RU2179861C2 - Vaccine against streptococcosis in cattle - Google Patents

Abstract

FIELD: veterinary microbiology. SUBSTANCE: the vaccine proposed additionally contains adjuvant from aluminum hydroxide and as a culture antigen an inactivated cellular suspension of pathogenic streptococcal strains of B and D serogroups which have pathogenicity degree being not lower than DLM 10^-5 and strain K cells of serogroup C in a culture medium at each strain content being 4x104•10⁹ cells/ml. EFFECT: increased efficiency to prevent the highest percentage of animals against death and streptococcosis in cattle, decreased quantity of infected calves and, also, that of abortions, metritis and mastitis cases in adult animals. 1 tbl

Description

 The invention relates to veterinary microbiology, namely to the design of bacterial vaccines for the prevention of streptococcosis in cattle.

 Cattle streptococcosis is everywhere registered in livestock farms and causes great damage. In adult animals, streptococcosis is manifested by abortion, metritis and mastitis. Calves are sick from the first days of birth: umbilical sepsis, joint damage, enteritis, pneumonia. Mortality of young animals can reach 70%. At the same time, high costs are supplemented by a violation of growing technology, reduced productivity, as well as through labor-intensive and expensive veterinary and sanitary measures to eliminate this disease.

 The known vaccine against diplococcal infection contains an antigen only from pathogenic D. Septicus strains, which currently belong to streptococci of serological group D and does not protect against streptococci of serological groups B and C, which are often the etiological factor of the disease not only in calves, but also in cows (Biological and chemotherapeutic veterinary drugs, Moscow, publishing house of agricultural literature, magazines and posters, 1963, pp. 221-226).

 The technical result of the invention is that the vaccine according to the invention protects against streptococci of serological groups B, C and D, as a result of which the number of diseased calves, abortions, metritis and mastitis in adult animals is reduced.

The inventive vaccine additionally contains an adjuvant of aluminum hydroxide, and as a culture antigen, an inactivated suspension of cells of pathogenic strains of streptococci of serogroups B and D having a pathogenicity of at least DLM 10 ^-5 and cells of strain K of serogroup C in a culture medium containing each strain 4 • 10 ⁹ cells / ml.

For the manufacture of experimental series of vaccines against streptococcosis in cattle, ampoules with a lyophilized culture of streptococcus serogroups B, D and C are used. The contents of each ampoule are individually seeded into 3-4 Petri dishes with MPA containing 1% glucose according to the following procedure: 0.1 -0.2 ml of a suspension of culture is applied to the surface of the agar and distributed with a glass spatula on the surface of the agar. The same spatula is carried out on the surface of the agar sequentially another 3 cups. Crops on MPA are incubated for 48 hours at a temperature of 37-37.5 ^o C. At the same time, crops are made in MPB, MPPB and on Saburo medium.

 At the end of the incubation period, streptococcus cultures of serogroups B, D and C are isolated, which are then checked for pathogenicity.

For the preparation of the vaccine, any strains of streptococci of serogroups B and D isolated from farms with a degree of pathogenicity in white mice of at least DLM 10 ^{-5 are used} .

 As streptococcus serogroup C, strain K is used (patent RU 2097422).

To prepare a matrix seedling from vials or flasks, the daily broth culture of each series is subcultured into 2.5-liter cylinders at a temperature of 37-37.5 ^o С MPB with glucose at a rate of 80-100 ml per 10 liters. Crops are cultivated at a temperature of 37-37.5 ^o C for 18-24 hours. By this time, the concentration of the bacterial mass should not be lower than 4 • 10 ⁹ m. To. In 1 ml.

 After ascertaining the purity of growth, streptococcal biomass (each serogroup separately) is poured into 20 liter bottles and commercial formalin is added.

Inactivation of cultures is carried out for 48 hours at a temperature of 16-18 ^o C, subjecting to agitation each bottle up to 3-4 times for 60 minutes.

 After inactivation, samples are taken from each bottle individually to control the completeness of inactivation. For this purpose, inoculations are carried out in test tubes with MPA, MPB, MPPB under vaseline oil.

 After the incubation period has expired and the required results of monitoring the completeness of inactivation have been obtained, a sterile adjuvant 3% aluminum hydroxide solution is added to the bacterial mass.

 The adjuvant is added with continuous agitation of the bacterial mass.

Note: a 3% solution of aluminum hydroxide in physiological saline is sterilized in an autoclave at a temperature of 120 ^o C for 30 minutes.

 Three days after the adjuvant is introduced, a series of vaccines is formed, the pH of the finished vaccine should be in the range of 7.0-7.2.

 The vaccine is packaged in sterile vials, which are sealed with aluminum caps, ensuring tightness of the corking of the contents of the vial.

The resulting vaccine contains pathogenic streptococcal strains of serological groups B and D having a pathogenicity of at least DLM ^10-5 and serological group C (strain K) in the culture medium.

 To determine the quality of the aluminum hydroxide formaldehyde, a sample is taken from various places in the series in the amount of 10 200 ml bottles, of which 5 are used for testing, 5 are stored in the archive for 12 months.

 To determine the appearance, color, presence of impurities, flakes, mold, non-breaking sediment, the vaccine vials are thoroughly shaken and viewed visually in transmitted light. At the same time, the vials are checked for tightness of the corking and the correct labeling.

 The concentration of hydrogen ions is determined by the electrometric method using a potentiometer brand LPU-01 or another device of the same accuracy class. For testing, use 3 bottles of vaccine. The contents of each vial are tested separately. Determination of the pH of the vaccine is carried out according to the instructions attached to the potentiometer. The vaccine should have a pH in the range of 7.0-7.2.

 To carry out a sterility test, 5 vaccine bottles are used. Crops are carried out from each bottle in two test tubes with MPB, MPA, MPPB and Saburo medium. Crops are carried out in a volume of 0.2-0.3 ml of the vaccine. Culture media with crops can withstand for 10 days, while there should be no growth.

 The safety of the vaccine is tested on guinea pigs weighing 300-350 g and on white mice - 16-18 g. Three vials of the vaccine are shaken and 20-30 ml of the drug are taken from each, sterilized, transferred to a sterile vial, and the total sample is used to determine the safety and immunogenic activity.

 To determine the safety, the contents of the vial are thoroughly shaken and five guinea pigs are injected subcutaneously in the back in a volume of 2 ml and ten white mice subcutaneously in the back in a volume of 0.5 ml. The vaccine does not cause disease and death of guinea pigs and white mice during the 10-day observation period.

 To determine the immunogenic activity, the total vaccine sample in the vial was shaken and administered to 60 white mice weighing 16-18 g subcutaneously from the outside of the thigh at a dose of 0.2 ml. 14 days after immunization, 20 vaccinated and 20 control white mice of the same mass were infected subcutaneously in the thigh with a five-fold dose of vaccine streptococcus vaccine strains of serological groups B, D and C separately. The observation period is 10 days.

 A vaccine is considered immunogenic if it protects the disease and the death of at least 18 immunized mice, with the disease of all and the death of at least 19 white mice in control when infected with vaccine strains of streptococci of serogroups B, D and C separately.

 We tested a series of vaccines against streptococcosis in cattle, we tested in a production environment (see table).

 Commercial antibiotics were used to treat calves of the first group. The animals of the second and third groups were administered biologics intramuscularly according to the instructions for the use of the vaccine. The table shows the results of tests of the effectiveness of the developed vaccines for the prevention of streptococcosis in cattle compared with the known vaccine and drug treatment.

 The developed vaccine based on the proposed streptococcal strains of serological groups B, D and C provides the preparation of the preparation, the use of which allows to protect the largest percentage of animals from the death and disease of cattle streptococcosis compared with the known vaccine and drug treatment.

Claims (1)

A vaccine against cattle streptococcosis containing a cultural antigen from pathogenic pathogen strains and a formalin-based inactivator, characterized in that it additionally contains an aluminum hydroxide adjuvant, and an inactivated cell suspension of pathogenic strains of serogroup B and D streptococci having culture antigen having the degree of pathogenicity is not lower than DLM 10 ^-5 and cells of strain K of serogroup C in a culture medium with the content of each strain of 4x10 ⁹ cells / ml.

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