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    Studies on Mycoplasma ovipneumoniae in New Zealand sheep : epidemiology and comparison of isolates : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Microbiology at Massey University, New Zealand
    (Massey University, 1983) Ionas, George
    As part of a larger study to examine the role of M. ovipneumoniae in chronic non-progressive pneumonia (CNP) of sheep, the colonisation of the respiratory tract by mycoplasmas was examined in two flocks of lambs over . a nine month period. In both farms M. ovipneumoniae was detected in the ewes at the time of first swabbing of the lambs. The two flocks of lambs differed in the time when the nasal cavity first became colonised by M. ovipneumoniae: thus in Flock 2 M. ovipneumoniae was detected in the nasal cavity relatively early and also became disseminated throughout the flock some months earlier than occurred in Flock 1. Nevertheless, M. ovipneumoniae was widespread in both flocks of lambs by March i.e. at• or before peak seasonal prevalence of CNP. At slaughter in May, Flock 2 (colonised early) had a much higher prevalence of CNP than Flock 1. These findings are consistant with the hypothesis that M. ovipneumoniae colonises the nasal tract of lambs and subsequently invades the lung possibly in response to the stress of exposure to hot dry weather. The second part of this thesis is concerned with the adaptation of Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) to distinguish strains of M. ovipneumoniae with the ultimate objective of comparing M. ovipneumoniae strains isolated from pneumonic lungs to nasal isolates and isolates from apparently normal lungs. Isolates from different sources were heterogeneous when examined by SDS-PAGE, but comparisons were made difficult because of the excessive number of protein bands. In response to this problem fractions of M. ovipneumoniae were examined. Membrane preparations conserved the unique protein bands which in principle allow discrimination between strains, but because the number of protein bands was still excessive we examined surface proteins by labelling intact cells with Fluorescein isothiocyanate (FITC). This approach still gave gels with too many protein bands for convenient comparisons to be made, but had the advantage of allowing the identification of surface proteins, some of which were unique to individual isolates. This encouraged us to combine SDS-PAGE with a classic immunological approach to strain identification i.e. we investigated the possibility of excising protein bands from gels for subsequent use as immunising antigens. One common protein band was excised, it was found to be antigenic and the antisera crossreacted with a single line of identity in gel precipititin tests with all the strains tested. While within the time limit available we have examined only one common protein band, the result suggests that the excision of individual strain-specific protein bands from SDS-PAGE for use as immunising antigens will provide strain-specific antisera which should allow the development of a simple approach to strain identification.
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    Studies of Pasteurella haemolytica : (1) comparison of serotyping techniques, (2) prevalence of serotypes in New Zealand sheep : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Microbiology at Massey University
    (Massey University, 1985) Prince, Diane Valerie
    P.haemolytica, which is the aetiological agent of pasteurellosis, is the major cause of mortalities in sheep in Great Britain and is a problem in several other countries including New Zealand. Furthermore, P.haemolytica, acting as a secondary invader, exacerbates lesions of chronic non-progressive pneumonia (CNP) initiated by Mycoplasma ovipneumoniae and this disease causes considerable economic loss to the New Zealand sheep farming industry. P.haemolytica exists as 15 serotypes and immunity is serotype specific. P.haemolytica vaccines are marketed overseas and their use in New Zealand is under active consideration. It is logical however, to establish which serotypes are present in New Zealand before a vaccine is produced, but there is at present, no information on this point. This is largely because of technical difficulties in typing isolates. This situation stimulated the present investigation which has 2 major aims: To develop an improved method of typing P.haemolytica and to gather some data on the prevalence of the various serotypes in Ne>..J Zealand. Hith respect to the first aim, several approaches \..Jere taken to the problem of differentiating between serotypes. (1) Indirect heamagglutination (IHA), the standard method by which P.haemolytica is serotyped, was found to be laborious and gave many cross-reactions. (2) SDS-PAGE of total protein showed similar patterns for all serotypes within a biotype, whereas the 2 biotypes had different patterns. Thus, SDS-PAGE cannot be used to identify the serotype, but could be useful for identifying the species and the biotype. (3) Latex beads, coated with antibody pre~ared against whole cells, agglutinated homologous cells, but also gave many cross-reactions. This test should, in principle, become type-specific if purified capsule were used as the immunising antigen, but further work is required to prepare capsular polysaccharide free from endotoxin which stimulates the production of cross-reacting antibody. (4) Gel precipitation was simple to perform and was serotype specific. However, serotype A2 required a concentrated antigen preparation for the detection of a precipitation line. The concentrated antigen could not be routinely used with the other types because of cross-reactions between antigens. (5) Counter immunoelectrophoresis (CIE) was not specific due to endotoxin causing cross-reactions. serotype (6) Bacterial agglutination was a rapid and simple test to perform and showed some limited cross-reactions. Since IHA, gel precipitation and agglutination showed some potential, they were then compared using 40 isolates from the lungs of 60 sheep with CNP These findings reinforced the conclusions drawn when prototype strains were used. We initially proposed that the serotype of isolates should be primarily determined by agglutination tests, but since this is not 100% specific, the results must be confirmed by gel precipitation. This approach was investigated using 110 isolates from the nasopharynx of sheep and it was satisfactory for all serotypes except A2. Strains within the A2 serotype showed some heterogeneity at least when examined by gel precipitation and a hypothesis is presented to explain this. We conclude that at present, the best method of serotyping isolates is by gel precipitation, but that all isolates which are not positively serotyped by this approach should be retested by IHA. Information concerning the prevalence of serotypes was obtained during the above studies. 110 isolates were obtained from the nasopharynx of 50 sheep from each of 4 farms. All nAn serotypes, except A12 and A1 u, were isolated. up 35% of these isolates. 60 lungs with Serotype A2 made CNP lesions were obtained from 20 farms in the Manawatu region and 40 isolates of P.haemolytica were obtained. All were serotyped and they were: A1 (25%), A2 (55%), A6 (7.5%), A7 (7.5%), A8 (2.5%), and A9 (2.5%). No "Ttt types were found in either the nasopharynx or the lungs. The implications of these findings for vaccine production are discussed.
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    Studies on Pasteurella haemolytica : comparison of serotyping techniques and surveys of the prevalence of serotypes in sheep and goats in New Zealand : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Microbiology at Massey University, New Zealand
    (Massey University, 1987) Midwinter, Anne Camilla
    P.haemolytica is the aetiological agent of pneumonic pasteurellosis in sheep and goats, and, as a secondry invader it also exacerbates lesions of chronic non-progressive pneumonia (CNP) . These diseases cause considerable economic loss to the New Zealand farming industry. P. haemolytica exists as 15 serotypes and immunity is serotype specific. Vaccines against P .haemolytica are produced, but it is not known if the serotypes contained in the vaccine are the same as those causing disease in New Zealand as there is a lack of information on the prevalence and distribution of the serotypes of P .haemolytica in this country. This is largely due to the technical difficulties involved in typing isolates because the standard method, the indirect haemagglutination assay, ( IHA), is laborious and may give anomalous results due to cross-reactions. The present investigation was undertaken with two major aims: to replace IHA with a more convenient typing system, viz. agar gel immunodiffusion, (AGID), and to use AGID to survey the serotypes of P.haemolytica present in CNP lesions of sheep, pneumonic pasteurellosis of sheep, pneumonic pasteurellosis of goats and the nasal cavities of goats. Difficulties were encountered in the preparation of rabbit antisera to some of the 15 prototype strains. These difficulties were overcome by using domestic hens when necessary. Using these sera it was possible to distinguish the 15 prototype strains by IHA, and following absorption of sera, by AGID. The results obtained by IHA and AGID were in agreement, at least when prototype strains were examined. It was necasary to show that AGID is able to correctly establish the serotype of field isolates of P. haemolytica. Hence 2 5 caprine isolates of P. haemolytica from field cases of pneumonic pasteurellosis were serotyped by both IHA and AGID. In 24 cases the results from the two tests agreed. In the remaining case IHA indicated that the isolate was serotype A2 or All. We were able to show that this isolate gave a line of identity with antigen prepared from the prototype strain of All, but showed no line of identity in the AGID with any other antigen preparation. Taking this as the critical criterion we concluded that this isolate was serotype All, although IHA showed a 2- fold preference for A2 over All. Since AGID was shown to be a reliable test we used it alone for future serotyping, for two reasons: it is more convenient, and any cross-reactions that do occur may be resolved by looking for a line of identity between antigens of the isolate and a prototype antigen. In the case of two serotypes involved in many crossreactions, namely Al and A7, the capsular polysaccharide was purified by organic solvent precipitation. This purified polysaccharide was used to test for a line of identity with reacting isolates. This eliminates the possibility that the line of identity seen was due to a non-serotypespecific antigen. Four surveys (two in sheep, two in goats) of the serotypes of P.haemolytica present in New Zealand were undertaken. The first involved 139 isolates derived from ovine lesions of CNP collected from 4 areas of New Zealand. A total of 9 serotypes were found. Serotypes Al (31.7%), A2 (47.8%) and A7 (10%) made up 89.5% of the total. A smaller survey of 18 isolates from pneumonic pasteurellosis of sheep revealed 6 serotypes, including 1 isolate of TlO, a serotype and biotype not previously found in New Zealand. Al (11.1%) and A2 (61.1%) were the predominant serotypes present and represented 72.2% of the total. The 25 isolates of P.haemolytica from caprine pneumonic pasteurellosis contained only 4 serotypes. A2 represented 8 0% of the total. 14 isolates of P.haemolytica were obtained from the nasal cavities of 109 goats. Only 2 serotypes were isolated. 13 isolates were A2 and the remaining isolate was All. The implications of these results for vaccine manafucture were discussed and it was suggested that a vaccine containing A2, Al and A7 (in order of importance) should control CNP in sheep and pneumonic pasteurellosis in both sheep and goats. Field isolates of P.haemolytica were compared with prototype strains for capsule production (using Laurell Rocket test), and antibiotic sensitivities. The total proteins of caprine and ovine strains were also compared, using SDS-PAGE. Laurell Rocket tests showed that the prototype strains produced more capsular polysaccharide than did any of our field isolates. All isolates of P. haemolytica showed some resistance to streptomycin while none were resistant to more than 4f.1g/ml chloramphenicol or penicillin so these are the drugs of choice. No difference was found within a serotype between the total proteins of caprine and ovine isolates by SDS-PAGE.
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    Colonisation of the ovine respiratory tract by Pasteurella (Mannheimia) haemolytica : a thesis presented in partial fulfilment of the requirements for the degree of Master of Veterinary Studies at Massey University
    (Massey University, 2003) Ahmed, Muftikhar
    Pasteurella (Mannheimia) haemolytica is a member of the normal bacterial flora of the nasopharyngeal, tonsillar and oral mucous membranes of sheep. The history, characteristics and pathogenicity of this organism are reviewed and the associated diseases of the ovine respiratory tract are discussed. In New Zealand, P. haemolytica is associated with two major disease entities; acute pneumonic pasteurellosis and chronic non-progressive pneumonia (CNP). Clinical or acute pneumonic pasteurellosis occurs as a sporadic disease with low prevalence on certain farms whereas CNP is very widespread and economically important as it causes poor growth rates and downgrading of carcasses during slaughter. The epidemiological relationship between the nasal carriage of P. haemolytica in healthy ewes and their lambs was investigated and it was found that although lambs occasionally became infected from their dams they were more commonly infected from other sources. A very significant difference between the rate of nasal carriage on four farms in the Manawatu district was observed and a peak prevalence of P. haemolytica was seen in the February-March period. A close relationship between nasal carriage and pneumonia was found on one farm (Farm 4), which initially had a pure and vigorous growth of P. haemolytica from the nasal swabs obtained from young lambs. When 6 lambs were kept in close contact for a period on one farm, all developed a high rate of nasal carriage of P. haemolytica within 5 days. DNA fingerprinting of the isolates from ewes and their offspring showed a variety of restriction endonuclease patterns using pulsed field gel electrophoresis (PFGE). The pulsed field profiles of isolates from the nasal cavity of ewes and their new-born lambs showed that lambs are more likely to obtain the first strain of P. haemolytica from in-contact ewes, lambs or the environment rather than from their mothers. The pattern of isolation of P. haemolytica in lambs on three farms without pneumonia showed that most strains of the organism were present on only one occasion and within two months the nasal cavity was occupied by other strains. On one farm (Farm 4), some strains of P. haemolytica were present throughout the whole life of the lambs and one these strains was later isolated from pneumonic lesions at slaughter.
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    The tracheobronchial airways of normal and pneumonic sheep : cytology and cytopathology : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy at Massey University
    (Massey University, 1986) Al-Kaissi, Ayad
    As a basis for subsequent pathological studies the histology, topographical morphology and ultrastructure of the normal ovine tracheobronchial epithelium at five different levels were investigated. In addition,the topographical studies were extended to involve the normal alveolus. The lining epithelium of the trachea and bronchi consisted of pseudostratified ciliated columnar and goblet cells, while from the small bronchi distally, the airways were lined by low columnar or cuboidal ciliated and non-ciliated cells. A slightly lower proportion of mucous cells were present in the upper trachea compared to the lower trachea which also contained Clara cells with PAS-positive granules. Topographically, there was a marked change from a predominance of ciliated cells in the trachea and bronchi to non-ciliated cells in the distal bronchioli. Ten different types of epithelial cell were identified ultrastructurally. These were; two types each of ciliated, goblet and unknown secretory cells together with Clara, brush, basal and intermediate cells. Several cell types of unknown function which have not been previously described were observed. It was concluded that the ovine lung was similar to that of cattle but different from other mammals in two important features. Firstly, interalveolar pores of Kohn were uncommon in young sheep and secondly, there was a relative paucity of alveolar macrophages in alveolar spaces. It is thought that these features may have an influence on the pathophysiology and pathogenesis of pneumonia in sheep and the resistance of the ovine lung to infection. The pathological changes which occurred in the tracheobronchial epithelium at five different levels were studied in both early and advanced lesions of chronic non-progressive pneumonia (CNP) in lambs 3 to 10 months old. In addition, the alveolar topographical changes were investigated. The most common topographical finding was loss of cilia from the epithelial surface which was more severe in early lesions. The tracheobronchial epithelium in advanced pneumonic lesions showed large areas of squamous metaplasia, while focal areas were observed in early lesions. Extensive inflammatory cell infiltration of the tracheobronchial epithelium was one of the main histological features seen in both early and advanced pneumonic lesions indicating that active inflammatory changes were occurring at all stages of the disease. Aggregations of lymphoid cells together with submucosal gland hyperplasia and metaplasia were more extensive in advanced cases. Striking changes to Clara cells were observed by scanning electron microscopy in bronchioli in both stages of the disease. Mycoplasmas were commonly found attached by means of pili-like structures to the cilia of epithelial cells of the trachea and bronchi in early lesions and to tracheal and bronchiolar epithelial cilia in advanced lesions. Their presence in early pneumonic lesions suggested that they may compromise the effectiveness of the mucociliary system, allowing other destructive bacteria normally resident in the upper respiratory tract to penetrate into the pulmonary parenchyma and produce more severe lesions. To quantitate the proliferative changes observed the epithelial and submucosal thicknesses of the tracheobronchial airways of sheep affected with CNP were measured at 6 levels and submucosal gland size and number were measured at 4 levels. The mean thickness of the tracheobronchial mucosal layers of normal sheep decreased regularly from the upper trachea to the distal bronchioli, while in pneumonic lesions the decrease in mucosal thickness was more irregular. Small bronchi and bronchioli were the most severely affected and the percentage increase above normal was 146.5% and 268.2% respectively.Comparative statistical analysis of the results showed that in early lesions the epithelium of the trachea and bronchi were worst affected, whereas in advanced lesions the epithelium of the peripheral airways showed the most severe change. It is thought that the increase in the thickness of the wall of peripheral airways together with the accumulation of inflammatory cells and mucus may result in partial or complete obstruction of the lumen of small airways in affected areas. Statistical analysis of sectional areas of submucosal gland of normal sheep showed that they decreased regularly from the upper trachea to the small bronchi, but this pattern became irregular in the pneumonic lesions. The most significant changes in submucosal gland parameters of early pneumonic sheep occurred in the intrapulmonary bronchi. In sheep with advanced pneumonic lesions changes were most severe in both intrapulmonary and extrapulmonary bronchi. Enlargement of the submucosal glands in pneumonic lesions was found to be due to both hyperplasia and hypertrophy and these changes were more severe in advanced than early lesions. The histochemistry of the submucosal gland glycoproteins in normal and pneumonic sheep was also studied and statistical analysis of the results showed a change in the types present. It was found that most mucous cells of submucosal gland at all levels of the normal ovine tracheobronchial tree contained either neutral or mixed types of glycoprotein and very few contained the acid type. The submucosal glands of normal bronchi contained significantly more neutral glycoprotein and less mixed and acid glycoproteins than those of the trachea. In pneumonic lungs there were no significant differences in the amount and types of glycoprotein between levels. Comparative statistical analysis showed that in the intrapulmonary bronchi, acid glycoprotein increased and neutral glycoprotein decreased in advanced pneumonic lesions when compared to normal and early pneumonic sheep. It was concluded that the ovine tracheobronchial airways respond to unspecified noxious agents by changing the chemical and physical nature of their mucous secretions. Ovine tracheal organ cultures were used to investigate the pathogenicity of Mycoplasma ovipneumoniae, Bordetella parapertussis, Pasteurella haemolytica and Neisseria catarrhalis. The ciliary activity, histology, topographical morphology, ultrastructure and microbiology of these experiments are described in detail. Four different titres of each microorganism were used. It was found that all the microorganisms caused cytopathological changes and the ciliostasis produced was dose dependent. Mycoplasma ovipneumoniae and B. parapertussis attached to cilia at 30 min and 1 hr respectively and produced ciliostasis as early as 13 hrs and 1 hr respectively. The means of attachment of both organisms was investigated with both scanning and transmission electron microscopes. A fimbria or pili-like structure was found in close proximity to cilia with both microorganisms. Pasteurella haemolytica and N. catarrhalis failed to attach to cilia but they produced cytopathological changes and the ciliostatic effect was achieved as early as 3 hrs and 4 hrs respectively. Although both of these organisms behaved in a similar manner in organ culture and produced similar cytopathological changes, P. haemolytica was more destructive and produced ciliostatic effects faster than N. catarrhalis. Of the four microorganisms used it was found that only M. ovipneumoniae and B. parapertussis had both an affinity for tracheal epithelial cells and the ability to produce destructive changes in organ culture. On the basis of this work both M. ovipneumoniae and B. parapertussis could be considered as likely candidates for organisms which in vivo could initiate bronchiolar disease and thus allow the development of CNP. This hypothesis in regard to M. ovipneumoniae is strongly supported by several other workers. The role of B. parapertussis remains to be more fully investigated.