Vaccine to protect a ruminant against pneumonia caused by mannheimia haemolytica

Abstract

The present invention pertains to a vaccine for administration to the upper respiratory tract of a ruminant to protect said ruminant against pneumonia caused by Mannheimia haemolytica, the vaccine comprising in combination live attenuated Mannheimia haemolytica bacteria, live attenuated parainfluenza-3 virus and live attenuated bovine respiratory syncytial virus, wherein the vaccine is for administration to the upper respiratory tract of the ruminant via intranasal atomization of the vaccine.

Claims

1. A method to protect a ruminant against pneumonia caused by Mannheimia haemolytica bacteria comprising administering a vaccine to the upper respiratory tract of the ruminant by intranasal atomisation of said vaccine, wherein said vaccine comprises live attenuated Mannheimia haemolytica bacteria, live attenuated parainfluenza-3 virus, and live attenuated bovine respiratory syncytial virus.

2. The method of claim 1, wherein the vaccine further comprises live attenuated infectious bovine rhinotracheitis virus.

3. The method of claim 1, wherein the atomisation provides a mist of vaccine particles having an average size below 50 m in diameter.

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

5. The method of claim 4, wherein the vaccine further comprises live attenuated infectious bovine rhinotracheitis virus.

6. The method of claim 3, wherein the vaccine further comprises live attenuated infectious bovine rhinotracheitis virus.

7. The method of claim 1, wherein the administration is to bovines at an age of less than 4 weeks.

8. The method of claim 1, wherein the vaccine is applied by a single administration.

9. The method of claim 8, wherein the volume of the single administration is divided over both nostrils.

10. The method of claim 3, wherein the administration is to bovines at an age of less than 4 weeks.

11. The method of claim 3, wherein the vaccine is applied by a single administration.

12. The method of claim 11, wherein the volume of the single administration is divided over both nostrils.

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 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 average size Standard deviation Cannula 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) The objective of this experiment was to test the efficacy of intranasal vaccination with an atomization device with a live MH-BRSV-Pi3 vaccine in 2-week-old calves against Mannheimia haemolytica M 7/2 challenge. The vaccine contains live Mannheimia haemolytica (lktA), live BRSV (the Jencine strain; the same strain as in the product Jencine 4, available from Merck Animal Health, Summit, N.J., USA)) and live Pi3 (the strain INT2; the same strain as in the product Bovilis IBR-PI3 live, available from MSD Animal Health, Boxmeer, The Netherlands).

Experimental Design

(6) Twenty clean catch and colostrum deprived calves (2-3 weeks of age at day of vaccination) were used for the experiment. Ten calves were vaccinated once intranasally with the live MH-BRSV-Pi3 vaccine and ten calves were left as unvaccinated control. Three weeks after vaccination all calves were challenged intratracheally with Mannheimia haemolytica. During 7 days after challenge, the calves were observed for the development of clinical signs of respiratory disease. At 7 days post-challenge (or earlier in case of severe clinical signs), the calves were killed and necropsied, and examined for lung lesions, pleuritis and bacterial load.

Vaccine

(7) The vaccine was constituted based on the following compounds: freeze dried Mannheimia haemolytica lktA, strain D153, serotype A1, which does not produce active leukotoxin, in Rho BACF stabilizer. frozen live BRSV strain Jencine, diluted to a titer of 5.7 log.sub.10 TCID.sub.50/ml at day of vaccination and put on ice, and frozen live Pi3 strain INT2, diluted to a titer of 5.7 log.sub.10 TCID.sub.50/ml at day of vaccination and put on ice.

(8) Right before vaccination, the freeze dried Mannheimia haemolytica cake was dissolved in Unisolve to aim at 1.510.sup.8 CFU/ml. The three compounds were mixed in equal volumes. The vaccine was administered intranasally into both nostrils (1 ml per nostril) using the MAD atomisation device. The bacterial titre in the vaccine was verified immediately after use (life count). The titre was 8.410.sup.7 CFU per 2 ml dose.

Challenge Culture

(9) Mannheimia haemolytica M7/2 (serotype A1), was inoculated on bloodagar and incubated 16 hours at 37 C. Subsequently, one inoculation loop was inoculated in 100 ml TSB and incubated for 4-5 hours at 37 C. This culture was diluted, aiming at 210.sup.7 CFU/ml. The actual titre of the challenge culture was verified by plate counting. The titre appeared to be 2.410.sup.7 CFU per ml.

Vaccination

(10) The calves were divided into two groups of 10 animals. Group 1 was vaccinated once intranasally with 2 ml (1 ml per nostril) using a syringe equipped with the MAD device. Group 2 was left as unvaccinated control. Vaccinates and controls were housed in separate rooms/compartments.

Challenge

(11) Three weeks after vaccination all calves were challenged intratracheally with 30 ml challenge culture aiming at a challenge dose of approximately 610.sup.8 CFU per animal. The actual challenge dose was 710.sup.8 CFU.

Clinical Examination

(12) After vaccination the calves were daily observed for any abnormalities of general health and/or behaviour.

Post-Mortem Examination

(13) Seven days after challenge or earlier in case of severe clinical signs the calves were killed by electric sedation and subsequent bleeding to death, immediately followed by a post-mortem examination with special attention to the lungs. For each of the six lung lobes the % consolidation was estimated and recorded. The total lung score was obtained by addition of the % lung consolidations of the individual lung lobes (and thus theoretically is between 0 and 600). The lung consolidation is representative of pneumonia.

(14) For each lung lobe fibrinous pleuritis was scored absent (0), mild (1), moderate (2) or severe (3) and recorded.

(15) With regard to isolation of M. haemolytica from post-mortem samples, 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.

Statistical Analysis

(16) The lung lesion scores (% consolidation) and pleuritis scores were evaluated by means of Generalized Estimating Equations (GEE, Agresti, 2002), taking into account the repeated measurement structure of the data. The percentage lung consolidation was converted into classes 0%=0, >0% to 5%=1, >5%10%=2 and then every 10% increase an increase of 1 unit. Re-isolation data were evaluated by the ANOVA for repeated measurements accounting for the correlation in the measurements of the lung parts in an animal.

(17) All parameters were tested two-sided with the level of significance () set at 0.05. Statistical analysis was carried out in the statistical programme SAS V9.1 (SAS Institute Inc. Cary N.C., USA).

Results

(18) After vaccination (until challenge) all calves remained in good health and no vaccine related abnormalities were observed (data not shown). After challenge the control calves developed clear respiratory disease signs whereas the vaccinated calves developed much less signs. One control calf was found dead on day 2 after challenge and three control calves had to be euthanized on day 1 or 2 post-challenge because of severe clinical signs. None of the vaccinated calves died or had to be euthanized.

Post-mortem

Lung Consolidations

(19) The lung consolidations in the vaccinates were significantly (74%) reduced compared to the controls: 40 versus 152 (p=0.0015, GEE). Of the 10 vaccinated animals, 7 had a lung consolidation lower than 30 (meaning less than about 5% consolidation per lobe), one had a score of 45 and two animals had a consolidation score of 125 and 175 respectively which resulted in the mean value of 40. Of the control animals, all had a score above 40 and seven had a score above 100.

Pleuritis

(20) The pleuritis scores in the vaccinates were significantly (95%) reduced compared to the controls: 0.1 versus 2.0 (p=0.0040, GEE). In effect, hardly any pleuritis could be detected in the vaccinated animals. Even in the two vaccinated animals that had a consolidation score of 125 and 175 respectively, no pleuritis could be detected

Bacterial Re-Isolation of M. haemolytica from Lungs

(21) The bacterial load in the vaccinates was significantly (about 5 log.sub.10) lower compared to that in the controls: 2.4 log.sub.10/ml versus 7.1 log.sub.10/ml (p=0.0001, ANOVA repeated measurements). This remarkable lowering of the bacterial load indeed corresponds to the significant effects seen with regard to lung consolidation and pleuritis.

(22) The overall effect against the infection with Mannheimia haemolytica is believed to be markedly better than what is known from the prior art, since not only the lung consolidation is reduced by 74%, the pleuritis is virtually down to zero, and the bacterial load is down almost five orders.