NL8005083A - Infectious bronchitis vaccines for poultry - derived from novel virus serotype(s) isolated from chickens vaccinated with H-type vaccines - Google Patents

Abstract

Infectious bronchitis virus strains of a novel serotype are indicated by the following notations: Utrecht. 101 (deposited at the Czechoslovak National Collection of Type Cultures as CNCTC A07/80; and at the Pasteur Institute as I.111); Utrecht. 102 (CNCTC A08/80; I. 112); Drente. 201 (CNCTC A09/80; I-113); Limburg. 501 (CNCTC A010/80; I. 109); Limburg. 502 (CNCTC A011/80; I. 110); Brabant. 801 (CNCTC A016/80; I. 132); Limburg. 536 (CNCTC A014/80; I. 134); Overijssel. 728 (CNCTC A015/80; I. 135); and Utrecht. 121 (CNCTC A013/80; I. 133). Live and inactivated vaccines for infectious bronchitis in poultry, derived from these new serotypes, are also claimed. These vaccines may be combined with known infectious bronchitis vaccines derived from IBV-H120 or IBV H52 Massachusetts strain; and may further include vaccines for totally different virus types such as Newcastle disease virus, adeno-virus or reo virus. The new viruses were isolated by the trachae swab method from (a) flocks of laying chickens which had been vaccinated 2 and 3 times resp. with IB H120 and IB H52 vaccine and which showed high humoral antibody titres as to the Massachusetts type of the IB virus at the beginning of the occurrence of respiratory symptoms and/or drops in egg prodn.; or from (b) broilers in the second half of the fattening period which showed respiratory symptoms after vaccination with an IBV vaccine of the H type.

Description

, NED-231211 & - 1 -

Gist-Brocades N.V. in Delft.

The Applicant names Manfred M. Krasselt and Peter Apontoweil as inventors.

Infectious poultry bronchitis vaccines and method for preparing such vaccines.

The invention relates to infectious bronchitis (IB) vaccines for poultry and a 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 of the viruses known so far.

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 epizootics of this disease.

The infectious bronchitis still causes a high mortality especially in young poultry. In addition to mortality and more or less strong respiratory symptoms, damage to the laying device and consequent production decreases result from an IB infection. In addition, IB virus infections can stimulate latent virus or bacterial infections and thus lead to serious economic losses, especially in the broiler sector.

Vaccines derived from inactivated virus and so-called living virus were used to combat 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. (1965) 615).

Because normal healthy chicks are also often killed by vaccination with a live, not or little attenuated virus vaccine, the danger being especially serious for animals less than 2 to 3 weeks old or for 8005083 * * - 2 - chickens shortly before the start or during the leq, there is a clear preference in the art for using dead vaccines or live vaccines, attempting to increase the harmlessness of such vaccines by sensing original IB field virus isolates.

For example, the H strain now widely used worldwide and isolated and attenuated by Bijlenga, Hoekstra and Rispens, is described in Tijdschr. Veterinary. 81:43 10 "Infectious bronchitis in chicks in the Netherlands", (1956), Tijdschr. Veterinary Medicine, 85: 320 (1960), Magazine. Diergeneesk., 85: 279 (1960) and Tijdschr. Veterinary Medicine, 85: 398 (1960).

For such modified vaccines have hitherto been used with viruses that have undergone 25 or more embryo passages to reduce their pathogenicity and spreading potential, such as, for example, the Massachusetts type and more particularly the IBV W48, M41, 82828 or the H52 and H120 strains thereof (which have been passaged 52 and 120 times on chicken eggs, respectively), a Connecticut isolate 20 eg A5968 or the Beaudette IBV (IBV-42). Although the use of vaccines of these modified strains is nowadays quite safe and effective However, under certain circumstances these vaccines appear to be unable to sufficiently prevent outbreaks of infectious bronchitis, as shown, for example, in Avian Diseases Vol. 20, no. 1, p. 42 and 177 and Avian Diseases vol. 19, No. 2, p. 323 and 583.

This shortcoming of the current infectious bronchitis vaccines is largely attributed to the occurrence of antigenic variations of the virus, as is also shown, for example, in 3.0 Archiv für die Gesamte Virusforschung 34, p. 32 (1971) and Cunningham CH; Develop.Biol. Standard, 33, 311 (1976).

Therefore, efforts have been 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 difficulty encountered has been found to be the reduction of immunogenic properties of the respective starting 8005083 '- 3 - 4 * viruses by interaction, as shown, for example, in Am.J. Vet.Res. 36, 4, 524 and 525 (1965) and Avian Diseases 1-2757 (1968).

Therefore, there is still a great need for infectious bronchitis vaccines with effective immunizing properties. It will be understood that the sought improvement of these vaccines is still severely hampered by the changing antigenic, immunogenic and other properties of existing IB viruses after a large number of passages in embryonated chicken eggs, respectively. the occurrence of new serotypes and the lack of sufficiently well applicable serological and immunological test procedures.

For example, reference may be made in this regard to Avian Diseases, 19, 2, 323, and 324 (1975).

Surprisingly, as a result of extensive research, a number of new IB viruses could be obtained which were used in cross-neutralization tests (virus neutralization tests), for example, the method described in American Association of Avian Pathologists, "Isolation and Identifreaction of Avian Pathogens", p. 184 (1975) provided that use is made of 1: 5 diluted antisera - and in challenge experiments with subsequent virus isolation tests, deviating from the most commonly used H-type IB viruses so far (eg IB Hl20 and IB H52) Cd, i.e., inoculation with an H type virus, the animals in question are not protected against the virus replication in the mucosa of the respiratory system after a challenge with aforementioned abnormal new IB viruses.

Nor do humoral antibodies against the H strain 30 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 cause respiratory symptoms in animals with high antibody titres against the H-strain of the IB virus and in production animals already laying down.

Examples of such new viruses are those identified internally by Utrecht.101 (U.101), Utrecht 102 (U.

102), Drente. 201 (D.201), Limburg.501 (L.501), Limburg.502 (L.502), Limburg.503 (L.503), Brabant.801 (B.801), Limburg.

8005083 - 4 - 536 (L.536), Overijssel. 728 (0.728) and Utrecht. 121 (U.121), as deposited at the Czechoslovak National Collection of Type Cultures of the Institute of Hygiene and Epidemiology in Prague under the resp. nos. CNCTC AQ7 / 8Q, CNCTC A08 / 80, CNCTC 5 A09 / 80, CNCTC A010 / 80, CNCTC A011 / 80, CNCTC A012 / 80 ,.

CNCTC A016 / 80, CNCTC A014 / 80, CNCTC A015 / 80 and CNCTC A013 / 80 while the virus strains are indicated by Limburg. 501, Limburg. 502, Utrecht.101, Utrecht.102 and Drente.201 also were deposited under the resp. nos. 1-109, 1-110, 1-111, 1-112 and 1-113 'at the Collection Nationale de Cultures de Microorganis-mes Institut Pasteur, Paris, on November 14, 1979.

These viruses were isolated using the trachea swab method in flocks of laying animals, which have 2 resp. Had been inoculated 3 times with 15 IB H120 and IB H52 vaccine and who had high humoral antibody titres against the Massachusetts 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 prior vaccinations with H120 type IB vaccination, showed respiratory symptoms, with both types of animals at the time of isolation being located in the provinces listed in the internal designations used above.

It has now been found that, by attenuation to SPF-25 chicken embryos, the virus strains found have largely lost their pathogenicity to SPF chicks, despite the fact that their immunization potential remains.

For example, the virus strain with internal designation IBV Utrecht 101 showed these aforementioned properties 50 after 85 SPF type I chicken embryo passages and the virus strain IBV Limburg.503 showed these properties after 56 SPF type I chicken embryo passages.

Surprisingly, in a comparative study with conventional H120 resp. H52 vaccine found that the protection against Massachusetts type IB viruses - measured by the amount of virus-neutralizing antibodies - need not be significantly reduced if instead of the H-type vaccine, an H-type vaccine combined with, for example, 8005083 4 - 5 - image the new IB isolate Utrecht-101 was administered.

This study was based on intranasal application of the relevant vaccines.

For example, the following neutralization indices were determined 4 weeks after administration of the different vaccines, respectively. vaccine combinations.

Table 1 Immunization potential of different vaccines measured by the humoral immune response (neutralization index):

10 Vaccine Test Virus / IBV IBV

N.I. Utrecht. Limburg.

H (B222) 101 503 1. H120 6.5 1.7 0.8 2. IBV u.101 (82nd egg passage) 2.1 6.8 N.D.

15 3. H120 + IBV li.101 6.2 6.5 N.D.

4. IBV L.503 (53rd egg passage) 0.9 N.D. 5.8 5. H120 + IBV L.503. 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 isolation technique described in "Specifications for the production and control of avian live virus vaccines" of the Ministry of Agriculture, Fisheries and Food of the United Kingdom Central Veterinary 25 Laboratory of Biological Products and Standards Department,

New Haw, Weybridge, Surrey KT 153 NB, 2nd Edition (1977) biz. 12.

Cross-neutralization tests in SPF chicken embryos and cross-infection tests on SPF chickens were performed cf.

30 tables below: 8005083 «w - 6 -

Table 2

Cross neutralization tests in SPF chicken embryos: a '* 1 "1 ™ 1 virus isolate against virus type H o.101 0.728 L.536 5 H ^ 7.2 neg. Neg. Neg.

ü.101 neg. 5.3 neg. neg.

No -no c O N.I. (10) 0.728 neg. neg. 5.8 neg.

L.536 neg. neg. neg. > 6.2 10 In this context, the designation "neg." means that no significant positive neutralization index (N.I.),. o 0 i.e. N.I. ^ KT 'are obtained.

Table 3

Cross-infection tests performed on SPF chicks; 15 results of virus isolation tests.

vaccine virus challenge virus "FOOT" ü.101 0.728 L.536 type Massachusetts H neg. + + + 20 120th egg passage U.101 + neg. + + 50th egg passage 25 0: ° 827. + + neg. + 100th ex-passage L.536 + + + neg.

80th egg-30 passage

Under the phrase "neg." in this context, it is understood that none of the SPF eggs injected with tracheal swab material exhibited symptoms which can be traced back to an infection with IB virus.

8005083 * - - 7 - From the results of Tables 2 and 3 (neutralization and cross-infection tests) it appears that the four serotypes of the IB virus used, namely those of the H, U.101, 0.728 and L. 536 type, not only differ substantially in terms of antigenicity according to the research methods used, but also - in view of the results of the infection tests with subsequent virus isolation - show good immunogenic properties with respect to the homologous virus.

Practical studies have shown that flocks of broilers, reproductive animals and laying animals in IQ sera have antibodies against the virus types Utrecht.101, Limburg.536 and Overijssel. 728 occur very frequently.

It will therefore be clear that the new IB virus types are not only significantly different in antigenicity from the H virus hitherto mainly used, but also exhibit significantly different properties from each other.

It will be clear that the properties of the new virus types described above make the virus strains found particularly suitable for the preparation of both inactivated and live poultry vaccines for more effective control of infectious bronchitis, especially in areas or countries where, in addition to IB viruses, the so-called H-type, the described deviating serotypes according to the invention occur.

More specifically, virus strains of the serotypes of the aforementioned new virus strains can be successfully used for the preparation of mixed live and inactivated vaccines derived from one or more H-type virus strains as well as from one or more of the new IB virus strains.

The new IBV vaccines according to the invention can be obtained by propagating, and optionally inactivating, one or more of the new virus strains, according to methods known per se for this purpose, acc. methods known per se.

For example, the virus can be propagated in fertilized SPF chicken eggs or suitable cell cultures such as SPF chick kidney cell cultures, but care must be taken that the antigenic properties do not change to such an extent that the viruses in question are no longer or only less suitable for vaccination purposes. The cultured virus material is then collected and purified. Finally, one or more stabilizers and optionally antibiotics, such as sodium penicillin G, streptomycin or natamycin, may optionally be added and the mixture freeze-dried.

More particularly, the seed virus in question is inoculated under sterile conditions into the allantoic cavity of 10-11 days pre-hatched type I SPF chicken eggs.

After incubation at + 37 ° C for 28 to 34 hours, the amnion allantoic fluid of the then living and the embryos that have died specifically (ie after 24 hours after seed virus inoculation) is harvested, purified and after any addition of stabilizers and / or antibiotics freeze-dried. According to this method, single vaccines could be prepared containing, after freeze-drying, £ 10 'EID ^ 0 per dose of the virus concerned, while, for example, thus prepared combined vaccines of a new virus strain and a known H strain or several new virus strains have a virus content X 2 x 10 ^ EID (-n per dose, preferably of each of the virus components ^ -10% EID5q Per dose.

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

Live vaccines derived from one or more H-type viruses and one or more of the new viruses are preferably used. Preferably live vaccines are derived from the H120 or H52 virus strain and from one or more IB viruses selected from the group consisting of U.101, L.536 and 0.728. The vaccines can also be used in young animals and more particularly broilers. The vaccines can be administered by the so-called eye or nose 35 drip, drinking water or spray method for live vaccines. Vaccination by means of the new live vaccines acc. the invention should preferably take place in poultry from 1 day to 18 weeks of age. Preferably, new inactivated vaccines are administered subcutaneously or intramuscularly to animals at least 8005083-9-4 weeks old.

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

For the preparation of inactivated IBV vaccines acc. for example, the present invention can be started from an amnion allantoic fluid, which, after inactivation acc. the usual methods for this, e.g.

-propiolactone or formalin a suitable carrier is added. Preferably, the virus fluid of a suitable titer is processed into an oil-in-water emulsion vaccine, derived from a mineral or vegetable oil and one or more emulsifiers i.v.v. nonionic surfactants, such as esters and ester ethers, derived from alkene oxide and / or hexahydric alcohols and / or from higher natural fatty acids (C ^ q-^ q). Examples of the latter are 20 mannide monooleate (Span® 80. Arlacel 80®, Arlacel A and (S) polyoxyethylene (20) sorbitan monooleate (eg Tween 80).

The volume ratio between the water phase (virus fluid) and the oil phase can range from 3: 7 to 1: 1 and is preferably about 7:13.

8005083 - 10 -

The invention is illustrated by the following examples, without, however, limiting the subject matter of the invention to these specific embodiments.

Example 1 Preparation IB virus vaccine from strain U.101 A. Culture virus.

10 to 11 days of incubated type I SPF chicken eggs are inoculated with 10 ^ to 10 ^ * ^ EIDj.q U.101 seed virus (0.2 ml per egg) in the allantoic cavity. 20 to 24 hours after the virus inoculation, the eggs are candled for the first time and all non-specific embryos that have died are removed. After an incubation period of a total of 28 hours at + 37 ° C, the amnion allantoic 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 at least 3% wt. albumin and / or mannitol. The stabilized bulk virus material is then frozen to a minimum of -35 ° C and stored at this temperature until the further processing stage. Samples of this material are now being investigated for virus content using the EID ^ q (Egg Infectious Dose 50%) determination method. After this information has become known, the virus material is thawed again and filled in freeze-drying bottles.

The virus content (volume) is hereby set so that at the end of the subsequent freeze-drying process, at least 30 10 ^ * ^ EID ^ g of the virus concerned per dose are still present in the vaccine. The vials are closed under vacuum at the end of the freeze drying. When preparing the multivalent (mixed) vaccine, care should be taken to achieve the minimum virus content for all virus components.

8005083 5 * - 11 - Example 2

Preparation IB virus vaccine of the strain L.536_ A. Breeding virus.

10 to 11 days of incubated type I SPF chicken eggs are inoculated with 10 ^ to 10 ^ EID ^ q L.536 seed virus (0.2 ml per egg) in the allantoic cavity. The eggs are candled for the first time 20 to 24 hours after the virus inoculation and all non-specific embryos that have died are removed. After a total incubation period of 32 hours at + 37 ° C, the AAV is harvested.

B. Treatment of the virus suspension.

After purification of the AAV by centrifugation at 2,000 rpm. in a cooling centrifuge and / or filtration 6 x 10 ^ E sodium penicillin G and 900 mg strep-15 tomycin / liter are added to this AAV.

The virus material is then stabilized by adding about 5% wt. albumin and / or mannitol. For example, bovine albumin can be used as albumin. The stabilized bulk virus material is then frozen to a minimum of 20 -35 ° C and stored at this temperature until the further processing stage. Samples of this material are now being tested for virus content using the EID ^ q (Egg Infectious Dose 50%) method. After this information has become known, the virus material is thawed again and filled into 25 freeze-drying bottles.

The virus content (volume) is hereby * adjusted so that after the subsequent freeze-drying process at least 4 5 10 * EIDj-q of the virus concerned per dose are still present in the vaccine. The vials are closed under vacuum at the end of the freeze drying. When preparing the multivalent (mixed) vaccine, care should be taken to ensure that the minimum virus content for all virus components is achieved.

Example 3

Preparation IB virus, vaccine of the strain O. 723 35 A. Culture virus 10 to 11 days pre-incubated type I SPF chicken eggs are inoculated with 103-104-10 EID. IBV O. 728 seed virus 8005083 - 12 - (0/2 ml per egg) in the allantoic cavity. 20 to 24 hours after the virus inoculation, the eggs are candled for the first time and all non-specific embryos that have died are removed. After a total incubation period of 32 hours at + 37 ° C, the AAV is harvested.

B. Treatment of the virus suspension.

After purification of the AAV by centrifugation at 2,200

r.p.m. in a cooling centrifuge and / or filtration at the AAV

5 8 x 10 E sodium penicillin G and 1100 mg streptomycin / 10 liters added.

The virus material is then stabilized by adding about 7 wt. % albumin and / or mannitol.

The stabilized bulk virus material is then frozen to a minimum of -35 ° C and stored at this temperature until the further processing stage. Samples of this material are now being tested for virus content using the EID ^ q (Egg Infectious 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 hereby set in such a way that at the end of the subsequent freeze-drying process, at least 4 5 10 "EIDCrt of the virus in question are still present in the vaccine. The vials are closed under vacuum at the end of the freeze drying. When preparing the multi-25 (mixed) vaccine, care should be taken to ensure that the minimum virus content for all virus components is achieved.

Example 4

Preparation of combined IB virus vaccine of strains U.101 / L. 536 and 0.728 ___ 50 A. Culture virus 10 to 11 days pre-incubated type I SPF chicken eggs are inoculated with 103 * 0 to 104 * 0 EID5Q IBVU.101, L. 536 and O. 728 seed virus (0.2 ml total per egg) in the allantoic cavity. 20 to 24 hours after virus inoculation, the 35 eggs are candled for the first time and all non-specific embryos that have died are removed. After a total incubation period of 32 hours at + 37 ° C, the AAV is harvested.

B. Treatment of the virus suspension.

8005083 - 13 -

After purification of the AAV by centrifugation at 2,000 rpm. in a cooling centrifuge and / or filtration, 8 x 10 ^ E Na-penicillin G and 1000 mg of streptomycin / liter are added to this AAV. The virus material is then stabilized by adding a minimum of 3 wt. % albumin and / or mannitol.

The stabilized bulk virus material is then frozen to a minimum of -35 ° C and stored at this temperature until the further processing stage.

Samples of this material are now being examined for virus content using the EID ^ q determination method. The virus material is thawed again and filled in freeze-drying flasks. The virus volume is hereby set so that at the end of the subsequent freeze-drying process 4 5 15, at least 10 * EIDjjq of each virus type per dose are still present in the vaccine. The vials are closed under vacuum at the end of the freeze drying.

Example 5

Preparation of inactivated combined IB virus vaccine of the 20 strains H 120, U.101, L.536 and 0.728. __

In an analogous manner as in Example 4A, the virus was grown in SPF eggs and the resulting virus suspension was treated in an analogous manner as in Example 4Bf until the frozen phase, but without addition of antibiotics and stabilizers.

The frozen AAV is thawed and inactivated in a water bath with 0.1% i-propiolactone for 90 minutes at 37 ° C.

The virus suspension is stored overnight at -f-4 ° C. The inactivation is checked by inoculating incubated embryonated SPF chicken eggs with the inactivated virus material and then incubating.

The inactivated AAV is optionally diluted with PBS + 0.3% formalin, depending on the EID, -n of each virus type, determined in the non-inactivated AAV (minimum 10 for U.101.

35 l.536 and 0.728 and a minimum of 10 ^ for H 120). jfëi

3.5% Tween is added to the virus suspension of the four strains.

8005083 Τ '- 14 -

The inactivated virus suspension is mixed with an oil phase in the ratio 6.5 parts oil / 3.5 parts virus liquid and emulsified so that the average particle size of the water phase is about 0.5 h. Emulsification 5 is carried out using an Ultra Turrax homogenizer or by means of dooT * passing the starting mixture through a colloid mill.

The oil phase used has the following composition:

Marcol 5 2j ^ (white paraffinic Esso Oil) 93.5% 10 Arlacel A Arlacel 80 ® or Span 80 ® (mannide monooleate) 6/5%

The oil phase components are autoclaved separately at 110 ° C or the mixture is sterile filtered.

8005083

Claims (19)

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, Utrecht. 102, Drente. 201, Limburg.501, Limburg.502, Limburg.503, Brabant.801, Limburg.536, Overijssel.728 and Utrecht.121 and deposited at Czechoslovak National Collection of Type Cultures of the Institute of Hygiene and Epidemiology in 10 Prague under the resp. numbers CNCTC A 07/80, CNCTC A 08/80, CNCTC A 09/80, CNCTC A 010/80, CNCTC A 011/80, CNCTC A 012/80, CNCTC A 016/80, CNCTC A 014/80, CNCTC A 015/80 and CNCTC A 013 / 80- 2. Infectious bronchitis vaccines acc. 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 and deposited with the Czechoslovak National Collection of Type Cultures of the Institute of Hygiene and Epidemiology in Prague under CNCTC 20 A 07/80 and at the Collection d'Institut Pasteur, Paris under no. 1-111. 3. Infectious bronchitis vaccines acc. claim 1, characterized in that they are derived from at least one virus strain of a new serotype of the infectious bronchial virus designated Limburg.536 and deposited with the Czechoslovak National Collection of Type Cultures of the Institute of Hygiene and Epidemiology in Prague under no. CNCTC A 014/80. 4. Infectious bronchitis vaccines acc. claim 1, characterized in that they are derived from at least one virus strain of a new serotype of the infectious bronchitis virus designated Overijssel.728 and deposited with the Czechoslovak National Collection of Type Cultures of the Institute of Hygiene and Epidemiology in Prague under no. . 35 CNCTC A 015/80. Combined infectious bronchitis vaccines according to claims 1 and 2, characterized in that they are additionally derived from the IBV H 120 or the IBV H 52 of the Massachusetts type. 8005083 "V- - 16 - Infectious bronchitis vaccines according to claim 5, characterized in that they are additionally derived from the Massachusetts type I3V H 120. Live infectious bronchitis vaccines according to claims 1-6, characterized in that after freeze-drying v Δη they contain a virus content of # 10 EID per dose of each of the virus strains. Infectious bronchitis vaccines according to claim 7, characterized in that after lyophilization, they contain a total virus content of 2 2 x 100% EID / g per dose of each of the virus types. Inactivated infectious bronchitis vaccine according to claims 1-6, characterized in that it contains an oil phase, which as essential constituents contains at least a mineral or vegetable oil and one or more suitable emulsifiers i.v.v. non-ionic surfactants, such as esters or ester ethers, derived from alkylene oxide and / or from hexahydric alcohols and / or from higher natural fatty acids (C 1 -5-q). 10. Infectious bronchitis virus strains of a new serotype, indicated by means of Utrecht.101, Utrecht.102, Drente.201, Limburg.501. Limburg. 502, Limburg. 503, Brabant.801, Limburg.536, Overijssel.728 and Utrecht. 121 and deposited with Czechoslovak National Collection 25 of Type Cultures of the Institute of Hygiene and Epidemiology in Prague under resp. numbers CNCTC A 07/80, CNCTC A 08/80, CNCTC A 09/80, CNCTC A 010/80, CNCTC A 011/80, CNCTC A 012/80, CNCTC A 016/80, CNCTC A 014/80, CNCTC A 015/80 and CNCTC A 013/80 '30 11. Infectious bronchitis virus strain, designated by means of Utrecht.101 and deposited with Czechoslovak National Collection of Type Cultures of the Institute of Hygiene and Epidemiology in Prague under no. CNCTC A 07/80 35 and with the Collection d'Institut Pasteur, Paris under no. 1.111. 12. Infectious bronchitis virus strain, indicated by means of Limburg.536 and deposited with Czechoslovak National Collection of Type Cultures of the Institute of 40 Hygiene and Epidemiology in Prague under no. CNCTC A 014/80. 8005083 - 17 - 13. Infectious bronchitis virus strain, designated by means of Overijssel.801 and deposited with Czechoslovak National Collection of Type Cultures of the Institute of Hygiene and Epidemiology in Prague under no. CNCTC A 016/80. 14. 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 chicken eggs or suitable cell cultures such as SPF chick kidney cell cultures, after which the cultured virus material is collected and purified, followed by freeze drying of the mixture, optionally after addition of stabilizers and / or antibiotics. Method according to claim 14, characterized in that the seed virus is inoculated under sterile conditions into the allan-15 oats cavity of 10-31 days pre-incubated type I SPF chicken eggs, the amniotic allantoic fluid of living and specifically died embryos after 28 to Incubation at + 37 ° C for 34 hours is harvested, purified and, after addition of stabilizers, and optionally antibiotics, freeze-dried. 16. A method according to claims 14 and 15, characterized in that single vaccines are prepared which contain, after freeze-drying, 10% EID 50 per dose of the virus concerned. 17. Method for the preparation of combined vaccines according to claims 5 and 6, characterized in that a virus liquid obtained by cultivating at least one virus strain of a new serotype of the infectious bronchitis viruses indicated by means of Utrecht. 101, Limburg. 501, Limburg. 502, Limburg. 503, Utrecht. 102, Drenthe. 30 201, Brabant.801, Limburg.536, Overijssel.728 and Utrecht.121, and subsequent purification and after any addition of stabilizers and / or antibiotics, is mixed with a virus liquid, obtained by cultivating an IBV H-120 or a IBV H-52, purification and optional addition of stabilizers and possibly antibiotics, followed by lyophilization. Process for the preparation of combined vaccines according to claim 17, characterized in that vaccines 4o are prepared, which after lyophilization>. 2 x 10 'EID (- n per dose' 4 o and preferably of each of the virus components q 10 '8005083 - 18 - EID q per dose. A method for the prevention of infectious bronchitis in poultry by vaccinating it in a manner known per se with a vaccine according to claims 1-9.