RU2116082C1 - Method of preparing gamma-globulin for treatment of horses with epizootic lymphangoitis - Google Patents

Abstract

FIELD: veterinary science, biotechnology. SUBSTANCE: gamma-globulin is prepared from cattle serum blood (age is 1.5-2 years, weight is 250-280 kg). Hyperimmunization of animals with disease pathogen is carried out for two cycles at 30 days interval. During the first cycle antigen is administrated to animals at dose 4 ml for 3 days being dose consists of 1 ml fungus an aqueous suspension (50 mln/ml), 1 ml physiological solution and 2 ml 1% saponin solution. During the second cycle antigen is administrated to animals at dose 8 ml for 2 days being dose consists of 2 ml fungus an aqueous suspension, 2 ml solution and 4 ml 1% saponin solution. After 5 days of the second hyperimmunization cycle termination animals are subjected for bleeding. Hyperimmune serum is separated from obtained blood and pH is brought about at the range 6.9-7.2. Polyethylene glycol as 50% an aqueous solution is added to serum up to final concentration 20% and after 2 h exposition at 10 C serum is centrifuged at 2 500 rev/min for 30 min to isolate gamma-globulin followed by its lyophilization to provide its stability during storage. Before using dry gamma-globulin is diluted in 10 ml physiological solution. Gamma-globulin solution (10 ml) is administrated to sick horses by subcutaneous injection in site of lower one-third of neck (two-fold every other day). EFFECT: high activity and safety of preparation. 4 cl, 1 tbl

Description

 The invention relates to veterinary medicine and biotechnology and relates to a method for producing gamma globulin for the treatment of equine epizootic lymphangitis (ELL).

 ELL (synonyms - African glanders, Japanese skin ulcers, pseudosap, epizootic inflammation of the lymphatic vessels, blastomycosis, histoplasmosis, pantushko) is a contagious chronic disease of one-hoofed animals, characterized by damage (inflammation) of the lymphatic vessels of the skin, lymph nodes and subcutaneous tissue, mucous membranes, mucous membranes, mucous membranes, mucous membranes, mucous membranes, ulcers and abscesses, keratitis and pneumonia.

 The causative agent of the disease is the yeast fungus Histoplasma farciminosus (synonym for Cryptococcus farciminosus) was first described in 1873 by the Italian scientist Rivolta. The disease was most widespread in Africa and Asia, it was also registered in various European countries, including Russia during the first and second world wars, which was associated with contact of animals from different epizootic countries, frequent movements of sick horses, and non-compliance with the necessary veterinary Sanitary events.

 The main source of infection is a sick animal. However, infected soil and care products can also cause infection. The fungus enters the animal’s body through damaged areas of the skin and is localized in the lymphatic vessels, subcutaneous tissue and the skin itself. The clinical manifestation of ELL is characterized by the appearance of ulcers on the skin, inflammation of the lymphatic vessels and the formation of nodules. The degree of damage depends on the virulence of the pathogen and the level of the body's defenses. In the USSR, various drugs have been successfully tested for the treatment of ELL: formalin, turpentine, novarsenol, ichthyol, crystal violet, iodine, and many others. Along with local treatment for mild lesions, surgical treatment and intravenous administration of a 4% aqueous solution of formalin and mercury chloride at a concentration of 1: 1000 are used. For the prevention of ELL, diagnostic and veterinary and sanitary measures were taken to identify sick animals, timely treatment of sick animals, thorough disinfection of care items and dysfunctional premises. A lot of work carried out in our country by veterinary specialists contributed to the elimination of ELL in the USSR. However, at present, there is a threat of its occurrence in the southern regions of Russia, which necessitated the search and development of specific preventive measures for ELL, which have not yet been obtained [1].

 There is a method of treating horses with ELL by subcutaneous injection into the region of the upper third of the neck 100 ml of blood of other horses suffering from the same disease, and treating the affected areas with copper sulfate [2].

 The disadvantages of this method are that for the treatment they use the blood of sick animals, which may contain the causative agent of the disease, which significantly reduces the value of the proposal.

 No solutions similar to the present invention were found in accessible sources of information.

 The objective of the invention is to develop a method for producing gamma globulin for the treatment of ELL. The technical result from the use of the invention is to ensure the safety and high activity of the drug.

 The specified technical result is achieved by the creation of the invention, characterized by the following set of features: a method for producing gamma globulin for the treatment of ELL from biological fluid; hyperimmune blood serum of animal producers is used as biological fluid; cattle (cattle) are used as producer animals; Cattle is used at the age of 1.2 - 2.0 years, weighing 250 - 280 kg; Cattle are subjected to preliminary multiple hyperimmunization with the causative agent of the disease in the middle third of the neck in two cycles with an interval of 29 to 31 days; during the first cycle of cattle hyperimmunize for 3 to 4 days in a dose of 3-5 ml once; during the second cycle - for 2 to 3 days in a dose of 7 to 9 ml once; 4 to 6 days after the end of hyperimmunization, cattle are bled; hyperimmune serum is separated from the obtained blood; the pH of the hyperimmune serum is set between 6.9 and 7.2; 50% aqueous solution of polyethylene glycol is added to hyperimmune serum to a final concentration of 20%; after two hours of exposure, the hyperimmune serum is centrifuged at 2500 rpm for 30 minutes to isolate gamma globulin; gamma globulin is dried by lyophilization to ensure the stability of the properties of the drug.

 The invention includes the following set of essential features providing a technical result in all cases for which the scope of legal protection is claimed: a method for producing gamma globulin for the treatment of ELL from biological fluid; as a biological fluid use hyperimmune blood serum of animal producers; cattle are used as animal producers; Cattle are subjected to preliminary multiple hyperimmunization with the causative agent of the disease in two cycles with an interval of 29 to 31 days; during the first cycle of cattle hyperimmunize for 3 to 4 days in a dose of 3-5 ml once; during the second cycle - for 2 to 3 days in a dose of 7 to 9 ml once; 4 to 6 days after the end of hyperimmunization, cattle are bled; hyperimmune serum is separated from the obtained blood; the pH of the hyperimmune serum is set between 6.9 and 7.2; 50% aqueous solution of polyethylene glycol is added to hyperimmune serum to a final concentration of 20%; after two hours of exposure, the hyperimmune serum is centrifuged to isolate gamma globulin.

 The achievement of the technical result of the proposed method can be explained as follows.

 The use of hyperimmune blood serum as a biological fluid and cattle as animal producers of this serum ensure the safety of gamma globulin for the treatment of ELL, since cattle is not susceptible to this disease.

 The proposed cattle hyperimmunization scheme allows to obtain a high titer of antibodies in the blood of animals.

 Processing cattle hyperimmune blood serum with polyethylene glycol at a pH of 6.9 - 7.2, followed by centrifugation, allows for the complete selective isolation of high purity and activity gamma globulin.

 The resulting gamma globulin contains a high titer of highly purified antibodies that interact with the pathogen and neutralize it, which prevents the further reproduction of cryptococcus in the animal body. The use of gamma globulin obtained by the proposed method on patients with epizootic lymphangitis horses ensures their recovery in 85 - 90% of cases after 30 - 40 days.

 The proposed method is also characterized by other features expressing specific forms of its implementation: cattle are used at the age of 1.5 - 2 years, weighing 250 - 280 kg; to isolate gamma globulin, hyperimmune serum is centrifuged at 2500 rpm for 25 - 35 minutes; the obtained gamma globulin is dried by lyophilization to ensure the stability of the properties of the drug during storage.

 In the study of the set of essential features characterizing the proposed method, no similar known solutions were found regarding methods for producing gamma globulin for the treatment of ELL.

 The analysis of the prior art, including a search by patent and scientific and technical sources of information, and the lack of sources containing information about analogues of the present invention, indicate that the invention meets the condition of patentability "novelty."

 To verify the conformity of the proposed method to the patentability condition "inventive step", an additional search for known solutions was carried out to identify the features included in the characterizing part of the claims. The search results showed that, the proposed method does not follow explicitly from the prior art set forth in the corresponding section of the description (no solutions having features matching the distinguishing features of the claimed invention have been identified), and the effect of the essential features of the proposed invention is not revealed transformations to achieve the technical result set forth in clause 19.5.3 of the Rules for the preparation, filing and consideration of applications for the grant of a patent for an invention.

 Therefore, the proposed method meets the condition of patentability "inventive step".

Example 1. To obtain gamma globulin for the treatment of ELL, hyperimmune blood serum of animal producers is used as a biological fluid, which is used as cattle at the age of 1.5 - 2 years old, weighing 250 - 280 kg. For cattle hyperimmunization, an aqueous suspension of the fungus microconidia of the epizootic strain Cryptococcus farciminosus Rivolta at a concentration of 50 million / ml is used as antigen. Hyperimmunization of animals with the causative agent of the disease is carried out subcutaneously in the middle third of the neck in two cycles with an interval of 30 days. During the first cycle, the antigen is administered to animals for 3 days once in a dose of 4 ml, including 1 ml of an aqueous suspension of the fungus, 1 ml of physiological saline and 2 ml of a 1% saponin solution. After 30 days, a second cycle of cattle hyperimmunization is carried out. During the second cycle, the antigen is also administered to animals subcutaneously in the middle third of the neck for 2 days once in a dose of 8 ml, including 2 ml of an aqueous suspension of the fungus, 2 ml of physiological saline and 4 ml of a 1% saponin solution. 5 days after completion of the second cycle of hyperimmunization, the animals are bled. From one head of cattle receive - 12 liters of blood, from which you can prepare ≈250 doses of gamma globulin. Hyperimmune serum is separated from the obtained blood. serum pH is reduced to 6.9 - 7.2. Then, a 50% aqueous solution of polyethylene glycol is added to it to a final concentration of 20%. The serum is mixed and, after a two-hour exposure at 10 ^° C., it is centrifuged on a Mistral 6 L centrifuge at 2500 rpm for 30 minutes to separate the precipitate containing gamma globulin. The resulting gamma globulin has a pasty consistency of yellowish-brown color. Gamma globulin is resuspended in physiological saline in 1/50 of the original volume. The obtained gamma globulin is dried by lyophilization to ensure the stability of the properties of the drug during storage. Before drying, 4 ml of gamma globulin concentrate are placed in each ampoule. The activity of the obtained preparation in RDP is 1: 64, in ELISA - 1: 64000. The protein content in the preparation is determined by the Lowry method and its amount is ≈30 mg / ml. Before use, dry gamma globulin is diluted in 10 ml of physiological saline. Sick horses are injected with 10 ml of gamma globulin twice every other day subcutaneously in the lower third of the neck.

 Example 2. Verification of the therapeutic and prophylactic properties of gamma globulin against ELL in laboratory animals (mice) was carried out as follows.

 For the experiment, live microconidia of cryptococcus fungus with a concentration of 20 million / ml and lyophilized gamma globulin with an activity in ELISA of 1: 1000, prepared as described in example 1, were prepared. 35 mice were taken for the experiment to determine the therapeutic and prophylactic properties of gamma globulin which was introduced live microconidia of cryptococcus at a dose of 0.1 ml with a concentration of 20 million / ml After 14 days, 10 control mice were decapitated and inoculations were made on lung schools with CDA from lung tissue. The remaining mice, divided into two groups, were injected with gamma-depthin at a dose of 0.1 ml and 0.2 ml (0.1 ml every other day). After 7 days of incubation, the mice were decapitated, and KDA was seeded from lung tissue of mice. Observation from the time of seeding was conducted for 15 days. The properties of gamma globulin were evaluated by the percentage of the occupied area of the grown cryptococcus colonies. The results of the experiment are shown in the table.

 According to the results given in the table, it is clear that a single administration of gamma globulin has a positive therapeutic effect in 89% of cases, a double - in 93% of cases. It should be noted that the number of infected mice decreased from 90% (control) to 50%.

 Thus, we can conclude that cattle gamma globulin has a therapeutic effect against ELL, and a double administration of the drug gives better results.

 Example 3. Checking the therapeutic and prophylactic properties of gamma globulin against ELL in sick animals was carried out as follows. Gamma globulin was prepared as described in example 1.

 12 sick horses that had not previously undergone drug treatment were taken into the experiment. Four horses were in the early stages of the disease, seven were of moderate severity, and one was in a severe form of the disease. Gamma globulin in a dose of 10 ml was administered twice and every other day to the lower third of the neck to horses with an early and moderate severity of the disease. In the latter case, a double dose of gamma globulin was used. Three days after the administration of the drug, an improvement in the condition of the first two groups of animals was observed. They began the healing process of ulcers. Complete recovery of animals of these groups occurred after 40 days. Despite the introduction of a double dose of gamma globulin, a horse with a severe form of the disease fell.

 Example 4. Checking the therapeutic and prophylactic properties of gamma globulin against ELL on sick horses was carried out as follows. Gamma globulin was prepared as described in example 1.

 12 horses with epizootic lymphangitis in various stages of the disease and not previously treated were taken into the experiment: 8 horses were in the early stages of the disease, 2 horses were of moderate severity and 2 horses were in severe form of the disease. Gamma-globulin in a dose of 10 ml was administered twice and every other day to the lower third of the neck to horses with an early and moderate severity of the disease. In the latter case, a double dose of gamma globulin was used. Three days after administration of the drug, an improvement in the condition of animals in the early and moderate severity of the disease was observed. They began the healing process of ulcers. Complete recovery of animals of these groups occurred after 40 days. Horses with severe forms of the disease fell, despite the introduction of double doses of gamma globulin. The control group consisted of 15 horses treated with antibiotics and ASD-2 fraction. Of these, 9 horses identified in the initial stage of the disease underwent treatment and were considered recovered, the rest fell.

Thus, the above information indicates that when using the proposed method the following set of conditions:
- gamma globulin against ELL, embodying the proposed method in its implementation, is intended for use in agriculture, namely in veterinary medicine;
- gamma globulin obtained by the proposed method has high therapeutic and prophylactic properties;
- for the proposed method in the form described in the independent claim, the possibility of its implementation using the means and methods described in the application or known prior to the priority date is confirmed.

 Therefore, the invention meets the patentability condition "industrial applicability".

Sources of information
1. Petrovich S.V. Epizootic lymphangitis. Mycosis of animals. / M., 1989, 79 - 82.

 2. Lopatnikov G.I. Experience in the treatment of horses lymphangitis // Veterinary medicine, 1954, 8, 32.

 3. Naulainen A.I. Therapy of epizootic lymphangitis of horses with sulfoetrol and monoseptol // Veterinary Medicine, 1951, 9, 61.

 4. Reference. Infectious and invasive diseases of horses. Epizootic lymphangitis. - M .: Kolos, 1976, 209 - 219.

 5. Bayakhunov Y.K. The search for an effective method of treatment and the development of a set of measures to eliminate the epizootic lymphangitis of horses / Tr. Alma-Ata Institute of Veterinary, 1955, 8, 177 - 188.

 6. Higelevsky B.N. Epizootic lymphangitis. Veterinary Encyclopedia, - M., 1976, 676 - 681.

Claims (4)

 1. A method of producing gamma globulin for the treatment of horses' epizootic lymphangitis from a biological fluid, characterized in that hyperimmune blood serum of animal producers is used as biological fluid, and cattle that are subjected to repeated hyperimmunization with the pathogen in two cycles with an interval of 29 - 31 days, while during the first cycle, cattle are hyperimmunized for 3 to 4 days in a dose of 3-5 ml once, and during the second of the cycle - for 2 to 3 days in a dose of 7 to 9 ml once, 4 to 6 days after the end of hyperimmunization, the animals are bled, the hyperimmune serum is separated from the blood obtained, the pH of which is set in the range of 6.9 - 7.2 and a 50% aqueous solution of polyethylene glycol is added to it to a final concentration of 20%, after a two-hour exposure, the hyperimmune serum is centrifuged to isolate the desired product.  2. The method according to claim 1, characterized in that cattle is used at the age of 1.5-2.0 years, weighing 250-280 kg. 3. The method according to claim 1, characterized in that to isolate gamma globulin, hyperimmune serum is centrifuged at 2500 min ^-1 for 25 - 35 minutes  4. The method according to claim 1, characterized in that the obtained gamma globulin is dried by lyophilization to ensure the stability of the properties of the drug during storage.

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