RU1789218C - Method for determining immunogenic activity of vaccine against anthrax in cattle - Google Patents

Abstract

Usage: veterinarians, the evaluation of means of prevention, Siberian call. SUMMARY OF THE INVENTION: Before and after the use of test means of specific prophylaxis of anthrax, animals are orally administered with feed an encapsulated suspension of spores of a virulent strain of anthrax in a dose of (0.75, 90-109) spores. The spore suspension is encapsulated in a gelatin capsule and placed in a piece of bread of size (5x5) -

Description

 The invention relates to veterinary medicine, in particular to methods for evaluating the effectiveness of specific prophylaxis of anthrax, animals.

A method is known for determining the immunogenic activity of STI anti-virus vaccine by controlling subcutaneous infection of a virulent strain of anthrax pathogen by immunized guinea pigs (GOST 15991-86) and sheep (GOST 15991-71) and counting the number of surviving animals.

However, the known method, performed on laboratory animals and sheep and empirically extrapolated to cattle, does not allow to reliably evaluate the effectiveness of immunization prophylaxis of anthrax in large animals, since in the control subcutaneous infection of even non-immunized cattle the causative agent of anthrax, get flying the effect is not successful.

Taking into account that the deadly infection of animals with the causative agent of anthrax in natural conditions (on pasture) occurs through food (oral), they test the oral method of infection control in cattle, which would provide a 100% lethal effect in intact (non-immune to Siberian star) animals. In the case of the development of the method, it is used to evaluate the effectiveness of the tested anthrax vaccines in cattle.

An object of the invention is to provide a reliable and reliable method for determining the immunogenic activity of anthrax vaccine in cattle.

To do this, the animals are orally administered an encapsulated suspension of spores of a virulent strain of bos (L

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Siberian ulcer wakeup with food before and after application of the Siberian Eva vaccine and the effect is evaluated by the criterion of PZ (percentage of protection).

A method for evaluating the effectiveness of the tested anti-siberia cattle vaccine in cattle consists of the following steps ;.

1. Immunization of animals with test vaccines against anthrax.

2. Perral infection of control (non-immunized) and immunized animals with the causative agent of anthrax in doses that provide a 100% lethal effect in non-immunized animals.

3. Evaluation of the effectiveness of the test vaccine by the criterion of PZ (percentage of protection).

At the first stage, animals of the same age (young from 3 to 6 months) or adult animals (12 months and older) are selected, the corresponding groups are formed from them (6 animals each).

One group of animals is subcutaneously immunized with a tested anti-sir vaccine unit vaccine (instructions for the use of the drug), and the other group of animals is not immunized, it serves as an emergency. Animals of both groups are kept under identical conditions.

2nd stage. After the appropriate post-vaccination period (15, 21, 30 days, 2, 3, 8, 9, 12 months after immunization, depending on the conditions and objectives of the study), the animals are subjected to control oral infection with a virulent strain of the causative agent of anthrax. The oral infection method includes the following sub-steps:

a) the preparation of infectious material;

b) determination of concentration and viable spores;

c) encapsulation of infectious material and its introduction to animals through the mouth (oral).

To prepare infectious material, a spore culture of a virulent reference strain of anthrax pathogen 4-7 is seeded on ordinary nutrient media (MPA and MPB), grown for 18-24 hours at 3 ° C, 5-37.5 ° C. The grown cultures are identified according to the methods generally accepted in anthrax microbiology (morphologists, motility, capsule formation, spore formation).

For spore formation, the culture grown in an incubator at 3–5.5–37.5 ° C for 18–24 h is subsequently incubated

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until complete spore formation in a thermostat at 32-33 ° C.

After 5-6 days of incubation in an incubator at 32-33 ° C, the initial culture usually turns into a spore (95-98% spore), which is suitable for further use. The degree of spore formation and the concentration of live spores in the culture are determined according to GOST 15991-86 using the optical turbidity standard GISK named after L.A. Tarasovich number 10.

Encapsulation of infectious material is carried out as follows.

The initial spore suspension is diluted with sterile saline to a concentration of 0.5x109 spores / ml. The suspension is filled into 1-2 cm3 sterile pipettes and 0.5 cm3 each is added to the instant gelatin capsules used in medical practice for internal use of drugs manufactured by the domestic industry. Capsules are gelatin cylinders with a capacity of

5 0.6-0.9 cm3, with a diameter of 7-10 mm and a height of 22-30 mm.

To prevent premature dissolution of the capsules in the animal's oral cavity and secondary dispersion of spores

0 anthrax causative agent capsules are enclosed in pieces of bread measuring (5x5) - (10x10) cm.

Pieces of bread containing encapsulated spores in a dose of 0.25x109 spores each,

5 feed animals 3 pieces per head. The total dose of spores in this case is 0.75x109 spores per head, which is optimal, causing 100% lethal outcome of non-immunized

0 (control) animals. Animals are fed bread with encapsulated spore material before they are fed and fed water.

At the 3rd stage, the effectiveness of the tested anti-siren vaccine is assessed by the criterion of PZ (percentage of protection). For this purpose, three groups of cattle of the same age and breed are used. One group of animals (6 animals) is immunized with the standard anti-virus vaccine STI vaccine, the second (6 animals) is immunized with the test vaccine (VGNKI, VNIIVViM, etc.) according to the instructions for the use of biological preparations,

5 the third group of animals (3 heads) are not immunized and it serves as a control.

After a certain period of time (15. 21, 30 days, 2, 3, 6, 9, 12 months after immunization) animals of all troupes (vaccinated and unvaccinated)

they infect an oral reference strain of the causative agent of anthrax in an absolutely lethal dose (, 75 x 109 spores). All animals are monitored throughout the entire period, having studied their clinic / measured body temperature, pulse, respiration. Take into account the number of dead and survivors in each group of animals.

The effectiveness of the test vaccine is evaluated by the criterion of PZ (percentage of protection), using the formula

. .; - :: -.

where is the PZ percentage of protection,

Kp - the number of surviving animals, goals .; . ... / .- :; Ki - the number of animals used in the experiments, goals .;

100 - percentage conversion rate. : ...: .- ....; -; ::::

So, if in the group of animals immunized with STI anti-virus vaccine, 5 out of 6 goals survived in the experiment survived, then substituting the corresponding values in the above formula, we get P3 of 83.4% ..; -:

Example. Absolutely lethal doses of the causative agent of anthrax are determined by oral administration to cattle that are not immunized against anthrax. For this purpose, a spore suspension of the pathogen is poured into gelatin capsules at 0.10; 0.15; 0.20; 0.25; 0.30; 0.45; 0.50 x 109 spores in 1 cm3, capsules are enclosed in pieces of bread and fed animals one, two and three pieces per head. The animals are monitored for 7 days, while taking into account the number of dead animals from each DBZA. The fact of death of animals from anthrax is determined by preparing blood smears from an incision in the ears of dead animals and microscopy them under a light and luminescent microscope.

: The results of experiments to determine the optimal dose of oral infection of cattle with spores of the causative agent of anthrax are presented in Table. 1. From the materials of the table it can be seen that the oral administration of the pathogen spores in one dose, even in large doses (0.90-1.80 billion spores), does not give a 100% lethal effect, while a dose of 0.75x109 a dispute in three doses (in 3 pieces) provides a 100% lethal effect. Thus, an absolutely lethal dose of spore of the causative agent of anthrax for cattle when administered orally

infection is (0.75-0.90) x109 spores per animal.

Example 2. Evaluation of the effectiveness of a single subcutaneous immunization of 5 cattle with standard STI anti-vaccine vaccine by oral infection of animals after 15, 21, 30 days or after 2, 3, 6, 9, 12 months after immunization with spores, the causative agent of anthrax in an absolutely lethal dose of LD, 75x109 spores. Animals are immunized at the same age (3-6 months or 6-12 months) in accordance with existing instructions for

5 to the use of a biological product, the Group of control animals is not immunized, but contain them (immunized and non-immunized) under identical conditions, ensuring the same feeding,

0 drinking, caring, etc.

The results of testing the effectiveness of a single subcutaneous immunization of 3-4-month-old young cattle with anti-siren vaccine

5 STI, orally infected after 15 days, 7 and 13 months after vaccination, are presented in table. 2.

From the table. Figure 2 shows that the standard anti-vaccine STI vaccine, administered to cattle subcutaneously at the age of 3-4 months once, creates intense immunity after 15 days, after 6-7 months in young animals immunized with the STI vaccine

5 is weakened (PZ no more than 67%), in adult animals, once immunized with the STI vaccine, a rather high anti-siren immunity is generated ().

0 Example Z. A comparative assessment of the effectiveness of the proposed anti-sir chain (strains VGNKI, VNIIVViM-55, etc.) and standard vaccine STI. The experiments are carried out as in

5 example 2, but only with the difference that in

experience take three groups of cattle

cattle: control (unvaccinated),

immunized with standard vaccine

STI and vaccinated with the tested anti-virus vaccine (VGNKI, VNI-IVViM-55). Immunization of animals of the same age is carried out in accordance with the instructions for the use of drugs, feeding and care - in identical

5 conditions, oral infection of animals is carried out according to example 2.

Based on the results of the experiment, PP is taken into account and the advantages or disadvantages of the proposed anti-sir anti-sir vaccine are determined. Experiments Show that a single

the use of the STI vaccine provides reliable anti-Siberian immunity in adult cattle for 13; Thousand can hardly be vaccinated ;; which indicates the inappropriateness of double immunization of an adult herd of this species against anthrax. In contrast to adult animals, it is young to cattle that have been immunized once with the STI vaccine and does not acquire sufficient immunity against anthrax (PZ after 7 months after the STI vaccine is used does not exceed 67%), which suggests re-vaccination (revaccination citation) blnrd kyGB this 6zrasta.

The technical and economic advantages of the proposed method compared with the prototype are as follows:

1. The use of oral control infection of cattle in the proposed method provides 100% death of control (unvaccinated) animals, which allows to reliably and / or not extrapolate, as in the well-known bribery, to evaluate the effectiveness of the tested anti-sir vaccine .

2. The proposed method allows us to develop a rational scheme for the use of STI antiviral vaccine in cattle, which is currently used twice without taking into account age, which implies an overuse of labor and material means.

3. The application of the differential scheme of immunization with the STI vaccine, taking into account the results of the control oral infection of the immunized animals in different periods after the vaccine is used, will save a significant amount of labor and material costs, given that currently in the country according to the existing scheme, twice a dozen of STIs are vaccinated million head of cattle, a large proportion of which is an adult population. The transition to a single immunization with the STI vaccine of adult cattle, justified by the results of testing the proposed method, will save 50% of the costly STI vaccine produced by the domestic bio-industry. .....

F6 formula: Method for determining the immunogenic activity of a vaccine against anthrax in cattle, which includes immunizing the animals with the vaccine studied, followed by infection with the spore culture of the pathogen and evaluating the results by the number of animals that are excreted, so that aiming higher30

nor the reliability of the method, cattle are immunized, and infection is carried out by oral spore culture in a dose of 0.75 109 - 0.90 -109 spores, enclosed in a quick-dissolving gelatin capsule, embedded in a piece of bread 5x5-10x10 cm in size.

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