Histol Histopath (1 989) 4: 77-84 Histology and Histopathology

Rabbit pasteurellosis: Respiratory and renal pathology of control and immunized rabbits after challenge with Pasteurella multocida

Z.S. Al-Lebbanl, S. Kruckenberg2and E.H. Coles1 'Department of Laboratory Medicine and 2Department of Pathology, College of Veterinary Medicine, Kansas State University, Manhattan, KS 66506, USA

Surnrnary. Gross and microscopic lesions of lntroduction pasteurellosis were studied in control and immunized pasteurella-free rabbits after challenge with virulent Respiratory diseases are common in domesticated Pasteurella multocida. Pathologic responses were rabbits. This may vary from a mild, chronic mucopurulent compared in rabbits immunized intravenously or upper respiratory infection (snuffles) to more acute or mucosally with P. multocidaor with J5, a cross protective subacute bronchopneumonia leading to high mortality core LPS mutant of E. coli Al1 rabbits were challenged (Hagen, 1958). Epizootics and enzootics of acute fatal conjunctivally with approximately 2xLD,, of P. pneumonia have been reported (Alexander et al., 1952; multocida. Rabbits were necropsied and examined for Hagen, 1959; Flatt and Dungworth. 1971). histopathology of the respiratory tract and kidneys. Lung It has been reported that Pasteurella multocida lesions varied in severity depending on the duration of frequently causes either acute bronchopneumonia in the the disease, the route of vaccination, and the vaccine cranio-ventral portion of the lungs of rabbits dying of used. The most severe lung lesions occurred in rabbits prieumonia or chronic purulent to fibrinopurulent vaccinated intravenously with P. multocida and bronchopneumonia (Weisbroth, 1979). challenged with the same strain. Some of these rabbits This study is part of a project to compare the had purulent bronchopneumonia and pleuropneumonia. effectiveness of vaccines administered intravenously, by Lung lesions were absent or less severe in rabbits aerosol. and in the conjunctival sac. This includes the vaccinated by a mucosal (aerosol, conjunctival) roiite gross and microscopic changes in lungs and kidneys of and in unvaccinated controls. In these animals there was rontrol and immunized rabbits after challenge with no bronchopneumonia or pleuropneumonia, and P~r+surellamultocida. bronchiolitis, if present, was less severe. Kidney lesions were found only in rabbits vaccinated intravenously. Materials and methods There was an interstitial nephritis. some collagen deposition. mononuclear cell infiltration, and a loss of Rabbits tubular architecture in the cortex. Some glomeruli were affected. Ninety-six male Pasteurella free (Pf) rabbits These results indicate that intravenous immunization (Dutchland Laboratories. Inc., Denver, PA), 8 to 9 contributes to the formation of lesions whereas mucosal weeks of age and weighing 1.5-2.5 kg, were used for immunization prevented lesion formation to some vaccination and challenge as has been described (Al- degree. Lebban et al., 1988). Briefly, 58 Pf rabbits were vaccinated intravenous!~ with boiled cells (at 22% Key words: Pasteurella multocida, Rabbit, Pneumonic transmission) of P. multocida 1059, P. multocida 1062, E. Pasteurellosis, Immuniza tion col¡ J5, or sterile saline solution. The rabbits were given six I.V. injections, 1.0 mllinjection, at 2-day intervals. Two routes were used for mucosal vaccination with P. multocida 1062, E. coliJ5, or sterile saline. Aerosols were created by an ultrasonic nebulizer (Ultramist 111, Offprint requests to: Z.S. Al-Lebban, BVMS, MS, Ph.D., Department Macrosonics Corp, Rahway, NJ) with approximately 3 of Environmental Practice, College of Veterinary Medicine, University m1 of aerosolized vaccine exposure and given to 18 of Tennessee 37901, USA rabbits. Each rabbit was exposed for 15 minutes, 3 times, 78 lmmunopathology of rabbit pasteurellosis

at 5-day intervals. Conjunctival vaccination (20 rabbits) was done by dropping 0.1 m1 of the vaccine into both conjunctival sacs six times at 2-day intervals. Twenty-one days after the first vaccination, al1 rabbits were challenged by placing 0.1 m1 of a suspension of P. multocida (containing approximately 2x the LD,,) into the conjunctival sac. Additional groups of four rabbits immunized intravenously and mucosally with P. multocida, E. coliJ5, and saline were necropsied without being challenged.

Necropsy Examination

Al1 rabbits were necropsied; sections of trachea, lungs, and kidney no thicker than 0.5 cm were taken and fixed in 10% buffered neutral formalin. The tissues were dehydrated through graded ethanols, cleared in xylene, and infiltrated with embedding paraffin in a routine automated processor cycle (Autotechnicon, Technicon Corporation, Chauncey, N.Y.). cut at 6 p. mounted on glass slides, stained by hematoxylin and eosin (H & E) using an automated slide stainer (Histotech, Ames Company, Div. Miles Laboratories, Inc., Elkhart, IN.), and covered with glass coverslips. The periodic acid Schiff (PAS) stain on kidney tissues was done by hand (Luna, 1968).

Results

Macroscopic Findings Fig. 1. Lung of a Pf-rabbit vaccinated intravenously with P. Trachea: There were few abnormalities except for a multocida 1062 and sacrificed 7 days after challenge with P. diffusely hyperemic mucosa in rabbits that died 12 to 144 multocida 1062. The alveoli are filled with heterophils, few hours post-challenge. Survivors that were necropsied 180 rnononuclear cells and fibrin. Hematoxylin-eosin stain. x 31 3 hours post-challenge had no tracheal lesions or patches of hyperemic mucosa. by the intravenous route. There were scattered dark red foci on the outer surfaces of the kidney that varied in size Lungs: The severity of lung lesions varied depending and number. These lesions were the most frequent and on how soon the rabbit died after challenge. the route of numerous in rabbits vaccinated with E. coliJ5. vaccination and the type of antigen used. Rabbits that were vaccinated but not challenged had no lung lesions. Microscopic findings Rabbits that died 12-24 hours post-challenge generally had few lesions. The lungs from rabbits that died latter Trachea: There were no marked differences in lesions had either no lesions, various degrees of hyperemia. or of the trachea with respect to route of immunization. multifocal to diffuse areas of consolidation of the apical antigen used, or saline. Tracheal lesions in rabbits that and cardiac lobes. Survivors that were necropsied 180 died during the first 24 hours post-challenge varied from hours post-challenge had more severe lung lesions. The no microscopic change to a severely congested mucosa. lesions were most severe in rabbits vaccinated Most rabbits that died later or were killed 180 hours post- intravenously. Lesions were less severe in rabbits challenge developed an inflammatory response that vaccinated by conjunctival and aerosol routes and in varied from a mild to a moderate catarrhal inflammation. unvaccinated rabbits. In some, cuboidal epithelial cells had detached while the Severe lung lesions varied from bilateral diffuse basa1 layer remained intact, and there was some fibrinous pleuritis to consolidated areas of lung that were accumulation of mucus and heterophils in the lumen. firm and varied from red to grey or black. Cross-sections Tracheal blood vessels of rabbits that died after through such altered lobes were alternately dark red to challenge were moderately to severely congested. greywhite. Some survivors had no changes, small red or Tracheal lesions varied in rabbits that survived one week grey areas, or slightly enlarged lungs. post-challenge. In some, there were no marked changes, and in others, there was severe catarrhal inflammation Kidneys: Macroscopic changes in the kidney were with desquamation of the epithelial lining. and the lumen found in challenged and unchallenged rabbits vaccinated was filled with heterophils and mucus. lmmunopathology of rabbit pasteurellosis

Table 1. A surnrnary of the histopathology of the respiratory tract of Pasteurella-free New Zealand white rabbits challenged with 2 x LD,, of two strains of Pateurella multocida following vaccination with P. multocida or Escherichia col; J5.

Number of Rabbits with lesions

No. of lnterstitial Broncho- Pleuro- Rabbits 1 Pneumonitis Pneumonia Pneumonia Bronchiolitis 1

P. multocida hornologuos

heterologuos m 3 ? a, > m 4-L E. col¡ l = 1 control O O O O O 7 O I lb ' O o o o o o o o

pp P. multocida hornologous 8a 7 O O O O O 4 O - 7b m 6 O O O O O 1 O O O 2 E. coli J5 7a 5 O O O O O 6 O 8b 4 O O O O O 3 O

Control 8a 2 O O O O O 4 O

a. Moderate to rnarked b. Slight c. Death occurred < 96 hours post inoculation d. Euthanatized 180 hours post inoculation.

Table 2. lncidence of renal pathology according to route of vaccination

Rabbits with renal pathology Vaccine Route Positive/Total Percent Positive ~one i All routes l 0123 1 Pasteurella multocida 1 lntravenous I 11/28 39.3 Pasteurella multocida I Mucosal 1 011 5 0.0 1 Escherichia coli J5 lntravenous 811 5 53.3 l I I 1 Escherichia coli J5 1 Mucosal 1 011 5 0.0 1

Lungs: The major lung lesions were interstitial were variable, and severity was correlated with the route pneumonia, bronchopneumonia, pleuropneumonia, and of vaccination and the antigen used. bronchiolitis. The frequency and severity of these lesions Rabbits vaccinated intravenously developed lung in the various groups is shown in Table 1. lesions of variable severity. The condition of the Rabbits that died during the first 24 hours post- bronchioles ranged from relatively normal to severe challenge had few microscopic lesions in the epithelial epithelial desquamation that included the basa1 layer lining of the bronchioles or terminal bronchioles. There cells. The lumen of the bronchioles in these rabbits was some mucus in the lumen. The alveolar wall was contained copious amounts of fibrin, heterophils, and normal or slightly thickened and infiltrated with noncellular debris. The terminal bronchioles generally heterophils. Alveolar spaces were clear or filled with had the same response as the bronchioles, but were more serous fluid, and the blood vessels were congested. frequently affected. The alveolar walls were thickened in Lung lesions in rabbits killed 180 hours post-challenge rabbits of most groups with a marked increase in severity 80 lmmunopathology o f rabbit pasteurellosís

Fig. 2. Lung of a Pf-rabbit vaccinated intravenously with P. multocida 1059. The alveoli contain many heterophils, red blood cells, fibrin and edema. Hematoxylin- eosin stain (A) x 112and (B) x 313

Fig. 3. Lung of a Pf-rabbit vaccinated ntravenously with P. multocida 1059 and sacrificed 7 days after challenge wi th P. multocida 1059. The alveoli are filled with tieterophils, proteinaceous material, fibrin, and edema. Hernatoxylin- eosin stain (A) x 112and (B) x 313 Fig. 4. Fibrinous p,europneurnonia of a Pf-rabbit vaccinated ini:ravenously with P. multocida 1059, and sacrificed 7 days after chailenae witn P. niultocida 1059. The pleura is thickened and there is extensive marginaGon of the heterophils. Hernatoxylin-eosin stain. x 112

Fig. 5. The kidney of Pf- rabbit vaccinated intravenously with P. multocida 1062 and challenged with P. multocida 1062. There is a focal interstitial nephritis and mononuclear cell infiltration. Hernatoxylin- eosin stain. x 112

Fig. 6 The kidney of a Pf-rabbit vaccinated intravenously with E. coli J5 and challenged with P. multocida 1059. There is a diffuse interstitial nephritis and nfiltration of mononuclear cells. Periodic Acid Schiff Stain (A) x 112 and (B) X 313 82 lmmunopathology o f rabbit pasteurellosis

in rabbits vaccinated intravenously and challenged with lung, presumably mediated by systemic immunity, was the autologous strain. The alveoli were filled with protective. But it was at the cost of considerable damage heterophils. Edema and fibrin were prominent (Fig. 1). to the host in the form of moderate to severe pneumonia. The alveolar space of these rabbits was either clear or filled This is comparable to Markham & Wilkie's findings of with heterophils, fibrin and noncellular debris (Fig. 2). more severe pneun~oniain vaccinated calves than in Some rabbits developed hemorrhagic lesions in which the controls (Markham and Wilkie. 1980a). They suggested interstitial tissue and the alveolar spaces were filled with that since immunized serum enhances phagocytosis of P. erythrocytes (Fig. 3). It was striking that bronchopneumonia hemolytica in challenged animals (Markham and Wilkie, occurred only in intravenously iinmunized animals. 1980b) and that since P. hemolytica is cytotoxic for The pleura varied in response to challenge. In some alveolar macrophages, the destruction of macrophages rabbits the pleura was relatively normal; in others the and release of enzymes in immunized animals may account pleura was thickened and infiltrated with fibrin, for the more severe pneumonia in this group (Kaehler heterophils, a few mononuclear cells, and bacteria1 et al., 1080). Similar phenomena may be occurring in colonies (Fig. 4). These severe changes were seen only in systemically immunized rabbits in our study. Immune rabbits vaccinated intravenously with P. multocida and complex deposition could be involved also as suggested challenged with the autologous strain. by Wilkie et al. (1974). Physiologically. the predominance Rabbits vaccinated by the aerosol or conjunctival of IgG antibody at the alveolar leve1 does not hold in the route developed an interstitial pneumonia of variable upper airways, where IgA prevails (Bienenstock. 1985). severity. None of the rabbits in these two experiments Most natural immune complex-mediated intra-alveolar developed a fibrinous pneumonia, and the pleura was injury is likely to be induced by IgG complexes or those unaffected. of both 1gG and IgA. Also, it is well established that IgG immune complex injury requires an intact complement Kidney Kidney lesions were restricted to challenged system (Henson and Cochrane, 1971; Johnson and Ward, or unchallenged rabbits vaccinated intravenously. Other 1974; Larsen et al., 1981), and the role of complement rabbits in this study that were vaccinated by aerosol and appears to relate to the intrapulmonary activation of conjunctival routes including the controls did not develop complement and subsequent immigration of heterophils kidney pathology (Table 2). Multifocal interstitial into the site. Recent evidence suggests that IgG immune nephritis with mononuclear cell infiltration and complex-induced lung injury is dependent on neutrophils fibroblast proliferation was frequently observed (Fig. 5). and the production of toxic oxygen metabolites (Johnson In some kidneys, diffuse interstitial nephritis with and Ward, 1981: McCormick et al., 1981: Morganroth, collagen deposition was present (Fig. 6). The lesions 1986). This phenomenon may also occur in systemically were prominent in the cortex with lesser lesions found in immunized rabbits in our study: where their serum IgG the medulla. In affected glomeruli, there was a loss of titers are high, they have the most severe pneumonia, glomerular architecture, debris in Bowman's space, and and the affected areas of the lungs are infiltrated almost a mononuclear cell infiltrate. Heterophils were absent entirely with heterophils. from most lesions. The epithelium of the proximal The picture seems quite different in mucosally convoluted tubule had retracted from the basement immunized animals, however. Protection against lethal membrane, and there was a loss of tubular architecture. bacteremia with these very invasive strains of P. In some there was a segmental loss of the brush border, multocida was not as good as with homologous P. and in others there was mononuclear cell infiltration. multocida systemic immunization. Even so, protected Lesions in the dista1 convoluted tubules were generaIly animals did not have severe or even moderate broncho- the same as those in the proximal tubules. The tubular pneumonia or fibrinous pneumonia. Bronchiolitis, if epithelial nuclei were hyperchromatic. Two rabbits had present, was relatively mild. This may be due to the areas of mononuclear and heterophil aggregation in the higher IgAIIgG ratio produced in bronchoalveolar collecting tubules. washes of the rabbits as compared with serum IgAIIgG Discussion ratio (Wilkie et al., 1980). Mucosa1 immunization would be expected to produce more IgA antibodies than would It is clear from the results presented above that lesions systemic imn~unization. were correlated with the route of immunization and the It has been suggested that IgA may serve to prevent antigen as well as the time of death post challenge. antigen adherence to mucosa1 surfaces (Williams and The most severe lung lesions were found in animals Ribbons, 1972), is a more efficient agglutinator than IgG vaccinated intravenously with P. multocida and sacrificed (Reynolds and Thompson, 1973), and is bactericidal in one week following challenge, probably because there the presence of complement and lysozymes (Newhouse was time for lesions to develop. It was interesting that the et al., 1976), and is much less phlogistic than the IgG and rabbits protected by irnmunization were the ones that IgM classes, which are more profound complement developed the most severe pneumonia. This points out activators (Lamm, 1976). This low phlogistic potential of the paradox of the positive and negative sides of the the IgA class is an important factor for protection of systemic immune response. Bacteremia and death were mucous membranes, where antigen-antibody reactions prevented in most of these animals (Al-Lxbban et al., along the lining of the respiratory tract are continous 1988). In that sense. the inflammatory response in the occurrences. Any or al1 of these factors could have been lmmunopathology of rabbit pasteurellosis

important in reducing the severity of the pathology in References those groups vaccinated by aerosol or into the conjunctival sac. Protection may also be related to cell-mediated Alexander M.M., Sawin P.B. and Rochirn D.A. (1952). Respiratoty immunity andlor IgA initiation of adherence resulting in infection in the rabbit: An enzootic caused by P. lepeseptica less colonization. and atternpts to control it by vaccination. J. Infect. Dis. 90, The lower incidence of pneumonia in animals syste- 30-33. mically immunized with J5 than in those systemically Al-Lebban Z.S., Corbeil L.B. and Coles E.H. (1988). Rabbit immunized with P. multocida is interesting. It may be pasteurellosis: lnduced disease and vaccination. Arn. J. Vet. that lower antibody titers with cross reacting antigen Res. 49,312-31 6. produce less opsonization and fewer immune complexes. Bienenstock J. (1985). Bronchus-associated lyrnphoid tissue. In: As renal lesions were observed only in rabbits that lrnrnunology of the Lung and Upper Respiratoty Tract, by were vaccinated intravenously, the pathology may have Bienenstock J., (ed). New York, McGraw-Hill. been caused by endotoxic damage or immunologic injury. Flatt R.E. and Dungworth D.L. (1979). Enzootic pneurnonia in

Endotoxins administered intravenouslv in rabbits are- - rabbits: Naturally occurring lesions in lungs of apparently known to produce severe disseminated intravascular healthy young rabbits. Arn. J. Vet. Res. 32, 621-627. coagulation (DIC) that culminates in bilateral renal Hagen K.W. (1958). Enzootic pasteurellosis in dornestic rabbits. l. cortical necrosis (Thomas and Good, 1952; Lee. 1963). Pathology and bacteriology. JAVMA 1952, 77-80. Most rabbits die as consequence of the severe kidney Hagen K.W. Jr. (1959). Chronic respiratoty infection in the damage. In the typical Schwartzman reaction. there are dornestic rabbit. Proc. Animal Care Panel 9, 55-60. widespread occlusions of the glomerular capillaries by Henson P.M. and Cochrane C.G. (1971). Acute irnrnune cornplex dense fibrin deposits. Although some of the rabbits disease in rabbit. The role of cornplement and of a leukocyte- vaccinated intravenously did have abnormal glomeruli, dependent release of vasoactive arnines frorn platelets. J. the lesions were not seen throuehout" the cortex. Althoueh" Exp. Med. 133,554-571. we did not see severe generalized Schwartzman Johnson K.J. and Ward P.A. (1974). Acute irnrnunologic reactions, there may have been some «Schwartzman- pulrnonary alveolitis. J. Clin. Invest. 54,349-357. like» damage after vaccination. Affected rabbits were Johnson K.J. and Ward P.A. (1981). Role of oxygen rnetabolites in necropsied 10 to 17 days following the last vaccination irnrnunecornplex injury of lungs. J. Irnrnunol. 125,2365-2369. and 20 to 27 days following the second intravenous Kaehler K.L., Markharn R.J.F. and Muscoplat C.C. (1980). injection of bacteria1 antigen. Since Schwartzman Evidence of cytocidal effects of Pasteurella hemolyfica on reactions are usually demonstrated 24 hours after the bovine peripheral blood rnononuclear leukocytes. Arn. J. Vet. second injection of endotoxin, the chronic inflammation Res. 41,18-22. we observed may represent an attempt to repair the Larnrn M.E. (1976). Cellular aspects of irnrnunoglobulin. Adv. initial damage. The intravenous injections of an antigen lrnrnunol22,223-290. into an animal with a humoral antibody response can Larsen G.L., Mitchell B.C. and Henson P.M. (1981). The result in glomerulo-nephritis due to deposition of pulrnonaty response of C5 sufficient and deficient rnice to immune complexes in the glomeruli. Unanue and Dixon irnrnune cornplexes. Arn. Rev. Respir. Dis. 123,434-439. suggested that immune complexes may be involved in the Lee L. (1963). Antigen-antibody reaction in the pathogenesis of pathogenesis of any nephritis induced by repeated bilateral renal cortical necrosis. J. Exp. Med. 117,365-377. injection of antigenic materials (Unanue and Dixon. Luna L.G. (1968). Manual of histologic staining rnethods of the 1963). The continous formation of complexes between Arrned Forces lnstitute of Pathology. 3rd edition. McGraw-Hill injected antigens and host antibody might lead to renal Book Cornpany, NY. pp 156-168. injury. As renal lesions were observed in both challenged Markharn R.J.K. and Wilkie B.N. (1980a). lnfluence of broncho- and unchallenged rabbits vaccinated intravenously. it is alveolar washing supernatants and stirnulated lyrnphocyte possible that the initial damage was produced by immune supernatants on uptake of Pasteurella hemolyfica by cultured complex deposition. This possibility needs to be bovine alveolar rnacrophages. Arn. J. Vet. Res. 41,433-466. confirmed by additional tests for the presence of Markharn R.J.K. and Wilkie B.N. (1980b). lnteraction between immune-complexes. Pasteurella hemolytica and bovine alveolar rnacrophages: In conclusion. it is clear that although intravenous Cytotoxic effect on rnacrophages and impaired phagocytosis. immunization provided the highest serum antibody titers Arn. J. Vet. Res. 41,18-22. and the best protection against bacteremia, it also McCorrnick J.R., Harkin M.M., Johnson K.J. and Ward P.A. produced the greatest immunopathology in the lung and (1981). Suppression by superoxide disrnutase of irnrnune- kidney. Protection by mucosa1 immunization is much less cornplex-induced pulrnonaty alveolitis and derrnal inflarnrnation. costly in terms of immune damages to the host. Arn. J. Pathol. 102, 55. Morganroth M.L., Till G.O., Kunkel R.G. and Ward P.A. (1986). Acknowledgernents. The authors would like to thank Drs. Erby Cornplernent and neutrophil-rnediated injury of pet-íused rat Wilkinson and Doris Govefor theireditorial work; Mr. Kreis Weiael and Mr. Brian Rice for preparing the rnicrographs; and Ms. sheila~anis lungs. Lab. Invest. 54, 507-514. and Ms. Betsy Cagle for their secretaria1 assistance. Newhouse M., Sanchis J. and Bienenstock J. (1976). Lung This study was supported in part by Animal Health and Disease defense rnechanisrns. N. Engl. .l. Med. 295, 1045-1052, Pt. 2. Formula Funds; contribution No. 82-513-j frorn Kansas Agricultura1 Reynolds H.Y. and Thornpson R.E. (1973). Pulrnonaty host Experimental Station. defenses 11. lnteraction of respiratoty antibodies with lmmunopathology o f rabbit pasteurellosis

Pseudomonas aeruginosa and alveolar macrophages. J. studies of bone hypersensitivity pneumonitis. Can. J. Comp. Immunol. 11,369-380. Med. 38,475-479. Thornas L. and Good R.A. (1952). Studies on the generalized Wilkie B.N., Markharn R.F.J. and Shewen P.E. (1980). Response Schwartzman reaction. l. General obsewations concerning of calves to lung challenge exposure with Pasteurella the phenornenon. J. Exp. Med. 96,605-625. hemolyticaafterparenteral or pulmonary immunization. Arn. J. Unanue E.R. and Dixon F.J. (1963). Experimental glomerulo- Vet. Res. 41,1773-1 778. nephritis: lrnrnunological events and pathogenetic mechanisrns. Williams R.C. and Ribbons R.J. (1972). lnhibition of bacteria1 Adv. Immunol. 6, 1-78. adherence by secretory irnmunoglobulin A: A Mechanism of Weisbroth S.H. (1979). Workshop on pasteurellosis in rabbits. antigen disposal. Science 177,697-699. Pasteurellosis in Rabbits, March 1, NIH Bethesda, Maryland 20014. Wilkie B.N., Gygax M. and Pauli B. (1974). Irnrnuno-fluorescent Accepted August 12,1988