A drug for prevention and treatment of infectious diseases

 

(57) Abstract:

The invention relates to biotechnology, veterinary science, medicine and can be used for the prevention and treatment of infectious diseases of fish, animals and humans, caused by microorganisms. Offered product containing enzymes of microbial and animal origin with lysozyme activity, in the following ratio of components: egg white lysozyme: microbial lysozyme - 1:1-2 activity. The drug is made on the proposed ratio of ingredients, allows you to lyse the cell wall not only bacteria and fungi, and protozoa. 12 table.

The invention relates to the field of biotechnology, veterinary science and medicine and can be used for the prevention and treatment of infectious diseases of fish, animals and humans, caused by microorganisms.

The use of egg white lysozyme as a bacteriolytic enzyme is widely known [1, 2].

The disadvantages of the use of lysozyme can be attributed to the narrow range of his actions, because he has no political action on a wide range of microorganisms, including pathogenic.

Closest to the proposed technical the m of the drug can be attributed to the narrow range of its effects on microorganisms, especially bacteria.

The aim of the invention is to increase the activity and expansion of the spectrum of antimicrobial action of enzyme preparation.

To achieve this objective, it is suggested preparation containing a mixture of egg white lysozyme with drugs of microbial lysozyme.

A certain ratio of these ingredients can dramatically increase the activity of the drug and to expand the range of its validity.

The proposed ratio of ingredients in the preparation of the following:

egg white lysozyme: microbial lysozyme - 1 : 1-2 (activity).

The proposal demonstrates the following examples.

Example 1.

1.1. Determination of the activity of the drug

Lysozyme activity was determined by the reduction of the optical density of the suspension of the microorganism Micrococcus lysodeikticus.

The microorganism Micrococcus lysodeikticus suspended in 0.05 M Tris buffer pH 7.0, was made solutions enzyme concentration in the reaction mixture of 0.5 μg/ml, and incubated at temperature 37oC for 60 minutes

The optical density of the suspension was measured at KFK-2 at a wavelength of 590 nm in a cuvette with the distance between the walls of the Naya optical density of the reaction mixture;

D2- the final optical density of the reaction mixture;

t is the incubation time of the reaction mixture, min;

the enzyme concentration of the drug in the mixture, µg/ml;

1 000 000 - conversion factor from ág,

The measurement results are shown in table 1.

1.2. The choice of the optimum ratio of ingredients.

To determine the optimal ratio of ingredients in the preparation of the microorganism Micrococcus lysodeikticus suspended in 0.05 M Tris-buffer pH 7.0; contributed solutions of mixtures of enzymes in various ratios (activity) concentration enzyme mixture in the reaction medium was 1.0 µg/ml and incubated at a temperature of 37oC for 60 minutes

The results of the measurements are presented in table 2.

The data show that all the studied enzyme mixture in the ratio 1: 1 -2 (activity) is several times greater than on lysozyme activity used for their compilation ingredients (see tab. 1).

Example 2.

Preparation of a drug for prevention and treatment of infectious diseases.

A drug for prevention and treatment of infectious diseases was prepared by simple mixing of lysozyme existing scheme:

2.1. 10 g of crystalline egg white lysozyme activity 14400 u/g was mixed with 13 g of protocolin GH with lysozyme activity 1100 IU/d (the ratio of the components of activity - 1:1).

The result obtained 23 g of a product with the activity of 20,000 units/year

The drug was estimated based on the yield factor activity, which was calculated as follows.

A. Calculation of theoretical yield activity of the drug:

At= (AlPl) + (AmlPml), ed,

where Attheoretical output activity, ed;

Althe activity of lysozyme, egg white, ed;

Plthe number of egg white lysozyme taken to obtain the drug, g;

Amlthe drug activity of microbial lysozyme, ed;

Pmlthe number of microbial lysozyme taken to obtain the drug,

B. Calculation of experimental activity release:

Ae= AopPop, ed,

where Aeexperimental release of activity, ed;

Aopthe activity of the obtained enzyme preparation, ed;

Pop- the number of the obtained enzyme preparation,

C. calculation of the coefficient of the output activity is t theoretical output activity unit

2.1.1. Characteristics of the obtained product:

At= (14400 10) + (1100 13) = 158300 unit

Ae= 20000 23 = 460000 unit

K = 460000 / 158300 = 2,9.

Thus, from the above calculations it follows that the output activity of the drug is 2.9 times greater than the total activity of the components taken for its preparation.

2.2. 10 g of crystalline egg white lysozyme activity 14400 u/g was mixed with 19.6 g of protocolin GH with lysozyme activity 1100 IU/d (the ratio of the components of activity - 1:1,5).

She got up 29.6 g of the drug with activity 20100 u/,

2.2.1. Characteristics of the obtained product:

At= (14400 10) + (1100 19,6) = 165560 unit

Ae= 20100 29,6 = 594960 unit

K = 594960 / 165560 = 3,6.

Thus, from the above calculations it follows that the output activity of the drug is 3.6 times higher than the total activity of the components taken for its preparation.

2.3. 10 g of crystalline egg white lysozyme activity 14400 u/g was mixed with 26 g of protocolin GH with lysozyme activity 1100 IU/d (the ratio of the components of activity - 1:2).

The result was 36 g Ave the 1100 26) = 172600 unit

Ae= 20100 36 = 723600 unit

K = 723600 / 172600 = 4,2.

Thus, from the above calculations it follows that the output activity of the drug is 4.2 times greater than the total activity of the components taken for its preparation.

In further tests were carried out with the use of the drug with activity 20100 u/g, obtained by simple mixing of the ingredients with a ratio of lysozyme: it 1:2 activity.

Example 3.

The study of the spectrum of antimicrobial action of enzyme preparation and its ingredients.

To study the spectrum of antimicrobial action of enzyme preparation and its ingredients were lysis of cells of microorganisms used as test objects, in 0.05 M Tris-buffer pH 7.0; temperature 25oC for 5 hours.

The optical density of the suspension of microorganisms was determined on KFK-2 at a wavelength of 590 nm in a cuvette with the distance between the walls 1 see

The calculation of the degree of lysis of the cells (L) in % was performed according to the formula

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where D1the initial optical density of the reaction mixture;

D2- the final optical density of the reaction medium;

100 - conversion factor for expressing rezago protein 0, 02 IU/ml (1.5 μg/ml);

The it GH - 0.02 units/ml (18 mg/ml);

The preparation of 0.02 units/ml (1 μg/ml).

The results are given in table 3.

Example 4.

Test the action of the drug on the eggs of aquarium fish, prevention miksobakteriosis.

The miksobakteriosis caviar brings great damage to aquatic and fishery fish culture. The causative agent of the disease is the fungus Saprolegnia. Particularly large losses from this disease is fish farming with the cultivation of fish with a long term incubation of eggs.

The first series of experiments were conducted on aquarium fish the minors (red Tetra) Hyphessobrycon callistus (Boul, 1900) in aquariums with a volume of 17.5 liters and sizes 35 x 25 x 20 cm at a temperature of 20oC. the Aquaria were supplied with a mesh through which failed caviar otheractivity fish, avoid eating producers. One day the producers otsuzhivalis and by this time was hatching eggs. The drug was introduced in the experimental aquaria immediately after spawning.

The results of the experiment are presented in table 4.

The second series of experiments with relatively long inkubiruemykh caviar was conducted on the speckled catfish ukes observation palea producers laid and fertilized eggs portions, attaching it to the walls of the aquarium. After spawning, was otsuzhivalis. The period of incubation of eggs lasted from 6 to 7 days.

The drug was introduced in the experimental aquaria immediately after spawning.

The results of the experiment are presented in table 5.

As can be seen from the above data, the drug has mycocide effect on pathogen miksobakteriosis and no adverse effects on eggs and on the exit of the larvae.

Example 5

Trials of drug action on a live feed aquarium fish, infected with pathogenic for fish pathogens.

It is known that for feeding aquarium and juveniles of commercial fish species most often used crustaceans-feeders, often captured in various water bodies, including those that may be sick fish. Therefore, crustaceans filter-feeding, accumulating and concentrating of infectious diseases, can serve as their vectors.

The experiment was carried out with Daphnia Daphnia magna (4-day culture) and Minami Moina macrocoma (2-day culture).

As the test cultures used a water suspension of the bacteria: Aeromonas punctatum (the causative agent of rubella carp) and Aeromonas salmonicida (the causative agent of furriery (VNIRO).

Given the optimum conditions for the existence of crustaceans, the experiments were conducted at concentrations of bacteria in water, which contained Daphnia and Moina, about 1 million cells/ml

In glass cups with a volume of 50 ml was made by culture of bacteria and approximately 10 individuals of Daphnia and MES.

In the experimental glasses were additionally made a solution of the product of the calculation of its final concentration in the environment of 10 mg/ml

In the control and the drug is not made.

Cups with amphipods were incubated at room temperature for 3 hours.

Scheme of the experiment is presented in table 6.

After 3 hours of exposure from all cups were planting water in Petri dishes the lawn on a solid nutrient medium. Then were caught using sieves crustaceans filter feeders and were pulverized in a sterile mortar, to which was added 10 ml of sterile water. The chopped stir and produced inoculation of 0.1 ml of the material on a solid nutrient medium in Petri dishes.

All cups were incubated in a thermostat at a temperature of 27oC for 5 days and watched. The results of the crops are given in table 7.

From the table 7 data shows that bakterii Aeromonas punctatum not exceed 3-4 dozen colonies in water and 100 colonies in crustaceans.

The scatter in the results, the control should probably explain digesting bacteria in the digestive tract Daphnia and MES.

Example 6

Test the action of the drug on the fish hit by Matt" disease (presumably flexibacter).

Flexibility fish - common diseases of ornamental and commercial fish. Cause these diseases multiple species of bacteria of the genus Flexibacter.

Effective control measures flexibilitythe to date no.

Experiments to test the validity of the drug to "dull" the disease of aquarium fish was carried out on the Swordsmen, shape and red's černoplavnikovyj (Xiphophorus helleri; Heckel, 1848) by the method of "bathing" in several versions and demo aquariums.

The tests were carried out according to the following scheme:

1. "swimming", the drug concentration is 10-50 mg/l, exposure time 2 hours;

2. Demonstration aquariums:

control;

- drug - 10 mg/l

As a result of experiments, it was found:

- the drug regardless of the concentration (in the range from 10 to 50 mg/l) has a therapeutic effect in the treatment of "matte" disease Swordtails;

- weakly affected Ribet becomes inconspicuous.

Example 7

Testing of therapeutic action of the drug at ichthyosporidium and chilodonella fish.

7.1. Test the action of the drug on the parasite disease can fish and fish patients ichthyosporidium.

The disease can is a widespread parasitic disease as aquarium and fishes. Pathogen - reverencia ciliate Ichthyophthirius multifiliis. The development cycle of the parasite consists of several stages: Mature trufant, localized beneath the epithelium of the body of the host fish; cyst, inside which is a division of the parasite, and the tramp is released from cysts young parasites (from 1 to 2 thousand from each of cysts), freely floating in the water up to 3 days. Once on the body of the fish, the crawlers are implemented under the epithelium, where they grow and Mature.

A. The study of drug action on the tramp (in vitro).

The object of studies were sick with the disease can fish Tetra von Rio (Hyphessobrycon flammeus; Myers, 1924). In the scrapings from the surface of the fish detection under the microscope of crawlers made a drop of aqueous solution preparation with a concentration of 10 mg/ml

When the observation of the behavior of crawlers under a microscope it is seen that in all cases within 1 min came lysis of the membranes of the crawlers and their gingili patients the disease can fish Neon blue (Paracheirodon innesi; Myers, 1936). As in the previous experience with the body surface of the fish were taken scrapings and detection in the field of view of the microscope adult stages of the parasite - Trifonov made a drop of solution preparation with a concentration of 10 mg/ml

Observed result is similar to the above - treponti reacted to the drug as a tramp - within 1 min lysis of the membrane and death of the parasite.

C. effect of the drug on the parasite disease can method "bathing" (in vivo).

The object of studies were fish Kerry (Inpaichthys kerri; Cery-Junk, 1977), affected by the disease can. Method of scrapings and direct microscopy was determined by the intensity of infection. For "bathing" was selected in each experiment for 10 individuals with a degree of destruction - 7-8 instances of parasites on scraping in the field of view of the microscope. The tramp and travanti was approximately 50%, respectively. After 1 hour exposure bathing in water containing the drug (10 mg/ml), none scrapings taken again, in the field of view of the microscope of live parasites were not found.

, the Study of the direct action of the drug on the infected with the disease can aquasystem.

The study included a Small blue whale (Liocassis braschnikowi; Berg). In the three demonstration AK and 6 trofanov on scraping. The aquariums were treated with preparation of the calculation of its final concentration in water of 10 mg/l

In the next day none of the scrapings of parasites were found. Fish kills were observed.

Example 8

Test the action of the drug on pathogen chilodonella fish (in vitro) and on fish, patients chilodonella (in vivo).

Chilodonella fish brings great damage to both the aquarium and commercial fish farming. The spectrum of disease includes not only a large number of fish species, but also a wide temperature range. The disease is caused by parasitic representatives of the genus Chilodonella, which belong to ravnovesnym the ciliates SEM. Chlamidodontidae squad Holotricha.

The test drug to combat chilodonella was conducted according to the same scheme as for ichthyosporidium, that is, the action of the drug (10 mg/ml) on the pathogen in vitro, the effect of the drug (10 mg/ml) in fish infected by the parasite by "bathing" (in vivo) c one-hour exposure and the effect of the drug (10 mg/l) on the sick fish in the demo aquarium.

The work was carried out on Gold fish and Black telescopes (Carassus carassus). Evaluation of test results was performed by microscopy of scrapings from telatar, received at ichthyosporidium: 1-minute exposure of the drug concentration of 10 mg/ml of the pathogen of the disease caused his death; "bathing" patients chilodonella fish in the solution preparation with a concentration of 10 mg/ml for 1 hour at taken after processing the scrapings parasites were not detected; sample aquariums in a day after making the drug (10 mg/l) none of the scraping living chilodonella discover never failed.

Example 9

The study of therapeutic action of the drug in model epizootics fish stocks in the closed-aquasystem.

For modeling of the emergence and development of epizootics fish stocks in the conditions of their detention in a closed-aquasystem used 3 aquarium volume 20, 10 and 5 liters each.

In these aquariums were released on 19 healthy fish - Cardinal (Tanichthys albonubes; Lin Shu Yen, 1932). Then in each of the aquariums was released one fish infected by the parasite (ichthyosporidium or chilodonella) with intensity of infection - 6-8 instances of parasites on scraping.

Thus, we studied three models of a population of fish Cardinal with density 1, 2, and 4 copies/l, respectively. The exposure of fish populations conducted during 7ptx2">

The following experiment was put on a similar scheme, but after 4 days in aquariums made the drug at the rate of 10 mg/L. Exposure of the fish population was carried out for 7 days and studied the dynamics of fish mortality.

The research results are summarized in tables 10 and 11.

From the experimental data it follows that the proposed drug is an effective therapeutic and prophylactic agent for combating infectious diseases of fish.

Example 10

The test drug to combat the pathogen furuncular angina man - Streptococcus-29.

To study the antimicrobial activity of the enzyme preparation and its ingredients were lysis of cells of the microorganism Streptococcus-29.

The microorganism suspended in 0.05 M Tris-buffer pH 7.0; contributed solutions of the drug and its ingredients, and incubated at temperature 37oC for 5 hours.

The enzyme concentration of the drug and its ingredients in the reaction mixture:

Egg white lysozyme - 0, 02 IU/ml (1.5 μg/ml);

The it GH - 0.02 units/ml (18 mg/ml);

The preparation of 0.02 units/ml (1 μg/ml).

The results are given in table 12.

From the above data should ctoi angina person.

The presented examples demonstrate the achievement of the objectives of the proposal, i.e. drugs, based on the proposed ingredients in them a certain combination, have a high activity and broad spectrum of antimicrobial activity and can be used for the prevention and treatment of infectious diseases caused by bacteria, fungi and protozoa.

Sources of information

1. Jolles P. the Relationship between chemical structure and biological activity of lysozyme, chicken eggs and litzkow from other sources. In Proc.: protein Chemistry, M.: Mir, 1969, S. 5-22.

2. Podbornov C. M. comparison of litzkow different origin. Antibiotics and chemotherapy, 1990, T. 35, N 8, p 22-25.

3. D. Knorr, K. J. Shetty, L. F. Hood, J. E. Kinsell An enzymatic method for yeast autolysis - J. Food Science, 1979, v. 44, N 5, p. 1362-1365.

A drug for prevention and treatment of infectious diseases of animals and humans, including egg white lysozyme, characterized in that it further comprises microbial lysozyme in the following ratio of ingredients: egg white lysozyme : microbial lysozyme - 1 : 1 - 2 activity.

 

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