Prevention of experimental haemorrhagic septicaemia with a live vaccine

Pasteurella multocida serotype B:3,4 isolated from a fallow deer in England was used as a vaccine to prevent haemorrhagic septicaemia. The deer strain was less virulent for calves than typical serotype B:2 of haemorrhagic septicaemia strains. It elicited antibodies in cattle that protected mice against serotype B:2 infection. The live deer vaccine containing 2 X 10(7) viable organisms per dose was used to immunise calves. Six months after vaccination, five of six calves were protected against serotype B:2 challenge. Two calves challenged nine months after vaccination survived the same challenge. The live vaccine was more efficacious than an alum precipitated vaccine in protecting calves against B:2 challenge.     

Safety, efficacy and cross-protectivity of a live intranasal aerosol haemorrhagic septicaemia vaccine

The safety, efficacy and cross-protectivity of a live intranasal aerosol haemorrhagic septicaemia vaccine containing Pasteurella multocida serotype B:3,4 were tested in young cattle and buffaloes in Myanmar, where more than 1.5 million animals had been inoculated with this vaccine between 1989 and 1999. A recommended dose of 2 x 10(7) viable organisms was used for the efficacy test. The administration of 100 times the recommended dose to 50 cattle and 39 buffalo calves was innocuous. Seven months after they were vaccinated, three of three buffaloes were protected and 12 months after they were vaccinated, three of four buffaloes were protected against a subcutaneous challenge with serotype B:2 which killed three of three unvaccinated buffaloes. Twelve months after they were vaccinated, eight of eight cattle survived a serotype B:2 challenge, which killed four of four unvaccinated controls. The vaccinated cattle had developed serum antibodies detectable by the passive mouse protection test. Indirect haemagglutination tests on sera taken from cattle 10 days and five weeks after they were vaccinated showed high titres of antibodies. The serum of vaccinated cattle cross-protected passively immunised mice against infection with P. multocida serotypes E:2, F:3,4 and A:3,4.     

Molecular Variability among Strains of Pasteurella multocida Isolated from an Outbreak of Haemorrhagic Septicaemia in India

The applicability of conventional and molecular methods for rapid detection and differentiation of Pasteurella multocida serogroup B isolates involved in an outbreak of haemorrhagic septicaemia affecting Indian buffaloes, was studied. Five isolates were obtained and were subjected to phenotypic and genotypic characterization. None of the five isolates could be differentiated on the basis of cultural, biochemical, pathogenicity and antimicrobial sensitivity patterns. Polymerase chain reaction (PCR)-based techniques were found to be specific and sensitive for rapid detection and differentiation of isolates. Repetitive extragenic palindromic (REP-) PCR, enterobacterial repetitive intergenic consensus (ERIC-) PCR and single-primer PCR differentiated all the five isolates into different profiles. All the isolates involved in the outbreak were found to have a genetic profile different from standard P. multocida strain (P52). However, three isolates had similar profiles, whereas each of the remaining two had a different profile. The study indicates the involvement of multiple strains of P. multocida in a single outbreak of haemorrhagic septicaemia in buffaloes. The results also indicate that molecular methods of detection and typing are superior to conventional methods for rapid epidemiological investigations of haemorrhagic septicaemia.     

Experimental transmission of Pasteurella multocida 6:B in goats

Haemorrhagic septicaemia (HS) is an acute disease of cattle and buffaloes caused by Pasteurella multocida 6:B. Outbreaks of the disease have been closely associated with carrier animals that transmit the organism to susceptible animals during stressful condition. This study was conducted to determine whether goats exposed intranasally to P. multocida 6:B can transmit the organism to contact goats. Thirty-six healthy local Katjang goats were divided into four groups and goats of groups 1 and 3 were each inoculated intranasally with a 1-ml inoculum that contained 1 x 10(9) CFU/ml of live P. multocida 6:B. Following the exposure, all goats of groups 3 and 4 were injected with dexamethasone at the rate of 1 mg/kg for three consecutive days. At the end of the dexamethasone treatment, goats of groups 1 and 2 were commingled but kept separate from goats of groups 3 and 4, which were commingled in another pen. Three surviving goats from each group were killed on days 7, 14 and 21 post-exposure for postmortem examination. Naso-pharyngeal mucus and heart blood were collected on swabs. Tissues from lungs, lymph nodes and tonsils were collected for bacteriological isolation and identification. Only one goat of group 3 died 6 days post-exposure showing clinical signs and lesions typical of HS. Other goats showed mild signs of upper respiratory tract infection. Goats of all groups developed acute mild pneumonic lesions, however, those treated with dexamethasone had significantly (P < 0.05) more extensive lesion scoring based on the lesion scoring system. P. multocida 6:B was isolated from the nasal mucosa and lung lesions of exposed and contact goats not treated with dexamethasone. Exposed and contact goats treated with dexamethasone carried the organism for 21 days. P. multocida isolation from heart blood was made only from exposed and contact goats treated with dexamethasone. P. multocida was isolated from the lymph node of the goat that died during the experiment.     

Protective effect following intranasal exposure of goats to live Pasteurella multocida B:2

This study aimed to determine the effect of intranasal exposure to low doses of Pasteurella multocida B:2 on survival of goats challenged with high doses of the same organism. Eighteen goats were selected and divided into three groups. Goats of group 1 were exposed intranasally twice, with a two-week interval, to 7× 10⁶ cfu/ml of live P. multocida B:2. Goats of group 2 were not exposed to P. multocida B:2 but were kept together with the exposed group 1. Goats of group 3 remained as unexposed controls and were kept separated from the other two groups. Serum samples were collected at weekly intervals to determine the antibody levels. At week 5 post exposure, all goats were challenged subcutaneously with 3.7× 10¹⁰ cfu/ml of live P. multocida B:2. Following challenge exposure, 8 (67%) goats (4 goats from each of groups 1 and 2) were killed owing to haemorrhagic septicaemia. Four goats were killed peracutely within 48 h post challenge, while the other four goats were killed acutely between 2 and 4 days post challenge. None of the goats of group 3 were killed for haemorrhagic septicaemia. Goats of groups 1 and 2 showed significantly (p<0.05) higher antibody levels following the first intranasal exposure to P. multocida B:2. However, only group 1 retained the significantly (p<0.05) high antibody levels following a second intranasal exposure, and remained significantly (p<0.05) higher than groups 2 and 3 at the time of challenge. P. multocida B:2 was successfully isolated from various organs of goats that were killed between 1 and 4 days post challenge.     

Safety and efficacy of an oil-adjuvant vaccine against haemorrhagic septicaemia in buffalo calves: cross-protection between the serotypes B:2,5 and E:2,5.

The safety, efficacy and duration of immunity of an improved oil-adjuvant vaccine against haemorrhagic septicaemia, containing inactivated cells of Pasteurella multocida serotype B:2,5, were tested in young buffalo calves in Pakistan. For safety testing, five buffalo calves were vaccinated intramuscularly with twice the normal dose, and six weeks later with a normal dose. Except for a transient rise in rectal temperature at six hours after the vaccinations, no systemic reactions were observed. The buffaloes remained in good condition and had a normal appetite. No local reactions were observed at the injection site. For efficacy testing two trials were carried out. In the first, buffalo calves were vaccinated intramuscularly either with two doses two-and-a-half months apart, or with a single dose, or left unvaccinated. They were challenged subcutaneously with virulent P multocida after eight, 13 or 15 months. After challenge at eight months the four buffaloes given two doses and the buffalo given one dose were protected, whereas the control animal developed the typical signs of the disease. After the challenges at 13 and 15 months, the vaccinated animals were still protected whereas the control animals died. In the second trial, buffalo calves were vaccinated intramuscularly either with two doses two months apart, or with a single dose at two months or left unvaccinated. The buffaloes were challenged after eight or 14 months. After challenge at eight months the four control animals died, whereas three of the four buffaloes given a single dose were protected. After challenge at 14 months, the three control animals died, whereas four of the five buffaloes given two doses and both the buffaloes given a single dose were protected. To test for cross-protection against the heterologous serotypes E:2,5 and B:3,4, groups of mice were vaccinated once or left unvaccinated. Four weeks later, the vaccinated and control groups were challenged with a dilution series of the different challenge cultures. The vaccine appeared to induce protection against challenge with different strains of serotypes B:2,5 and E:2,5 but not against strains of serotype B:3,4.     

Immune response of chickens to various routes of administration of Australian infectious bronchitis vaccine

Groups of 100 two-week-old cockerels were vaccinated with the A₃ strain of IB vaccine by conjunctival, intranasal, in-contact, drinking water, or aerosol routes, or were left as unvaccinated controls. Three weeks after vaccination, each group of chickens was challenged with the Appin strain of IBV.
Vaccination by the conjunctiva!, intranasal and in-contact routes induced a good resistance to challenge, concurring with an obvious stimulation of the Harderian gland, while the drinking water route led to a low resistance to challenge, with minor changes in the gland. The results of no immune response and no resistance to challenge in the birds vaccinated by aerosol route was due to unsuccessful vaccination in the group. Application of the vaccine by the conjunctival route would appear to be a most effective route for the application of Australian IB vaccines, while the in-contact method appears worthy of further study.     

Comparative sequence analysis of 16S rRNA gene of Pasteurella multocida serogroup B isolates from different animal species

The phylogenetic relationships of five isolates of Pasteurella multocida serotype B:2 belonging to buffalo, cattle, pig, sheep and goat were investigated by comparative sequence analysis of 16S rRNA gene. The 1468bp fragment of 16S rRNA gene sequence comparison showed that the isolates of cattle (PM75), pig (PM49) and sheep (PM82) shared 99.9% homology with the buffalo isolate (vaccine strain P52) whereas, the goat isolate (PM86) shared 99.8% homology with the vaccine strain. The 16S rRNA gene sequences of these isolates were also found monophyletic with type B reference strain NCTC 10323 of P. multocida subsp. multocida. The present study indicated the close relationships of haemorrhagic septicaemia causing P. multocida serotype B:2 isolates of buffalo and cattle with other uncommon hosts (pig, sheep and goat).     

An evaluation in pigs of Nobi-Vac AR and an experimental atrophic rhinitis vaccine containing P multocida DNT-toxoid and B bronchiseptica

The trial involved eight large white sows obtained from a closed experimental specific pathogen free herd. Four sows (two each for an experimental vaccine and for Nobi-Vac AR) were vaccinated twice (eight weeks and two weeks before parturition) with 2 ml of vaccine administered intramuscularly. Two unvaccinated sows were used as an infected control group and two unvaccinated sows served as an uninfected control group. Forty-six piglets (28 from vaccinated sows and 18 from unvaccinated sows) were challenged by intranasal instillation of Bordetella bronchiseptica at two days of age and Pasteurella multocida type D, dermonecrotic toxin at seven days of age. Among the infected control group some piglets died and there were clinical signs of pneumonia and severe turbinate atrophy. In the vaccinated groups the results showed that immunisation of the pregnant sows had provided a good level of antibodies, which were transmitted to their offspring. There was a significant reduction in the clinical signs and no lesions were observed in the group vaccinated with the experimental vaccine and only moderate atrophy of the turbinates in the Nobi-Vac AR group. B bronchiseptica and P multocida were never recovered from the lungs of the vaccinated groups and in the nasal cavities their frequency declined with age.     

Prevention of haemorrhagic septicaemia in buffaloes and cattle with a live vaccine

Young cattle and buffaloes were vaccinated subcutaneously and intradermally with a live vaccine containing Pasteurella multocida serotype B:3,4. Twelve months after vaccination three of five young cattle in the subcutaneously vaccinated group and three of four in the intradermally vaccinated group were protected against serotype B:2 challenge. Eleven buffaloes vaccinated subcutaneously and two vaccinated intradermally survived the same challenge 13 months after vaccination.     

Pasteurella multocida antigen-induced in vitro lymphocyte immunostimulation, using whole blood from cattle and turkeys.

A whole blood lymphocyte stimulation assay to study cell-mediated immune responses in bovine pasteurellosis was developed. Peripheral blood lymphocytes from cattle artificially immunised with three Asiatic haemorrhagic septicaemia strains of Pasteurella multocida exhibited higher stimulation indices when incubated with antigen preparations from homologous strains than with the heterologous shipping fever strain. Lymphocytes from cattle immunised with the shipping fever strain of P multocida exhibited a higher stimulation index when incubated with an antigen preparation from the homolgous strain than with antigen preparations from heterologous haemorrhagic septicaemia strains. These results suggest that immunogenic differences exist between the haemorrhagic septicaemia strains and the shipping fever strain of P multocida. An assay using turkey whole blood lymphocytes was also developed. The use of small amounts of whole blood, microtitre plates, either 125I iododeoxyuridine or 3H-thymidine as the labelling agent, and a multiple cell-culture harvester makes the method simple, rapid and suitable for the study of immune competence and cell-mediated immune responses in turkeys on a flock basis.     

Immunization with outer membrane proteins of Pasteurella multocida (6:B) provides protection in mice

The immunoprotective efficacy of Pasteurella multocida (6:B) outer membrane proteins (OMPs) was examined in the mouse model. Bacterial OMPs were extracted using sarkosyl method and analysed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and immunoblotting. Prototype vaccines were prepared using OMPs with adjuvants including dioleoyl phosphatidyl choline-based liposome and Montanide ISA206 water-in oil-in water emulsion. Antibody response to the vaccine was monitored using indirect enzyme linked immunosorbent assay. The results of the study showed that immunized mice had high titre with both the formulations. The vaccinated mice were able to survive a live virulent bacterial challenge. Based on the findings of the study it can be inferred that OMPs are important determinants of immunoprotection hence can serve as vaccine candidates against haemorrhagic septicaemia.     

Cloning and characterisation of the ahpA gene of Pasteurella multocida serogroup b:2 (strain P52): short communication

Pasteurella multocida B:2 is responsible for haemorrhagic septicaemia in cattle and buffaloes, causing severe economic losses in the developing countries. In the present study, the ahpA gene of P. multocida B:2 (P52) was cloned, sequenced and compared with the previously reported ahpA gene sequence in P. multocida A:1, which is responsible for its haemolytic phenotype. E. coli DH5a cells were further transformed with recombinant plasmid carrying the ahpA gene from P. multocida B:2 (P52) but SDS-PAGE analysis failed to show the expression of haemolysin protein. Slight haemolysis was albeit observed in horse blood agar plates streaked with recombinant E. coli carrying the ahpA gene. Our study indicates that there is 99.6% similarity and 0.4% divergence between ahpA gene of P. multocida B:2 (P52) and P. multocida A: 1, while membrane topology analysis has predicted that ahpA is an inner membrane protein with two strong hydrophobic regions at the N and C terminals. The presence of significant homology in ahpA sequence in A: 1 and B:2 perhaps suggests a common mechanism of pathogenesis in different species of animals.     

Induction of antibodies to glycoprotein I in pigs exposed to different doses of a mildly virulent strain of Aujeszky's disease virus

It has recently been shown that the antibody response to glycoprotein I (gI) of Aujeszky's disease virus can be used to distinguish infected from vaccinated pigs. To examine whether pigs exposed to low doses of a mildly virulent strain of Aujeszky's disease virus produce antibody to gI four groups of four pigs were inoculated intranasally with 10, 10(2), 10(3) or 10(4) plaque forming units (PFU) of the Sterksel strain. Two unvaccinated pigs and two pigs vaccinated intranasally with Bartha's K strain, a gI-negative vaccine, were placed in contact with each group. The pigs given 10 PFU and the in-contact pigs in this group did not become infected. The inoculated and the unvaccinated in-contact pigs in the other groups developed mild signs of illness and produced antibody to gI. Four of six vaccinated in-contact pigs that became infected showed neither clinical signs nor virus shedding and still produced antibody to gI. The other two vaccinated pigs appeared to be resistant to contact-challenge. The antibody response to gI persisted for at least seven months. These results support the idea that Aujeszky's disease virus may be eradicated by a programme based on vaccination with gI-negative vaccines, in conjunction with the detection and subsequent removal of gI-antibody positive, infected, pigs.     

Mucosal immunization provides better protection than subcutaneous immunization against <Emphasis Type="Italic">Pasteurella multocida</Emphasis> (B:2) in mice preimmunized with the outer membrane proteins

To investigate the effect of boosting immunity via mucosal route vis-a-vis parenteral route in the mouse model of haemorrhagic septicaemia, mice preimmunized with OMP of Pasteurella multocida (B:2) were immunized with 10² cfu of P. multocida via intranasal and subcutaneous routes. Mice were challenged through intranasal route (natural route of infection) with 10⁸ cfu 14 days after immunization. Group of mice which were immunized intranasally showed significant protection (P < 0.05) of 88% as compared to 50% protection in group of mice immunized subcutaneously. In the control group of mice, 100% mortality occurred within 48 h. of challenge. The results of present study indicated that boosting of immunity via mucosal route in mice preimmunized with OMP provided better protection against P. multocida. This study may have implications for developing better vaccination strategies for the natural host.     

Efficacy of an inactivated recombinant vaccine encoding a fimbrial protein of Pasteurella multocida B:2 against hemorrhagic septicemia in goats

This study was carried out to determine the antibody responses and protective capacity of an inactivated recombinant vaccine expressing the fimbrial protein of Pasteurella multocida B:2 following intranasal vaccination against hemorrhagic septicemia in goats. Goats were vaccinated intranasal with 10⁶ CFU/mL of the recombinant vaccine (vaccinated group) and 10⁶ CFU/mL of pET32/LIC vector without fimbrial protein (control group). All three groups were kept separated before all goats in the three groups were challenged with 10⁹ CFU/mL of live pathogenic P. multocida B:2. During the course of study, both serum and lung lavage fluid were collected to evaluate the antibody levels via enzyme-linked immunosorbent assay. It was found that goats immunized with the inactivated recombinant vaccine developed a strong and significantly (p < 0.05) higher specific IgA and IgG responses in both serum and lung lavage fluid samples compared to the control and unvaccinated groups. Following intratracheal challenge, the rate of isolation was 17% for the vaccinated group, 67% for the control group and 100% for the unvaccinated group. However, none of the goat from the vaccinated group had P. multocida B:2 in the liver, tonsil and heart. Therefore, the study revealed that an inactivated recombinant vaccine significantly provides significant protection against high dose challenge and enhances the stimulation of the local and systemic immunities.     

Proliferation and transmission patterns of <Emphasis Type="Italic">Pasteurella multocida</Emphasis> B:2 in goats

This report describes the proliferation and transmission patterns of Pasteurella multocida B:2 among stressful goats, created through dexamethasone injections. Thirty seven clinically healthy adult goats were divided into three groups consisted of 15 goats in group A, 11 goats in group B and the remaining 11 in group C. At the start of the study, all goats of group A were exposed intranasally to 1.97 × 10¹⁰ CFU/ml of live P. multocida B:2. Dexamethasone was immediately administered intramuscularly for 3 consecutive days at a dosage rate of 1 mg/kg. The exposed goats were observed for signs of HS for a period of 1 month. At the end of the 1-month period, 11 goats from group B were introduced into and commingled with the surviving goats of group A before all goats from both groups were immediately injected intramuscularly with dexamethasone for 3 consecutive days. The treatment with dexamethasone was then carried out at monthly interval throughout the 3-month study period. Goats of group C were kept separately as negative control. Three surviving goats from each group were killed at 2-week interval for a complete post-mortem examination. Two (13%) goats of group A were killed within 24 hours after intranasal exposure to P. multocida B:2 while another two (13%) goats from the same group were killed on day 40, approximately 10 days after the second dexamethasone injection. All four goats showed signs and lesions typical of haemorrhagic septicaemia. Bacteraemia was detected in 3 goats of group A that were having rectal temperature higher than 41°C. The P. multocida B:2 isolation pattern was closely associated with dexamethasone injections when significantly (p < 0.05) higher rate of isolations from both groups were observed after each dexamethasone injection. Transmission of P. multocida B:2 from goats of group A to group B was successful when P. multocida B:2 was isolated from goats of group B for a period of 28 days. There was a strong correlation between dexamethasone injections, rate of bacterial isolation and serum cortisol level. The IgG level showed an increasing trend 2 weeks after exposure to P. multocida B:2 and remained high throughout the study period.     

Serological response of cattle to simultaneous vaccinations against foot-and-mouth disease and haemorrhagic septicaemia

In Malaysia, where vaccination campaigns against foot-and-mouth disease and haemorrhagic septicaemia are routinely carried out, it was desirable to determine whether it was safe and efficacious to administer both vaccines simultaneously. A trial group of 104 cattle was divided into three groups; group 1 animals received both vaccines simultaneously, group 2 animals received only foot-and-mouth disease vaccine and group 3 animals received only haemorrhagic septicaemia vaccine. The serological response to vaccinations was monitored at 0, 21 and 35 days by the virus neutralisation test for foot-and-mouth disease and the mouse-protection and indirect haemagglutination tests for haemorrhagic septicaemia. The simultaneous administration of the two inactivated vaccines produced no adverse effects and the serological response did not differ from the response to either vaccine given separately, thus indicating that cattle may be safely and effectively vaccinated simultaneously in this way.     

Preliminary studies with a live streptomycin-dependent Pasteurella multocida and Pasteurella haemolytica vaccine for the prevention of bovine pneumonic pasteurellosis.

Twelve Pasteurella-free Holstein-Friesian calves were used in a study to test the efficacy of a live streptomycin-dependent Pasteurella multocida A:3 and streptomycin-dependent Pasteurella haemolytica A1 vaccine. The calves were inoculated intramuscularly twice at 14-day intervals with either the streptomycin-dependent vaccine, containing 1 X 10(6) colony forming units/mL P. multocida and 4 X 10(8) colony forming units/mL P. haemolytica, commercial bacterin, or phosphate buffered saline. Two weeks following the second vaccination, all calves were challenged by intranasal inoculation of 10(8) TCID50/4.0 mL infectious bovine rhinotracheitis virus followed three days later by intratracheal injection with 2.3 X 10(7) colony forming units/mL of a 16 hour culture of P. multocida A:3 and 2.6 X 10(8) colony forming units/mL of an 8 hour culture of P. haemolytica A1. Seven days after challenge with Pasteurella, calves were killed for collection of tissues at necropsy. Each calf was given a score based on macroscopic and microscopic lesions. The scores for the calves receiving live vaccines were significantly lower (p less than 0.025) than those for the controls. Also, the calves receiving live vaccines had a significant (p less than 0.05) increase in the level of serum antibody to P. haemolytica. The results of this preliminary study showed that the streptomycin-dependent vaccine offered better protection than the commercial bacterin against a virulent homologous challenge.