RU2276995C2 - Method for preventing infectious bursal hen disease - Google Patents

Abstract

FIELD: veterinary medicine.

SUBSTANCE: method involves vaccinating chickens with vaccine comprising virus-containing material produced from small-plaque CT virus strain of infectious bursal disease occurring in hens. The chickens are vaccinated twice at the age of 14 and 21 days. The vaccine is introduced per os at a dose of 10^3.5 TCD₅₀ given in 10 ml of water.

EFFECT: enhanced effectiveness in creating expressed immunity; increased daily weight increment.

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Description

The invention relates to poultry farming and can be, first of all, used for the prevention of infectious bursal disease of hens in both safe and dysfunctional poultry farms.

Known virus vaccines from the BG strains produced by ARRIAH, D-78 manufactured by Intervet Holland, Bür-706 manufactured by Ron-Merier (France) are virus vaccines prepared from medium strains, and they are ineffective, the immune response when using them insufficient to retain highly contagious field IBD strains.

The emergence of highly virulent strains of the IBD virus, causing outbreaks of the disease under repeated use of live vaccines, served as the basis for the search for new strains.

The problem is that in highly pathogenic virus spread areas, infection of birds occurs before moderately immunogenic vaccine strains that are used can cross the maternal antibody barrier.

Therefore, the developed vaccine virus from the more reactogenic strain "ST" is of particular interest in the prevention of IBD.

The purpose of the invention is a virus vaccine from strain "ST" is effective in vaccinating chickens with a high level of maternal antibodies.

This goal is achieved by a series of sequential measures, the first of which is the determination of the biological activity of the IBD virus at the level of different passages on SPF chicken embryos.

Passaging on SPF embryos of chickens was carried out for up to 10 passages. Infection of SPF embryos on Chao showed that the studied strains, of which 52/70 and 27/94 are reference and epizootic, and the strain ST for the production of the vaccine virus are pathogenic for 10-day-old SPF embryos. 80-100% death of embryos on 3-6 days after infection with their reference and vaccine strain at a 1:10 dilution is characteristic.

The results of studies of the biological activity of IBD virus strains at the level of different passages on chicken SPF embryos are shown in Table 1.

Table 1
Biological activity of the IBB virus at the level of different passages on EC (lg EID ₅₀ / ml) No. Strains Passage number one 2 3 four 5 6 7 8 9 10 one "52/70" 4.52 ± 0.14 5.0 ± 0.20 5.47 ± 0.24 6.00 ± 0.29 6.00 ± 0.31 5.93 ± 0.31 6.07 ± 0.30 6.02 ± 0.23 6.00 ± 0.22 5.97 ± 0.23 2 "ST" 3.98 ± 0.21 4.00 ± 0.19 4.42 ± 0.35 4.49 ± 0.28 5.55 ± 0.27 5.50 ± 0.3 5.50 ± 0.39 5.43 ± 0.37 5.39 ± 0.41 5.5 ± 0.23 3 "27/94" 2.97 ± 0.19 3.00 ± 0.18 3,50 ± 0,23 4.00 ± 0.36 4.50 ± 0.11 5.00 ± 0.19 5.5 ± 0.16 5.40 ± 0.41 5.50 ± 0.16 5.47 ± 0.31

The data presented in the table showed that with an increase in the number of passages of the virus on embryos, its titer increases, which is associated with its adaptation to this system. However, the biological activity of the virus varied depending on the studied strain and the number of passages.

The maximum accumulation of the virus (strain 52/70 and CT) was at the 5th passage, respectively 10 ^6.5 and 10 ^5.5 EID ₅₀ / ml.

In order to determine the sensitivity of cell cultures to the studied strains of the IBD virus, they were passaged in the primary culture of FEC and PEC and in transplantable cells of the VERO and BCM-70 type using media and serums of domestic production.

The starting material for infection of the cell culture was insufficient fluid of the homogenate of the embryos of SPF embryos infected with each of the studied strains.

It was found that the strains (52/70, ST, and 27/94) of the IBD virus in all 5 passages in the culture of FEC and PEC multiplied with a pronounced cytopathic effect and accumulated in high titers. However, they varied significantly in their ability to reproduce in primary transplantable cell cultures.

As a result of 5 consecutive passages on FEC and PEC, strain adaptation was observed. In the culture of VERO and BCM-70, although the titer of the virus increased from passage to passage, it did not reach the level established in the culture of FEC and PEC.

Strain differences also occurred during their reproduction in the culture of FEC and PEC. The highest titers of the virus were detected in the culture of FEC and PEC and amounted to 6.68 and 5.98 TCD ₅₀ / ml, respectively.

Table 2.

The results of a study of the intracellular and extracellular accumulation of the ST virus in the FEC culture with a multiplicity of infection of 0.01 TCD ₅₀ per cell showed that the multiplication of the IBD virus in cells begins 4 hours after infection. The amount of the virus is constantly growing and at 12 o’clock is 5.5 TCD ₅₀ / ml, but no further increase in the cell-bound virus was observed, and its activity remained at approximately the same level, slightly decreasing by 48 hours.

In the culture fluid, the virus was detected 2 hours after the start of its active reproduction in cells; the amount of the virus gradually increased, and after 16 hours its activity was at the level of the intracellular virus.

table 2
The biological activity of IBD virus strains in various cell cultures No. Virus strain Virus titer (lg TCD ₅₀ / ml) FEK PEK Vero In G M - 70 one 2 3 four 5 one 2 3 four 5 one 2 3 four 5 one 2 3 four 5 one "ST" 5.00 ± 0.27 5.18 ± 0.00 6.02 ± 0.22 6.32 ± 0.12 6.68 ± 0.11 3.98 ± 0.23 4.40 ± 0.12 4.96 ± 0.18 5.60 ± 0.18 5.98 ± 0.15 2.00 ± 0.45 2.6 ± 0.08 3.02 ± 0.16 4.00 ± 0.14 4.00 ± 0.12 2.00 ± 0.31 1.98 ± 0.18 2.52 ± 0.11 3.00 ± 0.08 3.98 ± 0.23 2 "52/70" 3.00 ± 0.06 3.00 ± 0.17 3.44 ± 0.12 3.98 ± 0.19 4.04 ± 0.20 2.00 ± 0.18 2.54 ± 0.18 2,50 ± 0,10 3.00 ± 0.25 4.96 ± 0.20 2.06 ± 0.26 2.02 ± 0.10 2.00 ± 0.15 3.00 ± 0.14 3.00 ± 0.19 2,50 ± 0,14 2.42 ± 0.16 2.46 ± 0.10 3.00 ± 0.23 3.00 ± 0.18 3 "27/94" 2.00 ± 0.30 1.95 ± 0.11 2,50 ± 0,13 2.54 ± 0.05 3.00 ± 0.14 2.00 ± 0.30 2,50 ± 0,12 2,50 ± 0,18 3.00 ± 0.09 3.00 ± 0.17 2.00 ± 0.15 2.51 ± 0.10 2.52 ± 0.21 3.00 ± 0.36 3.00 ± 0.20 2.00 ± 0.17 2,50 ± 0,13 2.52 ± 0.11 3.00 ± 0.11 3,50 ± 0,14

However, even on the 4th day, when the cytopathogenic effect of the virus, its content spread to all cells. it turned out to be more in the culture fluid than in the cell suspension.

In some cell cultures, the IBD virus is known to cause plaque formation under a medium containing agarose.

To determine the optimal conditions for obtaining plaques under agar in cultures infected with the IBD virus, Difco agar was used, as well as additional components: aminopeptide and chymopepsin.

The results showed that during infection of chicken fibroblasts with IBD virus, plaques of 0.5-3 mm in size formed.

They usually appeared 3-4 days after infection of the culture.

When studying the IBD virus populations, its heterogeneity was revealed by the size of the plaques. The frequency of distribution of plaques of various diameters was such that approximately 70% were plaques with a diameter of 0.5-1.5 mm and about 30% with a diameter of 3 mm, that is, the viral population had mainly a small plaque characteristic.

In this regard, to determine the correlation between the size of the plaques of the virus formed under the agar coating and its pathogenicity for chickens, cloning variants of the IBD virus, forming large and small plaques under the agar, were cloned.

Plaques with a diameter of 1 ± 0.5 mm were considered small and denoted S ^- . Plaques with a diameter of 2.5 ± 0.5 mm were considered large and denoted S ⁺ .

Six clones of each variant of the virus were studied during 5 passages.

The clone was considered homogeneous if the passivated variant of the virus induced the formation of only large or small plaques.

The results of checking the pathogenicity of variants of the IBD culture virus in comparison with the pathogenic strain “27/94” on sensitive chickens are shown in table 3.

Table 3
Pathogenicity for SPF - chickens of the epizootic strain "27/94" and two variants of the strain "ST" in FEC cultures No. Indicators The percentage of damage to chickens infected with different strains of the virus control "27/94" PC. "ST" S ^- PC. "ST" S ⁺ one Death - 2 - - 2 Clinic - 80 - - 3 Pathanatomy - one hundred - 10 four Pathomorphology of the cloacal bag - one hundred - 60

The table shows that coarse plaque variants, as a rule, cause lesions of the cloacal bag, which are detected when the infected chickens are opened in 10% of cases and about 60% of cases during histological examination. Variants of the IBD virus, forming small plaques, were pathogenic for SPF chickens, and were subsequently used to prepare the vaccine virus against IBD.

Subsequently, the ST vaccine strain passed control for sterility, contamination with mycoplasmas, hemagglutinating and other viruses, determination of genetic uniformity and control for reversibility, as well as the stability of all its properties during prolonged cultivation, storage and transportation.

The reactogenic and immunogenic properties of the ST strain were studied on 14-day-old SPF chickens who were orally injected with the native virus (passage 5 in the FEC culture) at doses of 10 ^4.0 -10 ^5.0 TCD ₅₀ / ml.

14 days after immunization, all chickens of the experimental and control groups were injected with a virulent strain "52/70" at a dose of 100 ID ₅₀ / ml.

The research results showed that the strain "ST" of the IBD virus in doses of 10 ^4.0 -10 ^5.0 did not cause post-vaccination complications in SPF chickens for 14 days.

All chickens vaccinated with the ST strain were resistant to control infection, whereas unvaccinated chickens were ill with the characteristic clinic of the disease and 3 chickens died on the 4th day after the challenge.

At the autopsy, fallen chickens observed hemorrhages in the pectoral muscles.

The cloacal bags were enlarged and many point hemorrhages were observed in the section. The results are shown in table 4.

Table 4
Reactogenic and immunogenic properties of the strain "ST" of the IBD virus No. Group Dose vaccination TCD ₅₀ Number of chickens in gr. Vaccine response Control infection results got sick fell % protection one Experienced 10/4 twenty no - - one hundred 2 Experienced 10/5 twenty no - - one hundred 3 Control - twenty - twenty 3 -

It is known that the passage of the virus in cell culture can lead to a decrease or complete loss of its immunogenic properties. Therefore, to determine the stability of immunogenic properties during prolonged cultivation, the strain "ST" was sequentially passaged in the FEC culture. The virus at the levels of 5-, 10-, 15-, and 20th passage was tested for infectious and immunogenic activity.

The biological and immunogenic activity of the ST strain of various passages was determined in duplicate (n = 2).

Table 5
Biological and immunogenic activity of the ST strain of the virus at the level of different passages in the FEC culture No. Virus activity Passage number M ± T 5 10 fifteen twenty one Biological (lg) (TCD ₅₀ / ml) 5.8 6.05 6.2 6.1 6.04 ± 0.03 2 Immunogenic. (ImD ₅₀ / ml) 5.7 5.7 5,6 5.5 5.6 ± 0.12

The data shown in table 5 show that the strain "ST" of the IBD virus retains biological and immunogenic activity for 20 consecutive passages in the culture of FEC.

Thus, the data obtained indicate that the strain "ST" is areactogenic for SPF chickens, has a pronounced immunogenic activity and is stable when passaged in cell culture.

Based on the foregoing, we carried out work on the manufacture of an experimental series of vaccines from the strain "ST", its control and testing in laboratory and production conditions.

Laboratory tests were carried out in accordance with the work program approved by the director of VNINIP, which fully confirmed the high immunogenicity and harmlessness of the vaccine from strain "ST".

In experimental conditions, we established the safety and high immunogenic activity of the virus vaccine against infectious bursal disease. This served as the basis for testing the vaccine from ST in the production environment at the Ryazan poultry farm in the Ryazan Region (see, for example, Collection of Scientific Works, Vladikavkaz, ANVSh LLC, No. 1 (11), pp. 81-82).

Laboratory studies have ruled out known viral and bacterial infections of birds, as well as the benign quality of feed components included in the diet of broiler chickens.

The farm is preventing Newcastle disease according to the scheme of a successful farm using a vaccine from the LA-SOTA strain.

At the age of one day, chickens are vaccinated against Marek's disease. On the basis of epizootic, clinical, pathological and anatomical data and laboratory studies conducted in the Virology Department of VNIVIP, a diagnosis of infectious bursal disease was made. In this regard, the farm was offered the use of an experimental virus vaccine against IBD.

Prior to vaccination, the serum of the chickens was examined for the presence of passive immunity. Serological parameters of the investigated blood serum from chickens before vaccination are presented in table 6.

Table 6
The results of the study of blood serum in chickens before vaccination according to the RDP and pH Age of chickens (days) Number of samples The number of positive samples Geometric average titer of virus-neutralizing antibodies log 2 RDP % Virus neutralizing antibodies log2 0 one 2 3 four 5 6 7 8 9 10 one 10 10 one hundred one 2 2 2 one 2 5.8 3 10 8 80 2 2 2 one one one 5,0 5 10 5 40 one 3 four one one 3.8 7 10 2 twenty 2 one 2 2 one one one 2.0 10 10 0 0 3 2 one 3 one 1.7 fifteen 10 0 0 four one one one one 1,0 21 10 0 0 four 2 2 2 2 1,2 thirty 10 0 0 0 10 1,0

From the data of table 6 it is seen that up to 7 days of age, chickens have a fairly high level of passive antibodies to the IBD virus.

Virus neutralizing antibodies were detected in their titer of 5.8 log 2. Moreover, in individual chickens the antibody titer was 7.0 log 2.

A decrease in the titer of neutralizing antibodies to 1.7 was observed by the 15th day of their growth, in chickens at 21 days of age, specific antibodies were found in a dilution of 1: 2-1: 16. No antibodies were detected in chickens of 30 days of age.

At the poultry farm, the test of the virus vaccine from the strain "ST" was carried out with the simultaneous use (to compare the effectiveness) of the imported vaccine from the strain "P-2" (Germany).

For preliminary testing, three groups of broilers were created. The experiments were performed on 64381 chicken.

Chickens of the first group in the amount of 22951 animals were vaccinated twice at the 14- and 21-day-old age with the virus vaccine from the strain "ST" (room 3). Chickens of the second group in the amount of 21430 animals were vaccinated twice at the 15- and 25-day-olds with the vaccine produced in Germany (room 2). The third group of 20,000 chickens served as a control (Room 4).

The virus vaccine from the strain "ST" was drunk at a dose of 10 ^3.5 TCD ₅₀ in 10 ml of water from 3-liter glass drinkers. Before vaccination, the drinkers were thoroughly washed.

The P-2 vaccine was administered as directed.

There were no post-vaccination complications among chickens in both groups after vaccination.

The feeding and keeping conditions were the same in all groups of chickens. According to the existing technology, the duration of fattening broiler chickens at the poultry farm was 57 days.

During the growing period, 826 chickens fell in the first group, which amounted to 3.6%, in the second group - 1307 chickens or 6.1%, in the third group 1400 chickens or 7%. The average weight of one head during slaughter in the first group was 1676 g, in the second - 1621 g, and in the control - 1610 g.

The average daily gain in the 1, 2, 3 groups, respectively, was 34.2, 32.1, and 31.3 g.

From the above data it follows that the safety of poultry vaccinated with the domestic vaccine from the ST strain was 3.4% higher compared to the control and 2.5% compared to the safety of chickens vaccinated with the vaccine produced in Germany.

Thus, the production tests of the experimental series of the virus vaccine against infectious bursal disease from the strain "ST" showed its safety and economic efficiency.

Claims (1)

A method for the prevention of infectious bursal disease of hens, comprising vaccinating chickens with a vaccine from the virus-containing material of an avirulent strain of the bursal disease of the chicken, characterized in that the vaccination of chickens is carried out twice at 14- and 21 days of age with a vaccine comprising the virus-containing material from the small-scalar strain "ST" of the bursal virus chicken disease, which is administered orally at a dose of 10 ^3.5 TCD ₅₀ in 10 ml of water.