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    Experimental pneumonia induced by a Bordetella parapertussis-like organism in the ovine and murine lung : a thesis presented in partial fulfillment (70%) of the requirements for the degree of Master of Philosophy in Veterinary Pathology at Massey University
    (Massey University, 1987) Chen, Wangxue
    Thirty-four specific pathogen-free (SPF) Swiss mice were intranasally inoculated with a suspension containing about 3 x 10 7 colony-forming units (CFU)/ml of a B. parapertussis-like organism isolated from pneumonic ovine lung. Eleven per cent of the animals died between 2 and 3 days of inoculation and over 90% of infected mice developed a subacute bronchopneumonia morphologically similar to early lesions of naturally-occurring ovine chronic non-progressive pneumonia (CNP). The sequential pulmonary changes were examined by light microscopy and transmission electronmicroscopy from 12 hr to 29 days after inoculation. The early stages were characterized by alveolar septal congestion and oedema, focal intra-alveolar haemorrhage, and intra-alveolar and septal infiltration by neutrophils and macrophages. Later, hyperplasia of perivascular and peribronchiolar lymphoid tissue and the deposition of collagen in the interalveolar septa were prominent. The bronchial and bronchiolar epithelium remained intact throughout the experiment, but bronchiolar lumina became occluded by inflammatory exudate at an early stage. Ultrastructural changes consisted of the degeneration of the alveolar type I and type II epithelial cells and marked degeneration of alveolar macrophages. Pure cultures of the B. parapertussis-like organism were consistently recovered from mouse lungs 12 hr to 6 days after inoculation. Both intact and degenerating organisms were found free in alveolar spaces and within phagocytic vacuoles of alveolar macrophages. However, replication of organisms was not observed at any stage of infection and no special association was observed between organisms and the ciliated or non-ciliated respiratory epithelium. Injury to ovine respiratory tract was demonstrated when a similar bacterial suspension to that given to the mice was given by intratracheally to colostrum-deprived lambs. The lesions produced in the pulmonary parenchyma of the lambs were similar to those seen in both early naturally-occurring ovine CNP and the experimental infection with this organism in mice. They consisted of an acute mild tracheobronchitis, severe alveolar collapse and acute bronchopneumonia which developed within 24 hr and was most severe at 1 to 3 days after inoculation. Ultrastructurally, the alveolar epithelium exhibited extensive degenerative changes and necrosis of individual epithelial cells. Topographical studies revealed extensive coverage of the infected tracheobronchial epithelium with a dense layer of inflammatory cells mixed with mucus, and focal extrusion of ciliated cells. Occasionally, moderate numbers of the B. parapertussis-like coccobacilli were seen closely associated with cilia. Inoculated lambs showed a marked elevation in the numbers of cells in bronchoalveolar lavage 24 hr after infection. Up to 93% of the cells in the lavage at 24 hr were neutrophils. However, no close interation between phagocytic cells and the organises was detected. Many of the macrophages in the lavage exhibited cytoplasmic vacuolation from five days after inoculation onwards. Blood leucocyte and neutrophil counts in infected lambs gradually rose to reach peaks at five and three days after inoculation, respectively. The B. parapertussis-like organism was recovered in pure culture from the nasal cavity of lambs killed on days one, three, five and nine. The viable bacterial count in bronchoalveolar lavage fluid decreased from 24 hr to 5 days with almost complete elimination of organisms nine days after inoculation. The retention of the B. parapertussis-like organism in the mouse trachea was compared to that in the mouse lung from 0 to 48 hr after intranasal inoculation. Although there was greater bacterial deposition in the trachea than the lung there was a faster clearance from the trachea. At 48 hr after instillation, almost all organisms were eliminated from the trachea but about 45% of organisms were retained in the lung. The current investigation has shown that the B. parapertussis-like organism can infect SPF mice and colostrum-deprived lambs and induce a subacute bronchopneumonia. The morphological changes seen suggest that this organism has the potential to predispose the ovine respiratory tract to further infection by other microorganisms and that the organism itself may also be able to cause severe pulmonary damage. The relevance of these observations to the problem of CNP in sheep in the field has yet to be determined.
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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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    Cytological studies of ovine alveolar macrophages : interaction with Mycoplasma ovipneumoniae in vitro : this thesis is presented in partial fulfilment (30%) of the requirements for the degree of Master of Philosophy in Veterinary Pathology at Massey University
    (Massey University, 1981) Al-Kaissi, Ayad
    The attachment between Mycoplasma ovipneumoniae organisms and ovine alveolar macrophages was studied in culture for a 24 hour period and antibody-mediated phagocytosis of M. ovipneumoniae organisms was observed by both scanning and transmission electron microscopy. Mycoplasma ovipneumoniae organisms have the ability to attach to the alveolar macrophage membrane without inducing phagocytosis although they stimulated mitotic division in early cultured cells. The addition of specific antibody to the mycoplasma-macrophage cultures provoked phagocytosis of surface attached and surrounding M. ovipneumoniae organisms. Alveolar macrophages stimulated by specific antibody showed rapid and extensive spreading on the glass coverslip and prominent membrane ruffling and filopodia. Many exterior openings and fine cytoplasmic pits were also evident which may represent pinocytotic vesicle formation sites. With transmission electron microscopy M. ovipneumoniae organisms were observed surrounded by macrophage filopodia 2 hours after the addition of specific antibody and numerous micro-organisms were seen within phagocytic vacuoles. Some of the intracellular M. ovipneumoniae organisms appeared normal while others appeared partially or completely degraded. Twenty four hours after the addition of specific antibody, intracellular M. ovipneumoniae organisms had been digested. A new procedure for collection of alveolar macrophages was developed. The procedure provides an alternative to other methods and may be particularly useful for collecting alveolar macrophages from the lungs of large animal species such as sheep and cattle. Acetone was used to dehydrate macrophages for SEM with excellent results. In conclusion, it was found that the addition of specific antibody to an M. ovipneumoniae -macrophage culture stimulated phagocytosis of these micro-organisms. This suggests that if sheep gain high titres to M. ovipneumoniae, their alveolar macrophages will be able to destroy inhaled M. ovipneumoniae organisms quickly and effectively; a possibility which should be tested further in vivo.
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    The pathogenesis of pneumonia in sheep : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy at Massey University
    (Massey University, 1975) Alley, Maurice Rewi
    The pathology of pneumonia in sheep in New Zealand is described in a study of over 400 naturally-occurring cases obtained from field and abattoir sources. The common forms of enzootic pneumonia consist of two distinct pathological and epidemiological entities; an acute pneumonia affecting sheep of all ages and a subacute or chronic, non-progressive pneumonia affecting lambs from approximately 3 to 10 months of age. Acute pneumonia is characterised by intense congestion, alveolar haemorrhage, fibrinous exudation and ventral consolidation of both lungs. Ultrastructurally the cellular exudate consists of a mixture of neutrophils, macrophages and detached alveolar epithelial cells with which bacteria are closely associated. Subacute and chronic pneumonia is characterised by varying degrees of dull red to grey consolidation of the anterior lobes. Ultrastructural studies reveal a variety of degenerative changes in the alveolar epithelium including several subcellular changes not previously recorded. Repair is by type II cell hyperplasia and this has been studied ultrastructurally and histochemically. Undifferentiated type II cells resembling those found in the foetal lamb and cells transitional between type II and type I have been observed. The significance of these findings in relation to the origin and dynamics of alveolar epithelial repair is discussed. The major factor underlying the pathological differences between acute and chronic pneumonia is considered to be the degree of damage to the alveolar epithelium which is universal in the former disease and less severe and localised in the latter. Experimental injury to the ovine lung produced by the endobronchial instillation of dilute (1%) nitric acid with India ink as a marker was studied at periods from 2 hours to 10 days after administration. Alveolar collapse and neutrophil infiltration were the earliest changes seen but few neutrophils remained after 3 days. Large macrophages which were active from 3 hours were joined by smaller macrophages which migrated from interstitial tissues from 12 hours until 3 days after administration. The ultrastructural changes observed in the alveolar epithelium were similar to those encountered in naturally-occurring pneumonia. Proliferation of Clara cells and type II cells was detected one day after administration and partial "epithelialization" of some alveoli at 5 days. There was complete loss of pulmonary surfactant from affected areas by 12 hours and return to normal activity was irregular. Parentally administered Paraquat and oral dosing with busulphan were also tested for their value as agents for producing experimental pulmonary injury in sheep. Maximum pulmonary involvement occurred at between 6 to 10 mg/Kg of Paraquat but death appeared to result from liver and kidney toxicity. Paraquat pre-treatment did not affect pulmonary resitance to endobronchially inoculated bacteria in pure or mixed cutures, however lesions similar in nature to those of acute enzootic pneumonia were produced by Staphylococcus aureus. No significant pulmonary effects were produced with busulphan at high dose rates. To investigate the bacterial flora of the respiratory tract of normal and pneumonic sheep, 184 normal sheep and 246 sheep aged 6 to 9 months with chronic or subacute pneumonia were examined at slaughter over a 2 year period. Pasteurella haemolytica was present in the nasal cavities of 73% of normal sheep and 78% of sheep with pneumonia, while Neisseria catarrhalis was also commonly isolated from both classes. Pneumonic lungs characterised by alveolar collapse yielded few bacteria whereas those in which cellular exudate predominated contained P. haemolytica in 75% of cases. In lungs with severe proliferative changes P. haemolytica was recovered in over 60% of cases and N. catarrhalis in 25 to 33%. The prevalence of Mycoplasma ovipneumoniae and Mycoplasma arginini was also investigated in the respiratory tract of normal and pneumonic 6 to 9-month-old sheep. Both organisms were ubiquitous in the nasal cavity but M. ovipneumoniae was recovered more frequently than M. arginini. The recovery rate and titre of M. ovipneumoniae in pneumonic lungs were substantially higher than in normal lungs and several proliferative histological features were found to be associated with these titres. Cellular exudation and epithelial hyperplasia were associated with combined high titres of M. ovipneumoniae and bacteria. Lymphoid hyperplasia and mucus secretion were associated with low bacterial titres. Transmission experiments with lung homogenate derived from cases of acute pneumonia succeeded in producing lesions similar to the natural disease when inoculated endobronchially into worm-free, housed lambs whereas cultures of P. haemolytica, M. arginini or pneumonic lung homogenised in medium containing antibiotic produced minimal or no effect. However, the excessive amount of inoculum and unnatural means of inoculation required suggested that host and environmental factors have a major role in the pathogenesis of the acute form of the natural disease. Serial transmission of subacute and chronic pneumonia was achieved by intranasal aerosol inoculation of lung homogenate derived from abattoir cases. The clinical signs and pathological lesions were similar in most respects to the naturally-occurring disease. The pathological development of the lesions was studied in a further transmission experiment in which 12 lambs were slaughtered sequentially from 2 to 12 days after inoculation. In studying the effect of various chemotherapeutic agents on the development of chronic pneumonia it was found that both ronidazole at 100 mg/Kg and oxytetracycline suppressed the development of the disease while tylosin and penicillin suppressed the development of the lesions without completely inhibiting the growth of micro-organisms. A controlled experiment to assess the effect of pneumonia transmission on weight gain produced a significant reduction in the weight gain of treated animals but there was no correlation between the weight gain of individuals and pneumonic lesions. It was presumed that the result was due to a transitory systemic effect immediately following inoculation. Intranasal inoculation of M. ovipneumoniae cultures produced lesions in 2 caesarian-derived lambs but inoculation of 9 worm-free housed lambs was unsuccessful. The balance of evidence indicates that pneumonia in sheep, as it occurs in this country, results from the interaction of host and environmental factors with infectious agents. In acute pneumonia, bacterial multiplication in alveoli, presumably damaged by systemic agents, is responsible for the destructive changes which occur. In chronic pneumonia bacteria from the nasal cavity actively contribute to the severity of the lesions but it is unlikely that they initiate the disease process. M. ovipneumoniae is also closely associated with the lesions of chronic pneumonia but further inoculation experiments and epidemiological studies are needed to define this organism's role more closely.