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Experimental pneumonia in mice produced by combined administration of Bordetella parapertussis and Pasteurella haemolytica isolated from sheep
j. Comp. Path. 1991 Vol. 104
Experimental Pneumonia in Mice Produced by Combined Administration of Bordetella parapertussis and Pasteurella haemolytica Isolated from Sheep Z. Jian, M. R. Alley and B. W. Manktelow Department of VeterinaryPathology and Public Health, Massey University, PalmerstonNorth, New Zealand
Summary One-hundred-and-thirty-seven, 3-week-old, Swiss mice were inoculated intranasally with Bordetella parapertussis and Pasteurellahaemolytica which had been isolated from naturally occurring cases of chronic non-progressive pneumonia in sheep. The combined administration produced a significantly more severe bronchopneumonia which occurred earlier, persisted for a longer period and involved a higher percentage of mice than that which was produced with B. parapertussis or P. haemolytica alone. These findings demonstrate an additive or synergic action between the two agents or their metabolic products, and provide indirect evidence that such interaction may occur in ovine chronic non-progressive pneumonia.
Introduction Chronic non-progressive pneumonia is an important disease of sheep in many parts of the world (Gilmour and Brotherston, 1963; St. George and Sullivan, 1973; Alley, 1987). Although Pasteurella haemolytica, Mycoplasma ovipneumoniae and several viruses have been recovered from clinical cases of the disease, experimental reproduction of a comparable condition with any of these agents alone has proved difficult (Smith, 1964; Biberstein, Nisbet and Thompson 1967; Davies, Dungworth, Humphreys and Johnson, 1977; Sharp, Gilmour, Thompson and Rushton, 1978; Alley and Clarke, 1979; Rushton, Sharp, Gilmour and Thompson, 1979; Davies, Herceg, Jones and Thurley, 1981; Davies, Herceg and Thurley, 1982) giving rise to the current view that the disease has a multiple aetiology. Recently Bordetella parapertussis, which is one of the causative agents of whooping cough in man (Linnemann and Perry, 1977), was recovered from the airways of healthy lambs and lambs affected with chronic non-progressive pneumonia in New Zealand (Cullinane, Alley, Marshall and Manktelow, 1987). Studies of the pathogenicity of this organism have shown that it can attach itself to ovine ciliated epithelium and caused rapid ciliostasis in vitro (A1-Kaissi, 1986) and that the organism or its metabolic products can cause subacute bronchopneumonia in m i c e ( C h e n , Alley and Manktelow, 1989). 0021-9975/91/030233 + 11 $03.00/0
© 1991 Academic Press Limited
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When inoculated into 3-week-old lambs, the organism produced a mild to moderate tracheobronchitis and focal acute bronchopneumonia (Chen, Alley and Manktelow, 1988). The aim of the present study was to investigate the possibility of a synergic effect between B. parapertussis and P, haemolytica in the murine respiratory tract. Macroscopic, microscopic and ultrastructural methods were used to assess the nature and degree of severity of infection. Materials
and Methods
Experimental Animals
One-hundred-and-thirty-seven, 3-week-old, Swiss mice were used in two experiments. T h e source colony was free of enzootic respiratory disease. T h e y were kept in several cages, fed a standard diet and water ad l i b i t u m and observed for 1 week before the experiments commenced. Inoeula and Challenge Procedures
Field isolates ofP. haemolylica type A1 and B. parapertussis were cultured on blood agar plates, the growth being scraped off after i n c u b a t i o n at 36°C overnight and for 72 h, and evenly suspended in phosphate-buffered saline (PBS) at p H 7"4. T h e mice were anaesthetized with halothane and inoculated intranasally by the method described by R u s h t o n (1978). Experimental Design
Experiment 1: I n order to decide on the dose of i n o c u l u m suitable fbr the m a i n experiment, a pilot experiment was carried out with 60 mice. The mice were r a n d o m l y divided into six groups of 10. Each group received one of three different doses of B. parapertussis or P. haemolytica intranasally (Table 1). Four days after inoculation, all the mice were killed. T h e severity of p n e u m o n i a was assessed by gross observation a n d histopathological examination, and the dose of each organism capable of p r o d u c i n g p n e u m o n i a in approximately 30 per cent of mice at 4 days was regarded as suitable for Experiment 2. Experiment 2: Seventy-eight mice were r a n d o m l y divided into four groups. All the mice received two intranasal inoculations of culture or PBS at a 30 m i n interval. T h e groups, doses a n d types of inocula are shown in Table. 2. After inoculation, all mice Table 1 Experiment 1: Severity of lung lesions in mice inoculated with various doses ofP. haemolytica or B. parapertussis
Number of mice with histological lesions Group
hzocula
Dose
1 2 3 4 5 6
P, haem P. haem P. haem B. parap B, parap B. parap
1'7 x 1'7 x 1.7 x 1.6 x 1.6 x 1'6 x
l0n 10? 105 I0~° 10~ 108
PB
BP +
BP + +
Nil
Total number of mice
6 5 2 2 6 3
0 0 0 6 2 0
1 0 0 I 0 0
3 5 8 1 2 6
10 10 10 10 10 9*
* One mouse died under anaesthesia. Dose= Colony-formingunits (CFU) per 0'025 ml, PB = peribronchiolltis, BP+ = mild bronehopneumonia,BP+ + = bronchopneumonla.
235
Induced Pneumonia in Mice
Experimentally
Table 2 Experiment 2: Experimental design for the combined intranasal inoculation of B.parapertussis and P. haemolytica. Number of mice killed on days Group
Number of mice
First inoculnm
18" 20 20 20
PBS P. haem B. parap B. parap
1 2 3 4
Dose +
6-2 × 107 7'5 x 108 7'5 x l0 s
Second inoculum PBS PBS PBS P. haem 6"2 x I0 ~
2
6 6 6 6
(0) (2) (1) (3)
4
7 8 8 8
(0) (0) (3) (5)
6
5 6 6 6
(1) (0) (1) (5)
*---Two mice died under anaesthesia. + = Colony-forming units (CFU) per 0.025 ml, PBS = Phosphate buffered saline 0'025 ml. N u m b e r of mice with macroscopic lesions given in parentheses.
were observed twice daily and clinical signs recorded. Six to eight mice from each group were randomly selected and killed at 2, 4 and 6 days after inoculation. At necropsy the lungs were removed and the gross lesions were recorded and photographed. The left lung from each mouse was used for bacteriological culture and the right lung was fixed fbr transmission electron microscopy (TEM) and histological examination.
Bacterial Viable Counts and Bacteriology Ten-fold dilutions of suspensions of each of the two organisms were made in PBS by transferring 1 ml of suspension into 9 ml of diluent. A 0'1 ml suspension of each organism in No. 10 dilution tubules was placed on the surface of blood agar plates. Three replicate counts were performed on each organism and an average taken as the true count. To recover bacteria from the inoculated mice, fluid from the cut surface of the left lung was inoculated onto blood agar and incubated at 36°C overnight for P. haemolytica and for 1 week for B. parapertussis. The number of colonies of each organism was semi-quantitatively recorded as large ( + + + ), small ( + +), very small ( + ) and none
(-).
Pathology The entire right lung from each mouse was fixed in 10 per cent formol-saline, except for the small portions fixed for T E M . The lungs were embedded in paraffin wax, sectioned and stained with haematoxylin and eosin (HE). Histological examination and scoring of lesions in each lung was undertaken without prior knowledge of challenge groups and the pathological features observed were graded on a 4 point scale of severity (PB = peribronchiolitis; B P + = bronchopneumonia, involving less than 20 per cent of lobe; B P + + = involving 20 to 40 per cent of a lobe; B P + + + involving > 40 per cent of a lobe). Samples of tissue for T E M were fixed in modified Karnovsky's fixative and post-fixed with osmium tetroxide. Resin-embedded sections, 0"5 gm thick, were cut for light microscopy and 6000 nm thick for electron microscopy, The thick sections were stained with toluidine blue and thin sections were stained with uranyl acetate and lead citrate and examined on a Philips EM201c transmission electron microscope.
Results
Experiment 1 Clinical Signs. W i t h i n the first 48 h all mice showed a r o u g h e n e d coat, variable degrees of depression a n d were h u d d l e d together. R e d u c t i o n in the consump-
236
Z. Jian et aL
tion of water and food was also noted. These signs decreased in severity and after a further 48 h were present in only a few mice.
Gross Pathology. Macroscopic lesions were seen only in the lungs of mice in groups 1, 4 and 5. They consisted of very small irregular red areas mainly in the anterior portion of the left lung and the cranial and middle lobes of the right lung. Histopathology.
The lesions are summarized in Table 1. The most severe were seen in groups 4 and 5 and consisted of multifocal pneumonic areas in which numerous neutrophils and moderate numbers ofmacrophages were present in alveoli, and alveolar septa were thickened by infiltrating mononuclear ceils. There were variable numbers of neutrophils in the lamina propria of bronchi or in the lumen of bronchi and bronchioles, some of which also contained sloughed epithelium and excess mucus.
Experiment 2 Clinical Signs. The mice in groups 2 and 3 showed clinical signs similar to those seen in Experiment 1. Those in group 4 also showed signs of a similar type to those in Experiment 1 but of greater severity. Obvious signs of illness persisted in several mice in this group until the end of the experiment. No mice in group 1 showed signs of ill health. Gross Pathology. Two days after inoculation, three of the six mice killed from group 4 (B. parapertussis and P. haemolyt!ca) showed multifocal, consolidated areas which occupied 20 to 50 per cent of the lung surface. They were red brown, slightly firm and delineated fi'om the surrounding tissue. T w o mice in group 2 (P. haemolylica alone) developed moderate areas of consolidation (Fig. 1) while one mouse in group 3 (B. parapertussis alone) had a small consolidated lesion on the right cranial lobe. Four days after inoculation, p u l m o n a r y involvement was more extensive than at two days. In group 4 the consolidated areas were grey-red, firm and dense, while those in group 3 remained dark red. No lesions were ~bund in either group 1 or group 2. Six days after inoculation, the consolidated areas of lung in group 4 occupied more than 50 per cent of some lobes (Fig. 2) and were grey-red to grey in colour. They were located irregularly throughout the cranial lobes of both lungs, and in the middle and caudal lobes of the right lung. The distribution of lung lesions in group 3 was similar to those in group 4 but the lesions were smaller than those seen at 4 days after inoculation. No significant gross lesions were found in groups 1 and 2, except for one mouse in group 1 with a very small reddened area. Histopathology.
The main results of the histopathological examination of the lungs are summarized in Table 3. T h e most severe lesions in the mice inoculated with either P. haemolytica (group 2) or B. parapertussis (group 3) alone were at 2 and 4 days after inoculation, respectively. In group 3, the n u m b e r of mice showing pneumonia and the severity of the lung lesions decreased at d a y 6 whereas an increasing number of mice with severe bronchopneumonia was
Experimentally
Fig. 1, Fig. 2.
Induced Pneumonia
in Mice
237
Pneumonic area on day 2 in a mouse inoculated with P. haemolytica (group 2, experiment 2). HE x15. Large confluent pneumonic lesion on clay 6 in a mouse inoculated with P. haemolytica and B.
paraperlussis (group 4, experiment 2). HE x 15.
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Table 3 E x p e r i m e n t 2: S e v e r i t y o f h i s t o l d g i e a l l e s i o n s p r o d u c e d f o l l o w i n g the i n t r a n a s a l i n o c u l a t i o n o f m i c e w i t h B. p a r a p e r t u s s i s a n d P. h a e m o l y t i e a
Numbers of mice with histological lun~ lesions when killed after 2 to 6 days Group number
Nil
PB
1 (PBS) 2 3 4
16 15 6 l
1 2 3 4
:
BP +
BP + +
t l 7 3
0 2 2 l
BP +
-
0 0 2 l1
+
Total number 18 20 20 20
See T ab le 2 for inocula. PBS = Phosphate buffered saline, BP = peribroncholitis, BP + = mild bronchopneumonia, BP + + = moderate bronchopneumonia, BP + + + = severe bronchopneumonia.
observed in group 4 (combined administration). In group 4, there were numerous ncutrophils with mucin and sloughed epithelium in the lumina of bronchi and bronchioles on day 2. In some areas, the inflammatory reaction extended into adjacent alveoli, and involved almost whole lobes. The affected alveoli contained a sero-fibrinous exudate with excessive numbers of neutrophils (Fig. 3). The endothelial cells lining blood vessels were swollen, and there was margination of neutrophils. On days 4 and 6, the lesions were more extensive with increased numbers of macrophages with a foamy cytoplasm, numerous degenerating and necrotic neutrophils, hypercellularity of interalveolar septa (Figs. 4 and 5) and peribronchiolar and perivascular lymphocytic cuffs. A confluent type of bronchopneumonia was commonly seen at this stage. In group 2, a moderate focal bronchopneumonia was found in two mice killed on day 2. In group 1 (PBS only) one mouse showing a mild focal bronchopneumonia was found on day 2 and one mouse showing a mild peribroncholitis was found on day 4. T h e significance of the differences in the numbers of mice showing histological lesions in the various groups was tested by the Chi squared test. Significant differences were found between mice given the combined infection and mice receiving B. parapertussis or P. haemolylica alone (P< 0"05 and P < 0"01, respectively). No significant differences could be found between mice inoculated with P. haemolytica and mice receiving PBS only.
Electron Microscopy. Group 2 (P. haemolytica alone): O n day 2 there was variable collapse of alveolar spaces, increased numbers of erythrocytes a n d monocytes in capillaries, and slight oedema of interalveolar septa. Occasional intact or degenerating bacteria were found in collapsed alveolar spaces. O n day 6, no ultrastructural changes or bacteria were observed. Group 3 (B. paraperlussis): The ultrastrUctural changes seen in lungs of mice in this group were similar to those described previously by Chen et al. (1989). Group 4 (combined administration): On day 2, pulmonary alveoli in consolidated areas contained mainly neutrophils with increased numbers of phagosomes. There was also some necrotic cellular debris and a few active macrophages which occasionally contained phagocytosed neutrophils. Some of the alveolar macrophages were degenerating and contained vesiculated endoplasmic reticulum, distended nuclear envelope and showed increased electron
Experimentally Induced Pneumonia in Mice
239
Fig. 3.
Sero-fibrinous exudate and neutrophils in alveolar spaces on day 2 in a mouse inoculated with P. haemolytica and B. parapertussis (group 4, experiment 2). HE x 150.
Fig. 4.
Acute pneumonia with numerous neutrophils in alveolar spaces on day 4 in a mouse inoculated with P. haemolylica and B. par@ertussg (group 4, experiment 2). HE x 75.
Fig. 5.
Damaged bronchlole and extensive consolklation o1" surrounding alveoli on day 6 in a mouse inoculated with P. haemolytiea and P, parapertussis (group 4, experiment 2), HE x 75.
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e t al.
density of the nucleus. The interalveolar septa were oedematous and type I alveolar epithelial cells had a.swollen electron-lucent cytoplasm. Type II cells were slightly increased in number and many had increased focal electron density of the cytoplasm, swollen mitochondria and a distended endoplasmic reticulum. On day 6, neutrophils still predominated in alveolar spaces and there was a large amount of necrotic electron-dense cellular debris present. Macrophages were increased in number and contained numerous large phagosomes of variable density. Type II cells contained a multifocal electron-dense layer within the cytoplasm, and laminated bodies were replaced by varying sized vesicles. The oedema of interalveolar septa had increased in severity and large irregularly-shaped electron-lucent areas were often seen. Degenerate bacteria-like structures were often seen both within alveolar spaces and the cytoplasm of macrophages, but living organisms were difficult to identify.
Bacteriology. Pasteurella haemo~tica was not recovered, except in small numbers from the lungs of two mice in group 2, 48 h after inoculation. On days 2, 4 and 6, B. parapertussis was recovered in small numbers from 4 (of6), 7 (of8), and 6 (of 6) group 3 mice respectively; and in larger and progressively increasing numbers from 5 (of 6), 6 (of 8) and 6 (of 6) group 4 mice, respectively.
Discussion
Parainfluenza type 3 virus (PI3), adenovirus, bovine respiratory syncytial virus (RSV) and mycoplasmas have been used with P. haemolytica in experimental combined infections of the respiratory tract in sheep (Sharp el al., 1978; Rushton et al., 1979; Davies et al., 1981, 1982; Jones, Gilmour and Rae 1982; Al-Darraji, Cutlip and Graham, 1982a; A1-Darraji, Cutlip, Lehmkuhl, Graham, Kluge and Frank, 1982b), but this is the first report of combined administration with B. parapertussis. In the present investigation, the combined administration produced pneumonic lesions which occurred earlier, were significantly more severe, persisted for a longer period and involved a significantly higher percentage of mice than in mice given either B. parapertussis or P. haemolytica alone. Although both gross and microscopic lesions in the lungs were recorded in both experiments, the irregularity of distribution of gross lesions made quantitative scoring difficult. Scoring of severity of lesions observed histologically was therefore used as the basis for analysis. Previous work has shown that pulmonary clearance ofP. haemolylica in mice occurs rapidly from 4 h after inoculation to 72 h (Rushton, 1978; Rodriguez, Lopez, Merno-Moncada, Martinez-Burnes and Mondragon, 1985). The same phenomenon was seen in the present study in the group inoculated with P. haemolytica alone. However, no P. haemolytica was recovered from the mice in group 4, receiving both agents, at any of the three sampling periods. The failure to recover the organism may result, in part, from the localization of bacteria (Davies and Penwarden, 1981), sites of sampling the lungs
Experimentally Induced pneumonia in Mice
241
(Al-Darraji et al., 1982b) and intervals between inoculating the two agents (Sharp et al., 1978; Davies et al., 1982; Al-Darraji et al., 1982b). A relationship between the recovery rate of B. parapertussis and severity of lung lesions was noted in the present investigation. However, the consistency of recovery of both agents did not correlate completely with the severity of pulmonary lesions, indicating that cytotoxins of the agents may play a role in establishing and prolonging the inflammatory response. A recent study of the interaction between Bordetella bronchiseptica and PasteureUa multocida in experimental atrophic rhinitis of pigs (Chanter, Magyar and Rutter, 1989) has shown that toxin production by some strains of B. bronchiseptica is the key factor in allowing persistent colonization & t h e nasal cavity by large numbers of P. multocida. Although our results suggest that in experimental pneumonia in mice, Pasteurella toxins may be responsible for an increase in the pathogenicity of B. paraperlussis, the possibility of an interaction similar to that seen in atrophic rhinitis cannot be discounted in ovine pneumonia. Ovine isolates of B. parapertussis have been shown to produce extracellular toxic substance(s) which are capable of inducing a subacute bronchopneumoni.a when inoculated intranasally into mice (Chen, Alley and Manktelow, 1990). Pasteurella haemolytica type A1 also produces a cell-free factor believed to be an exotoxin, which is cytotoxic for both ovine and bovine alveolar macrophages (Markham and Wilke, 1980; Sutherland, Gray and Wells, 1983; Lopez, Yong and Shewen, 1987). It is possible that this factor was responsible for exacerbating the B. parapertussis bronchopneumonia in the present study. Experimental work with purified bacterial toxins will be necessary to clarify the nature of the interaction between the organisms in these species. From the present study, it is concluded that respiratory infection with B. parapertussis and P. haemolytica in mice produced a more severe acute respiratory disease than that produced with either B. parapertussis or P. haemolytica alone and that the higher morbidity and greater severity of lung lesions after the combined administration reflect a synergic or, at least, additive action between the two agents or their metabolic products. It is possible that similar phenomena play a role in the pathogenesis of ovine chronic non-progressive pneumonia. Acknowledgments
The authors wish to thank DrJ. K. Clarke for providing the Pasteurella haemolytica type A1 and Miss L. C. Cullinane for the Bordetellaparapertussis. Mrs P. M. Slack, Mr P. M. Davey and Ms. D. McMillan provided technical assistance and Mrs A. Scott typed the manuscript. References
AI-Darraji, A. M., Cutlip, R. C. and Graham, D. L. (1982a). Experimental infection of lambs with bovine respiratory syneytial virus and Pasteurella haemolytica: Pathologic studies. American Journal of Veterinary Research, 42, 224-229. A1-Darraji, A. M., Cutlip, R. C., Lehmkuhl, H. D., Graham, D. L., Kluge, J. P. and Frank, G. H. (1982b). Experimental inl~ction of'lambs with bovine respiratory syncytial virus and Pasteurella haemolytica: Clinical and microbiologic studies. American Journal of Veterinary Research, 40, 236-240.
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A1-Kaissi, A. (:1986). The tracheobronchial airways &normal and pneumonic sheep: Cytology and cytopath01ogy. Ph.D. thesis. Massey University, Palmerston North, New Zealand. Alley, M. R. (1987). The effect of chronic non-progressive pneumonia on weight gain of pasture-fed lambs. New Zealand VeterinaryJournal, 35, 163-166. Alley, M. R. and Clarke, J. K.(1979). The experimental transmission ofovine chronic non-progressive pneumonia. New Zealand VeterinaryJournal, 27, 217-220. Biberstein, E. L., Nisbet, D:,I. and Thompson, D. A. (1967). Experimental pneumonia in sheep. Journal of Comparative Pathology, 77, 181-192. Chanter, N., Magyar, N. C. and Rutter, J. M. (1989). Interactions between Bordetella bronchiseptica and toxigenic Pasteurella multocida in atrophic rhinitis of pigs. Research in Veterinary Science, 47, 48-53. Chen, W., Alley, M. R. and Manktelow, B. W. (1988). Pneumonia in lambs inoculated with Bordetella parapertussis: Clinical and pathological studies. New Zealand Veterinary Journal, 36, 138-142. Chen, W.: Alley, M. R. and Manktelow, B. W. (1989). Experimental induction of pneumonia in mice with Bordetella parapertussis isolated from sheep. Journal of Comparative Pathology, 100, 77-89. Chen, W., Alley, M. R. and Manktelow, B..W. (1990). Pneumonia in mice produced by cell-free extract of cultures of BordeteUaparapertussis. Research in VeterinaryScience, 48, 18-22. Cullinane, L. C., Alley, M. R., Marshall, R. B, and Manktelow, B. W. 11987). Bordetella paraperlussis in lambs. New Zealand VeterinaryJournal, 35, 175. Davies, D. H. and Penwarden, R. A. (1981). The phagocytic cell response of the ovine lungs to Pasteurella haemolytica. Veterinary Microbiology, 6, 183-189. Davies, D. H., Dungworth, D. L., Humphreys, S. and Johnson, A. J. (1977). Concurrent infection of lambs with parainfluenza virus type 3 and Pasteurella haemolytica. New Zealand VeterinaryJournal, 25, 263-265. Davies, D. H., Herceg, M. and Thurley, D. C. (1982). Experimental infection of lambs with an adenovirus followed by Pasleurella haemolytica. Veterinary Microbiology, 7, 369-381. Davies, D. H., Herceg, M., Jones, B. A. H. and Thurley, D. C. (1981). The pathogenesis of sequential infection with parainfluenza virus type 3 and Pasteurella haemolytica in sheep. Veterinary Microbiology, 6, 173-182. Gihnour, N. J. L. and Brotherston, J. G. (1963). Clinical and pathological findings in an outbreak of pneumonia in sheep. Journal of Comparative Pathology, 73, 329-336. Jones, G. E., Gilmour, J. S. and Rae, A. G. (1982). The effect of Mycoplasma ovipneumoniae and Pasteurella haemolytica on specific-pathogen-free lambs. Journal of Comparative Pathology, 92, 261-266. Linnemann, C. C. and Perry, E. B. (1977). BordeteUaparapertussis: Recent experience and review of the literature. American Journal of Diseases of Children, 131, 560-563. Lopez, A., Yong, S. and Shewen, P. (1987). Effect of intratracheal inoculation of Pasteurella haemolytica cytotoxin on the integrity of rat lung. Canadian Journal of Veterinary Research, 51, 533-535. Markham, R . J . F . and Wilke, B. N. (1980). Interaction between Pasteurella haemolytica and bovine alveolar macrophages: Cytotoxic effect on macrophages and impaired phagocytosis. American Journal of Veterinary Research, 41, 18-22. Rodriguez, L. M., Lopez, A., Merno-Moncada, M., Martinez-Burnes, J. and Mondragon, I. (1985). Tracheal versus pulmonary deposition and clearance of inhaled Pasteurella haemolytica or Staphylococcus aureus in mice. Canadian Journal of Comparative Medicine, 49, 323-326. Rushton, B. (1978). Induction &pneumonia in mice with Pasleurella haemolytica. Journal of Comparative Pathology, 88, 477-480. Rushton, B., Sharp, J. M., Gilmour, N . J . L . and Thompson, D. A. (1979). Pathology of an experimental infection of specific-pathogen-free lambs with parainfluenza virus type 3 and Pasteurellahaemolytica. Journal of ComparativePathology, 89, 321-329.
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Sharp, J. M., Gilmour, J. L., Thompson, D. A. and Rushton, B. (1978). Experimental infection of specific-pathogen-free lambs with parainfluenza virus type 3 and Pasteurella haemolytica. Journal of Comparative Pathology, 88, 237-243. Smith, G. R. (1964). Production of pneumonia in adult sheep with cultures of Pasteurella haemolytica. Journal of Comparative Pathology, 74, 241-249. St. George, T. D. and Sullivan, N. D. (1973). Pneumonias of sheep in Australia. Veterinary Review No. 13. The Post-Graduate Foundation of Veterinary Science. University of Sydney. Sutherland, A. D., Gray, E. and Wells, P. W. (1983). Cytotoxic effect of Pasteurella haemolytica on ovine bronchoalveolar macrophages in vitro. VeterinaryMicrobiology, 8, 3-15.
E Received, July 1st, 1990 7 Accepted, November 3rd, 1990.J
Experimental Pneumonia in Mice Produced by Combined Administration of Bordetella parapertussis and Pasteurella haemolytica Isolated from Sheep Z. Jian, M. R. Alley and B. W. Manktelow Department of VeterinaryPathology and Public Health, Massey University, PalmerstonNorth, New Zealand
Summary One-hundred-and-thirty-seven, 3-week-old, Swiss mice were inoculated intranasally with Bordetella parapertussis and Pasteurellahaemolytica which had been isolated from naturally occurring cases of chronic non-progressive pneumonia in sheep. The combined administration produced a significantly more severe bronchopneumonia which occurred earlier, persisted for a longer period and involved a higher percentage of mice than that which was produced with B. parapertussis or P. haemolytica alone. These findings demonstrate an additive or synergic action between the two agents or their metabolic products, and provide indirect evidence that such interaction may occur in ovine chronic non-progressive pneumonia.
Introduction Chronic non-progressive pneumonia is an important disease of sheep in many parts of the world (Gilmour and Brotherston, 1963; St. George and Sullivan, 1973; Alley, 1987). Although Pasteurella haemolytica, Mycoplasma ovipneumoniae and several viruses have been recovered from clinical cases of the disease, experimental reproduction of a comparable condition with any of these agents alone has proved difficult (Smith, 1964; Biberstein, Nisbet and Thompson 1967; Davies, Dungworth, Humphreys and Johnson, 1977; Sharp, Gilmour, Thompson and Rushton, 1978; Alley and Clarke, 1979; Rushton, Sharp, Gilmour and Thompson, 1979; Davies, Herceg, Jones and Thurley, 1981; Davies, Herceg and Thurley, 1982) giving rise to the current view that the disease has a multiple aetiology. Recently Bordetella parapertussis, which is one of the causative agents of whooping cough in man (Linnemann and Perry, 1977), was recovered from the airways of healthy lambs and lambs affected with chronic non-progressive pneumonia in New Zealand (Cullinane, Alley, Marshall and Manktelow, 1987). Studies of the pathogenicity of this organism have shown that it can attach itself to ovine ciliated epithelium and caused rapid ciliostasis in vitro (A1-Kaissi, 1986) and that the organism or its metabolic products can cause subacute bronchopneumonia in m i c e ( C h e n , Alley and Manktelow, 1989). 0021-9975/91/030233 + 11 $03.00/0
© 1991 Academic Press Limited
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Z. J i a n et al.
When inoculated into 3-week-old lambs, the organism produced a mild to moderate tracheobronchitis and focal acute bronchopneumonia (Chen, Alley and Manktelow, 1988). The aim of the present study was to investigate the possibility of a synergic effect between B. parapertussis and P, haemolytica in the murine respiratory tract. Macroscopic, microscopic and ultrastructural methods were used to assess the nature and degree of severity of infection. Materials
and Methods
Experimental Animals
One-hundred-and-thirty-seven, 3-week-old, Swiss mice were used in two experiments. T h e source colony was free of enzootic respiratory disease. T h e y were kept in several cages, fed a standard diet and water ad l i b i t u m and observed for 1 week before the experiments commenced. Inoeula and Challenge Procedures
Field isolates ofP. haemolylica type A1 and B. parapertussis were cultured on blood agar plates, the growth being scraped off after i n c u b a t i o n at 36°C overnight and for 72 h, and evenly suspended in phosphate-buffered saline (PBS) at p H 7"4. T h e mice were anaesthetized with halothane and inoculated intranasally by the method described by R u s h t o n (1978). Experimental Design
Experiment 1: I n order to decide on the dose of i n o c u l u m suitable fbr the m a i n experiment, a pilot experiment was carried out with 60 mice. The mice were r a n d o m l y divided into six groups of 10. Each group received one of three different doses of B. parapertussis or P. haemolytica intranasally (Table 1). Four days after inoculation, all the mice were killed. T h e severity of p n e u m o n i a was assessed by gross observation a n d histopathological examination, and the dose of each organism capable of p r o d u c i n g p n e u m o n i a in approximately 30 per cent of mice at 4 days was regarded as suitable for Experiment 2. Experiment 2: Seventy-eight mice were r a n d o m l y divided into four groups. All the mice received two intranasal inoculations of culture or PBS at a 30 m i n interval. T h e groups, doses a n d types of inocula are shown in Table. 2. After inoculation, all mice Table 1 Experiment 1: Severity of lung lesions in mice inoculated with various doses ofP. haemolytica or B. parapertussis
Number of mice with histological lesions Group
hzocula
Dose
1 2 3 4 5 6
P, haem P. haem P. haem B. parap B, parap B. parap
1'7 x 1'7 x 1.7 x 1.6 x 1.6 x 1'6 x
l0n 10? 105 I0~° 10~ 108
PB
BP +
BP + +
Nil
Total number of mice
6 5 2 2 6 3
0 0 0 6 2 0
1 0 0 I 0 0
3 5 8 1 2 6
10 10 10 10 10 9*
* One mouse died under anaesthesia. Dose= Colony-formingunits (CFU) per 0'025 ml, PB = peribronchiolltis, BP+ = mild bronehopneumonia,BP+ + = bronchopneumonla.
235
Induced Pneumonia in Mice
Experimentally
Table 2 Experiment 2: Experimental design for the combined intranasal inoculation of B.parapertussis and P. haemolytica. Number of mice killed on days Group
Number of mice
First inoculnm
18" 20 20 20
PBS P. haem B. parap B. parap
1 2 3 4
Dose +
6-2 × 107 7'5 x 108 7'5 x l0 s
Second inoculum PBS PBS PBS P. haem 6"2 x I0 ~
2
6 6 6 6
(0) (2) (1) (3)
4
7 8 8 8
(0) (0) (3) (5)
6
5 6 6 6
(1) (0) (1) (5)
*---Two mice died under anaesthesia. + = Colony-forming units (CFU) per 0.025 ml, PBS = Phosphate buffered saline 0'025 ml. N u m b e r of mice with macroscopic lesions given in parentheses.
were observed twice daily and clinical signs recorded. Six to eight mice from each group were randomly selected and killed at 2, 4 and 6 days after inoculation. At necropsy the lungs were removed and the gross lesions were recorded and photographed. The left lung from each mouse was used for bacteriological culture and the right lung was fixed fbr transmission electron microscopy (TEM) and histological examination.
Bacterial Viable Counts and Bacteriology Ten-fold dilutions of suspensions of each of the two organisms were made in PBS by transferring 1 ml of suspension into 9 ml of diluent. A 0'1 ml suspension of each organism in No. 10 dilution tubules was placed on the surface of blood agar plates. Three replicate counts were performed on each organism and an average taken as the true count. To recover bacteria from the inoculated mice, fluid from the cut surface of the left lung was inoculated onto blood agar and incubated at 36°C overnight for P. haemolytica and for 1 week for B. parapertussis. The number of colonies of each organism was semi-quantitatively recorded as large ( + + + ), small ( + +), very small ( + ) and none
(-).
Pathology The entire right lung from each mouse was fixed in 10 per cent formol-saline, except for the small portions fixed for T E M . The lungs were embedded in paraffin wax, sectioned and stained with haematoxylin and eosin (HE). Histological examination and scoring of lesions in each lung was undertaken without prior knowledge of challenge groups and the pathological features observed were graded on a 4 point scale of severity (PB = peribronchiolitis; B P + = bronchopneumonia, involving less than 20 per cent of lobe; B P + + = involving 20 to 40 per cent of a lobe; B P + + + involving > 40 per cent of a lobe). Samples of tissue for T E M were fixed in modified Karnovsky's fixative and post-fixed with osmium tetroxide. Resin-embedded sections, 0"5 gm thick, were cut for light microscopy and 6000 nm thick for electron microscopy, The thick sections were stained with toluidine blue and thin sections were stained with uranyl acetate and lead citrate and examined on a Philips EM201c transmission electron microscope.
Results
Experiment 1 Clinical Signs. W i t h i n the first 48 h all mice showed a r o u g h e n e d coat, variable degrees of depression a n d were h u d d l e d together. R e d u c t i o n in the consump-
236
Z. Jian et aL
tion of water and food was also noted. These signs decreased in severity and after a further 48 h were present in only a few mice.
Gross Pathology. Macroscopic lesions were seen only in the lungs of mice in groups 1, 4 and 5. They consisted of very small irregular red areas mainly in the anterior portion of the left lung and the cranial and middle lobes of the right lung. Histopathology.
The lesions are summarized in Table 1. The most severe were seen in groups 4 and 5 and consisted of multifocal pneumonic areas in which numerous neutrophils and moderate numbers ofmacrophages were present in alveoli, and alveolar septa were thickened by infiltrating mononuclear ceils. There were variable numbers of neutrophils in the lamina propria of bronchi or in the lumen of bronchi and bronchioles, some of which also contained sloughed epithelium and excess mucus.
Experiment 2 Clinical Signs. The mice in groups 2 and 3 showed clinical signs similar to those seen in Experiment 1. Those in group 4 also showed signs of a similar type to those in Experiment 1 but of greater severity. Obvious signs of illness persisted in several mice in this group until the end of the experiment. No mice in group 1 showed signs of ill health. Gross Pathology. Two days after inoculation, three of the six mice killed from group 4 (B. parapertussis and P. haemolyt!ca) showed multifocal, consolidated areas which occupied 20 to 50 per cent of the lung surface. They were red brown, slightly firm and delineated fi'om the surrounding tissue. T w o mice in group 2 (P. haemolylica alone) developed moderate areas of consolidation (Fig. 1) while one mouse in group 3 (B. parapertussis alone) had a small consolidated lesion on the right cranial lobe. Four days after inoculation, p u l m o n a r y involvement was more extensive than at two days. In group 4 the consolidated areas were grey-red, firm and dense, while those in group 3 remained dark red. No lesions were ~bund in either group 1 or group 2. Six days after inoculation, the consolidated areas of lung in group 4 occupied more than 50 per cent of some lobes (Fig. 2) and were grey-red to grey in colour. They were located irregularly throughout the cranial lobes of both lungs, and in the middle and caudal lobes of the right lung. The distribution of lung lesions in group 3 was similar to those in group 4 but the lesions were smaller than those seen at 4 days after inoculation. No significant gross lesions were found in groups 1 and 2, except for one mouse in group 1 with a very small reddened area. Histopathology.
The main results of the histopathological examination of the lungs are summarized in Table 3. T h e most severe lesions in the mice inoculated with either P. haemolytica (group 2) or B. parapertussis (group 3) alone were at 2 and 4 days after inoculation, respectively. In group 3, the n u m b e r of mice showing pneumonia and the severity of the lung lesions decreased at d a y 6 whereas an increasing number of mice with severe bronchopneumonia was
Experimentally
Fig. 1, Fig. 2.
Induced Pneumonia
in Mice
237
Pneumonic area on day 2 in a mouse inoculated with P. haemolytica (group 2, experiment 2). HE x15. Large confluent pneumonic lesion on clay 6 in a mouse inoculated with P. haemolytica and B.
paraperlussis (group 4, experiment 2). HE x 15.
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Z. J i a n e t al.
Table 3 E x p e r i m e n t 2: S e v e r i t y o f h i s t o l d g i e a l l e s i o n s p r o d u c e d f o l l o w i n g the i n t r a n a s a l i n o c u l a t i o n o f m i c e w i t h B. p a r a p e r t u s s i s a n d P. h a e m o l y t i e a
Numbers of mice with histological lun~ lesions when killed after 2 to 6 days Group number
Nil
PB
1 (PBS) 2 3 4
16 15 6 l
1 2 3 4
:
BP +
BP + +
t l 7 3
0 2 2 l
BP +
-
0 0 2 l1
+
Total number 18 20 20 20
See T ab le 2 for inocula. PBS = Phosphate buffered saline, BP = peribroncholitis, BP + = mild bronchopneumonia, BP + + = moderate bronchopneumonia, BP + + + = severe bronchopneumonia.
observed in group 4 (combined administration). In group 4, there were numerous ncutrophils with mucin and sloughed epithelium in the lumina of bronchi and bronchioles on day 2. In some areas, the inflammatory reaction extended into adjacent alveoli, and involved almost whole lobes. The affected alveoli contained a sero-fibrinous exudate with excessive numbers of neutrophils (Fig. 3). The endothelial cells lining blood vessels were swollen, and there was margination of neutrophils. On days 4 and 6, the lesions were more extensive with increased numbers of macrophages with a foamy cytoplasm, numerous degenerating and necrotic neutrophils, hypercellularity of interalveolar septa (Figs. 4 and 5) and peribronchiolar and perivascular lymphocytic cuffs. A confluent type of bronchopneumonia was commonly seen at this stage. In group 2, a moderate focal bronchopneumonia was found in two mice killed on day 2. In group 1 (PBS only) one mouse showing a mild focal bronchopneumonia was found on day 2 and one mouse showing a mild peribroncholitis was found on day 4. T h e significance of the differences in the numbers of mice showing histological lesions in the various groups was tested by the Chi squared test. Significant differences were found between mice given the combined infection and mice receiving B. parapertussis or P. haemolylica alone (P< 0"05 and P < 0"01, respectively). No significant differences could be found between mice inoculated with P. haemolytica and mice receiving PBS only.
Electron Microscopy. Group 2 (P. haemolytica alone): O n day 2 there was variable collapse of alveolar spaces, increased numbers of erythrocytes a n d monocytes in capillaries, and slight oedema of interalveolar septa. Occasional intact or degenerating bacteria were found in collapsed alveolar spaces. O n day 6, no ultrastructural changes or bacteria were observed. Group 3 (B. paraperlussis): The ultrastrUctural changes seen in lungs of mice in this group were similar to those described previously by Chen et al. (1989). Group 4 (combined administration): On day 2, pulmonary alveoli in consolidated areas contained mainly neutrophils with increased numbers of phagosomes. There was also some necrotic cellular debris and a few active macrophages which occasionally contained phagocytosed neutrophils. Some of the alveolar macrophages were degenerating and contained vesiculated endoplasmic reticulum, distended nuclear envelope and showed increased electron
Experimentally Induced Pneumonia in Mice
239
Fig. 3.
Sero-fibrinous exudate and neutrophils in alveolar spaces on day 2 in a mouse inoculated with P. haemolytica and B. parapertussis (group 4, experiment 2). HE x 150.
Fig. 4.
Acute pneumonia with numerous neutrophils in alveolar spaces on day 4 in a mouse inoculated with P. haemolylica and B. par@ertussg (group 4, experiment 2). HE x 75.
Fig. 5.
Damaged bronchlole and extensive consolklation o1" surrounding alveoli on day 6 in a mouse inoculated with P. haemolytiea and P, parapertussis (group 4, experiment 2), HE x 75.
240
z. Jian
e t al.
density of the nucleus. The interalveolar septa were oedematous and type I alveolar epithelial cells had a.swollen electron-lucent cytoplasm. Type II cells were slightly increased in number and many had increased focal electron density of the cytoplasm, swollen mitochondria and a distended endoplasmic reticulum. On day 6, neutrophils still predominated in alveolar spaces and there was a large amount of necrotic electron-dense cellular debris present. Macrophages were increased in number and contained numerous large phagosomes of variable density. Type II cells contained a multifocal electron-dense layer within the cytoplasm, and laminated bodies were replaced by varying sized vesicles. The oedema of interalveolar septa had increased in severity and large irregularly-shaped electron-lucent areas were often seen. Degenerate bacteria-like structures were often seen both within alveolar spaces and the cytoplasm of macrophages, but living organisms were difficult to identify.
Bacteriology. Pasteurella haemo~tica was not recovered, except in small numbers from the lungs of two mice in group 2, 48 h after inoculation. On days 2, 4 and 6, B. parapertussis was recovered in small numbers from 4 (of6), 7 (of8), and 6 (of 6) group 3 mice respectively; and in larger and progressively increasing numbers from 5 (of 6), 6 (of 8) and 6 (of 6) group 4 mice, respectively.
Discussion
Parainfluenza type 3 virus (PI3), adenovirus, bovine respiratory syncytial virus (RSV) and mycoplasmas have been used with P. haemolytica in experimental combined infections of the respiratory tract in sheep (Sharp el al., 1978; Rushton et al., 1979; Davies et al., 1981, 1982; Jones, Gilmour and Rae 1982; Al-Darraji, Cutlip and Graham, 1982a; A1-Darraji, Cutlip, Lehmkuhl, Graham, Kluge and Frank, 1982b), but this is the first report of combined administration with B. parapertussis. In the present investigation, the combined administration produced pneumonic lesions which occurred earlier, were significantly more severe, persisted for a longer period and involved a significantly higher percentage of mice than in mice given either B. parapertussis or P. haemolytica alone. Although both gross and microscopic lesions in the lungs were recorded in both experiments, the irregularity of distribution of gross lesions made quantitative scoring difficult. Scoring of severity of lesions observed histologically was therefore used as the basis for analysis. Previous work has shown that pulmonary clearance ofP. haemolylica in mice occurs rapidly from 4 h after inoculation to 72 h (Rushton, 1978; Rodriguez, Lopez, Merno-Moncada, Martinez-Burnes and Mondragon, 1985). The same phenomenon was seen in the present study in the group inoculated with P. haemolytica alone. However, no P. haemolytica was recovered from the mice in group 4, receiving both agents, at any of the three sampling periods. The failure to recover the organism may result, in part, from the localization of bacteria (Davies and Penwarden, 1981), sites of sampling the lungs
Experimentally Induced pneumonia in Mice
241
(Al-Darraji et al., 1982b) and intervals between inoculating the two agents (Sharp et al., 1978; Davies et al., 1982; Al-Darraji et al., 1982b). A relationship between the recovery rate of B. parapertussis and severity of lung lesions was noted in the present investigation. However, the consistency of recovery of both agents did not correlate completely with the severity of pulmonary lesions, indicating that cytotoxins of the agents may play a role in establishing and prolonging the inflammatory response. A recent study of the interaction between Bordetella bronchiseptica and PasteureUa multocida in experimental atrophic rhinitis of pigs (Chanter, Magyar and Rutter, 1989) has shown that toxin production by some strains of B. bronchiseptica is the key factor in allowing persistent colonization & t h e nasal cavity by large numbers of P. multocida. Although our results suggest that in experimental pneumonia in mice, Pasteurella toxins may be responsible for an increase in the pathogenicity of B. paraperlussis, the possibility of an interaction similar to that seen in atrophic rhinitis cannot be discounted in ovine pneumonia. Ovine isolates of B. parapertussis have been shown to produce extracellular toxic substance(s) which are capable of inducing a subacute bronchopneumoni.a when inoculated intranasally into mice (Chen, Alley and Manktelow, 1990). Pasteurella haemolytica type A1 also produces a cell-free factor believed to be an exotoxin, which is cytotoxic for both ovine and bovine alveolar macrophages (Markham and Wilke, 1980; Sutherland, Gray and Wells, 1983; Lopez, Yong and Shewen, 1987). It is possible that this factor was responsible for exacerbating the B. parapertussis bronchopneumonia in the present study. Experimental work with purified bacterial toxins will be necessary to clarify the nature of the interaction between the organisms in these species. From the present study, it is concluded that respiratory infection with B. parapertussis and P. haemolytica in mice produced a more severe acute respiratory disease than that produced with either B. parapertussis or P. haemolytica alone and that the higher morbidity and greater severity of lung lesions after the combined administration reflect a synergic or, at least, additive action between the two agents or their metabolic products. It is possible that similar phenomena play a role in the pathogenesis of ovine chronic non-progressive pneumonia. Acknowledgments
The authors wish to thank DrJ. K. Clarke for providing the Pasteurella haemolytica type A1 and Miss L. C. Cullinane for the Bordetellaparapertussis. Mrs P. M. Slack, Mr P. M. Davey and Ms. D. McMillan provided technical assistance and Mrs A. Scott typed the manuscript. References
AI-Darraji, A. M., Cutlip, R. C. and Graham, D. L. (1982a). Experimental infection of lambs with bovine respiratory syneytial virus and Pasteurella haemolytica: Pathologic studies. American Journal of Veterinary Research, 42, 224-229. A1-Darraji, A. M., Cutlip, R. C., Lehmkuhl, H. D., Graham, D. L., Kluge, J. P. and Frank, G. H. (1982b). Experimental inl~ction of'lambs with bovine respiratory syncytial virus and Pasteurella haemolytica: Clinical and microbiologic studies. American Journal of Veterinary Research, 40, 236-240.
242
Z. Jian et al.
A1-Kaissi, A. (:1986). The tracheobronchial airways &normal and pneumonic sheep: Cytology and cytopath01ogy. Ph.D. thesis. Massey University, Palmerston North, New Zealand. Alley, M. R. (1987). The effect of chronic non-progressive pneumonia on weight gain of pasture-fed lambs. New Zealand VeterinaryJournal, 35, 163-166. Alley, M. R. and Clarke, J. K.(1979). The experimental transmission ofovine chronic non-progressive pneumonia. New Zealand VeterinaryJournal, 27, 217-220. Biberstein, E. L., Nisbet, D:,I. and Thompson, D. A. (1967). Experimental pneumonia in sheep. Journal of Comparative Pathology, 77, 181-192. Chanter, N., Magyar, N. C. and Rutter, J. M. (1989). Interactions between Bordetella bronchiseptica and toxigenic Pasteurella multocida in atrophic rhinitis of pigs. Research in Veterinary Science, 47, 48-53. Chen, W., Alley, M. R. and Manktelow, B. W. (1988). Pneumonia in lambs inoculated with Bordetella parapertussis: Clinical and pathological studies. New Zealand Veterinary Journal, 36, 138-142. Chen, W.: Alley, M. R. and Manktelow, B. W. (1989). Experimental induction of pneumonia in mice with Bordetella parapertussis isolated from sheep. Journal of Comparative Pathology, 100, 77-89. Chen, W., Alley, M. R. and Manktelow, B..W. (1990). Pneumonia in mice produced by cell-free extract of cultures of BordeteUaparapertussis. Research in VeterinaryScience, 48, 18-22. Cullinane, L. C., Alley, M. R., Marshall, R. B, and Manktelow, B. W. 11987). Bordetella paraperlussis in lambs. New Zealand VeterinaryJournal, 35, 175. Davies, D. H. and Penwarden, R. A. (1981). The phagocytic cell response of the ovine lungs to Pasteurella haemolytica. Veterinary Microbiology, 6, 183-189. Davies, D. H., Dungworth, D. L., Humphreys, S. and Johnson, A. J. (1977). Concurrent infection of lambs with parainfluenza virus type 3 and Pasteurella haemolytica. New Zealand VeterinaryJournal, 25, 263-265. Davies, D. H., Herceg, M. and Thurley, D. C. (1982). Experimental infection of lambs with an adenovirus followed by Pasleurella haemolytica. Veterinary Microbiology, 7, 369-381. Davies, D. H., Herceg, M., Jones, B. A. H. and Thurley, D. C. (1981). The pathogenesis of sequential infection with parainfluenza virus type 3 and Pasteurella haemolytica in sheep. Veterinary Microbiology, 6, 173-182. Gihnour, N. J. L. and Brotherston, J. G. (1963). Clinical and pathological findings in an outbreak of pneumonia in sheep. Journal of Comparative Pathology, 73, 329-336. Jones, G. E., Gilmour, J. S. and Rae, A. G. (1982). The effect of Mycoplasma ovipneumoniae and Pasteurella haemolytica on specific-pathogen-free lambs. Journal of Comparative Pathology, 92, 261-266. Linnemann, C. C. and Perry, E. B. (1977). BordeteUaparapertussis: Recent experience and review of the literature. American Journal of Diseases of Children, 131, 560-563. Lopez, A., Yong, S. and Shewen, P. (1987). Effect of intratracheal inoculation of Pasteurella haemolytica cytotoxin on the integrity of rat lung. Canadian Journal of Veterinary Research, 51, 533-535. Markham, R . J . F . and Wilke, B. N. (1980). Interaction between Pasteurella haemolytica and bovine alveolar macrophages: Cytotoxic effect on macrophages and impaired phagocytosis. American Journal of Veterinary Research, 41, 18-22. Rodriguez, L. M., Lopez, A., Merno-Moncada, M., Martinez-Burnes, J. and Mondragon, I. (1985). Tracheal versus pulmonary deposition and clearance of inhaled Pasteurella haemolytica or Staphylococcus aureus in mice. Canadian Journal of Comparative Medicine, 49, 323-326. Rushton, B. (1978). Induction &pneumonia in mice with Pasleurella haemolytica. Journal of Comparative Pathology, 88, 477-480. Rushton, B., Sharp, J. M., Gilmour, N . J . L . and Thompson, D. A. (1979). Pathology of an experimental infection of specific-pathogen-free lambs with parainfluenza virus type 3 and Pasteurellahaemolytica. Journal of ComparativePathology, 89, 321-329.
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Sharp, J. M., Gilmour, J. L., Thompson, D. A. and Rushton, B. (1978). Experimental infection of specific-pathogen-free lambs with parainfluenza virus type 3 and Pasteurella haemolytica. Journal of Comparative Pathology, 88, 237-243. Smith, G. R. (1964). Production of pneumonia in adult sheep with cultures of Pasteurella haemolytica. Journal of Comparative Pathology, 74, 241-249. St. George, T. D. and Sullivan, N. D. (1973). Pneumonias of sheep in Australia. Veterinary Review No. 13. The Post-Graduate Foundation of Veterinary Science. University of Sydney. Sutherland, A. D., Gray, E. and Wells, P. W. (1983). Cytotoxic effect of Pasteurella haemolytica on ovine bronchoalveolar macrophages in vitro. VeterinaryMicrobiology, 8, 3-15.
E Received, July 1st, 1990 7 Accepted, November 3rd, 1990.J