Molecular characterization of Mycoplasma gallisepticum strains from South African poultry

[摘要] English: Mycoplasma gallisepticum is the most pathogenic avian Mycoplasma species and leads to great economic losses worldwide (Kleven, 2003). Prevention and control of this organism has been achieved by vaccination and antibiotic administration. Ferraz and Danelli (2003) reported on the most widely used live vaccines which are MG-F, Ts-11 and 6/85. The MG-F strain is currently not registered for use in South Africa. M. imitans is the most closely related organism to M. gallisepticum and was tentatively identified as M. gallisepticum until Bradbury and co-workers (1993) defined it as a different species. However, this species is still misidentified as M. gallisepticum due to serological cross-reactivity and M. gallisepticum specific primers also detecting M. imitans (Markham et al., 1999). While M. gallisepticum has been characterized in some countries, very little information is documented on the South African isolates. It therefore became the aim of this study to investigate the presence and diversity of this organism in Southern Africa, investigate the presence of M. imitans as well as to establish the relatedness of the Southern African strains to those isolated outside Africa. Samples were collected from serologically positive birds from different farms in South Africa and Zimbabwe. Two PCR (Polymerase Chain Reaction) assays were optimized, one to detect M. gallisepticum of a specific gene Mgc2, the other to detect both M. gallisepticum and M. imitans. A total of five samples were detected with the Mgc2-PCR, while almost all samples could be detected with the 16S rRNA gene-PCR. This is a possible indication of a different M. gallisepticum isolate that can be detected on the 16S rRNA gene level but not at the Mgc2 level, or the isolate could indeed be M. imitans. In a study by Woese and co-workers (1985), the Mgc2 gene was implicated as one of the rapidly mutating genes due to adaptation, a possible reason for the non- detection of Mgc2 but the detection of the 16S rRNA gene. Of the sequenced 16S rRNA gene-PCR amplicons, M. gallisepticum and M. imitans had the highest homology. However, there are only two complete sequences of the 16S rRNA genes that belong to two M. gallisepticum strains deposited in Genbank, thus limiting the information on the isolates detected. Restriction Fragment Length Polymorphisms (RFLPs) were performed and well optimized. Ts-11 gave the expected profile while tested samples could not be placed in any of the reference groups. It was observed, however, that the RFLP profile of one of the amplicons was similar to the 6/85 vaccine strain and subsequent results correlated with this finding. Two of the amplicons could be sequenced and further analyzed. A phylogenetic tree showed one of the amplicons clustering away from the other Mycoplasma species though its sequence was found to be that of M. gallisepticum or M. imitans. In another tree, one of the amplicons showed more homology to the pathogenic strains while the other showed more homology to the vaccine strain 6/85. The DGGE analyses showed that the amplicons consist of a mixed template which was the reason why some samples could not be properly sequenced. This might be an indication of mixed infection within the flocks. However, it was expected of the 16S rRNA to give these results. Furthermore, the DGGE results indicated that the vaccine strain Ts-11 is used to vaccinate some of the flocks, while other flocks are infected with wild-type Mycoplasma. The results also suggest the possibility of the presence of two copies of the amplified region of the 16S rRNA gene in this vaccine strain. The study has confirmed the presence of M. gallisepticum and the possible presence of M. imitans. Different yet closely related Mycoplasma isolates are also present in South Africa. These could represent novel strains or species and warrant further investigation.

[发布机构] University of the Free State