CZ574889A3 - preparation for non-specific immunotherapy of domestic animals endomethritis and process for preparing thereof - Google Patents

Abstract

Therapy for endometritis in farm animals using a preparation for the non-specific immunostimulation of the uterine immunity system, containing in 1 ml of the nutrient medium inactivated bacteria Bordetella bronchiseptica CAMP 6229 and CAMP 6230 in a mutual ratio of 1:1 and a total number of at least 2.5.10<9>, a surfactant preferably from the group of saponines the quantity of which corresponds to the haemolytic activity within a range of 8 to 16 units in 1 ml, and 0.1 to 0.15 mg of a preservative, such as sodium salt of 2-(ethylmercurythio)-benzoic acid known under the trivial name of sodium merthiolate. The preparation is obtained by the submerged cultivation of either of the two mentioned strains in 50 parts by weight of the nutrient medium, inactivation by heating to a temperature of 60 to 70 degrees C, mixing the bacterial suspensions, adding the surfactant and preservative, and modifying the pH to the final value of 7.0 to 7.4. By the proposed immunostimulation after intrauterine application it is possible to reach a considerable therapeutic effect of 70 to 90%.

Description

Inflammatory uterine diseases of farm animals - endometritis - are a serious health and economic problem due to their high incidence. The upward trend of their occurrence in recent years, especially in cattle, has been alaught, due to the increasing performance and unsecured increased demands on the welfare of their breeding.

Presently, antibiotic-based ballet formulations such as penicillin, streptoraycin, bacitracin, neoraycine, chloramphenicol, tetracycline, and others, and chemotherapeutic agents such as sulfonamides, iodine-based preparations, and others are used for the treatment of endoraethritis. Hormonal preparations and substances with an effect on the vegetative nervous system are used for supportive or complementary treatment.

The treatment of uterine inflammations is accompanied by an increasing resistance of pathogenic and conditionally pathogenic microorganisms to these antibiotics and chemotherapeutic agents (Gustafsson,

J.Am.Veter.Med.Ass. , 185.1 9θ ^, p.119 ^ -1198), despite the demonstrated immunosuppressivity of some broad spectrum antibiotics (Dreux, Bull.Soc.Veter.Pract.de France, 68,1984, p.81-102).

The use of said therapeutic agents is subject to hygienic withdrawal periods due to their residues in foodstuffs, in particular in meat and milk. Limiting the enormous use of antibiotics in livestock production is a current societal current requirement in terms of food chain protection and human health.

For these reasons, new methods of endometritis therapy by non-specific immunomodulation have recently been sought

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nonspecific defenses. Generally unspecified, respectively. paraspecific effects of stimulants have long been discussed by Mayr (Zbl. Vater. Med., B. 29, 1982, p. 5-23} Mh. Veter. Med., 4θ, 1985, p. 12-15) and others. Several preparations have been described in the literature for non-specific stimulation of farm animal immunity against respiratory and enteral tract infections, such as PIND-AVI and PEI-ORF, which are based on paramyxoviruses and parapoxviruses. parvum and BCG. In veterinary gynecology, Henschelchen (inaug. Diss., Munchen 1981, 94 p. - Ludwig-Maxmilians-Univesitat) studied the effect of the PIND-AVI paraimunity inducer in cows after delivery on the level of phagocytic activity of blood leukocytes after several days of parenteral administration. Jablonsky and Prigara (Veterinariya, Moscow 1985, 10, pp. 59-60) again used Filatov tissue preparation parenterally with a positive preventive effect on the course of puerperium. Data on the local, intrauterine use of non-specific immunostimulants for the treatment of inflammatory diseases of the uterus are rare in the litarature. N _{and the} example of Asbury (Theriogenology, 21, 1984, pp. 387-393) verified the effect of homologous blood serum in mares affected by endometritis.

In this context, the object of the present invention is to provide a preparation for intrauterine administration with both preventive and therapeutic effects, which stimulates the local defensive, reticuloendothelial system of the uterus and thereby directly affects the healing of the inflammatory process and indirectly the involutional processes of the genital organs after delivery.

The object is achieved and the subject of the invention is a preparation for non-specific immunotherapy of farm animal endometritis, consisting of inactivated bacteria with an immunostimulatory effect, a non-ionic surfactant and a hygienically safe preservative in a nutrient medium containing a carbon source, nitrogen source, mineral salts and buffers. containing, per ml of nutrient medium with a final pH in the range of 7.0 to 7.4, preferably a nutrient medium of 17.0 g of enzymatic casein hydrolyzate, 3.0 g of soya peptone, 5.0 g of chloride sodium phosphate, 5.0 g disodium hydrogen phosphate and 2.5 S glucose in 1 liter aqueous solution, inactivated bacteria of the Bordetella bronchiseptica strains CAMP 6229 and CAMP 6230,

an active substance, preferably from the group of saponins, in an amount corresponding to hemolytic activity in the range of 8 to 16 units per ml of preparation, and 0.1 to 0.15 mg of a preservative, for example sodium 2- (ethylhydrargiothio) -benzoic acid, known under trivial called sodium merthiolate.

The composition of the nutrient medium does not affect the quality of the bacterin, but its density.

The amount of saponin per 1 L of bacterial suspension depends on the degree of its activity, which varies not only according to the species but also the provenance of the saponin. Therefore, the amount required is always recalculated according to the degree of activity of the saponin used to the tested known standard. A unit of hernolysis activity is the amount by weight of saponin that causes hemolysis of a 10% ram cell suspension in solution at a 1: 16 dilution,

The invention also relates to a process for the preparation

Its essence lies in the fact that strains Bordetella bronchiseptica

CAMP 6229 and CAMP 6230 are each cultivated submergedly, preferably 1 part by weight of the strain in 50 parts by weight of the culture medium, inactivated after cultivation, the suspension density is adjusted to a final content of at least 2.5 by adding sterile saline. 10 bacteria per ml and inactivated suspensions are mixed together, then the surfactant and preservative 5 are added to the mixed suspension, preferably in the form of an aqueous solution, and the pH of the formulation is adjusted to a final value of 7.0 to 7. The inactivation of the bacteria in the nutrient medium is carried out preferably by heating the suspension to a temperature in the range of 60 to 70 ° C for one to two hours.

The novelty of the adhesive according to the invention lies in the selection of the bacterial species, its strains, the composition and the method of preparation. The advantage is the use of well-known properties of the available bacterial species, which provide a higher stimulating effect on the immune response of the organism than in the above-mentioned viral preparations. Said immunostimulation achieves a significant, 70-90% therapeutic effect after intrauterine administration.

In addition to the advantages of eliminating the drawbacks of existing methods of treating endometritis in livestock with antibiotics and chemotherapeutic agents, the formulation of the invention has other significant advantages:

By using the product, the body's own defense mechanisms are stimulated, neither suppressed nor resistant pathogenic germ strains.

Introducing the product in veterinary practice will currently limit the enormous use of broad spectrum antibiotics and thus their penetration into the food chain, which will significantly contribute to the protection of human population health.

Practical use of the preparation contributes to the protection of the environment as it is harmless to the animal and human population.

The preparation has a high therapeutic effect and its expected cost is higher than that of existing antibiotic foams.

Exemplary embodiment

1. Preparation of the preparation a / Selection of bacterial strains

Two Bordetella bronchiseptica strains CAMP 6229 and CAMP 6230, which repeatedly showed the best immunostimulatory effects, were selected for the inninostimulatory formulation.

b / Preparation of Bordetella bronchiseptica

Bordetella bronchiseptica CAMP 6229 and CAM? 6230 are cultivated submerged in a fermenter in a liquid nutrient medium of the following composition:

enzymatic casein hydrolyzate 17.0 S soya peptone 3.0 g sodium chloride 5.0 g disodium hydrogenphosphate. (5.0 g Na ₂ HPO _{fourth 12H?} 0) 2.5 g glucose.

Make up with distilled water to a total volume of 11 and autoclave at 121 ° C for twenty minutes, adjust the pH to 7.2 to 7.

Two-seed batches of one strain were used for cultivation in the fermenter. Each strain is cultivated separately.

After the cultivation is complete, the heat is inactivated by the bacterin for 1.5 hours at 60-70 ° C.

Suspensions of bacteria whose density is adjusted by adding sterile physiological saline to a minimum of 2.5 · 10 ^ bacteria / 1 ml are mixed together c / Adding saponin and preservative

After mixing, saponin is added to the inactivated bacterial suspension at a dose of 0.5 grams of the bacterial suspension.

The preservation of the preparation is carried out by adding 2.6 µl of the aqueous solution of 2- (ethylhydrargyriotliio) -benzoic acid sodium salt, known under the trivial name sodium mortliolate, which is 10 mg of the substance, per 11 preparations. After preservation and sterility control, the pH of the formulation is adjusted to 7.2.

d / Sterility test

The sterility of the finished preparation is tested according to CSL 3 “Sterility Test. The sterility test of the inactivated bacterial suspension is performed in a standard nutrient medium and thioglycolate broth at a 1: 100 dilution at 37 ° C for five days.

(e) Harmless test

K The product is tested for harmfulness in 10 mice weighing 15 to 20 grams and administered intraperitoneally in 0.1 ml portions. Within 7 days of application, the mice must not die as a result of application. Bordetella bronchiseptica must not be cultured in dead mice. The test should be repeated if more than two out of 10 mice died.

f / Efficiency test

The essence of the efficacy of the product is the determination of sufficient density of bacteria, which is performed by comparison with the standard. The hemolytic activity of the saponin component is then checked according to the following procedure:

Centrifuge ml at 10 000 ml. g for 30 minutes. The supernatant obtained is diluted with the standard nutrient medium by the binary method up to a 1:64 dilution. Simultaneously, a control solution of saponin is prepared in a standard nutrient medium at a concentration of 5 mg / 10 ml and a dilute dilution is performed.

A pre-prepared 10% ram erythrocyte suspension (washed three times with buffered saline) was added to each dilution tube in an amount of 0.1 ml of erythrocyte suspension per ml of each dilution.

The tubes are placed in a thermostat at 37 ° C for 30 minutes. The hemolysis of the blood cells in the individual dilutions is then checked. The appropriate dilution, where complete hemolysis appears, indicates the number of hernolytic units. Hemolysis at the same dilution as the control or one dilution lower is considered to be satisfactory (when using Merck saponin, complete hernolysis is at a dilution of 1: 8 to: 16 and a control solution of 1: 16).

In addition, the efficacy test is carried out on white mice, which is based on the demonstration of increased resistance of the stimulated mice to infection by a virulent strain of Pasteurella multocida. Ten mice weighing 15 to 20 grams are challenged intraperitoneally with 0.1 ml of a 1: 1 dilution of saline and ten mice remain as controls. After 48 hours, both groups were challenged with 0.2 ml of Pasteurella multocida strain 6254 suspension, each of five mice at 10 dilution and five mice at 10 dilution.

After challenge in mice stimulated with the preparation of the invention, 5θ mice should survive at a dose ten times higher than the control (10th LD50). The evaluation is carried out after five days.

2. Pr ale. technical use of the preparation

The composition according to the invention is used for the treatment of cow's puerperal endometritis and in cases of uterine subinvolution with a delayed self-cleaning process. The temperature-controlled preparation is administered intrauterine in a dose of 20 ml. In case of a larger uterine filling with pathological contents, it is desirable to evacuate by administration of uterotonics before the actual application of the bacterin.

The course of therapy is checked one week after treatment of the breed. If there is an obvious improvement in the outflow from the genital organs, no further treatment is performed. In the event of a persistent pathological process, bacterin treatment is repeated, and in the case of marked deterioration, a different therapeutic approach should be selected.

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In the case of individual treatment of DC, it varies between 70 and 100 in individual cows.

Other possible uses of the invention may be summarized as follows:

In cow breeding, the product can also be used for the treatment of postpuorperal endometritis.

The product can be applied preventively in cows after the retention of the seconds and in the case of severe births where increased bacterial contamination of the genital organs and reduced defense of the animals can be expected.

The preparation can be used in the treatment of endometritis in heifers or other livestock.

Claims (3)

PATENTOVÉ NÁROICL D f> O O < | O - <! <- · ´m >> ie r— - <m N < Composition for non-specific immunotherapy of farm animal endometritis, consisting of inactivated bacteria having an immunostimulating effect, a non-ionic surfactant and a non-toxic preservative in a nutrient medium comprising a carbon source, a nitrogen source, a mineral salt and a buffering agent, characterized in that a nutrient medium having a final pH in the range of 7.0 to 7.4, preferably a nutrient medium having a composition of 17.0 S enzymatic casein hydrolyzate, 3.0 g of soy peptone, 5.0 g of sodium chloride, 5.0 g of disodium hydrogen phosphate and 2.5 S glucose in one liter of aqueous solution contains inactivated bacteria of the Bordetella bronchiseptica strains CAMP 6229 and CAMP 6230 deposited in Cs. a collection of microorganisms in Brno, in a relative weight ratio of 1: 1 to a total of at least 2.5 · 10 ^, a surfactant preferably of the saponin group in an amount corresponding to a herniolytic activity ranging from 8 to 16 units per ml of formulation, and 0.1 to 0.15 ^m s> preservative, such as sodium 2- (hydrargiothio ethyl) benzoic acid, known under the trivial name sodium merthiolate · 2 A process for preparing a preparation according to the invention. 1, characterized in that the Bordetella bronchiseptica strains CAMP 6229 and CAMP 6230 are each cultivated submergedly, preferably 1 part by weight of the strain in 50% by weight. After the cultivation is completed, they are inactivated, the density of the suspension is adjusted by adding sterile saline to a final content of at least 2.5 · 10 ^ bacteria per ml, and the inactivated suspensions are mixed together, then the surfactant is added to the mixed suspension and the preservative is preferably in the form of a 5% aqueous solution and the pH of the formulation is adjusted to a final value in the range of 7θ to 7.4. The method according to claim 2, characterized in that the inactivation of the bacteria in the nutrient medium is carried out by heating the suspension for 1 to 2 hours to a temperature in the range of 60 to 70 ° C. In power of attorney