A COMBINATION VACCINE AGAINST PCV2 VIRUS AND MYCOPLASMA HYOPNEUMONIAE INFECTION

Abstract

The present invention pertains to a vaccine comprising in combination non-replicating immunogen of porcine circo virus type 2 and non-replicating immunogen of Mycoplasma hyopneumoniae and an adjuvant containing a nano-emulsion of mineral oil in water, for use in prophylactically treating an animal against an infection with porcine circovirus type 2 (PCV2) and an infection with Mycoplasma hyopneumoniae by administration of the vaccine into the dermis of the animal.

Claims

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16. A vaccine comprising in combination a non-replicating immunogen of porcine circovirus type 2 (PCV2), a non-replicating immunogen of Mycoplasma hyopneumoniae, and an adjuvant comprising a nano-emulsion of mineral oil in water, for use in prophylactically treating an animal against an infection of PCV2, Mycoplasma hyopneumoniae, or both PCV2 and Mycoplasma hyopneumoniae, wherein the vaccine is administered into the dermis of the animal.

17. A vaccine of claim 16, wherein the vaccine is administered by a single dose.

18. A vaccine of claim 16, wherein the vaccine is administered with a needle-less vaccination device.

19. A vaccine of claim 16, wherein the non-replicating immunogen of PCV2 is recombinantly expressed ORF2 protein of PCV2.

20. A vaccine of claim 16, wherein the non-replicating immunogen of PCV2 is baculovirus expressed ORF2 protein of PCV2.

21. A vaccine of claim 16, wherein the non-replicating immunogen of Mycoplasma hyopneumoniae is a bacterin.

22. A vaccine of claim 16, wherein the vaccine additionally comprises live attenuated porcine reproductive and respiratory syndrome (PRRS) virus.

23. A vaccine of claim 22, wherein the live attenuated PRRS virus is combined with the immunogen of PCV2 and Mycoplasma hyopneumoniae within 24 hours before administration.

24. A vaccine of claim 23, wherein the live attenuated PRRS virus is combined with the immunogen of PCV2 and Mycoplasma hyopneumoniae within 6 hours before administration.

25. A vaccine of claim 16, wherein the vaccine additionally comprises non-replicating immunogen of Lawsonia intracellularis.

26. A vaccine of claim 25, wherein the immunogen of Lawsonia intracellularis is combined with the immunogen of PCV2 and Mycoplasma hyopneumoniae within 24 hours before administration.

27. A vaccine of claim 26, wherein the immunogen of Lawsonia intracellularis is combined with the immunogen of PCV2 and Mycoplasma hyopneumoniae within 6 hours before administration.

28. A vaccine of claim 25, wherein the immunogen of Lawsonia intracellularis is added to the vaccine in the form of a freeze-dried composition of Lawsonia intracellularis bacterin.

29. A method for prophylactically treating an animal against an infection with porcine circovirus type 2 (PCV2), an infection with Mycoplasma hyopneumoniae, or an infection of both PCV2 and Mycoplasma hyopneumoniae, by intradermally administrating to the animal a vaccine comprising in combination non-replicating immunogen of PCV2, non-replicating immunogen of Mycoplasma hyopneumoniae, and an adjuvant containing a nano-emulsion of mineral oil in water.

30. A method of manufacturing a vaccine comprising the non-replicating immunogen of porcine circovirus type 2 (PCV2), the non-replicating immunogen of Mycoplasma hyopneumoniae, and an adjuvant containing a nano-emulsion of mineral oil in water, for intradermal administration to an animal to prophylactically treat the animal against an infection with PCV2, an infection with Mycoplasma hyopneumoniae, or an infection of both PCV2 and Mycoplasma hyopneumoniae.

Description

EMBODIMENTS OF THE INVENTION

[0031] In a first embodiment the vaccine is administered by a single dose. It was found that a single dose administration led to an effective vaccine. This provides for a very convenient and economical way to protect animals against both pathogens.

[0032] In a next embodiment the vaccine is administered with a needle-less vaccination device, using a jet of the vaccine to reach the dermis through the skin of the animal. Vaccination into the dermis is in this embodiment provided by a needle-less vaccination device using a liquid jet of the vaccine (a high pressurized fluid stream), typically using a very low volume of vaccine in the range of 0.05 to 0.2 ml. This further increases the safety of the vaccine and method of administration.

[0033] In another embodiment the non-replicating immunogen is recombinantly expressed ORF2 protein of porcine circo virus type 2, for example expressed by baculo virus as known in the art. This recombinant protein has proven to be suitable for application in the present invention. In particular, the ORF2 protein can be expressed in a baculo virus expression system such as described in WO2007/028823, WO 2007/094893 or WO2008/076915.

[0034] In yet another embodiment the non-replicating immunogen of Mycoplasma hyopneumoniae is a bacterin. Such Mhyo antigen is relatively easy to produce and has a good track record of efficacy in the everyday swine industry practice.

[0035] In still another embodiment the vaccine in addition comprises live attenuated PRRS virus. In this embodiment the vaccine is capable of providing protection against three major swine pathogens by using just one vaccine.

[0036] In again another embodiment the live attenuated PRRS virus is combined in the vaccine within 24 hours, preferably within 6 hours before administration. Combining the antigens right before administration provides more freedom to choose the excipients since long-term stability, although known for many pharmaceutical compositions, even for combination vaccines including PCV2 ORF2 antigen (for example Porcilis PCV M Hyo, available from MSD Animal Health), as such is known, might still not be straightforward to achieve, at least not for any and all pharmaceutically acceptable carrier compositions.

[0037] In another embodiment the vaccine in addition comprises non-replicating immunogen of Lawsonia intracellularis. In this embodiment the vaccine is capable of providing protection against three major swine pathogens by using just one vaccine. Corresponding to what was said herein before regarding the addition of PRRS immunogen to the vaccine, the immunogen of Lawsonia intracellularis in an embodiment is combined with the immunogen of PCV2 and Mycoplasma hyopneumoniae within 24 hours before administration, preferably within 6 hours before administration. In a further embodiment the immunogen of Lawsonia intracellularis is added to the vaccine in the form of a freeze-dried composition of Lawsonia intracellularis bacterin.

[0038] The invention will now be explained further using the following examples.

EXAMPLES

[0039] Study 1

[0040] Objective

[0041] The objective of this study was to assess the safety and efficacy of different experimental PCV2/Mhyo combination vaccines for intradermal application in piglets.

[0042] Experimental Design

[0043] The progeny of approximately 10 sows were available for this study. The animals had maternally derived antibodies (MDA) against PCV2. A total of 50 piglets were allotted to 4 treatment groups of 10 piglets each and 2 control groups of 5 piglets each. PCV2 antigen was produced by two different concentration processes, a first one using ultrafiltration (UF) and a second one using gravitational forces (G). Vaccines were formulated by the addition of two different adjuvants using the commercially available micro-emulsion adjuvant XSolve (average particle size slightly below 0.5 m; obtainable from MSD Animal Health, Boxmeer, The Netherlands), differing in final concentration of the oil. XSolve 50 contained 20.8% mineral oil and X-solve 30 contained 12.5% mineral oil.

[0044] Piglets from groups 1 to 4 were intradermally vaccinated with a single dose of 0.2 ml vaccine, applied with the IDAL vaccinator (MSD Animal Health) when they were approximately 3 weeks old. Piglets from groups 5 and 6 were injected with a placebo in the same manner. The combination vaccine contained PCV2 ORF2 protein at a final concentration of 9 g per dose and Mhyo bacterin at 1 PCVU/dose (the same as in the commercially available vaccine Porcilis M Hyo ID ONCE of MSD Animal Health). Here below the different groups are indicated:

[0045] Group 1: PCV/Mhyo, XSolve 30, UF

[0046] Group 2: PCV/Mhyo, XSolve 50, UF

[0047] Group 3: PCV/Mhyo, XSolve 30, G

[0048] Group 4: PCV/Mhyo, XSolve 50, G

[0049] Group 5: Placebo, XSolve 30

[0050] Group 6: Placebo, XSolve 50

[0051] All piglets were observed daily for clinical signs. On the day of vaccination clinical signs were monitored in all animals one and four hours post vaccination. The body temperature was taken from all animals, daily from day 1 until day +4, and at 4 hours post vaccination. Local reactions were monitored by palpation starting on the day of vaccination until 4 weeks after vaccination or until regression of the reaction. Blood samples were collected from all animals the day before vaccination, and 2, 3, 5 and 10 weeks after vaccination. Serum samples from each animal taken before vaccination and 2, 3, 5 and 10 weeks thereafter were tested for antibodies against Porcine Circovirus Type 2 (PCV2).

[0052] Results

[0053] At the start of the experiment, all animals were found to be healthy.

[0054] At the time of vaccination and onwards from one day after vaccination all groups had comparable temperatures. At four hours after vaccination all groups vaccinated with a combination vaccine showed a considerable increase in body temperature when compared to the control groups. No significant differences were seen between the UF and G groups. The mean body temperature increase in the vaccinated groups was up to 1.0 C. higher than the mean body temperature in the control animals. Peak values were well above 41 C. (up to 41.6 C.) whereas values in the control groups were in the 39-40 C. range, which peak values of 40.2 C.

[0055] Regarding local reactions as assessed by palpation, in the control groups, no animals in the XSolve 30 group had any local reactions. In the XSolve 50 group, 60% of the animals had local reactions but the maximum size stayed below 1.5 cm. For the vaccinated groups the situation was completely different. Of the animals receiving the XSolve 30 vaccine, 60-90% showed local reactions, with maximum sizes around 5 cm. In the XSolve 50 groups 70-80% of the animals showed local reactions, with maximum sizes around 6 cm, the local reactions being blue and/or painful to the animal.

[0056] Although serological responses to the vaccines were good (results not shown), the vaccines were generally regarded as not being safe in particular due to severe local reactions.

[0057] Study 2

[0058] Objective

[0059] With respect to Study 1, the objective of this second study was to assess whether or not the combination vaccines could be made safer when lowering the amount of the micro-emulsion, while retaining efficacy, and also to test other potential adjuvants with the aim at arriving at a safe and efficacious combination vaccine.

[0060] Experimental Design

[0061] A total of 60 piglets was allotted to 4 treatment groups of 15 piglets each. The piglets were vaccinated when they were approximately three weeks old. Piglets from groups 1 to 3 were vaccinated with a single dose of 0.2 ml vaccine formulated with PCV2-Mhyo antigen as used in Study 1, using the XSolve adjuvant at a concentration of 5% for the mineral oil (XSolve 12), or in the alternative the same vaccine using in addition to the XSolve micro emulsion the commonly accepted alum adjuvant, or a vaccine using an adjuvant based on squalane (hydrogenated shark liver oil) adjuvant (0.2 ml). Piglets from group 4 were vaccinated with a single dose of Porcilis Mhyo ID ONCE (0.2 ml). All piglets were vaccinated intradermally in the right side of the neck. Here below the different groups are indicated:

[0062] Group 1: PCV/Mhyo, XSolve 12

[0063] Group 2: PCV/Mhyo, XSolve 12 plus Alum (Al(OH)3

[0064] Group 3: PCV/Mhyo, Squalane

[0065] Group 4: Porcilis M Hyo ID ONCE, positive control

[0066] On study day (SD) 29 all animals were challenge infected intratracheally on two consecutive days with 10 ml of a virulent Mhyo strain. On SD 52 or 53 all animals were necropsied.

[0067] All piglets were observed daily after vaccination for clinical signs. Local reactions were monitored by palpation every second day, starting on the day of vaccination until 29 days post vaccination, or until local reactions had resolved. Serum samples were collected from all animals on the day of vaccination as well as on SD 22, 29 and 52/53.

[0068] Samples were tested for antibodies against PCV2 and Mhyo and were compared with each other. At necropsy, the Mhyo specific lung lesions were scored.

[0069] Results

[0070] At the start of the experiment all animals were found to be healthy.

[0071] Following vaccination the severity of local reactions (average maximum size, number of animals with local reactions) was the lowest in the Group 1 (XSolve 12) and Group 3 (Squalane). In these groups the average maximum size of the local reactions was 1.1 and 0.9 cm respectively, while the percentage of animals with local reactions were 40 and 65%. In the other groups most animals had local reactions (90-100% of piglets) and the average maximum size was between 1.5 and 1.8 cm. All these figures are acceptable for vaccines authorised for use in practice.

[0072] Regarding PCV serology, at the time of vaccination (SDO) all animals were negative for PCV2 IgM antibodies. Following vaccination no clear difference in average PCV2 total Ig antibody response could be observed between the test groups 1, 2 and 3. Three weeks post vaccination the percentage of PCV2 IgM positive animals was 80-100% per test group and 0% for the controls. This shows that the combination vaccines were able to actively induce an anti-ORF2 antibody titer.

[0073] Regarding Mhyo, at the start of the experiment none of the piglets were positive for antibodies against Mhyo. Four weeks post vaccination (SD29) there was minimal response against Mhyo in the test groups 1 to 3. Only 1 animal in group 2 was positive. In the positive control group (Porcilis Mhyo ID Once) 10 animals (67%) of the piglets had a Mhyo antibody response. Following Mhyo challenge almost all animals had a positive Mhyo serological response. This indicated that the combination vaccines would not have an acceptable efficacy, which indication was confirmed by assessing the lung lesion scores which are summarised in Table 1.

TABLE-US-00001 TABLE 1 Median and mean percentage of Lung Lesions Group Median Mean 1. PCV2/Mhyo Xsolve 12 2.20 4.69 2. PCV2/Mhyo Xsolve 12 + Al(OH)3 1.90 3.01 3. PCV2/Mhyo squalane 3.99 5.15 4. Porcilis Mhyo ID Once (XSolve 50) 0.90 1.97

[0074] In conclusion, by using a less concentrated micro emulsion of a mineral oil, optionally with the addition of an alum adjuvant, or by using squalane as an adjuvant, safe PCV2/Mhyo combination vaccines could be devised, but these vaccines were regarded as not sufficiently efficacious for combating an infection with Mycoplasma hyopneumoniae.

[0075] Study 3

[0076] Objective

[0077] The objective of the third study was to find an alternative adjuvant that could be used to make the PCV2/Mhyo combination vaccine safe for ID use, while at the same time retaining efficacy and optionally be suitable for admixing non-replicating immunogen of Lawsonia intracellularis.

[0078] Experimental Design

[0079] A total of 75 piglets was allotted to 5 treatment groups of 15 piglets each. The piglets were vaccinated when they were approximately three weeks old. Piglets from groups 1 to 3 were vaccinated with a single dose of 0.2 ml vaccine formulated with PCV2-Mhyo antigen as used in Study 1, using the nano-emulsion of mineral oil Montanide IMS251C (available from SEPPIC France) according to manufacturer's instructions. The vaccine for Group 1 contained a half dose of Mhyo antigen (0.5 PCVU/dose) as compared to the other groups. Before vaccination, freeze-dried Lawsonia bacterin (at a dose of approximately 10.sup.8 cells) was added to the vaccine for Group 3. Piglets from group 4 were vaccinated with a single dose of Porcilis Mhyo ID Once (0.2 ml). All piglets were vaccinated intradermally in the right side of the neck. Piglets from group 5 were not vaccinated (negative control group). Here below the different groups are indicated:

[0080] Group 1: PCV/Mhyo, 0.5 PCVU Mhyo, Montanide

[0081] Group 2: PCV/Mhyo, 1 PCVU Mhyo, Montanide

[0082] Group 3: PCV/Mhyo, 1 PCVU Mhyo plus Lawsonia, Montanide

[0083] Group 4: Porcilis M Hyo ID ONCE, positive control

[0084] Group 5: No vaccination, negative control

[0085] On study day (SD) 35 all animals were challenge infected intratracheally on two consecutive days with 10 ml of a virulent Mhyo strain. On SD 59 or 60 all animals were necropsied.

[0086] All piglets were observed daily after vaccination for clinical signs. Body temperatures were monitored as well as local reactions. The latter by palpation every second day, starting on the day of vaccination until 26 days post vaccination, or until local reactions had resolved. Serum samples were collected from all animals on the day of vaccination as well as on SD 22, 29 and 35. Samples were tested for antibodies against PCV2, Mhyo and Lawsonia (groups 3 and 5 only) and were compared with each other. At necropsy, the Mhyo specific lung lesions were scored.

[0087] Results

[0088] At the start of the experiment all animals were found to be healthy.

[0089] At the time of vaccination (SDO) all groups had comparable average rectal temperatures. At four hours post vaccination only a slight increase was observed in the average rectal temperatures for all treatment groups (average increase of 0.4-0.6 C.). The maximum temperature increase of 1.8 C. for one animal was observed in group 2. These temperature rises are acceptable for a swine vaccine.

[0090] In the vaccinated groups most of the animals had local reactions (80-100% of piglets), but the average size was low, viz. 2 cm for all vaccinated groups, including the positive control. The lowest percentage of animals having local reactions and the lowest average size could be found in Group 2, viz. 80% and 1.8 cm respectively. These figures are acceptable for vaccines authorised for use in practice.

[0091] Regarding PCV serologie, at the time of vaccination (SDO) all animals were negative for PCV2 IgM antibodies. Following vaccination no clear difference in average PCV2 total Ig antibody response could be observed between the test groups 1, 2 and 3. Three weeks post vaccination the percentage of PCV2 IgM positive animals was 93% in Groups 1 to 3 and 0% for the controls. This shows that the combination vaccines were able to actively induce an anti-ORF2 antibody titer.

[0092] Regarding Mhyo, at the start of the experiment none of the piglets were positive for antibodies against Mhyo. Four weeks post vaccination (SD30) in the test groups 1, 2 and 3 between 93 and 100% of the animals were Mhyo positive. In the positive control group this was 40%. Lung lesion scores confirmed a good efficacy against an Mhyo infection as depicted below in Table 2.

[0093] Regarding Lawsonia serology, at the start of the study all animals in groups 3 and 5 were negative for antibodies against Lawsonia. During the course of the study the percentage of positive animals in the LFD vaccinated group increased from 60% at SD30 to 100% at the end of the study. None of the animals in the control group had a Lawsonia serological response until the end of the study.

TABLE-US-00002 TABLE 2 Median Lung Lesions score and % of animals with a low score (<5% lesions) Group Median <5% 1. PCV/Mhyo, 0.5 PCVU Mhyo, Montanide 1.2 80% 2. PCV/Mhyo, 1 PCVU Mhyo, Montanide 1.6 80% 3. PCV/Mhyo, 1 PCVU Mhyo plus Lawsonia, Montanide 1.6 72% 4. Porcilis M Hyo ID ONCE, positive control 0 85% 5. No vaccination, negative control 10.3 20%

[0094] Study 4

[0095] Objective

[0096] The objective of the fourth study was to evaluate efficacy and safety of PCV2/Mhyo combination vaccines using a nano emulsion adjuvant as described in Study 3, to which combination vaccines live attenuated PRRS virus was added to arrive at a triple combination vaccine. The efficacy towards protection against infection with PCV2 is evaluated by assessing anti-ORF2 serology. The efficacy against infection with Mycoplasma hyopneumoniae is evaluated by comparing the serological response with that of the commercially available Mhyo vaccine Porcilis Mhyo (MSD Animal Health, Boxmeer, The Netherlands). The efficacy against an infection with PRRS virus is evaluated by assessing the PRRs viraemia upon challenge with a pathogenic PRRS strain, 4 weeks post vaccination.

[0097] Experimental Design

[0098] The progeny of 10 sows was available for this study. A total of 40 animals were allotted to 4 groups of 10 piglets each. All animals were transferred to an animal facility when they were approximately 4 weeks old. Groups 1 to 4 were intradermally vaccinated using the IDAL vaccinator into the right side of the neck. Groups 1 and 2 each received an ORF2 protein based PCV2 vaccine comprising in addition Mhyo bacterin (the same antigen as in the commercially available product Porcilis M Hyo), in which combination vaccine a live PRRS virus vaccine (Porcilis PRRS) was reconstituted. The vaccine for group 1 was based on Montanide IMS 251, available from SEPPIC, France) to which 3% ovalbumin was added. The vaccine of group 2 contained the same adjuvant but no ovalbumin was added. Each vaccine contained 9 g/dose of the ORF2 protein, and Mhyo antigen at 1-2 times the concentration of the M Hyo antigen in the commercially available vaccine Porcilis M Hyo ID ONCE. The PRRS vaccine was a freeze-dried vaccine and was reconstituted immediately before administration to contain 10.sup.4.5 TCID.sub.50 of virus per dose of 200 l using the appropriate PCV2 vaccine or a diluent. Group 3 only received the PRRS vaccine and group 4 remained unvaccinated and served as control. All piglets were observed daily for clinical signs. The animals were challenge-infected with pathogenic PRRS virus (type I) when they were approximately 8 weeks old (day 28). The challenge material contained (a calculated dose of) 5.3 log 10 TCID50 of the virus in 2 ml. The material was intra-nasally administered, 1 ml per nostril. At the end of the observation period (49 days after vaccination corresponding to 21 days post challenge) all pigs were sacrificed. Blood samples (via v. jugularis) will be were taken from all animals individually on day 0, 14, 28 (right before challenge), 31, 35, 38, 42 and 49 and tested for the presence of PRRS virus, for antibodies against PRRSV, PCV2 and Mhyo.

[0099] Results

[0100] No animals showed any clinical signs due to vaccination and rectal temperatures remained within 1.5 C. from controls. The vaccines are thus regarded safe.

[0101] Regarding Mhyo, the serological response of the combination vaccine appears to be comparable to that as obtainable with the commercially available vaccine Porcilis M Hyo (no numerical results depicted in a figure). It may thus be assumed that the vaccine protects against infection with Mhyo.

[0102] The results of the PCV2 serological response are given in FIG. 1. It appears that the two combination vaccines induce a positive anti-ORF2 antibody response which means that the vaccines induce protection against infection with wild-type PCV2.

[0103] The results of the PRRS serological response are given in FIG. 2. It appears that the two combination vaccines, like the commercially available PRRS vaccine, induce a positive anti-PRRS antibody response before challenge. This is an indication that the vaccines provide protection against PRRS virus infection. In FIG. 3 the viraemia data are given. It appears that all three vaccines provide protection against PRRS virus infection since viraemia levels are lower than the level in the positive control animals (group 4) at each point in time.

[0104] Study 5

[0105] The objective of the fifth study was to evaluate efficacy and safety of PCV2/Mhyo combination vaccines using alternative nano emulsion adjuvants, when compared to the nano emulsion adjuvant as described in Study 3. Regarding PCV2, the efficacy was evaluated by assessing anti-ORF2 serology. Regarding Mycoplasma hyopneumoniae, efficacy was evaluated by comparing the serological response with that of the commercially available Mhyo vaccine Porcilis M Hyo ID ONCE (MSD Animal Health, Boxmeer, The Netherlands).

[0106] The experimental design was largely the same as described here above. In particular, a total of 50 piglets were allotted to 5 treatment groups: 5 groups of 10 piglets each. The piglets were vaccinated intradermally when they were approximately three weeks old. Piglets from groups 1 to 3 were vaccinated with a single dose of vaccine (0.2 ml) formulated with PCV2-Mhyo antigen as used in Study 3 and three different adjuvants based on nano emulsions of mineral oils in water (the fact that the emulsions were actually nano emulsions was established microscopically). The first adjuvant (received by group 1) was formulated as an Amphigen like nano-emulsion, viz. the same adjuvant emulsion as Amphigen (obtainable from Zoetis), but containing the mineral oil as a nano emulsion by subjecting the adjuvant to microfluidisation. The second adjuvant (received by group 2) was formulated as a Metastim (Boehringer Ingelheim) like adjuvant, made by adding vitamin E-acetate to the commercially available adjuvant and emulsifying the adjuvant to become a nano-emulsion by microfluidisation. The third adjuvant (received by Group 3) resembled IMS251 as used in Example 3, apart from the fact that vitamin E-acetate was added, and the method of obtaining the emulsion was different, namely via an aqueous intermediate emulsion (i.e. an aqueous concentrate to be used for formulating the vaccine), instead of a pure oil/surfactant intermediate (i.e. an oily concentrate to be used to formulate the vaccine). Piglets from group 4 were vaccinated with Porcilis M Hyo ID ONCE. Piglets from group 5 were not vaccinated.

[0107] The results indicated that the three experimental vaccines based on alternative nano-emulsions of mineral oil in water were completely safe: no increase in body temperature was noticed after vaccination and the average local reactions were less than or equal to the average local reactions as observed with the commercial vaccine Porcilis M Hyo ID ONCE. PCV2 serology showed that in all three groups receiving the combined PCV2/Mhyo vaccine (groups 1-3) the anti-ORF2 titer increased due to vaccination, whereas in groups 4 and 5 a continuous decrease was seen in the same period. Regarding Mhyo, all animals (groups 1-5) were challenged. The percentage of positive animals after challenge was 90-100% in groups 1-3, the same as in the positive control group 4 (receiving the commercial M Hyo vaccine). In the negative control group 5 the percentage of anti-body positive animals was 0%.