RU2201758C2 - Method for prophylaxis and treatment of mastitis in cows - Google Patents

Abstract

FIELD: veterinary science. SUBSTANCE: for preventing and treating cows against mastitis they are introduced with tissue preparation as filtrate of mammary gland extract of healthy animals stabilized with mannitol. The method enables to increase efficiency of prophylaxis and treatment of mastitis in cows. EFFECT: improved results of therapy and prophylaxis. 2 cl, 3 ex, 5 tbl

Description

 The invention relates to veterinary medicine, and more specifically to biological preparations having the property to adequately stimulate (activate) the general nonspecific resistance of an organism and factors of local (local) protection of breast tissue in cows.

 It is known that mastitis is a widespread disease of dairy cows in all countries of the world (V.I. Mutovin. Fighting with mastitis of cows. - M .: Kolos, 1974; V.P. Goncharov, V.A. Karpov, I.L. Yakimchuk. Prevention and treatment of mastitis in animals. - M.: Rosselkhozizdat, 1987; AI Ivashura. System of measures to combat mastitis in cows. - M .: Rosselkhozizdat, 1991).

 The works of G.V. Zvereva, 1988; I.N. Afanasyeva, 1989; V.P. Parikova, 1990, it is proved that each cow with mastitis loses from 7.8 to 9.3% of the daily milk yield, and the average milk yield from the diseased share compared to the healthy one is 23% less (V. Velikachevina, 1992). It is established that milk obtained from cows with mastitis is unsuitable for the preparation of fermented milk products and cheese making. In addition, there are reports that calves obtained from mastitis cows suffer from dyspepsia and other disorders of the gastrointestinal tract 2-3 times more often and die 4-5 times more often than calves from healthy mothers.

 It is also known that the most dangerous periods for the occurrence and development of mastitis in cows are the periods of start-up and dead wood, the degree of animal incidence is directly dependent on the level of the general natural resistance of the organism and directly local (local) immunity of the mammary gland.

 To stimulate the body's natural resistance, many scientists recommend using biogenic preparations, among which the most effective are tissue preparations (Fibs, peat, aloe extract, peloiododistylate, suspension and placenta extract, L-103 hydrolysin, agar-tissue preparation, hemolysate, enzyme hydrolysin) made according to the method of V.P. Filatov from the tissues of animals and plants (I. A. Kalashnik. Tissue preparations and their use. - M., 1976, 198 S.). These drugs have a wide range of biological effects on the body, contribute to the restoration of physiological processes, increase defenses.

 However, these drugs are intended mainly for medical purposes, their industrial production for veterinary and livestock needs has not yet been established. Therefore, in a number of zones and regions of the Russian Federation, they are trying to prepare them on their own (handicraft) in laboratories and meat processing plants, as a result of which they differ in biological activity and naturally effectiveness.

 The disadvantages of these drugs include the lack of methods and methods for their intended use - for the prevention and treatment of breast pathology in cows, which requires additional financial resources for their development.

 A biogenic tissue preparation of veterinary use is also known - biostimulgin (Instructions for the preparation and control of biostimulgin, approved by the GUV of the Ministry of Agriculture of the USSR, 03/13/79).

 Biostimulgin has a general stimulating effect on the animal organism, activates the manifestation of the stage of the reproductive cycle, and in some cases helps to suspend the development of inflammatory processes in the reproductive organs of females.

 However, biostimulgin has a weakly expressed specific orientation associated with a direct effect on the factors of local immunity of organs and tissues.

 In addition, the preparation does not contain mucopolysaccharides, which are known to have bactericidal, bacteriostatic, virus-inhibiting and virus-neutralizing properties. The disadvantages of this prototype include low biological activity compared with the claimed drug, the remoteness of the therapeutic effect, as well as the lack of stabilization, so the shelf life and use of biostimulgin are limited (no more than 6 months from the date of manufacture). In this regard, the drug is not widely used in obstetric and gynecological practice.

 The closest prototype to the mechanism of action of the claimed invention is the application for Russian patent 2141832, "Uteroline for the prevention and treatment of symptomatic infertility in cows." Authors: I.N. Zyubin, M.F. Zyubina, V.G. Chernykh, and O.N. Samokrutova (publication November 27, 1999, BI 33). According to this prototype, a targeted tissue preparation is proposed - uterolin, which contributes to the rapid restoration of disorders in humoral and cellular immunity, stimulates the mechanisms of general nonspecific immunobiological reactivity of the body, and activates the natural factors of local (local) immunity of the reproductive organs.

 However, according to this prototype, which is practically devoid of drawbacks, it is impossible to successfully carry out highly effective prophylaxis and therapy of the pathology of the mammary gland, since, having high selective specificity with respect to factors of local (local) immunity of the genital organs, uterolin native does not significantly indicators of local protection of the mammary gland in cows.

 The aim of the present invention is the creation of a new, more effective biological product with the property to stimulate both general nonspecific resistance of the body and activate the natural factors of local (local) immunity of breast tissue in cows during the prevention and treatment of mastitis.

 This goal is achieved by using a new tissue preparation of directed action - native ureralin (from the Latin word uber - udder) made from the tissues of the mammary gland of cows that had previously had nonspecific postpartum mastitis.

 The technology for obtaining the drug is based on the methodology of academician V.P. Filatov with some additions by the authors (stabilization and sterilization of the drug).

The manufacture of the drug includes the following steps:
- procurement and processing of fabric raw materials;
- cold preservation;
- homogenization (tissue grinding);
- extraction and filtration;
- stabilization of the drug and its sterilization with fluid vapor;
- packaging and evaluation of the biological activity of the drug.

 The raw material for the manufacture of native uberalin is the mammary gland of cows, which does not have pathological changes.

The processing of raw materials is as follows: the mammary gland of a cow is taken from a meat processing plant or directly from the farm from animals that were just slaughtered for meat, placed in a clean glass jar or an enameled container, tightly closed and wrapped with parchment paper. Then placed in a refrigerator for preservation and maturation at a temperature of 2-4 ^o C and incubated for 6-7 days, after which the tissue is passed through a meat grinder and optionally through a homogenizer 302.

The resulting homogenate (minced meat) is weighed and poured with an extractant - physiological saline in a ratio of 1: 5, thoroughly mixed, poured into a glass bottle, corked, wrapped with parchment paper and placed in a refrigerator for extraction at a temperature of 2-4 ^o C for 24 hours.

 After daily extraction, the tissue mass is filtered through gauze, squeezing slightly, and additionally through a dense sterile cotton-gauze filter (filter paper or a multilayer filter can be used).

 The resulting filtrate is stabilized with mannitol at the rate of 2% per 100 ml of the drug.

 Stabilized in this way, the drug is packaged in bottles, closed with rubber stoppers with aluminum caps and subjected to fractional sterilization with fluid steam (for 3 days for 30 minutes).

By chromatography and using an amino acid analyzer in the prepared preparation, all irreplaceable amino acids, complexes of polypeptides, modified components of nucleic and carboxylic acids with high biological activity were found and identified. Carbohydrates, macro- and microelements, which include calcium, phosphorus, manganese, zinc, copper, nickel, and iron, have been identified. In addition, the preparation contains vitamins of the group A, B, B ₂ , B ₁₂ . Therefore, the active principle in the preparation is not only biogenic stimulants (“resistance” substances) formed during the process of limited autolysis during its manufacture, but also a protein complex with micro-macroelements and vitamins.

The resulting preparation has the following properties necessary to achieve the objective of the invention:
1. Long-term storage stability for at least 2 years (observation period) with 100% biological activity.

 2. Promotes the rapid restoration of disorders in humoral and cellular immunity, stimulates the mechanisms of general nonspecific natural resistance of the body; under the influence of uberalin, the content of hemoglobin, erythrocytes and leukocytes significantly increases in the blood of animals, the level of bactericidal and lysozyme activity of blood serum increases, the phagocytic-absorbing and protective functions of RES are activated (see examples of specific performance, table 1-2).

 3. It has a pronounced specific orientation of action on the mammary glands, while factors of local (local) protection of mammary gland tissues are activated, their antimicrobial properties and resistance to the microbial factor are enhanced, which impedes the development of the inflammatory process, and if there is a faster resolution.

 4. It is adequately effective when used for prophylaxis and therapy in combination with etiotropic agents for obstetric and gynecological pathology in cows that cause symptomatic infertility (see examples of specific use, acts and the report of the production test of the drug).

Case Studies
Example 1. The study of the influence of native Uberalin on factors of the general non-specific resistance of the animal organism
The research was conducted on the basis of the closed enterprise "Union" of the Uletovsky district of the Chita region.

 Thirty clinically healthy black-and-white cows were taken into the experiment. They were divided according to the principle of analogues into three equal groups (10 animals each).

 Before the introduction (to establish baseline indicators), and then on the 5th and 10th days after the administration of drugs from all cows, blood was taken for examination for hematological and immunological parameters.

 The first (experimental) group of animals was treated with the proposed tissue preparation - native uberalin, the second (experimental) group of cows - uterolin (prototype). The drugs were administered subcutaneously, at a dose of 0.1 ml per kg of animal body weight. The specified dose is taken from the calculation of the dosage established for uterolin (prototype).

 Animals of the third group served the purpose of control, they were injected with saline in the same dose as the test drugs. The results are presented in table. 1 and 2.

 From the data table. 1 shows that in the cows of the control group on the 10th day, the blood counts did not differ significantly from those before the use of tissue preparations in the experimental groups.

 In the 1st experimental group, where uberalin native was used, after 10 days the hemoglobin content increased by 10.5% compared to baseline and control, the number of red blood cells - 11.55%, white blood cells - 9.42%, and lymphocytes - by 11.93%.

 The number of young, stab, segmented neutrophils and eosinophils decreased by 18.04, respectively; 20.3; 8.22 and 9.27% at P <0.0001. The number of basophils and monocytes has not changed.

 In the 2nd experimental group, where uteroline was used, the hematological indices on the 11th day increased slightly: hemoglobin - by 1.36%, which is 13.19% lower than in cows of the 1st experimental group, erythrocytes - by 2 , 62% or 15.78% less than in the 1st experimental group, and leukocytes - 0.68% or 10.76% lower than in the cows of the 1st experimental group. The number of lymphocytes remained unchanged.

 The number of young, stab, segmented neutrophils and eosinophils decreased by 4.48, respectively; 0.4; 0.18 and 0.5% in relation to the initial (background) data and control (at P <0.05).

 Animals of the 1st experimental group treated with native uberalin had higher indices of natural resistance than the control and cows of the 2nd experimental group (Table 2).

 The total number of immunoglobulins in the serum of cows was: in the 1st experimental group, 39.8 ± 0.7 g / l against 24.4 ± 0.21 g / l in the 2nd group treated with uterolin. The lowest content of immunoglobulins was observed in the serum of cows of the control group - 22.7 ± 0.11 g / l.

 After the use of native uberalin on day 11, in cows of the first group, the total protein content in the blood in relation to the control is higher by 23.7 g / l and 22.6 g / l than in cows of the second experimental group.

 The bactericidal activity of blood serum on the 11th day increased to 62.3%, which is 41.4% higher than that of cows in the control group.

 In the 2nd experimental group of animals where uteroline was used, this indicator was 23.9% less active than in animals of the 1st experimental group.

 Uberalin native also contributed to a significant increase in cellular factors of the body's immune defense, which manifested itself in the activation of the phagocytic activity of neutrophils. So, in the cows of the 1st experimental group after the use of the drug, the phagocytic activity of neutrophils was the highest - 68.8%, which is 17.9% more activity than in the cows of the control group (P <0.001). In cows of the 2nd experimental group, phagocytic activity was 50.1%, which is 17.5% less than in the 1st experimental group of animals (P <0.05).

 Thus, the obtained results indicate that the proposed tissue preparation, uberalin native, reliably activates both humoral and cellular factors of non-specific resistance of the animal organism.

 The immunomodulatory and stimulating effects of native uberalin on cellular and humoral defense mechanisms in the body of cows reach a maximum 10 days after the use of the drug.

 Example 2. The effect of native ureralin on factors of local protection of the mammary gland in cows.

 It is known that the mammary gland (udder) of females has all the necessary non-specific and specific factors (mechanisms) of local (local) immunity. The main factors of local protection of the mammary gland include: poly- and mononuclear phagocytes, natural oxonins (lactoglobulins), as well as the immunoreactive enzyme - lysozyme- "M".

 Unfortunately, while studying the etiopathogenesis of inflammatory processes in the mammary gland and developing therapeutic and prophylactic agents, very little attention has been paid to the state of factors of local protection of the mammary gland to date.

 Observations show that the use of drugs for the prevention and treatment of cows with mastitis patients without taking into account their effect (effect) on the immunobiological activity of the mammary gland often does not have the desired effect. Many drugs suppress the tension of local protective reactions of the mammary gland, destroy mucopolysaccharides of the mucous membrane and often lead to serious consequences. Therefore, it is of great interest to take into account the state of factors of local (local) breast protection and their activation in the provision of therapeutic and preventive care to animals with mastitis.

 Based on the foregoing, and in a set of measures to develop schemes and methods for the treatment and prevention of mastitis in cows using the biological tissue preparation uberalin native, the authors conducted studies to study the effect of this drug on factors of local (local) resistance of breast tissue in cows.

 Due to the lack of background indicators of local mammary gland resistance in cows of the study zone, the authors studied these parameters in healthy and mastitis-infected cows during various periods of lactation.

 For these purposes, on the basis of the Shishkinskoye GUSHP two groups of black-and-white analogue cows were formed with 10 animals in each group.

 The 1st group included healthy animals, the 2nd - with the presence of subclinical and clinically pronounced forms of mastitis.

 At the beginning, middle and end of lactation, animals from both groups were taken - colostrum, milk, breast secretion - and subjected to laboratory studies on the main indicators of local resistance of the breast - the number of somatic cells, the content of the immunoreactive enzyme lysozyme-M and the level of lactoglobulins.

 During the experiment, animals were regularly monitored with periodic medical examination, with particular attention to the condition of the mammary gland. The results of these studies are shown in table.3.

 Comparative analysis of the materials of the table. 3 shows that regardless of the lactation period (period) in colostrum, milk and breast secretion of cows with mastitis, the content of somatic cells is significantly higher in comparison with healthy animals. At the same time, the titer (activity) of the immunoreactive enzyme lysozyme-M and the immune fraction of the protein -χ-lactoglobulin, on the contrary, is reduced.

 These data were used by the authors in further work as a criterion for assessing background indicators.

 A study of the influence of native uberalin on the main indicators of local immunity of the mammary gland was carried out in the above farm on two groups of analog cows with 15 animals each, located in the dry period. Before the formation of groups, all animals underwent clinical examination and checked for the presence of subclinical (hidden) forms of mastitis. Animals with negative tests for subclinical mastitis were taken in the experiment.

 The first experimental group of cows was treated with native uberalin three times, subcutaneously, at a dose of 0.1 ml per kg of body weight according to the following scheme: 30-20 days and the 10th day before birth. The second (control) group of cows was not processed.

 Before and after drug administration, colostrum (milk) was taken from animals of both groups and studied for the main indicators of breast resistance. The research results are given in table. 4.

 From the data table. Figure 4 shows that the main indicators of local protection of the mammary gland in cows of the experimental group after treatment with uberaline significantly differ from those of the control group of animals.

 So the activity of the main criterion for local protection of lysozyme-M in the cows of the experimental group was 28.8 mm versus 16.9 mm in the control.

The number of somatic cells, on the contrary, sharply increases in animals of the control group and reaches 7520 thousand / ml ³ against 207 thousand / ml ³ in cows of the experimental group, which indicates the inhibition of nonspecific resistance of breast tissue in cows of the control group.

 Significant changes occur in the cytological picture of colostrum. So, in cows of the experimental group, the number of lymphocytes from 38.8 ± 0.85 per day of labor by 5-7 days after the birth decreased to 31.0 ± 0.02%, the number of eosinophils and monocytes, respectively, from 3.46 ± 0.2% up to 0.33 ± 0.3% and from 25.0 ± 0.4% to 14.82 ± 0.88%.

 In animals of the control group, for the same period of studies, the number of eosinophils, neutrophils and lymphocytes is greater, and monocytes are less.

 Colostrum eosinophilic and neutrophilic leukocytosis in animals of the control group indicates a significant influx of RES cells from the blood into the mammary gland tissue, which is a protective factor for the suppression of nonspecific resistance of this organ. The same, according to the authors, explains the fact of a sharp increase in the number of somatic cells in the colostrum of the control group of cows.

 During the final dispensary examination of animals at the end of the experiment, 3 cows of the control group showed acute serous-catarrhal, and 4 - subclinical (hidden) mastitis.

 Thus, the developed scheme for the phased use of native uberalin provides a high level of basic indicators of local (local) protection of breast tissue and increases their resistance to the microbial factor, which allows us to recommend this scheme for the prevention of breast pathology in cows. During 1999, 97 cows were processed at the Shishkinskoye State Farm for Agricultural Control, of which 99.3% of the animals had a preventive effect.

 The authors studied the therapeutic efficacy of native uberalin both in a monovariant and in combination and comparison with etiotropic (antimicrobial) drugs - bacteriofungicide (NIIV VS preparation, copyright certificate 1050155), mastisan-A and masticide.

To achieve this goal, 4 groups of analog cows were formed with the presence of subclinical forms of mastitis:
The 1st (experimental) group of cows (35 animals) was treated with uberaline in combination with a bacteriofungicide according to the following scheme: native uberaline was injected subcutaneously at a dose of 0.1-0.2 ml per kg of animal body weight, and the bacteriofungicide was retrograde into the notched space , under the base of the affected udder lobes in a dose of 25-30 ml;
The 2nd (experimental) group of cows (20 animals) was treated with mastisan-A (a complex preparation consisting of benzylpenicillin potassium salt, streptomycin sulfate, sulfadimizine or norsulfazole sodium, sunflower oil and emulsifier), which were administered intracisternally at a dose of 5-10 ml two once a day with an interval of 12 hours;
3rd (experimental) group of cows (35 goals) - masticide (complex preparation containing antibiotics and sulfonamides) - intracisternally, at a dose of 10 ml, with an interval of 24 hours between injections;
The 4th (control) group of cows (35 animals) was not treated.

 The results of the therapeutic effectiveness of the tested drugs and schemes (methods) of their use are shown in table. 5.

 From the above table. 5 data shows that the highest therapeutic effect was obtained in the 1st experimental group, where the tissue preparation uberalin was used in combination with an etiotropic agent - bacteriofungicide, while the overall therapeutic effect in this group was 97.1%, which is 17 , 1% higher than in the 2nd and 14.2% than in the 3rd experimental group.

 Relatively good results were also obtained in the treatment of mastitis in cows of the 2nd and 3rd groups, where the basic complex preparations Mastisan and Masticide were used, while the therapeutic efficacy in the groups was 80.0 and 82.9%, respectively. However, the methodology for the use of these drugs is very laborious, since both of them are administered via the nipple canal intracisternally. In addition, the active principle of these drugs are antibiotics, which have an inhibitory effect on the body and are excreted in milk for a long time after their use. According to GOST 3264-88 "Cow's milk" the presence of inhibitors is not allowed, such milk is subject to culling.

 The complex scheme (methodology) for the treatment of mastitis in cows developed by the authors using an environmentally friendly tissue preparation of native and etiotropic (antimicrobial) uberalin bacteriofungicide according to the technique of implementation, therapeutic and prophylactic efficacy and the obtained indicators compares favorably with the basic methods of treatment and prophylaxis of mastitis with antibiotic-containing drugs like mastisan and masticide.

 Example 3. Scientific and industrial experience on the use of native uberalin for the prevention and treatment of mastitis in cows.

 In accordance with a temporary permit approved by the veterinary department of the Agro-Union of the Chita Region (now KSHiP) dated November 15, 1992 to Uberalin Native, during 1992-1999. the specified preparation was tested on 4562 cows and heifers in 12 farms of the whole dairy zone of the Chita region (Beklemishevsky, Kenon, Makkaveevsky, Zastepinsky state farms; the ZabNIISKh Kolocho experimental farm, the Way to Communism collective farms, named after Chapaev, JSC Rodina, named after 40 years of October, "Association", "Victory" of the Uletovsky district and the collective farm "Talachinsky" of the Karymsky district).

 To carry out this work, 286 liters of native uberalin were prepared and used. The work was carried out with the participation of professional specialists of the aforementioned farms.

 1. For prophylactic purposes, native Uberaline was prescribed during dead wood (run) to 1697 pregnant cows and heifers according to the scheme: 30-20 and 10 days before the birth and in the first 5 days after the birth. The drug was administered subcutaneously according to the method of its use in a dose of 0.1-0.2 ml per kg of animal body weight.

 The obtained results confirmed the data of previous experiments on the development of methods for the use of native uberalin. The appointment of native ureralin to stylish animals in the last trimester of pregnancy provides prevention of postpartum mastitis in 99.8% of cows and heifers.

 2. For therapeutic purposes, native Uberalin was used in combination with a bacteriofungicide (complex etiotropic preparation - authors MF Zyubina, GM Larionov, IN Zyubin, copyright certificate 1050155 dated July 23, 1983) on 2865 cows with acute postpartum mastitis.

 At the same time, native uberaline was injected subcutaneously at a dose of 0.1-0.2 ml per kg of animal body weight, and bacteriofungicide in the notched space was retrograde at a dose of 25-30 ml with an interval of 24 hours between injections.

 The combined use of uterine native and antimicrobial bacteriofungicide in subclinical and acute postpartum mastitis in cows exceeds the results of treatment of animals with basic methods (antibiotic therapy) by 24.5%. At the same time, the overall therapeutic effect was 92.3%, including with a catarrhal course of the pathological process - 91.5%, serous - 95.0% and catarrhal-purulent - 82.0%.

 The obtained positive results on the use of native uberalin for the prevention of subclinical and postpartum mastitis in cows and in combination with an etiotropic drug for the treatment of sick animals gives reason to recommend it for widespread use in obstetric and gynecological practice.

 According to the mechanism of action, native uberalin has a pronounced directed action, since the organs and tissues from which it is prepared (mammary gland) are directly related to the function of the mammary gland, metabolism and the regulatory mechanism.

 According to the mechanism of action, native Uberalin is a natural stimulant and regulator of the body's defenses and those organs from which it is made.

 From an environmental point of view, this is a biologically pure, completely harmless native drug, does not have side effects on the animal organism, does not have anaphylactogenic properties, histamine-like effect and switching.

 The economic efficiency of native ureralin is presented in examples of specific performance and a report on the production test of an experimental batch of native ureralin (see the appendix).

Claims (3)

 1. A method for the prevention and treatment of mastitis in cows, comprising administering a tissue preparation, characterized in that a mannitol stabilized filtrate of the breast extract of healthy animals is used as the tissue preparation.  2. The method according to claim 1, characterized in that for prophylactic purposes, the tissue preparation is administered twice for 30-20 and 10 days before calving, subcutaneously at a dose of 0.1-0.2 ml per kg of body weight.  3. The method according to claim 1, characterized in that for therapeutic purposes, bacteriofungicide is additionally introduced into the indented space at a dose of 25-30 ml twice with an interval of 24 hours

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