Br. vet.
1.
(1991) . 147, 43 7
EVALUATION OF COMBINED PASTEURELLA VACCINES IN CONTROL OF SHEEP PNEUMONIA
S . CHANDRASEKARAN * , KAMAL HIZATt, ZAMRI SAADt, M . Y . JOHARA * and P . C . YEAP* *Veterinary Research Institute, P. 0. Box 369, 30740-Ipoh, Malaysia; tMalaysian Agricultural Research and Development Institute (MARDI), 43400-Serdang, Selangor, Malaysia ; $Faculty of Veterinary Science, Malaysian Agricultural University, 43400-Serdang, Selangor, Malaysia
SUMMARY The effectiveness of an oil adjuvant vaccine (OAV) incorporating locally isolated strains of Pasteurella haemolytica type 7 and Pasteurella multocida types A and D was compared with that of Carovax (Wellcome Laboratories) in imported cross-bred lambs . The criterion of efficacy was the ability of the vaccines to reduce the extent of pneumonic lesions in vaccinated as against unvaccinated control lambs. The OAV produced at this Institute significantly reduced the lung lesions at P< 0 .05 level compared with its control group when challenged with P. haemolytica alone . However, the vaccine was unsatisfactory against P . multocida or combined P. multocida P. haemolytica challenge . Carovax did not produce any significant reduction in the lung lesions caused by P. haemolytica and/or P. multocida.
INTRODUCTION The Department of Veterinary Services, Malaysia, has embarked on large scale sheep farming in rubber plantation . Pure and cross-bred sheep were imported to achieve the set target of one million sheep by the year 2000 . However, high mortality was recorded amongst imported pure bred as well as cross-bred sheep at the onset of this import programme . These were mainly due to pasteurellosis and haemonchosis . Hadi (1988) reported that more than 50% of the deaths was due to bluetongue-pneumonia complex . Pneumonic pasteurellosis (PP) is an important disease of sheep . The aetiological agent for this condition has been tentatively identified as Pasteurella haemolytica biotype A and Pasteurella multocida serotypes A and D while attempts at elucidating predisposing causes and the role of other pathogens are underway . Some of the uncontrollable predisposing factors are the hot, tropical climate of Malaysia with unpredictable convectional rainy spells and two definite monsoons which cause changes in the environment . In addition, management practices such as docking, drenching, castration, etc . cause additional stress . It was, therefore, found necessary to examine measures for control including vaccination as one means of reducing losses . Initially several kinds of commercially prepared broth vaccines were used to control
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this disease but with little success (Wan Mohamed et al, 1988) . One such vaccine used widely was Carovax (Wellcome Laboratories, UK) . This necessitated the development of a pasteurella vaccine using locally isolated strains of P. haemolytica and .P. mullocida types A and D . The aim of this trial was to compare the efficacy of the locally prepared oil adjuvant vaccine (OAV) with Carovax which was the only readily available commercial vaccine at that time . The parameter used in the evaluation was the ability of the vaccines to reduce the extent of lung lesions caused by experimentally induced PP in cross-bred lambs .
MATERIALS AND METHODS Animals
Thirty-five clinically healthy imported cross-bred lambs were selected from a government farm . They were aged between 4 and 6 months and weighed on average 12 .5 kg each . Blood samples and nasal swabs were taken from these animals . The former was used to determine the antibody levels to P. haemolytica and P. multocida by the indirect haemagglutination assay (IHA) as followed at this Institute (Chandrasekaran et al., 1981) . The nasal swabs were used to evaluate the bacterial flora involved . The animals were randomly assigned to three major groups (Table I) . All animals were subjected to a semiintensive management system, grazing during the day and being housed at night, with concentrate supplementation . Clean drinking water was available ad libitum . Table I Experimental design for vaccination trial Group
1 2 3
Vaccine/No . of animals
Challenge organism
P. haemolytica type 7 P. multocida types A & D Combined P. haemolytica type 7 and P. multocida A & D
VRI OA V
Carovax
Control
5 5 5
4
ND
4 4 4
4
ND, not done . Preparation of vaccine and vaccination
The OAV used in this trial was produced at the Veterinary Research Institute (VRI), Ipoh, Malaysia . It contained locally isolated strains of P. haemolytica type A7 and P. multocida types A and D . The details of the method of production have been given elsewhere (Chandrasekaran et al., 1987) . Briefly, the seed organisms were obtained from field outbreaks of PP in sheep . Separate dense broth aliquots were prepared for each organism by intermittent aeration, churning and incubation at 37°C for about 22-24 h . The average dry weight per ml of eight separate aliquots determined spectrophotometrically (Beckman instruments, California, USA) was 0 .67 mg of P. haemolytica and 0 .94 mg P. multocida type A and 0.85 mg type D .
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The aliquots were then incorporated with the adjuvant, a light mineral oil (Ondina grade 15) and an emulsifying agent (lanolin), using an industrial model emulsifier (Silverson emulsifier/mixer, Model EX, Bucks, England) . This yielded a water-in-oil type of emulsion . Lambs aged between 3 and 5 months and those above 5 months were intramuscularly given 3 and 5 ml respectively of the vaccine each . Annual revaccination was recommended .
Induction of pneumonic pasteurellosis The lambs were challenged 4 weeks post-vaccination . The procedure followed has been previously described by Chandrasekaran (1988) . Briefly, the animals were stressed by transportation for about 250 km . At the end of the journey, each animal was given 10 mg dexamethasone (Dexasone, Atlantis, Germany) intramuscularly, followed by two similar doses on consecutive days . Immediately after the last dexamethasone injection experimental PP was induced through aerosol administration of the infective organisms, using a modified version of that of Gilmour et al. (1983) . In this trial the tip of the nebulizer was placed against the nostrils . The experimental design is given in Table I .
Challenge The animals in group I were given an aerosal spray of P. haemolytica type 7 prepared from a 24-h broth culture . On average each animal in this group was given 1 .82X10' colony forming units (c .f.u .) . The animals in group 2 were given 2 .5X10" c .f.u . of P. multocida type A combined with 7 .2X10' c .f.u . of P. multocida type D prepared from separate 18-h broth cultures . For the combined challenge of the animals in group 3 each animal was given 9 .8X 10' c .f.u . of P. haemolytica, 1 .42X 10' c .f.u . of P. multocida type A and 3 .22X 10- c .f.u . of P. multocida type D .
Clinical observations and necropsy The animals were observed twice daily (8 :00 and 16 :00) following challenge . The clinical signs recorded were the rectal temperature, lethargy, anorexia, abnormal respiration (stertorous and abdominal respiration, polypnoea and dyspnoea) and mortality . All dead lambs were necropsied within 24 h of death . The extent of pneumonic lesions was recorded according to the method described by Gilmour et al. (1983) . A portion of the affected lung, the retropharyngeal, submandibular and mesenteric lymph nodes were collected for bacteriological examination . Blood was collected on the 3rd, 5th, 7th and 9th days post-challenge to assess the antibody status against P . haemolytica and P. multocida by the IHA (Chandrasekaran et al., 1981) . All surviving animals were euthanized 9 days post-challenge by intravenous administration of 12 ml per animal of a saturated solution of magnesium sulphate . Necropsy was performed by an independent team of veterinarians and the extent of lung lesions recorded .
RESULTS Serology
The sera of all the animals pre- and post-challenge were found to be negative for antibodies to P. haemolytica and P. multocida by the IHA test .
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Clinical observations None of the vaccinated (VRI OAV and Carovax) animals showed any adverse effects to vaccination . Seven animals died following challenge-four from the control group and three from the Carovax vaccinated group . There were no obvious clinical signs in the VRI OAV vaccinated animals as opposed to those in the other two groups . The mortalities and the clinical signs observed in the three groups are summarized in Table II .
Table II Summary of clinical signs and mortalities post-infection in vaccinated and unvaccinated lambs
Group
Challenge organism
VRI OA V
Carovax
Control
Number of animals in each group with 1
2
3
P. haemolytica type 7
P. multocida types A & D
P. haemolytica type 7 and P. mullocida types A & D
No . of deaths
0
No . of deaths
1
No . of deaths
1
No . with clinical signs
0
No . with clinical signs
0
No . with clinical signs
3
No . with 5 no clinical signs
No . with 3 no clinical signs
No . with 0 no clinical signs
No . of deaths
0
ND
No . of deaths
1
No . with clinical signs
2
ND
No . with clinical signs
3
No . with 3 no clinical signs
ND
No . with 0 no clinical signs
No . of deaths
0
No . of deaths
2
No . of deaths
2
No . with clinical signs
4
No . with clinical signs
2
No . with clinical signs
2
No . with 1 no clinical signs
No . with 0 no clinical signs
No . with 0 no clinical signs
ND, not done ; clinical signs : temperature > 40 .5 ° C and/or dyspnoea, polypnoea, and stertorous respiration .
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Bacteriology
The bacteriological culture of the prevaccination nasal swabs evaluated at the outset of the experiment and again prior to challenge were negative for P. haemolytica and P. multocida . Profuse, pure growth of the respective species used in the challenge was consistently isolated from the lung and/or lymph nodes of the lambs except in six (17%) animals from group 1 where three each were vaccinated with the VRI OAV and Carovax . However, the predominant organism reisolated in the mixed challenge group was P . multocida type A . Macroscopic lung lesions
Most lambs (62%) in this trial had lung lesions . The lesions were consolidation with red hepatization of the anteroventral portions of the apical, cardiac and intermediate lobes . Consolidation was also present along the borders of the diaphragmatic lobes . Fibrin deposition with adhesion to the parietal pleura was present in all the control animals and three dead animals vaccinated with Carovax . The lung lesions were more pronounced and extensive in the control group . The lambs vaccinated with the VRI OAV in group 1 had a significant reduction (P< 0 .05) in the lung lesion score compared to its control group . However, the animals in groups 2 and 3 did not show any significant difference in comparison with their respective control groups . A summary of the average lung lesion score is given in Table III . Table III Average lung lesion score-in vaccinated and control animals challenged with P. haemolytica type 7 and/or P. multocida types A & D Vaccination status
Control
Challenge organism Pasteurella haemolytica Pasteurella multocida
Mixed infection VRI OAV
Pasteurella haemolytica Pasteurella multocida
Mixed infection Corovax
Pasteurella haemolytica
Mixed infection
Lung lesion (%) Average
AD
9 .83 5 .63 8 .18
6 .23 5 .42 5 .25
0 .3 * 3 .68 2 .88
2.11 1 .83 1 .91
3 .95 11 .97
3 .10 4 .23
*Significantly different at P< 0 .05 when compared with the control group of similar treatment .
DISCUSSION Pneumonic pasteurellosis has been successfully reproduced in specific pathogen free sheep (Sharp et al., 1978 ; Gilmour et al., 1983) . In this study the disease was experimentally reproduced in conventionally reared cross-bred lambs . Experimental infection of sheep with aerosol of P. haemolytica was not always successful as inductions of
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pneumonic lesions were inconsistent . A similar observation has been made by Gilmour et al. (1975) . Prior to infection some form of stress was necessary . Other workers (Sharp et al., 1978 ; Gilmour et al., 1983) achieved this by first infecting with parainfluenza virus type 3 followed by aerosol infection of P. haemolytica 7 days later. In this trial the animals were initially stressed by transport and then further predisposed to infection by steroids . The 10 mg administered was double the therapeutic dose for sheep . Steroids have been presumed to exert their immunosuppressive effect by interfering with the cell-mediated immune (CMI) response (Tizard, 1982) . While it is possible that it affected the elicitation of secondary CMI response it is unlikely to have affected the primary response which could already have occurred in response to vaccination 28 days previously . This primary response would probably still exist . Since the vaccinated and control animals would presumably be similarly suppressed by the drug, the difference seen in the lung lesions could probably be attributed to the effect of the vaccines . The prechallenge bacterial flora in the nasal cavity of these animals did not correlate with the lung lesions following infection . This indicated that the lesions observed in this study most probably developed from the experimental infection . This was further confirmed by recovery of the respective organisms used for the challenge . The results obtained in this trial indicate that it is possible to immunize sheep against PP caused by P. haemolytica with the locally produced formalin-killed OAV . The efficacy of this vaccine against pneumonia caused by P. multocida alone or in combination with P. haemolytica was low . Mosier et al. (1989) observed a similar phenomenon in bovine PP where P. haemolytica OAV effectively reduced lesions of pneumonia caused by that organism . They, however, recorded little or no effect when P. multocida OAV or P. multocida in combination with P. haemolytica in aluminium hydroxide adjuvant were administered . Unsatisfactory protection in sheep vaccinated with pasteurella vaccines incorporating sodium salicylate and heat-killed organisms has also been reported (Gilmour et al., 1983) . In a separate trial Zamri Saad et al. (1989) observed that the VRI OAV was ineffective in controlling naturally occurring ovine PP caused by either P . haemolytica or P. multocida in imported pure bred sheep as opposed to cross-breds used in this trial . A private agricultural organization (Ngah Mohammed, personal communication) experienced a similar outcome with the VRI OAV . These could probably be attributed to the following . 1 . Strain differences : P. haemolytica exists as two main biotypes and 15 serotypes and protection appears to be serotype specific (Gilmour & Gilmour, 1989) . P. multocida can be classified into four capsular and 16 somatic types (Carter & Chengappa, 1981) . Of these, serotypes A and D have been isolated from cases of PP in sheep and goats . Thompson et al. (1977) found that varying serotypes of P. haemolytica were isolated during different seasons and suggested their incorporation in vaccines . On a similar note, Fraser et al. (1982) stressed the importance of continually monitoring the prevalence of the different serotypes, investigating the pathogenicity of new serotypes so that the appropriate strains could be incorporated in the vaccines . The organisms incorporated into the VRI OAV were P. haemolytica type A7 and P. multocida capsular types A and D and of undetermined somatic types . It is postulated that the natural infection reported could have been caused by other type/types of pasteurellae . 2 . The trial being reported was conducted in cross-bred sheep whereas the natural infection observed by Zamri Saad et al (1989), was in pure bred sheep .
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3 . The pure bred sheep could have been harbouring the aetiological agents in their respiratory tract as the bacterial status of the nasal orifices was not determined by swabbing and subculture . The stress of transport and the new environment could have triggered their multiplication before optimum protection could be elicited by the vaccine . No adverse side effects to vaccination were encountered in all the animals used in this trial . The IHA titre would seem to indicate that the antibody levels did not increase either as a result of vaccination and/or challenge . The IHA was probably an unsuitable in-vitro quantitative test for antibody estimation against P. haemolytica and P. multocida types A and D in sheep sera .
ACKNOWLEDGEMENTS The authors would like to thank the Director-General of Veterinary Services, Malaysia, for permission to publish this paper and to the Directors of VRI, Ipoh, and MARDI, Serdang, for providing the facilities and the animals respectively . The technical assistance of Mrs Rohani Saad is acknowledged .
REFERENCES CARTER, G . R . & CHENCAPPA, M . M . (1981) . 24th Annual Meeting of the American Association of Veterinary Laboratory Diagnosis p . 37 . CHANDRASEKARAN, S . (1988) . Proceedings of 1st Congress of the Veterinary Association of Malaysia p . 53 . CHANDRASEKARAN, S ., YEAP, P . C . & CHUINK, B . H . (1981) . British Veterinary Journal 137, 361 . CHANDRASEKARAN, S ., JOSEPH, P . G ., YEAP, P . C . & ROHANI SAAD . (1987) . Annual Report of the Veterinary Research Institute, Ipoh, Malaysia p . 23 . FRASER, J ., GILMOUR, N . J . L ., LAIRD, S . & DONACHIE, W . (1982). Veterinary Record 110, 560 . GILMOUR, N . J . L . & GILMOUR, J . S . (1989) . In Pasteurella and Pasteurellosis, eds C . Adlam & J .M . Rutter, p . 223 . London : Academic Press. GILMOUR, N . J . L ., THOMPSON, D . A ., SMITH, W . D . & ANCUS, K . W . (1975). Research in Veterinary Science 18, 340 . GILMOUR, N . J . L ., MARTIN, W . B ., SHARP, J . M ., THOMPSON, D . A., WELLS, P . W . & DONACHIE, W. (1982) . Research in Veterinary Science 35, 80 . HADI, H . (1988) . Proceedings of Symposium on Sheep Production in Malaysia p . 62 . MOSIER, D . A ., CONFER, A. W . & PANCEIRA, R . J . (1989) . Research in Veterinary Science 47, 1 . SHARP, J . M ., GILMOUR, N . J . L., THOMPSON, D . A . & RUSHTON, B . (1978) . Journal of Comparative Pathology 88, 237 . THOMPSON, D . A ., FRASER, J . & GILMOUR, N . J. L . (1977) . Research in Veterinary Science 22, 130 . TIZARD, I . (1982) . In Introduction to Veterinary Immunology, 2nd edn, p . 251 . Philadelphia : W .B . Saunders Company . WAN MOHAMED, W . E ., MOHAMED, N . & ABDUL RAHMAN, A. (1988) . Proceedings of Symposium on Sheep Production in Malaysia p. 98 . ZAMRI, SAAD, M ., KA9-IIL, W . M . & MUTALIB, A . R. (1989) . Jernal Veterinar Malaysia 1, 91 . (Accepted for publication 17 January 1991)
1.
(1991) . 147, 43 7
EVALUATION OF COMBINED PASTEURELLA VACCINES IN CONTROL OF SHEEP PNEUMONIA
S . CHANDRASEKARAN * , KAMAL HIZATt, ZAMRI SAADt, M . Y . JOHARA * and P . C . YEAP* *Veterinary Research Institute, P. 0. Box 369, 30740-Ipoh, Malaysia; tMalaysian Agricultural Research and Development Institute (MARDI), 43400-Serdang, Selangor, Malaysia ; $Faculty of Veterinary Science, Malaysian Agricultural University, 43400-Serdang, Selangor, Malaysia
SUMMARY The effectiveness of an oil adjuvant vaccine (OAV) incorporating locally isolated strains of Pasteurella haemolytica type 7 and Pasteurella multocida types A and D was compared with that of Carovax (Wellcome Laboratories) in imported cross-bred lambs . The criterion of efficacy was the ability of the vaccines to reduce the extent of pneumonic lesions in vaccinated as against unvaccinated control lambs. The OAV produced at this Institute significantly reduced the lung lesions at P< 0 .05 level compared with its control group when challenged with P. haemolytica alone . However, the vaccine was unsatisfactory against P . multocida or combined P. multocida P. haemolytica challenge . Carovax did not produce any significant reduction in the lung lesions caused by P. haemolytica and/or P. multocida.
INTRODUCTION The Department of Veterinary Services, Malaysia, has embarked on large scale sheep farming in rubber plantation . Pure and cross-bred sheep were imported to achieve the set target of one million sheep by the year 2000 . However, high mortality was recorded amongst imported pure bred as well as cross-bred sheep at the onset of this import programme . These were mainly due to pasteurellosis and haemonchosis . Hadi (1988) reported that more than 50% of the deaths was due to bluetongue-pneumonia complex . Pneumonic pasteurellosis (PP) is an important disease of sheep . The aetiological agent for this condition has been tentatively identified as Pasteurella haemolytica biotype A and Pasteurella multocida serotypes A and D while attempts at elucidating predisposing causes and the role of other pathogens are underway . Some of the uncontrollable predisposing factors are the hot, tropical climate of Malaysia with unpredictable convectional rainy spells and two definite monsoons which cause changes in the environment . In addition, management practices such as docking, drenching, castration, etc . cause additional stress . It was, therefore, found necessary to examine measures for control including vaccination as one means of reducing losses . Initially several kinds of commercially prepared broth vaccines were used to control
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this disease but with little success (Wan Mohamed et al, 1988) . One such vaccine used widely was Carovax (Wellcome Laboratories, UK) . This necessitated the development of a pasteurella vaccine using locally isolated strains of P. haemolytica and .P. mullocida types A and D . The aim of this trial was to compare the efficacy of the locally prepared oil adjuvant vaccine (OAV) with Carovax which was the only readily available commercial vaccine at that time . The parameter used in the evaluation was the ability of the vaccines to reduce the extent of lung lesions caused by experimentally induced PP in cross-bred lambs .
MATERIALS AND METHODS Animals
Thirty-five clinically healthy imported cross-bred lambs were selected from a government farm . They were aged between 4 and 6 months and weighed on average 12 .5 kg each . Blood samples and nasal swabs were taken from these animals . The former was used to determine the antibody levels to P. haemolytica and P. multocida by the indirect haemagglutination assay (IHA) as followed at this Institute (Chandrasekaran et al., 1981) . The nasal swabs were used to evaluate the bacterial flora involved . The animals were randomly assigned to three major groups (Table I) . All animals were subjected to a semiintensive management system, grazing during the day and being housed at night, with concentrate supplementation . Clean drinking water was available ad libitum . Table I Experimental design for vaccination trial Group
1 2 3
Vaccine/No . of animals
Challenge organism
P. haemolytica type 7 P. multocida types A & D Combined P. haemolytica type 7 and P. multocida A & D
VRI OA V
Carovax
Control
5 5 5
4
ND
4 4 4
4
ND, not done . Preparation of vaccine and vaccination
The OAV used in this trial was produced at the Veterinary Research Institute (VRI), Ipoh, Malaysia . It contained locally isolated strains of P. haemolytica type A7 and P. multocida types A and D . The details of the method of production have been given elsewhere (Chandrasekaran et al., 1987) . Briefly, the seed organisms were obtained from field outbreaks of PP in sheep . Separate dense broth aliquots were prepared for each organism by intermittent aeration, churning and incubation at 37°C for about 22-24 h . The average dry weight per ml of eight separate aliquots determined spectrophotometrically (Beckman instruments, California, USA) was 0 .67 mg of P. haemolytica and 0 .94 mg P. multocida type A and 0.85 mg type D .
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The aliquots were then incorporated with the adjuvant, a light mineral oil (Ondina grade 15) and an emulsifying agent (lanolin), using an industrial model emulsifier (Silverson emulsifier/mixer, Model EX, Bucks, England) . This yielded a water-in-oil type of emulsion . Lambs aged between 3 and 5 months and those above 5 months were intramuscularly given 3 and 5 ml respectively of the vaccine each . Annual revaccination was recommended .
Induction of pneumonic pasteurellosis The lambs were challenged 4 weeks post-vaccination . The procedure followed has been previously described by Chandrasekaran (1988) . Briefly, the animals were stressed by transportation for about 250 km . At the end of the journey, each animal was given 10 mg dexamethasone (Dexasone, Atlantis, Germany) intramuscularly, followed by two similar doses on consecutive days . Immediately after the last dexamethasone injection experimental PP was induced through aerosol administration of the infective organisms, using a modified version of that of Gilmour et al. (1983) . In this trial the tip of the nebulizer was placed against the nostrils . The experimental design is given in Table I .
Challenge The animals in group I were given an aerosal spray of P. haemolytica type 7 prepared from a 24-h broth culture . On average each animal in this group was given 1 .82X10' colony forming units (c .f.u .) . The animals in group 2 were given 2 .5X10" c .f.u . of P. multocida type A combined with 7 .2X10' c .f.u . of P. multocida type D prepared from separate 18-h broth cultures . For the combined challenge of the animals in group 3 each animal was given 9 .8X 10' c .f.u . of P. haemolytica, 1 .42X 10' c .f.u . of P. multocida type A and 3 .22X 10- c .f.u . of P. multocida type D .
Clinical observations and necropsy The animals were observed twice daily (8 :00 and 16 :00) following challenge . The clinical signs recorded were the rectal temperature, lethargy, anorexia, abnormal respiration (stertorous and abdominal respiration, polypnoea and dyspnoea) and mortality . All dead lambs were necropsied within 24 h of death . The extent of pneumonic lesions was recorded according to the method described by Gilmour et al. (1983) . A portion of the affected lung, the retropharyngeal, submandibular and mesenteric lymph nodes were collected for bacteriological examination . Blood was collected on the 3rd, 5th, 7th and 9th days post-challenge to assess the antibody status against P . haemolytica and P. multocida by the IHA (Chandrasekaran et al., 1981) . All surviving animals were euthanized 9 days post-challenge by intravenous administration of 12 ml per animal of a saturated solution of magnesium sulphate . Necropsy was performed by an independent team of veterinarians and the extent of lung lesions recorded .
RESULTS Serology
The sera of all the animals pre- and post-challenge were found to be negative for antibodies to P. haemolytica and P. multocida by the IHA test .
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Clinical observations None of the vaccinated (VRI OAV and Carovax) animals showed any adverse effects to vaccination . Seven animals died following challenge-four from the control group and three from the Carovax vaccinated group . There were no obvious clinical signs in the VRI OAV vaccinated animals as opposed to those in the other two groups . The mortalities and the clinical signs observed in the three groups are summarized in Table II .
Table II Summary of clinical signs and mortalities post-infection in vaccinated and unvaccinated lambs
Group
Challenge organism
VRI OA V
Carovax
Control
Number of animals in each group with 1
2
3
P. haemolytica type 7
P. multocida types A & D
P. haemolytica type 7 and P. mullocida types A & D
No . of deaths
0
No . of deaths
1
No . of deaths
1
No . with clinical signs
0
No . with clinical signs
0
No . with clinical signs
3
No . with 5 no clinical signs
No . with 3 no clinical signs
No . with 0 no clinical signs
No . of deaths
0
ND
No . of deaths
1
No . with clinical signs
2
ND
No . with clinical signs
3
No . with 3 no clinical signs
ND
No . with 0 no clinical signs
No . of deaths
0
No . of deaths
2
No . of deaths
2
No . with clinical signs
4
No . with clinical signs
2
No . with clinical signs
2
No . with 1 no clinical signs
No . with 0 no clinical signs
No . with 0 no clinical signs
ND, not done ; clinical signs : temperature > 40 .5 ° C and/or dyspnoea, polypnoea, and stertorous respiration .
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Bacteriology
The bacteriological culture of the prevaccination nasal swabs evaluated at the outset of the experiment and again prior to challenge were negative for P. haemolytica and P. multocida . Profuse, pure growth of the respective species used in the challenge was consistently isolated from the lung and/or lymph nodes of the lambs except in six (17%) animals from group 1 where three each were vaccinated with the VRI OAV and Carovax . However, the predominant organism reisolated in the mixed challenge group was P . multocida type A . Macroscopic lung lesions
Most lambs (62%) in this trial had lung lesions . The lesions were consolidation with red hepatization of the anteroventral portions of the apical, cardiac and intermediate lobes . Consolidation was also present along the borders of the diaphragmatic lobes . Fibrin deposition with adhesion to the parietal pleura was present in all the control animals and three dead animals vaccinated with Carovax . The lung lesions were more pronounced and extensive in the control group . The lambs vaccinated with the VRI OAV in group 1 had a significant reduction (P< 0 .05) in the lung lesion score compared to its control group . However, the animals in groups 2 and 3 did not show any significant difference in comparison with their respective control groups . A summary of the average lung lesion score is given in Table III . Table III Average lung lesion score-in vaccinated and control animals challenged with P. haemolytica type 7 and/or P. multocida types A & D Vaccination status
Control
Challenge organism Pasteurella haemolytica Pasteurella multocida
Mixed infection VRI OAV
Pasteurella haemolytica Pasteurella multocida
Mixed infection Corovax
Pasteurella haemolytica
Mixed infection
Lung lesion (%) Average
AD
9 .83 5 .63 8 .18
6 .23 5 .42 5 .25
0 .3 * 3 .68 2 .88
2.11 1 .83 1 .91
3 .95 11 .97
3 .10 4 .23
*Significantly different at P< 0 .05 when compared with the control group of similar treatment .
DISCUSSION Pneumonic pasteurellosis has been successfully reproduced in specific pathogen free sheep (Sharp et al., 1978 ; Gilmour et al., 1983) . In this study the disease was experimentally reproduced in conventionally reared cross-bred lambs . Experimental infection of sheep with aerosol of P. haemolytica was not always successful as inductions of
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pneumonic lesions were inconsistent . A similar observation has been made by Gilmour et al. (1975) . Prior to infection some form of stress was necessary . Other workers (Sharp et al., 1978 ; Gilmour et al., 1983) achieved this by first infecting with parainfluenza virus type 3 followed by aerosol infection of P. haemolytica 7 days later. In this trial the animals were initially stressed by transport and then further predisposed to infection by steroids . The 10 mg administered was double the therapeutic dose for sheep . Steroids have been presumed to exert their immunosuppressive effect by interfering with the cell-mediated immune (CMI) response (Tizard, 1982) . While it is possible that it affected the elicitation of secondary CMI response it is unlikely to have affected the primary response which could already have occurred in response to vaccination 28 days previously . This primary response would probably still exist . Since the vaccinated and control animals would presumably be similarly suppressed by the drug, the difference seen in the lung lesions could probably be attributed to the effect of the vaccines . The prechallenge bacterial flora in the nasal cavity of these animals did not correlate with the lung lesions following infection . This indicated that the lesions observed in this study most probably developed from the experimental infection . This was further confirmed by recovery of the respective organisms used for the challenge . The results obtained in this trial indicate that it is possible to immunize sheep against PP caused by P. haemolytica with the locally produced formalin-killed OAV . The efficacy of this vaccine against pneumonia caused by P. multocida alone or in combination with P. haemolytica was low . Mosier et al. (1989) observed a similar phenomenon in bovine PP where P. haemolytica OAV effectively reduced lesions of pneumonia caused by that organism . They, however, recorded little or no effect when P. multocida OAV or P. multocida in combination with P. haemolytica in aluminium hydroxide adjuvant were administered . Unsatisfactory protection in sheep vaccinated with pasteurella vaccines incorporating sodium salicylate and heat-killed organisms has also been reported (Gilmour et al., 1983) . In a separate trial Zamri Saad et al. (1989) observed that the VRI OAV was ineffective in controlling naturally occurring ovine PP caused by either P . haemolytica or P. multocida in imported pure bred sheep as opposed to cross-breds used in this trial . A private agricultural organization (Ngah Mohammed, personal communication) experienced a similar outcome with the VRI OAV . These could probably be attributed to the following . 1 . Strain differences : P. haemolytica exists as two main biotypes and 15 serotypes and protection appears to be serotype specific (Gilmour & Gilmour, 1989) . P. multocida can be classified into four capsular and 16 somatic types (Carter & Chengappa, 1981) . Of these, serotypes A and D have been isolated from cases of PP in sheep and goats . Thompson et al. (1977) found that varying serotypes of P. haemolytica were isolated during different seasons and suggested their incorporation in vaccines . On a similar note, Fraser et al. (1982) stressed the importance of continually monitoring the prevalence of the different serotypes, investigating the pathogenicity of new serotypes so that the appropriate strains could be incorporated in the vaccines . The organisms incorporated into the VRI OAV were P. haemolytica type A7 and P. multocida capsular types A and D and of undetermined somatic types . It is postulated that the natural infection reported could have been caused by other type/types of pasteurellae . 2 . The trial being reported was conducted in cross-bred sheep whereas the natural infection observed by Zamri Saad et al (1989), was in pure bred sheep .
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3 . The pure bred sheep could have been harbouring the aetiological agents in their respiratory tract as the bacterial status of the nasal orifices was not determined by swabbing and subculture . The stress of transport and the new environment could have triggered their multiplication before optimum protection could be elicited by the vaccine . No adverse side effects to vaccination were encountered in all the animals used in this trial . The IHA titre would seem to indicate that the antibody levels did not increase either as a result of vaccination and/or challenge . The IHA was probably an unsuitable in-vitro quantitative test for antibody estimation against P. haemolytica and P. multocida types A and D in sheep sera .
ACKNOWLEDGEMENTS The authors would like to thank the Director-General of Veterinary Services, Malaysia, for permission to publish this paper and to the Directors of VRI, Ipoh, and MARDI, Serdang, for providing the facilities and the animals respectively . The technical assistance of Mrs Rohani Saad is acknowledged .
REFERENCES CARTER, G . R . & CHENCAPPA, M . M . (1981) . 24th Annual Meeting of the American Association of Veterinary Laboratory Diagnosis p . 37 . CHANDRASEKARAN, S . (1988) . Proceedings of 1st Congress of the Veterinary Association of Malaysia p . 53 . CHANDRASEKARAN, S ., YEAP, P . C . & CHUINK, B . H . (1981) . British Veterinary Journal 137, 361 . CHANDRASEKARAN, S ., JOSEPH, P . G ., YEAP, P . C . & ROHANI SAAD . (1987) . Annual Report of the Veterinary Research Institute, Ipoh, Malaysia p . 23 . FRASER, J ., GILMOUR, N . J . L ., LAIRD, S . & DONACHIE, W . (1982). Veterinary Record 110, 560 . GILMOUR, N . J . L . & GILMOUR, J . S . (1989) . In Pasteurella and Pasteurellosis, eds C . Adlam & J .M . Rutter, p . 223 . London : Academic Press. GILMOUR, N . J . L ., THOMPSON, D . A ., SMITH, W . D . & ANCUS, K . W . (1975). Research in Veterinary Science 18, 340 . GILMOUR, N . J . L ., MARTIN, W . B ., SHARP, J . M ., THOMPSON, D . A., WELLS, P . W . & DONACHIE, W. (1982) . Research in Veterinary Science 35, 80 . HADI, H . (1988) . Proceedings of Symposium on Sheep Production in Malaysia p . 62 . MOSIER, D . A ., CONFER, A. W . & PANCEIRA, R . J . (1989) . Research in Veterinary Science 47, 1 . SHARP, J . M ., GILMOUR, N . J . L., THOMPSON, D . A . & RUSHTON, B . (1978) . Journal of Comparative Pathology 88, 237 . THOMPSON, D . A ., FRASER, J . & GILMOUR, N . J. L . (1977) . Research in Veterinary Science 22, 130 . TIZARD, I . (1982) . In Introduction to Veterinary Immunology, 2nd edn, p . 251 . Philadelphia : W .B . Saunders Company . WAN MOHAMED, W . E ., MOHAMED, N . & ABDUL RAHMAN, A. (1988) . Proceedings of Symposium on Sheep Production in Malaysia p. 98 . ZAMRI, SAAD, M ., KA9-IIL, W . M . & MUTALIB, A . R. (1989) . Jernal Veterinar Malaysia 1, 91 . (Accepted for publication 17 January 1991)
