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Experimental infection of dexamethasone-treated goats with Pasteurella haemolytica A2
Br. vet . J. (1991) . 147, 5 65
EXPERIMENTAL INFECTION OF DEXAMETHASONETREATED GOATS WITH PASTEURELLA HAEMOLYTICA A2
M . ZAMRI-SAAD, S . JASNI, A. B . NURIDA and A. R. SHEIKH-OMAR Faculty of Veterinary Medicine and Animal Science, Universiti Pertanian Malaysia, 43400 Serdang, Selangor, Malaysia
SUMMARY Sixteen goats either subjected to transport stress or without transport stress were treated with dexamethasone for 3 days prior to infection with P. haemolytica serotype A2 intranasally . The transport-stressed and dexamethasone-treated goats in the first group had various degrees of pulmonary lesions and the organism was re-isolated from the nasal cavity, lymph nodes and lungs . None of the goats treated with dexamethasone only were infected with P. haemolytica and had no lesions of pneumonic pasteurellosis . Treatment with dexamethasone alone failed to induce experimental infection by P. haemolytica except in combination with another stress factor .
INTRODUCTION Experimental pneumonic pasteurellosis has been reproduced in small ruminants usually by combined viral and Pasteurella infection . Viruses such as parainfluenza type 3 virus, adenovirus and herpes virus have been successfully used (Davies et al., 1981, 1982 ; Buddle et al., 1990) . However, dexamethasone has also been used to mimic stress resulting in more severe lesions than in the absence of dexamethasone treatment (Chiang et al ., 1990) . The present experiment was designed to study the experimental establishment and lesions of pneumonic pasteurellosis in dexamethasone-treated goats with and without transport stress .
MATERIALS AND METHODS Sixteen local goats about 6 months old were used . They were divided into two equal groups, fed daily with cut grass, supplemented feed and drinking water . Nasal swabs were collected weekly in a period of 4 weeks for isolation of P. haemolytica to ensure that none of the goats had the organism in their nasal cavity prior to the experiment . At the start of the experiment, goats in the first group were transport stressed by conveying them by road for a distance of about 200 km followed by intramuscular injections of dexamethasone at the rate of 1 mg/kg/day
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BRITISH VETERINARY JOURNAL, 147, 6
for 3 days . The dexamethasone treatment alone was also given to the second group . One day following the first dexamethasone treatment, 1 ml suspension containing 10 7 cfu- of P. haemolytica serotype A2 isolated earlier from the pneumonic lung of a goat and grown for 36 h in infusion broth was introduced intranasally (0 .5 ml per nostril) to all 16 goats . Nasal swabs for bacterial isolation were collected and body temperatures were recorded daily following the infection until the end of the 12-day study period . At days 1, 3, 7 and 12 post-infection with P. haemolytica, two goats from each group were killed and detailed post-mortem examinations were carried out on the respiratory tract . The extent of pulmonary lesions were determined according to the method described by Gilmour et al. (1983) . Samples from the nasal mucosa, trachea, lung and submandibular lymph nodes were cultured in blood agar and incubated at 37°C for 24 h before colonies suspected of P. haemolytica were re-cultured in MacConkey agar and identified using the triple sugar ion, oxidase, urease and mortility tests (Lennette et al., 1974) . Similar samples were also fixed in 10% buffered formalin, embedded in paraffin, sectioned at 4-6 tm and stained with haematoxylin and eosin for histological examination .
RESULTS
Clinical signs Seven goats in the first group were depressed, showing nasal discharge and having body temperature of 40 .5 ° C on day 3 post-inoculation with P. haemolytica A2 . Two goats died on days 4 and 7 respectively . Three goats in the second group showed mucoid nasal discharge but were alert and had normal body temperature of 38 ° C .
Bacteriology P. haemolytica was isolated mostly in pure cultures from the nasal cavity, lungs and submandibular lymph nodes of the seven goats in the first group killed either at day 1, 3, 7 or 12 post-infection . One goat had mixed P. haemolytica growth in the nasal cavity but the organism was absent from the submandibular lymph node and lung . Isolations from goats in the second group were made only from the nasal cavity at day 1 post-inoculation, mostly in mixed cultures . Following these isolations, none of the goats had the organism either in the nasal cavity, submandibular lymph node or lungs .
Gross lesions Eight goats (four from each group) killed at days 1 and 3 following inoculation showed severe hyperaemia and had mucoid discharge covering the nasal mucosa. Similar but less severe lesions were observed in the two goats from group 1 that were killed at day 7 post-inoculation but the lesions were absent from the remaining goats of both groups . Five goats in the first group killed at days 3, 7 and 12 had lung lesions typical of pneumonic pasteurollosis . An average of 19% (1-36%) of the lung area of these
P. HAEMOLYTICA IN GOATS
5 67
animals were affected, mostly involving the anterior and ventral parts of the lungs . Affected parts appeared dark red and firm with distinct demarcation between the lesions and the normal lung areas . None of the goats in the second group developed pneumonic lesions .
Microscopic lesions The main microscopic lesions observed in the nasal mucosa of goats in groups 1 and 2 killed at days 1 and 3 post-inoculation included excessive mucus production that was found covering the hyperaemic and eroded nasal mucosa. There were numerous goblet cells in some areas . Various degrees of rhinitis with accumulation of mononuclear cells was noted . The microscopic lesions in lungs of the five group 1 goats that had pneumonia consisted mainly of acute exudative bronchopneumonia . Most interalveolar septa were thickened by accumulation of oedema fluid, mononuclear cells and blood congestion . The associated alveoli were filled by exudate consisting of a mixture of numerous neutrophils and macrophages . Some exudate was observed in the lumen of some bronchi and bronchioles which also showed various degrees of subepithelial plasma cell and lymphocytic accumulations . Similar subepithelial accumulation of mononuclear cells and neutrophils were observed in the trachea as well as the loss of epithelial cilia.
DISCUSSION There are two categories of lesions observed in experimental infection of lambs with parainfluenza type 3 virus combined with P. haemolytica: necrotic pneumonia and purulent bronchopneumonia (Davies et al., 1981) . The purulent bronchopneumonia type of lesions is considered to be less severe, presumably because of the low virulence of the P. haemolytica strain used (Davies et al., 1982) . However, the presence of large numbers of spindle-shaped cells with intensely basophilic nuclei (oat cells) is considered the pathognomonic feature of the lesion (Gilmour, 1990) . Goats in this study developed only the less severe purulent bronchopneumonia type of lesion without oat cells . A similar experimental study in goats using herpesvirus and P. haemolytica serotype A7 reported the occurrence of mainly necrotizing pneumonia (Buddle et al., 1990) . Combined dexamethasone and transport stress influenced the development of pneumonic pasteurellosis by causing immunosuppression (Chiang et al., 1990) rather than by initiating pulmonary lesions as observed in viral infection (Davies et al., 1982) . Immunosuppression caused by dexamethasone alone, however, was insufficient to initiate experimental pneumonic pasteurellosis .
ACKNOWLEDGEMENTS The authors thank Dr Zulkifli Ahmad, Dr Abdul Rani Bahaman and Mr Abdullah Misron for the technical assistance . The financial support was provided by IRPA grant 50245 .
568
BRITISH VETERINARY JOURNAL, 147, 6 REFERENCES
BUDDLE, B . M., PFEFFER, A ., COLE, D . J. W., PULFORD, H . D . & RuLSTON, M . J. (1990) . New Zealand Veterinary Journal38, 22 . CHIANG, Y . W ., RoTH, J. A . & ANDREWS, J. J . (1990) . American Journal of Veterinary Research 51, 759 . DAVIES, D . H ., HERCEL, M ., JONES, B . A . H . & THURLEY, D . C . (1981) . Veterinary Microbiology 6, 173 . DAVIES, D . H ., HERCEL, M . & THURLEY, D . C . (1982) . Veterinary Microbiology 7, 369 . GILMOUR, N . J. L . (1980) . Veterinary Quarterly 2, 191 . 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 . LENNETTE, E . H ., SPAULDING, E . H . & TRUANT, J. P . (1974) . Manual of Clinical Microbiology, p . 246. Washington : American Society for Microbiology . (Accepted for publication 15 March 1991)
EXPERIMENTAL INFECTION OF DEXAMETHASONETREATED GOATS WITH PASTEURELLA HAEMOLYTICA A2
M . ZAMRI-SAAD, S . JASNI, A. B . NURIDA and A. R. SHEIKH-OMAR Faculty of Veterinary Medicine and Animal Science, Universiti Pertanian Malaysia, 43400 Serdang, Selangor, Malaysia
SUMMARY Sixteen goats either subjected to transport stress or without transport stress were treated with dexamethasone for 3 days prior to infection with P. haemolytica serotype A2 intranasally . The transport-stressed and dexamethasone-treated goats in the first group had various degrees of pulmonary lesions and the organism was re-isolated from the nasal cavity, lymph nodes and lungs . None of the goats treated with dexamethasone only were infected with P. haemolytica and had no lesions of pneumonic pasteurellosis . Treatment with dexamethasone alone failed to induce experimental infection by P. haemolytica except in combination with another stress factor .
INTRODUCTION Experimental pneumonic pasteurellosis has been reproduced in small ruminants usually by combined viral and Pasteurella infection . Viruses such as parainfluenza type 3 virus, adenovirus and herpes virus have been successfully used (Davies et al., 1981, 1982 ; Buddle et al., 1990) . However, dexamethasone has also been used to mimic stress resulting in more severe lesions than in the absence of dexamethasone treatment (Chiang et al ., 1990) . The present experiment was designed to study the experimental establishment and lesions of pneumonic pasteurellosis in dexamethasone-treated goats with and without transport stress .
MATERIALS AND METHODS Sixteen local goats about 6 months old were used . They were divided into two equal groups, fed daily with cut grass, supplemented feed and drinking water . Nasal swabs were collected weekly in a period of 4 weeks for isolation of P. haemolytica to ensure that none of the goats had the organism in their nasal cavity prior to the experiment . At the start of the experiment, goats in the first group were transport stressed by conveying them by road for a distance of about 200 km followed by intramuscular injections of dexamethasone at the rate of 1 mg/kg/day
5 66
BRITISH VETERINARY JOURNAL, 147, 6
for 3 days . The dexamethasone treatment alone was also given to the second group . One day following the first dexamethasone treatment, 1 ml suspension containing 10 7 cfu- of P. haemolytica serotype A2 isolated earlier from the pneumonic lung of a goat and grown for 36 h in infusion broth was introduced intranasally (0 .5 ml per nostril) to all 16 goats . Nasal swabs for bacterial isolation were collected and body temperatures were recorded daily following the infection until the end of the 12-day study period . At days 1, 3, 7 and 12 post-infection with P. haemolytica, two goats from each group were killed and detailed post-mortem examinations were carried out on the respiratory tract . The extent of pulmonary lesions were determined according to the method described by Gilmour et al. (1983) . Samples from the nasal mucosa, trachea, lung and submandibular lymph nodes were cultured in blood agar and incubated at 37°C for 24 h before colonies suspected of P. haemolytica were re-cultured in MacConkey agar and identified using the triple sugar ion, oxidase, urease and mortility tests (Lennette et al., 1974) . Similar samples were also fixed in 10% buffered formalin, embedded in paraffin, sectioned at 4-6 tm and stained with haematoxylin and eosin for histological examination .
RESULTS
Clinical signs Seven goats in the first group were depressed, showing nasal discharge and having body temperature of 40 .5 ° C on day 3 post-inoculation with P. haemolytica A2 . Two goats died on days 4 and 7 respectively . Three goats in the second group showed mucoid nasal discharge but were alert and had normal body temperature of 38 ° C .
Bacteriology P. haemolytica was isolated mostly in pure cultures from the nasal cavity, lungs and submandibular lymph nodes of the seven goats in the first group killed either at day 1, 3, 7 or 12 post-infection . One goat had mixed P. haemolytica growth in the nasal cavity but the organism was absent from the submandibular lymph node and lung . Isolations from goats in the second group were made only from the nasal cavity at day 1 post-inoculation, mostly in mixed cultures . Following these isolations, none of the goats had the organism either in the nasal cavity, submandibular lymph node or lungs .
Gross lesions Eight goats (four from each group) killed at days 1 and 3 following inoculation showed severe hyperaemia and had mucoid discharge covering the nasal mucosa. Similar but less severe lesions were observed in the two goats from group 1 that were killed at day 7 post-inoculation but the lesions were absent from the remaining goats of both groups . Five goats in the first group killed at days 3, 7 and 12 had lung lesions typical of pneumonic pasteurollosis . An average of 19% (1-36%) of the lung area of these
P. HAEMOLYTICA IN GOATS
5 67
animals were affected, mostly involving the anterior and ventral parts of the lungs . Affected parts appeared dark red and firm with distinct demarcation between the lesions and the normal lung areas . None of the goats in the second group developed pneumonic lesions .
Microscopic lesions The main microscopic lesions observed in the nasal mucosa of goats in groups 1 and 2 killed at days 1 and 3 post-inoculation included excessive mucus production that was found covering the hyperaemic and eroded nasal mucosa. There were numerous goblet cells in some areas . Various degrees of rhinitis with accumulation of mononuclear cells was noted . The microscopic lesions in lungs of the five group 1 goats that had pneumonia consisted mainly of acute exudative bronchopneumonia . Most interalveolar septa were thickened by accumulation of oedema fluid, mononuclear cells and blood congestion . The associated alveoli were filled by exudate consisting of a mixture of numerous neutrophils and macrophages . Some exudate was observed in the lumen of some bronchi and bronchioles which also showed various degrees of subepithelial plasma cell and lymphocytic accumulations . Similar subepithelial accumulation of mononuclear cells and neutrophils were observed in the trachea as well as the loss of epithelial cilia.
DISCUSSION There are two categories of lesions observed in experimental infection of lambs with parainfluenza type 3 virus combined with P. haemolytica: necrotic pneumonia and purulent bronchopneumonia (Davies et al., 1981) . The purulent bronchopneumonia type of lesions is considered to be less severe, presumably because of the low virulence of the P. haemolytica strain used (Davies et al., 1982) . However, the presence of large numbers of spindle-shaped cells with intensely basophilic nuclei (oat cells) is considered the pathognomonic feature of the lesion (Gilmour, 1990) . Goats in this study developed only the less severe purulent bronchopneumonia type of lesion without oat cells . A similar experimental study in goats using herpesvirus and P. haemolytica serotype A7 reported the occurrence of mainly necrotizing pneumonia (Buddle et al., 1990) . Combined dexamethasone and transport stress influenced the development of pneumonic pasteurellosis by causing immunosuppression (Chiang et al., 1990) rather than by initiating pulmonary lesions as observed in viral infection (Davies et al., 1982) . Immunosuppression caused by dexamethasone alone, however, was insufficient to initiate experimental pneumonic pasteurellosis .
ACKNOWLEDGEMENTS The authors thank Dr Zulkifli Ahmad, Dr Abdul Rani Bahaman and Mr Abdullah Misron for the technical assistance . The financial support was provided by IRPA grant 50245 .
568
BRITISH VETERINARY JOURNAL, 147, 6 REFERENCES
BUDDLE, B . M., PFEFFER, A ., COLE, D . J. W., PULFORD, H . D . & RuLSTON, M . J. (1990) . New Zealand Veterinary Journal38, 22 . CHIANG, Y . W ., RoTH, J. A . & ANDREWS, J. J . (1990) . American Journal of Veterinary Research 51, 759 . DAVIES, D . H ., HERCEL, M ., JONES, B . A . H . & THURLEY, D . C . (1981) . Veterinary Microbiology 6, 173 . DAVIES, D . H ., HERCEL, M . & THURLEY, D . C . (1982) . Veterinary Microbiology 7, 369 . GILMOUR, N . J. L . (1980) . Veterinary Quarterly 2, 191 . 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 . LENNETTE, E . H ., SPAULDING, E . H . & TRUANT, J. P . (1974) . Manual of Clinical Microbiology, p . 246. Washington : American Society for Microbiology . (Accepted for publication 15 March 1991)