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    The diagnosis of an outbreak of Mycoplasma bovis clinical mastitis in a multi-farm North Otago farming operation : a thesis presented in partial fulfilment of the requirements for the degree of Master of Veterinary Science at Massey University, Manawatū, New Zealand
    (Massey University, 2021) Kearney, Kevin Patrick
    Mycoplasma bovis (M. bovis) causes a multitude of disease syndromes in dairy cattle including clinical mastitis (CM), arthritis and pneumonia. The detection in July 2017 of M. bovis, for the first time in New Zealand (NZ), on a South Island dairy farm, prompted a national animal disease response. This descriptive study aims to describe the clinical and diagnostic test findings of an outbreak of M. bovis CM, on a large multi-farm dairy enterprise where there was a single hypothesised infection source and date. Samples were collected as part of surveillance activities on-farm and at slaughter, together with farmer-selected CM cows, to provide results from real-time polymerase chain reaction (qPCR) and enzyme-linked immunosorbent assay (ELISA) tests of bulk tank milk (BTM), individual cow serum ELISA tests, quarter milk samples (QMS), and palatine tonsils qPCR tests. Post-mortem sampling of the mammary glands of M. bovis CM cases was also performed. Positive BTM PCR, supported by BTM ELISA, confirmed infection in two of the four dairy herds in the enterprise and herd-level serology (serum ELISA) confirmed infection in a third herd. There was a common clinical presentation in infected herds of an unusually high incidence of apparent treatment failure (ATF) of non-systemically ill, multiple quarter CM cases, from some of which M. bovis was detected. Individual CM cases were found in the main to be QMS M. bovis qPCR positive, serum ELISA positive and palatine tonsil qPCR positive. In approximately 70% of M. bovis CM cases, M. bovis was found to be the sole pathogen. A smoothed function model between serum ELISA and time from first diagnosis of CM, from which M. bovis was detected, predicted that the average interval between clinical diagnosis and a serum ELISA test positive result was five days. The higher observed agreement between the serum ELISA and palatine tonsil qPCR, was for M. bovis CM cows sampled on-farm compared with cows sampled at slaughter. Gross lesions of fibrosis, caseous necrosis and cystic dilation in the udders of M. bovis positive CM cows were seen together with granulomatous and suppurative inflammatory patterns histologically. High immunoreactivity in immunohistochemistry for the M. bovis antigen was also present. From the key diagnostic test findings, M. bovis was likely to have been one of several pathogens which caused individual cases of CM on the farming enterprise, and in many cases may have been the sole cause of CM cases. The results of this study can raise awareness of and provide information to aid dairy farmers and veterinarians determine if M. bovis has a role in CM outbreaks with unexpectedly increased numbers of treatment failures and can inform the regulatory response for surveillance and testing of herds and individual cattle for M. bovis.
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    Molecular and immunological analysis of New Zealand isolates of Mycoplasma ovipneumoniae : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Veterinary Science at Massey University, Palmerston North, New Zealand
    (Massey University, 2021) Bridgeman, Benjamin
    Mycoplasma ovipneumoniae is a pathogen of caprinae expected to infect the majority of domestic sheep in early life. M. ovipneumoniae is suspected to be the primary cause of chronic non-progressive pneumoniae (CNP) in lambs. Currently there is no effective vaccine against M. ovipneumoniae, and many commonly used antibiotics have limited effect. Diagnosis of M. ovipneumoniae infections can be difficult, due in part to the limited overt symptoms seen in cases of CNP. Despite the significance of M. ovipneumoniae infections there is limited information on the molecular and immunological data of M. ovipneumoniae. Thus, the major focus of this thesis was to identify potential immunogenic proteins for a vaccine or diagnostic purposes and construct genomes from New Zealand isolates of M. ovipneumoniae. Draft genomes were produced for three New Zealand isolates of M. ovipneumoniae, (isolates 16, 90, and 103), and annotated. Using antisera produced against these isolates, immunogenic proteins were identified from hydrophobic membrane protein fractions of M. ovipneumoniae using Western blotting. Those protein bands were subjected to mass spectrometry analysis, and by comparing mass spectrometry data to the genomes, six novel proteins, GAPDH, P146, MATE, hypothetical protein 1, hypothetical protein 2, hypothetical protein 3, and two previously described immunogenic proteins, EF-Tu and HSP70, were identified. Five of the proteins were produced as recombinant proteins for further study. The nucleotide sequence of EF-Tu was edited using an overlap polymerase chain reaction (PCR) to convert the sequence to Escherichia coli codon preference. This sequence was then cloned into the pET22b (+) vector for expression of histidine-tagged fusion protein. Additionally, EF-Tu, GAPDH, P146, hypothetical protein 3 and an epitope of hypothetical protein 1 were produced as recombinant proteins by GenScript. Antisera were produced in sheep against each of the recombinant proteins for further studies. Enzyme-linked immunosorbent assay (ELISA) confirmed that each protein was immunogenic and elicited antibody responses in sheep. Immunofluorescence microscopy confirmed the localisation of native EF-Tu and GAPDH proteins on the surface of M. ovipneumoniae cells. Antisera produced against whole cell antigens from M. ovipneumoniae cross-reacted with P146 and hypothetical protein 3 suggesting a potential role for these proteins to be used to detect animals infected with M. ovipneumoniae. Sheep vaccinated with M. ovipneumoniae produced weak IFN-γ responses but stronger IL-17 responses in whole blood cultures stimulated with whole cell antigens. Of the five proteins tested as recall antigens in the IL-17A assay, recombinant GAPDH produced a strong IL-17A response in sheep vaccinated with M. ovipneumoniae suggesting a potential role for GAPDH in a diagnostic assay for measuring IL-17A responses in M. ovipneumoniae infected sheep. Mycoplasmas are known to adhere to host epithelial cells as part of the pathogenesis process. A bovine endometrial epithelial cell line was established as a model to study adherence of M. ovipneumoniae to epithelial cells and determine if antibodies against the surface proteins could interfere with adherence. M. ovipneumoniae cells adhered to the endometrial epithelial cells. Adherence was inhibited by antibodies directed against whole cells of M. ovipneumoniae but not against the recombinant proteins.
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    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
    (Massey University, 1985) Norman, Nicholas Grant
    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.
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    A study of mycoplasmas of the ovine lung and their relationship to chronic non-progressive pneumonia of sheep in New Zealand : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Microbiology at Massey University
    (Massey University, 1980) Brian, Peter Norman
    The relationship of mycoplasmas to diseases of the lower respiratory tract in a variety of animals was reviewed and investigations were undertaken to determine the role of micro-organisms, with particular reference to mycoplasmas, in the aetiology of ovine chronic nonprogressive pneumonia (CNP). A survey of the prevalence of mycoplasmas in pneumonic sheep lungs revealed that Mycoplasma ovipneumoniae was present in 98% of the lungs tested, whereas Mycoplasma arginini was present in 4%. Ureaplasmas were not detected in any lungs. To facilitate further investigations into the significance of M. arginini in ovine GNP, the in•~vitro growth of the organism was investigated and its ultrastructure was determined and compared with that of M. ovipneumoniae. Although ultrastructural differences between M. arginini and M. ovipneumoniae were found~ these wouid probably not allow all cells of each of the two species to be unequivocally identified in thin sections of lung material. M. ovipneumoniae, M. arginini and parainfluenza type 3 virus were shown to be sensitive to digitonin when suspended in either conventional laboratory medium, or in lung homogenate. Furthermore, treatment of pneumonic lung homogenate with 10 mg/cm3 digitonin destroyed its ability to transmit ovine CNP. Viruses (in particular PB virus) were not detected in aliquots of the pool of lung homogenate used to transmit CNP so it is likely that the necessary digitonin sensitive component is a mycoplasma. Since M. arginini has a consistently low prevalence in pneumonic lesions, whereas M. ovipneumoniae is found in the vast majority of such lesions, it was concluded that M. ovipneumoniae is responsible for initiating primary lesions of the disease. This however does not imply that M. ovipneumoniae on its own is capable of causing lesions comparable in severity to the fully developed "field" cases. The inactivation of M. ovipneumoniae by formalin, with a view to making a vaccine, was investigated.
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    The characterisation of strains of Mycoplasma ovipneumoniae by restriction endonuclease analysis : a thesis presented in partial fulfillment of the requirements for the degree of Master of Science in Microbiology at Massey University, New Zealand
    (Massey University, 1982) Mew, Alison Jane
    Studies of the pathogenicity of Mycoplasma ovipneumoniae would be facilitated by an in vitro method of identifying and classifying isolates of this organism, and it is with the development of such a method, and its possible applications, that this thesis is mainly concerned. The method chosen was Restriction Endonuclease Analysis (REA) which has already been successfully applied to the identification of some viruses and bacteria. Initially the REA patterns of 2 strains of M. ovipneumoniae were observed. They differed markedly in restriction pattern, but the pattern exhibited by any one of them was recognisable and reproducible. Furthermore, patterns did not change significantly after limited passage in vitro. An extension of this study to 8 isolates from widely differing sources, showed that all gave markedly different patterns. It was concluded that, unlike the findings for Leptospira and Rhizobium, REA could not be used for the identification of species, but is an extremely powerful method for identifying strains of M.ovipneumoniae. Despite the marked heterogeneity of isolates from different sources, the relative stability of pattern of an individual isolate, suggested that REA could be used to examine the epidemiology of individual strains of M. ovipneumoniae within a flock of sheep. Hence, we undertook a study of M. ovipneumoniae isolates obtained by serial swabbing of the nasal cavities of a flock of lambs over a 6-month period, and from the lungs of the same lambs at slaughter. It was shown that 54 isolates from the nasal cavities fill into 7 major groups with respect to restriction pattern (although minor differences were detected within a group). There was a tendency for these groups to occur sequentially. None of the isolates were shown to persist for long periods, but a later strain could replace an earlier one. The isolates from the lungs were more homogeneous and the predominant strain fell within one of the 7 "nasal" groups. This suggests that nasal isolates may vary in their pathogenicity for the lungs, although other explanations are possible (see General Discussion). Notwithstanding the apparent stability of mass cultures of M. ovipneumoniae following limited passage in vitro, the unexpectedly large number of restriction patterns found with field strains, led us to re-examine, in more detail, the stability of cloned isolates. A multiply-cloned isolate was propagated in vitro and 8 sub-clones selected before and, a further 8 sub-clones after 20 passages. Some limited heterogeneity was detected among the 8 sub-clones selected before passage, and a somewhat greater degree of heterogeneity was detected among sub-clones selected after passage. It should, however, be emphasised that these differences were small compared to the total lack of similarities seen when isolates from different sources were examined. Limited passage in the presence of sub-lethal concentrations of antibody did not increase the heterogeneity of patterns - if anything, the reverse is true. Explanations for these findings and Future experiments to confirm or deny these possibilities are discussed.
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    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
    (Massey University, 1989) Ionas, George
    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.