Ultrastructural pathology of nasal and tracheal mucosa of rabbits experimentally infected with Pasteurella multocida serotype D:1

Sixteen 8- to 9-week-old Pasteurella multocida-free New Zealand White rabbits were divided into two equal groups. The first group was inoculated intranasally with P multocida serotype D:1 strain and the second group that was inoculated with phosphate-buffered saline (PBS) only was used as a control group. Pasteurella multocida was isolated from the nasal cavity of all infected rabbits in group 1 and from tracheal swabs of seven rabbits in this group. Four rabbits in group 1 died with clinical signs of septicaemia, two rabbits had mucopurulent nasal discharge and pneumonic lesions and the other two did not show any clinical signs or gross lesions. The ultrastructural changes detected were deciliation or clumping of cilia of ciliated epithelium, cellular swelling, vacuolation and sloughing. The subepithelial capillaries showed congestion, intravascular fibrin deposition, platelets aggregation and endothelial injury. Pasteurella multocida was observed attached to the injured endothelial cells. Heterophils, mast cells, vacuolated monocytes and macrophages infiltrated the lamina propria and between the degenerated epithelial cells.     

Polypeptides associated with Pasteurella multocida infection in rabbits.

Polypeptides from whole cell preparations of Pasteurella multocida serotypes A:12 and A:3 were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to nitrocellulose paper. Antigens were detected by immunoblot analysis, using sera from 3 groups of rabbits. Sera were obtained from rabbits inoculated intranasally with P multocida serotype A:12 or A:3, from rabbits maintained in a rabbitry with enzootic P multocida A:12 infection, and from rabbits maintained in a rabbitry with enzootic P multocida A:3 infection. Immunoblot analyses of pre- and postinoculation sera from experimentally infected rabbits, using serotype A:12 antigen, revealed 3 polypeptides with approximate molecular mass of 28, 30, and 37 kDa that consistently detected antibodies after P multocida-induced infection. Sera from rabbits naturally infected with either serotype, tested against serotype A:12 and A:3 antigens, detected the same polypeptides in both serotypes. Thus, immunologic reactivity to these polypeptides may be useful for serologic detection of P multocida infection.     

Pasteurella multocida-associated sinusitis in khaki Campbell ducks (Anas platyrhynchos)

Pasteurella multocida group B, serotype 3, was isolated from sinusitis-affected khaki Campbell ducks. To study the role of P. multocida in sinusitis, commercial khaki Campbell ducks were experimentally infected with P. multocida alone or combined with Escherichia coli. In Expt. 1, experimental ducks were infected with P. multocida intranasally or ocularly. A comparison was done by intranasal inoculation with pooled nasal discharge from the affected ducks or phosphate-buffered saline. The ducks intranasally inoculated with the nasal discharge or P. multocida showed sinusitis. In Expt. 2, E. coli alone or a combination of P. multocida and E. coli was intranasally inoculated into experimental ducks. The ducks intranasally inoculated with the combination of P. multocida and E. coli had sinusitis, the same as found in the field but less severe than that of the field cases. Pasteurella multocida was already present in litter/floor of duck farms. We concluded that P. multocida played a role in induction of sinusitis. However, the sinusitis in ducks may be initiated by poor management, especially in the brooding period of ducks.     

Ultrastructural Observation of Nasal and Pulmonary Intracellular Pasteurella multocida A:3 in Rabbits

Sixteen 8- to 9-week-old Pasteurella multocida-free rabbits were divided into two equal groups. Eight rabbits in one group were inoculated intranasally with P. multocida type A:3. The other eight were inoculated intranasally with phosphate-buffered saline and used as controls. Nasal swabs taken before and after inoculation were cultured for bacterial isolation. Post-mortem nasal swabs and lung samples were cultured for bacteriological isolation. Nasal mucosa and lung samples were collected and processed for transmission electron microscopy. Pasteurella multocida was isolated from the nasal cavity of all infected rabbits and from the lungs of four infected rabbits. Degenerative ultrastructural changes in epithelial cells and endothelial cells were seen in the infected rabbits. Deciliation of the cilated epithelium and hyperplasia of the goblet cells in the nasal mucosa were noted. Thickening of the alveolar septa due to hyperplasia of type II pneumocytes, swelling of the endothelial lining of capillaries and infiltration of inflammatory cells were also observed. Intracellular invasion of the nasal epithelial cells and of type II pneumocytes by the organism was observed. Coccobacilli were observed in membrane-bound vacuoles in the cytoplasm of these cells. The vacuoles were adjacent to the host-cell mitochondria and some of these vacuoles appeared to be fused to the mitochondrial membrane. Some type I pneumocytes with intracellular membrane-bound vacuoles containing bacterial cells showed protrusions, which appeared to detach into the alveolar lumina. These results indicated that P. multocida serotype A:3 in rabbits can invade the epithelial cell and cause structural changes in the interstitium, epithelium and endothelium. Heterophils and macrophages appear to play important roles in tissue injury.     

Pasteurella multocida infection in the domestic rabbit: immunization with a streptomycin-dependent mutant.

Fourteen Pasteurella multocida-free rabbits were inoculated intranasally with a streptomycin-dependent mutant of P. multocida serotype 12:A. Vaccinations with approximately 10(8) colony forming units were done on days 0, 14 and 28. Two weeks later the animals were separated into groups, which included 12 rabbits divided into two control groups of six unvaccinated Pasteurella-free animals. Seven vaccinated rabbits were challenged intranasally with the homologous virulent parent strain and the other seven vaccinates were challenged with a virulent strain of serotype 3:A. Rabbits were necropsied two weeks later. The vaccinated group challenged with the parent strain showed a more rapid nasal clearance of the organism than the vaccinated group challenged with the heterologous strain. However, the number of positive cultures of P. multocida recovered from tissues post-challenge were similar in vaccinated and control animals. In a significant number of animals, vaccination with serotype 12:A induced detectable antibody production to somatic antigens of both 12:A and heterologous strain 3:A.     

Severe pleuritis associated with certain strains of Pasteurella multocida in swine

Pasteurella multocida was isolated from 2 farms on which grower or finisher pigs had problems of severe emaciation and high death loss (greater than 5%). At necropsy, the pigs had extensive suppurative pleuritis and pericarditis, with adhesions over the lung surface. On one farm, the pigs also had multiple lung abscesses. Histologic findings included polymorphonuclear cell infiltration in bronchial and alveolar spaces, thickening of alveolar walls, pleuritis, and in some cases, abscesses. From all pigs, P multocida was isolated. The strains (A52, A59) were serotype A and were nontoxigenic. Experimental reproduction of the disease was achieved by sequentially infecting conventionally weaned pigs intranasally with pseudorabies virus; 7 days later, infection with selected P multocida laboratory strains (A50 and D82, A52 and A59) was achieved. At necropsy, pigs inoculated with strains A59 and A52 (serotype A, pleurotropic) had more severe lesions (P less than 0.05) than those inoculated with strain A50 (serotype A, pneumotropic). Also, pigs infected with strains A59 and A52 had extensive pleuritis and abscessation, which were not observed in the other groups. Strain D82 (serotype D) was not capable of producing pneumonia or pleuritis. Pleuritis and abscessation may be associated with certain P multocida strains that are serotype A, but not with others. These pleurotropic strains seem to be more virulent than pneumotropic strains, and infection with the former may result in extensive pleuritis and abscess formation.     

Induction of pneumonia in rabbits by use of a purified protein toxin from Pasteurella multocida.

Heat-labile toxin from a cell sonicate of a virulent type-D strain of Pasteurella multocida was purified by ammonium sulfate precipitation followed by ion exchange chromatography, gel filtration chromatography, and polyacrylamide gel electrophoresis. Toxic activity was assayed during toxin purification by cytopathic effect in Vero or bovine embryonic lung cell cultures. Toxicity for cells correlated with dermonecrosis in guinea pig skin. Toxicity was accounted for by a single protein with a molecular weight of 149,000, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Rabbits were inoculated intranasally with purified toxin to determine whether toxin had a role in the induction of pneumonia in rabbits infected with P multocida. Pneumonia, pleuritis, acute hepatic necrosis, and splenic lymphoid atrophy were found in 4 of 5 rabbits. One of 5 rabbits had bilateral turbinate atrophy. Western blotting with monoclonal antibodies to toxin from a P multocida isolate causing atrophic rhinitis in pigs revealed the toxin that induces pleuritis and pneumonia in rabbits to be the same or a closely related toxin.     

Experimental pneumonia in rabbits inoculated with strains of Pasteurella multocida.

Domestic rabbits were inoculated with either a 3:A or 3:D serotype of Pasteurella multocida by aerosol, intravenous, or intratracheal inoculation. Different colony forming units of P. multocida were used. Animals which died or were killed after the 14 day observation period were examined macroscopically and microscopically for lesions in the lower respiratory tract. Pneumonic lesions were most consistently produced in rabbits inoculated intratracheally with serotype 3:A. Pulmonary and pleural lesions were observed in some animals inoculated intravenously with serotype 3:A. Lesions were minimal in rabbits inoculated with serotype 3:D. Of the three routes of inoculation evaluated, the intratracheal route appeared to be the best method to produce Pasteurella-associated lesions in the lower respiratory tract.     

The pathology of experimental respiratory infection with Pasteurella multocida and Bordetella bronchiseptica in rabbits

Groups of female New Zealand White rabbits, 8-10 weeks old, were inoculated intranasally with three different Pasteurella multocida serotypes (A:3, A:4 and A:12) or one of three Bordetella bronchiseptica strains of rabbit origin. Seven out of 18 rabbits died of experimental infection with P. multocida. B. bronchiseptica killed 3 out of the 8 animals inoculated with it. Deaths occurred between 3 and 6 days postinoculation (PI). In the rabbits that died of P. multocida inoculation, necropsy and histology revealed severe pleuritis with the accumulation of a remarkable amount of fibrinopurulent exudate in the thoracic cavity, serous rhinitis and tracheitis, acute hepatitis with necrotic foci in the parenchyma, and atrophy of the lymphoid organs and tissues. Rabbits killed 10 days PI developed only subacute serous rhinitis and hyperplasia of the lymphoid tissues. Rabbits that died of B. bronchiseptica inoculation showed acute serous rhinitis, acute catarrhal-fibrinopurulent pneumonia and mild pleuritis. As opposed to P. multocida inoculated animals, hepatitis and atrophy of the lymphoid tissues were not characteristic of these rabbits. Rabbits killed 10 days PI developed subacute purulent and necrotic pneumonia with remarkable macrophage proliferation, involving all lobes, and hyperplasia of the lymphoid tissues.     

Intranasally inoculated Mycoplasma hyorhinis causes eustachitis in pigs

Specific-pathogen-free pigs were experimentally inoculated with Mycoplasma hyorhinis, Pasteurella multocida, or both bacterial isolates to evaluate the role of these bacteria in the pathogenesis of otitis media. Six pigs were inoculated intranasally with 4.4 X 10(8) colony-forming units (CFU) of M. hyorhinis. Twenty-one days later, three of these six pigs were inoculated intranasally with 5.0 X 10(8) CFU of P. multocida. Three additional pigs were also inoculated intranasally at the time with P. multocida alone. Two pigs served as uninoculated controls. Seven days later, all pigs were euthanatized. Histologically, subacute inflammation was found in 10 auditory tubes of six pigs and two tympanic cavities of two pigs inoculated with M. hyorhinis. Immunohistochemically, M. hyorhinis antigens were detected on the luminal surface of eight of 10 inflamed auditory tubes, and ultrastructural examination confirmed mycoplasmal organisms in two pigs. M. hyorhinis was isolated from the inflamed tympanic cavities of two pigs. None of the pigs inoculated only with P. multocida had otitis, and P. multocida was not isolated from the tympanic cavity. These findings indicate that M. hyorhinis can cause eustachitis but rarely otitis media in specific-pathogen-free pigs.     

The influence of growth conditions of Pasteurella multocida on its ability to colonise the nasal mucosa of SPF piglets

Colonisation of type D Pasteurella multocida was studied in five groups of seven SPF piglets each. Piglets of Group 1 were kept together with seven 5-week-old piglets obtained from a large herd infected with toxigenic P. multocida for 16 weeks (contact infection). These piglets were made free from toxigenic Bordetella bronchiseptica by local immunisation. Piglets of Group 2 were inoculated with 5 x 10(7) colony-forming units (cfu) of P. multocida washed from the nasal mucosa of piglets free from toxigenic B. bronchiseptica with fetal calf serum. Piglets of Group 3 were inoculated intranasally with 5 x 10(7) cfu of P. multocida washed from yeast-extract proteose-peptone cystine (YPC)-blood agar with fetal calf serum. Piglets of Group 4 were inoculated with 5 x 10(7) cfu of P. multocida grown in a YPC-based broth without blood. Piglets of Group 5 served as controls. The piglets of Group 1 did not contract P. multocida infection from infected contact piglets. After a single inoculation one of four, while after three inoculations two of three piglets of Group 2 became infected by P. multocida. After a single inoculation none of four, while after three inoculations one of three piglets of Group 3 were colonised by P. multocida. Both single and repeated inoculation failed in piglets of Group 4.     

Host response in rabbits to infection with Pasteurella multocida serogroup F strains originating from fowl cholera

Although Pasteurella multocida serogroup F has been described as an avian-adapted serogroup, it was recently found in rabbit nests in the Czech Republic. Therefore, the ability of 2 avian P. multocida serogroup F strains to induce disease in rabbits was investigated. Two groups of 18 Pasteurella-free rabbits were intranasally challenged with strains isolated from chickens and turkeys. Half of the animals in each challenge group were immunosuppressed using dexamethasone. All of the challenged rabbits exhibited clinical signs of peracute septicemic disease, ending with shock, and died or were euthanized in the terminal stages of the disease 1 to 2 d post-infection. Gross pathological changes included systemic vascular collapse and vascular leak syndrome. Hyperemia, hemorrhage, edema, inflammatory cell infiltrates, focal necrosis, and degenerative changes were observed histologically in parenchymatous organs. This is the first study directly demonstrating that avian P. multocida serogroup F strains are highly virulent in rabbits and that avian hosts cannot be excluded as a possible source of rabbit infection with serogroup F.     

Colonization of the pharyngeal tonsil and respiratory tract of the gnotobiotic pig by a toxigenic strain of Pasteurella multocida type D

Seven-day-old gnotobiotic pigs were inoculated intranasally with Pasteurella multocida and euthanatized 2, 5, 9, and 14 days after inoculation. Tissues from the oropharynx and respiratory tract of pigs were cultured quantitatively and analyzed microscopically. Pigs remained afebrile and alert, except one that died of acute fibrinopurulent pneumonia. Pasteurella multocida was isolated in greatest numbers from the pharyngeal tonsils, but only in low numbers from turbinate, trachea, lung, spleen, and liver. Significant histologic changes were limited to the tonsil. Infected pigs developed mild tonsillitis with lymphocytic hyperplasia, and accumulation of cell debris and bacteria in crypts. Capsular antigens of P. multocida, identified on tissue sections with rabbit anti-capsular polysaccharide antibody and immunocytochemical reagents, were confined to the crypt lumen. Ultrastructurally, bacteria were free within crypt material or within phagosomes of macrophages or neutrophils. In a second experiment, 5-day-old pigs were infected with Streptococcus suis type 2, followed by toxigenic Pasteurella multocida at 7 days of age; one pig died of streptococcal septicemia. Pigs developed a mild tonsillitis, and both bacteria were cultured from the tonsillar crypts for up to 14 days after infection. These studies show that a toxigenic strain of Pasteurella multocida, which is a causative agent of atrophic rhinitis, can colonize the tonsil and respiratory tract of gnotobiotic pigs for up to 14 days. In addition, colonization can occur concurrently with Streptococcus suis type 2.     

Expression of the dermonecrotic toxin by Bordetella bronchiseptica is not necessary for predisposing to infection with toxigenic Pasteurella multocida

This experiment was designed to determine whether a Bordetella bronchiseptica mutant that does not produce dermonecrotic toxin (DNT) is still capable of predisposing pigs to infection with toxigenic Pasteurella multocida. Three groups of pigs were initially inoculated intranasally with a wild type B. bronchiseptica that produces DNT, an isogenic mutant of B. bronchiseptica that does not produce DNT, or PBS. All pigs were then challenged intranasally with a toxigenic strain of P. multocida 4 days later. P. multocida was recovered infrequently and in low numbers from pigs initially inoculated with PBS, and no turbinate atrophy was present in these pigs. P. multocida was isolated in similar numbers from the pigs initially inoculated with either the wild type or the DNT mutant of B. bronchiseptica, and turbinate atrophy of a similar magnitude was also seen in pigs from both of these groups. Thus, although the DNT has been shown to be responsible for much of the pathology seen during infection with B. bronchiseptica by itself, infection with non-DNT-producing strains can still predispose to secondary respiratory infections with P. multocida.     

Detection of antibodies against Pasteurella multocida using immunohistochemical staining in an outbreak of rabbit pasteurellosis

To detect serum antibody against Pasteurella multocida (P. multocida) in infected rabbits. a modified immunoperoxidase assay was applied. An outbreak of P. multocida infection in rabbits started from sudden death. The infected rabbits had severe fibrinous and purulent pneumonia with hemorrhage, and a large number of P. multocida (A:12) was isolated from the trachea and lungs of the animals. Antibodies of IgM and IgG to P. multocida were assessed by immunohistochemical staining using the sera of the animals as primary antibodies and applying them to formalin-fixed, paraffin-embedded sections of P. multocida attached to calf fibrin. IgM antibodies to P. multocida were first detected 7 days after the onset of the disease. IgG antibodies began to rise on the 7th or 14th day. These results suggested that the modified immunoperoxidase assay could detect antibodies against P. multocida.     

Bordetella bronchiseptica and toxigenic type D Pasteurella multocida as agents of severe atrophic rhinitis of swine

Bordetella bronchiseptica and toxigenic type-D Pasteurella multocida were cultured from pigs in each of five herds diagnosed as having severe atrophic rhinitis (AR). B. bronchiseptica alone, P. multocida alone, or both organisms isolated from four herds were inoculated intranasally into 1-week-old gnotobiotic pigs which were necropsied 4 weeks post-inoculation (PI). Nasal turbinate atrophy in B. bronchiseptica-inoculated pigs was moderate to severe, while P. multocida-inoculated pigs had slight to severe atrophy. Pigs inoculated with both organisms had moderate to complete turbinate atrophy. P. multocida was reisolated at necropsy from all pigs receiving the organism except those having no turbinate damage. B. bronchiseptica and P. multocida from a fifth herd were simultaneously inoculated into six naturally farrowed 6-day-old SPF pigs. Necropsy performed 4 weeks PI revealed severe to complete turbinate atrophy. Nasal turbinates were normal for control pigs in both experiments.     

Atrophic rhinitis in New Zealand white rabbits infected with Pasteurella multocida

Atrophic rhinitis was detected in New Zealand White rabbits when upper respiratory tract disease was evaluated during a vaccine field trial for the prevention of pasteurellosis. Of 52 adult rabbits euthanatized and necropsied, 26 (50%) had evidence of turbinate atrophy. Atrophy was detected in 77% of rabbits with Pasteurella multocida infection only, 71% of rabbits with concurrent P multocida and Bordetella bronchiseptica infections, and 6% of rabbits with B bronchiseptica infection only. Grossly, turbinate atrophy was characterized by a mild to severe loss or diminution in the maxilloturbinates. Histologically, turbinate bones were small and irregular in thickness and had numerous osteoclasts and osteoblasts. A neutrophilic exudate filled the nasal passages, and infiltrates of neutrophils and lymphocytes were detected in the mucosa and submucosa of the nasal turbinates. Rhinitis was significantly (P less than 0.001) associated with turbinate atrophy. Isolates of P multocida from rabbits with turbinate atrophy were serotype A:12.     

10日龄肉鸭混合感染新型鸭肝炎病毒和多杀性巴氏杆菌的病原学研究

对1例疑似鸭肝炎病毒和多杀性巴氏杆菌混合感染的10日龄肉鸭采用常规的病毒、细菌鉴定方法和RT-PCR、PCR方法分别进行病毒、细菌的分离与鉴定。病毒鉴定为新型鸭肝炎病毒,细菌鉴定为荚膜血清A型多杀性巴氏杆菌多杀亚种。细菌对SPF鸡的毒力试验结果显示,分离的巴氏杆菌与强毒标准株C48-1毒力相近,为强毒株。细菌对10日龄肉鸭的致病性回归试验结果表明,一定数量的该株巴氏杆菌可导致10日龄雏鸭的感染死亡。结果表明,该批肉鸭为新型鸭肝炎病毒和A型多杀性巴氏杆菌混合感染。这是国内首例从感染鸭肝炎病毒10日龄雏鸭肝脏中分离到多杀性巴氏杆菌。     

Intranasal vaccination of rabbits with Pasteurella multocida A:3 outer membranes that express iron-regulated proteins

OBJECTIVE: To determine efficacy of intranasal vaccination of rabbits with Pasteurella multocida A:3 outer membrane proteins (OMP) expressing iron-regulated OMP (IROMP) in conferring protection against experimental challenge exposure. ANIMALS: 52 male New Zealand White rabbits. PROCEDURE: Rabbits were vaccinated intranasally on days 0, 7, and 14; some vaccines included cholera toxin (CT) as an adjuvant. Concentrations of intranasal IgA and serum IgG antibodies against P multocida OMP were determined. In experiment A, rabbits were vaccinated with either phospate-buffered saline solution (PBSS), PBSS-CT, OMP-CT, or IROMP-CT, challenge-exposed intranasally on day 16, and euthanatized and necropsied on day 28. Rabbits were also vaccinated with OMP or IROMP without CT and were not challenge-exposed. In experiment B, rabbits were vaccinated with PBSS, PBSS-CT, IROMP, or IROMP-CT. On day 17, rabbits were challenge-exposed intranasally. Nasal bacteria and antibodies were determined on day 24. RESULTS: In experiment A, OMP-CT vaccination stimulated mucosal and systemic antibody responses to the bacterium and enhanced resistance against challenge exposure. Intranasal bacterial counts were not significantly reduced. Vaccination with IROMP-CT stimulated mucosal and systemic antibodies, enhanced resistance to challenge exposure, and significantly reduced nasal bacterial counts. In experiment B, natural infection was detected in several rabbits at challenge exposure; however, IROMP-CT-vaccinated rabbits had significantly higher serum and nasal antibody responses, compared with other rabbits IROMP-CT-vaccinated rabbits had significantly lower nasal bacterial counts compared to control rabbits. CONCLUSIONS AND CLINICAL RELEVANCE: Intranasal vaccination of rabbits with P multocida outer membranes containing IROMP and CT stimulated immunity against experimental pneumonic pasteurellosis.     

Experimental study of pathogenicity of Pasteurella multocida serogroup F in rabbits

The role of Pasteurella multocida serogroup F in inducing disease in rabbits was investigated in this study. Three groups of 12 Pasteurella-free rabbits each were intranasally (i.n.), subcutaneously (s.c.), and perorally (p.o.) challenged, respectively. Six rabbits of each group were immunosuppressed using dexamethasone. Eight rabbits (four of them immunosuppressed) inoculated i.n. showed symptoms of respiratory distress resulting in respiratory failure and died or were euthanized in the terminal stage of the disease 3-6 days post-infection (p.i.). The main pathological findings were fibrinopurulent pleuropneumonia (immunocompetent rabbits) or diffuse haemorrhagic pneumonia (immunosuppressed rabbits). Septicemic syndrome ending with shock occurred in 11 rabbits (6 of them immunosuppressed) inoculated s.c., which died or were euthanized in the terminal stage of the disease 2-3 days p.i. The most significant pathological findings were extensive cutaneous and subcutaneous lesions. All of the p.o. inoculated rabbits survived the challenge showing no clinical signs of the disease and no macroscopic lesions. The observations in this study indicate that in addition to serogroups A and D of P. multocida, serogroup F also can be highly pathogenic for rabbits and therefore might be a cause of considerable economic loss in commercial rabbit production.