HU203983B - Process for producing living vaccine against fowlpox - Google Patents

Abstract

The invention relates to a live vaccine against Newcastle disease comprising a virus strain NDV-6/10. The vaccine administered in the form of a spray or aerosol or in the drinking water to chickens at an age of several days-weeks is harmless and ensures a prolonged immunity.

Description

The present invention relates to a live vaccine against avian influenza from strain NDV-6/10.

There is an abundance of literature on the use of avian influenza, its virus and vaccines against the disease, and the way in which they are administered, in D.J. Alexander et al., Newcastle Disease (Kluwer Ácademic Publishers, 1988). In the following, we will highlight information from this work.

Two very low virulence strains of avian influenza virus (NDV) are used worldwide as vaccines to prevent Newcastle disease (NDV): LaSota strain of America and NDV strain B-1 (lentogenic).

The B-1 strain was found in 1948 to induce immunity in non-pathogenic and susceptible chickens. The LaSota strain was isolated in 1946, and researchers agree that it provides better protection than the B-1 strain. Vaccines from both strains are prepared in the same way. For details of this well-known method, reference is made to the summary of Allan, W. H., Lancaster, J. B. and Tóth, B. "The Production and Use of Newcastle Disease Vaccines" (FAO, Rome, 1973).

Vaccines made from these two strains are used in many countries around the world. Because of its low pathogenicity (day-old chickens have a brain index of 0.2 and an average kill time of 120 hours), strain B-1 is usually used for the first immunization of young chickens, usually at 1-2 weeks of age. Namely, if the serum of the animals to be immunized contains antibodies against poultry plague (even eggs from the ovaries), the B-1 strain will not reproduce sufficiently in the body and thus its immunogenic effect will not be satisfactory. Therefore, it is usually used when the level of spiky antibodies decreases.

LaSota strain slightly more pathogenic than B-1 strain (brain index 0.4; average death time in vaccinated embryos 103 hours), but respiratory symptoms were observed after immunization of animals 3 weeks or younger (especially by spray or aerosol) , may show a vaccination reaction in the event of a temporary decrease in weight gain. Especially in flocks infected with mycoplasmosis, its economic impact can be significant. Therefore, a vaccine prepared from the LaSota strain e.g. They are not allowed in England. The preparation of a live vaccine from LaSota strain, namely strain P / 76/5, is described in U.S. Pat. Hungarian patent specification. Clones of the LaSota strain have been selected to reduce vaccine reactions, and "Clon 30" is a commercially available vaccine (manufactured by Intervet, The Netherlands). Foreign and domestic data show slightly fewer vaccine reactions after Clon 30 vaccine, but the data show a large variation per herd.

Vaccines of the B-1 and LaSota strains are most commonly administered in drinking water. Because spiked antibodies inhibit the proliferation of oral virus (B-1 strain is more potent than LaSota), first watering is recommended at 1-3 weeks of age. Vaccines made with these strains can also be immunized by injection into the conjunctiva or the omircum, but it is very labor-intensive. Immunization in the form of spray or aerosol is recommended to reduce the need for work. It is clear that this route provides better protection than after booster vaccination, but it also carries a higher risk of a vaccine reaction. Especially after the LaSota vaccine and generally when the size of the droplets containing the virus is small. The smaller the size of the droplets, the deeper the airways are, which favors reproduction but increases the risk of a vaccination reaction (especially in non-mycoplasmosis-free herds). This is mainly due to the fact that the B-1 and LaSota vaccines are sprayed mainly with larger droplets (> 30 µm), whereas in aerosol form (droplet size 1-10 µm) usually only adult and adult (preferably mycoplasmosis free) immunization is recommended. Moreover, Allan and Borland, two internationally renowned experts on the subject, found the LaSota strain aerosol for chicks to be downright dangerous (1. page 325).

Examining the respiratory pathogenicity of previous vaccines, a new phenomenon has been observed: both LaSota and strain B-1 can be recovered from the disease with rhinitis-like symptoms 8 to 10 days after the vaccination, even after the less dangerous watering method. The virus could only be identified using a monoclonal antibody that recognizes the LaSota virus (Judit Erdei et al., Magy. Áo. Lapja, 1988, 43,267). For this reason, it would be advisable to replace their LaSota and B-1 and their derivative variants with a weaker strain.

A further disadvantage of vaccination with the above strains is that immunization at two to three weeks of age will delay the rate of weight gain for days, which is a major loss in large-scale rearing.

It is an object of the present invention to overcome the above disadvantages by developing a novel live vaccine against avian influenza.

This object is achieved by producing a vaccine using a lentogenic strain NDV, namely strain NDV-6/10.

The present invention therefore relates to a process for the preparation of a live vaccine against avian influenza by inoculating hatched eggs with strain NDV-6/10, killing embryos and obtaining the allantoic fluid after reaching the appropriate virus titer.

Preferably, the hen eggs are raised for about 9 days and, after inoculation with NDV-6/10, for 60 to 110 hours, preferably 72 to 96 hours, at 35 to 40 ° C, preferably 37 ° C. incubate until the appropriate vimit is reached. A suitable virus titer is at least 10 ⁸ EID ₅₀ , determined by titration in an egg embryo and a haemagglutination inhibition assay. Once the appropriate virus titer has been reached, the embryos in the incubated eggs are killed, preferably by cooling, and the allantoic fluid is aspirated and, after testing for bacterial purity, formulated as a vaccine.

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In the selection of the strain NDV6 / 10 used in the method of the invention, it was considered that the virus strain would have the following advantageous properties for aerosol immunization: combination of good conception and low pathogenicity, and in vitro correlation with the latter.

A more detailed examination of the known strain NDV-6 (Hungarian Patent No. 190,620) revealed that it consists of subpopulations with different properties. One of its subpopulations selected by cladding showed a qualitative difference in vitro. behaviors typical of other avirulent strains: after infection of epithelial cultures (kidney, lungs) of chicken4 embryos, not the avirulent strains / giant cells were formed but only cell proliferation. This in vitro hallmark of lower pathogenicity is one aspect that has led to the use of the subpopulation as a live vaccine. However, when tested with watering, it was found to be too low in propagation: some animals were not consistently tuned. At this time, this variant was passaged ten times in growing chickens by isolating the oral virus from the cloaca and then re-infecting it. After adaptation to the intestinal epithelium, the tuning rate increased, but the fine aerosol-induced stress did not increase because no respiratory symptoms were present. The NDV-6/10 strain is thus a subpopulation of the NDV-6 strain, which shows signs of further attenuation (parental embryonic epithelial cell culture) in the parent and other avirulent strains in vitro; due to its increased adaptation to chicken intestinal epithelium, it has a better spread than the parent.

The strain NDV-6/10 used as starting material for the live vaccine produced by the process of the invention under the number NCAIM V (P) 001075 December 1988

We deposited it at the International Collection of Agricultural and Industrial Microorganisms (Budapest).

Advantages of the NDV6 / 10 strain used in the method of the invention are as follows:

1. About 100 EID ₅₀ (50% of infectious dose of embryo) virus does not kill within 5 days inoculated with allantoic cavity 1 day old chickens (at least 10 chickens per test) inoculated with 10 days of chicken embryoL 2.10 ⁷ EID ₅₀ remain healthy and normal evolving.

3. Chickens of all ages, even freshly raised, will be asymptomatic at 100 times the immunization dose after any vaccination (with iris, eyelid, or mucosa).

4. The immunizing dose of NDV-6/10 when used in viral mist (aerosol) is 10 ⁷ · ³ EID _{5 O} / m ³ , which is not less expensive than the old vaccine strains.

5. Extremely heat stable so that the amount of water intended for chickens is not reduced during vaccination (when used in drinking water or fog). But compared to the old vaccines, there is less loss of wine even during virus production, lyophilization and storage.

6. Relatively lower levels of haemagglutination inhibition (HAG) serum have at least the same protection (immunity) as the old vaccines. The advantage of lower serum HAG values is that it is more reliable to distinguish only vaccinated animals (flocks) from those that have been infected with wild boar but have not become infected after vaccination, because in these latter cases the HAG values increase rapidly.

7. Causes moderate respiratory reaction. The risk was also reduced by partially adapting NDV-6/10 to intestinal epithelial cells.

8. With very high specificity immunological reagent (monoclonal antibodies) and highly sensitive immunoassay (ELISA), NDV-6/10 can be distinguished from all avirulent (lentogen) NDV strains known in the literature, including the old vaccine strains (LaSota and B-1). ), and even NDV-6/10 is uniquely recognizable. This device makes it easy to determine whether or not an avirulent water strain isolated from any respiratory condition is the same as the NDV-6/10 vaccine strain.

Unique recognition of a vaccine strain is a basic requirement for a modem vaccine. Is NDV-6/10 the first vaccine against poultry ·? a strain that fulfills this requirement.

The strain NDV-6/10 is based on the above properties for various forms of immunization against avian influenza.

Administration of the vaccine of the invention may be effected by aerosolization, in the form of a virus code, in drinking water, by instillation into the conjunctiva or peritoneum.

The first immunization of the poultry flock can take place at day-to-week weeks, and the long-term protection of juvenile and adult animals can be achieved by repeated immunization. The duration of the protection can be significantly prolonged by repeated immunization and thus the protection against avian influenza can be achieved in the poultry population through the use of NDV-6/10 strain alone.

The live vaccine of the invention is also suitable for use in combination. The NDV-6/10 vaccine can be used to immunize young and young flocks if they are to be immunized with an inactivated or possibly associated vaccine before the laying season. The good booster effect of the inactivated vaccine against avian influenza is provided by the NDV-6/10 vaccine.

The vaccine prepared by the method of the present invention also provides adequate protection when other vaccines (e.g., Marek's disease, Fer55 sting bronchitis, Gumboro's disease) are used in immunized herds, either simultaneously or within a short period of time.

Avian influenza virus has good interferol inducing activity. Thus, strain NDV-6/10 can be used to produce interferon or to produce the same effect, all the more since this strain is completely harmless.

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Immunization experiments

A) Immunization of broilers

The first immunization of NDV-6/10 strain in day-to-week-old animals is particularly recommended as it does not induce a vaccination reaction. Reproduction of a live vaccine strain is a prerequisite for the development of immunity. However, this growth is inhibited by egg-derived mathematical antibodies in the chicks. Especially if the level of mathematical antibodies is high at the time of immunization. The following options are available to reduce this inhibitory effect.

a) If the first immunization is given at 2-3 weeks of age, the inhibitory effect is less pronounced due to the already reduced antibody level. However, as age increases, chickens become more susceptible to natural infection, and if this occurs, a severe outbreak may occur.

b) When the virus is applied to the conjunctiva of the eye or to the nasal mucosa, the growth of the virus at the entrance port is not significantly inhibited by mathematical antibodies. However, this method of immunization is very labor-intensive and time-consuming and is therefore very cumbersome to use in high capacity hatcheries.

c) A significant part of the vaccine virus in the form of large droplets (30-100nm) of spray is also transmitted to the conjunctiva or the mucosa of the ovid, which is very advantageous. However, some of the water particle particles are inhaled by the animals and, depending on the vaccine strain's ability to infect, may cause vaccination reactions. This is not to be feared for the NDV-6/10 strain.

d) The simultaneous injection of 0, 2, 2 ml of an inactivated vaccine into the conjunctiva or nasal mucosa increases the immunity, but the procedure is very labor-intensive.

e) The droplet (> nm) nebulization of the vaccine virus-containing fluid is the most advantageous in terms of triggered immunity, since the aerosol injects the vesicles into the trachea and lungs, in addition to the conjunctiva and the peritoneum. At a young age, vaccine strains used to date can cause severe vaccination reactions. One of the major advantages of NDV-6/10 strain is that a

Before the age of 2-3 weeks (even in the hatchery) it can be used not only as a spray but also as an aerosol.

The above statements are based on extensive experiments conducted by our institute and industry. LaSota (Phylavac, PHYLAXIA) virus was administered in 10 ⁶ · ² and NDV-6/10 virus in 10 ⁶ · ⁵ EIDjq in drinking water for 3 weeks old chickens. Following vaccination, the chickens in the two vaccinated groups survived the 1 million lethal infections in the following percentages:

Phylavac (Control) NDV 6/10 3 weeks later 40% 100% In 6 weeks 18% 93%

In our repeated comparative experiments, we investigated whether the protection afforded by the conjunctiva-only NDV-6/10 strain (50,000 EID ₅₀ ) at daytime was different from the immunization of chickens receiving at the same time 0, 2 ml of inactivated vaccine (Phylapest, PHYLAXIA). In a similar arrangement, this experiment was set up with groups vaccinated with strain NDV-6/10. At four weeks of age, chicks from all groups survived the artificial infection. During artificial infections at 6-7 weeks of age, 10 animals immunized with conjunctiva and immunized with the conjunctiva also received 100% protection.

After verifying the safety and efficacy of NDV-6/10 strain in the form of drinking, dripping and spraying, we began to test the aqueous aerosol. This was done using a MASTERDROP (MD-2500) generator patented by Géza Tamási et al. (Manufactured by Atentszolg. Small Cooperative) and also a patented aerosol stabilizer (DROY'S DROPS) (manufactured by Patent Patent Cooperative). This device emits droplets smaller than 7 nm into the air and floats there for 25-30 minutes. The 10 ⁷ · ³ EIC _5O / m ³ is applied to the inhaled chicks for 30 minutes. Hereinafter, NDV-6/10 aerosol was used at this concentration.

Aerosol of the first experiment, 47 days, free of Newcastle disease antibody being 45 chickens only NDV 6/10 marked VINIS immunized (1ml 1O ^9.08 EID 50 / ml NDV 6/10 VIMS kiporlasztva 500 ml of water), consisting of another 45 chicks and inhaled aerosolized ml containing NDV-6/10 and Bronchovac I. (Phylaxia) virus (10 ⁶ · ⁸⁴ EID50 / ml Bronchovac I. vaccine, sprayed in 500 ml water). Three weeks after vaccination, chickens in the inhaled group of NDV-6/10 only had a serum serum of 1: 27.8, while the inhaled group of both calves had a geometric mean of 1: 33.8. (Detection was performed by a haemagglutination inhibition test based on DJ Alexander, Id.). At day 40 postinfection, only 1 out of 90 animals (99% protection) was killed in the superinfection with viable avian influenza virus, also from the group vaccinated with avian influenza only.

Based on these favorable experiments, large-scale groups were vaccinated at day-to-day age. In our experiments to date on nearly 80,000 broiler chickens, it was found that the serum of immunized chickens containing the mathematical antibody contained 3.4 to 9.4 geometric mean titres against avian influenza at day 30. They injected 98 million to 100% protection against the 1 million lethal viruses injected under the skin. In two groups co-infected with aerosol containing infectious bronchitis strain H, ₂₀ strain vaccine (Intervet, The Netherlands) at the dose recommended by the manufacturer, protection was 100% in one group and 92% in the other. Chicks of the latter group had a 75% resistance rate to artificial transfection 50 days after immunization. The rest

EN 203 983 Β group 50-54 after vaccination. 1 million feline infections at daily doses survived 90-100%.

These experiments clearly demonstrate that NDV-6/10 strain provides good protection for broiler chicks throughout the entire rearing period (weeks 7-8) with a single immunization at day-10 days. This protection is also sufficient when the broilers are simultaneously immunized with a vaccine strain which is harmless to infectious bronchitis day-old chicks. Comparative studies show that aerosol immunization is best, but it also provides good protection in the form of a spray or a conjunctiva, and NDV-6/10 virus delivered to the body by drinking water. At the same time, 0.110

0.2 ml of inactivated vaccine enhances protection, but does not appear to be justified in broilers. It is of particular importance that the animals immunized up to 1 to 10 days of age not only did not show a vaccination reaction but also had an unbroken weight gain. Daily body weight gain compared to the previous day, a

3-4. week 2 did not occur, which is often observed in conventionally immunized flocks during weeks two to three. Body weight at slaughter varied according to circumstances but was always satisfactory and mortality rates were below 5%.

The results are shown in Table 1.

/. spreadsheet

Indicators of immunized broilers

The animals Vaccination mode First 7th 14th 21st 28th 35th 42nd 49th Tak. reached. ELH. % number on the sun because average os you s t t ö in e g g -in The group 170 (fi One day NDV-6/10 aerosol 34.1 105.5 251.5 515.0 891.6 1356.6 1798.5 2205.4 1.78 ² 1 ⁵ ^ 170 (Youth One day NDV-6/10 aerosol 35.4 99.6 268.3 532.3 947.1 1390.8 1882.0 2201.8 1.84 4.70 - 170 (Π) One day NDV-6/10 Aerosol + Phylapest 37.4 106.1 259.5 510.1 985.9 1361.0 1880.6 2155.1 1.82 4.11 72 170 (ARC) Day 19 Phylavac 38.0 110.0 290.0 570.0 900.0 1260.0 1620.0 1938.0 1.97 3.50 170 (V) Day 19 Phylavac 37.4 105.2 256.3 504.5 894.8 1354.0 1848.7 1901.6 1.96 2, 3 - Group B) 4250 One day NDV-6/10 aerosol 44 127.5 302.5 557.6 834.3 1191.3 1514.8 1836.2 2.42 4.9 170 One day NDV-6/10 aerosol 44 122.0 277.2 496.3 880.0 1113.3 1403.9 1811.3 2.55 4.7 170 One day NDV-6/10 aerosol 44 116.6 257.0 519.3 877.5 1207.6 1558.0 1788.7 2.44 4.7 170 One day NDV-6/10 aerosol 44 130.4 297.9 549.3 917.3 1343.0 1603.0 1829.0 2.33 1.7 170 One day NDV-6/10 aerosol 44 133.0 306.4 545.6 918.8 1314.0 1579.1 1905.3 2.72 3.5 170 One day NDV-6/10 aerosol 44 135.0 301.4 533.0 931.3 1300.1 1627.0 1920.9 2.20 3.5 Group C) 4350 Day 17 Clon 30 39.6 110.1 274.3 487.6 761.9 1121.6 1411.4 1720.8 2.29 7.1 170 Day 17 Clon 30 41.3 120.9 256.7 496.0 800.6 1151.3 1563.0 1757.2 2.32 8.8 170 Day 17 Clon 30 41.1 129.0 266.9 494.0 697.0 1051.3 1335.3 1616.9 2.28 8.8 170 Day 17 Clon 30 40.7 127.2 275.3 556.6 868.6 1084.6 1411.3 1613.3 2.41 3.5 170 Day 17 Clon 30 41.0 118.3 244.3 534.0 838.0 1169.3 1594.0 1633.3 2.59 6.5 170 Day 17 Clon 30 40.3 117.3 253.5 372.6 643.0 888.7 1335.3 1505.3 2.38 9.4 Group D) 260 Day 19 Phylavac watering 48.0 119.8 278.0 542.0 848.2 1145.6 1454.6 1677.5 2.71 14.5 253 Day 19 Phylavac watering 46.4 123.3 302.8 545.6 928.6 1199.3 1507.3 1660.4 2.42 4.3 255 Day 19 Phylavac watering 47.5 116.6 306.0 546.1 789.4 1083.2 1485.1 1672.1 2.72 14.9

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B) Immunization of breeding or laying flocks

Our experience in this field of about 90,000 animals can be summarized as follows:

The first immunization was performed as described for the broilers. Because breeding or table eggs are also immunized against Marek's disease at daytime (hatchery), our experimental groups received this vaccine in the hatchery (Rismavac, manufactured by Intervet). The chicks of our two large-scale experimental flocks were also immunized against infectious bronchitis in the hatchery, and received the H ₁₂₀ vaccine strain (Intervet) in the form of a large drop spray by Gloria back spray (manufactured by Vegyépszer). Chicks delivered to the site were immunized with NDV-6/10 aerosol at 2-10 days of age. Your vaccine was administered at the dose recommended by the manufacturer. No vaccination reaction was observed and the mortality rate during the first 2 months of rearing was below 3%.

at day of age, antibody titers were higher than those of broiler chickens of the same age (probably due to improved housing conditions and non-forced rearing), and 100% protection was observed in the superinfection. In one flock (where they were not immunized against infectious bronchitis in the hatchery), chicks were immunized by infecting the H ₁₂₀ vaccine strain (Intervet) at 34-37 days of age. Superinfection at 60 days of age showed 84% protection against avian influenza.

In a further herd, after vaccination against scapular or spray vaccine against Marek's disease and infectious bronchitis, the chickens received a Gumboroi vaccine (Intervet) at 1 month of age in drinking water and then at 5 or 6 weeks of age with strain H ₁₂₀ (Intervet). also immunized them with drinking water beu. At the age of 45-53 days, chickens infected with 1 million deadly doses of avian influenza virus showed 96% protection,

One of the flocks to be maintained was re-immunized with NDV-6/10 in aerosol at about 7 weeks of age. After 3 weeks, the serum antibody levels increased significantly (1: 24 geometric mean titer) and was still 1:12 at 4 months, which is a sign of good immunity.

836 juvenile pullets received only NDV6 / 10 vaccine against Marek's disease and against avian influenza at day age, then at 5 weeks of age they were given the weaker Bronchovac I (Phylaxia) vaccine against infectious bronchitis, followed by the stronger Bronchovac II (Phylaxia) vaccine. At the age of 10 weeks, the geometric mean of the HAG titres of each group ranged from 4.4 to 7.8. The serum liter of poultry increased from 1: 4.4 to 1: 9.2 and from 1: 7.8 to 1: 13.2.

Before the laying season, at 4-5 months of age, we had two options. A third time, the egg producing flock was vaccinated by aerosolization. Depending on the evolution of the titers, aerosol immunization may be performed again.

In the other two breeding flocks, after two day aerosol vaccinations and around 7 weeks of age, pre-lay immunization was carried out with the combined inactivated vaccine (Intervet) against Avian Infectious Bronchitis-Gumboroi.

In summary, aerial immunization with the NDV-6/10 strain following vaccination against Marek's disease and possibly infectious bronchitis in laying flocks can be performed without any vaccination reaction on any of the days following hatching. However, HAG titers will be higher when aerosolized immunization against avian influenza is performed at 7-10 days of age. If there is no risk of epidemics, vaccination should be carried out as described in section 2-3. wait for weeks.

In flocks immunized with the NDV-6/10 vaccine, protection against avian influenza will also be good if the animals receive other vaccines in due course depending on the local immunization technology. The NDV-6/10 aerosol vaccine given at week 7 provides adequate protection until the beginning of the laying season. The protection can then be maintained either by further immunization by similar means, or the herd may receive a (preferably trivalent) inactivated vaccine.

It has also been found during aerosol vaccination that HAG titers are lower than those under optimal conditions under unfavorable housing and feeding conditions. It is therefore advisable to regularly test immunized flocks with the HAG test and, if appropriate, repeat the immunization.

The titers after the NDV-6/10 aerosol vaccine are generally lower than those after the LaSota vaccine. However, cellular immunity is very pronounced. This is due to the fact that -1 million lethal infections caused by virulent poultry infection resulted in the death of only animals with no or only traces of antibodies prior to infection (except for one animal with a titre of 1: 4). However, aerosol-immunized, but due to the long time since immunization, nearly 75% of HAG-negative animals survived the superinfection. Most surviving animals also had a positive serum serum dilution of approximately 1: 2000. Only the eg. The exception was mycoplasmosis-weakened animals.

The vaccine prepared from the avian influenza strain NDV-6/10 according to the present invention has the advantage that it is harmless, does not cause a vaccination reaction, and can be used to immunize poultry of any age. It does not adversely affect weight gain. Its application is simple, it can be administered in drinking water or sprayed to a large number of herds, so the animals do not need to be individually handled. Immunization with other vaccines has not been shown to impair immunity against avian influenza. Thus, any herd can be programmed into an immunization program.

The process of the present invention is illustrated by the following example, but is not intended to be limiting.

Example

Preparation of live vaccine

Nine-day-old embryonated hen eggs were inoculated with virus strain NDV-6/10 deposited with NCAIM V (P) 001075 and incubated for 72-96 hours at 37 ° C. Titer of wine by titration of egg embryo and

EN 203 983 Β haemagglutination inhibition assay. Once a minimum titer of 10 'EID _S0 / ml is reached, the embryos are killed by cooling, the allantoic fluid is sterile aspirated and, after checking for bacterial purity, is filled in sterile ampoules as a vaccine. For details on production and quality control, see Allan W. H. "Lancaster JB, Tóth B .:" Production and Use of Newcastle Disease Vaccines "(FAO, Roma, 1973).

Claims (3)

PATENT CLAIMS A method for producing a live vaccine against avian influenza by inoculating pre-incubated hen eggs with ND virus, culturing to the appropriate virus titer, and collecting allantoic fluid, characterized in that the incubated hen eggs are designated NDV-6/10 (NCAIM V). the Agricultural and Industrial Microorganisms National Inoculate with 5 strains in his collection (Budapest). 2. The method of claim 1, wherein the allantoic fluid is cultured for at least 10 ⁸ hours after culturing at 3540 ° C for 60-110 hours. Collect when 10 EID _S0 / ml virus titers are reached. 3. A process for the preparation of a combination vaccine, wherein the vaccine produced by the method of claim 1 is a vaccine against other poultry diseases, preferably Gumboro's disease, bronchitis, 15 vaccines against Marek's disease - combined.