An investigation of the heterogeneity of isolates of Mycoplasma ovipneumoniae using restriction endonuclease analysis : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Microbiology at Massey University
Previous studies of Mycoplasma ovipneumoniae by Restriction Endonuclease Analysis (REA) (Mew, 1982) indicated that the species shows exceptional heterogeneity as compared to other species of pathogenic Mycoplasmas. This thesis further investigates this heterogeneity. To get confirmation of the heterogeneity of M. ovipneumoniae, sixty isolates derived from three sheep on each of twenty farms, were examined by REA. All twenty independant isolates (i.e. isolates originating from sheep on different farms) gave REA patterns that were markedly different, with at most, only 5% of bands in common. Isolates from sheep on the same farm were found to be either indistinguishable, similar (i.e. at least 95% of bands in common) or markedly different (i.e. less than 5% of bands in common). Having confirmed the heterogeneity of M. ovipneumoniae isolates from sheep on different farms further study was directed at providing an explanation for this heterogeneity. The stability of the M. ovipneumoniae genome was investigated by serial passage of a multiply cloned isolate in vitro. Three REA patterns, A, B and C (pattern A was the original pattern) were observed. These pattern changes were non-random in that they were reversible. Thus it appears that an internal rearrangement system is present in M. ovipneumoniae. No non-reversed REA pattern changes were seen. It was concluded that the pattern changes seen after serial in vitro passage were minimal, and that genomic instability could not explain the heterogeneity seen in M. ovipneumoniae. Changed REA patterns must represent DNA changes which in turn may mean changes in proteins. To attempt to detect protein changes, 3 clones which showed patterns A, B and C respectively were examined by SDS-Polyacrylamide gel electrophoresis of total cellular proteins. No differences were detected. There remains the possibility that antigenic changes occurred which might not be demonstrable by this method. A second possible explanation for the heterogeneity seen in M. ovipneumoniae is that frequent genetic interchange between initially distinct REA strains might result in the generation of many new REA types that differ markedly from both parental strains. Three approaches were taken to investigate this possibility: 1. "Classical crosses" detected by antibiotic resistance markers. 2. Mixtures of two cultures of M. ovipneumoniae with different REA patterns were mixed and propagated together. (a) Clones were selected from a mixed culture after it had been passaged for about 30 generations and examined by REA. (b) "Presumptive recombinants", i.e. clones of M. ovipneumoniae which were resistant to two antibiotics, recovered from mixtures of singly resistant clones were examined by REA. 3. M. ovipneumoniae was examined for the presence of extrachromosomal DNA which, if present, could facilitate genetic interchange. Using these three approaches, we were unable to demonstrate genetic interchange in M. ovipneumoniae so it is unlikely that genetic interchange accounts for the considerable heterogeneity seen in the species. It was concluded that the heterogeneity seen in the species is due to the presence of a large number of strains that are genetically stable with respect to REA, which have evolved over a long time period and which are independantly maintained. We estimated the minimum number of strains of M. ovipneumoniae that must exist in a population so that when 29 independent isolates are examined, all will be different. With 95% certainty, this minimum number is 150. The possibility that at least 150 M. ovipneumoniae strains could be maintained in New Zealand was discussed. By applying general epidemiological principles to M. ovipneumoniae, we concluded that many more than 150 could be independently maintained.