LV15071B - Composition for treatment of subclinical mastitis in cows - Google Patents

Abstract

The invention relates to veterinary medicine and biotechnology, especially with new, natural composition development for the treatment of subclinical mastitis in cows. The aim of invention is to create the effective compositions for the treatment of subclinical mastitis in cows, that can provide milk product quality and safety, which is not required for making of synthetic antibacterial agents. The aim is achieved with developing a new composition, which contains lactic acid, lysozyme, glycopeptides and 0,15M Na Cl physiological solution.

Description

DESCRIPTION OF THE INVENTION

The invention relates to veterinary medicine and biotechnology and relates to the development of a novel composition of naturally occurring components for the treatment of subclinical mastitis in cows.

TECHNICAL LEVEL

Mastitis is an inflammation of the parenchyma of the mammary gland. No visible abnormal udder changes (not clinically detectable) in subclinical mastitis, but milk is microscopically altered or contains bacteria and other inflammatory products, resulting in significant release of leukocytes and pro-inflammatory cytokines [1]. number changes in milk. Regulation (EC) No 1049/2001 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 the requirements that the total bacterial count (KBS) of the milk marketed at 30 ° C (per 1 ml) must not exceed 100 000 CFU and somatic cell count (SCC) 400 000 (per 1 ml) [2].

Typically, subclinical mastitis is detected in up to 75% of animals in the herd [3] and is mediated by a number of factors, such as housing, feeding, the immune status of the animal, and the inflammatory response to udder microorganisms. Streptococcus agalactiae, Staphylococcus aureus and other Gram-positive trees, in particular Streptococcus dysgalactiae, Streptococcus uberis, enterococci, coagulase-negative staphylococci, including S. hyicus, are mentioned as the most important causes of subclinical mastitis in the literature. epidermidis, S. xylosus un 5. intermedius [3].

Mastitis in cows is a serious problem in livestock farming, because the quality and safety of dairy products depend on this disease. Acute mastitis in cows can be detected by the following external signs: udder swollen, hot and painful. The animal raises body temperature and protein clots appear in the milk. However, in 90-95% of cases, mastitis occurs in a subclinical (chronic) form, which is characterized by signs of allergy, i.e., the inflammation of the mammary gland is invisible and the milk visually appears normal. To detect subclinical mastitis, udder quarterly milk is counted with somatic cell count and microflora analysis. Treatment of mastitis focuses on eradicating infection and increasing immunity.

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

Ciprofloxacin is ground in a mortar, dissolved in 100.0 ml of distilled water, then combined with the other components of the preparation and dissolved in 1 liter of water. After the milk is removed from the cow's udder, the solution is administered intracystemally (intramammary) at a dose of 50.0 ml once daily until recovery over a period of 14 to 20 days. Milk from the affected udder is disposed of [4]. However, the use of synthetic antibacterial and chemotherapeutic agents for the production of environmentally friendly products is permitted only in extreme cases, as milk containing synthetic antibacterial agents is not harmful to human health. Synthetic antibacterial and chemotherapeutic agents retained in milk cause violations of dairy technology. special caution should be exercised in the administration of medicinal products that are intended to be administered via the bean duct (intracystal), as this may cause an allergic reaction in humans and worsen the disease.

The use of therapeutic phytotherapeutic preparations in the treatment of mastitis in cows is also known. Use garlic, sage, root of the clove [5], ginseng, aloe, echo [6] and other herbal remedies. Known herbal remedies for the preparation of anti-inflammatory ointments using dry calendula flowers or calendula flower extract [7].

Use of a known antimicrobial agent anavidine [8], pine needle extract [9], guanidine [10] in the production of polymeric antimicrobial films to be applied to cow udder beans. The film in the bean duct prevents microflora from entering and promotes softening of the bean end skin.

However, the use of phytotherapeutic agents should be prolonged and may affect the organoleptic properties of the milk.

The development of an effective agent for the treatment of 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 pressing issue.

The object of the invention is:

= to provide an effective composition for the treatment of subclinical mastitis in cows, which is able to ensure the quality and safety of dairy products without the use of synthetic antibacterial and chemotherapeutic agents;

- search for the natural constituents of the composition and their relationship;

- Development of a new solution formulation containing natural components and capable of preserving its technological properties and activity for 12 months without causing side effects up to 3 times.

The object is achieved by the development of a composition consisting of lactic acid, lysozyme, glycopeptides and saline. In addition, the composition contains lactic acid, lysozyme, glycopeptides and saline 0.15M NaCl in the following weight ratios (mg):

lactic acid lysozyme glycopepfidi

0.15M NaCl saline

200.0 - 500.0;

100.0-300.0;

2.10-10.0;

to 10.0 ml.

In addition, the composition comprises glycopeptides from Lactobacillus spp.

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

Lysozyme (Lysozyme) - a hydrolase class enzyme that breaks down bacterial cell walls by hydrolysing bacterial cell wall (murein) with peptidoglycan. Lysozyme is obtained from chicken egg protein. The enzyme is present in both human and animal body: gastrointestinal mucosa, tear fluid, saliva. Lysozyme has antibacterial properties [12]. Lysozyme is used in the preparation of a suppository antifungal preparation for children [13], in the preparation of a transdermal composition for the treatment of eye infections [14].

Glycopeptides. Known synthetic glycopeptides (vancomycin, teicoplanin, glimuride) - a class of antibiotics used to treat septic infections. However, the use of synthetic glycopeptides can lead to increased levels of histamine in the blood 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 viral septic infections [16]. Natural glycopeptides have antiviral and immunomodulatory properties and are used in the production of organic food supplements containing colostrum [17].

BEST MODES FOR CARRYING OUT THE INVENTION

The experimental composition in vitro and in vivo selected and investigated the components of the novel composition and their relationship.

Examples of new composition

Example I - П:

lactic acid lysozyme 200.0-500.0; 100.0-300.0; glycopeptides 0.15M NaCl saline 2.0-10.0; to 10.0 ml.

If the 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 doses of the components above 500 mg (lactic acid), 300 mg (lysozyme) and 10 mg (glycopeptides), respectively, did not increase the antimicrobial effect.

METHOD OF PREPARING A NEW COMPOSITION

Example 1:

200.0 mg of lactic acid was continuously stirred on a 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 a pore size of 0.22 µm.

100.0 mg of lysozyme and 2.0 mg of glycopeptide were dissolved in 8 ml of distilled water, stirring constantly on a magnetic stirrer at room temperature, brought to pH 6.5 with 0.5 M lanes and sterilized by filtration through a membrane filter with a pore diameter of 0, 22 pm. The resulting solutions were pooled, made up to 10.0 ml with saline, the final pH checked, and filtered again through a membrane filter with a pore size of 0.22 µm. The reconstituted 10.0 ml solution was sterile filled into a sterile Falcon tube and sealed.

Example 2:

A 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 preparation method of Example 1.

RESULTS OF RESEARCH

In vitro

The antimicrobial activity of the solution of the compositions of Example 1 was tested using the well diffusion method [18,19]. Bacterial cultures were prepared in peptone saline at a concentration of 1.5x10 * kw / ml (0.5 Mc Farland). Bacterial cultures were plated in 0.1 ml in sterile 100 mm Petri plates and transferred to 20 ml Miller Hinton agar medium. The agar formed wells (6 mm 0) and filled with 60 µl of the test solution of the first composition. Plates were prepared in 3 replicates, incubated for 24 hours at 37 ° C and the diameter of the zone of inhibition of bacterial growth (or antagonistic activity). In vitro results are shown in Table 1.

Table 1

Damping zone diameters (mm) on Miller Hinton agar for first composition *

Cultures of microorganisms Depression (or antagonistic activity) zone (mm) S. saprophyticus 18th S. haemolyticus 19th S. aureus 19th E. coli 20th S. liquefaciens 18th C. freundii 19th

* The composition contained 500 mg lactic acid, 300 mg lysozyme and 10 mg glycopeptide per 10 ml saline.

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

The doses of the components above 500 mg (lactic acid), 300 mg (lysozyme) and 10 mg (glycopeptides), respectively, did not increase the antimicrobial effect.

The composition of the solution of Example 2 was as follows: 200 mg lactic acid,

100 mg of lysozyme and 2 mg of glycopeptide and was prepared analogously to Example 1. The results of the in vitro antimicrobial activity assay are shown in Table 2.

Table 2

Suppression zone diameters (mm) for Miller Hinton agar for a second ** composition

Cultures of microorganisms Depression (or antagonistic activity) zone (mm) 5. saprophyticus 14th S. haemolyticus 14th S. aureus 12th E. coli 12th S. liquefaciens 11th C. freundii 8th

** The composition consisted of 200 mg lactic acid, 100 mg lysozyme and 2 mg glycopeptide per 10 ml of saline.

From the results of Table 2, it can be concluded that the tested composition of 200 mg of lactic acid, 100 mg of isozyme and 2 mg of glycopeptide in vitro shows an 8-14 mm inhibition against all tested bacterial cultures, which means that the composition also has an antagonistic effect against mastitis agents, but less than in the first example, when the components are present in higher concentrations.

If the 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 solutions were tested for non-molar activity and sterility on a blood agar medium containing 50 g of Г ¹ cow's blood. The test solution was streaked onto blood agar medium, incubated for 24 hours at 37 ° C, and determined by hemolysis - β-hemolysis (clear and transparent area around the colonies), α-hemolysis (greenish area around the colonies) or γ-hemolysis (no haemolysis) [1.17]. None of the solutions tested resulted in haemolysis on blood agar (γ-haemolysis) and were sterile.

In vivo

At mid-lactation, 10 cows with a history of increased somatic cell count in milk were selected in the dairy cowshed. The cows were divided into 2 groups, i.e. 5 cows in each group. In the control group, 5 cows (20 quarters) were given an intramammary injection of a sterile 0.9% NaCl solution in 10 ml volume once a day, 1 day after milking, 3 times at 48 hour intervals. Experimental groups of 5 cows (20 quarters) received a sterile, new infusion of 10 ml of the composition for the treatment of subchiectal mastitis, once daily after milking, 3 times at 48 hour intervals. The composition consisted of: 10 ml saline, 500 mg lactic acid, 300 mg lysozyme and 10 mg glycopeptide. The composition was prepared as in Example 1 by the method of making an example.

Total bacterial count, somatic cell count in milk, and milk quality parameters (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 first administration.

TOTAL BACTERIA

In the udder quarters affected by subclinical mastitis (see Table 3) (SCC 1, day 2000 thousand / ml and above), the total bacterial count decreased significantly after administration of the composition, ie from 3167 thousand / ml on day 1 to 55 thousand / ml 3, per day and 7 thousand / ml 14, per day,

Table 3

Changes in the total bacterial count in milk of cows under study, thousand / ml

Total bacterial count (KBS) IBS, thousand / 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 -

In udder quarters, with moderately elevated somatic cell count (SCC) (4012000 thous / ml), the bacterial count increased moderately during the experiment, ie from 107 thous / ml on day 1 to 157 thous / ml on day 3 and 244 thous / ml 7 on day 14, but on day 14, the bacterial count in these quarters was lower than before the administration (95 thousand / ml).

In healthy quarters with an IBS of less than 400 thousand / ml, the total bacterial count at baseline 14 days after the start of the experiment was 12 thousand / ml.

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

NUMBER OF SOMATIC CELLS

Somatic cell count (SCC) is a key indicator of the quality of milk and its secretion status in the mammary gland [2]. A sharp increase in somatic cell counts is frequently observed, suggesting a positive immunogenic response in the animal. For the most part, CSFs fall within a few days.

During the experiment, irrespective of the initial somatic cell count in udder quarters (see Table 4), SCC was approximately 4000 thousand / ml on day 3, 2000-3000 thousand / ml on day 5 and approximately 3000 thousand on day 7. / ml. Significant differences were observed 14 days after the start of the experiment, with CSF being 54-fold lower in the quarters affected by subclinical mastitis initially greater than 2,000 thousand / ml, times higher in the quarters with initially elevated SCC, and 0.4-fold higher in the healthy quarters.

Table 4

Changes in somatic cell count (SCC) in milk of experimental group cows, thousand / ml

IBS, thousand / 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. 9th 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 ± 0 1366 ± 292.9 - > 2000 3556 ± 488.9 4823 ± 1353. 8th 3046 ± 2089. 9th 3215 ± 2119. 4 -

The tested composition, 14 days after the start of treatment, resulted in significant changes in milk quality in quarters that initially had somatic cell counts of more than 2,000,000 cells / ml, with an average 54-fold reduction in SCF, resulting in SCF in milk meeting the milk quality criteria. The experimental groups in the mammary glands, which initially had SCCs up to 2000,000 cells / ml, had elevated SCCs during the experiment and were 3.1-4.2 times higher on day 14 than on day 1. We can conclude that the tested composition has a beneficial effect on the health of the mammary glands with SCCs of 2000,000 cells / ml and above.

Quality of milk. Subclinical mastitis results in changes in the composition and quality of milk, which vary with the severity of the inflammatory process. In the acute case, reduced lactose synthesis, increased fat and lipase activity, and somatic cell (leukocyte) elevation may occur.

pH. At baseline, the mean pH of the milk in both groups was 6.58. Day 3 post-test, pH decreased by 0.6 in the experimental group and by 0.06 in the control group. The milk pH of the experimental group was 6.53 on the 7th day and 6.55 on the control group. Conclusion: On average, the pH of the milk decreased by 0.6, which did not affect the quality of the milk at all.

Fat content. Initially, the mean fat content of milk was 1.83% in the experimental group and 1.88% in the control group. On day 3, the fat content in the experimental group increased by 0.65% and in the control group increased by 0.11%. On day 7, the cows' milk fat content was 1.86% in the experimental group and 2.12% in the control group. Conclusion: The use of the composition did not cause a significant change in the milk fat content.

Lactose content. Initially, the mean lactose content in milk was 4.57% in the experimental group and 4.16% in the control group. On day 3, the lactose content decreased by 0.65% in the experimental group and 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 a significant change in the lactose content of the milk.

Protein content. Initially, the mean milk protein content was 3.80% in the experimental group and 3.46% in the control group. Protein content in the experimental group was reduced by 0.21% on the third day after the drug test and by 0.06% in the control group. On the seventh day the milk protein content was 3.73% in the experimental group and 3.36% in the control group.

Conclusion: The use of the composition did not cause a significant change in the milk protein content.

The number of pathogenic microorganisms was determined on blood agar medium after 24 hours incubation at 37 ° C. These 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 in 20 quarters (5 cows).

In subclinical mastitis-affected udder quarters (400,000 IU / ml on day 1), the mean pathogen count decreased significantly from 102.0-181.7 CFU / ml on day 1 to 0.0-4.0 KW / day after administration of the composition. ml on day 7 (see

Table 5). In healthy udder quarters, the number of microorganisms decreased slightly during the experiment, from 8.7 to 5.2 CFU / ml.

Table 5

Number of pathogenic micro-organisms in milk samples (CFU / ml)

IBS, thousand / ml Day 1 Day 3 Day 7 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

Conclusion: The performed studies confirmed the efficacy of the new composition in the treatment of subclinical mastitis in cows. The new composition does not contain synthetic antibiotics, it has biocompatibility. Application of the composition (up to 3 times every other day, once daily) does not cause an inflammatory reaction and a decrease in milk yield.

The use of the new composition does not cause significant changes in milk quality, i.e. milk fat, protein and lactose content.

Thus, in vitro and in vivo studies showed a beneficial effect of the proposed composition in the treatment of subclinical mastitis in cows.

INDUSTRIAL USE

An industrial solution formulation for the treatment of subclinical mastitis in cows pre-injected with 10.0 ml has been developed. The composition is for administration to udder-lactating cows for the treatment of subclinical mastitis caused by Staphylococcus spp., Streptococcus spp., Enterobacteriaceae and the like. microorganisms.

Administration is done after milking (morning or evening) - 3 times with 48 hour intervals. One injector (10.0 ml) is injected into each quarter of the udder affected by mastitis by wiping the tip of the bean with a disinfectant solution. Restrictions on the use of animal production: meat and by-products: zero days, milk: zero hours.

In some cases, clots may form in the milk while using the composition, but this does not change the overall consistency of the milk.

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

Sources of information

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Claims (3)

CLAIMS 1. A composition for the treatment of subclinical mastitis in cows, characterized in that it contains lactic acid, lysozyme, glycopeptides and saline. The composition for the treatment of subclinical mastitis in cows according to claim 1, characterized in that it contains lactic acid, lysozyme, glycopeptides and 0.15M NaCl saline in the following weight ratios (mg): lactic acid 200.0 - 500.0; lysozyme 100.0 -000.0; glycopeptides 2.0-Ю, 0; 0.15M NaCl saline to 10.0 ml. The composition for the treatment of subclinical mastitis in cows according to claims 1 and 2, characterized in that it contains glycopeptides from Lactobacillus spp.