Remedy for bovine leukemia prophylaxis and use thereof

Abstract

The invention generally relates to the field of veterinary medicine and can be used for bovine leukemia prevention. The invention expands the range of means of the claimed application and provides lifelong resistance of animals to the leukemia virus. The invention is aimed at solving the problem of expanding the range of preventive medications and forming new approaches to the problem of preventing bovine leukemia, which allow ensuring lifelong resistance of the animals to leukemia infection. The technical result consists in the design of a remedy and use thereof for bovine leukemia prevention, which would allow providing a targeted immune response, efficiently and with minimal costs, by activating cellular immunity against bovine leukemia virus by increasing the number of killer cells. The remedy for bovine leukemia prevention contains a water-soluble protein fraction with a molecular weight of 18-20 kDa, isolated from the tuberculosis mycobacterium destruction products, characterized by the presence of peaks at a wavelength of 214 nm in the UV region, a phosphate-buffered saline, an aqueous formaldehyde solution, and an isotonic sodium chloride solution. The use consists in administering the said remedy to 1 to 9 days old calves with a single intramuscular injection at a dose of 4.0-6.0 ml per capita.

Claims

1. Remedy for bovine leukemia prevention characterized in that it contains: a water-soluble protein fraction with a molecular weight of 18-20 kDa, isolated from the destruction products of tuberculosis mycobacterium from the strain M. bovis No. 8 and characterized by the presence of peaks near the wavelength of 214 nm in the UV region, a phosphate-buffered saline, an aqueous formaldehyde solution, and an isotonic sodium chloride solution at the following ratio of the components, wt %: TABLE-US-00007 water-soluble protein fraction with the molecular 0.05-0.125; weight of 18-20 kD, isolated from the destruction products of the tuberculosis mycobacterium from the strain M. bovis No. 8 phosphate-buffered saline 9.95-24.875; formaldehyde aqueous solution of 0.025-0.046; concentration within 36.5-37.5% isotonic sodium chloride solution the rest. of concentration within 0.85-0.95%

2. Remedy for bovine leukemia prevention characterized in that it contains: a water-soluble protein fraction with a molecular weight of 18-20 kDa, isolated from the destruction products of the tuberculosis mycobacterium from the strain M. bovis No. 8 and characterized by the presence of peaks near the wavelength of 214 nm in the UV region, a phosphate-buffered saline, an aqueous formaldehyde solution, and an isotonic sodium chloride solution at the following ratio of the components, wt %: TABLE-US-00008 water-soluble protein fraction with the molecular 0.125; weight of 18-20 kD, isolated from the destruction products of the tuberculosis mycobacterium from the strain M. bovis No. 8 phosphate-buffered saline 24.875; formaldehyde aqueous solution of concentration within 36.5-37.5% 0.037; isotonic sodium chloride solution the rest. of concentration within 0.85-0.95%

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) The disclosure is illustrated in FIG. 1, which shows the chromatogram of the protein fraction isolated from the destruction products of the causative agent of tuberculosis in cattle M. bovis (strain No. 8).

DETAILED DESCRIPTION

(2) The remedy to prevent bovine leukemia is prepared as follows. As the destruction products of the causative agent of tuberculosis, PPD tuberculin for mammals is used, obtained from the strain M. bovis No. 8 and representing a clear liquid of light brown color.

(3) To obtain the protein fraction, the liquid with the destruction products of the pathogen is precipitated with a saline solution (for example, ammonium sulfate) and centrifuged. The supernatant is then removed and the precipitate is redissolved in a saline buffer solution.

(4) Dialysis is carried out, and the pH of the solution is adjusted to 7.0-7.4, after which the protein concentration is analyses by conventional methods, in particular, by the Lowry method.

(5) The choice of such pH values of the solvent is explained by the corresponding value in the body cells. It is well known that pH in the body cells and the extracellular fluid is about 7.0 and 7.4, respectively.

(6) The molecular weight of the protein was determined by high pressure liquid chromatography (HPLC), which was carried out, in particular, on a Stayer liquid chromatograph, using a spectrophotometric detector A214 (the wavelength 214 nm). A BioSep-SEC-S 2000 column (5 m 145 A, 3007.8 mm) was used for separation. 0.01 M phosphate-buffered saline was used as the eluent, the flow rate was 1 ml/min, or 0.01 M phosphate-buffered saline with 0.025% sodium azide solution (pH 6.8) and at a flow rate of 2 ml/min.

(7) Bovine serum albumin with a molecular weight within 64-70 kDa, lactoferrin with a molecular weight of 75-80 kDa, and papain with a molecular weight of 20 kDa were used as standards.

(8) Standard values were taken into account, such as the retention time of the sample on the column, or the retraction time (RT, min), the peak intensity by height (mAU), and the relative quantitative content of the substance, which was calculated as the percentage ratio of each peak to the total area of the peaks (A, %).

(9) The major proteins in the fraction isolated from the destruction products of the causative agent of bovine tuberculosis M. bovis (strain No. 8) had molecular weights within 18-20 kDa (see FIG. 1).

(10) Table 1 shows the result of chromatography of the composition based on the protein fraction isolated from the destruction products of the causative agent of bovine tuberculosis.

(11) TABLE-US-00003 TABLE 1 Mol. Time, Height, Area, Resolution, n, weight, No. min mAU mAU .Math. s n + 1 A, % kDa 1. 18.54 100.18 3234.48 0.00 100 18-20

(12) Next, the resulting protein fraction with the molecular weight of 18-20 kDa is mixed, in a buffer solution containing 4.8-5.2 mg/ml of the protein (i.e., 0.48-0.52%), with an isotonic sodium chloride solution, of a concentration within 0.85-0.95%.

(13) To prepare a 50% solution of the protein fraction, 1 part of the buffer solution with the protein is taken and 1 part of the isotonic solution is added.

(14) The composition of the resulting mixture is as follows:

(15) the protein fraction0.24-0.26%,

(16) the phosphate-buffered saline49.74-49.76%, and

(17) the sodium chloride solutionthe rest.

(18) When preparing a 20% solution of the protein fraction, the composition of the resulting mixture will be as follows:

(19) the protein fraction0.096-0.104%,

(20) the phosphate buffered saline49.896-49.904%, and

(21) the sodium chloride solutionthe rest.

(22) Next, a mixture of an aqueous formaldehyde solution with an isotonic sodium chloride solution is prepared.

(23) To do this, 0.15-0.25 ml of a 36.5-37.5% medical solution of formic aldehyde (formaldehyde) is taken and added to 99.75-99.85 ml of an isotonic 0.85% (or 0.95%) solution of sodium chloride. The mixture of these solutions is stirred thoroughly.

(24) The ratio of the components of the formaldehyde and isotonic sodium chloride solutions was selected experimentally.

(25) When preparing the remedy from 37% formaldehyde solution, the final formaldehyde concentration in the resulting composition will be 0.055-0.099 wt %.

(26) Then the resulting mixture of the solutions of the protein fraction in the buffer and isotonic solutions is mixed with the mixture of the formaldehyde and isotonic sodium chloride solutions.

Example 1. Preparation of the Remedy for Leukemia Prophylaxis

(27) 100 ml of PPD tuberculin for mammals, obtained from M. bovis strain No. 8 is taken. The protein fraction is prepared therefrom as follows.

(28) 42 g of ammonium sulfate is added to 100 ml of tuberculin to precipitate the protein, centrifuged at 4,500 rpm during 20 min. The supernatant is removed and the precipitate is redissolved in 1 ml of a 0.01 M phosphate-buffered saline. The system is dialyzed by means of a dialysis membrane with a pore diameter of 3,500 Da in a 0.01 M phosphate-buffered saline during 24 h, the pH of the solution is adjusted to 7.2-7.4, after which the protein concentration is determined by the Lowry method using a StatFax 3300 biochemical analyzer.

(29) The concentration of the protein fraction from the destruction products of the causative agent of bovine tuberculosis was 5.0 mg/ml.

(30) Analogously to Example 1, protein fractions from other batches of PPD tuberculin were obtained. The amount of protein everywhere varied from 4.8 to 5.2 mg/ml.

(31) Next, the resulting protein fraction with the molecular weight of 18-20 kDa in the buffer solution containing 5 mg/ml protein (i.e. 0.5%) is mixed with the isotonic sodium chloride solution (0.85-0.95%).

(32) A mixture of the aqueous formaldehyde solution and the isotonic sodium chloride solution is then prepared.

(33) To do this, 0.2 ml of a 37% medical solution of formic aldehyde (formaldehyde) is taken and added to 99.8 ml of an isotonic (0.85%) sodium chloride solution. The mixture of these solutions is thoroughly stirred. The final formaldehyde concentration in the resulting composition will be 0.074 wt %.

(34) The protein fraction in the phosphate-buffered saline is mixed with the mixture of solutions of formaldehyde and sodium chloride in equal proportions (1:1).

(35) The contents of the components in the composition will be as follows, wt %:

(36) TABLE-US-00004 the water-soluble protein fraction 0.125 with the molecular weight of 18-20 kD, isolated from the destruction products of mycobacterium of M. bovis strain No. 8 the phosphate-buffered saline 24.875 the aqueous formaldehyde solution 0.037, the isotonic sodium chloride solution the rest.

(37) Similarly, a remedy was prepared from protein fractions with protein contents of 0.48-0.52%.

Example 2. Substantiation of the Quantitative Content of Components in the Remedy

(38) 120 two to nine days old calves were used in our experiments, of which 108 were injected at doses of 4 to 6 ml to prevent leukemia with different quantitative contents of the components.

(39) 12 animals formed a reference group, which were injected pure saline in a dose of 5 ml per capita. The results are shown in Table 2.

(40) TABLE-US-00005 TABLE 2 Quantitative contents of components, wt % Phosphate- Formal- Isotonic Dose of Protein buffered dehyde NaCl preparation, Number Died Survived fraction saline solution solution mL of calves Nos % Nos % 0.05 9.95 0.037 Rest 4 12 0 0 12 100 to 5 12 1 8.3 11 91.7 100% 6 12 3 25.0 9 75.0 0.075 14.925 0.037 Rest 4 12 2 16.7 10 83.3 to 5 12 0 0 12 100 100% 6 12 2 16.7 10 83.3 0.125 24.875 0.037 Rest 4 12 0 0 12 100 to 5 12 0 0 12 100 100% 6 12 1 8.3 11 91.7

(41) 4 animals of 12 were lost in the reference group, which was 33.3%.

Example 3

(42) To substantiate the increased number of killer cells in the body of animals (heifers and cows), immunological studies of the blood of animals treated at the age of 2-9 days with our remedy to prevent bovine leukemia were carried out:

(43) 2.5 years old heifers, and

(44) 5 years old cows.

(45) The results are shown in Table 3.

(46) TABLE-US-00006 TABLE 3 Immunological indicators of 2.5 years old heifers and 5 years old cows Group T-lymph. Th-lymph. Ts-lymph. Th/Ts T-active. B-lymph. Th0 2.5 years old HEIFERS Non- .sup.3343 286.07 .sup.2054 178.18 .sup.1295 113.84 1.6 0.07 1876 159.95 459 38.29 .sup.877 97.93 treated Treated 3081 249.9 1522 133.1 1559 132.3 1.0 0.14 1631 133.51 .sup.517 39.5 1616 242.7 5 years old COWS Non- 2421.14 279.34 1489.0 157.85 931.29 127.07 1.66 0.11 1315.43 154.77 288.43 32.89 744.43 110.62 treated Treated 4028.0 586.89 2369.57 345.5 1660.71 241.39 1.43 0.04 2167.86 318.2 528.29 68.81 1406.0 203.93

(47) From Table 3 it follows that the 2.5 years old heifers show a decreased ratio of T-helpers to T-suppressors (Th/Ts) and an increased number of the third subpopulation of T-lymphocytes (Th0).

(48) In the 5 years old (experimental) cows, a significant increase in the contents of all lymphocyte fractions (T and B) was recorded while maintaining a high content of Th0 (killers).

(49) The results of our serological tests for the presence of antibodies to BLV, carried out as the immunodiffusion reaction, in the heifers and cows aged 2.5 and 5 years, respectively, were negative.

(50) Thus, the claimed remedy for bovine leukemia prevention, as well as the method of use thereof, ensures lifelong resistance of animals to leukemia infection by activating the cellular immunity against BLV by increasing the number of killer cells.