LV15071A - Composition for treatment of subclinical mastitis in cows - Google Patents

Abstract

Invention relates to veterinary medicine and biotechnology. Composition containing components of natural origin is proposed for treatment of subclinical mastitis in cows. The composition contains lactic acid, lysozyme and glycopeptides produced by lactobacilli.

Description

LV 15071

DESCRIPTION OF THE INVENTION

The invention relates to veterinary medicine and biotechnology, and relates to the development of a new composition of natural origin components for the treatment of subclinical mastitis in cows.

TECHNICAL LEVEL

Mastitis is an inflammation of the parenchyma of the milk gland. Subclinical mastitis does not show any abnormal changes in the udder (not clinically detectable), but the milk is microscopically altered or contains bacteria and other inflammatory products, meaning that there is a significant release of leukocytes and pro-inflammatory cytokines [1], Somatic Cells Essential for Subclinical Mastitis changes in the number of milk. Regulation (EC) No 1782/2003 of the European Parliament and of the Council Commission Regulation (EC) No 853/2004 of 29 April 2004 laying down specific hygiene rules for food of animal origin lays down requirements under which the total quantity of bacteria (KBS) at 30 ° C (per ml) in raw milk to be disposed of may not exceed 100 000 KW and somatic cell count (SCC) 400,000 (1 ml) [2].

Usually, up to 75% of animals are found in subclinical mastitis in the herd [3] and its formation is facilitated by several factors - keeping animals, feeding, the status of the animal's immune system and the inflammatory reaction caused by the microorganisms entering the udder tissue. The most important sources of subclinical mastitis in the literature include Streptococcus agalactiae, Staphylococcus aureus and other gram-positive trees, especially Streptococcus dysgalactiae, Streptococcus uberis, enterococci, coagulase negative staphylococci, including S. hyicus, S. epidermidis, S. xylosus and S. intermedius [3].

Mastitis in cows is a serious livestock problem, as the quality and safety of dairy products depend on this disease. In acute mastitis cows, it is possible to determine the following external signs: udder swollen, hot and painful. The body raises body temperature, protein clots appear in the milk. However, in 90-95% of cases, mastitis occurs in a subclinical (chronic) form characterized by signs of allergy, i.e., the inflammation of the milk gland is invisible, the milk visually looks normal. To determine the subclinical massif, somatic cell count is made for the quarterly milk of the udder and a microflora analysis is performed. Mastitis treatment is aimed at eliminating infection and increasing immunity.

The use of various synthetic antibacterial and anti-inflammatory drugs is known, in particular the use of ciprofloxacin and metronidazole in the treatment of mastitis in cows. 2

Cyprofloxacin is pulverized, dissolved in 100.0 ml of distilled water, then combined with other components of the preparation and dissolved in 1 liter of water. After milk from the cow of the cow is wiped, the solution is injected intracistically (intramammary), the dose is 50.0 ml once a day until recovery takes place for 14-20 days. The milk obtained from the udder is recovered [4]. However, the use of synthetic antibacterials and chemotherapeutic agents is only permitted in exceptional cases for the production of environmentally friendly products, as milk containing synthetic antibacterials is not harmful to human health. Synthetic antibacterial and chemotherapeutic preparations that remain in the milk cause irregularities in the dairy production technology. special care should be taken when using medicinal products for intraperitoneal administration, as this may cause an allergic reaction in humans and worsen the course of the disease.

The use of healing phytotherapeutic agents in the treatment of mastitis in cows is also known. Used for garlic, sage, shoehorn root [5], ginseng, aloe, echo [6] and other3 herbal remedies. Use of known herbal remedies for anti-inflammatory grease using dry calendula flowers or calendula flower extract [7].

The use of a known antimicrobial agent anavidin [8], pine cone extract [9], guanidine [10] in the production of polymeric antimicrobial films to be applied to cow's udder beans. The film in the bean channel prevents the microflora from penetrating and facilitates softening of the bean ends.

However, the use of phytotherapeutic agents should be long-lasting and may affect the organoleptic characteristics of the milk.

The development of an effective means of treating subclinical mastitis in cows, which could ensure the quality and safety of dairy products that do not require the use of synthetic antibacterial and chemotherapeutic agents - is a topical issue.

The object of the invention is to: - provide an effective composition for treating subclinical mastitis in cows capable of ensuring the quality and safety of dairy products, without the use of synthetic antibacterial and chemotherapeutic agents; - searching for the natural components of the composition and their mutual relationship; - Developing a new solution that contains natural components and is able to retain its technological properties and activity for 12 months, as well as no side effects when used up to 3 times. 3LV 15071

The stated goal is achieved by developing a composition consisting of lactic acid, lysozyme, glycopeptides and physiological saline. In addition, the composition contains lactic acid, lysozyme, glycopeptides, and 0.15M NaCl physiological saline in the following component weight ratios (mg): 200.0-500.0; 100.0-300.0; 2.0 - 10.0; up to 10.0 ml. lactic acid lysozyme glycopeptides 0.15M NaCl physiological solution

In addition, the composition contains glycopeptides from Lactobacillus spp.

Lactic acid (Lacticacid) - acid of α-oxypropanoic acid, chemical formula CH3CH (OH) COOH or C3H6O3. It is formed in the process of lactic acid fermentation of sugar, fermented milk, wine and beer fermentation process. In the human and animal body, lactic acid is formed by the breakdown of glucose, the main source of the body's carbohydrates, providing energy for many of the body's chemical reactions. In the food industry, lactic acid is used as a preservative, food additive E270 [11].

Lysozyme (Lysozyme) - a hydrolase class enzyme that destroys the walls of bacterial cells, with the help of hydrolysis of the peptidoglycan peptide of bacterial cell wall (murein). Lysozyme is derived from chicken egg protein. The enzyme is present in both the human and animal body: gastrointestinal mucosa, tear fluid, saliva. Lysozyme has antibacterial properties [12]. Lysozyme is used in the manufacture of a suppository-type antifungal preparation for children [13], in the manufacture of a mesh composition for treating eye infections [14].

Glycopeptides. Known synthetic glycopeptides (vancomycin, teicoplanin, glimuride) - a group of antibiotics used in septic infections. However, the use of synthetic glycopeptides can lead to increased blood levels of histamine and various allergic reactions. Known natural glycopeptides that do not cause side effects are derived from lactic bacteria, mainly from Lactobacillus spp [15]. Natural glycopeptides also have antibacterial properties against microbial and vomit-induced septic infections [16]. Natural glycopeptides have antiviral and immunomodulatory properties, and are used in the manufacture of food supplements containing colostrum [17].

BEST EXAMPLES OF CARRYING OUT THE INVENTION

In the in vitro and in vivo experimental studies, the components of the newly developed composition and their relationship were selected and studied. 4

New Composition Examples Example I-II: 200.0- 500.0; 100.0-300.0; 2.0 - 10.0; up to 10.0 ml. lactic acid lysozyme glycopeptides 0.15M NaCl physiological solution

If component doses were less than 200 mg (lactic acid), 100 mg (lysozyme) and 2 mg (glycopeptides), respectively, the desired antimicrobial effect was not achieved.

If component doses were greater than 500 mg (lactic acid), 300 mg (lysozyme) and 10 mg (glycopeptides), respectively, the resulting antimicrobial effect did not increase.

METHOD FOR MANUFACTURING THE NEW COMPOSITION COMPOSITION Example 1 200.0 mg of lactic acid was continuously stirred at the magnetic stirrer at room temperature, brought to pH 6.5 with 2 mL of 15 N NaOH. The resulting solution was sterilized by filtration through a membrane filter with pore diameter of 0.22 μχη. 100.0 mg of lysozyme and 2.0 mg of glycopeptide dissolved in 8 ml of distilled water, stirring continuously, to a magnetic stirrer at room temperature, brought to pH 6.5 with 0.5 M NaHCO 3 and sterilized by filtration through a membrane filter with a pore diameter of 0.22 μπ / ml. . The resulting solutions were pooled together, the volume was brought to 10.0 ml with physiological solution, the final pH was checked, and again filtered through a membrane filter with a pore diameter of 0.22 µm. The prepared 10.0 ml solution was sterilized into a sterile Falcon tube and sealed tightly. Example 2:

The composition comprising the following components: 500.0 mg of lactic acid, 300.0 mg of lysozyme and 10.0 mg of glycopeptide, was prepared analogously to the production method of Example 1.

RESEARCH RESULTS

Here, the antimicrobial activity of the vtiro Example 1 composition solution was tested using the diffusion method of the wells [18,19]. Bacterial cultures were prepared in peptone salt solution at a concentration of 1.5x10 * kv / ml (0.5 Mc Farland). Bacterial cultures were introduced in 0.1 ml sterile 100 mm Petri plates and transferred with 20 ml of Miller Hinton agar medium. Holes (6 mm 0) were formed in the agar and 60 µL of the first 5LV 15071 test solution was added. The plates were prepared in 3 replicates, incubated for 24 hours at 37 ° C and measured the diameter of the bacterial growth inhibition (or antagonistic activity) zone. Results in vitro are shown in Table 1. Table 1

Diameter of depression zones (mm) in Miller Hinton's agar for the first composition *

Culture of microorganisms Depression (or antagonistic activity) area (mm) S. saprophyticus 18 S. haemolyticus 19 S. aureus 19 E. coli 20 S. liquefaciens 18 C. freundii 19 * 500 mg of lactic acid in 300 ml of physiological solution mg lysozyme and 10 mg glycopeptide.

From the results of Table 1 we can conclude that the tested composition containing 500 mg of lactic acid, 300 mg of lysozyme and 10 mg of glycopeptide in vitro shows an inhibition of 18-20 mm against all tested bacterial cultures (S.saprophyticus, S.haemolyticus. S. aureus E.coli, S.liquefaciens and C.freundii), which means that this composition has an active antagonistic action against mastitis agents.

If component doses were greater than 500 mg (lactic acid), 300 mg (lysozyme) and 10 mg (glycopeptides), respectively, the resulting antimicrobial effect did not increase. The composition of the components of Example 2 was as follows: 200 mg lactic acid, 100 mg lysozyme and 2 mg glycopeptide, and was prepared analogously to Example 1. The results of in vitro antimicrobial activity detection are shown in Table 2. Table 2

Diameter of the depression zones (mm) for Miller Hinton agar for the second ** composition

Microorganism culture Area of lesion (or antagonist activity) (mm) S. saprophyticus 14 S. haemolyticus 14 S. aureus 12 E. coli 12 S. liquefaciens 11 C. freundii 8 ** 200 mg of lactic acid in 10 ml of physiological solution 100 mg lysozyme and 2 mg glycopeptide. 6

From the results of Table 2 we can conclude that the tested composition contains 200 mg of lactic acid, 100 mg of lysozyme and 2 mg of glycopeptide in wool, showing an inhibition of 8-14 mm against all tested bacterial cultures, which means that the composition also has antagonistic effects in such composition. against mastitis agents, but it is less than in the first example, when the components are at a higher concentration.

If component doses were less than 200 mg (lactic acid), 100 mg (lysozyme) and 2 mg (glycopeptides), respectively, the desired antimicrobial effect was not achieved. The hemolytic activity and sterility of the solutions were tested on a blood agar medium containing 50 g Γ1 of cow's blood. Test strips were administered in strips on blood agar media, incubated for 24 hours at 37 ° C and determined by hemolysis - β-haemolysis (clear and translucent area around the colonies), α-hemolysis (green area around colonies) or γ-haemolysis (no haemolysis) [1.17]. None of the tested solutions produced haemolysis on blood agar (γ-hemolysis) and were sterile.

In vivo

In the mid-lactation period, 10 cows with an increased number of somatic cells in milk were selected for dairy cows. The cows were divided into 2 groups, i.e. 5 cows per group. In the control group, 5 cows (20 quarters) were given intramammary sterile 0.9% NaCl solution in 10 ml each quarter - once a day after milking, 3 times 48 hours apart. Experimental groups of 5 cows (20 quarters) were given a sterile new infusion of 10 ml subcutaneous mastitis for the treatment of subclinical mastitis, once a day after milking, 3 times at 48 hour intervals. The composition contained: 10 ml of physiological saline, 500 mg of lactic acid, 300 mg of lysozyme and 10 mg of glycopeptide. The composition was prepared as mentioned in the production method of Example 1.

The total number of bacteria, the number of somatic cells in the milk and the milk quality (pH, fat, lactose and protein content) were determined at the start of the experiment (day 1), 3 days, 5 days, 7 days, and 14 days after the first administration.

TOTAL BACTERIA

In subclinical mastitis affected udder quarters (see Table 3) (CHD Day 1, 2000 thousand / ml and above), the total number of bacteria decreased significantly after administration of the composition, ie from 3167 thousand / ml on Day 1 to 55 thousand / ml 3. day and 7 ths / ml 14 «day. 7 7LV 15071 Table 3

Total change in bacterial count in milk of test cows, thsd / ml

Total bacterial count (KBS) CHC, thsd / ml Day 1 Day 3 Day 5 Day 7 Day 14 Experimental group < 400 10 ± 1.3 516 ± 288.6 524 ± 317.6 503 ± 362.0 12 ± 2.1 401-2000 107 ± 63.0 157 ± 1.1 199 ± 118.5 244 ± 89.6 95 ± 61.2 > 2000 3167 ± 1343.3 55 ± 0 170 ± 109.2 536 ± 269.6 7 ± 0 Control group < 400 389 ± 358.0 12 ± 2.66 104 ± 81.6 9 ± 1.3 - 401-2000 13 ± 1.2 9 ± 0.82 16 ± 6.4 12 ± 4.4 - > 2000 449 ± 258.1 1545 ± 766.4 1029 ± 446.0 2066 ± 1679.5 -

The number of bacteria increased moderately during the experiment, ie from 107,000 / ml on day 1 to 157,000 / ml on day 3, and 244,000 / ml on the thymus quarters, with a moderate increase in somatic cell count (401-2000,000 / ml). ml on day 7, but on day 14, the number of bacteria in these quarters was lower than before the administration (95 thousand / ml).

In healthy quarters, where the SCC was less than 400,000 / ml, the total number of bacteria at 14 days after the start of the experiment was equal to the baseline and was 12,000 / ml.

Conclusion: The tested composition, acting on the subclinical mastitis affected mammary glands, has a beneficial effect on the number of bacteria in the milk by reducing it from 400 to 12,000 / ml. We can conclude that the tested composition significantly reduced the total number of bacteria in the milk.

NUMBER OF SOMATIC CELLS

Somatic Cell Number (CPC) is a key indicator of milk quality and milk secretion status in milk glands [2]. The sharp increase in somatic cell count is common, suggesting a positive immunogenic response in the animal's body. Most of the time the SCS falls within a few days.

During the experiment, irrespective of the initial somatic cell count of the udder's quarterly milk (see Table 4), SCC was about 4000 thsd / day on day 3, -2000-3000 thsd / day on day 5, and about 3000 on day 7 thousand / ml. Significant differences were observed 14 days after the start of the experiment, when the SCC was 54 times less in the quarters of subclinical mastitis, which were initially more than 2000 thousand / ml, 8 4 times in quarters with the initially mean SCC, and 0.4 times in the healthy quarters. . Table 4

Changes in somatic cell count (CPC) in experimental group cows' milk, thsd / ml CHF, thsd / mL Day 1 Day 3 Day 5 Day 7 Day 14 Experimental group < 400 67 ± 22.7 4887 ± 778.3 2262 ± 431.9 3188 ± 543.8 209 ± 71.1 401-2000 1026 ± 218.2 4648 ± 1063. 1 3419 ± 724.9 3257 ± 443.5 4294 ± 1728. 4 > 2000 8585 ± 1923. 9 4241 ± 825.5 2649 ± 310.2 3953 ± 561.7 157 ± 81.7 Control group < 400 77 ± 19.4 1106 ± 754.0 1050 ± 482.0 305 ± 115.6 - 401-2000 1444 ± 117.2 1031 ± 93.7 1174 = t0 1366 ± 292.9 - > 2000 3556 ± 488.9 4823 ± 1353. 8 3046 ± 2089. 9,315 ± 2119. 4 "The tested composition induced significant changes in milk quality in quarters, with an initial somatic cell count of more than 2000,000 cells / ml, 14 days after the start of treatment, with a mean reduction of 54 times the SCC resulting in milk quality criteria for SCC. In experimental milk mammals, where SCC was initially up to 2000,000 cells / ml, SCC increased during the experiment and on day 14 it was 3.1-4.2 times higher than on day 1. \ t We can conclude that the tested composition has a beneficial effect on the health of the mammary glands, where SCC was 2000,000 cells / ml and more.

Milk quality. Subclinical mastitis results in changes in the composition and quality of milk, which varies with the severity of the inflammatory process. In acute cases, reduced lactose synthesis, increased fat and lipase activity, and somatic cell (leukocyte) increase can be observed. pH. At the start of the study, the average pH of the milk in both groups was 6.58. On day 3 after the test, the pH in the experimental group decreased by 0.6 and in the control group decreased by 0.06. In the experimental group cows, the milk pH was 6.53 on day 7 and 6.55 in the control group. Conclusion: During the use of the preparation, the pH of the milk decreased by an average of 0.6, which generally did not affect the quality of the milk.

Fat content. Initially the milk fat content in the experimental group was 1.83% and in the control group 1.88%. On day 3, after the test, the fat content in the experimental group increased by 0.65% and in the control group increased by 0.11%. On day 7 9 9LV 15071 experimental group cows had 1.86% milk fat content and -2.12% control group. Conclusion: The use of the composition did not cause significant changes in the milk fat content.

Lactose content. Initially, the average lactose content in the experimental group was 4.57% and in the control group 4.16%. On day 3, the lactose content decreased by 0.65% in the experimental group and decreased by 0.12% in the control group. On day 7, the lactose content in the experimental group was 4.14% and in the control group 4.19%. Conclusion: The use of the composition did not cause significant changes in the lactose content of the milk.

Protein content. Initially the milk protein content in the experimental group was 3.80% and in the control group - 3.46%. In the experimental group on the third day after the test the protein content decreased by 0.21%, but in the control group it decreased by 0.06%. On the seventh day in the experimental group the milk protein content was 3.73% and in the control group - 3.36%. Conclusion: The use of the composition did not lead to significant changes in the milk protein content.

The number of pathogenic microorganisms was determined on a blood agar medium after incubation for 24 hours at 37 ° C. This included coagulase-free staphylococci, S.aureus, Streptococcus spp., E.coli, Enterobacteriaceae, and other mastitis agents.

The number of pathogenic microorganisms in the experimental group was determined from subclinical mastitis udder in 20 quarters (5 cows) and control group in healthy udder 20 quarters (5 cows).

In subclinical mastitis affected udder quarters (SCS on day 1 400 thousand / ml and more) after administration of the composition, the average number of pathogenic microorganisms decreased significantly from 102.0-181.7 CFU / ml on day 1 to 0.0-4.0 CFU / day ml on day 7 (see Table 5). In healthy udder quarters, the number of microorganisms in the experiment decreased slightly from 8.7 to 5.2 CFU / ml. Table 5

Number of pathogenic microorganisms in milk samples (CFU / ml) CHC, thsd / ml Day 1 3rd day 7th day Control group < 400 8.7 ± 2.0 8.5 ± 11.5 5.2 ± 6.4 Experimental group 2000 < 102.0 ± 26.6 4.7 ± 0.3 4.0 ± 26.6 10

Conclusion: The research carried out showed the effectiveness of the new composition in the treatment of subclinical mastitis in cows. The new composition does not contain synthetic antibiotics, it has biocompatibility. Using the composition (up to 3 times, every other day, once a day) does not cause an inflammatory reaction and a decrease in milk yield.

The use of the new composition does not lead to significant changes in the quality of the milk, i.e. the content of milk fat, protein and lactose. Thus, in vitro and in vivo studies showed a positive effect of the proposed composition on the treatment of subclinical mastitis in cows.

INDUSTRIAL USE

An industrial form of solution for treating subclinical mastitis in cows put up in the pen injector - 10.0 ml has been developed. The composition is for administration to the udder lactating cows for the treatment of subclinical mastitis caused by Staphylococcus spp., Streptococcus spp., Enterobacteriaceae, etc. microorganism.

Intake is expected after milking (morning or evening) - 3 times at 48 hour intervals. The contents of one injector (10.0 ml) are injected into each mastectomized quarter of the udder, wiping the end of the bean with a disinfectant solution. Restriction period for the use of animal products: meat and by-products - zero days, milk - zero hours.

In some cases, during the use of the composition, milk may form clots, but it does not change the overall consistency of the milk.

The technological properties of the new composition and the activity of the consistency remain for 12 months at 4-8 ° C.

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Claims (3)

CLAIMS 1. The composition for treating subclinical mastitis in cows is characterized by containing lactic acid, lysozyme, glycopeptides, and physiological saline. Composition for treating subclinical mastitis in cows according to the first claim, comprising lactic acid, lysozyme, glycopeptides, and 0.15M NaCl physiological solution in the following weight ratio of components (mg): lactic acid 200.0-500.0; lysozyme 100.0-300.0; glycopeptides 2.0-10.0; 0.15M NaCl saline to 10.0 ml. 3. A composition for treating subclinical mastitis in cows according to the first and second claims, comprising containing glycopeptides from Lactobacillus spp.