MXPA99007134A - Infectious bursitis vaccine - Google Patents

Abstract

The present invention is concerned with a vaccine which is capable of protecting poultry against Infectious Bursitis Infections, characterized in that it contains an Infectious Bursitis virus which has the combined properties of upon administration to a chicken causing a reduction in the size of the bursal size, expressed as bursa/body weight ratio, of less than 55%and the capability to protect poultry having an ELISA antibody titer of at least about 500 and with viruses having the above characteristics.

Description

VACCINE AGAINST INFECTIOUS BURSITIS BACKGROUND OF THE INVENTION The present invention relates to a vaccine that is capable of protecting poultry against infectious bursal infections and new viruses useful for preparing such vaccines. Infectious bursal disease is an infectious disease that can affect poultry and is caused by the infectious bursal disease virus.

(VEBI) The causative agent of infectious bursitis belongs to the family Birnaviridae, genus Birnavirus and mainly affects the Fabricio Exchange. This causes atrophy of the bag. Infection by virulent viruses found in the field, generally causes the reduction of the weight of the bag to approximately one third of the normal weight. This is usually expressed as the bag / body weight ratio (RBC). The disease causes degeneration and necrosis of the population of B lymphocytes in the pouch. This can cause immunosuppression. The degree of atrophy (and subsequent degeneration) of the pouch depends on the virulence of the strain involved. The EBBI serologically can be classified into two different types: type 1 is found in chickens, REF .: 30850 while «that type 2 is found in turkeys. The prevention of infectious bursitis is based on vaccines. Currently used vaccine strains are divided into highly virulent strains (= intermediate plus, examples: Bursine Plus, Bursine 3), intermediate virulent (e.g. Bursine 2, D78, LZ228TC) and mildly attenuated (= virulent; e.g. Bursine 1). The problem facing poultry farmers is that in a regimen in which poultry are regularly vaccinated, the new offspring are born with a high titer of maternal antibodies. It is known that maternal antibody titers interfere with the vaccine virus. Highly virulent, intermediate and avirulent strains produce virus-neutralizing antibody titers in the mother at levels of 1: 500, 1: 250 or less than 1: 100, respectively ("Diseases of Poultry", 9th edition, editor: Calnek et al. al., Iowa State University Press, 1991, p.659). Using the ELISA system (enzyme linked immunosorbent assay or ELISA), to monitor maternal antibodies, comparable results have been obtained. Virulent vaccines (intermediate plus) produce IDDEX-ELISA titers of approximately 5000. Intermediate and mild strains produce much lower titers to induce a serum response.

The virulence of the strains is monitored by the effect on the weight of the bag (RBC) as already described above, or by the microscopic changes. The following table (Table 1) shows the virulence level of intermediate plus (Poul ac Bursa Plus, Delvax LZ228E) and virulent vaccines in field (isolation D6948E) on the weight of the bag (expressed as a percentage of RBC compared to normal) ). TABLE 1 The comparison shows that the intermediate plus vaccines affect the weight of the bag as much as the virulent virus in the field. As a consequence, those who wish to vaccinate young chickens with high maternal antibody titers have to accept the negative consequences of the more "hot" strains (such as severe tracheal lesions and a high mortality rate). Therefore, poultry farmers lose chickens with > 1: 500 antibody titre when using intermediate vaccines, because they do not produce this titer and when they use a virulent vaccine, poultry farmers lose chickens due to the effect of the virus. Thus, there is a need for a vaccine that is less virulent but capable of producing high maternal antibody titers at the same time. Surprisingly, the VEBI with this feature set has been found. Thus, in accordance with the present invention, a vaccine is provided which is capable of protecting poultry against infectious bursal infections., characterized in that it contains an infectious bursal disease virus having the combined properties of an intermediate virulence and the ability to protect poultry with an antibody titer by ELISA of at least about 500. A particular example of this new kind of virus. The new strain VEBI is internally known as strain 9793, a sample of which has been deposited in the National Collection of Cultures of Microorganisms of the Pasteur Institute in Paris (France) under number 1-1810, on January 22, 1997. The deposited material 1-1810 is in the form of master seeds of viruses. The vaccine virus is prepared from a seed virus by one or more passes in a suitable medium. Preferably, all vaccine viruses are derived from a single stock of virus - * the Master Seed virus. Optionally, a combination of Working Seed Virus is prepared from this Master Seed by one or more passes and the final vaccine virus is prepared from this seed by one or more passes as well. The vaccine usually contains a stabilizer, such as inositol or mannitol, which is used in the preparation process. If required, a diluent for the vaccine can be any physiological solution. Suitable means for making the passes during these respective propagation steps from the Master Seed to the vaccine virus are, for example, specific pathogen-free eggs (LPE), eggs negative to specific antibodies (NAE), mainly cells of chicken or an avian cell line. The vaccine according to the present invention preferably contains live viruses of the new EBBI class described above. The effective dose for chickens can vary between about 102 and about 10 EID50, preferably between about 103 and 105 EID50. The vaccine can be safely administered to chicks a day old or in ovo. The vaccine according to the present invention based on live-VEGBI, preferably is shipped in lyophilized form and dissolved in water before use. The vaccine according to the present invention is preferably administered enterally, e.g. orally (in drinking water or individually with a pipette), by oculonasal or by spraying. The VEBI 9793 strain is characterized by the ability to induce antibodies in birds with a maternal antibody titre of >; 500 (measured by IDDEX-ELISA) and because it is much less harmful to the bag than hot vaccines. The induction of antibodies in birds with a maternal antibody level of > 500, normally only possible with one of the so-called "hot" vaccines and / or with virulent field strains. These hot vaccines and field strains will damage the bursa of Fabricio causing a depletion of B lymphocytes. The strain VEBI 9793 is much less damaging to the pouch, which has been measured by the size of the pouch after infection. Thus, in other aspects of the present invention, there is provided a vaccine comprising a virus of strain 9793 having the general or specific characteristics of deposit No. 1-1810, or a vaccine comprising a virus derived or developed from from strain 9793, ie derived or developed from any of the current deposits or obtained from the natural virus. An infectious bursal disease virus (IBD) is also provided which has the general or specific characteristics of deposit 1-1810 or a vaccine comprising a virus derived or developed from strain 9793, i.e. derived or developed from either the actual deposit or obtained from the natural virus. The present invention is illustrated, but not limited, by the following Examples. EXAMPLE 1 Preparation of Master Seed Virus Two vials of stock material of strain 9793 were resuspended in 2.0 ml of distilled water. This virus suspension was diluted 1/10 by transferring 1 ml of it in 9 ml of saline. After mixing, the solution was diluted 1/30 by transferring 2 ml to 58 ml of saline to obtain a suspension containing approximately 500 EID50 per 0.1 ml. 400 embryos of NAE eggs each were inoculated with 0.1 ml of the previous virus suspension, via the yolk sac using a 1 ml syringe with a 23 G x 1"needle.The eggs were incubated at 37 ° C and were observed with light after 24 hours, 49 dead embryos were found and discarded, After 48 and 72 hours after inoculation, the dead embryos (340 eggs in total) were separated from the other eggs and decapitated. groups of approximately 50 embryos Each group was homogenized separately using a Ystral mixer equipped with a small sterilizable rod Each homogenate was emptied through two layers of gauze over a vessel and each filtrate was transferred to a Duran bottle separately. To each was added a volume of stabilizer containing inositol and mannitol, equivalent to approximately one third of the volume of the filtrate, samples of 2 or 3 ml of each b were taken. bottle for its titration or for sterility tests, respectively. The bottles and samples were frozen and stored at -70 ° C until further processing. The sterilized bottles were thawed, combined and mixed well using a magnetic stirrer. A total volume of 496 ml of master seed suspension was obtained, which was emptied into bottles, 0.5 ml per bottle, placed in an Edwards Lyomaster 4000 kit and lyophilized. The bottles were sealed under vacuum with rubber stoppers and sealed with aluminum capsules.

EXAMPLE 2 Preparation of the working seed A vial of the master seed virus of strain 9793 was resuspended in 1.0 ml of distilled water. This solution was diluted 1/10 by transferring 0.2 ml of it to 1.8 ml of saline. After mixing, the suspension was diluted 1/200 by transferring 1 ml to 199 ml of saline, to obtain a suspension containing approximately 10 EID59 per 0.1 ml. Six hundred and fifty NAE egg embryos were each inoculated with 0.1 ml of the previous viral suspension via the yolk sac using a 1 ml syringe with a 23 G x 1"needle.The eggs were incubated at 37 ° C. they observed with light after 24 and 48 hours.12 dead embryos were found after 24 hours and 11 after 48 hours.These were all discarded.After 72 hours after inoculation, the embryos were separated from the remaining eggs and decapitated. They were combined into 10 groups of approximately 60 embryos Each group was homogenized separately using a Ystral mixer equipped with a small sterilizable rod Each homogenate was emptied through two layers of gauze into a container and each filtrate was transferred to a bottle of They last separately Each bottle was filled with a volume of stabilizer containing inositol and mannitol, equivalent to approximately one third of the volume of the filtrate. The final volume of each bottle was between 50 and 60 ml. One ml of each bottle of Duran was removed and a combination was made. After mixing this combination, it was distributed in 1 ml aliquots in Nunc cryotubes and these were stored at -70 ° C for subsequent titration. Another 1 ml aliquot was removed from each bottle of Duran and inoculated individually into 5% blood agar plates to verify sterility. All Duran bottles were stored at -70 ° C until required. There was no growth in any of the plates after 14 days, therefore the 10 aliquots were included in the work seed. Lyophilization and testing of the working seed - Each of the working seed aliquots was thawed, combined and mixed well on a magnetic stirrer using a moving part. 420 vials each were filled with 20 ml of the virus suspension. The bottles were placed in a Modulyo freeze dryer. The lyophilization was carried out for 14 hours before the bottles were sealed and removed from the cleaning room. Each bottle was fitted with an interlocked lid and all were tested with vacuum using an Edwards spark tester.

Preparation of the vaccine The vaccine material was prepared according to the method indicated above from the working seed. A stabilizer (inositol, mannitol) was added to the vaccine material. EXAMPLE 3 Vaccine Safety The safety of the vaccine prepared according to Example 1 was tested after vaccination of 1-day-old chickens according to two methods that are explained below. Weight ratio of bag / body The size of the bag is expressed as the weight ratio of the bag / body (RBC) according to the following formula: RBC = weight of the bag (g) x 1000 body weight (g ) Classification of lesions of the bag The lesions observed by microscopic examination were judged as follows: After weighing the bags, they were removed and fixed in 4% formalin for microscopic examination in a PHC team, Doorn, The Netherlands. The records of the results are summarized in Table 2. TABLE 2 Severe fibrosis and epithelial proliferation. The aforementioned cavities are dispersed throughout the fabric of the bag.

Groups of 30 birds were vaccinated with the 9793 vaccine by spraying, pt controls. Three weeks after vaccination, half of the birds in each group were sacrificed and RBC was determined. The other half of the birds in each group were challenged three weeks after vaccination. This challenge was carried out using a highly virulent field strain (D6948E, obtained at the Animal Health Institute in Deventer, The Netherlands). After 10 to 12 days of challenge, these birds were sacrificed and RBC was determined. From a selected number of groups both before and after the challenge, the Fabricio pouch was examined microscopically and lesion scores were determined. RBC results The results of the RBC determinations are summarized in Table 3. These results show that the% RBC compared to the controls after vaccination on average, is approximately 60. Thus, it can be concluded that the Damage to the bag caused by the vaccine virus is very moderate. In addition, it is clear that the effect of the challenge with the virus for the bag of vaccinated birds is absent, while in the control birds considerable damage to the bag is prominent. These latter data from the control animals, in fact confirm the data previously presented for the field viruses and the virulent vaccine strains. TABLE 3 * unvaccinated controls Microscopic examination The microscopically visible damage to the bag is summarized in Table 4. The score of the lesions after the vaccine is very low and is at an acceptable level for a vaccine, while a very high score is found. in animals not vaccinated after the challenge. The bag of vaccinated animals again showed that it is not affected by the challenge virus. TABLE 4 * unvaccinated controls EXAMPLE 4 Serum response after vaccination These studies were carried out with the birds used in Example 3. Blood samples were taken from the birds at approximately 5 6 6 days of age. The individual titles were calculated after 2 to 5 days in accordance with the formula of Kouwenhoven (B. Kouwenhoven and J. van den Bosch, in: Proceedings of 19th World's Poultry Congress, 1992, pp. 465-468). Subsequently, the birds were divided into different title groups and vaccinated. During 3 or 4 weeks they took blood samples to examine the seroconversion. Results The results of these studies are summarized in Table 5. These data confirm the high efficacy of the vaccine according to the present invention. High levels of maternal antibodies do not interfere with vaccination. Kouwenhoven (supra) provides ELISA titers of approximately 500 that the intermediate plus strains are capable of producing. The vaccine according to the present invention easily produces high levels of maternal antibodies to induce seroconversion. TABLE 5 * unvaccinated controls; ND "means not determined.

EXAMPLE 5 Blood samples were taken from one day old chickens in order to calculate the vaccination day using the Kouwenhoven formula previously mentioned. Two studies, Study A and Study B, were prepared to determine the effects of strain 9793 on mortality. For study A, blood samples were taken at 1 day of age, at 14 days (day of vaccination with strain 9793), 21, 28 and 35 days of age. Blood samples were taken at 1 day of age and at 11 days (day of vaccination with strain 9793), at 21, 28 and 35 days of age for study B. In both experiments a group was vaccinated for EBI and the other one worked as a control. Each group consisted of 1500 chickens. All the chickens were vaccinated against Newcastle disease (EN) and for infectious bronchitis (Bl) on the day of vaccination. The vaccines used were Poulvac NDW (vaccine against Newcastle disease) and Psulvac IB Primer (vaccine against infectious bronchitis containing strains H120 and D274). Vaccination was done by coarse spray. The serology was performed using the Hl test for EN and Bl and the IDEXX-ELISA test for EBI. Finally, mortality was recorded. The serology data are presented in Tables 6A and 6B (studies 97-62 A and 97-62 B). Mortality was as follows: Study A: Vaccine against IBD (group 1) = 7% Unvaccinated controls (group 2) = 6.4% Study B: Vaccine against EBI (group 2) = 2.8% Unvaccinated controls (group 1) = 5.1% Study B shows that EBI 9793 produces maternal antibodies > 500. In study A, maternal antibodies were below 500 on the day of vaccination. The serology data for Bl and EN does not show any immunosuppression caused by EBI 9793 in the humoral antibody response against Bl or EN. The mortality data also show "that the vaccine is safe.

TABLE 6A. Titers of average Hl antibodies against NDV, IBV M41 and IBV D274 and average titers of ELISA antibodies against EBVI. statistically significant difference (P <0.05) between the groups using one-way ANOVA with the Group Factor.

TABLE 6B. Average antibody titers against EN, Bl M41, Bl D274 and EBI. 1: statistically significant difference (P <0.05) between the groups using one-way ANOVA with the Group Factor.

It is noted that in relation to this date, the best method known to the applicant to carry out the aforementioned invention, is that which is clear from the present description of the invention.

Claims (12)

1. CLAIMS Having described the invention as an antecedent, the content of the following claims is claimed as property: 1. A vaccine that is capable of protecting poultry against infections of infectious bursitis, characterized in that it comprises an infectious bursal virus which has the combined properties after its administration to chickens, to cause a reduction in the size of the Fabric Bag, expressed as the weight ratio of bag / body, less than 55% and the ability to protect poultry that have an antibody titer by ELISA of at least about 500.
2. 2. A vaccine comprising an infectious bursal virus having the characteristics of strain 9793, a sample of which is deposited at the Pasteur Institute in the National Crop Collection of Microorganisms, under number 1-1810.
3. 3. The vaccine according to claim 1, characterized in that the infectious bursal virus has the characteristics of deposit No. 1-1810. .
4. The vaccine according to any one of claims 1 or 2, characterized in that the infectious bursal virus is a virus of strain 9793, a sample of which is deposited in the Pasteur Institute in the National Collection of Microorganism Crops, under the number 1-1810.
5. The vaccine according to any of claims 1 to 3, characterized in that the infectious bursal virus is a virus derived or developed from strain 9793, a sample of which is deposited in the Pasteur Institute in the Collection National Institute of Microorganism Crops, under number 1-1810.
6. 6. A vaccine according to any of the preceding claims, characterized in that it contains the virus in an amount between 10 and 10 EID50 per dose.
7. A vaccine according to claim 6, characterized in that it contains the virus in an amount between 10 3 and 105 EID50 per dose.
8. 8. An infectious bursal virus characterized in that it has the combined properties after being administered to chickens, that it causes a reduction in the size of the pouch expressed as body / body weight ratio, less than 55% and the ability to protect to poultry that have an ELISA antibody titer of at least about 500.
9. 9. An infectious bursal virus characterized because it has the characteristics of a virus of strain 9793, a sample of which is deposited in the Institute. Pasteur- in the National Collection of Microorganism Crops, under number 1-1810.
10. 10. The infectious bursal virus according to claim 8, characterized in that the infectious bursal virus has the characteristics of deposit No. 1-1810.
11. 11. The infectious bursal virus according to any of claims 8 or 9, characterized in that the infectious bursal virus is a virus of strain 9793, a sample of which is deposited in the Pasteur Institute in the National Collection of Microorganism Crops, under number 1-1810.
12. 12. The infectious bursal virus according to any of claims 8 to 10, characterized in that the infectious bursal virus is a virus derived or developed from strain 9793, a sample of which is deposited in the Institute. Pasteur in the National Collection of Microorganism Crops, under number 1-1810.