Comparison of single strains of four serotypes of Pasteurella haemolytica biotype A in experimental pneumonia of sheep

SHORT COMMUNICAnONS Research in Veterinary Science /986, 40. /36-/37

Comparison of single strains of four serotypes of Pasteurella haemolytica biotype A in experimental pneumonia of sheep J. S. GILMOUR, G. E. JONES, A. G. RAE, M. QUIRIE, Moredun Research Institute. 408 Gilmerton Road. Edinburgh EH17 7JH

Single strains of serotypes AI, A2, A7 and A9 of Pasteurella haemolytica were separately used in combination with Mycoplasma ovipneumoniae to reproduce pneumonia. Macroscopically and microscopically the pneumonias associated with individual serotypes were similar and it is concluded that serotypes of P haemolytica isolated with low frequency in field disease may be equally virulent to common serotypes. ELEVEN serotypes of Pasteurella haemolytica biotype A are currently defined and others may await recognition, because I I per cent of isolates in a recent survey were classified as untypable (Fraser et al 1982). Some vaccines produced currently to combat ovine pasteurellosis have incorporated antigens from a range of serotypes of the bacterium as an immune serotype specificity has been demonstrated (Evans and Wells 1979, Gilmour et al 1979). Also because differences between serotypes in the antigen preparation and the adjuvant type necessary for optimal stimulation of protective immunity have been shown (Gilmour et aI1983). However, there is wide variation in the prevalence of individual serotypes isolated from lungs of sheep with pneumonic pasteurellosis (Thompson et al 1977, Fraser et a11982) and inconsistent differences were observed in the relative frequency of serotypes isolated from nasopharyngeal mucus between flocks affected with pneumonic pasteurellosis and those unaffected (Biberstein and Thompson 1966). P haemolytica is also implicated in the aetiology of atypical pneumonia in the sheep and, again, different serotypes are isolated from affected lungs with a wide variation in frequency (Jones and Gilmour 1983). Differences in pathogenicity of serotypes might account for some of these variations and if present could influence vaccine development but so far this possibility has not been directly examined. A small scale examination of potential differences in the pathogenicity of serotypes is described here, using common (A I and A2) and less common (A7 and A9) serotypes in experimental chronic pneumonia of sheep. Twenty Cheviot lambs, conventionally reared on a farm with no history of outbreaks of pneumonia over the previous 10 years, were housed at eight to 10 weeks old. Four groups of five animals each, randomised except for separation of siblings, were accommodated in separate loose boxes with no common airspace. A homogenate of pneumonic tissue was prepared as described previously (Jones et al 1978). This was treated with 2 mg ml- 1 ampicillin (Penbritin; Beecham) for four hours at 37°C immediately before injection. The only microorganism demonstrable in the lung homogenate after this

treatment was Mycoplasma ovipneumoniae, present at 1()6 colour changing units (ccu) per 0·2 ml. Each of the four serotypes of P haemolytica studied (AI, A2, A7 and A9) was the only bacterium isolated from a field case of acute ovine pneumonia and had undergone. only two passes in 7 per cent sheep blood agar before storage at -70°C in nutrient broth. No other organisms were sought in these cases. Five-hour shaken cultures were diluted in phosphate buffered saline and adjusted to similar optical densities. Titres of each were AI: I, 2 X 107 colony forming units (cfu) ml l; A2: 2· 8 x 107 cfu ml t; A7: 8· 3 x 1()6 cfu ml l; and A9: 1·4x 107 cfu ml- I • Nasal swabs were collected immediately before the first treatment (day 0), which consisted of 8 ml pneumonic lung homogenate administered intratracheally and 5 mg ampicillin (kg bodyweight) "! injected intramuscularly. A further injection of 125 mg ampicillin was given to all lambs on each of the subsequent two days. On day 6, each serotype of P haemolytica was administered to a group of lambs, each lamb receiving 2 ml intranasally and 2 ml intratracheally. The animals were killed on days 14 or 15. Diagrams of the pulmonary lesions provided a quantitative assessment of the extent of macroscopic pneumonia (lung lesion score) visible on the dorsal and ventral surfaces (Jones et al 1978). Microbiological and histopathological examinations of pulmonary tissues were done as described previously (J ones et a11978, Gilmour et aI1982). Neither P haemolytica nor mycoplasmas were isolated from the pre-treatment nasal swabs. One animal developed an afebrile hyperpnoea between days 0 and 6. Clinical examinations of individual sheep were discontinued from day 6 to prevent the possibility of serotype cross infection. M ovipneumoniae was the only mycoplasma isolated from all lungs in titres ranging from IW to more than 108 ccu g-I of tissue at necropsy. The only bacterium isolated, P haemolytica biotype A, was found in the lungs of all lambs except one in each of three groups in numbers varying from 102 to 105 cfu g - 1 of tissue. Only the serotype administered was recovered from each animal. There were no significant inter-group differences in mean bacterial numbers or number of animals positive for either microorganism. Neither viruses, in particular parainfluenza type 3 virus, nor chlamydia were isolated. All animals except one given serotype A9 had macroscopic lesions in the apical and dependent areas of the lung. The mean lung lesion score and other pathological features are recorded in Table I. Histological examination revealed a proliferative exudative pneumonia in 19 lambs, characterised by brochiolar epithelial hyperplasia, exudation of r

r

136

r

137

P haemolytica pneumonia of sheep TABLE 1: Pathological findings in four groups of five lambs. each given lung homogenate and a different serotype of Pasteurella haemofytica biotype A

Serotype of P haemofytica

biotype A

lesionscore 1%)

24 ± 23 ± 23 ± 34 ±

1

2 7 9 PE

Mean ± sd consolidated lung

Number of lambs with 'PE pneumonia with PE pneumonia necrotic/organised alone lesions

3·5 (14-34)' 6·0 (8-39) 7'5110-43) 9·7 (0-53)

Pleurisy

1 4 3 3 2 2 1 4 2 3 1 3

Range in brackets Proliferative exudative

macrophages and neutrophils and lymphocytic cuffs of bronchioles and blood vessels. Lymphoid nodular hyperplasia occurred in one lamb (given P haemolytica A7), and a further three (two given A9 and one given A7) had hyaline scars. No significant difference was observed between groups with respect to the extent of these changes. However, additional changes comprising prominent foci of necrotic alveolitis and, or, organised post necrotic inflammatory lesions of the form commonly seen in experimental pneumonic pasteurellosis (Rushton et al1979) were seen in II lambs. Fibrocellular pleurisy was present in 10 lambs. Both the pleurisy and the necrotic lesion were seen in at least one lamb in every group. Only a very mild perivascular infiltrate of lymphocytes was noted in the single lamb with no gross lesions. These results clearly associated each of the four serotypes of P haemolytica with the production of pneumonia. In each group, the pneumonia had the same range of characteristics, namely a proliferative exudative pneumonia closely resembling the early stages of atypical pneumonia (Gilmour et al 1982), or a proliferative exudative pneumonia accompanied by the necrotising changes more commonly seen in pneumonic pasteurellosis. The small numbers per group and the presence within each group of the complete range of pathology seen prohibited statistical comparison of individual groups. Furthermore, the close similarity between groups in the frequency of pleurisy and in mean lung lesion score supports the conclusion that there is n,o clear difference in pathogenicity between these serotypes of P haemolytica. Thus the differences in frequency of isolation of these serotypes in field outbreaks is not reflected by differences in virulence. M ovipneumoniae alone is capable of producing a chronic pneumonia (Gilmour et al 1979) and this may account for the variation observed within the groups in this study. On the other hand, this was unlikely to have masked an important difference between groups since at least some animals in each group displayed severe tissue damage. The possibility of. strain differences within serotypes may, however, be relevant. This aspect has not yet been extensively investigated, though recent work (Donachie et al 1984) showed that two untypable strains of P haemolytica were ineffective when given in conjunction with parainfluenza type 3 virus in attempts to reproduce pneumonic pasteurellosis experimentally. The strains employed in the present study as well as those

described in the prevalence reports (Thompson et al 1977, Fraser et al 1982) originated from outbreaks of pneumonic pasteurellosis. Virulence, however, cannot be assumed in such circumstances since multiple serotypes and untypable strains are frequently isolated from lungs in the naturally occurring disease. Nevertheless, even if strain differences within serotypes do exist, this report confirms the potential pathogenicity of two of the less frequently isolated serotypes (A7 and A9) and thus supports the decision to incorporate such serotypes into vaccines. Acknowledgements The authors gratefully acknowledge the contributions of Mr M. Mcl.auchlan, Miss D. McCusker and Miss A. Wood. References BIBERSTEIN, E. L. & THOMPSON, D. A. (1966) Journal of Comparative Pathology 76, 83-94 DONACHIE, W., FRASER, J., QUIRIE, M. & GILMOUR, N. J. L. (1984) Research in Veterinary Science 37, 188-193 EYANS, H. B. & WELLS, P. W. (1979) Research in Veterinary Science 23,213-217 FRASER, J., GILMOUR, N. J. L., LAIRD, S. & DONACHIE, W. (1982) Veterinary Record 110, 560-561 GILMOUR, J. S., JONES, G. E.& RAE, A. G. (1979) Comparative Immunology, Microbiology and Infectious Diseases I, 285-293 GILMOUR, J. S., JONES, G. E., KEIR, W. A. & RAE, A. G. (1982) Journal of Comparative Pathology 92, 229-238 GILMOUR, N. J. L., MARTIN, W. B., SHARP, J. M., THOMPSON, D.1. & WELLS, P. W. (1979) Veterinary Record 104,15 GILMOUR, N. J. L., MARTIN, W. B., SHARP, J. M., THOMPSON, D. A., WELLS, P. W. & DONACHIE, W. (1983) Research in Veterinary Science 35,80-86 JONES, G. E. & GILMOUR, J. S. (1983) Diseases of Sheep. Ed W. B. Martin. Oxford, Blackwell. pp 17-23 JONES, G. E., GILMOUR, J. S. & RAE, A. G. (1978) Journal of Comparative Pathology 88, 85-% JONES, G. E., GILMOUR, J. S. & RAE, A. G. (1982) Journal of Comparative Pathology 92, 261-266 RUSHTON, B., SHARP, J. M., GILMOUR, N. J. L. & THOMPSON, D. A. (1979) Journal of Comparative Pathology 89,321-329 THOMPSON, D. A., FRASER, J. & GILMOUR, N. J. L. (1977) Research in Veterinary Science 22, 130-131

Received for publication May 7, 1985 Accepted September 10, 1985