RU2205021C2 - Vaccine against infectious bursal disease in poultry - Google Patents

Abstract

FIELD: veterinary virology. SUBSTANCE: the suggested vaccine contains virus- bearing material out of avirulent large-platelet strain at biological activity of 5.5-6.5 Lg U D₅₀/ml and stabilizer. Vaccine in question is of high immunogenic activity and harmful. EFFECT: higher efficiency of vaccine. 10 tbl

Description

 The invention relates to the field of veterinary virology and biotechnology and can be used in the manufacture of a vaccine against infectious bursal disease (IBD).

 Infectious bursal disease is a widespread highly contagious viral disease of chickens mainly aged 15-80 days, characterized by damage to the factory bag, kidneys, intramuscular hemorrhages and diarrhea. Over the past 10 years, IBB has become widespread and registered in almost most large poultry farms in Russia.

 The economic damage from IBD, caused by the death of chickens up to 20-70% in the acute course of the disease, a significant culling of poultry, a decrease in productivity, and also due to the occurrence of secondary infections, is very large.

 Currently, it is universally recognized that the only correct and reliable direction for the prevention of this infection is the use of vaccines. For specific prevention of IBD, live and inactivated vaccines are used.

 The advantages of live vaccines include their low cost, high performance, the ability to form a high level of immunity for a longer period.

 Known vaccine against IBD birds, including avirulent genetically homogeneous viral material from the strain "VNIVIP" virus IBD birds. To prevent the disease, a bird is immunized from day old, since the vaccine is harmless to birds of any age [1].

 The disadvantage of this vaccine is that it is intended for the prevention of the disease in successful farms, and in conditions of poor farms, as well as in the presence of a high level of maternal antibodies, the effectiveness of its use is extremely low.

Also known is a vaccine against IBD in birds based on the "BG" strain adapted to SPF chicken embryos with biological activity of 5.25-6.0 lg EID ₅₀ / ml. Chicks are vaccinated from 10-14 days of age twice with an interval of 10-14 days at a dose of 100-1000 EID ₅₀ / ml by the method of feeding. The vaccine is able to overcome maternal antibodies against IBD [2].

 The experience of its application in practice revealed a deficiency in the attenuation of this strain. The vaccine from the strain "BG" negatively affects the overall resistance of the body, a decrease in the bird's immune reactivity to vaccines against other infections, and also has a negative effect on the increase in live weight of the bird. According to Scootar I.G. et al. in farms where the prevention of IBD is carried out using a vaccine from the “BG” strain, severe clinical signs of the disease and death of up to 5% of birds are noted on the 5-6th day after immunization [3].

 In studies of Reece R.L. et al. [4] found that a direct correlation between the ability of the vaccine strain to overcome maternal immunity and cause atrophy of the factory bag, ie the manifestation of the subclinical course of the disease, accompanied by severe immunosuppression of the vaccinated bird.

The closest to the proposed invention in terms of essential features is a vaccine against IBD of birds from the Winterfield 84-DEP strain, the original virus for which was used the Winterfield 2512 strain isolated from a sick bird in the USA in 1965. The Winterfield 84-DEP strain is reproduced both in FEC cell culture and on 10-day-old SPF chicken embryos with a biological activity titer of 6.5-8 lg TCD ₅₀ / ml and 5.00-6.00 lg EID ₅₀ / ml, respectively. Chicks are vaccinated from 7-10 days of age twice with an interval of 10 days by the method of feeding [5].

 The disadvantage of this vaccine is its low immunogenic activity and instability of the main immunobiological properties.

 Haffer K. [6] conducted a comparative study of the basic properties of vaccine strains of the IBD virus from the Winterfield group used to produce the IBD vaccine in various countries. These studies showed that all the Winterfield class strains during their long-term use in production underwent multiple (more than 150) passages under different cultivation conditions, which caused the stability of the main properties of these strains to be lost (immunogenicity, antigenicity, genetic uniformity). As a result, the very possibility of producing vaccines from these strains that meet existing standards is doubtful.

 The aim of the invention is to obtain a highly immunogenic, effective, safe vaccine, as well as reducing funds for vaccine prophylaxis of IBD.

To achieve the goal in the manufacture of the vaccine, the BCF strain, an avirulent large plaque clone adapted to 10-day incubation SPF embryos with a biological activity titer of 5.5-6.5 lg EID ₅₀ / ml, is used as a vaccine strain.

For the prevention of IBD, the bird is immunized subcutaneously from day old at the age of 2.5-2.7 EID ₅₀ / ml.

 Previously, the vaccine strain “BCF” of the IBD virus was cultured in a BGM-70 cell culture and a clone was isolated that forms 4-5 mm diameter plaques in the cell culture [7]. In the future, a systematic adaptation of the isolated clone to SPF chicken embryos was carried out by conducting 50 consecutive passages. The degree of adaptation of the virus to chicken SPF embryos was evaluated by determining the biological activity of the virus-containing material after 5-, 10-, 20-, 30-, 40- and 50 passages (Table 1).

To obtain the vaccine, matte material was prepared from the BCF strain of the IBD virus of birds. The resulting virus-containing liquid was used for mass infection of 10-day-old SPF chicken embryos. Viral material in a volume of 0.2 ml was introduced onto the chorioallantoic membrane (HAO) and infected SPF embryos were incubated at 37.7-38.0 ^° C for 48-125 hours. Fallen embryos were homogenized, frozen three times and thawed with phosphate-buffered saline with a pH of 7.4-7.5 and centrifuged for 30 minutes at 5000 rpm. A virus-containing supernatant with a biological activity of at least 5.5-6.5 lg EID ₅₀ / ml was carefully poured under sterile conditions, and then a stabilizer (skim milk, peptone, lactose, sucrose) was added in a ratio of 1: 1 and subjected to lyophilization. Lyophilized vaccine is stored at a temperature of 4-8 ^o C for 1 year to 1.5 years.

Example 1. Determination of the optimal immunizing dose of the vaccine from the strain "BSC"
Experiments to study the optimal immunizing dose of a vaccine from the BCF strain were performed on 14-day old SPF chickens using the 40th passage virus with 6.0 lg EID ₅₀ / ml virus activity. Ten-fold dilutions of the virus were previously prepared, and 10 SPF chickens were vaccinated with each dilution of the virus. The vaccine was drunk with water.

In the control, 10 unvaccinated SPF chickens were used. Control infection of chickens in all groups was carried out with the virulent strain 52/70-M of the IBD virus at a dose of 100 ID ₅₀ / ml 21 days after vaccination. The experiments were carried out in triplicate. The experimental protocols for testing the immunogenic activity of the vaccine strain "KBK" are given in table.2.

The results of the experiments indicate that vaccination of SPF chickens with the KBK virus at a dose of 2.5 lg EID ₅₀ / ml by the method of feeding provides 100% immunity. However, given that vaccination of chickens with passive immunity requires some “reserve” of the virus, 2.5 lg EID ₅₀ / ml was chosen as the immunizing dose.

Example 2. Determination of the harmlessness of the vaccine from the strain "KBK"
The safety of the vaccine strain "KBK" was tested on SPF-chickens of 14 days of age. For this, 15 chickens were orally injected with a virus at a dose of 3.5 lg EID ₅₀ / ml. After the observation period (15 days), the chickens of the experimental and control groups were killed in order to identify pathological changes characteristic of IBD. In all internal organs and the factory bag, pathological changes were not detected. The safety of the structure of the factory bag was confirmed by histological examination.

Example 3. Immunogenic activity of the vaccine from the strain "KBK" at the level of different passages on SPF-embryos of chickens
To assess the immunogenic activity on SPF chickens of 14 days old, the virus of the 10th, 30th, 40th and 50th passages was used. It was found that the “KBK” strain of all passage levels when used once with water at a dose of 2.0 lg EID ₅₀ / ml provides 100% protection against control of infection with a virulent strain 21 days after vaccination, which indicates the stability of its immunogenic properties (table. 3).

Example 4. The study of the timing of the development and duration of immunity with subcutaneous injection of the strain "KBK"
The production period and duration of post-vaccination immunity was studied in chickens hatched from SPF eggs. For this purpose, chickens were vaccinated once subcutaneously with a BCF strain of IBD virus at the age of 14 days at a dose of 2.5 lg EID ₅₀ / ml. The immune status of the chickens was judged by the results of the challenge with the virulent strain 52/70-M of the IBD virus, as well as by serological studies of blood serum in the RPD and pH 7, 14, 21, 28 and 42 days (observation period) after immunization (table 4).

 It was found that 14 days after vaccination in the RDP, antibodies were detected in the blood serum of 70% of chickens, and in all subsequent periods of the study in 100% of cases. The neutralization index was 2.8 lg after 14 days, reached its maximum value by 28 days after immunization, and remained at that level until the end of the experiment.

 During the control infection with a virulent strain of the IBD virus after 14, 21, 28, 42 days, the safety of the chickens was 100%. There were also no clinical and pathological signs characteristic of infectious bursal disease, while in the control groups in 100% of cases there was a clinical manifestation of the disease and the death of chickens from IBD. The data obtained indicate a high immunogenicity of the BSC virus.

Example 5. Checking the strain "KBK" for reversibility. Verification of the vaccine strain of the IBD virus for reversibility was carried out by 8 consecutive passages on 21-day-old SPF chickens (10 animals per passage). For this, 0.5 ml vaccine strain was administered orally to SPF chickens at a dose of 4.5 lg EID ₅₀ / ml. In the experiment used the virus of the 40th passage with an activity of 5.5 lg EID ₅₀ / ml

 On the 4th day after the introduction of the vaccine strain, half of the chickens were killed and the factory bags were taken, and the rest were monitored for 15 days, after which the material from them was examined histologically, and the sera were checked in the RDP with the IBD virus antigen. The second and subsequent 6 passages were carried out according to the same scheme with the only difference being that the homogenate of the factory bags of chickens of the previous passage at a 1:10 dilution was taken as the virus-containing material. At the same time, the material of each passage was examined histologically. The result of this series of experiments is shown in Table 5.

 Studies have shown that with an 8-fold passivation on SPF chickens, the vaccine strain did not cause a clinical picture and disease pattern in the experimental bird, i.e. the passage of the virus did not increase its virulence.

Example 6. Checking the strain "KBK" for immunosuppressive activity
In order to test for immunosuppressive activity, the vaccine strain “KBK” was administered subcutaneously to the daily SPF chickens at the recommended dose of 2.5 lg EID ₅₀ / ml for immunization. Non-vaccinated chickens and SPF chickens infected with the 52/70-M virulent virus (40 chickens per group) served as a control.

 At the age of 14 days, chickens of the experimental and control groups (20 goals) were vaccinated against Newcastle disease with a vaccine from the La Sota strain according to the instructions for its use.

The vaccine was manufactured on September 17, 2000 at the Kursk Biofactory, series 7, virus titer 10 ¹⁰ EID ₅₀ / ml. Shelf life 12 months.

 Similar experiments using vaccine and virulent strains of the IBD virus were carried out on day old chickens with passive antibodies.

 The results obtained during these studies are presented in table.6.

 From the data given in Table 6, it follows that the average titer of antihemagglutinins in the group where the chickens were injected with the vaccine strain was 7.3 log2, which is slightly lower compared to the performance of intact chickens. However, the difference is not statistically significant. The introduction of a virulent strain to daily SPF chickens caused a significant decrease in the body's immune response to the subsequent administration of the La Sota vaccine. Thus, the titer of antihemagglutinins in RHCA in chickens after recurring IBD was 2.4 log2 at 8.6 log2 in intact chickens.

 Infection of chickens with passive immunity with the virulent strain of IBD, as well as the introduction of a vaccine strain of the virus, did not have a significant immunosuppressive effect and this suggests that passive antibodies to the IBD virus prevent not only the clinical manifestation of the disease, but also protect the bird from the immunosuppressive effect of the IBD virus.

Example 7. Comparative evaluation of the immunogenicity of the vaccine strain "KBK" and vaccines manufactured by the company "TAD"
The next stage of our work was the study of the immunogenicity of this strain in comparison with the vaccine company "TAD" (Germany). In the experiments, we used a vaccine strain of the IBD virus, which passed 50 passages on chicken SPF embryos with a biological activity titer of 5.75 lg EID ₅₀ / ml.

For control infection, the virulent strain 52/70-M of the IBD virus was used at a dose of 100 ID ₅₀ / ml.

 The vaccine of the company "TAD" was used according to the instructions for its use. Two experiments were performed (Table 7).

In the first experiment, SPF chickens in one group were immunized with an attenuated strain of this virus once subcutaneously at a dose of 2.5 lg EID ₅₀ / ml. The chickens in the second group were vaccinated with a foreign vaccine. The control was unvaccinated chickens contained in isolated boxes. Control infection of the experimental and control groups of chickens was carried out at 28 days of age. Chickens were observed for 10 days after challenge. As can be seen from the table. 13, a single subcutaneous immunization with an attenuated BCF strain of the IBD virus, as well as vaccination with the TAD vaccine, ensured 100% safety in the control infection of SPF chickens and prevented the clinical manifestation of the disease. In the control, the incidence of chickens was 100%, and the mortality rate was 13.3%.

Example 8. The effectiveness of the vaccine from the strain "KBK" in the presence of maternal immunity
To study the effect of maternal antibodies on the formation of post-vaccination immunity, an experiment was conducted on chickens aged 14, 21, and 28 days with maternal immunity. One group of chickens was immunized twice at a dose of 2.5 lg EID ₅₀ / ml at the age of 14 and 35 days. Control infection of chickens from all groups was performed with a virulent strain 52/70-M of the IBD virus at a dose of 100 ID ₅₀ / ml 21 days after vaccination. The experiment was carried out in comparison with a commercial vaccine manufactured by TAD. The results of these studies are given in table.8.

 From the table it follows that immunization of chickens of different ages with the vaccine strain "KBK" in the same dose provides a different percentage of protection. The vaccine strain provides the greatest immunological effect (95%) when immunizing chickens at the age of 28 days. The introduction of the same dose of vaccine to chickens aged 14 and 21 days creates immunity in 75 and 80% of the bird, respectively.

 The efficiency of double vaccination of chickens at the age of 14 and 35 days was 100%.

Example 9. The effectiveness of the vaccine from the strain "KBK" depending on the method of administration
Evaluation of the effectiveness of various methods of vaccination against IBD was carried out on SPF chickens aged 5-6 days.

 The chickens of the first group were given the vaccine intranasally, the second - orally, the third were immunized ocularly, the fourth - subcutaneously in the upper third of the neck, and the fifth group was vaccinated intramuscularly. The control was unvaccinated chickens. Immunity against IBD was determined by the level of virus-precipitating antibodies in the blood serum 21 days after immunization, as well as by the resistance of the vaccinated bird to infection with a virulent virus. The data of these experiments are given in table.9.

 The data in the table indicate that with all methods of introducing the virus to SPF chickens, a sufficiently high post-vaccination immunity is formed, which once again confirms the effectiveness of the proposed strain.

 Based on the positive results of experiments to evaluate the immunogenic activity of the vaccine from the strain “KBK” in the laboratory, we tested the drug in the production environment of ZAO BCP Bolshevik. The data of this experiment are given in table. 10.

 Based on the results of serological studies in the RPD and ELISA of blood serum of chickens vaccinated with experimental series of vaccines from the strain "KBK", it should be concluded: the use of the vaccine causes active seroconversion in the vaccinated bird. The effectiveness of the drug can also be judged by the results of the bioassay, where the vaccinated bird had 90-100% resistance to control infection.

 At present, the epizootic situation of Marek's disease and infectious bursal disease in most poultry farms remains difficult due to the circulation of highly pathogenic strains of the causative agents of these infections. The effectiveness of specific preventive measures against BM and IBD largely depends on the level of formation of active immunity at the time of contact of the bird with the field virus. The degree of this risk in dysfunctional farms is quite high due to the high concentration of pathogens of these infections and their stability in the external environment. At the same time, vaccinated chickens form a tense immunity against both infections.

 We conducted a production experiment to study the antigenic compatibility of the vaccine strain "KBK" of the IBD virus and the multi-strain vaccine of Marek's disease on day-old chickens of meat breeds.

 A total of 450 thousand chickens were vaccinated. Vaccination of chickens was carried out by the method of feeding twice at the age of 7 and 17 days (150 thousand animals) by subcutaneous single injection of 150 thousand animals and simultaneously with vaccination against Marek’s disease 150 thousand animals. With the simultaneous method of vaccination, vaccines were diluted in the diluent of the Marek's disease vaccine in one bottle before vaccination.

 The effectiveness of vaccination was judged by the results of studies of blood serum for the presence of specific antibodies to the IBD virus in the RPD and ELISA, the well-being of the IBD farm, the productivity and safety of young animals, and the level of post-vaccination immunity after immunization of chickens with the vaccine from the La Sota strain, and also by the number of detected cases of Marek’s disease during pathological autopsy of birds up to 180 days of age.

 The experiments showed that, regardless of the method of using the vaccine from the strain “KBK”, there were no signs characteristic of infectious bursal disease. In case of double vaccination by the method of drinking, the positive reaction to the RDP in different batches of birds ranged from 80 to 95% at the age of 35 days.

 With a single subcutaneous use of the vaccine, the chickens at the age of one day had positive results of RDP in 100% of cases at the age of 25 days.

 With simultaneous vaccination against Marek's disease and infectious bursal disease, antibodies to the IBD virus in the RDP were detected in 100% of cases at the age of 25 days.

 Antibody titers in ELISA were the same both during mono-vaccination against IBD, and with the simultaneous method of immunization (geometric mean titer in ELISA - 2214, correlation percentage - 25-27).

 The number of cases of Marek’s disease is pathologically registered from 200 to 220 per 100 thousand birds during 180 days of keeping, which is 20-35 cases lower than when vaccinating birds with a vaccine only against Marek’s disease.

 The use of the vaccine against IBD did not adversely affect the level of immunity against Newcastle disease. The titer of antibodies in RHCA to the Newcastle disease virus after a single vaccination after 15 days ranged from 1: 16 to 1:64 and in isolated cases 1: 8 and 1: 128 (with intranasal vaccination). The percentage of group immunity is from 92 to 98.

 The simultaneous method of immunization against IBD and Marek’s disease, in our opinion, is the most promising (in the presence of passive immunity no more than 30% according to our observations), since it provides reliable protection against IBD and reduces the cost of vaccination by at least 2 times. The percentage of poultry safety in the experimental groups for the observation period was 96.7%, the daily gain in body weight for 36 days (before the bird was placed on limited feeding) was 38-39.5 g.

SOURCES OF INFORMATION
1. USSR copyright certificate 1824443; IPC ⁵ C 12 N 7/00, A 61 K 39/12 06/30/93

2. RF patent 2127602, IPC ⁶ A 61 K 39/12, C 12 N 7/006 03/20/99,

 3. Scooter I. G. et al. Prevention of infectious diseases of birds in the Republic of Moldova. // Veterinary medicine. - 1997. - 2. - p. 12-13.

 4. Reece R. L., Gould J. A., Hindmarsh M. Studies on a vaccine against infectious bursal disease / // Australian veterinary journal, 1982, Vol. 59, 1, p. 27-29.

5. RF patent 2127604, IPC ⁶ 6 A 61 K 39/12, C 12 N 7/00, 03.20.99.

 6. Haffer K. Field test studies of the 2512 strain of infectious bursal disease. // Avian Dis, 1982, Vol. 26, 4, p. 847-851.

7. RF patent 2054038, IPC ⁶ C 12 N 7/00, C 12 R 1/92, 02/10/96.

Claims (1)

Vaccine against infectious bursal disease of birds, including virus-containing material from an avirulent strain adapted to 10-day incubation SPF chicken embryos and a stabilizer used in equal proportions, characterized in that the large-plaque strain “BSC” with biological activity is used as a vaccine strain 5.5-6.5 lg EID ₅₀ / ml.

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