Laboratory Animals (1990) 25, 337-341


Pathogenicity of Pasteurella multocida A:3 in Flemish Giant and New Zealand White rabbits D. L. DILLEHAyl, K. S. PAUL2, R. F. DIGIACOM03 & M. M. CHENGAPPA4 IDepartment of Pathology; and 1,2Division of Animal Resources, Emory University School of Medicine, Atlanta, GA 30322; 3Department of Comparative Medicine, School of Medicine, University of Washington, Seattle, WA 98195; and 4Department of Laboratory Medicine, College of Veterinary Medicine, Kansas State University, Manhattan, KS 66506, USA

Summary Pasteurella multocida A:3 was isolated during an outbreak of pasteurellosis in Flemish Giant (FG) rabbits. Since New Zealand White (NZW) rabbits housed in the same room were not as severely affected as FG rabbits, experimental inoculation was undertaken to determine if FG rabbits were more susceptible than NZW rabbits to pasteurellosis induced by this isolate. Rabbits of each breed were inoculated with P. multocida A:3 and observed for 3 weeks. Four of 5 FG' rabbits developed severe clinical disease on days 6, 9, 12 and 14 after inoculation; whereas, the one affected NZW rabbit became ill 14 days after inoculation. AlI rabbits with clinical disease developed fibrinosuppurative pleuritis, pyothorax and pneumonia which was more severe in FG than NZW rabbits. At necropsy, P. multocida A:3 was isolated from multiple sites of the diseased rabbits. No significant difference (P = O· 099) in the prevalence of

lesions between the two breeds was found; however, the score of pneumonia and pleuritis was 3 times greater in FG rabbits than NZW rabbits. Keywords: Pasteurellosis; Breed comparison; Rabbits

Correspondence to: Dr Dirck L Dillehay, Emory University School of Medicine, GOn, Rollins Research Center, 1510 Clifton Road, NE, Atlanta, GA 30322, USA. Received 3 December 1990; accepted 8 April 1991

Pasteurellosis is caused by infection with Pasteurella multocida and is one of the most significant diseases of domestic rabbits. Pasteurellosis in laboratory rabbits can cause acute epizootics of pneumonia with high morbidity and mortality. Chronic infection may result in rhinitis, bronchopneumonia, otitis media, pyometra, abscessation and encephalomyelitis (Manning et al., 1989). Furthermore, the high morbidity due to P. multocida infections in laboratory rabbit colonies results in animals that are unsuitable for research. Five capsular (A, B, D, E, F) and 16 somatic (1-16) serotypes of P. multocida have been described (Manning, 1982; RimIer & Rhoades, 1987). Capsular types A and D and somatic types 1, 3, 4, 11, 12 have been isolated from rabbits (Brogden, 1980; Lu et 01., 1983). Various studies showed that A: 12 and A:3 were the most important serotypes associated with pasteurellosis in rabbits (Lu et al., 1978; DiGiacomo et 01., 1983). Serotype A:3 is reported to be more virulent in rabbits than A: 12 (Lu & Pakes, 1981; DiGiacomo et al., 1983). Our colony of Flemish Giant (FG) rabbits experienced an outbreak of respiratory disease caused by P. multocida A:3. The disease in FG rabbits was more severe than that observed in New Zealand White (NZW) rabbits housed in the same room. The disease in FG rabbits was characterized by a sudden onset of anorexia, fever and rhinitis that was unresponsive to treatment. All affected rabbits died or became moribund and were euthanized within 2-4 days after onset of clinical signs. At necropsy, most FG rabbits had severe pyothorax, pleuritis or

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Dillehay, Paul, DiGiacomo & Chengappa

338 septicaemia. Pasteurellosis in NZW rabbits was associated with mild rhinitis, and rarely death. Either the FG rabbits were more susceptible to pasteurellosis or a more pathogenic P. multocida was involved. Hence, the purpose of this study was to investigate the pathogenicity of the P. multocida A:3 isolate recovered from FG rabbits in FG and NZW rabbits. Materials and methods Animals P. multocida-free, male and female, FG 16month-old rabbits (Oryctolagus cuniculus) were from our caesarean-derived colony. P. multocidafree NZW 16-month-old male and female, rabbits were obtained from a commercial vendor (Myrtle's Rabbitry, Thompson Station, TN, USA). Rabbits, 4 females and 3 males of each breed, were housed individually in stainless steel cages in a barrier-maintained room and given commercial rabbit chow (Laboratory Rabbit Chow High Fiber 5326, Purina Mills, Inc., Richmond, IN, USA) and water ad libitum. The rabbit rooms were on a 12: 12 h light: dark cycle. The animal rooms were kept at 18-20 °C with a 50-60070humidity and 15 air changes per hour. All rabbits had two negative nasal cultures to P. multocida and were free of serum antibodies (IgG) for P. multocida as determined by enzyme linked immunosorbent assay (ELISA). Rabbits inoculated with P. multocida were housed in a separate room from rabbits inoculated with saline (control). The 4 control rabbits consisted of 2 FG and 2 NZW rabbits of each sex. The experimentally inoculated rabbits consisted of 5 FG and 5 NZW rabbits, 3 females and 2 males of each breed. Experimental design Rabbits were inoculated intranasally with 1.0 ml of P. multocida A:3 at a dose of 4,0 X 108 colony forming units (cfu)/ml in phosphate buffered saline (PBS). Sham inoculated rabbits were given 1.0 ml of sterile PBS intranasally. On days 6,9 and 12 after inoculation, one FG rabbit each either died or was moribund and euthanized. One inoculated NZW rabbit was moribund and

euthanized 14 days after inoculation. One inoculated FG and two NZW rabbits were euthanized and necropsied 14 days after inoculation. One NZW and one FG rabbit from the sham inoculated groups were necropsied at 14 days after inoculation. Two rabbits from the sham inoculated groups and 3 rabbits from the experimentally inoculated groups were necropsied 21 days after inoculation. Each animal that was euthanized was examined pathologically, bacteriologically, and serologically. Rabbits were euthanized by an intravenous overdose of pentobarbital. Rabbits that were found dead were cultured and examined for gross and microscopic lesions. Inoculum preparation P. multocida A:3 was previously isolated from a FG rabbit that died with pneumonic pasteurellosis. The isolate was identified by hyaluronidase decapsulation and agar gel precipitin tests (Chengappa et al., 1980). The isolate was stored in 10% glycerol and brain heart infusion (BHI) at -70 °C in 1·0 ml samples. Bacteria were quantitated by diluting a l' a ml thawed sample lO-fold in sterile PBS, pH 7·4. Each dilution was streaked on BHI plates and counted after incubation for 24 hat 37°C in 5% CO2, Samples for inoculation were diluted in PBS to 4 x 108 cfu/ml. Bacterial isolation At necropsy, swabs from the nasal cavity and tissue from lungs, liver, spleen, heart blood and pleural and pericardial fluid were cultured from all dead and euthanized rabbits. Selected P. multocida isolates were typed by the hyaluronidase decapsulation and agar gel precipitin tests using rabbit type specific anti-sera. Serologic tests Blood was collected from the marginal ear vein in anaesthetized rabbits prior to inoculation and from the brachial plexus at necropsy. Sera were obtained by centrifugation and stored at -70 °C. Sera were diluted 1: 100 and assayed for classspecific IgG antibodies by ELISA as described

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Comparison of pasteurellosis in rabbits Table 1. Histopathological,



and serological

findings in Flemish Giant and New Zealand

White rabbits inoculated

with Pasteurella multocida A:3



Died/ euthanized on dayS'


P. multocida P. multocida P. multocida P. multocida P. multocida P. multocida P. multocida P. multocida P. multocida P. multocida Saline Saline Saline Saline

6 9 12 14 21 ]4 ]4 ]4 21 21 14 2] 14 21

Pneumonia Lesion pleuritis Rhinitis indeX'

P. multocida isolation

+++ +++ +++ + 0 +++ 0 0 0 0 0 0 0 0

Liver, pleura, peridcardium Liver, pleura, lung, blood, spleen 0'239 Liver, lung, blood, spleen, pericardium O· 396 Pleura 0'581 0'629 I'] 13 Liver, pleura Nasal cavity 0'379 0'585 0'381 0·628 o· 313 0'323 o· ]53 0'273

+ + + + 0 + 0 0 0 0





Rabbits were inoculated intranasally with 4'0 x lOS cfu of serotype P. multocida A:3 or saline (controls) and necropsied 14 or 21 days after inoculation. Three Flemish Giant (FG) rabbits were necropsied before day ]4 after inoculation due to severe illness or death. bIndicates lesion severity of the group. A lesion index of 1·0 represents the most severe disease possible. 'Optical density (00) of ELISA obtained on sera at I: 100 dilution. No serum was collected from the FG rabbit found dead on day 6. AIl sera was tested at the same time. 00 values greater than O' 350 were considered positive for antibodies to P. multocida. a

previously (DiGiacomo et al., 1987). Blood was obtained at necropsy from thirteen rabbits instead of 14 because one rabbit died unexpectedly during the night. All sera were tested at the same time.

Pathological evaluation Sections of nasal turbinates, trachea, heart, lungs (all lobes), liver, spleen and genital organs were fIXed in 10070 neutral buffered formalin, embedded in paraffin, sectioned at 611, stained with haematoxylin-eosin, and examined for microscopic lesions. Nasal turbinates were decalcified before embedding. Lesions in the lungs were scored on a severity scale of: 0 (no lesions); 1 + (mild lesions characterized by minimal thickening of alveolar septa by heterophils and/or mononuclear inflammatory cells); 2 + (moderate lesions characterized by inflammatory cells in alveoli, bronchioles and interstitium with or without pleuritis); 3 + (severe lesions characterized by extensive infiltration of inflammatory cells, pleuritis and pyothorax). To compare groups, the sum of lesion scores for the lungs from individual rabbits within a group' was divided by the sum of

maximum possible scores to arrive at the group lesion index. A group lesion index of 1 .0 was the most severe change possible for a group.

Statistical analysis Fishers-exact test was used to determine significant differences of disease incidence between groups at the level of P 103 OF), anorectic and lethargic one to two days prior to death. At necropsy, lesions were observed in 4 of 5 FG rabbits and one of 5 NZW rabbits. The three FG rabbits necropsied within 12 days after inoculation had pyothorax, pleuritis, pericarditis, and pulmonary abscesses. The NZW rabbit which was euthanized 14 days after inoculation had pyothorax and pleuritis.

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Dillehay, Paul, DiGiacomo & Chengappa


Microscopically, the FG rabbits had severe fibrinosuppurative pleuritis and mild to moderate fibrinosuppurative pericarditis. Affected FG rabbits also had multifocal interstitial pneumonia affecting predominantly the subpleural region associated with pleuritis. One FG rabbit inoculated with P. multocida and the two sham inoculated FG rabbits remained clinically normal and had no significant lesions. One NZW rabbit had severe fibrinosuppurative pleuritis with adjacent mild interstitial pneumonia. The other four NZW rabbits inoculated with P. multocida remained clinically normal and had mild to moderate hyperplasia of bronchial associated lymphoid tissue. The two sham inoculated NZW rabbits had no significant lesions. Histologically, mild rhinitis was observed in 4 FG and one NZW rabbits (Table 1). There was no significant difference (P=0'099) in the prevalence of lesions between these two breeds; however, the group lesion index scores of pneumonia and pleuritis in the FG rabbits was 3' 3 times greater than that of the NZW rabbits (Table 1). P. multocida A:3 was isolated from multiple sites from the 3 severely affected FG rabbits (Table 1). In the one affected NZW rabbit, P. multocida was isolated from the liver and pleural fluid. All inoculated rabbits that lived longer than 11days after inoculation developed IgO antibodies to P. multocida (Table 1).There was no difference in tit res between the breeds. All sham inoculated rabbits failed to develop tit res to P. multocida. Discussion Although considerable data have been accumulated on the prevalence of different serotypes of P. multocida in rabbits, little is known about differences in pathogenicity of various serotypes and the comparative susceptibility of different breeds. Webster (1927)reported that the prevalence of rhinitis and mortality due to P. multocida was considerably lower in the Blue Beveren than in the chinchilla or Havana breeds. Similarly, the carriage of P. multocida in the nares of healthy rabbits was significantly greater in rabbits of the chinchilla than the Blue Beveren breed; 60 of 142 (42070)of the former were infected compared to 20 of 76 (20070)of the latter breed. Hence, the

Blue Beveren breed was more resistant to infection and disease caused by P. multocida. The present study suggests that our isolate recovered from a FG rabbit was more pathogenic for FG than NZW rabbits. Three FG rabbits developed severe clinical pasteurellosis 14 days after inoculation. Infection in FG rabbits was detected at more disseminated sites, including the blood, than in NZW rabbits. Furthermore, more severe disease developed in FG than NZW rabbits as shown by the 3' 3-fold increase in the lesion score of FG rabbits. The cause of the more severe lower respiratory disease in FG than NZW rabbits infected with P. multocida was not determined in this study; however, another study has shown that more severe disease due to P. multocida resulted when NZW rabbits were immunocompromised by injection of hydrocortisone (Lu et al., 1982). Hydrocortisone inhibits the immune response by decreasing the phagocytic and killing capacity of neutrophils (Mandall et al., 1970). Thus, the increase in clinical disease and severity of lesions in the FG rabbits may have been due to a depressed local immune response to P. multocida. In most species, P. multocida infections result in the production of protective opsonizing antibodies in conjunction with phagocytic neutrophils (Collins, 1977). In the rabbit, however, nonspecific opsonins, such as complement, play a more important role in promoting phagocytosis of P. multocida by granulocytes than specific antibody (Hofing et al., 1979). Bacterial infections are more common and more severe in man and animals with diseases associated with . defects in granulocyte function, i.e. chronic granulomatous disease, than in animals with normal granulocytes (Quie et al., 1976). Acknowledgement We thank Dr Patti Fields, Centers for Disease Control, Atlanta, GA, USA, for her technical expertise and Ms Susan Mars for her secretarial assistance in the preparation of this manuscript. Supported in part by grant RR01203 from the Division of Research Resources, National Institutes of Health, Bethesda, MD, USA.

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Comparison of pasteurellosis in rabbits


References Brogden KA (1980) Physiological and serologica] characteristics of 48 Pasteurella mu/tocida cultures from rabbits. Journal of Clinical Microbiology 11, 646-649 Chengappa MM, Myers RC & Carter GR (]980) A streptomycin dependent live Pasteurella multocida vaccine for the prevention of rabbit pasteurellosis. Laboratory Animal Science 30, 5]5-5]8 Collins FM (1977) Mechanisms of acquired resistance to Pasteurellamultocida infection: a review.Cornell Veterinarian 67, 103-138 DiGiacomo RF, Deeb BJ, Bernard BL, Kloassen JM & Chengappa MM (1987) Safety and efficacy of a streptomycin dependent live Pasteurella multocida vaccine in rabbits. Laboratory Animal Science 37, ]87-190 DiGiacomo RF, Garlinghouse LE & Van Hoosier GL (]983) Natura] history of infection with Pasteurella multocida in rabbits. Journal of the American Veterinary Medical Association 183, I] 72-1175 Horing GL, Rush HG, Petkus AR & Glorioso JC (]979) In vitro killing of Pasteurella mu/tocida: the effect of rabbit granulocyte and specific antibody source. Animal Journal of Veterinary Research 40, 679-683 Lu YS & Pakes SP (1981) Protection of rabbits against experimental pasteurellosis by a streptomycin-dependent Pasteurella multocida serotype 3:A live mutant vaccine. Infection and Immunity 34, 1018-1024 Lu YS, Pakes SP, Rehg JE & Ringler DH (1982) Pathogenicity of a serotype 12:A Pasteurella multocida in hydrocortisone treated and nontreated rabbits. Laboratory Animal Science 32, 258-2~2

Lu YS, Pakes SP & Stefanu C (1983) Capsular and somatic serotypes of Pasteurella multocida isolates recovered from healthy and diseased rabbits in Texas. Journal of Clinical Microbiology 18, 292-295 Lu YS, Ringler DH & Park JS (1978) Characterization of Pasteurella multocida isolates from the nares of healthy rabbits and rabbits with pneumonia. Laboratory Animal Science 28, 69]-697 Mandall GL, Rubin LW & Hook EW (1970) The effect of an NADH oxidase inhibitor (hydrocortisone) on polymorphonuclear leukocyte bactericidal activity. Journal of Clinical Investigation 49, 1381-1388 Manning PJ (1982) Serology of Pasteurella multocida in laboratory rabbits: a review. Laboratory Animal Science 32,666-671 Manning P J, DiGiacomo RF & DeLong D (1989)Pasteurellosis in Laboratory Animals. In Pasteurellaand Pasteurellosis.New York, NY: Academic Press, Inc, pp.265-302 Quie PG, White JG, Holmes G et 01. (1976) In vitro bactericidal capacity of human polymorphonuclear leukocytes: Diminished activity in chronic granulomatous disease. Journal of Clinical Investigation 46, 668-679 Rimier RB & Rhoades KR (1987) Serogroup F, a new capsule serogroup of Pasteurella multocida. Journal of Clinical Microbiology 25, 615-618 Webster LT (1927) Epidemio]ogical studies on respiratory infections of the rabbit. IX. The spread of Bacterium lepisepticum infeciton at a rabbit farm in New York City, NY. Journal of Experimental Medicine 45, 529-551

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Pathogenicity of Pasteurella multocida A:3 in Flemish giant and New Zealand white rabbits.

Pasteurella multocida A:3 was isolated during an outbreak of pasteurellosis in Flemish Giant (FG) rabbits. Since New Zealand White (NZW) rabbits house...
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