RU2742160C1 - Method for treatment of aleutian mink disease - Google Patents

Abstract

FIELD: veterinary medicine; immunology.

SUBSTANCE: invention relates to veterinary medicine and immunology and relates to the treatment of Aleutian mink disease. Animals are injected subcutaneously with the oligopeptide alloferon at a dose of 0.5 mg per injection twice with an interval of 6 days.

EFFECT: invention provides effective treatment of the disease and reduces mortality among minks due to the antiviral and immunomodulatory properties of alloferon.

3 cl, 8 dwg, 2 tbl

Description

The invention relates to veterinary medicine, fur farming and immunopharmacology and can be used for the treatment of Aleutian mink disease.

Aleutian Mink Disease (ALD) is a viral disease of the marten family that causes a large death in animals. ABN is widespread in the USA, Canada, Norway, Sweden, Finland, Denmark, Great Britain, etc. In Russia it is registered in all areas where minks are bred

Aleutian mink disease (viral plasmacytosis of minks) is a contagious, slowly developing infectious disease characterized by widespread plasma cell proliferation (plasmacytosis), hypergamma globulinemia, hemorrhagic diathesis, arteritis, hepatitis, anemia, and progressive exhaustion of animals; it is an immunological disease of minks resulting from persistent viral infection. The incubation period lasts from 6 to 150 days, usually 10-15 days. The acute course of ABN lasts from 10 to 30 days, chronic from 2 to 24 months with a slow inevitable progressive development and death.

The ABN-causing virus belongs to the Parvoviridae family and the Parvovirus genus. Virions are isometric, 18-22 nm in diameter and do not have a supercapsid membrane. They consist of a capsid of a cubic symmetry type and a genome. The genome is represented by a single-stranded linear DNA molecule.

The virus is stable in the external environment. In untreated tissues of diseased minks it retains infectivity for a long time. Does not collapse under the influence of high temperatures, formalin. Low temperatures preserve the virus. Resistant to organic solvents and cellular enzymes (protease, nuclease).

Infection of animals occurs both horizontally and vertically. The virus can be transmitted by contact, sexual, nutritional and aerogenic routes, carried by insects. It retains infectious activity on wool and cells for 40 days.

Clinical signs of the disease are detected shortly before the death of the animal, 1-24 months after infection. Typical of them are - progressive emaciation, recurrent bleeding from the nose and mouth (on the mucous membrane of the oral cavity - small bleeding sores), increasing thirst (midday), anemia, fever. The animals are inactive - "drowsy look", the wool is tousled and dull, the molt is delayed. The tip of the nose and crumbs of the fingers become yellow in diseased minks. The animals have diarrhea, tarry feces. In some individuals, signs of damage to the nervous system are observed - balance, coordination of movements are disturbed, paresis and paralysis develop (more often of the pelvic limbs). Complication of secondary infections is possible.

In females infected before the rutting period, abortion, emptying, resorption of fetuses, and loss of maternal instinct are considered typical. In some animals, clinical signs do not appear for several years, but the indicators of whelping decline sharply.

In the course and outcome of the Aleutian disease, kidney damage is important. A feature of the Aleutian mink disease is the fact that the initial cycle that has begun continues until the end of the mink's life, since the formed antibodies are not able to neutralize the invading virus. This ensures the persistent circulation of the virus in the body and the continuous formation of immune complexes.

The prognosis is poor. In the asymptomatic form, up to 20% of minks die: in the progressive form - during the first year after infection - more than 50%, and in the second year - up to 80%. Most often, the disease is recorded among young minks in summer or autumn.

There are no specific means of prevention and treatment for ABN. In practice, in the case of a diagnosis of ABN, only symptomatic treatment with the use of antibiotics, vitamins, protein hydrolysates, immunosuppressants, hormones is indicated. The spread of the disease is fought mainly by the destruction of infected livestock and the burning of used equipment.

Thus, the creation of an effective method for the prevention and treatment of ABN, which could be introduced into fur farming practice, is extremely important.

The number of works on the creation of drugs for the treatment of ABN is extremely small. So Poletaev A.B. and Budykina T.S. a vaccine preparation for the prevention of ABN is proposed (RF patent 2218354 "Method for the specific prevention of Aleutian mink disease"). Anti-idiotypic antibodies or their F (ab) -fragments, which are immunochemical analogs of the main antigens of ABE viruses, are used as a vaccine preparation. The technical result of the patent is a significant reduction in the incidence and mortality of minks.

Employees of the St. Petersburg State Academy of Veterinary Medicine A.F. Kuznetsov and V.M. Korotkov for the treatment of ABN tested the antiviral drug Viturid, the active principle of which is an organic mercury complex. Studies conducted on the broodstock and slaughter livestock of young animals with ABN confirmed the positive effect of Viturid (oral) on biochemical processes, morphology of cells and tissues. Within 3-4 months, 40% of minks were healed from ABN, which indicates a fairly active immunological restructuring of the body of animals under the action of the drug. However, Viturid is currently not produced.

There is a patent RU 2036654, publ .: 09.06.1995. (Grishin Yu.I., Kovalevskaya A.M., Stonik V.A., Avilov S.A., Vlaznev V.P., Slugin V.S. "Means for the prevention and treatment of Aleutian mink disease"). The authors proposed the use of cucumarioside triterpene glycosides as a means for the prevention and treatment ABN. The amount of triterpene glycosides is obtained from natural raw materials-decoction of sea cucumber Cucumaria joponica by a series of physical and chemical procedures. Experiments on female minks have shown that the use of the proposed tool allows you to increase the yield of puppies in comparison with the control group. This decision can be taken as a prototype...

There is a wide range of antiviral drugs that are effectively used to treat diseases in humans. However, no information was found about their use for the treatment of ABN.

One of the antiviral drugs used in clinical practice is the drug "Allokin-alpha" (P N002829 / 01-210610). "Allokin-alpha" is effective in the treatment of patients suffering from chronic papillomavirus infection caused by oncogenic human papillomaviruses; in the complex therapy of chronic recurrent herpes of types 1 and 2; as part of the complex therapy of moderate (icteric) forms of acute hepatitis B.

The active principle of this drug is the linear oligopeptide histidyl-glycyl-valyl-seryl-glycyl-histidyl-glycyl-glutaminyl-histidyl-glycyl-valyl-histidyl-glycine Alloferon. Alloferon was originally isolated from a natural object - the tissues of the immune system of calliphorid fly larvae ( Calliphora vicina , Calliphoridea family) - the so-called "surgical larvae" with a long history of use in medicine. Currently, Alloferon is produced synthetically, using the methods of classical peptide synthesis. In terms of its chemical structure and properties, the synthetic product is completely identical to the natural Alloferon secreted from the tissues of the larvae. Alloferon is registered as a pharmaceutical substance (P N002830 / 02-240510) and is produced by Russian enterprises.

The object of the invention is a method for the treatment of Aleutian mink disease using the Alloferon oligopeptide or preparations based on it.

The technical result of the invention is a method that allows reducing the incidence and mortality of minks.

The specified technical result is achieved due to the fact that the claimed method for the treatment of Aleutian mink disease, in which therapy is carried out by subcutaneous injections of an antiviral and immunomodulatory agent, characterized in that the oligopeptide Alloferon is used as an antiviral and immunomodulatory agent, which is administered twice with an interval of 6 days in the amount of 0.5 mg per injection.

It is preferable that the drug "Allokin-alpha" is used as the preparation containing Alloferon.

Preferably, subcutaneous injections of the antiviral and immunomodulatory agent are administered to minks aged 30 days and older.

Implementation of the invention

The effectiveness of the proposed method is confirmed by the data of the experiment, which was carried out in one of the fur farms of the Leningrad region. Work planning was carried out by employees of the St. Petersburg State Academy of Veterinary Medicine (SPbGAVM). Immunological studies of mink blood serum were carried out in the clinical and biochemical laboratory of the SPbGAVM. The experiment starts on May 27, 2019, ends on December 10, 2019, and lasts 6.5 months.

The experiments used "Blue" minks (standard). The animals were kept in two-row sheds, with a longitudinal working passage, which are sheds with a gable roof, with cages located inside the doors. Sheds are equipped with running water, electric lighting. Feeding and watering of animals was carried out, according to the existing technology, manually. The animals were fed according to standard diets.

Two groups of minks were formed, in which the ABN virus was detected according to the results of diagnostics: 1 - experimental group - 5 males; 25 females, 2 - control group - 5 males; 25 females. The minks are 30 days old.

Minks of group 1 were injected subcutaneously with 0.5 ml of Alloferon solution 2 times with an interval of 6 days (Alloferon dose 0.5 mg). To prepare the solution, an ampoule of the drug "Allokin-alpha" containing 1 mg of Alloferon was diluted in 1 ml of physical. solution (0.9% NaCl).

Minks of the second group (control) were injected with 0.5 ml of sterile saline solution.

The state of the minks was assessed by clinical, laboratory (biochemical, molecular genetic, morphological) studies and economic indicators.

Clinical researches. The clinical condition, mobility of animals, appetite, body weight and other parameters were taken into account. To examine the mucous membranes of the animals, they were fixed by conventional methods, the mouth was opened with the help of ribbons. The study of mucous membranes in the dynamics of the experiment was carried out selectively for 3 - 5 minks from each experimental and control groups. According to the results of examination, the mucous membranes of the oral cavity of the experimental minks had a pale pink color. The color of the mucous membranes remained unchanged throughout the experiment.

The subject's minks were very mobile during the entire experiment compared to those of the control group. They reacted vividly to external stimuli (the appearance of people at the cage, shouting, knocking, distribution of food). Among the minks of the control group, there were individuals with a reduced response to stimuli. In general, animals from the control group by the end of the study showed signs of malaise - lethargy, decreased appetite, drowsiness. The physique of the experimental minks was strong and proportional, in contrast to the animals of the control group. The body weight of the control minks was reduced in comparison with the experimental ones. It was found (Table 1) that the average weight in the group of females receiving the drug was 15.1% higher than in the control group. In males, the average weight in the experimental group was 2,570 g, compared to 2,250 g in the control group, which is 12.5% higher.

Table 1.

Mink weight indicators as of October 01, 2019

Control group Colors Females Males Qty Total weight Average weight Qty Total weight Average weight Sergo 25 33750 five 11 250 2250 Test group Sergo 25 39750 1590 five 12 850 2570

The safety of animals in the experimental group by the end of the experiment was 100%. In the control group, 79%.

On the basis of clinical observations, it is obvious that the administration of Allokin-alpha to minks had a positive effect on their bodies.

Biochemical blood tests.

In the blood of minks of the control and experimental groups, total protein was measured, g / l; albumin, g / l; globulin, g / l; ALT, AST (Table 2, Figure 1).

As a result of biochemical blood tests, characteristic changes were revealed, consisting in the fact that with AB in minks of the control group, the level of globulins, urea and creatinine significantly increases compared with the group of minks receiving the drug.

The total protein content in the blood of minks increased due to the development of hypergammaglobulinemia. At the same time, there was a redistribution of proteins, a sharp increase in the level of globulins and a slight decrease in albumin. This suggests that a large number of protein compounds - antibodies are present in the blood. They form virus-antibody immune complexes, which are fixed in the glomeruli of the kidneys, causing the development of glomerulonephritis. In turn, the development of renal failure leads to an increase in the content of metabolic end products (urea and creatinine). When phagocytosing immune complexes by the reticulo-endothelial system, the virus is released in the nuclei of the macrophages of the liver and causes its destruction. Destructive changes in the liver cause an increase in the amount of aspartate aminotransferase (ACT) and alanine aminotransferase (ALT).

The results of biochemical blood tests are shown in Fig. 1.

Destructive changes in blood parameters revealed in the study are inherent to a much greater extent in minks of the control group. The difference between the groups in the concentration of urea and creatinine is especially great, which indicates the positive effect of Alloferon on the functioning of the animal body.

Molecular genetic research. PCR studies by gel electrophoresis confirm the presence of the Aleutian mink disease causative agent in all animals in the experiment.

Morphological studies. When studying the morphology of organs in the kidneys of the animals of the control group, interstitial nephritis, glomerulonephritis and glomerular edema were found (see Fig. 2, which shows glomerulonephritis in the kidneys of females of the control group. Uv. 40x. Coloring GE). In the medulla there are focal, in places merging hemorrhages. There are also microscopic areas of calcification (see Fig. 3, which shows hemorrhages and a site of calcification in the kidneys of females of the control group. Uv. 10x. Coloring of GE). In the kidneys, lymphoplasmacytic infiltration was revealed (see Fig. 4, which shows plasmacytes in the kidneys of females of the control group. Uv. 1000x. Coloring GE).

In the experimental group, males and females have plethora of blood vessels, there are no foci of hemorrhage, mild glomerulonephritis, and lymphatic-plasma infiltration is less pronounced.

In the liver of animals of the control group, total hydropic degeneration of hepatocytes was found (see Fig. 5, which shows the hydropic dystrophy of hepatocytes in females of the control group. Moderate hyperplasia of the epithelium of the bile ducts occurs. Revealed focal lymph-plasmacytic infiltrates not only of the stroma, but also of the bile ducts (Fig. 6, which shows plasmacytes in the liver of females of the control group. Uv. 100x. Coloring GE). Sinusoidal capillaries are filled with blood.

In the experimental group, the changes were less pronounced in males and females.

In the ovaries, the basis of the cortex and medulla are interstitial cells, which are similar in structure to epithelial cells. They are located in cords, resembling the excretory ducts and end sections of the tubular-alveolar glands. A large number of arthretic bodies (dead oocytes) were found in the control group. Primordial, primary, secondary and tertiary follicles are found (see Fig. 7, where primordial, primary and secondary ovarian follicles of females of the control group are shown. Magnification 10x. Coloring of GE). One animal has an ovarian cyst.

In the experimental group, the number of primordial follicles in females increases with a decrease in atretic ones. Lymphocytic-plasma infiltration is less pronounced (see Fig. 8, which shows the primordial and primary follicles of the ovaries of females in the experimental group. Magnification. 10x. Coloring of GE). (Approximately 2.5 - 3 times). There are microscopic foci of lymph - plasmacytic infiltration. In general, the ovaries of females in the experimental group appear to be significantly more preserved.

As a result of the studies of organ morphology, it can be concluded that the change in the control group is characteristic of the Aleutian mink disease. In the experimental group, the changes are less pronounced and more typical for convalescents.

As a result of the studies, it was found that the use of Alloferon ("Allokin-alpha") positively affects the physiological state of animals, improves the digestion processes in sick minks, bringing it closer to physiologically normal, has a beneficial effect on the morphological and biochemical parameters of blood in sick minks.

Economic indicators. The experimental group, in which Alloferon was used, was visually noted by an increase in the density of the fur; when blowing hair into the parting, no free skin areas were found. The hairs were strong and elastic. When stroking, the silkiness of the coat was noted. The color was uniform and uniform throughout the body with a pronounced sheen.

Thus, a 2-fold injection of 0.5 mg of Alloferon 0.5 ml subcutaneously after 6 days at the age of 30 days has a positive effect both on the symptomatology of minks in patients with Aleutian disease and on the final economic indicators.

Claims (3)

1. A method for the treatment of Aleutian mink disease, in which therapy is carried out by subcutaneous injections of an antiviral and immunomodulatory agent, characterized in that the oligopeptide Alloferon is used as an antiviral and immunomodulatory agent, which is administered twice with an interval of 6 days in an amount of 0.5 mg per injection ... 2. The method according to claim 1, characterized in that the drug "Allokin-alpha" is used as the preparation containing Alloferon. 3. The method according to claim 1 or 2, characterized in that subcutaneous injections of the antiviral and immunomodulating agent are performed in minks aged 30 days and older.

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