A study of the heterogeneity of Mycoplasma ovipneumoniae isolates by examination of their proteins and deoxyribonucleic acid : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Microbiology at Massey University, New Zealand

Abstract

Chronic non-progressive pneumonia is an important disease of three to ten month old sheep in New Zealand. The lesions invariably contain M. ovipneumoniae yet the disease, or at least its more severe manifestations, cannot be consistently produced by intranasal inoculation of isolates of this organism. This may be due to the existance of different strains of this organism. This in vitro study attempts to establish whether or not there is a large number of distinguishable strains of this organism. Comparison of M. ovipneumoniae isolates by the metabolic inhibition test showed that they differed but did not allow the recognition of well defined groups. The heterogeneity of M. ovipneumoniae was then examined using SDS-PAGE of total proteins and it was found that all isolates derived from sheep on twenty different farms differed except for three isolated from one farm. Protein bands on SDS-PAGE gels could be common to all isolates, shared by some isolates, or unique to individual isolates. Identification of surface proteins by partial proteolytic digestion of intact organisms indicated that about five proteins were present on the surface, some were common to all isolates, some were shared by several isolates and others were unique to one isolate. Several proteins were investigated by "immuno-blotting". Most showed a serological relationship to one or more proteins of other isolates but antiserum to one such band reacted with only a proportion of the isolates. This band is a surface glycoprotein as indicated by proteolytic digestion and by periodic acid-silver staining. Antiserum to it could have the potential to divide M. ovipneumoniae isolates into two meaningful groups. This could best be done by using monoclonal antibodies, however this thesis pursues an alternative approach to comparing isolates namely the examination of the DNA of M. ovipneumoniae. Examination of M. ovipneumoniae DNA, using digests produced by EcoRI, showed that isolates from sheep on different farms were totally distinct. This conclusion was extended to digests produced by a range of restriction endonucleases. However, some endonucleases gave only partial or no digestion. Such enzymes had cytosine-rich recognition sequences which suggested that M. ovipneumoniae may contain 5-methylcytosine. This possibility was supported by using two endonucleases which cut the same sequence but which are, or are not, indifferent to cytosine methylation. The presence of 5-methylcytosine was then confirmed with one isolate by analysis of the DNA using HPLC. The DNA of this isolate had about 25% of its cytosine methylated. Since 5-methylcytosine occurs in M. ovipneumoniae it is possible that the different restriction endonuclease patterns seen with different isolates may be due to differences in methylation rather than to differences in DNA sequence. To investigate this we undertook a comparison of the DNA of isolates using DNA-DNA hybridization. This confirmed that M. ovipneumoniae isolates were heterogeneous because they showed a range of homology of 70-100%. This is a wide range but can be accommodated within one species. Thus it confirms that M. ovipneumoniae is one species despite its heterogeneity and does not give support to the existence of two or more subspecies within the species. The conclusion that the heterogeneity of the restriction endonuclease cleavage patterns depends on DNA sequences and not methylation was further confirmed by showing that adenine was not methylated. This was followed by digestion of DNA by Dral which cleaves a sequence lacking cytosine (5'-TTTv AAA-3'). Like other enzymes, this gave heterogeneous digestion patterns which indicated that methylation cannot account for the heterogeneity. The remarkable heterogeneity of M. ovipneumoniae strains led us to ask the ultimate question in this context, namely "are isolates from the lung of one sheep heterogeneous?" Six lungs were investigated. Three contained four different strains, one contained three strains and two contained two strains. The four lungs containing three or four strains had large lesions and the other two had smaller lesions. We suggest that the classical view that one strain of a micro-organism colonizes a lesion to cause a disease may need to be modified, that is mixtures of micro-organisms of the same species may have a potentiating effect on pathogenicity.