RU2545986C1 - Preparation trametin for treating gastrointestinal diseases in calves and method for using it - Google Patents

Abstract

FIELD: veterinary science.

SUBSTANCE: declared are a preparation for treating calves' gastrointestinal diseases and a method of treating using the same. The preparation represents a sterile culture fluid produced by culturing the fungus Trametes pubescens (strain 0663) on standard solid and fluid media with sodium selenite and zinc sulphate additionally included into the nutrient medium; that is following by lyophilisation. A method of treating involves adding the preparation to feeding milk. The preparation is introduced in a dose of 180-300 mg per 1 kg of animal's body weight once a day for 4-5 days.

EFFECT: high therapeutic effect of the preparation.

3 cl, 18 tbl, 4 ex

Description

The invention relates to veterinary medicine, namely, to preparations and methods for treating gastrointestinal diseases of calves.

Known use in medicine and veterinary medicine of drugs with antimicrobial and immunostimulating effects and obtained on the basis of xylotroph fungi of the genus Trametes - (Chkhenkeli, V.A., Baltukhaeva T.A., Vlasov B.Ya., Goryaeva N.A., Kalinovich A.E., Kukharenko N.S., Martynova A.Yu. Antioxidant properties of the latest generation of products - biotechnology products: effects detected, mechanisms of action // Bulletin of the Irkutsk State Agricultural Academy. 2012. No. 49. P. 81-91).

The closest solution adopted for the prototype is a method of treating gastrointestinal diseases of calves using a preparation that is a sterile culture fluid obtained by cultivating the fungus Trametes pubescens (strain 0663) on standard solid and liquid nutrient media with microcrystalline cellulose further included in them. . The drug is added to milk for drinking at a dose of 1.5-2.5 ml per 1 kg of live weight of the animal 1 time per day for 4-5 days (patent RU 2429871, A61K 36/07).

The disadvantage of the prototype is the low immunostimulating activity of the drug, as well as the insufficiently long shelf life of the drug and the inconvenience associated with its transportation over long distances.

The task of the invention is to expand the arsenal of tools and methods for treating gastrointestinal diseases of young farm animals.

The problem is solved in that the preparation of trametin for the treatment of gastrointestinal diseases of calves, including sterile culture fluid obtained by cultivating the fungus Trametes pubescens (strain 0663) on standard solid and liquid nutrient media, according to the invention, a sterile culture fluid is obtained by cultivating the fungus Trametes pubescens ( strain 0663) on standard solid and liquid nutrient media with sodium selenic acid and zinc sulfate additionally included in them, followed by lyophilization.

The essence of the invention lies in the fact that in the preparation of trametin for the treatment of gastrointestinal diseases of calves, according to the invention, sodium selenic acid is added in an amount of 3.0 mg / l in the form of a solution, zinc sulfate is added in an amount of 1.0 g / l.

The problem is also solved by the fact that in the method of treating gastrointestinal diseases of calves, including applying the drug to milk for feeding, according to the invention, the drug is administered in a dose of 180-300 mg per 1 kg of live weight of the animal 1 time per day for 4-5 days.

Obtaining a drug for the treatment of gastrointestinal diseases of calves.

The initial step in obtaining the drug is to maintain the producer, i.e. culture of the fungus Trametes pubescens (strain 0663), on solid nutrient medium - on wort agar. The seed is grown on Saburo standard solid nutrient medium, into which microcrystalline cellulose, which activates the producer’s natural metabolic activity, is preliminarily added. Next, seed is grown in a liquid nutrient medium. For seed and fermentation, a medium of the following composition (g / l) is used:

Potassium phosphate monosubstituted 1.88 Potassium chloride 0.94 Magnesium Sulphate 0.94 Sucrose 40,0 Microcrystalline cellulose 12.5 Peptone enzymatic 12.5 Sodium selenic acid 0.003 Zinc Sulfate 1,0

pH 6.0 ± 0.2.

Selenium was introduced into the culture medium for cultivation of the producer fungus in the form of sodium selenite in an amount of 3.0 mg per 1 liter of medium in the form of an aqueous solution of selenic acid sodium (1 ml of a 0.3% solution per 1 liter of medium).

Zinc was introduced in the form of zinc sulfate in an amount of 1.0 g per 1 liter of culture medium.

The optimal concentration of sodium selenous acid and zinc sulfate in the nutrient medium during the cultivation of the fungus T. pubescens was determined by the duration of fermentation and the yield of fungal biomass (Table 1).

Table 1 The salt content in the nutrient medium, g / l The biomass yield, g / l a.s. Fermentation time, days sodium selenic acid zinc sulfate - - 8.90 7 0.001 0.5 9.12 7 0.002 0.75 9.51 6 0.003 1,0 10.52 5 0.004 1.25 10.13 5 0.005 1,5 9.72 5 0.01 1.75 6.25 5 0.015 2.00 3.74 5 Note: a.s.b. - absolutely dry biomass.

The data in table 1 show that the introduction of zinc sulfate at a concentration of 1.0 g / l and sodium selenic acid at a concentration of 0.003 g / l in a nutrient medium reduces the fermentation time from 7 to 5 days, while the biomass yield increases from 8.90 to 10 52 g / l, i.e. by 18.3%.

The seed is grown in Erlenmeyer flasks in a shaker incubator at 220 rpm at a temperature of 28-30 ° C for 4 days. Seeds are introduced into the fermentation medium in an amount of 5-10% by volume.

Fermentation is carried out for 4-5 days at 220 rpm and a temperature of 28-30 ° C.

Then the culture fluid is separated from the biomass of the culture mycelium by centrifugation for 10 min at 6000 rpm, autoclaving at 0.8 atm for 30 min.

Lyophilization of a biological preparation is carried out using a laboratory freeze dryer LZ-9СР (Czech Republic). The process was carried out in 2 stages.

1. Freezing. The drug is poured into 2 ml in penicillin vials, which were installed in a duralumin cassette. Next, the cartridge was installed on a shelf pre-cooled to -40 ° C. Freezing was carried out for 3 hours. The temperature of the shelf at the end of the 1st hour from the start of freezing was -60 ° C, the temperature of the substance was -55 ° C.

2. Drying. Evacuation begins with the following parameters: shelf temperature -65 ° C, substance temperature -55 ° C, desublimator temperature -80 ° C. During the first 2 hours from the start of drying, the temperature of the shelf was raised to 0 ° C, each subsequent hour the temperature of the shelf was increased by 5 ° C. Upon reaching a shelf temperature of up to + 25 ° C, drying was carried out at a given temperature until the 20th hour from the start of drying. Draining was carried out at a shelf temperature of + 30 ° C. At pre-set heating values, the temperature of the preparation passed 0 ° C between the 16th and 17th hours from the start of drying. The total drying time was 25 hours. The temperature of the preparation varies from -55 ° C to + 28 ° C. The temperature of the desublimator is 83 ± 3 ° C, a vacuum of 13 Pa - 6 Pa (0.1 mm Hg - 0.05 mm Hg).

To determine the antimicrobial activity of the starting preparation (prototype) and the claimed preparation trametin, reference strains of microorganisms (Escherihia coli ATCC-5264; E. coli ATCC-35218; E. coli ATCC-1176; Enterococcus faecalis) obtained from FGBU GISK im. L.A. Tarasovich, pathogenic strains of microorganisms isolated from pathological material and identified at the Federal State Budget Institution Irkutsk Interregional Veterinary Laboratory (E. coli-TI from calf; E. coli-O4; E. coli-139-1; Salmonella enteritidis, S. ngor - from birds ) The results are presented in table 2.

table 2 Test culture The diameter of the zones of inhibition of growth of the test culture (in mm) Source drug (prototype) The inventive drug E. coli-TI 26.5 ± 0.5 28.0 ± 0.3 E. coli-O4 10.5 ± 0.3 12.0 ± 0.5 S. enteritidis 28.0 ± 0.5 27.5 ± 0.6 S. ngor 32.5 ± 0.2 25.5 ± 0.7 E. coli-139-1 34.0 ± 0.4 30.0 ± 0.5 E. coli- ATCC 526-4 12.0 ± 0.3 21.0 ± 0.4 E. coli ATCC 35218 16.0 ± 0.6 20.0 ± 0.3 E. coli ATCC 1176 15.0 ± 0.3 16.0 ± 0.4 Enterococcus faecalis 20.0 ± 0.2 17.0 ± 0.5

It was found that all drugs have antimicrobial activity to varying degrees. It was shown that the activity of the lyophilized form of the modified preparation against bacteria of the genera Escherichia and Salmonella remains at the level of activity of the original drug (prototype) and its lyophilized form.

Thus, the lyophilization of the drug does not reduce its biological activity, increases the shelf life of the drug and the possibility of its transportation over long distances.

The inventive preparation (trametin) is a lyophilized sterile culture fluid obtained by deep cultivation of Trametes pubescens (strain 0663). Trametin is a white powder with a yellowish tinge. The drug has low toxicity when introduced into the stomach and peritoneum in laboratory animals - white mice and rats. LD _5o is 8200 mg / kg. The local irritant and allergic effects of the drug are poorly expressed. The content of salts of heavy metals in it is at the level of trace amounts. There are no residual quantities of pesticides, mycotoxins and nitrosoamines.

The content of zinc and selenium in the starting preparation (prototype) and in the claimed preparation, as well as some toxic elements are presented in table 3.

Table 3 Trace element Content, mg / kg prototype the claimed drug (trametin) Zinc 2.55 ± 0.36 237.5 ± 3.2 Selenium 0.14 ± 0.05 0.75 ± 0.08 Copper 0.15 ± 0.07 0.23 ± 0.03 Lead 0.109 ± 0.002 0.068 ± 0.004 Mercury 0.0007 ± 0.00005 0.0004 ± 0.00007 Cadmium 0.007 ± 0.0003 0.004 ± 0.0002 Arsenic 0.0001 ± 0.00004 0.001 ± 0.0003

Calculations carried out in accordance with the accepted norms of trace elements for young cattle during rearing and fattening (Makartsev N.G. Feeding of farm animals. - Kaluga: GUP Oblizdat, 1999. 646 p.), Indicate that when using The claimed preparation of calves receive a daily norm of microelements - zinc and selenium.

Examples of the use of the drug.

Example 1. For the optimal method of drug administration, only oral was adopted, since intramuscular and subcutaneous methods are unacceptable due to the strong reactogenicity of the drug, irritability, and painful reaction at the injection site.

To determine the therapeutic doses of the drug for gastrointestinal diseases of newborn calves with a symptom complex of diarrhea, mainly toxic dyspepsia and colibacteriosis, 64 calves of 1-10 days old black-motley breed were selected, which were distributed in 8 groups of 8 goals (Table 4) .

Table 4 Groups of animals Dose of the drug Duration of treatment, days Therapeutic efficacy,% Preservation,% 1 experiment (trametin) 100 mg / kg weight 6-7 84.7 93,4 2 experience 180 mg / kg weight 4-5 88,20 100.0 3 experience 240 mg / kg weight 4-5 88.2 100.0 4 experience 300 mg / kg weight 4-5 88.2 100.0 5 experience 360 mg / kg weight 4-5 88.2 100.0 6 experience 480 mg / kg 4-5 88.2 100.0 7 Control with the original drug - prototype 2.5 ml / kg weight 6-7 87.4 100.0 8 control (without treatment) - - - 56.8

The first 7 groups are experienced and the 7th, 8th group are control. The calves of 6 experimental groups were given the drug once a day in the following doses: 0.8, 100, 180, 240, 300, 360, 480 mg per 1 kg of live weight of animals until recovery. The drug was added to milk before drinking. Calves of group 7 received the initial preparation (prototype) at a dose of 2.5 ml per 1 kg of live weight, which was introduced into milk before drinking. The calves were monitored for 10 days, the biochemical and morphological parameters of the blood were determined, the leukocyte formula was calculated, and the duration of the disease and safety were taken into account.

During bacteriological examination of pathological material, E. coli (non-pathogenic for mice during their experimental infection) and other opportunistic microflora (Staphylococcus aureus, Pseudomonas aeruginosa, Proteus vulgaris, Streptococcus faecalis, etc.) were isolated. At autopsy, it was found that the mucous membranes were anemic-cyanotic, there were no hemorrhages, the spleen and lymph nodes were pale in color and not enlarged.

When treating calves with a drug in doses of 180-480 mg per 1 kg of live weight, calves recover after 4-5 days, while the treatment efficiency is on average 88.2%. With a decrease in the dose of the drug, its therapeutic effectiveness decreases to 84.7%, the duration of treatment increases to 6-7 days. Tables 5, 6, 7 and 8 show the results of biochemical, morphological, immunological studies of the blood of calves of all groups, as well as leukograms.

The data in tables 5, 6, 7, 8 indicate that the optimal therapeutic doses of the drug should be considered doses of 180-300 mg per 1 kg of live weight of the animal.

When analyzing the biochemical parameters of calves (Table 6) treated with the drug, it was found that the level of total protein increased slightly (by 3.1-8.1%), no other significant differences were noted. The number of red blood cells in calves in groups 1-6 increased by 13.1-18.1, hemoglobin - by 14.0-19.5% (Table 5). The leukocyte count in all experimental groups, in comparison with the control group, remained practically unchanged (Table 6). The phagocytic activity increased by 5.5-10.8%, the phagocytic index - by 40.8-45.8%, the phagocytic number - by 23.8-37.0% (Table 8). Higher doses (> 300 mg per 1 kg of live weight) and lower (<180 mg per 1 kg of live weight) were not effective enough. The use of the drug before clinical recovery leads not only to a reduction in the duration of the disease, the exclusion of relapses, but also to further intensive growth and development of calves.

Example 2. To determine the therapeutic doses of the drug for colibacteriosis of calves, 64 calves of 10-15 days of age of black-and-white breed were selected, which were divided into 8 groups of 8 goals (Table 9).

The first 6 groups are experimental and 7, 8 control groups. The calves of the experimental groups were given the drug in the following doses: 100, 180, 240, 300, 360, 480 mg per 1 kg of live weight of animals until recovery. The calves were monitored for 10 days, the biochemical and morphological parameters of the blood were determined, the leukocyte formula was calculated, and the duration of the disease and safety were taken into account. In calves, depression, loss of appetite, increased intestinal motility, fluid, beige colored feces were observed, bowel movements were up to 7 times a day, lethargy, unsteady gait, and shortness of breath were also observed. The body temperature was 39.0-40.1 ° C, the respiratory rate was 28-32. Bacteriological investigation of the feces were isolated bacteria E. coli and serovars O ₉ O _26, pathogenic for white mice.

When treating calves with a drug in doses of 180-300 mg per 1 kg of live weight, calves recover after 4-5 days, while the treatment efficiency is at least 88.7%. With a decrease in the dose of the drug, its therapeutic effectiveness decreases to 80.4%, the duration of treatment increases to 6-7 days. The observational data are shown in tables 10, 11, 12, 13. The data in tables 10, 11, 12, 13 indicate that the optimal therapeutic doses of the drug should be considered doses of 180-300 mg per 1 kg of live weight of the animal.

When analyzing the biochemical parameters of calves (Table 11) treated with the drug, it was found that the level of total protein increased by 13.9-21.0%, other significant differences were not noted. The number of red blood cells in calves in 2-4 groups increased by 16.0-23.0%, hemoglobin - by 22.9-34.0% (Table 10). The content of leukocytes in all experimental groups, compared with the control group, decreased by 6.6-7.2%, mainly due to stab neutrophils and eosinophils (Table 11). The phagocytic activity increased by 2.7-11.6%, the phagocytic index - by 44.2-60.2%, the phagocytic number - by 16.4-39.7% (Table 13). Higher doses (> 300 mg per 1 kg of live weight) and lower (<180 mg per 1 kg of live weight) were not effective enough.

Example 3. To determine the therapeutic doses of the drug for calf salmonellosis, 64 calves of 80-90-day-old black-motley breed were selected, which were divided into 8 groups of 8 animals (Table 14).

In calves, depression was observed, a decrease in appetite, the temperature at the beginning of the disease was 39.9-40.9 ° C, and then in the majority it decreased to 39.1-39.5 ° C, feces were excreted with a high mucus content, from beige to dark color, watery, gruel-like consistency. During bacteriological examination of feces, cultures of Salmonella dublin and S. thyphymurium were isolated. The first 6 groups are experimental and 7, 8th groups are control. The calves of the experimental groups were given the drug in the following doses: 100, 180, 240, 300, 360, 480 mg per 1 kg of live weight of animals until recovery. The calves were monitored for 10 days, the biochemical and morphological parameters of the blood were determined, the leukocyte formula was calculated, and the duration of the disease and safety were taken into account. When treating calves in doses of 180-300 mg per 1 kg of live weight, calves recover after 4-5 days, while the treatment efficiency is 96.3%. With a decrease in the dose of the drug, its therapeutic effectiveness decreases to 92.4%, the duration of treatment increases to 6-7 days. Observation data are given in tables 14, 15, 16, 17, 18.

The data in the tables indicate that the optimal therapeutic doses should be considered doses of 180-300 mg per 1 kg of live weight. When analyzing the biochemical parameters of calves treated with the drug, it was found that the level of total protein increased by 5.2-6.5% (Table 15). The number of red blood cells in calves in the 2nd-4th groups increased by 16.7-19.2%, hemoglobin - by 13.6-16.6% (Table 14). The leukocyte content in all experimental groups, compared with the control group, decreased by 6.6-7.2% (Table 16), mainly due to stab neutrophils and eosinophils (Table 17). The phagocytic activity increased by 1.5-8.4%, the phagocytic index - by 32.1-35.6%, the phagocytic number - by 13.7-42.5% (Table 18). Higher doses (> 300 mg per 1 kg of live weight) and lower (<180 mg per 1 kg of live weight) were ineffective. This is due to the fact that the drug in doses of less than 180 mg per 1 kg of live weight has a lower antimicrobial effect, and at higher doses it has an irritating effect on the mucous membrane of the gastrointestinal tract.

The rationale for the treatment of calves for four to five days is the average duration of gastrointestinal diseases of calves, i.e. the most optimal period when the disappearance of clinical signs and recovery of the animal.

The most optimal option for the treatment of gastrointestinal diseases of calves caused by bacteria of the Enterobacteriaceae family - colibacteriosis and calf salmonellosis - is the introduction of the drug into milk during drinking in doses from 180 mg to 300 mg per 1 kg of live weight of the animal once a day. When the drug is added to milk during drinking at a dose of 180-300 mg per 1 kg of calf live weight 1 time per day, the treatment duration for colibacteriosis and calf salmonellosis is 4-5 days, the treatment efficiency is 96.3%.

Example 4. Scientific and production experiments on the use of the drug for therapeutic purposes were carried out in three farms of the Irkutsk region in coordination with the Veterinary Service of the Irkutsk region - at the MTF of the production department of the Research Institute of Agricultural Sciences (Irkutsk region) and at the MTF SC Belorechenskoye (Usolsky district) , FSUE “Elite” of the Russian Agricultural Academy (Ekhirit-Bulagat district) on 329 calves.

The drug was administered orally with milk in doses of 180-300 mg per 1 kg of calf live weight according to the method described above. 7.2-12.0 mg / goal. with colibacteriosis and at 14.4-24.0 mg / goal. with calf salmonellosis, because calves with colibacillosis were 10-15 days old, and calves with salmonellosis were 2.5-3 months old. The drug was administered once a day for 4-5 days. The therapeutic efficacy of the method is confirmed by studies: out of 234 calves that were treated with the drug, 228 animals recovered, which is 97.4% of the total. Of the total number of control animals (96 animals), which were treated according to the schemes generally accepted in farms, 74 animals recovered, or 77.1%. Thus, on the basis of the experiments, positive results were obtained, indicating a high therapeutic effect of the drug in this method of its use.

Using the proposed drug and method of treatment allows you to expand the arsenal of complex drugs used to treat gastrointestinal diseases of calves caused by bacteria of the Enterobacteriaceae family.

Claims (3)

1. A preparation for the treatment of gastrointestinal diseases of calves, representing a sterile culture fluid obtained by cultivating the fungus Trametes pubescens (strain 0663) on standard solid and liquid culture media with sodium selenic acid and zinc sulfate additionally added to the culture medium, followed by lyophilization. 2. The drug for the treatment of gastrointestinal diseases of calves according to claim 1, characterized in that sodium selenic acid is added in an amount of 3.0 mg / l in the form of a solution, zinc sulfate is added in an amount of 1.0 g / l. 3. A method of treating gastrointestinal diseases of calves, including the introduction of the drug into milk for drinking, characterized in that the drug according to claim 1 is administered at a dose of 180-300 mg per 1 kg of live weight of the animal 1 time per day for 4-5 days.