NL7908687A - INFECTIVE BRONCHITIS VACCINES FOR POULTRY AND METHOD FOR PREPARING SUCH VACCINES. - Google Patents

Description

NED-231 2 JACKET *: VJ

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Gist-Brocades N.V · in Delft ·

The applicants named Manfred ΜIrasselt and Peter Apontoveil as inventors.

Infectious poultry bronchitis vaccines and method of preparing such vaccines

The invention relates to infectious bronchitis (IB) vaccines for poultry and method of preparing such vaccines and more particularly to improved infectious bronchitis vaccines derived from at least one new infectious bronchitis virus strain of a new serotype. That of the hitherto known viruses

The use of live infectious bronchitis vaccines for poultry has been known for many years. Infectious bronchitis is a major respiratory disease in poultry caused by a corona virus. The poultry is seriously affected by epidemics of this disease

The infectious bronchitis still causes a high mortality especially in young poultry. Besides mortality and more or less strong respiratory symptoms, damage to the laying device occurs as a result of a 15 infectie · Β · infection. In addition, infections with Ι · Β · virus can be latent. stimulate virus or bacterial infections and thus to serious economic losses? especially in the broiler sector »lead ·

Both inactivated virus and so-called live virus derived vaccines were used to control infectious bronchitis, but it was found that a loss of immunogenic properties occurred after inactivation of these vaccines with, for example, formalin and ultra violet light (MS Hofstad, Diseases of Poultry, Biester and schwarte, Iowa State Univ · Press, Ames · (l965) 615) · 25 Because normal healthy chicks are often killed by vaccination with a live, little or no attenuated virus 7908687 V * * - 2 vaccine, whereby if the danger is especially serious for animals less than 2 to 3 weeks old or for breeding hens shortly before the start or during laying, there is a clear preference in professional circles for the use of live vaccines, in which the harmlessness of such increase vaccines by sensing original Ι · Β · field virus isolates ·

For example, the attenuated H strain, which is currently widely used worldwide and isolated by Bijlenga, Hoekstra and Rispens, is described in Tijdschr · Diergeneesk, 81:43 "Infectious bronchitis in chicks in the Netherlands, ( l956), Tijdschr · Diergeneesk ·, 85: 320 (i960), Tijdschr · Diergeneesk · 85: 279 (i960) and Tijdschr · Diergeneesk · 85: 398 (i960) · For the purpose of such modified vaccines, viruses have hitherto been used who have undergone 25 or more embryos 15 passages to reduce their pathogenicity and spreading capacity, such as, for example, the Massachusetts type and more particularly the IBV ¥ 48, M41, 82828 or the H52 and H 120 strains thereof (which 52 and 120 passages on chicken eggs, respectively), a Connecticut isolate e.g. A5968 or the Beau-20 dette IBV (lBV-42) .Although the use of vaccines of these modified strains is nowadays quite safe and effective, these vaccines, under certain circumstances, are unable to sufficiently prevent outbreaks of infectious bronchitis, as shown, for example, in Avian Diseases vol · 20, 25 no, 1, ρ · 42 and 177 and Avian Diseases vol · 19, no · 2, 323 and 583 · This lack of current infectious bronchitis vaccines is largely attributed to emerging antigenic variations of the virus, as evidenced, for example, by Archiv fllr die Gesamte Virusforschung 34, p32 (1971) and Cunningham CH; Develop30 Biol. Standard, 33 ,, 311 (1976)

Therefore, an attempt was also made to achieve adequate vaccination of poultry by preparing and using combined vaccines derived from multiple infectious bronchitis strains of different serotypes. However, a clear under-35 difficulty was found to be the reduction of immunogenic properties of the respective starting viruses by mutual interaction, as shown, for example, by Am · J · Vet · Res · 3,6, 4,524 and 525 (1965) and Avian Diseases 1.2 »577 (1968) 7908687 * - 3 -

Therefore, there is still a great need for infectious bronchitis vaccines with effective immunizing properties. It will be clear that the pursued improvement of these vaccines continues to be severely hampered. g.v. the altering antigenic, immunogenic and other properties of existing I.B. viruses after a large number of passages in embryonated chicken eggs resp. the occurrence of new serotypes and the lack of sufficiently well applicable serological and immunological test procedures. In this regard, reference can be made, for example, to Avian Diseases, 10 19, 2, 323 and 324 (1975).

Surprisingly, as a result of extensive studies, a number of new IB viruses could be obtained, which differ from the most commonly used H group of 15 IB (eg IB H-120 and IB H-52) in cross-neutralization tests (or virus neutralization tests) and in challenge experiments. viruses can be considered, ie, inoculation with H virus, the animals in question are not protected against the virus replication in the mucosa of the respiratory system after challenge with one of the different IB viruses.

Nor do humoral antibodies against the H strain of the IB virus appear to be able to neutralize significant amounts of IB virus of said aberrant types. It is of particular significance in practice that these newly found IB viruses in animals with high antibody titres against the H-strain of the IB virus cause clear respiratory symptoms and in production animals already laying down.

Examples of such new viruses are those identified internally by Utrecht 101-79, Limburg 501-79, Limburg 502-79, Limburg 503-79, Utrecht 102-79 and Drente 201-79 and deposited at the Collection d * Institut Pasteur, Paris under nos. 1,111, 1,109, 1,110, 1,112 and 1,113.

These viruses were isolated using the trachea swab method in flocks of laying animals, which have 2 resp. Had been vaccinated 3 times with IB H-120 and IB H-52 vaccine and who had high humoral antibody titres against the Massachusets type of the IB virus at the onset of respiratory symptoms and decreases in egg production and in broilers, which in the second half of the fattening period, after previously performed vaccinations with H-type IB vaccine, showed respiratory symptoms.

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It has now been found that, by attenuation, the virus strains found have largely lost their pathogenicity to SPF chicks, despite the fact that their immunization capacity is still present.

5 For example the virus strain with internal indication IBV

Utrecht 101-79 showed these properties after 85 SPF type I chicken embryo passages and the virus strain IBV Limburg 503-79 showed these properties after 56 SPF type I chicken embryo passages · 10 Surprisingly, it was found in a comparative study with conventional H 120 resp. H-52 vaccine, which does not significantly decrease protection against Massachusetts-type viruses - measured by the amount of virus-neutralizing antibodies - if instead of the H-type 15 vaccine, an H-type vaccine is combined with, for example, the new 101 · Β · isolate Utrecht 101-79 was administered · This study was based on intranasal application of the relevant vaccines · The neutralization indices were determined 4 weeks after administration of the different vaccines or vaccine combinations · 20 Table 1 Immunization potential of different vaccines measured »by the humoral immune response (neutralization index):

Vaccine Test Virus / N.I · IBV IBV

H (B222) Utrecht Limburg _101-79 503-79 25 1. H120 6.5 1.7 0.8 2. IBV Utr · 101-79 (82nd egg passage) 2.1 6.8 N.D.

3 · H120 + IBV Utr · 101-79 6.2 6.5 N.D · 4 · IBV Limburg 503-79 (53rd egg passage) 0.9 N.D · 5.8 5. H120 + IBV Limburg 503-79 6.7 N.D. 5.7 In addition to the immune response after administration of various vaccines resp. vaccine combinations, resistance to virus replication and persistence in and on the mucosa of the trachea was also determined by the virus travel isolation technique, as described in "Specifications for the production and control of avian live 35 virus vaccines" of the Ministry of Agriculture, Fisheries and Food of the United Kingdom, 2nd edition (1977) biz127908687 - 5 -

Table 2 Immune response of different vaccines measured by protection against the possibility of a virus travel isolation from the trachea 7 days after challenge.

Vaccine Challenge virus Result of the virus __rci.s, QU $ .ie- 5 FOOT (Massach. IBV Utrecht 101-79 H120 - + IBV Utrecht 101-79 N.D.

H120 + IBV Utrecht 101-79 - -

It will be clear that the above described properties of the new virus strains make them particularly suitable for the preparation of poultry vaccines t # b # v # more efficient control of infectious bronchitis »especially in regions or countries sailing next to the whole series Ϊ · Β Viruses covered by the H strain prevented the described aberrant serotypes of the virus

The new IBV vaccines according to the invention can be obtained by breeding one or more of the new virus strains by methods known per se for this purpose.

For example, the virus can be bred in fertilized SPP chicken eggs or suitable cell cultures such as SPP chick kidney cell cultures, however care must be taken to ensure that the antigenic properties do not change to such an extent. viruses no longer or less suitable for vaccination purposes # After this, the cultured virus material is collected and purified # Finally one or more stabilizers and possibly antibiotics are added and the mixture freeze-dried #

More specifically, the seed virus in question is inoculated under sterile conditions in the allantoic cavity of 10-11 days of pre-hatched SPP type I chicken eggs. # After incubation at + 37 ° C for 28 to 48 hours, the amniotic allantoic fluid ^ of the still living and the embryos specifically (d # w # z # 24 hours after seed virus inoculation) harvested harvested »purified and freeze-dried after addition of stabilizers and possibly anti-biotics # By this method, single vaccines could be prepared after freeze-drying" >> 104 * ^ EID ^ q per dose of the virus in question contain 35 whereas, for example, thus prepared combined vaccines of a new virus strain and a known H strain have a virus content of 2 x 104 ° BID per dose, for example each of 4 0 virus components ^ 10 7 EID ^ q Per dose # 79 0 8 6 8 7 - 6 -

It will be clear that the present invention also relates to new IBV vaccines which are at least derived from one of the new IB virus strains and to their use.

Live vaccines are preferably used, also in young animals and more particularly broilers. The vaccines can be administered by the so-called eye or nose drop »drinking water or spray method. Vaccination using the new vaccines according to the invention should preferably take place in poultry from 1 day to 18 weeks old.

It will be clear that also combined vaccines "derived from at least one of the new IB virus types and from one or more completely different syndromes causing virus types" such as, for example, Newcastle disease virus "are part of the invention.

The invention is illustrated by the following example.

Example

Preparation IB virus vaccine strain Utrecht 101-79.

A. Culture virus.

20 10 to 11 days pre-hatched SPF type I chicken eggs are inoculated with 103 * 0 to ,04.0 EID5q IBV Utrecht 101-79 seed virus (0.2 ral per egg) in the allantol cavity »20 to 24 hours after the virus inoculation, the eggs are examined for the first time and all non-specific embryos that have died have been removed. After an incubation period of a total of 28 hours at + 37 * 0, the amnion allantol fluid (AAV) is harvested.

B. Treatment of the virus suspension.

After purification of the AAV by centrifugation at 2000 r.p.m. in a cooling centrifuge and / or filtration 5 x 10 ^ E Na-penicillin G and 800 mg streptomycin / liter are added to this AAV.

The virus material is then stabilized by adding a minimum of 3% albumin or mannitol. The stabilized bulk virus material is then frozen to -35 * 0 and stored at this temperature until the further processing stage. Samples of this material are now being tested for virus content and stability using the EID ^ Q (Egg Infections 35 Dose 50%) method. After this information has become known, the virus material is thawed again and filled in freeze-drying bottles.

The virus content (volume) is set so that afterwards 4 5

of the subsequent freeze-drying process at least 10 * EID ^ Q

7908687 - 7 - * 3 s- of the virus is present in the vaccine per dose. The vials are closed by freeze-drying under vacuum. When preparing the multivalent (mixed) vaccine, care should be taken to ensure that the minimum virus content for both or all virus components remains above the minimum mentioned above.

$ 7908687

Claims (20)

1. Infectious poultry bronchitis vaccines, characterized in that they are derived from at least one virus strain of a new serotype of the infectious bronchitis viruses, indicated by means of Utrecht 101-79, Limburg 581-79, Limburg 502-79, Limburg 503-79, Utrecht 102-79 and Drente 201-79 and deposited at the Collection d * Institut Pasteur, Paris respectively under nos. 1,111, 1,109, 1,110, 1,112 and 1,113. Infectious bronchitis vaccines according to claim 1, characterized in that they are derived from at least one virus strain of a new serotype of the infectious bronchitis virus designated Utrecht 101-79 and deposited with the Collection d * Institut Pasteur, Paris, under no. 1.111 « Combined infectious bronchitis vaccines according to claims 1 and 2, characterized in that they are additionally derived from the IBV H-120 and / or the IBV H 52 of the Massachusetts type. Infectious bronchitis vaccines according to claim 3, characterized in that they are additionally derived from the Massachusetts type IBV H 120. Infectious bronchitis vaccines according to claims 1 to 4, characterized in that after lyophilization they contain a virus content of 10 ^ EID ^ q per dose of one of the new virus strains. 6. Infectious bronchitis vaccines according to claims 3-5, characterized in that after lyophilization they contain a total virus content of x 2 x 10 j EID 0 per per ° 3 ^. Infectious bronchitis vaccines according to claims 3-6, characterized in that after lyophilization of each of the viruses they contain 4 components 10 EID per dose. 8. Infectious bronchitis virus strains of a new serotype, indicated by means of Utrecht 101-79, Limburg 501-79, Limburg 502-79, Limburg 503-79, Utrecht 102-79 and Drente 201-79 and deposited with the Collection d Institut Pasteur, Paris, under nos. 1,111, 1,109, 1,110, 1,112 and 1,113. 9. Infectious bronchitis virus strain designated by Utrecht 101-79 and deposited at the Collection d Institut Pasteur, Paris, under No. 1.111. 7908687 - 9 - 10. Infeetienze bronchitis virus stamj denoted by Limburg 501-79f and deposited at the Collection d'Institut Pasteur, Paris, under no. 1,109 * 11. Infectious bronchitis virus strain, indicated by means of Limburg 502-79, and deposited at the Collection d * Institut Pasteur, Paris, under no. 1.110. 12. Infectious bronchitis virus strain, indicated by means of Limburg 503-79, - - '' '- - 13- Infectious bronchitis virus strain ^ designated by Utrecht 102-79, and deposited with the Collection d * Institut Pasteur, Paris, under no. 1.112. 14. Infectious bronchitis virus strain designated by Drente 201-79, and deposited with the Collection d * Institut Pasteur, Paris, under number 1.113. Method for the preparation of infectious bronchitis vaccines for poultry according to claim 1, characterized in that at least one of the virus strains is propagated as a seed virus in fertilized chickens or suitable cell cultures such as SPF chick kidney cell cultures, after which the cultured virus material is collected and purified, followed by addition of stabilizers and optionally antibiotics and freeze drying of the mixture. The method according to claim 15, characterized in that the seed virus is inoculated under sterile conditions in the allophantic cavity of 10-11 days of pre-incubated SPF type I chicken, the amniotic allantoic fluid of living and specifically died embryos after 28 up to 48 hours incubation at + 37 ° C is harvested, purified and, after the addition of stabilizers and optionally antibiotics, freeze-dried. 17. Method according to claims 15 and 16, characterized in that single vaccines are prepared which, after lyophilization, contain 10 EID g per dose of the virus concerned. Method for the preparation of combined vaccines according to claims 3 and 4, characterized in that a virus fluid obtained by propagating at least one virus strain of a new serotype of the infectious bronchitis viruses indicated by Utrecht 101-79, Limburg 501 -79, Limburg 502-79, Limburg 503-79, Utrecht 102-79 and Drente 201-79 and subsequent purification and addition of stabilizers and possibly antibiotics, 79 0: 7 ♦ -w - 10 - is mixed with a virus liquid. by propagation of an IBV H-120 or an IBV H-52, purification and addition of stabilizers and possibly antibiotics, followed by lyophilization. 19. Method for the preparation of combined vaccines according to claim 18, characterized in that vaccines are prepared which contain, after lyophilization, 5. 2 x 10 r EID, -n Per dose and preferably each of the virus components ^ 10 * EID ^ Per dose. 20. Method for preventing infectious bronchitis phenomena in poultry by vaccinating it in a manner known per se with a vaccine according to claims 1-7 * 7908687