Experimental Pasteurella multocida pneumonia in calves

Research in Veterinary Science /989, 47, /85-/89

Experimental Pasteurella multocida pneumonia in calves R. N. GOURLAY, L. H. THOMAS, S. G. WYLD, Agricultural and Food Research Council, Institute for Animal Health, Compton, Newbury, Berkshire, RGJ60NN

Pasteurella multocida was isolated from the lungs of about I em", from four different sites were triturated calves that died on a farm in the south of England. in phosphate buffered saline (PBS), pH 7· 2, using a This organism was inoculated experimentally into 13 pestle and mortar. Suspensions were serially diluted in calves by the intratracheal route: in all but two of the PBS and inoculated on to 5 per cent ox blood agar calves mild clinical disease resulted and at necropsy, plates. Plates were incubated aerobically at 37°C for three or four days later, pneumonic consolidation 24 to 48 hours and duplicate plates were incubated involving up to 22 per cent of the lung was observed. anaerobically in 90 per cent hydrogen and 10 per cent P multocida was isolated from all but two of the carbon dioxide for 48 hours. Bacteria present at more lungs. Of two calves inoculated intravenously with P than 103 colony forming units (cfu) s" tissue were multocida, one showed mild clinical disease and slight identified by the methods of Cowan and Steel (1974). pneumonic consolidation at necropsy and the other remained normal. Control calves inoculated intratracheally and intravenously with sterile broth showed no signs of illness and no pneumonic con- Preparation of P multocidafor calf inoculations solidation. Histologically the lung lesions comprised a A pool was prepared of four pure cultures of P fibrinous bronchopneumonia with variable sized multocida isolated from four calves that had died at areas of coagulative necrosis, extensive deposition of Warren Farm in 1985. This pool was used to inoculate fibrin and massive dilatation and oedema of the inter- a six-week-old calf (X448) intratracheally (9 ml; titre lobular and pleural lymphatics. It is concluded that P 7 x 107 cfu ml : I). At necropsy, six days after inoculamultocida should receive more recognition as a tion, the calf had pneumonic consolidation involving primary pathogen. 20 per cent of the lung and P multocida was isolated from four sites on the lungs in pure culture at titres of PASTEURELL~' multocida has been isolated from 1'1 to 8· 5 X 107 cfu g' '. The lung tissue from X448 many animal species, both wild and domesticated, as was stored in liquid nitrogen. Cultures for bacteriowell as from man. It has been isolated from both logy and calf inoculation were obtained by scraping a diseased and normal animals. This organism is the small piece of tissue off the frozen lung, grinding it up primary cause of certain major diseases of domestic in PBS and inoculating blood agar plates. Following animals, namely haemorrhagic septicaemia of cattle incubation at 37°C overnight a sweep of single and buffaloes, caused by serotypes Band E (Carter colonies was inoculated into brain-heart infusion 1981) and fowl cholera of chickens, turkeys, ducks, (BHI) broth and, after incubation, aliquots of culture geese and wild fowl, caused by serotype A. were stored in liquid nitrogen. For many years the authors have examined P multocida isolate (X448) was Gram-negative, pneumonic lungs from calves that died from non-motile, catalase and oxidase positive, indole pneumonia at Warren Farm, the Milk Marketing positive, produced acid from glucose, mannitol and Board's progeny testing station in the south of sorbitol, failed to grow on McConkey's medium and England. During 1984 and 1985 they isolated a par- appeared to be type A by its reaction with hyaluronticularly large number of P multocida strains from idase. The organism was non-toxigenic by tissue these lungs (25 strains from 73 lungs examined). It was culture assay (Chanter et al 1986). therefore decided to study this organism in more For calf inoculations an aliquot of X448 culture detail and examine its pathogenicity for the calf lung; was removed from storage in liquid nitrogen, a this paper gives details of those findings. loopful subinoculated into 10 ml BHI broth and incubated at 37°C overnight. One ml of the culture was subinoculated into 15 ml fresh, warm BHI broth Materials and methods (1:15 ml broth) and incubated at 37°C in an orbital Bacterial isolation and enumeration incubator at 100 rpm for three to four hours. This The surface of the lung was seared and samples, culture was used to inoculate the calves. 185

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R. N. Gourlay, L. H. Thomas, S. G. Wyld

FIG 1: Area of necrosis defined by dark band of pyknotic cells (top left quadrant). Extensive deposition of fibrin may be seen in alveoli outside the dark band: calf X436. Lissamine red blue x 25

FIG 2: Mixed inflammatory cell response in alveolar exudate. Large fibrin clot evident at right: calf X434. Haematoxylin and eosin x 145

Calves

solution), for histopathology and immunoperoxidase labelling (Thomas et al 1986) using antiserum to P multocida prepared in rabbits.

Thirteen conventionally reared Friesian cross calves were inoculated intratracheally with 10 ml of the P multocida culture. Three separate experiments were performed. In the first two, groups of four calves, aged five-and-a-half to six-and-a-half months were each inoculated with cultures containing I' 5 x 1()8 cfu ml : I and 6' 5 x I()8 cfu ml: I, respectively. In the third experiment, using five calves at two to two-and-a-half months old, the culture titre was I' 5 x 109 cfu ml- I • Two more calves, aged six months, were inoculated intravenously with 10 ml of a one in 10 dilution of P multocida culture (titre I' 5 x 107 cfu ml in PBS. Two control calves were inoculated intratracheally with 10 ml sterile BHI broth and another two intravenously with 10 ml of a one in 10 dilution of sterile BHI broth in PBS. Clinical scores were recorded daily for each calf thus: temperature >40°C = I; respiratory rate >50 min -I, dyspnoea or abnormal sounds on auscultation = I; apathy, depression or loss of appetite = I. A cumulative score was obtained for each calf. Calves were killed by intravenous pentobarbitone sodium (Euthatal; RMB Animal Health), three or four days after inoculation. The presence or extent of pneumonic consolidation was estimated as a percentage of the dorsal surface of the lung affected, calculated from drawings prepared on standard lung outlines. Samples of lung tissue were taken from four sites, which usually included the right apical and cardiac lobes and two other sites depending on the location of the lesions. Samples were examined for bacteria as described above. Samples of lung tissue, bronchial lymph nodes, trachea, nasal mucosa, tonsils, liver and kidney were fixed in formal sublimate (10 per cent formaldehyde solution in saturated mercuric chloride r

')

Results

Calf inoculations and disease Mild clinical illness was observed in nine of the 13 intratracheally inoculated calves. At necropsy pneumonic consolidation was observed in all but one animal (Table I, calf A659, experiment I). Another calf (BI23, experiment 3) had a minimal lesion. P multocida was isolated from all the lungs except those from A659 and B123. In the intravenously inoculated calves acute respiratory distress occurred in both animals within 10 to 20 minutes of inoculation; they were treated with flunixin meglumine (Finadyne; Schering) intravenously and recovered within 10 minutes. Subsequently one calf showed mild clinical illness (clinical score I) and 6 per cent pneumonic consolidation at necropsy four days later. P multocida was isolated from the lungs (0 to 1()3 cfu g The other calf showed no signs of illness, no pneumonic consolidation at necropsy and P multocida was not isolated from the lungs. The control calves inoculated intratracheally with BHI broth and the two inoculated intravenously with a 1/10 dilution of BHI broth in PBS showed no clinical illness and at necropsy, three and four days after inoculation, pneumonia was not observed in either of the intratracheally inoculated calves and only a very small area (I per cent) of consolidation was observed in one of those inoculated intravenously, the other being normal. P multocida or other bacteria were not isolated from any of these lungs. r

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Histopathology The histological reactions in the lungs of the intratracheally inoculated calves from all t~ree experiments were essentially similar and compnsed fibrinous bronchopneumonia with varying sized areas of lobular coagulative necrosis. The areas of necrosis were defined by characteristic rings of pyknotic c~lls (Fig I), within which clusters of rounded active macrophages packed the alveoli. Oat cell morph~lo~y was rarely seen in these macrophages. Fibrin deposition and neutrophils were not in evide~ce within the ring of necrotic cells but outside extensive deposition of fibrin was demonstrated; macrophages and neutrophils were present in equal numb~rs (Fig 2). A further striking lesion was massive dilatation and oedema of the interlobular and pleural lymphatic vessels, many of which were occluded by fibrin clots (Fig 3). Other microscopic lesions .were confined to the upper respiratory tract and associated lymphoid tissue. These comprised focal infiltration of

polymorphs into the lamina propria of the n.asal mucosa and hyperactivity of the lymph node folhcles and tonsils. Two calves (A659 and B123) had no significant lesions of the respiratory tract; only small areas of pulmonary collapse could be seen. In one calf (B648) the histological changes were essentially of an exudative bronchopneumonia with a mixed inflammatory cell response; areas of coagulative necrosis, fibrin deposition, clusters of rounded macrophages, interlobular oedema and lymphatic thrombosis were not seen. Macrophage giant cells were however especially prominent in the alveolar exudate. Necrotic fibrinous pneumonia was not seen in the two animals inoculated by the intravenous route, only patchy areas of collapse were present. In control animals no significant microscopic lesions were noted. P multocida organisms were located by immunoperoxidase labelling in the intratracheally inoculated calves towards the periphery of the necrotic areas (Fig 4) and infiltrating the epithelium and lamina

R. N. Gourlay, L. H. Thomas, S. G. Wyld

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TABLE 1: Details of calves inoculated intratracheally with PBstBurellB multocide culture and killed three or four days later

Lung cfu g-l % Consolidationt P multocida*

Calf

Inoculum cfu

Clinical score*

Experiment 1 A1Bl Al89 Al99 A659

1-5 x 109 1-5 x 109 1·5x 109 1-5 x 109

3 2 2 1

15 7 7 0

X435 X436 X439

6·5x 109 6·5x 109 6·5x 109 6·5 x 109

1 1 0 0

13 20 22 12

Experiment 3 8122 8123 8648 8649 8846

1·5x 1010 1·5x 1010 1·5x 1010 1·5x 1010 1·5x 1010

2 0 4 0 3

20 1 17 19 22

Experiment 2 X434

.

0§-107

o -107

0-107 0

o -107

0-107 10"-107 -107

o

10"-loB 0 -lOS 1()6·107

o o -loB

Temperature >40°C=l; respiratory rate >50 min-lor dyspnoea or abnormal sounds on auscultation = 1; apathy, depression or loss of appetite = 1. Sum of daily results t Pneumonic consolidation as a percentage of the lung dorsal surface :j: Range of titres from four sites § <5x 101 cfu g-1

propria of the large bronchioles, and penetrating the peribronchiolar lymphatics (Fig 5). Similarly, bacteria were seen in the subpleural lymphatic vessels (Fig 6) and in Kupffer cells of the liver.

Discussion Madsen et al(l985) tentatively designated 41 of 50 strains of P multocida-Iike organisms isolated from pneumonic calf lungs in Denmark as taxon 13, on differences in indole formation and acid production from mannitol and sorbitol. By these criteria the X448 isolate does not appear to be one of these organisms but a true P multocida. This isolate of P multocida induced pneumonia in 11 out of 13 conventionally reared calves following intratracheal inoculation. The extent of pneumonic consolidation averaged 13, 5 per cent and P multocida was isolated from all 11 lungs that showed pneumonic consolidation at titres of up to lOS cfu g-I (Table 1). The remaining two calves (A659 and B123) failed to reveal significant pneumonic consolidation by P multocida in their lungs at necropsy. The reason for this is not known but was not correlated with the number of P multocida inoculated since one of these animals received the lowest dose (I' 5 x 109 cfu) and one the highest dose (I, 5 x 1010 cfu). However, in general, the calves that received the lowest dose had less pneumonic consolidation, an average of 7 per cent compared, with 17per cent and 16per cent for the larger doses. The first and last figures were reduced by the two abnormal calves (A659 and BI23).

The group of calves that received the largest dose of pasteurella and, apart from B123, showed the most extensive pneumonic consolidation were also the youngest. These younger calves, however, did not appear to be particularly susceptible to P multocida infection compared with the older ones. The difference in extent of pneumonic consolidation could be accounted for by the larger inoculation dose. Clinical illness was not severe in any of the calves; even the most severely affected younger animals showed only transient pyrexia and transient respiratory signs. The authors' results with the X448 isolate support the conclusions of Ames et al (1985) that P multocida on its own produces less severe clinical disease and less extensive lung involvement than P haemolytica. In a concurrent study (Thomas et al 1989), intratracheal inoculation of 109 cfu of P haemolytica in calves induced severe clinical disease, with areas of pneumonic consolidation averaging 34 per cent. It does appear however that the X448 isolate of P multocida is more pathogenic than that used by Ames et al (1985). They reported that 109 P multocida induced only an average of 4·7 per cent lung involvement and furthermore their isolate, unlike X448, induced minimal or no necrosis. Histologically there was a striking similarity between the lesions induced by P multocida and those induced by P haemolytica (Friend et al 1977, Thomas et al 1989). Panciera and Corstvet (1984) who inoculated these two bacterial species into calves by transthoracic intrapulmonic injection did not distinguish histologically between the lesions induced

Experimental Pasteurella multocida pneumonia in calves by these two organisms. Ames et al (1985), however, reported that the lesions produced by P multocida inoculated intratracheally differed morphologically from those of P haemolytica. With P multocida there was a suppurative exudative component and minimal to no necrosis. In the present study deposition of fibrin was perhaps even greater in the P multocida infected calves than in the P haemolytica ones, but oat cell type macrophages were rarely seen in P multocida animals. Unlike Ames et al (1985) extensive necrosis was noted. The lack of oat cell macrophages was presumably because P multocida, unlike P haemolytica, does not produce a leucotoxin (Ames et al 1985). Further, unlike P haemolytica (Thomas et al 1989), P multocida does not induce significant pneumonic lesions following intravenous inoculation. The reason for this is not known but may also involve the absence of leucotoxin. The shock-like syndrome following intravenous inoculation of P multocida, which responded to treatment with f1unixin meglurnine, has also been observed following administration of P haemolytica via the intravenous route (Thomas et al 1989). In the latter case substantial lesions of pneumonic consolidation were produced. In both cases endotoxin seems a likely cause of the syndrome by triggering the arachidonic acid cascade but, in terms of overall development of pulmonary disease, this response would not appear to be significant. It is interesting that characteristic lesions of P multocida pneumonia were observed in the lungs of the calves that died at Warren Farm and from which this organism was isolated. This suggests that P multocida played a significant role in the aetiology of the pneumonia on the farm. In view of the many reported isolations of P multocida from cases of calf respiratory disease (Omar 1966, Gourlay et al 1970, Bitsch et al 1976, Allan 1978) perhaps this organism should receive more serious recognition as an important bovine respiratory disease pathogen.

189

Acknowledgements We thank Mr B. A. Turfrey and Mr K. R. Parsons for the histological preparations and immunoperoxidase labelling, respectively, Mr D. Hawkins for the photographic processing and the staff of the high security building for help with sampling and care of the animals. References ALLAN, E. M. (1978) Respiratory Diseases of Cattle. Ed W. B. Martin. The Hague, Martinus Nijhoff. pp 345-355 AMES, T. R., MARKHAM, R. J. F., OPUDA-ASIBO, J., LEININGER, J. R. & MAHESWARAN, S. K. (1985) Canadian Journal of Comparative Medicine 49, 395-400 BITSCH, V., FRIIS, N. F. & KROGH, H. V. (1976) Acta Veterinaria Scandinavica 17,32-42 CARTER, G. R. (1981) The Procaryotes, Vol 2. New York, Springer- Verlag. pp 1383-1391 CHANTER, N., RUTTER, J. M. & LUTHER, P. D. (1986) Veterinary Record 119, 629-630 COWAN, S. T. & STEEL, K. J. (1974) Manual for the Identification of Medical Bacteria, 2nd edn. Cambridge, Cambridge University Press FRIEND, S. c., THOMSON, R. G. & WILKIE, B. N. (1977) Canadian Journal of Comparative Medicine 41, 219-223 GOURLAY, R. N., MACKENZIE, A. & COOPER, J. E. (1970) Journal of Comparative Pathology SO,575-584 MADSEN, E. B., BISGAARD, M., MUTTERS, R. & PEDERSEN, K. B. (1985) Canadian Journal of Comparative Medicine 49, 63-67 OMAR, A. R. (1966) Veterinary Bulletin 36,259-273 PANCIERA, R. J. & CORSTVET, R. E. (1984) American Journal of Veterinary Research 45, 2532-2537 THOMAS, L. H., GOURLAY, R. N., WYLD, S. G., CHANTER, N. & PARSONS, K. R. (1989) Veterinary Pathology 26, 253-259 THOMAS, L. H., HOWARD, C. J., STOTT, E. J. & PARSONS, K. R. (1986) Veterinary Pathology 23,571-578

Received July 15. 1988 Accepted November 9. 1988