RU2532407C1 - Preparation for treating cows with subclinical mastitis and method for using it - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: group of inventions refers to veterinary science and aims at treating subclinical mastitis in cows. A preparation and a method for using it are declared. The preparation contains silver nanoparticles stabilised by submicrone titanium dioxide particles, poly-N-vinylpyrrolidone-2, and water in the following proportions, wt %: silver nanoparticles having a size of 200-300 nm 2.0×10^-3, titanium dioxide particles having a size of 2-4 mcm 60.0×10^-3, poly-N-vinylpyrrolidone-2 10.0, water - the rest up to 100.0. The method involves intracisternal introduction of the declared drug preparation in a dose of 10.0 ml twice a day for 2-4 days.

EFFECT: using the method enables providing the higher clinical effectiveness and reduces the length of recovery.

2 cl, 4 dwg, 7 tbl, 5 ex

Description

The invention relates to the field of veterinary medicine, in particular, can be used in the treatment of subclinical inflammation of the mammary gland in lactating cows.

Known drugs and their use in the treatment of cows with subclinical mastitis: masticide, mastisan A, B, E (Ivashura A.I. System of measures to combat mastitis in cows / A.I. Ivashura. - M., 1991. - C. 196-201. V. Yurkov, Antibiotics for the treatment of cows with mastitis / V. M. Yurkov, L. D. Demidova // Veterinary Medicine. - No. 10. - 1997. - P.30-32). The active ingredients in these preparations are combinations of antibiotics and sulfonamides stabilized in an oil base (vegetable oil). One therapeutic dose of the drug is 5 ml and contains the potassium salt of benzylpenicillin - 100,000 units; streptomycin sulfate - 100,000 units; soluble norsulfazole - 0.35 g; vegetable oil - 4.5 g and emulsifier - 0.05 g.

The essence of the method of administration consists in administering preparations to the cows intracisternally in the diseased quarter of the udder. Depending on the severity of the disease, 1-4 doses (5-20 ml) of the drug are administered, if necessary, repeated every 24 hours until the signs of the disease completely disappear.

However, for all the expediency and high efficiency of the use of these drugs, there are a number of disadvantages: the antibiotics, sulfonamides included in their composition contribute to the development of multiresistance in microorganism strains, and there is also a need to reject milk during and after treatment for 24-96 hours due to the selection of antibiotics with milk (Shakirov OF Control over the state of the udder of cows during lactation / O. F. Shakirov // Livestock. - No. 2. - 2006. - P.61-63); the oil base of the drug prevents the complete release and penetration of antibiotics into the tissues of the udder; the vegetable oils that make up the preparations are oxidized during prolonged storage.

The closest solution to the claimed adopted as a prototype is a water-soluble bactericidal composition containing highly dispersed metallic silver, a method for producing this composition (see RF patent application: 94042748/14 of 11/25/1994, IPC A61K 31/79, A61K 33/38 ) The method of obtaining such a composition according to the invention is realized by the interaction of silver nitrate with an aqueous-alcoholic solution of poly-N-vinylpyrrolidone-2 at the following concentrations of ingredients, wt.%:

poly-N-vinylpyrrolidone-2 0,065-11,0 silver nitrate 0.025-29.73 ethanol 5,0-3836 water the rest is up to 100.0

The disadvantages of the drug and the prototype method are: the method for producing the composition is not technologically advanced and is largely laborious, since the preparation process is carried out in the dark at a certain temperature (65-75 ° C), in an inert gas atmosphere. No data were found on the use of this composition in veterinary medicine, in particular for the treatment of subclinical mastitis in cows. The active substance - silver nitrate - may be an allergen; in the prototype method, alcohol is one of the components, which may also have an irritating effect on the mucous membrane of the udder when administered intracisternally.

The objective of the invention is the expansion of the arsenal of antibacterial drugs and methods for treating cows with subclinical mastitis.

The problem is solved in that in the preparation for the treatment of cows with subclinical mastitis, including an antibacterial substance and a filler, according to the invention, silver nanoparticles stabilized by submicron particles of titanium dioxide (hereinafter, Ag-TiO ₂ ) are used as an antibacterial substance, and use as a filler a polymer of synthetic origin poly-N-vinylpyrrolidone-2 in the following ratio of components, wt.%:

silver nanoparticles with sizes of 200-300 nm 2.0 × 10 ^-3 particles of titanium dioxide with a size of 2-4 microns 60.0 × 10 ^-3 poly-N-vinylpyrrolidone-2 10.0 water the rest is up to 100.0

The problem is solved in that in the method of using an antibacterial drug for the treatment of cows with subclinical mastitis, including intracisternal administration of the drug, according to the invention, 10.0 ml of the drug is administered to sick animals intracisternally twice a day for 2-4 days.

The method of preparation of the drug is as follows.

The complex, which is the active ingredient of the drug, is obtained by pre-mixing powdered silver with a particle size of 300-500 microns and powdered titanium dioxide with a particle size of 300-500 microns, the resulting mixture is mechanically activated in activators of the planetary, or vibrational, or vibrocentrifugal types, or roller mills providing acceleration of grinding media 200-400 m / s ² and a residence time of 5-15 minutes in the treatment area (see European Patent 0854765 B1 of 04.04.2001 Bulletin 2001/14).

After mechanical activation, the product is added to a solution of a high molecular weight polymer of synthetic origin with poly-N-vinylpyrrolidone-2 in the following ratio, wt.%:

silver nanoparticles with sizes of 200-300 nm 2.0 × 10 ^-3 particles of titanium dioxide with a size of 2-4 microns 60.0 × 10 ^-3 poly-N-vinylpyrrolidone-2 10.0 water the rest is up to 100.0

The use of silver nanoparticles provides the drug with antibacterial properties. Submicron particles of titanium dioxide, in combination with silver nanoparticles, contribute to the prolonged action of the drug, providing a gradual release of silver ions to the focus of inflammation.

The gel of poly-N-vinylpyrrolidone-2 is a more acceptable hydrophilic base that retains the activity of the etiotropic substance of the drug for a long time, as well as contributing to its better distribution in the tissues of the udder.

Preferably, the content of the high molecular weight compound in the solution is 10% by weight.

The rationale for the choice, the ratio of the components of the drug described in the description, is confirmed by examples that reveal the development of the optimal concentration with bactericidal, biocompatibility and not leading to cost overrun of the components of the drug.

The method of obtaining the drug is as follows.

The manufacture of a suspension containing silver nanoparticles stabilized by submicron particles of titanium dioxide.

Example 1. Powdered silver with a particle size of 300-500 microns is mixed with powdered titanium dioxide with a particle size of 300-500 microns in a ratio of 1:30. The mixture is mechanically activated in activators of the planetary, or vibrational, or vibrocentrifugal types, or roller mills, providing acceleration of grinding media 200 m / s ² and a residence time of 5 minutes in the treatment zone. The resulting product is added to a 10 wt.% Solution of polyvinylpyrrolidone to a silver concentration of 0.2 × 10 ^-3 %.

The final product contains silver nanoparticles, submicron particles of titanium dioxide, polyvinylpyrrolidone in the following ratio, wt.%:

- silver nanoparticles with sizes of 200-300 nm - 0.2 × 10 ^-3 ,

- particles of titanium dioxide with a size of 2-4 microns - 6.0 × 10 ^-3 ,

- polyvinylpyrrolidone - 10.0,

- water - the rest is up to 100.0.

Example 2. Powdered silver with a particle size of 300-500 microns is mixed with powdered titanium dioxide with a particle size of 300-500 microns in a ratio of 1:40. The mixture is mechanically activated in activators of the planetary, or vibrational, or vibrocentrifugal types, or roller mills, providing acceleration of grinding media 400 m / s ² and a residence time of 15 minutes in the treatment zone. The resulting product is added to a 20 wt.% Solution of polyvinylpyrrolidone to a silver concentration of 0.5 × 10 ^-3 %.

The final product contains silver nanoparticles, submicron particles of titanium dioxide, polyvinylpyrrolidone in the following ratio, wt.%:

- silver nanoparticles with sizes of 200-300 nm - 0.5 × 10 ^-3 ,

- particles of titanium dioxide with sizes of 2-4 microns - 20.0 × 10 ^-3 ,

- polyvinylpyrrolidone - 20.0,

- water - the rest is up to 100.0.

Example 3. Evaluation of the antimicrobial activity of the suspension, with different contents of silver nanoparticles stabilized by submicron particles of titanium dioxide

The antibacterial activity of the drug with different concentrations of a complex of silver nanoparticles stabilized by submicron particles of titanium dioxide (Ag-TiO ₂ ), in relation to cultures of Str. agalactiae, St. aureus, St. albus, Esh. coli, Enter, agglomerans was carried out by the method of serial dilutions in microplates. The working concentration of the culture suspension was 1 × 10 ⁶ cells / ml (standard turbidity GISK them. L.A. Tarasevich). Experience - composition + broth culture, control - broth culture. The culture plate and the preparation were incubated at 37 ± 1 ° C. After 24 hours from the start of the experiment, the optical density (units) of the liquid medium was measured on a Multiscan instrument (Table 1).

Table 1 Example Ag-TiO ₂ , wt.% Microorganism culture The optical density of the medium, units experience the control example 1 0.2 × 10 ^-3 Str. agalactiae 121.06 125.6 St. aureus 98.6 113.6 St. albus 102,4 152 Esh. coli 156.3 268.1 Enter agglomerans 219.6 290.3 example 2 1.0 × 10 ^-3 Str. agalactiae 76.9 125.6 St. aureus 35,2 113.6 St. albus 76,4 152 Esh. coli 98.7 268.1 Enter agglomerans 165.1 290.3 example 3 2.0 × 10 ^-3 Str. agalactiae 5.3 125.6 St. aureus 6.2 113.6 St. albus 9.7 152 Esh. coli 5,6 268.1 Enter agglomerans 15,2 290.3 example 4 4.0 × 10 ^-3 Str. agalactiae 4.48 125.6 St. aureus 1.3 113.6 St. albus 5,6 152 Esh. coli 5.2 268.1 Enter agglomerans 2,3 290.3

Based on the data presented in table 1, reflecting the change in the optical density of the medium, it follows that the maximum bactericidal effect is observed in the suspension with a concentration of the complex Ag-TiO ₂ (wt.%) Of 2.0 × 10 ^-3 and 4.0 × 10 ^-3 .

Example 4. Determination of the antibacterial activity of the drug, including silver nanoparticles stabilized by submicron particles of titanium dioxide, in the laboratory

To test the antimicrobial activity of the claimed drug in the dynamics of the experiment. The timing of the antimicrobial activity of the claimed drug was determined by the serial dilution method (Microbiological diagnosis of animal bacterial diseases / D.I. Skorodumov, V.V. Subbotin, M.A. Sidorov, and T.S. Kostenko - M .: Izograf, 2005. - 656 from.).

The results are presented in the form of figures 1-4.

From the presented data it can be seen that an antibacterial preparation containing silver nanoparticles stabilized by submicron particles of titanium dioxide has in vitro antibacterial activity against microorganisms that are an etiological factor in the occurrence of mastitis (growth curves of microorganisms of the experimental groups containing the drug in various concentrations, located below the growth curve of microorganisms in the control that does not contain the drug). Moreover, in the time period from twelve hours from the beginning of the experiment, the growth phase of the microbial culture is observed. From which we can conclude that the antibacterial activity of the claimed drug lasts 12 hours. Thus, the optimal frequency of use of an antibacterial drug in animals is twice a day.

Example 5. Checking the irritating effect of the drug, including silver nanoparticles based on submicron particles of titanium dioxide, stabilized by a polymer of synthetic origin of poly-N-vinylpyrrolidone-2, on the mammary gland of cows

Two groups of 10 goals of clinically healthy lactating cows were formed under the conditions of Example 1 and Example 2. A drug with a different ratio of components (a complex of silver nanoparticles and titanium dioxide) in an amount of 10.0 ml was injected intracisternally into the left front udder (experiment) after preliminary secretion. The drug was not injected into the right anterior lobe (control); it served as a control. The effect of the drug on breast tissue was judged by the change in the secretion of somatic cells, which was determined by the Prescott-Brid method in the following periods: before administration and 12, 24, 48 and 72 hours after drug administration. The control was symmetric healthy lobes of the udder, into which the drug was not administered. The results of the experiment are presented in table 2.

table 2 Example Ag-TiO ₂ , wt.% Study time, hours Shares of the udder, the number of somatic cells experience, thousand / ml control, thousand / ml example 1 2.0 × 10 ^-3 before introduction 468.8 ± 3.3 501.8 ± 3.8 12 241.05 ± 2.5 419.8 ± 2.2 24 265.8 ± 0.3 474.5 ± 6.8 48 295.2 ± 8.2 327.1 ± 7.1 72 455.2 ± 3.8 458.3 ± 2.0 example 2 4.0 × 10 ^-3 before introduction 328.8 ± 2.3 346.8 ± 1.8 12 168.5 ± 0.5 329.8 ± 2.4 24 206.8 ± 1.3 404.5 ± 3.8 48 298.2 ± 4.2 427.1 ± 1.1 72 365.2 ± 5.8 409.3 ± 2.1

It was found that 12 hours after the administration of the drug "Argo-mast", the general condition of the studied animals was within physiological boundaries. The local temperature of the experimental and control quarters of the mammary gland was not increased; the skin of the udder is elastic, painless. By 72 hours, a gradual increase in the number of somatic cells to a level not exceeding normative indicators was observed (GOST 52054-2003 “Natural cow's milk - raw materials”).

Example 6. Testing the optimal concentration of the active substance of an antibacterial drug

Based on previous studies, it was found that the maximum bactericidal effect is observed in a suspension with a concentration of the complex Ag-TiO ₂ (wt.%) Of 2.0 × 10 ^-3 and 4.0 × 10 ^-3 .

Two experimental groups of cows of black-motley breed of the second-fourth lactation were formed. The diagnosis of subclinical mastitis was made according to the results of the California mastitis test and laboratory counting of somatic cells in the Goryaev chamber. Cows of the 1-4th experimental groups were twice daily injected intracestrically with an antibacterial drug with different concentrations of the active substance Ag-TiO ₂ (wt.%) 2.0 × 10 ^-3 and 4.0 × 10 ^-3 . The drug was administered immediately after diagnosis twice a day until cure. Before use, the contents of the vial were thoroughly shaken until a uniform suspension was obtained; 10.0 ml was collected from the vial into a sterile syringe. The drug was administered via a milk catheter through the external opening of the nipple canal by pressing the syringe plunger into the teat cistern of the udder.

The animals were monitored. The therapeutic efficacy of the drug was determined by a negative test of the California mastitis test (negative reaction - the mixture in the well of the milk control plate is liquid, homogeneous, light lilac in color; the positive reaction is a mixture of jelly-like consistency of varying degrees, color from dark lilac to violet), and also by counting the number of somatic cells in the Goryaev’s chamber and a control bacteriological examination of milk samples for the presence of pathogenic microflora.

Table 3 Groups (concentration of active substance, mg / kg) Total Experience Recovered Recovery time, day cows affected lobes cows % share % 1st experienced (20.0) 16 17 16 one hundred 17 one hundred 4.18 ± 0.82 2nd experienced (40.0) fifteen fifteen fifteen one hundred fifteen one hundred 4.06 ± 0.69

From table 3 it follows that the therapeutic effect in the treatment of mastitis of cows was achieved in both groups. Recovery occurred on the 4th day.

Table 4 Groups The beginning of treatment, thousand / ml The end of treatment, thousand / ml Difference, thousand / ml 1st experienced (20.0) 787.5 ± 32.2 336.8 ± 24.6 * -450.7 ± 7.6 2nd experienced (40.0) 800.3 ± 37.8 313.4 ± 21.2 * -486.9 ± 16.6 Note: * P <0.05

As shown in table 4, the level of somatic cells in the milk of cows of the 1st experimental group decreased from 787.5 ± 32.2 to 336.8 ± 24.6 thousand / ml - by 57.2%; The 2nd experimental decreased from 800.3 ± 37.8 to 313.4 ± 21.2 thousand / ml - by 60.8%, respectively.

Table 5 Microflora End of treatment End of treatment Group Group O1 O2 O1 O2 Str. agalactiae + + - - St. aureus + + - - St. albus + + - - Esh. coli + - - - Prot. vulgaris - - - - Enter agglomerans + - - - Note: O1 ... O2 - 1st experienced ... 2nd experienced

During the control culture in the milk of animals of the 1st and 2nd experimental groups, the initial microflora was not found (Table 5).

As a result of the study, it was found that the use of the drug with the concentration of the active substance Ag-TiO ₂ (wt.%) 4.0 × 10 ^-3 animals in the 2nd experimental group did not reveal a significant increase in therapeutic efficacy and led to an overuse of the active substances of the drug. Therefore, in the future, a study was conducted of the therapeutic efficacy of an antibacterial drug with an active substance concentration Ag-TiO ₂ (wt.%) Of 2.0 × 10 ^-3 .

Example 7. Determination of therapeutic efficacy of an antibacterial drug with a concentration of the active substance Ag-TiO ₂ (wt.%) 2.0 × 10 ^-3 , in the treatment of subclinical mastitis in cows.

In order to determine therapeutic efficacy with the use of an antibacterial drug, 40 cows of patients with subclinical mastitis were selected. Animals were divided into two groups. An antibacterial preparation containing silver nanoparticles stabilized by submicron particles of titanium dioxide (Ag-TiO ₂ (mass%) 2.0 × 10 ^-3 ) at a dose of 10.0 ml twice daily until recovery was administered intracisternally to the experimental group. Similarly to the control group, an antibacterial drug, neotil, was introduced intracisternally, containing Tybocinum basicum 0.25 g 250,000 IU, Neomycinum sulfatum 0.1 g 100,000 IU.

Evaluation of the effectiveness of the treatment method was carried out by taking into account the results of a milk test by the California mastitis test (negative reaction - the mixture in the well of the milk control plate is liquid, homogeneous, light lilac in color; the positive reaction is a mixture of jelly-like consistency of varying degrees from dark lilac to violet ), by counting somatic cells in Goryaev’s chamber and by controlling bacteriological examination of milk samples, taking into account the timing of recovery.

As can be seen from table 6, the cure of the experimental animals occurred in 95% of cases (96% shares) on 4.45 ± 0.27 days of treatment with a preparation containing silver nanoparticles stabilized by submicron particles of titanium dioxide. In the control, 80% of the animals (81.8% of the shares) were cured within 8.95 ± 0.53 days.

Table 6 Group Total Experience Recovered Recovery period, day cows sick share cows % share % experienced (study drug) twenty 25 19 95 24 96 4.45 ± 0.27 control (neotil) twenty 22 16 80 eighteen 81.8 8.95 ± 0.53

From the data presented in table 7, it follows that after treatment in the milk of animals of the experimental group, the content of SC decreased from 873.6 ± 21.1 to 420.9 ± 36.0 thousand / ml (by 51.8% relative to the start of treatment), in the control, from 869.9 ± 22.3 to 576.2 ± 32.2 thousand / ml (by 33.8%).

Table 7 Group The number of somatic cells, thousand / ml Before treatment After treatment Difference experienced (study drug) 873.6 ± 21.1 420.9 ± 36.0 * 452.7 ± 14.9 control (neotil) 869.9 ± 22.3 576.2 ± 32.2 * 293.7 ± 9.9 Note: * P <0.05

The results of the studies indicate that the antibacterial preparation, including silver nanoparticles stabilized by submicron particles of titanium dioxide, accelerated the process of neutralizing microflora in patients with mastitis lobes of the mammary gland, which was manifested by a decrease in the number of somatic cells in milk and a reduction in the recovery time of animals.

To study the effect of drugs on the udder microflora, a bacteriological study of the secretion of the mammary gland of cows of both groups was performed. Secret samples were taken from the affected lobes in compliance with aseptic and antiseptic rules before drug administration and at the end of treatment.

Before the introduction of drugs in all cows found microflora in the amount of 6-96.2 × 10 ³ cells / ml. The isolated microflora was represented by microorganisms of the genera Streptococcus (Str. Agalactiae, Str. Dysagalactiae, Str. Spp.), Staphylococcus (St. albus, St. aureus), Escherichia (Esh. Coli), Klebsiella (Kleb. Pneumoniae), Enterobacter (Enter . agglomerans). After the end of therapy, in the secretion of the mammary gland in all cows of the experimental group (100.0%), the previously allocated microflora was absent, in the cows of the control group 4-65.9 × 10 ³ (68.6%) there were streptococci, staphylococci, E. coli and microorganisms genus Enterobacter.

Thus, the proposed method for the treatment of subclinical mastitis in cows using an antibacterial preparation containing silver nanoparticles stabilized by submicron particles of titanium dioxide, with a concentration of the active substance Ag-TiO ₂ (wt.%) Of 2.0 × 10 ^-3 at a dose of 10.0 ml twice a day with the intracisternal method of administration is more effective in comparison with the method of treatment with the drug neotil, which was manifested in a decrease in the duration of treatment, a decrease in somatic cells and sanitation of the udder.

Claims (2)

1. The drug for the treatment of cows with subclinical mastitis, including an antibacterial substance and an excipient, characterized in that silver nanoparticles stabilized by submicron particles of titanium dioxide act as an antibacterial substance, and a polymer of synthetic origin poly-N-vinylpyrrolidone-2 acts as an excipient the following ratio of components, wt.%:
silver nanoparticles with sizes of 200-300 nm 2.0 × 10 ^-3 particles of titanium dioxide with a size of 2-4 microns 60.0 × 10 ^-3 poly-N-vinylpyrrolidone-2 10.0 water the rest is up to 100.0 2. The method of using an antibacterial drug for the treatment of cows with subclinical mastitis, including intracisternal administration of the drug, characterized in that the antibacterial drug according to claim 1 is administered in a dose of 10.0 ml twice a day for 2-4 days.

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