Molecular characterization of mycoplasmas species isolated from the genital tract of Dorper sheep in South Africa

Abstract

Mycoplasmas are prokaryotic micro-organisms belonging to the class Mollicutes, which lacks rigid cell walls. Their genomic size ranges from 500-1500 bp. It causes a wide variety of different diseases in small ruminants and in particular ulcerative balanitis and vulvitis that affects Dorper Sheep in South Africa. The disease causes high economic losses to the Dorper sheep breeders in South Africa. The presence of the disease has been known in South Africa since 1979. Earlier publications have identified the causative agent of this disease as Mycoplasma mycoides mycoides LC (MmmLC). However, several Mycoplasma organisms isolated from cases of ulcerative balanitis have been shown not to be MmmLC. There is a need to characterize the organisms isolated from sheep suffering from this disease using conventional and genetic molecular methods. In this study, 16SrRNA gene-based PCR assays and gene sequencing was used for the detection and characterization of Mycoplasma species from cases of ulcerative vulvovaginitis and balanoposthitis in Dorper sheep in South Africa. This investigation was conducted on 34 stored field isolates of mycoplasmas collected between 2003-2009 from 15 different farms in the Northern and Western Cape provinces of South Africa. The isolates were screened and characterized by means of microbiological culture and biochemical methods and confirmed by PCR and sequencing. Evidence of involvement of these Mcoplasma idolates in ulcerative vulvovaginitis and balanoposthitis was obtained from the submission histories. All 34 isolates were analysed by means of PCR, cloning and sequencing of a 1 078 bp fragment length of 16S a rRNA gene and identified as Mycoplasma species. BLAST searches for sequence similarity from Genbank data revealed 18 isolates out of 34 four are 99 % similar to M. arginini, six out of 34 are 99 % similar to M. bovigenitalium, and two out of 34 were found to be 99 % similar to M. sp. ovine/caprine serogroup II. Two isolates out of 34 are 99 % similar to A. Laidlawii, and BLAST searches of two isolates gave 99 % similarity to M. sp. USP120. Two isolates were found to be 99 % similar to synthetic M. mycoides mycoides Jvc1. A last isolate gave 99 % similarity to M. canadense. Phylogenetic trees were drawn using the neighbour joining method and maximum parsimony analysis to compare the South African isolates with other GenBank reference strains to determine relationships between South African isolates with isolates in other parts of the world. This thesis is composed of five chapters. The first chapter deals with the historical background of ulcerative vulvovaginitis and balanoposthitis in Dorper sheep in South Africa and comparisons with findings from previous research. The chapter ends with the aims and objective of this research project. Chapter two contains a literature review that deals with ulcerative vulvovaginitis and balanoposthitis in various parts of the world and controversy about the views of researchers about the aetiology of ulcerative vulvovaginitis and balanoposthitis in sheep. Chapter three presents the first research on molecular characterization of mycoplasmas species isolated from cases of ulcerative vulvovaginitis and balanoposthitis in Dorper sheep in South Africa by means of PCR and gene sequencing. Chapter four provides the findings of the analyses of the various Mycoplasma species that were involved in ulcerative vulvovaginitis and balanoposthitis in Dorper sheep in South Africa. The chapter also gives the results of phylogenetic analysis of the various Mycoplasma species with their relationship to sequences from all over the world deposited by researchers in Genbank. Chapter five summarizes the research findings and provides conclusions. Copyright