplified regions of mgc2 gene of the isolated strains, compare protein profiles of these isolates with reference M. gallisepticum S6 and M. gallisepticum 6/85 vaccine strains and to detect specific antibodies produced against CRD by Western blotting (WB) in Şanlıurfa and its surroundings. Tracheal swabs and tissue samples which were belonged to 120 domestic birds were used to isolate of the agent in this work. Seven M. gallisepticum were isolated from 120 samples (5.8%). Three of the samples were isolated from chicken while four of them from turkeys. PCR analysis was positive for 65 (54.2%) samples for M. gallisepticum specific DNA. Thirty out of 62 (48.3%) chicken and 35 out of 58 (60.3%) turkeys were found positive by PCR with the species-specific primers. Forty two out of 84 (50%) tracheal swabs and 23 out of 36(63.9%) were positive by species specific PCR. PCR amplicons detected between 750-800 bp from Mgc2F and mgc2R target gene regions were 714 bp, 782bp, 721 bp, 716 bp, 716 bp, 712 bp, 782 bp, 718 bp and 787 bp for MG4, MG79, MG9, MG68, MG95, MG120, MG66, M. gallisepticum 6/85, and M. gallisepticum S6, strains, respectively. PCR products digested with AluI, HaeII, HinfI Restriction enzymes (RE) gave two different PCR-RFLP types. While most of the MG4, MG9, MG68, MG95 and MG120 isolates showed similar PCR-RFLP profile, MG66 and MG79 isolates gave different band profile. M. gallisepticum 6/85 vaccine strain showed quite high similar RFLP band profiles to MG4, MG9, MG68, MG95, and MG120 field isolates. On the other hand, M. gallisepticum S6 reference strain was found to have highly different digestion pattern. Phylogenetic analysis results of mgc2 gen region constituted two groups; 5 field isolates that are similar with each other (Group 1: MG4, MG9, MG68, MG95, MG120) and 2 similar field isolates (Group 2: MG66, MG79). Field isolates in Group 1 and 2 showed 96-99% similarity with those of South Africa and Brazil. This similarity was found between 96-100% with the isolates of India, America and Australia. Besides, group 1 and 2 isolates showed similarity to 6/85 vaccine strain, TS-11 vaccine strain and M. gallisepticum S6 with the percentages of 99.5-97.3%, 89-90-100%, %96-96.5 -97.2%, respectively. On the other hand, deletion of 63 nucleotides in base sequences of mgc2 gene was determined in only M. gallisepticum 6/85 and group 1 isolates. According to protein analysis of isolates by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), 120, 100, 70, 64, 67, 56, 53, 45, 43, 26 kDa molecular weight bands were clearly observed in all isolates of group 1 and group 2. Being different from others, MG68, MG95 and isolates in group 2 showed 28 kDa protein band. Also, 15 kDa protein band was observed in Group 2 isolates, which was not found in others. All the expected protein bands for M. gallisepticum 6/85 vaccine strain and reference M. gallisepticum S6 strains were observed by SDS-PAGE analysis. Antibodies against M. gallisepticum specific proteins were detected by WB analysis in all the isolates of group 1 and 2 at the molecular weight of 200, 120, 100, 98, 67, 64, 40, 35, 26 kDa. Group 1 isolates also showed 56 kDa protein band apart from rest of the isolates. Although using polyclonal sera for WB analysis might give rise to differentiation of some M. gallisepticum isolates antigenically, we predicted that using monoclonal antibodies could give more specific discrimination in terms of antigenicity and immunogenicity in WB analysis. At the end of study, we obtained valuable preliminary epidemiological data related to the presence of M. gallisepticum infection in domestic fowls manifesting respiratory problems in Şanlıurfa and its vicinity. We concluded that molecular tools can be used to differentiate between field and vaccine strains and these techniques can also be benefited to reveal some discrimination in field isolates. More work on different genes of M. gallisepticum for more specific discrimination between field isolates are needed with available data. As conclusion, our results will be effective to enhance the diagnosis and monitoring of CRD in the region. Besides, naturally infected flocks could be differentiated from vaccinated flocks, which is a very effective control and monitoring way of infection.