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

Abstract

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.