Sixteen goats either subjected to transport stress or without transport stress were treated with dexamethasone for 3 days prior to infection with P. haemolytica serotype A2 intranasally. The transport-stressed and dexamethasone-treated goats in the first group had various degrees of pulmonary lesions and the organism was re-isolated from the nasal cavity, lymph nodes and lungs. None of the goats treated with dexamethasone only were infected with P. haemolytica and had no lesions of pneumonic pasteurellosis. Treatment with dexamethasone alone failed to induce experimental infection by P. haemolytica except in combination with another stress factor.
Twenty transport-stressed goats were divided into two groups. The first group was further stressed with steroid. Pasteurella haemolytica was found at various sites in the nasal cavity of goats in this group as early as 2 weeks post-transportation. The successful isolations continued consistently with more goats having pure growth of P. haemolytica at later stages. Mild catarrh rhinitis, loss of epithelial cilia and erosions were the main lesions observed in the nasal cavity. Goats in the second group that were not given steroid injections had inconsistent bacterial isolation and less severe pathological lesions.
Twelve goats about 3 months of age were divided into 4 equal groups. Goats in Groups 1 and 2 were infected with orf virus followed by Corynebacterium pyogenes infection of Groups 1 and 3, 3 days after the first appearance of orf lesions. Goats in Group 4 were uninfected controls. Complicated orf lesions which consisted of wet suppurative scabs around the entire lips were observed in goats in Group 1. The lesions persisted for 24 days but were most severe from days 8 to 13. Goats in Group 2 developed lesions typical of orf virus infection that lasted 10 days, while goats in Group 3 developed small nodules of about 1 cm diameter, 48 hours following the introduction of C. pyogenes, which persisted for only 6 days. No lesion was observed in goats in Group 4. Two goats in Group 1 with complicated orf died after 16 and 22 days respectively.
A model of pneumonic pasteurellosis has been established in goats using Pasteurella multocida harvested from pneumonic lungs of goats (types A and D), rabbits (type A) and sheep (type D). The resultant infections were acute, subacute or chronic. The gross and histological lesions of the subacute and chronic infections were typical of pneumonic pasteurellosis. P. multocida type D produced significantly (P < 0.01) more severe lesions when compared with other isolates. There were strong correlations between the clinical signs and the severity of lesions.
Twenty goats of about 7 months of age were divided into five groups. The goats in groups 1 and 2 were exposed once, using an intranasal spray to 2 ml of an inoculum containing 10(6) colony-forming units/ml of living or dead Pasteurella haemolytica A2, respectively. The goats in groups 3 and 4 were similarly exposed twice at a 2-week interval. Group 5 was the untreated control. The number and size of the bronchus-associated lymphoid tissue (BALT) in goats exposed twice to either living or dead organisms were significantly (p < 0.05) increased compared with those exposed once and with the unexposed control. In vitro colonization by living P. haemolytica A2 onto the lung tissue in which the BALT had been stimulated by two exposures of either living or dead organisms was significantly (p < 0.05) reduced. The study indicates that stimulation of the respiratory mucosal immunity may prevent P. haemolytica A2 infection.
A trial was conducted to compare the efficacy of intranasal vaccination in protecting goats against pneumonic pasteurellosis with intramuscular vaccination using an oil adjuvant vaccine, and a combination of the two methods. Forty goats were divided into four equal groups. Group 1 was vaccinated twice intranasally with formalin-killed Pasteurella haemolytica A2, group 2 was vaccinated twice intramuscularly with an oil adjuvant vaccine containing P haemolytica A7, and group 3 was initially vaccinated intranasally with the formalin-killed P haemolytica A2 followed by intramuscular vaccination with the oil adjuvant vaccine. In each group the two vaccinations were carried out four weeks apart. Group 4 was the unvaccinated control group. All goats were challenged intratracheally with 4 ml of an inoculum containing live P haemolytica A2 at a concentration of 1.3 x 10(7) colony forming units/ml two weeks after the last vaccination and were killed 14 days after the challenge. Although group 2 showed the highest clinical score following the challenge, deaths were observed only in group 3. Three goats in group 1 had pneumonic lung lesions, compared with six goats in group 2 and all the goats in groups 3 and 4. The lung lesions in group 1 were significantly (P < 0.05) less severe than in groups 3 and 4. Similarly, the lesions in group 2 were markedly less severe than in groups 3 and 4, although the differences were not significant. The difference between the extent of the lung lesions in the goats in groups 1 and 2 was not significant. Antibody against P haemolytica A2 in group 1 reached peak levels and was significantly (P < 0.01) higher than in the control group one week after the second vaccination, before declining.
A study to determine the immunoglobulin and cellular responses in the respiratory tract of goats following intranasal exposures to formalin-killed Pasteurella haemolytica A2 was carried out. Forty-two goats were divided into two groups. Goats in Group 1 were subjected to double intranasal exposures to formalin-killed P. haemolytica A2 while goats in Group 2 were the unexposed control. Prior to and at weekly intervals post-exposure, three goats from each group were killed, serum samples were collected while the lungs were flushed with 50 ml normal saline before the right apical lobes were fixed in 10% buffered formalin. Both serum and lung lavage fluid were subjected to enzyme-linked immunosorbent assay (ELISA) to determine the levels of IgA, IgM and IgG while the formalin-fixed tissues were examined histologically. IgA levels in the lung lavage fluid increased rapidly to reach a significantly (p < 0.05) high level as early as Week 2 post-exposure and remained significantly (p < 0.05) high throughout the study period. The IgM levels increased at an intermediate rate to reach a significantly (p < 0.05) high level at Week 3 post-exposure before they decreased to an insignificant (p > 0.05) level the following week and the weeks thereafter. IgG levels increased gradually and only reached a significantly (p < 0.01) high level at Weeks 5 and 6 of the study. The size of the bronchus-associated lymphoid tissue (BALT) and the number of lymphocytes in BALT increased significantly from Week 2 and remained high thereafter. However, differences in the numbers of BALT were insignificant (p > 0.05) initially before becoming significantly (p < 0.05) high at Weeks 5 and 6. The BALT responses were parallel to those of imunoglobulins in the lung lavage fluid.
Twenty-four 8 to 9 week-old Pasteurella multocida -free rabbits were divided into three equal groups, the first group was pretreated with hydrocortisone and inoculated intranasally with pasteurella multocida serotype A:3. The second group was inoculated intranasally with P. multocida without hydrocortisone treatment. The third group was inoculated with phosphate buffered saline only and used as a control group. Pasteurella multocida was isolated from the nasal cavity of all infected rabbits in group 1 and 2 and from the trachea of seven rabbits in group 1 and five rabbits in group 2. This study was conducted to observe the ultrastructural changes of the upper respiratory tract of hydrocortisone treated and non-treated rabbits infected with P. multocida serotype A:3. The ultrastructural changes detected in infected rabbits were ciliary destruction and deciliation of the ciliated epithelial cells, cellular swelling, goblet cell hyperplasia and endothelial cell damage. Pasteurella multocida was observed attached to the degenerated cilia, microvilli and mucus. Pasteurella multocida infection was associated with inflammatory responses, which may have caused tissue damage. It is possible that hydrocortisone modulates the severity of infection as an immune suppressor and an inhibitor of goblet cell secretion.
Forty isolates of Pasteurella multocida from healthy (17 isolates) and diseased (23 isolates) rabbits were assayed for the presence of plasmids in seeking to determine whether any correlation exists between the presence of plasmids and health status, sensitivity to antimicrobial agents, capsular and somatic type, and the anatomic site of isolation. Six isolates were found harboring plasmids. A similar ladder pattern ranging from 18 to 3 megadalton (Mda) were found in three isolates recovered from diseased rabbits. One band of molecular weight 6.6 Mda was shared by four of five (4/5) isolates from the diseased rabbits. No correlation was found between the presence of the common plasmids and serotype, resistance to antimicrobial agents, and anatomic sites from which the bacteria were cultured. Random amplification polymorphic DNA was applied to subtype all the isolates of P. multocida. Two single primers were tested for their abilities to generate individual fingerprints by using PCR. Primer 1 grouped the isolates into 7 profiles, and primer 2 grouped them into 15. Random amplified polymorphic DNA-polymerase chain reaction (RAPD-PCR) results show the presence of a wide heterogeneity within P. multocida isolates. Therefore RAPD-PCR is an efficient technique to detect the DNA polymorphism and could be used to discriminate P. multocida of rabbit isolates together with serologic typing.
A trial was conducted to observe the immediate and chronic effects in goats of dexamethasone administration on the bronchus-associated lymphoid tissue (BALT) response to intranasal administration of formalin-killed Pasteurella haemolytica A2. Twenty-four goats were divided into four groups. Those in group 1 were injected intramuscularly with 1 mg/kg dexamethasone on three consecutive days, followed by intranasal exposure to formalin-killed P. haemolytica A2 one day after the last dexamethasone treatment. The goats in group 2 were similarly injected with dexamethasone followed by intranasal exposure to formalin-killed P. haemolytica A2 21 days after the last dexamethasone treatment. The animals in group 3 were exposed intranasally to formalin-killed P. haemolytica A2 without prior dexamethasone treatment. The animals in group 4 were untreated controls. The intranasal exposures to formalin-killed P. haemolytica A2 were repeated 2 weeks later. Intranasal exposure to formalin-killed P. haemolytica 1 day after dexamethasone treatment further reduced the number and size of BALT compared to the untreated control. Significantly (p < 0.01) more reduction of BALT occurred in goats exposed to formalin-killed P. haemolytica A2 21 days after dexamethasone treatment. On the other hand, intranasal exposure of goats without prior dexamethasone treatment stimulated the BALT compared to the untreated controls.
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. multoida 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 ciliated 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.
The outer membrane proteins (OMP) were extracted from the P. haemolytica A2, A7 and A9 to determine their potential as immunogens and their capability for cross-protection. Sixty lambs of approximately 9 months old were divided into four main groups. Animals in Group 1 were vaccinated with 2ml vaccine containing 100microg/ml of the outer membrane proteins of P. haemolytica A2. Animals in Group 2 were similarly vaccinated with the OMPs of P. haemolytica A7 while Group 3 with OMPs of P. haemolytica A9. Animals in Group 4 were unvaccinated control. During the course of the study, serum was collected to evaluate the antibody levels toward each OMP. There appeared to be good immune responses. However, high antibody levels did not necessarily result in good protection of the animals, particularly against cross-infection with P. haemolytica A9 in animals vaccinated with the OMPs of P. haemolytica A2. It seemed that the antibody responses were more specific toward the homologous challenge but generally did not cross-protect against heterologous serotype challenge. However, the OMPs of P. haemolytica A7 produced good in vivo cross-protection and excellent correlations when good antibody responses against all serotypes led to successful reductions of the extent of lung lesions following homologous and heterologous challenge exposures. Thus, the OMPs of P. haemolytica A7 was effective in protecting animals against homologous and heterologous infection by live P. haemolytica A2, A7 and A9.
In vitro experiments were undertaken to study the adhesion and colonization to tracheal mucosa, lung and aorta explants from freshly killed rabbits of two different strains of Pasteurella multocida. Serotype A:3 (capsulated, fimbriae +, haemagglutination -, dermonecrotic toxin -) isolated from a rabbit with rhinitis, and serotype D:1 (non-capsulated, fimbriae +, haemagglutination +, dermonecrotic toxin +) isolated from a dead rabbit with septicaemia, were used. When the explants were observed under the scanning electron microscope, the type D strain was highly adherent to trachea and aorta explants compared to the type A strain. Adhesion to lung explants was best achieved by the type A strain after 45 min incubation, but after 2 h incubation no significant difference was observed between the strains. Our data indicate that the presence of fimbriae and the absence of capsule seem to enhance the adherence of P. multocida type D strain to tracheal tissue. The capsular material of P. multocida type A strain and the toxin of the type D strain seem to influence the adherence to lung tissue in rabbit. Adhesion of strain D to aorta may indicate the expression of receptors on the endothelium to that strain and may also explain the ability of certain strains to cause septicaemia.
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.