RU2266753C1 - Method for preventing tuberculosis - Google Patents

Abstract

FIELD: veterinary immunology.

SUBSTANCE: the method deals with immunization in 10-20-d-aged animals with BCG vaccine followed by testing with allergic tuberculin sample and reimmunization at the age of 14-16 mo with BCG vaccine. Then, every 6-12 mo animals should be reimmunized with an antituberculosis cell-free vaccine TBC-2 subcutaneously at the dosage of about 5-7 mg protein/animal till complete recovery at the farm. Testing on tuberculosis should be carried out in 6 mo. The innovation suggested enables to develop permanent protective immunity in animals that helps to increase prophylactic effect by 1.5-2 times.

EFFECT: higher efficiency of prophylaxis.

2 ex, 6 tbl

Description

The invention relates to veterinary immunology, in particular to a method for specific prophylaxis, and can be used for the prevention and rehabilitation of livestock farms for tuberculosis.

A known method for the prevention of tuberculosis of young cattle (RF patent No. 2146533, class A 61 K 39/04, 31/115; V.N. Laskavy, E.D. Lakman, 03.20.2000), which provides for the introduction of calves in age 3-4 ml of tuberculin intramuscularly from 1 to 10 days, and after 30-40 minutes, 4-5 ml of the preparation containing formaldehyde as the main principle and distilled water as target additives are injected in the same way. Both drugs are administered once and intramuscularly.

The disadvantage of this method is that this kind of immunization does not create long-term anti-tuberculosis immunity in calves, and therefore, young people of older ages and cows will not be protected from tuberculosis in unsuccessful tuberculosis farms.

There is also a method for the prevention of animal tuberculosis (USSR patent No. 1790414, class A 61 K 39/04; A. S. Donchenko et al., 23.01.93. Bull. No. 3), which provides for the immunization of animals with BCG vaccine with an immunomodulator (high molecular weight RNA from yeast), then the immunomodulator is reintroduced without BCG vaccine after 13-15 days in the same amount (0.5-50 mg / kg).

The disadvantage of this method is that the use of an immunomodulator for immunization increases not only immunogenic, but also enhances the reactogenic properties of BCG vaccine, which reduces the duration of protective immunity in animals. Therefore, the known method of prevention for a long time does not protect animals from tuberculosis.

There is also known a method for specific prophylaxis of cattle tuberculosis in dysfunctional farms using BCG vaccine (Manual on the use of BCG vaccine in cattle tuberculosis, 02.26.1990), according to which BCG vaccine is vaccinated once calves at the age of 10-20 days. This allows you to reduce the recovery time of farms with less material costs. However, the known method for the prevention of tuberculosis protects animals for only one year.

In a number of households, the course of tuberculosis acquires a long, stationary character. The epizootic process of tuberculosis in such farms becomes uncontrollable and their improvement by the method of systematic allergic studies, as a rule, is not achieved. This was the basis for using the radical method of healing - by completely replacing the dysfunctional population with a healthy one at a time (Prevention and control of infectious diseases common to humans and animals // Collected Sanitary and Veterinary Rules. - M., 1996). The preparation of a dysfunctional farm for the placement of healthy livestock requires the implementation of a large amount of work on the rehabilitation of premises and territories, which is accompanied by high material costs.

The objective of the invention is to increase the effectiveness of the prevention of tuberculosis by creating in animals a permanent (continuous) immunity against tuberculosis.

The method of prevention is carried out by immunization with BCG vaccine in calves under the age of 20 days, by immunization with BCG vaccine in calves from 14-16 months old, then non-responsive animals are additionally immunized with TBP-2 tuberculosis vaccine-free vaccine at a dose of 5-7 mg protein per animal. Subsequent immunization with the TBTs-2 vaccine is carried out every 12 months for 4-6 years (the time of possible reinfection). Cows revaccinated with TBC-2 are tested for an allergic tuberculin test every 6 months.

Tuberculosis cell-free vaccine TBTs-2 is a new type of inanimate cell-free (molecular) vaccine. A method of obtaining a vaccine includes the cultivation of mycobacteria from BCG strain, the allocation of 2 fractions - cytoplasmic and fractions of cell membranes. The cell wall fraction was conjugated to the cellulose matrix in the presence of chromium trichloride, then the cytoplasmic fraction was added. Testing the drug in laboratory animals and young cattle showed that the new type of vaccine has high immunogenicity (patent 2217164, IPC A 61 K 39/04 from 11/27/2003, bull. No. 33).

Example 1. For the experiment, 20 heifers were selected and at the age of 10-20 days, vaccinated with BCG vaccine in the neck skin 1 mg each (group 1). For control, 20 heifers under the age of 20 days were also selected for whom BCG vaccine was not administered (group 2).

Then, 45 days after immunization, the calves of both groups were examined for tuberculosis using a single PPD-tuberculin test, patients are considered among vaccinated animals to thicken the skin fold by 15 mm or more (patent No. 1829929, IPC A 61 B 10/00 from 07.23.93 Bulletin No. 27) and among unvaccinated calves for thickening the skin fold by 3 mm or more (Manual on the use (PPD) of tuberculins for mammals and birds. Approved by the Department of Veterinary Medicine of the Ministry of Agriculture and the Russian Federation dated 02.16.1999).

In order to assess the immune status in the peripheral blood of animals of the experimental groups, the number of immunocompetent cells in absolute content (immunological parameters) was determined. The reaction of spontaneous rosette formation (E-rock) determined the number of T-lymphocytes; the reaction of indirect globulin rosette formation (EA-rock) - killer cells; complementary rosette formation (EAC-rock) - B-lymphocytes; lymphocytes reacting with mycobacterial antigens via rosette formation (ET-rock) with bovine erythrocytes adsorbing tuberculin. The count of leukocytes and leukograms was carried out according to generally accepted methods.

Further, immunological parameters were studied by the discrete-dynamic method, which is automated by a special computer program (application for the grant of patent No. 00669275 of January 21, 2003). Based on the results of these studies, differential prognostic tables have been compiled to evaluate the immune status of animals vaccinated with anti-TB drugs.

The studies established (Table 1) a high activity of immunocompetent cells in 75% of animals 5 months after immunization with BCG vaccine, in 40% of animals after 12 months (Table 2) and in 35% of animals after 14 months (Table 3) .

The results of these studies confirm the need for reimmunization of heifers at the age of 14 months with a BCG vaccine intradermally at a dose of 1 mg per animal.

The immune status in animals after revaccination was also determined using discrete-dynamic analysis. In 70% of animals (Table 4), increased activity of immunocompetent cells was established after 6 months and in 25% of animals 14 months after revaccination.

Subsequent re-immunization of the cows of the experimental group was carried out after 14 months with the acellular vaccine TBC-2. Before reimmunization of the animals, a tuberculin allergic test was examined and it was found that all cows had no skin reaction to PPD-tuberculin. The cows of the experimental group received the TBTs-2 vaccine subcutaneously at a dose of 6 mg of protein per animal.

6 months after the introduction of the TBTs-2 vaccine, the animals were examined with a tuberculin allergy test and the immune status of the cows was evaluated by a discrete-dynamic analysis. No tuberculin allergic reaction was detected in cows. The immune status of cows immunized with the TBTs-2 vaccine is described in Table 5, where 12 of 20 cows (60%) showed increased activity of immunocompetent cells.

Similar studies of cows were conducted 9 months after reimmunization with the TBTs-2 vaccine. No animals responding to PPD-tuberculin were detected; the immune status of cows is shown in the differential prognostic table 6.

Thus, in the experiment, a cattle vaccination scheme was developed, which allows creating permanent protective immunity in animals, thereby increasing the prophylactic effect by 1.5-2 times.

Example 2. The production test of the vaccination scheme for cattle was carried out in the OPH SibMIIS Tauride district of the Omsk region.

On the farm, due to tuberculosis in 1999, the number of cattle was slaughtered. After carrying out sanitary measures, 144 heifers aged 14-16 months from tuberculosis-free farms were placed in one of the prepared rooms. When tested for tuberculosis, the entire group received a negative result twice. In the study, 4 months after delivery, 18 animals that responded to tuberculin were isolated. Diagnostic slaughter of animals was carried out and tuberculosis was established in two pathological studies.

Then, twice the double tuberculin breakdown was examined of the remaining animals, the reactive PPD-tuberculin was separated and the non-reacting animals were inoculated with BCG vaccine intradermally at a dose of 1 mg per animal.

14 months after immunization with BCG vaccine, the cows were examined with a tuberculin allergy test and it was found that all animals had no reaction. Also, a discrete-dynamic analysis of immunological parameters showed a reduced number of relationships in the immune system, which was the basis for the reimmunization of cows with the acellular vaccine TBTs-2. Cows at 6 and 12 months after reimmunization were investigated by an allergic reaction and it was found that all animals did not respond to PPD-tuberculin.

Therefore, reimmunization with the TBTs-2 vaccine protects animals from tuberculosis and allows controlling a herd of cattle for tuberculosis with an allergic reaction 2 times a year (6 months after revaccination). Subsequently, according to the results of discrete-dynamic analysis, revaccination was carried out twice more with an interval of 12 months, which made it possible to maintain the welfare of the herd for more than 3 years (observation period) in the conditions of a cattle-unsuccessful cattle tuberculosis system, to prevent economic damage in the amount of 432 thousand only for one year . rubles.

In the future, born calves from cows of this herd are immunized with BCG vaccine at the age of 10-20 days, then 30-45 days after immunization they are additionally tested for tuberculosis using a single tuberculin test, non-responsive calves are doped, responsive to increase skin fold to 15 mm and more isolate, after feeding surrender to slaughter. Separate groups of sex and age are completed from vaccinated young animals and kept separately. A repeated study of a single tuberculin test of the vaccinated young animals is carried out after 14 months, the responders are handed over for slaughter, the unreacted heifers are formed into a separate herd (heifers before insemination) and BCG is revaccinated. Subsequently, these animals are treated as previously described.

Subsequent vaccination of cows with TBTs-2 is carried out annually for 4-6 years (the time of possible reinfection) after the rehabilitation of the farm (farm) from tuberculosis. A study of cows for tuberculosis is carried out 2 times a year after 6 months. Of the responders, 2-3 cows are selected for the control and diagnostic slaughter.

Table 1 Differential prognostic table for assessing the immune status of heifers 5 months after immunization with BCG vaccine, thousand / μl No. p / p Combination, variable parameter (basis) Intact Vaccinated n Student t-test variable basis n Student t-test variable basis 1 B-lymphocytes (T-killers) twenty 3.46 ⇐ 0.71 ⇐0.65 twenty 2,55 ⇐0.29 ⇐0.46 2 T-killers (white blood cells) 2.92 ⇐1.21 ⇐6.65 3 Antigen-reactive lymphocytes (leukocytes) 2,50 ⇐0.73 ⇐6.65 4 White blood cells (B-lymphocytes) 2.42 ⇐6.20 ⇐1.05 5 B - (T-lymphocytes) 2.29 ⇐0.76 ⇐ 1.01 6 White blood cells (T-killers) 2.27 ⇐6.20 ⇐0.65 7 T-lymphocytes (white blood cells) 4.29 ⇐ 0.80 ⇐6.20 8 T-lymphocytes (neutrophils) 3.63 ⇐0.53 ⇐1.10 nine Lymphocytes (neutrophils) 2.89 ⇐3.26 ⇐1.10 10 Antigenreactive lymphocytes (T-killers) 2.82 ⇐0.28 ⇐0.46 eleven Neutrophils (lymphocytes) 2.72 ⇐1.03 ⇐3.38 12 White blood cells (T-lymphocytes) 2,58 ⇐5.13 ⇐0.64 thirteen B - (antigen-reactive lymphocytes) 2.49 ⇐0.64 ⇐0.42 14 Neutrophils (T lymphocytes) 2.20 ⇐ 0.94 ⇐0.64 The number of immune animals 7 35% fifteen 75%

table 2 Differential prognostic table for assessing the immune status of heifers 12 months after immunization with BCG vaccine, thousand / μl No. p / p Combination, variable parameter (basis) Intact Vaccinated n Student t-test variable basis n Student t-test variable basis 1 Antigenreactive - (B-lymphocytes) twenty 3.31 ⇐0.12 ⇐1.85 twenty 2 White blood cells (B-lymphocytes) 4.26 ⇐6.92 ⇐1.85 3 White blood cells (T-killers) 2.75 ⇐6.23 ⇐0.99 3.08 > = 10.4 > = 2.12 4 T-killers (white blood cells) 3.14 > = 2.22 > = 8.50 The number of immune animals 6 thirty% 8 40%

Table 3 Differential prognostic table for assessing the immune status of heifers 14 months after immunization with BCG vaccine, thousand / μl No. p / p Combination, variable parameter (basis) Intact Vaccinated n Student t-test variable basis n Student t-test variable basis 1 E- (white blood cells) twenty 3.20 ⇐2.02 ⇐7.30 twenty 2 ET- (neutrophils) 2.17 ⇐1.26 ⇐1.55 3 T-killers (T-lymphocytes) 3.30 ⇐1.41 ⇐1.17 4 B-lymphocytes (T-killers) 2.44 ⇐1.06 ⇐1.64 5 B- (T-lymphocytes) 2.23 ⇐1.14 ⇐1.17 The number of immune animals 6 thirty% 7 35%

Table 4 Differential prognostic table for assessing the immune status of heifers and heifers vaccinated with BCG vaccine, thousand / μl 6 months after vaccination 14 months after vaccination No. p / p Combination, variable parameter (basis) n Student t-test variable Basis n Student t-test variable Basis 1 T-lymphocytes (white blood cells) twenty 2.71 ⇐0.90 ⇐6.40 twenty 4,56 ⇐0.79 ⇐ 7.80 2 White blood cells (T-lymphocytes) 4.29 ⇐8.10 ⇐1.30 2.73 ⇐7.93 ⇐0.69 3 Neutrophils (T-killers) 2.55 ⇐ 1.50 ⇐0.61 4 White blood cells (T-killers) 4.29 ⇐8.10 ⇐1.14 2.44 ⇐8.38 ⇐0.61 The number of immune animals 14 70% 5 25%

Table 5 Differential prognostic table for assessing the immune status of cows 6 months after revaccination of TBC-2, thousand / μl No. p / p Combination, variable parameter (basis) n Student t-test Variable Basis 1 B - (T-lymphocytes) twenty 5.00 ⇐0.42 ⇐0.36 2 T-killers (white blood cells) 4,50 ⇐1.07 ⇐ 4.90 3 Antigenreactive (B-lymphocytes) 4.45 ⇐0.10 ⇐0.44 4 White blood cells (T-killers) 4.11 ⇐ 4.87 ⇐1.14 5 Antigenreactive (T-lymphocytes) 3.80 ⇐0.08 ⇐0.36 6 White blood cells (T-lymphocytes) 3.76 ⇐ 4.87 ⇐0.36 7 T-lymphocytes (antigen-reactive) 3.46 ⇐0.40 ⇐0.07 8 T-lymphocytes (T-killers) 3.37 ⇐0.47 ⇐1.14 nine T-killers (T-lymphocytes) 3.35 ⇐1.16 ⇐0.36 10 T-lymphocytes (white blood cells) 3.30 ⇐0.40 ⇐ 4.90 eleven White blood cells (B-lymphocytes) 3.17 ⇐5.00 ⇐0.44 12 T - (B-lymphocytes) 3.13 ⇐0.42 ⇐0.44 thirteen B-lymphocytes (white blood cells) 2.60 ⇐0.51 ⇐ 4.90 14 White blood cells (antigen-reactive) 2,53 ⇐ 4.90 ⇐0.07 fifteen B-lymphocytes (antigen-reactive) 2,51 ⇐0.45 ⇐0.07 16 T-killers (antigen-reactive) 2.41 ⇐1.07 ⇐0.07 17 B-lymphocytes (T-killers) 2,38 ⇐0.59 ⇐1.14 eighteen T-killers (B-lymphocytes) 2,37 ⇐1.26 ⇐0.44 19 Antigenreactive (neutrophils) 2,36 ⇐0.13 ⇐0.68 twenty Antigenreactive (white blood cells) 2,30 ⇐0.09 ⇐ 4.90 21 Neutrophils (B lymphocytes) 2.24 ⇐ 0.80 ⇐0.44 The number of immune animals 12 60%

Table 6 Differential prognostic table for assessing the immune status of cows 9 months after revaccination of TBC-2, thousand / μl No. p / p Combination, variable parameter (basis) n Student t-test Variable Basis 1 T-lymphocytes (white blood cells) twenty 5.56 ⇐0 53 ⇐ 4.70 2 B-lymphocytes (white blood cells) 3.48 ⇐0.46 ⇐ 4.70 3 T - (B-lymphocytes) 3.26 ⇐0.60 ⇐0.49 4 White blood cells (T-lymphocytes) 3.25 ⇐ 4.82 ⇐0.51 5 White blood cells (B-lymphocytes) 3.15 ⇐ 4.83 ⇐0.49 6 White blood cells (T-killers) 2,57 ⇐ 4.90 ⇐0.97 7 B - (T-lymphocytes) 2,57 ⇐0.43 ⇐0.51 8 B-lymphocytes (T-killers) 2.27 ⇐0.44 ⇐0.97 nine B-lymphocytes (antigen-reactive) 2.23 ⇐0, 54 ⇐0.30 The number of immune animals nine 45%

Claims (1)

A method for the prevention of tuberculosis, including immunization of animals aged 10-20 days with BCG vaccine, followed by an allergic tuberculin test and reimmunization at 14-16 months of age with BCG vaccine, characterized in that then, after 12 months of non-tuberculin-responsive animals, they are immunized with the tuberculosis acellular TBC vaccine -2 subcutaneously at a dose of 5-7 mg of protein per animal, after which the TBC-2 vaccine is re-immunized every 12 months until the farm (farm) is healthy, and tuberculosis studies are conducted iat after 6 months.