CS275785B6 - Inactivated oily vaccine against rabbit haemorrhagic disease and its method of preparation - Google Patents

Abstract

The solution relates to an inactivated oil vaccine against hemorrhagic disease, suitable for prophylaxis of viral disease of rabbits and a method of its preparation in susceptible rabbits. The vaccine is intended for protective vaccination of susceptible animals against the said disease. The vaccine is liquid and is characterized in that 0.5 tin contains 1,000 to 5,000 hemagglutination units of inactivated rabbit hemorrhagic disease virus of strain BN 0788 CAMP V-363, emulsified in an oil adjuvant. The hemorrhagic disease virus is propagated in susceptible rabbits, infectious virus suspensions are inactivated with formaldehyde, betapropionlactone or 2-bromoethylamine hydrobromide and emulsified with a mixture of adjuvant oils.

Description

The invention relates to an inactivated oil vaccine against viral hemorrhagic disease in rabbits and to a process for its preparation in susceptible rabbits.

Hemorrhagic disease in rabbits is a new, acutely, viral, highly infectious disease that affects rabbits with varying intensity from the age of 5 weeks, regardless of their breeding relationship '. The first mass death of rabbits was recorded by LUI, S.S. - XUE, H.P. - at al .: A new viral disease in rabbits. Veterinary Medicine (Xumu yu Shouyi) 1984, 16 (6), 253255, China Nanjing Univ, in the fall and winter months of 1984 in Jinsau Province, China. The infection has an epizootic character, in 1985 to 1986 it was detected in the Soviet Union and at the beginning of 1987 it was transmitted to some rabbit farms located in Eastern Slovakia. Within two years, the disease spread throughout the USSR and caused significant losses in the affected farms.

LUI, S.S. - XUE, H.P. - et al .: A new viral disease in rabbits, Veterinary Medicine (Xumu yu Shouyi) 1984, 16 (6) 253-255, China Nanjing Univ, described by the inventors as a small unenveloped virus of icosahedral symmetry with a virion size of 18 to 33 nm, whose spherical nucleocapsids with a diameter of 20 nm contain single-stranded RNA. PU, B.Q. a koi .: Micro HA tests for the detection of antibody titers to so-called Haemorrhagic pneumonia in rabbits, Chin. Oourn. Vet. Med., 1985, XX, 10, 16-17 included the virus in the family Picornavirjdae, ŠMÍD, B. et al .: Reports on the results obtained in the detection of viral hemorrhagic pneumonia in rabbits (rabbit plague) 3242-3244 / 88/1 on the basis of detailed electron-optical studies in the genus Calicivirus. The virus agglutinates ovine, avian and erythrocyte group 0 erythrocytes, which are most sensitive to the virus in infected organ suspensions (LUI et al .: A new viral disease in rabbits, Veterinary Medicine (Xumu yu Shouyi) 1984, 16 (6) 253- 255, China Nanjing Univ.

Isolation of the pathogens and its identification paved the way for the study of the epizootiology, pathogenesis and immunogenesis of a new viral infection of rabbits.

Based on experimental infection of susceptible rabbits, LUI et al .: A new viral disease in rabbits, Veterinary Medicine (Xumu yu Shouyi) 1984, 16 (6) 253-255, China Nanjing Univ, determined the incubation period for hemorrhagic disease at 24-72 hours and the duration of the disease from a few hours to one to two days. The clinical signs of the disease are low, rabbits lose their shyness, stop eating, and the only typical symptom of the disease is a bloody, foamy runny nose, which is found in approximately 40% of infected animals shortly before death. The pathologist-anatomical picture after natural and experimental infection was first described by XU et al .: Study of pathology of viral hemorrhagic disease in rabbits, Anim. Husb. and Vet. Med., 1985, 17, 4, 153-155. Hemorrhagic hepatitis, hemorrhagic pulmonary edema, hemorrhagic tracheitis, nephritis, catarrhal gastroenteritis and gallbladder necrosis are detected macroscopically. Histologically, hemorrhages in all organs, membrane glomerulonephritis, hemorrhagic bronchopneumonia, non-purulent encephalitis, and demyelinating pupils and neurons were detected.

Infected rabbits produce hemagglutination-inhibiting antibodies against the hemorrhagic disease virus, which reach moderate to high values in infested animals (PU et al .: Micro HA test for the detection of antibody titers to so-called Haemorrhagic pneumonia in rabbits, Chin.Oourn. Vet. Med., 1985, II, 10, 16-17.

Attempts to actively immunize rabbits against hemorrhagic disease are based on the recognition that overcoming infection induces protection against the disease and practical immunization on the knowledge that inactivated virus structures responsible for inducing antibodies and immunity are native to the disease. The starting material for obtaining viral material for the preparation of vaccines is infectious organs obtained from experimentally infected animals. These are subjected to different purification and inactivation procedures in order to prepare highly effective and harmless vaccines. The organs were the first to prepare a vaccine inactivated with 0.4 weight percent formaldehyde LUI et al .: A new viral disease in rabbits,

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Veterinary Medicine (Xumu yu Shouyj) 1984, 16 (6) 253-255, China Nanjing Univ., Whose good immunization efficacy in laboratory and field conditions was confirmed by 8U et al .: An inactivated vaccine against hemorrhagic in rabbits, Chin.Courn. -VetJied., 1986, 12.2. 50-51. In 1987, an inactivated organ vaccine without adjuvant natures was prepared in the USSR, and in 1988, two inactivated vaccines based on different vaccine strains were prepared in the USSR.

The technology for the preparation of these vaccines is based on the preparation of viral suspensions! from the organs of infected rabbits which are inactivated in an appropriate manner. The immunostimulatory effect of the prepared virus suspensions can be potentiated by the addition of suitable adjuvant components. '

The presented solution consists in the elaboration of the method of infection of rabbits with our isolated strain BN 0788 CAMP V-363 of the heraororagic disease virus and the method of preparation of infectious organ fluids with high content of HA units, conditions of gentle inactivation of virus infectivity by selected inactivated substances efficacy of the prepared vaccine by the addition of adjuvant oils. By adhering to defined technological procedures, a vaccine with high quality parameters will be prepared.

An inactivated oil vaccine against viral haemorrhagic disease of rabbits was prepared, which contains 1,000 to 5,000 haemagglutination units of inactivated BN 0788 virus in 0.5 cm 3. It is prepared from roasts and spleens of experimentally infected rabbits, which are homogenized with buffered saline and supplemented. thiomersal. The resulting 10 to 20% by weight organ suspension, which contains in 1 cm 2 10,000 hemagglutination units, is chemically inactivated with formaldehyde, betapropionlactone or 2 bromethylamine hydrobromide. The inactivated mixture is then emulsified with 20 to 50% by weight of zinc adjuvant oils. The inactivated oil vaccine against viral haemorrhagic disease in rabbits thus prepared has the following immunobiological properties: a) it is harmless to animals of all ages, b) it induces the production of specific haemagglutination antibodies in the titre of 1: 160 to 1: 5120, c) induces early and long-term protection of vaccinated vaccinees. animals against disease and infection.

The invention is further illustrated by the following examples;

Method of preparing a virus suspension

Rabbits without antibodies to the hemorrhagic disease virus are used to produce viral material. Rabbits pre-examined by haemagglutination-inhibition test are infected parenterally, e.g. intra pulmonary at a dose of 1.0 cm ^ 10% by weight of the organ suspension of strain 80 0788 CAMP V-363. In 24 to 72 hours, there is a severe febrile reaction even with numerous deaths. At this time, the liver and spleen are removed from dead and clinically ill rabbits, which are individually checked for virus content by a hemagglutination test. For further processing, organs are used, of which 10% by weight of the organ suspension in physiological solution with the addition of 0.01% by weight of thiomersal contains at least 10,000 hemagglutination units in 1 cc /. The organs are kept frozen until processing. From selected organs with the desired virus content, a 10% by weight organ suspension with physiological buffer solution is prepared, which contains thiomersal in an amount of 0.01% by weight. The suspension is clarified by slow sedimentation and centrifugation at 2-8 ° C. Prior to inactivation, the suspension must contain at least 10 000 haemagglutination units of virus per cm 3.

Methods of virus inactivation

a) Inactivation of the virus by formaldehyde:

To the clarified 10% by weight of the organ suspension of BN 0788 virus, 0.2% by weight of

EN 275 785 3 6 muIde.hydu, can stand is thoroughly mixed and incubated at 30 ° C for 72 hours at 2 to 8 C. ^For over a further 72 hours.

(b) Inactivation of the virus by betapropionlactone:.

0.3% by weight of beta-propionone lactone was added to the clarified 10 .mu.l of organ suspension of BN 0788 virus, the mixture was mixed thoroughly and incubated at 37 DEG C. for 24 hours.

c) Inactivation of virus 2 by iromethylamine hydrobromide:

To the clarified 10% by weight of the organ suspension of 3M 0783 virus, 10% by weight of A 0.1 M solution of 2-bromoethylamine hydrobromide □ pH 7.6 and a mixture of virus and inactivating agent are stored at 25 ° C for 8 h. During inactivation, the virus suspension is mixed repeatedly.

Method of preparing an emulsion

The organ inactivated hemorrhagic disease virus suspension is stored at 2-8 ° C. 80 parts by weight of the organ suspension are emulsified with 20 parts by weight of adjuvant oils which contain light mineral oil and emulsifiers and form an oil-in-water emulsion with the organ suspension of doubles using an IKA-Ultra Thurax T / 45/6 emulsifier. Emulsification is performed at peak speed twice for five minutes with a ten minute break. The prepared emulsion is poured into sterling bottles, which are closed with rubber stoppers.

Example 1

Rabbits previously examined by the HI test are infected intra pulmonarily with a dose of 1.0 cnP 10% by weight, of an organ suspension of strain Bii 0788 CAMP V-363. In 48 hours there is a severe fever. reaction with numerous deaths. At this time, the livers are taken from dead and clinically ill rabbits and checked! for virus content by haemagglutination test. Organs are used for further processing, of which 10% by weight of the organ suspension in physiological saline supplemented with 0,01% by weight of thiomersal contains at least 10 000 haemagglutination units of virus per cm 2. The organs are kept frozen until processing. From the selected organs, a 10% by weight, organ suspension with physiological buffer solution containing thiomersal in an amount of 0.01 is prepared ^; ó mass. The suspension is clarified by slow sedimentation and centrifugation at 4 ° C. 0.2% by weight of formaldehyde is added to the clarified 10 'by weight of organ suspension containing at least 10,000 haemagglutination units of virus in 1 cm, the mixture is mixed thoroughly and incubated at 30 [deg.] C. for 72 h and at 4 [deg.] C. for another 72 h.

The complete vaccine is prepared by mixing 80 parts, parts of organ suspension with 20 parts, parts of adjuvant oils on an IKA-Ultra Thurax T / 45/6. The emulsification takes place at the highest speed, twice for five minutes with a ten-minute break.

Test results

The safety and good immunization efficacy of the rabbit haemorrhagic disease vaccine were confirmed by the results obtained after administration of 250,000 doses (i.e. verification production) of the vaccine to rabbits of different breeds and ages. The use of an oil adjuvant in a vaccine against haemorrhagic disease in rabbits was also approved by the Commission for Veterinary Biopreparations and Drugs on 6 June 1990 on the occasion of the defense of the final report on the vaccine in question.

The high efficacy of the inactivated vaccine is confirmed by the results of immunization and challenge. 45 rabbits without antibodies against the HChK virus were divided into trcoh groups of 15 animals and vaccinated with an inactivated oil vaccine against hemorrhagic disease of rabbits at a dose of 0.5 ml as follows:

Group 1 - vaccinated ood the skin behind the shoulder blade

Group 2 - vaccinated intramuscularly in the thigh

Group 3 - control, unvaccinated rabbits

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Blood samples were taken before vaccination and then at weekly intervals.

The rabbits of the first group produced HI antibodies at titers of 1:80 to 1: 2560 one week after vaccination with a mean titer expressed in binary logarithms of 1002 9.53. At 4 weeks post-vaccination, the mean group titer was 9.33 log2 ·

In rabbits of the second experimental group, antibodies in titers of 1: 160 to 1: 5120 were detected 7 days after vaccination with a mean group titer of 10.83 log2 ·

In rabbits of the third control group, no significant increase in specific antibodies to HChX virus was observed throughout the study period. FIG. 1.

At 4 weeks after vaccination, all rabbits were infected i. pulm. dose of 1 cm with virulent virus BN 0788. Infection results expressed in tab. 1 and FIG. 1 demonstrably confirm the reliable protective efficacy of an inactivated oil vaccine. Rabbits of the 1st and 2nd experimental groups, vaccinated with the inactivated vaccine responded to i. pul. application of virulent virus BN 0788 by further increase of HI antibodies, which reached values of 12 to 14 log ₂ 2 weeks after infection. · In the observation period, no death was recorded in any of the mentioned experimental groups of rabbits. Of the 15 vaccinated rabbits inspected, 11 died, i.e. 73.3%. Four rabbits that survived the infection contained the same HI antibody levels two weeks after infection as the vaccinated and subsequently infected rabbits.

The good protective effect of the vaccine is confirmed by the results of an experiment in which 6 rabbits were exposed to contact infection at the same time as vaccination. Two rabbits infected with i were placed in three cages together. pulm. a dose of 1 ml of virulent BN 0788 virus, and two rabbits vaccinated s.c. dose of 0.5 ml of inactivated oil vaccine. At the 14-day observation period, 4 of the six infected rabbits died, but all vaccinated rabbits exposed to the contact infection survived. The results are expressed in tab. 2.

The efficacy of inactivated oil vaccine, inactivated vaccine without oil adjuvant and Soviet vaccine is shown in Tab. 3. The inactivated oil vaccine showed the best efficacy (the highest values of average antibody titers in all time intervals monitored). The least effective was the Soviet vaccine (formolvaccine with aluminum hydroxides from organ homogenate). . ^a

Claims (3)

Blood samples were collected before vaccination and then at weekly intervals. The first group rabbits were created weekly after HI antibody inoculation at 1: 80-1: 2560 titers with an average titer expressed in log bits of log2 of 9.53. At 4 weeks of vaccination, the mean group titer was 9.33 log2- In the rabbits of the second experimental group, 7 days after vaccination, antibodies were found in the titers of 1: 160 to 1: 5120 with an average group titer of 10.83 log2. no significant increase in specific antibodies over HChX over the entire observation period. FIG. 1. Four weeks after vaccination, all rabbits were infected with i pulse. dose of 1 cm2 virulent BN 0788 virus. 1 and 1 show the reliable protective efficacy of the inactivated oil vaccine. Rabbits of the 1st and 2nd experimental groups vaccinated with the inactivated vaccine responded to i pulse. administration of viral BN 0788 virus to further upward HI antibodies, which reached 12-14 log2 at 2 weeks post-infection. No deaths were noted in either of the rabbit experimental groups during the observation period. Of the 15 non-vaccinated controlled rabbits, 11 died, ie 73.3%. Four weeks after the infection, the four rabbits contained the same HI antibody values as the vaccinated and subsequent infected rabbits. The good protective effect of the vaccine is confirmed by the results of an experiment in which 6 rabbits were exposed to contact infection simultaneously with vaccination. The three cages were co-located for two rabbits infected with the i pulm. 1 ml of virulent BN 0788 virus, apo two rabbits vaccinated with s.c. 0.5 ml of inactivated oil vaccine. In the observation period of 14 days, four of the infected rabbits died 4, but all vaccinated rabbits exposed to the contact infection survived. The results are shown in Tab. 2. The efficacy of an inactivated oil vaccine, an inactivated vaccine without a Soviet vaccine oil adjuvant, is shown in Tab. 3. The inactivated oil vaccine showed the best efficacy (peak mean antibody titres at all time points investigated). The least effective was the Soviet vaccine (formolvacin with hydroxide from organ homogenate). and PATENT CLAIMS What is claimed is: 1. An inactivated viral hemorrhagic rabbit vaccine oil vaccine comprising, in 0.5 cm @ 3, 1 000 to 5 000 hemagglutinating units of chemically inactivated rabbit hemorrhagic virus BN 0788 CAMP V-363 emulsified in an oil adjuvant. 2. A method for producing a vaccine according to claim 1 by homogenizing baking and spleens of infected perceptive rabbits with buffered saline containing 0.01-0.02% by weight. thiomersal and chemical inactivation, characterized in that the organ suspension of susceptible rabbits of rabbits infected with rabbits hemorrhagic virus BN 0783, CAMP V-363 containing 2 to 20% by weight of organ mass and containing at least 10,000 hema glutinating units in 1 cm @ 2 of emulsion with 20 to 50 wt. mixtures of adjuvant oils. 3. A method for producing a vaccine according to claim 2, wherein the adjuvant oil mixture comprises light mineral oil and emulsifiers.