Vaccine to protect a ruminant against pneumonia caused by pasteurella multocida

Abstract

The present invention pertains to a vaccine comprising live attenuated Pasteurella multocida bacteria for protection of a ruminant against pneumonia caused by P. multocida by administration of the vaccine to the upper respiratory tract of the ruminant via intranasal atomization of the vaccine. The invention also pertains to a method to use the vaccine to protect a ruminant against pneumonia caused by P. multocida bacteria.

Claims

1. A method to protect a ruminant against pneumonia caused by Pasteurella multocida bacteria, comprising administering by intranasal atomization a vaccine comprising live attenuated P. multocida bacteria to the upper respiratory tract of the ruminant.

2. The method of claim 1 wherein the intranasal atomization provides a mist of vaccine particles having an average size below 50 m in diameter.

3. The method of claim 2 wherein the average particle size is between 20 and 40 m in diameter.

4. The method of claim 3 wherein the vaccine additionally comprises live attenuated parainfluenza-3 virus and live attenuated bovine respiratory syncytial virus for protection against respiratory disease caused by parainfluenza virus and bovine respiratory syncytial virus.

5. The method of claim 4 wherein the vaccine further comprises live attenuated infectious bovine rhinotracheitis virus for protection against respiratory disease caused by infectious bovine rhinotracheitis virus.

6. The method of claim 2 wherein the vaccine additionally comprises live attenuated parainfluenza-3 virus and live attenuated bovine respiratory syncytial virus for protection against respiratory disease caused by parainfluenza virus and bovine respiratory syncytial virus.

7. The method of claim 6 wherein the vaccine further comprises live attenuated infectious bovine rhinotracheitis virus for protection against respiratory disease caused by infectious bovine rhinotracheitis virus.

8. The method of claim 1 wherein the vaccine additionally comprises live attenuated parainfluenza-3 virus and live attenuated bovine respiratory syncytial virus for protection against respiratory disease caused by parainfluenza virus and bovine respiratory syncytial virus.

9. The method of claim 8 wherein the vaccine further comprises live attenuated infectious bovine rhinotracheitis virus for protection against respiratory disease caused by infectious bovine rhinotracheitis virus.

Description

EXAMPLE 1

(1) Several atomization devices were assessed with respect to the obtained particle size. In this example, three cannulas were tested, which cannulas can be secured to standard syringes. The first cannula is the LMA MAD Nasal (MAD), available from LMA North America Inc, San Diego, Calif., USA. The second cannula is the Rispoval applicator (Pfizer), available from Pfizer Animal Health, Brussels, Belgium. The third cannula is a the 1 blue flex applicator nozzle available from Genesis Industries, Inc. Elmwood, Wis., USA (Genesis).

(2) These cannulas were tested with regular WFI (water-for-injection) and the obtained volume averaged droplet size was established using a Sympatec particle size analyser. The results are indicated below in Table 1.

(3) TABLE-US-00001 TABLE 1 Mean droplet size with various cannulas Volume Standard Cannula average size deviation type (diameter in m) (m) MAD 32.8 8.8 Pfizer 39.5 4.6 Genesis 197 Not determined

(4) It appears that with all three cannulas atomization of the WFI can be achieved. For the further experiments the MAD cannula was used.

EXAMPLE 2

(5) Two groups (called Group 1 and Group 2), consisting each of twenty 2-week-old calves (clean catch and colostrum deprived) were used for the experiments. Of each group, ten calves were vaccinated once intranasally with a live Pasteurella multocida vaccine (see below) and ten calves were left as unvaccinated control. At five weeks of age all calves were challenged intratracheally with wild-type P. multocida. During 7 days after challenge, the calves were observed for the development of clinical signs of respiratory disease, in particular pneumonia. At 7 days post-challenge (or earlier in case of severe clinical signs), the calves were killed and necropsied i.e. examined for lung lesions. The experiments with the second group took place several months after the experiments with the first group.

(6) Vaccine

(7) In both groups, a vaccine was used containing a Pasteurella multocida hyaE strain, derived from the wild type P1062 serotype A:3 strain which lacks the capsule (see Genbank EMBL AAK02858.1 for the gene). Just before administration, the bacteria were dissolved in WFI (water for injection) to reach about 510.sup.7 CFU/ml. The actual CFU/ml was 410.sup.7 for Group 1 and 810.sup.7 for Group 2.

(8) Another experiment is foreseen wherein the vaccine in addition to the above mentioned Pasteurella multocida strain, contains live attenuated BRSV (for example the same strains as in the product Jencine 4, available from Merck Animal Health, Summit, N.J., USA) and live attenuated Pi3 virus (for example the same strain as in the product Bovilis IBR-PI3 live, available from MSD Animal Health, Boxmeer, The Netherlands).

(9) Challenge Culture

(10) For the challenge with wild type Pasteurella two challenge cultures were made, one homologous with the vaccine strain, the other heterologous. For the homologous challenge culture Pasteurella multocida P1062 was inoculated on blood agar and incubated 16-24 hours at 37 C. Subsequently, one inoculation loop was inoculated in 100 ml TPB and incubated for 7-10 hours at 37 C. For the heterologous challenge culture Pasteurella multocida 971/90 was inoculated on blood agar and incubated 16 hours at 37 C. Subsequently, one inoculation loop was inoculated in 100 ml TPB and incubated for 4-5 hours at 37 C.

(11) Both cultures were diluted with PBS aiming at about 3.310.sup.8 CFU/ml. It is noted that since Pasteurella multocida is a secondary pathogen (i.e. in general it does not cause disease), a very high challenge dose, in combination with administration to the lower part of the respiratory tract (see below), was required to induce pneumonia.

(12) Vaccination

(13) For each group, the calves were divided into two sub-groups (groups A and B) of 10 animals. Group A was vaccinated once intranasally by administering 2 ml of the reconstituted live vaccine in one nostril, Group B was left as unvaccinated controls. In Group 1, the vaccine was administered using a normal plastic syringe, which led to the vaccine being administered as a fluid stream, breaking up in large droplets. In Group 2 the vaccine was administered using the MAD device, which lead to atomization of the vaccine.

(14) Challenge

(15) At 5 weeks of age (3 weeks after vaccination) all calves were challenged intratracheally with 30 ml challenge culture, thus aiming at a challenge dose of approximately 110.sup.10 CFU per animal. Group 1 was challenged with the homologous culture, Group 2 was challenged with the heterologous culture.

(16) Safety Examination

(17) In order to assess safety of the vaccine, the animals were daily observed for general health and behaviour.

(18) Post-Mortem Examination

(19) Seven days after challenge the animals were subjected to a post-mortem examination with special attention to the lungs. For each lung lobe the % consolidation was recorded, which corresponds to actual pneumonia. Also, Pasteurella multocida was reisolated from post-mortem samples in order to determine the bacterial load of the lungs with the bacterium. For this, tissue samples were excised from eight standard sites representative of the lobes of each half of the lung (4 sites per half); diseased tissue was preferentially selected for each site, if it was present. The mirror image samples (the two samples of the equivalent lobe on each half) were pooled to give 4 samples per calf. Each pooled sample was submerged in boiling water for 3 seconds, homogenized, serially 10-fold diluted and inoculated (100 l) on blood agar plates and then incubated for 16-24 hours at 37 C.

(20) Statistical Analysis

(21) Lung consolidation and re-isolation scores were evaluated by the Mann-Whitney U test using the statistical programme Statistix for Windows.

(22) Results

(23) Safety

(24) Vaccination with both vaccines appeared to be safe, and no clinical signs related to pneumonia or shock were observed.

(25) Pneumonia and Re-Isolation

(26) In Tables 2 and 3, the results for the post-mortem scores for pneumonia (percentage lung consolidation) and re-isolation of Pasteurella multocida (log.sub.10 CFU) are shown for Group 1. As can be seen, the results indicate that no substantial protective effects could be obtained with the live Pasteurella multocida vaccine by administration of the vaccine in the form of a liquid stream breaking up into large droplets. The actual uncertainty of any effect was 0.43 for the lung consolidation and 0.60 for the re-isolation scores.

(27) In Tables 4 and 5, corresponding results are indicated for Group 2, vaccinated with the live vaccine administered to the upper respiratory tract by atomization of the vaccine and challenged with a heterologous PM (P. multocida) strain (which when compared to a homologous challenge typically makes it more difficult to obtain protection). As can be seen, the lung lesion scores were substantially reduced by about 50%. Although the statistical analysis revealed that there is still an uncertainty of 0.12 (which is actually quite low given the small group of animals), it is clear that there is at least partial protection, despite the fact that the challenge was with a heterologous wild-type P. multocida. With respect to re-isolation, there appears to be a decrease in bacterial load by 1.5.sup.10 logs, which equals a decrease in bacterial load of about a factor 30 (that is a factor ten times as large as in Group 1). The statistical uncertainty is only 0.10 despite the fact that the experiment was carried out in such a small group.

(28) TABLE-US-00002 TABLE 2 % consolidation of lungs, Group 1 total average Group Vaccine score 1A live PM, 82.4 liquid 1B 105.6

(29) TABLE-US-00003 TABLE 3 Re-isolation (bacterial load in the lungs), Group 1 total average Group Vaccine score 1A live PM, 4.7 liquid 1B 5.2

(30) TABLE-US-00004 TABLE 4 % consolidation of lungs, Group 2 total average Group Vaccine score 2A live PM, 93 atomisation 2B 180

(31) TABLE-US-00005 TABLE 5 Re-isolation (bacterial load in the lungs), Group 2 total average Group Vaccine score 2A live PM, 2.8 atomisation 2B 4.3