Vaccine for use against subclinical <i>Lawsonia </i>infection in a pig

Abstract

The present invention pertains to a vaccine comprising non live Lawsonia intracellularis antigen and a pharmaceutically acceptable carrier for use in a method to reduce in a pig the negative impact on average daily weight gain (ADWG) associated with subclinical infection with Lawsonia intracellularis, by systemic administration of the vaccine to the pig.

Claims

1. A method to reduce the negative impact on average daily weight gain (ADWG) associated with a subclinical infection with Lawsonia intracellularis in a pig comprising a systemic administration of a vaccine comprising a killed whole cell Lawsonia intracellularis antigen and a pharmaceutically acceptable carrier to the pig, wherein the vaccine is administered prior to the pig being subclinically infected, and wherein said subclinical infection is due to a post vaccination infection with Lawsonia intracellularis.

2. The method of claim 1, wherein the systemic administration of the vaccine further results in the reduction of shedding of Lawsonia intracellularis bacteria by the pig as a result of the subclinical infection.

3. The method of claim 1, wherein the vaccine is administered only once.

4. The method of claim 1, wherein the vaccine is administered when the pig is 3-10 days of age.

5. The method of claim 3, wherein the vaccine is administered when the pig is 3 to 4 weeks old.

6. The method of claim 4, wherein 2-4 weeks after said systemic administration of the vaccine, a second dose of the vaccine is administered to the pig.

Description

EMBODIMENTS

(1) In a first embodiment of the vaccine, the method for its use is for reducing the negative impact on ADWG during a finishing period of the pig. In the finishing period most of the weight is added to the body of the animal. In this period a negative impact on ADWG has the most negative effect on end weight. Therefore, to be able and reduce the negative effect on ADWG in this period, as has been shown in the Examples section here beneath, is most advantageous.

(2) In a second embodiment of the vaccine according to the invention, the method in addition is for the reduction of shedding of Lawsonia intracellularis bacteria by the pig as a result of the subclinical infection. To a great surprise, even the shedding of Lawsonia bacteria of subclinically infected animals can be reduced with the current vaccine. So not only can the infection in the pig be affected such that there is a reduction of the negative impact on ADWG associated with subclinical Lawsonia infection, it has appeared that the infection is actually reduced such that significantly less bacteria are shed by the subclinically infected animal. This is an important advantage for any farmer trying to control the spread of the bacterium in a herd of animals.

(3) In another embodiment the vaccine is administered only once. A single administration of the vaccine has shown to be effective. A second dose of the vaccine, typically administered 2-4 weeks after the first dose, might improve the level of the immune response.

(4) In another embodiment the vaccine is administered when the pig is 3-10 days of age. By vaccinating the pigs at this young age, early protection can be provided, i.e. protection directly post weaning.

(5) In still another embodiment the non live Lawsonia intracellularis antigen comprises the carbohydrates that in live Lawsonia intracellularis bacteria are in association with the outer cell membrane of these bacteria. These carbohydrates have shown to be able and rise a specific anti-Lawsonia intracellularis immune response (see WO 2009/144088).

(6) In yet another embodiment the non live Lawsonia intracellularis antigen is antigen purified from a composition comprising killed Lawsonia intracellularis bacteria. By purification, non specific bacterial material can be removed from the actual antigen, for example in order to reduce, if present, any site reactions.

(7) In a practical embodiment the non live Lawsonia intracellularis antigen is killed whole cell Lawsonia intracellularis.

EXAMPLES

(8) Study 1

(9) Design of Study 1

(10) The study was carried out in a Dutch herd with a Lawsonia intracellularis (LI) infection in older finishing pigs and breeding gilts. The study followed a randomised, controlled and blinded design.

(11) One-hundred-and-fifty-eight piglets of 3-10 days of age (most of them being 3-5 days of age) were assigned randomly, within litters, to the test or control group. The piglets in the test group were vaccinated with an inactivated Lawsonia bacterin vaccine in an oil in water emulsion (see WO 2009/144088, Example 3) at admission and again 3 weeks later. The piglets in the control group received a placebo injection (emulsion without antigen) at the same days. No antibiotic group medication that was effective against LI (e.g. tylosin, lincomycin, tiamulin, tetracyclins) was allowed in the study animals.

(12) After weaning, i.e. in the nursery and finishing phase, the piglets of the test and control group were kept together. Further, during nursery and fattening non-experimental pigs were housed in the same rooms but in different pens as the experimental animals.

(13) The piglets were routinely checked for local and systemic reactions at 4 hours, 1 day, 3 days, 1 week and 2 weeks after each vaccination.

(14) From all pigs faeces samples were taken at one- or bi-weekly intervals during the period of expected exposure to a filed infection. Per treatment group, the faeces samples of ten animals were pooled and analysed for presence of LI bacteria by qPCR (from now on also called PCR). If a pooled faeces sample indicated presence of bacteria, the original samples from that pool were analysed individually. As soon as the first samples became positive, individual faeces samples were tested. At first vaccination, 10, 16 and 21-23 weeks after first vaccination, all animals were weighed.

(15) Results of Study 1

(16) During the study, a Lawsonia infection went through the herd as evidenced by PCR data on faeces (see below). Since none of the animals, except for one animal in the control group had any clinical symptoms, the group was affected by a subclinical infection resulting from a field exposure to of the (wild type) bacterium.

(17) Bacterial Load in Faeces

(18) The mean PCR-results are given in Table 1, number of positive animals and mean concentration of LI-DNA are given in Table 2.

(19) From week 13 onwards in a number of control animals, DNA from LI was found. The percentage of control animals with positive faecal samples gradually increased from zero at 12 weeks after first vaccination via 2-8% at week 13-15 up to around 18% at week 16-18, after which it decreased down to 0% after week 20.

(20) TABLE-US-00001 TABLE 1 Shedding of LI in the faeces (qPCR) Positive Duration animals positivity (weeks) Group number % mean range Controls 31 41.3 1.65 1-5 Vaccinated 1 1.4 1.00

(21) In the test (vaccine) group only a single PCR positive faeces sample was found at one collection day for a single animal (1 out of 72 pigs positive, i.e. 1.4%). In the Control group 31 out of 75 animals had at least one PCR positive faeces sample (41.3%). The difference between the groups was statistically significant (Fisher's exact test: p-value <0.001).

(22) TABLE-US-00002 TABLE 2 Number of animals with LI positive samples (and percentage) and mean concentration of LI in the positive fecal samples, by vaccination group and week after first vaccination. week pg DNA/5 l positive after 1st positive samples sample (Mean SD) vaccination Controls Vaccine Controls Vaccine 12 0 (0%) 0 (0%) 13 2 (2.7%) 0 (0%) 138 83 14 6 (8.0%) 0 (0%) 290 396 15 2 (2.7%) 0 (0%) 314 19 16 14 (18.7%) 1 (1.4%) 1262 2458 119 17 14 (19.7%) 0 (0%) 885 833 18 9 (17.6%) 0 (0%) 1627 3680 19 3 (5.7%) 0 (0%) 1387 1278 20 1 (3.0%) 0 (0%) 98 21 0 (0%) 0 (0%) 22 0 (0%) 0 (0%) 23 0 (0%) 0 (0%)

(23) Body Weight Gain

(24) Body weights and average daily weight gain are summarized in the Tables 3 and 4 below. ADWG is considered to be a relevant parameter that is often used to measure efficacy of Lawsonia vaccines. However, this parameter is non-specific as it is influenced by several different conditions (secondary infections, climate, feed, etc.). The preliminary calculated sample size to obtain statistical significant (p<0.05) values at an ADWG difference of 25 grams per day would need to be about 300 animals per group. For efficiency reasons, the groups actually used were made considerably smaller. In this pilot study group sizes were only 78 and 80 animals. So even if the difference in ADWG would be about 25 grams per day, the resulting p value was estimated to be above 0.05 in any case.

(25) TABLE-US-00003 TABLE 3 Mean bodyweight (kg, SD), by vaccination group and period. Control Vaccine Admission (3-10 days) 1.4 0.3 (n = 76) 1.5 0.3 (n = 80) At transfer to finishing 27.7 4.8 (n = 74) 28.5 4.1 (n = 73) (10 weeks) Third weighing (16 64.0 9.2 (n = 73) 65.6 9.1 (n = 71) weeks of age) Last weighing (18-24 89.4 16.8 (n = 72) 92.3 17.0 (n = 71) weeks of age)

(26) The treatment group comparison for the average daily weight gain during the finishing phase (see below) led to, as expected, no statistically significant difference (p-value 0.2042). If corrected for baseline values, the vaccinated group showed on average 21.4 grams per day higher daily weight gain in the finishing period than the control group, with a 90% confidence interval ranging from 6.4 grams per day lower to 49.2 grams per day higher. An average difference of 21.4 grams per day on itself is a substantial difference in ADWG. The Least Squares Means (LSMs) for the average daily weight gains of the two groups were 881 grams per day for the vaccinated group and 860 grams per day for the control group. These LSMs do fit the group comparison by ANCOVA but are slightly different from the ordinary means from Table 3 as they are corrected for the small differences in the covariate weight at vaccination between groups.

(27) TABLE-US-00004 TABLE 4 Mean average daily weight gain in grams*, by vaccination group and period. 90% confidence n LSM SEM int. pigs Finishing period Control 859.9 14.5 835.7-884.2 72 Vaccine 881.3 14.6 856.8-905.9 71 Control-Vaccine 21.4 16.8 49.2-6.4 Overall Control 621.6 10.1 604.6-638.5 72 Vaccine 634.0 10.2 616.9-651.2 71 Control-Vaccine 12.4 11.5 31.5-6.6 *Mean ADWG adjusted for litter, batch and weight at admission standard error of the mean (SEM) and 90% confidence interval.

(28) The p-value for the treatment effect on overall average daily weight gain was 0.2804. If corrected for baseline values, the Vaccinated group showed an average daily weight gain that was 12.5 g/day higher than the Control group. The 90% confidence interval for this estimate ranged from a growth that was 6.6 g/day lower to a growth that was 31.5 g/day higher in the Vaccinated group than in the Control group.

(29) Study 2

(30) Design of Study 2

(31) The objective of this study was to assess the efficacy of the same vaccine as used in the first study in finishing pigs under field conditions, but as a one shot vaccine. The study was conducted as a randomised, blinded, saline controlled clinical efficacy trial. About 750 3-4-week-old piglets were allocated randomly within litter to the test or to the control group. The piglets in the test group were vaccinated once intramuscularly with 2 ml of the vaccine and the piglets of the control group were injected with 2 ml of saline. At an age of 10 (1) weeks, 648 study piglets, 324 from each treatment group, were transported to the finishing farm. From then on, the control pigs were housed in separate pens from the vaccinated pigs in the same unit (8 pens of 8-11 animals per treatment group per unit) until slaughter (at an age of 25 weeks)

(32) A first parameter assessed was the average daily weight gain (ADWG) of the pigs between the different weighings: at admission, after transfer to the finishing farm and just prior to slaughter. A second parameter assessed was Lawsonia shedding: after transfer to the finishing farm, at 15 and 20 weeks of age and just prior to slaughter.

(33) Results of Study 2

(34) During this second study, a Lawsonia infection went through the herd as evidenced by PCR data on faeces. Only one animal in the control group had slight clinical symptoms (confirmed to be the result of a Lawsonia infection, as determined after necropsy). It can thus be confirmed that the group was affected by a subclinical infection resulting from a field exposure to of the (wild type) bacterium.

(35) The effect on ADWG is indicated in Table 5. As can be seen, the average daily weight gain during the nursery period was 319 grams per day in the test group, and 307 grams per day in the control group. The differences in average daily weight gain (ADWG) during the nursery period were significantly different between the control and the test group (ANOVA: p=0.0203). The ADWG in the finishing period, and the overall ADWG was also higher in the test group. Although the differences of 7 gram per day between the control and the test group are commercially relevant (leading to a difference in end weight of approximately 1.4 kg), they were not statistically significant.

(36) TABLE-US-00005 TABLE 5 Average daily weight gain (g/day), by vaccination group and period Control group Test group Nursery 307.sup.a 319.sup.b Finishing 764.sup.c 771.sup.d Overall 633.sup.e 640.sup.f .sup.abMixed model ANCOVA, p = 0.0203 .sup.cdMixed model ANCOVA, p = 0.6356 .sup.efMixed model ANCOVA, p = 0.4229

(37) Regarding Lawsonia shedding it appeared that in study week 6 all faecal samples were negative for Lawsonia intracellularis. In week 11, seven samples (19%) from the control group and none of the test group were positive. In week 16 seven samples (19%) from the control group and one of the test group were positive. In week 21 all animals were negative again. The number of pens with PCR Lawsonia positive faecal samples (for 1 or 2 consecutive weeks) during the study was significantly lower in the test group (Fisher exact test, p<0.001) compared to the control group.