RU2039571C1 - Method for manufacture of associated vaccine used against botulism and pseudomosis in minks - Google Patents

Abstract

FIELD: veterinary. SUBSTANCE: essence of this method resides in preparing botulinic antitoxin vaccine from strain No. 59, type C, having biological activity of about 400 to 600 thousands lethal doses of toxin for white mice (volume: 1.0 cu. cm), adding aluminum potassium sulfate for adsorbing botulinic antigen, treating whole stock with physiological salt solution, and finally admixing inactivated cultures of Pseudomonas aeruginosa of four strains Nos 40 (381-II-06), 48 (1-011), 63 (1677-08) and 77 (Dn 05) in certain proportions at microbial cell concentration of about 8 to 9 bln per 1 cu. cm. EFFECT: disclosed method allows manufacturing sterile biological preparation harmless for animals and possessing high immunogenic activity. 3 tbl

Description

 The invention relates to biotechnology and relates to an associated vaccine used for specific prophylaxis of botulism and pseudomonosis of minks.

 Botulism is an acute toxic infection of mink disease caused by Cl. Boulinum type C leads to 100% death of non-immunized animals, which causes significant economic damage to the country's animal husbandry. The leading place in the fight against botulism is specific prophylaxis using vaccines.

Known vaccine against botulism of minks, which is administered intramuscularly in a volume of 1.0-2.5 cm ³ puppies aged 40-45 days.

Pseudomoniasis is a widespread disease of animals, including fur animals, caused by Ps.aеruginosa. The most malignant occurs in minks, manifests itself enzootically and is accompanied by hemorrhagic inflammation and swelling of the respiratory system. The death of animals from pseudomonosis in the focus of infection reaches 50 percent or more. The causative agent of pseudomonosis has serotypic variability. The prevailing strains of Ps.aeruginosa isolated from minks are 05, 06 and 08 serological variants according to the Hubs classification. Strain 011 of the serological variant is rare. For specific prophylaxis, vaccines based on Ps.aeruginosa strains are used. Vaccines are administered to animals intramuscularly in a volume of 1.0-3.0 cm ³ at the age of 90 days and older.

 The aim of the invention is a method of manufacturing a vaccine and increasing the immunogenic activity of a biological product.

This is achieved by the fact that the associated vaccine against botulism and pseudomonosis of minks is prepared from strain N 59 Cl. botulinum type C with biological activity of 400-600 thousand lethal doses of toxin for white mice before inactivation, from strains Ps.aeruginosa NN 40 (381- P-06), 48 (1-011), 63 (1677-08) and 77 ("Dn" -05) with a concentration of 8-9 billion microbial cells in 1 cm ³ . As auxiliary substances, potassium alum and physiological saline are used.

 The vaccine is prepared in the following ratio of components in about.

Anatoxin vaccine from
strain N 59 Cl botulinum
with biological activity
400-600 thousand lethal doses
toxin for white mice up
inactivation 45-50
Mixture of inactivated
crops Ps aеruginosa from
strain NN 40, 48, 63 and 77
with microbial concentration
cells 8-9 billion in 1 cm ³ 25-30 Potassium alum 10-12
Saline
For the manufacture of the vaccine used:
strain N 59 Cl.botulinum type C; strains Ps.aeruginosa N 40, N 48, N 63, N 77. All strains were deposited in the collection of microorganisms VGNKI veterinary drugs.

 PRI me R 1. Obtaining the associated vaccine against botulism and pseudomonosis of minks is carried out first by separately preparing each antigen, then combine them in a certain proportion with the addition of potassium alum and saline.

To obtain botulinum toxin, either meat-peptone liver broth (Kitt-Tarrozi medium) or Hottinger growth medium are used. Immediately prior to sowing, 0.5-0.6% glucose is added to the growth medium, and then seeded 3-5% by volume of the growth medium is selected with the selected cl. Botulinum culture. After 6-8 day cultivation at 32 ^{° C} ± 0,5 checked microscopically growth culture purity, the resulting culture liquid was filtered and determine the biological activity of the botulinum toxin on white mice.

To inactivate the culture of C. botulinum, formalin (with a formaldehyde content of 37-38%) is introduced at the beginning of 0.4% after 6-7 days another 0.15%. Inactivation of the culture and the conversion of the toxin to toxoid is carried out for 30-35 days at a temperature of (37 ± 0.5) ^о С with daily stirring 2-4 times for 10-15 minutes. After this period, check for sterility by plating on nutrient media and the completeness of inactivation by subcutaneous administration to white mice in a volume of 1.0 cm ³ . A 10% solution of potassium alum in an amount of 10-12% to the volume of the medium is added with constant stirring, slowly cooled to a temperature of (19 ± 1) ^о С and left to precipitate the adsorbed toxoid and inactivated Cl. botulinum culture for 72-96 hours. After clarification of the supernatant, 50% of the supernatant is carefully drained, physiological saline is added to the original volume with constant stirring and left alone until the preparation of the associated vaccine.

 To obtain bacterial antigens of Ps.aeruginosa strains NN40, 48.63 and 77, each strain was grown separately, using Hottinger broth as a growth medium. Inoculated with matte seedlings in the amount of 8-10% of the volume of the medium, stirred for 3-5 minutes and left at rest for 4-6 hours.

Before sowing, vegetable oil is added to the growth medium to prevent foaming. After ^C for 20-24 hours to give 16-18 billion. Microbial cells per 1 cm ³ of said aeration medium term is carried out at a pressure of 30/50 kPa and continuous stirring with a mechanical stirrer at a temperature of (37 ± 0,5). Then stop aeration and stirring, leave for 10-15 hours, at which time there is a decrease in the concentration of microbial cells to 8-9 billion in 1 cm ³ .

Ps.aeruginosa cultures of all strains are combined in equal volumes in one reactor, formalin (formaldehyde content of at least 36%) is added at the rate of 0.3-0.4% to the total volume of the bacterial mass. For the inactivation mixture is maintained at 37 ± 0,5 ^{° C} for 18-24 days with occasional shaking (4-6 times daily for 15-20 min). Check for sterility and harmlessness for laboratory animals (white mice, guinea pigs).

 Before the manufacture of the associated vaccine, the botulinum toxoid vaccine is thoroughly mixed for one day, left at rest for 72-96 hours until the adsorbed botulinum toxoid is completely precipitated, and 50% of the supernatant are carefully drained. Then turn on the mechanical stirrer, with constant stirring add 25-30% of the total mixture of cultures of Ps aeruginosa and bring saline to the original volume of the botulinum toxoid vaccine.

 The resulting associated vaccine has the ratio of the components shown in table 1.

 PRI me R 2. To study the immunogenic activity of the proposed vaccines of example 1, white mice, guinea pigs and minks are immunized. In this case, take the associated vaccine against botulism and pseudomonosis of minks containing, about.

Anatoxin vaccine from
strain N 59 Cl
Botulinum type C with active
500 thousand lethal
white doses of toxin
mice in 1 cm ³ to inc
tivations 47.5
Mixture of inactivated
crops Ps aeruginosa
from strains NN 40, 48, 63
and 77 with a concentration of mic
robotic cells 8.5 billion
in 1 cm ³ 27.5
Alum Potassium Alum 11
Saline The rest is administered to white mice in a volume of 0.05-0.4 cm ³ , guinea pigs 0.4-0.6 cm ³ , minks 0.9-1.1 cm ³ and 21 days after immunization are infected with virulent strains of the corresponding pathogens. Mono vaccines against botulism and pseudomonosis are simultaneously tested.

 The research results are given in table.2.

 Based on the data of Table 2, we can conclude that the proposed associated vaccine has a fairly high immunogenic activity.

 PRI me R 3. The study of the safety of the vaccine. For this, animals are administered multiple volumes of the biological product by various methods. Mono vaccines are being tested in parallel. The research results are given in table.3.

 From the data in Table 3, it can be seen that, with the introduction of multiple doses of the associated vaccine, all animals did not deviate from the physiological norm during the 10-day observation and remained alive.

Claims (1)

METHOD FOR PRODUCING AN ASSOCIATED VACCINE AGAINST BOTULISM AND PSEUDOMONOSIS OF MURK, including the separate preparation of the toxoid vaccine from strain N 59 Cl. botulinum type C with biological activity of 400 600 thousand lethal doses of toxin for white mice in 1 cm ³ with the addition of potassium alum for adsorption of botulinum antigen and treatment with physiological saline and a mixture of inactivated cultures of Ps. aeruginosa from strains N 40, 48, 63 and 77 with a concentration of microbial cells of 8 9 billion in 1 cm ³ mixing them and obtaining the associated vaccine in the following ratio of components, vol. Anatoxin vaccine from strain N 59 Cl. botulinum type C with an activity of 400 600 thousand lethal doses of toxin for white mice in 1 cm ³ before inactivation 45 50
A mixture of inactivated cultures of Ps. aeruginosa from strains N 40, 48, 63 and 77 with a concentration of microbial cells of 8 9 billion in 1 cm ³ 25 30
Potassium Alum 10 12
Saline

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