RU2748168C1 - Means for disinfestation of objects of external environment - Google Patents

Abstract

FIELD: veterinary medicine.SUBSTANCE: invention relates to the field of veterinary medicine, namely veterinary parasitology, and can be used in poultry and pig farms, as well as in dog kennels for the prevention of animal parasitosis. The complex disinfestation agent contains the initial components in the following ratio (wt%): glutaraldehyde - 8, alkyldimethylbenzylammonium chloride - 5, isopropyl alcohol - 12, polyethylene glycol-400 - 30 and water - 45.EFFECT: proposed complex agent for disinfection of environmental objects allows it to be used both manually and mechanically, does not have a corrosive effect on the technological equipment available in livestock buildings, is highly effective against parasitic protozoan oocysts and helminth eggs, which significantly reduces the economic damage from animal parasites.1 cl, 2 tbl, 4 ex

Description

The invention relates to the field of veterinary medicine, namely, veterinary parasitology, and can be used in livestock farms, as well as in dog kennels with helminthiasis and protozoa.

It should be noted that parasitic diseases of animals in our country represent a serious problem for the intensive development of various industries, especially in farms with a large concentration of livestock. Among the various branches of animal husbandry in our country, in terms of production growth, poultry and pig breeding occupy the leading positions.

At the same time, along with the positive trends in modern poultry and pig farming in the country, there are still many problems that require a comprehensive solution. These include: the spread of parasitic diseases of birds, pigs and small domestic animals, the improvement of diagnostics of a number of diseases and the development of new effective technologies for the prevention and treatment of such dangerous and costly invasive bird diseases as eimeriosis, cryptosporidiosis, dermanissiosis, histomonosis, ascariasis, heterocytosis. and others, in pigs isosporosis, eimeriosis, balantidiosis, ascariasis, trichocephalosis, esophagostomosis, and in dogs - toxocariasis.

The practice of successful poultry farms and pig farms shows that the prevention of parasitosis of birds and pigs includes a set of therapeutic and prophylactic measures aimed both against exogenous (oocysts, cysts, eggs in the environment) and against endogenous stages of the pathogen (inside the body of poultry, pigs). and other animals) using modern antiparasitic drugs.

Despite the implementation of antiepizootic measures, complete prevention of parasitosis among the livestock of animals in poultry, pig and other farms is not achieved. This is evidenced by the work of domestic and foreign veterinary parasitologists.

In dysfunctional farms, the transmission of invasion occurs through contaminated with invasive elements (coccidial oocysts, balantidial cysts and helminth eggs) feeders, feed, water, bedding, inventory. Mechanical carriers of invasive elements are often insects, rodents, synanthropic birds, as well as service personnel - on shoes, clothes, and care items.

Various violations of the technology of rearing young animals contribute to the widespread spread of parasitic diseases of animals: overcrowding of livestock in the premises, high humidity and bedding, inadequate feeding and violations of veterinary and sanitary rules.

Studies have established that the invasive elements of many animal parasites are very stable in the external environment and retain their viability for a long time: oocystitis of bird eimeria and pig coccidia - more than a year, pig balantidia cysts - more than a year, pig roundworm eggs - more than four years, ascarid eggs and heterakis birds - more than a year, eggs of carnivorous toxocaras - more than two years.

It should be recalled that the epizootic process in parasitic diseases of animals, as in many infectious diseases, consists of three links: the source of invasion, transmission factors and susceptible animals. By influencing the factors of transmission of the invasion, it is possible to interrupt this chain, destroying the exogenous stage - the previously noted parasites.

To combat parasitic diseases of animals, a significant number of drugs have been proposed, both against endogenous and exogenous stages. The problem of parasitosis of animals poses tasks for researchers - to improve measures to combat invasion, to develop means and methods for disinfecting objects of the external environment against oocysts and cysts of parasitic protozoa and helminth eggs.

Of the known disinfestation agents that are used against the exogenous stages of parasitic protozoa and helminths, it is appropriate to note a 4% solution of caustic soda, which should have a temperature of at least 80 ° C on the surface of the object, 2% orthochlorophenol emulsion, 7% ammonia solution , 10% solution of iodine monochloride (1-5). The noted disinfestation agents are used in many livestock farms, but their effectiveness does not suit the demands of practice.

Considering the previously noted and special resistance of parasitic protozoan oocysts and helminth eggs in the external environment, it is possible to create an effective disinfectant against them using several active ingredients and excipients. Among such drugs, glutaraldehyde and alkyldimethylbenzylammonium chloride should be considered when used together at optimal concentrations.

It should be noted that for disinfestation of environmental objects against buxtonella cysts of cattle, a complex agent "Cystodesis" has been proposed. However, this agent has shown insufficient effectiveness against oocysts of coccidia of birds and eggs of porcine roundworm. Based on the above, we have created another complex tool "Cystodes-ultra" with a modified composition of the components that make up the composition, which had a significant impact on the effectiveness and expanded the indications for use for disinfestation against parasitic objects of animals.

The first of the noted drugs, glutaraldehyde, is a well-known disinfectant for external use, it is active against oocysts and cysts of parasitic protozoa, helminth eggs. The second of the mentioned - alkyldimethylbenzylammonium chloride - is known as a highly effective disinfectant, is a reliable antiseptic, antiprotozoal and antifungal agent. In the case of their combined use against oocysts and cysts of parasitic protozoa and helminth eggs, they have a synergistic effect on the parasitic object, and the auxiliary substances used ensure the cleaning of the floor, walls and technological equipment from biocontamination and evenly distribute the DV over the surface.

As an auxiliary substance, polyethylene glycol 400 (PEG-400) was used, which has high emulsifying properties, due to which it has an effective emollient effect on oocysts and cysts of parasitic protozoa and helminth eggs. Under the influence of polyethylene glycol, the cell membrane of the membrane of oocysts, cysts and eggs of the parasite partially dissolves, thereby opening access for the penetration of oocysts, cysts and eggs of disinfectants.

The task of our research was to develop an effective disinfestation agent against parasitic protozoa oocysts and animal helminth eggs with available means suitable for both manual and mechanical use, without corrosive effect on the technological equipment available in livestock buildings.

The solution to this problem is achieved by the fact that as a disinfestation agent against oocysts of parasitic protozoa and helminth eggs, we used a complex agent "Cystodes-ultra" compiled by us, which has the following composition: active ingredients - glutaraldehyde 8%, alkyldimethylbenzylammonium chloride 5% and auxiliary components - isopropyl alcohol 12%, polyethylene glycol 400 - 30% and water 45%, respectively, in a weight ratio,%: 8; five; 12; thirty; 45.

From the sources of information available to the applicant, the specified set of existing features that make it possible to obtain the specified technical result are not known, therefore the claimed invention meets the "novelty" condition of patentability.

The applicant is also not aware of the means of a similar purpose, in which the distinctive features of the claimed invention were disclosed with obtaining from its use in any set of features of the specified technical result. Consequently, the claimed invention meets the requirement of patentability inventive step.

Proofs of industrial applicability of the claimed are given in the following examples.

Examples of specific execution.

Example 1. Preparation of working solutions, dilutions of the culture of eimeria oocysts, eggs of pork roundworm and toxocara of dogs, the implementation of the lysis test and cultivation with different concentrations of the drug "Cystodes-ultra".

Research on this problem was carried out in the laboratory of the All-Russian Research Institute of Fundamental and Applied Parasitology of Animals and Plants - a branch of the Federal State Budgetary Scientific Institution FNTs VIEV RAS (Moscow).

The working solution with different concentrations of the combined agent "Cystodes-ultra" was prepared as follows. Took four glass flasks of 1 l and poured 800 ml of distilled water and the first three of them were added to 30; 40 and 50 ml of Cystodes-Ultra were thoroughly mixed for 5 minutes, the volume was brought to 1 liter, stirred again for 5 minutes and the solution was ready for use. In the fourth flask, 40 g of crystalline phenol was added to 800 ml of water, stirred, the volume was brought to 1 liter, stirred again, allowed to settle and the solution was ready for use.

Preparation of oocyst culture Eimeria spp.

To collect oocysts, feces of broilers of 3-4 weeks of age spontaneously infected with eimeria were used with the floor technology of keeping them in a poultry farm. Material from spontaneously infested broilers was examined by the Fülleborn flotation method. From samples of infected broilers using a parasitological loop, the surface film was removed and collected in a Petri dish with dichlorinated water, oocysts were washed three times from salt, their number was counted in 1 drop, then 1 ml was mixed with a layer of preservative - 2% potassium dichromate solution and used in further work.

When preparing the SWH buffer, 17.5 ml of a 10% solution of calcium chloride and 5 ml of a 10% solution of magnesium sulfate were added to 3300 ml of distilled water, autoclaved for 15 minutes at 120 ° C, the pH was determined using an Aquilon- 410 ".

To carry out the lysis test, the prepared disinfectants (3; 4 and 5% solutions "Cystodes-ultra" and 4% phenol) and the buffer were separately placed 50 ml in 250 ml Erlenmeyer flasks and added 50 ml of solution with oocysts coccidia at a concentration of 2000 oocysts / ml. Then these Erlenmeyer flasks were placed on a vibrating table with a rotation speed of 100 rpm for 2 hours. The contents of the flask were then poured into a 1500 ml screw cap plastic bottle. the flask with the remainder of the solution was rinsed several times and poured into a plastic bottle and the volume was adjusted with buffer to 1500 ml. Then the bottle was inverted several times (three times) and left at room temperature (20 ± 2 ° C) for 24 hours. After the time elapsed, the solution was poured out to the mark, 30 ml of the precipitate was poured into a new 100 ml container, the plastic bottle was rinsed several times using a buffer, bringing the volume to 50 ml.

Preparation of Ascaris suum egg culture

Pork roundworm eggs were collected from fresh feces of infected young pigs, using the Fülleborn flotation method. From samples of infected pigs using a parasitological loop, the surface film was removed and collected in a Petri dish with distilled water, eggs were washed from salt three times, their number was counted in one drop, then 1 ml was mixed with a layer of preservative - 1% solution of HC1 on distilled water and used in further work.

Toxocara canis egg culture preparation

Dog toxocar eggs were collected from fresh feces of infected young dogs (6 months of age) in a Fülleborn flotation study. The surface film was removed from the samples of the infested puppies using a parasitological loop and collected in a Petri dish with distilled water, the eggs were washed three times with water from salt, their number was counted in one drop and in 1 ml, then they were mixed with a layer of preservative - 1% HCl solution on distilled water and used in further work.

Example 2. Bioassay for experimental infection of broiler chickens to determine the effectiveness of the drug "Cystodes-ultra" for desivation.

The experiment on testing different concentrations of the drug "Cystodes-ultra" for desivasia was carried out in the vivarium of the All-Russian Research Institute of Fundamental and Applied Parasitology of Animals and Plants - a branch of the Federal State Budgetary Scientific Institution FSC VIEW RAS (Moscow) on 60 chickens 14 days of age free from coccidia and their food did not contain anti-coccidial drugs. In their work, to control the concentration of sporulated coccidial oocysts (2000 oocysts / ml), a Mac Master chamber and an MBS microscope were used, and SWH buffer was used for dilution so that it was possible to inject 1 ml of suspension into each chick. A magnetic stirrer was used to achieve good mixing of the material. All experimental chickens were subjected to clinical examination, individual numbering, weighing and, according to the principle of analogues, were divided into six groups of 10 chickens each.

Chickens of the first, second and third groups were given 1 ml of a suspension of eimeria oocysts, treated with 3; 4 and 5% concentration of the drug "Cystodes-ultra" inside with a micro-pipette gradually. Chickens of the fourth group were given 1 ml of eimeria oocyst suspension treated with 4% phenol solution (base preparation). Chickens of the fifth group received 1 ml of suspension containing 2000 oocysts / ml - infected control. Chickens of the sixth group received 1 ml of buffer solution and served as a "clean" control.

During the experiment, chickens of all six groups were kept in similar conditions, and they had the same diet. During the entire experiment, daily clinical observations of the general condition, their behavior, intake of food and water, visible physiological changes, and others were carried out for the chickens.

From chickens of each group separately to establish oocysts in feces from 6 to 12 days, all droppings were collected daily, weighed, water was added to a weight of 2000 g, and mixed with a mixer for 5 minutes. Samples were taken from each group for further studies in an amount of 25 g, which were preserved with a 4% potassium dichromate solution, stirring with a mixer, brought to a homogeneous mass, then transferred to microcontainers with a screw lid and stored in a refrigerator at a temperature of + 4 ° C.

Oocysts in 1 g of feces were determined by the Fülleborn flotation method using a saturated solution of sodium chloride with a density of 1.18 g / cm ³ , and their number was counted using a Mac Master chamber.

The effectiveness of disinfestation with the appointment of different concentrations of the drug "Cystodes-ultra", as well as 4% concentration of the basic phenol preparation was determined based on the percentage decrease in the release of eimeria oocysts after exposure to them with the marked concentrations of disinfectants compared to chickens of the infected control, which were given 2000 oocysts / ml.

Studies to determine oocysts in the feces of experimental chickens collected from 6 to 12 days after the administration of a suspension treated with disinfectants, a certain amount of them was found, but not in all groups. So, in the study of experimental chickens of the first group, who were prescribed a suspension of oocysts treated with a 3% concentration of "Cystodes-ultra", oocysts of eimeria in feces were found in all periods of research and the average indicator in one chamber for all periods of research was 1.53. The number of eimeria oocysts in 1 g of litter in this group was 306 specimens, which is 6.92% of the control. Hence, the intensity of "Cystodesis-ultra" in 3% concentration or the percentage decrease in the number of oocysts after exposure to the marked concentration of the drug is equal to 93.46% (Table 1).

The results of studies of samples of litter from chickens of the 2nd and 3rd groups, which received a suspension of oocysts treated with 4 and 5% concentration of Cystodes-ultra, in no case were oocysts found, which gives us reason to assert about 100% the effectiveness of the complex preparation "Cystodes-ultra" in the marked concentrations against oocysts of coccidia of birds.

In the fourth group of chickens, after administration of a suspension of oocysts treated with a 4% concentration of phenol (basic preparation), oocysts in the litter were found in all periods of research (from 1 to 7 days) in an amount of 0.5 to 10.1 specimens. in the chamber, and the average in the 1st chamber for the period of the study was 5.73 copies. The number of oocysts in 1 g of droppings in this group was 1146, which is 24.49% of the control.

Hence, the intensity of phenol at 4% concentration against coccidial oocysts was 75.51%. Chickens of the 5th group, receiving 2000 sporulated oocysts / ml at all periods of the study with droppings, isolated eimerian oocysts in an amount of 0.6 to 39.6 specimens. in the chamber and the average indicator in one chamber for the period of research was 23.4 copies. The number of oocysts in 1 year of droppings in the group of infected control was 4680 specimens. And this indicator was used as a baseline when calculating the percentage of reduction in the number of oocysts or the intensity of the tested preparations of their concentrations.

The chickens of the 6th group, which received the buffer without oocysts, served as uninfected control and remained free of invasion at all stages of the study.

The intensity of the disinfectants used in their studies or the percentage reduction in the number of eimeria oocysts was determined using the following formula:

IE = (KO _k - KO _d ) / KO _k × 100, where

IE - drug intensity,%;

KO _to - the number of oocysts in chickens of the control group;

KO _d - the number of oocysts in chickens that received oocysts treated with a disinfectant.

Using the data obtained by us in the experiment, we determined the intensity of the "Cystodes-ultra" agent in a 3% concentration

IE = (4680- 306) / 4680 × 100 = 93.46 (p <0.05)

At concentrations of 4 and 5% Cystodes-Ultra showed 100% effectiveness against eimeria oocysts.

The 4% phenol we used as a base preparation showed eimeria against oocysts:

IE = (4680 - 1146) / 4680 × 100 = 75.51% efficiency.

Example 3. Study of the ovocidal activity of different concentrations of the agent "Cystodes-ultra" ultra against the eggs of pork roundworm in laboratory experiments and bioassays for the infection of white mice.

The ovocidal activity of various concentrations of "Cystodes-ultra" and the recommended concentration of phenol was studied in an experiment on the cultivation of Ascaris suum eggs in a thermostat at 26-28 ° C in Petri dishes in a humid chamber for 30 days, in each of which 1000 specimens were placed. eggs. The first chamber was the control one, where cultivation took place in saline. In the second, third and fourth chambers, a solution was introduced containing 3, 4 and 5% concentration of the complex agent "Cystodes-ultra", respectively. After 24 h exposure, Ascaris suum eggs were washed three times with distilled water, microscoped to reveal structural changes, and placed on culture. During the period of egg cultivation, aeration was carried out once every two days, and embryogenesis was monitored. The viability of Ascaris suum eggs was determined by their appearance under light microscopy, staining, and bioassay.

When viewed under a microscope after a 24-hour exposure of Ascaris suum eggs to various disinfectants, no changes in their structure were revealed before being placed for cultivation. Whereas the eggs of the control group, which were in saline, were at the stage of two blastomeres. When viewed after 2; four; 6 and 8 days, in which the active development of blastomeres was observed - there were 4, 8, 16, 32, etc. In group 2, where Ascaris suum eggs were treated with a 3% solution of Cystodes-ultra in the marked the timing of blastomere development was observed in 5–10% of the eggs. In groups 3 and 4, where Ascaris suum eggs were treated with 4 and 5%, no cystodes-ultra solutions were observed during all periods of observation of the development of blastomeres and larvae in the eggs. In the 5th group, where Ascaris suum eggs were treated with the basic phenol preparation at 4%, the development of blastomeres, and then larvae, was observed in 10 - 17% of the eggs.

Our observations made after 10; 12; 14 days and further from the beginning of cultivation made it possible to see the development of larvae inside the eggs of Ascaris suum, which made active movements until the 24 and 28-day periods, and then they stopped moving.

It should be noted that the clearest picture of the development of blastomeres and larvae inside the invasive eggs of the porcine roundworm was observed in the control group.

In groups 2 and 6, blastomeres and larvae did not develop in all Ascaris suum eggs; they had to be searched for and isolated for further observation.

After 32 days of culturing Ascaris suum eggs in a thermostat, a final assessment was made, according to which, in the control group, 98% of Ascaris suum eggs had developed inside larvae. In the second group, after treatment with a 3% concentration of Cystodes-ultra, only 6% of Ascaris suum eggs had developed inside the larva. And in the sixth group after treatment with a 4% concentration of phenol, this figure was 11% (Table 2).

The intensity of "Cystodesis-ultra" against Ascaris suum eggs at a 3% concentration was; 93.6%, and in 4 and 5% concentrations - 100%. The basic drug phenol 4% provided 88.8% intensity efficiency.

Having received a high intensity of efficiency in laboratory experiments, we decided to confirm them by setting up a bioassay.

It should be noted that a bioassay is the most accurate method for determining the viability of invasive ascaris eggs by giving them inside to white mice.

A biological assay for experimental infection of white mice by giving eggs of pig roundworm after cultivation was carried out on 60 mice weighing 18 - 20 g, which were divided into six groups of 10 each. Mice of each group were kept isolated in cages and administered through a plastic tube inside 200 Ascaris suum eggs, which had previously been cultured for 32 days and taken from groups 1-6.

After the administration of the invasive material, the mice were clinically monitored, fed and watered according to the norms, and were kept in the conditions of the institute's vivarium.

After 10 days, the mice were individually sacrificed with ether anesthesia for each group. After dissection of the mice, their lungs and livers were crushed and examined according to Berman's method. The Berman apparatuses were filled with dichlorinated water, left at room temperature (+ 22 ° C) for 2 hours. When studying the sediment, migrating roundworm larvae were found in different numbers. So, in the first group of ascaris larvae were found in all infected mice, their number ranged from 17 to 135 specimens. In the second group of mice, which were given Ascaris suum eggs after exposure to a 3% solution of Cystodes-ultra, ascaris larvae were found in three in an amount of 11 to 32 individuals. In the third and fourth groups of mice, which were given eggs after exposure to 4 and 5% concentrations of the Cystodesis-ultra agent, no migrating Ascaris larvae were found in the lungs and liver. In the fifth group of mice that received ascaris eggs after exposure to 4% phenol solution, ascaris larvae were found in four in an amount from 17 to 56 individuals.

Thus, the complex agent "Cystodes-ultra" at a concentration of 4% showed high intensity in biological tests for experimental infection of broiler chickens with Eimeria, and a biological test for infection of white mice with eggs of pork roundworm, high intensity (IE - 100%).

At a 3% concentration, the agent "Cystodes-ultra" against oocysts of eimeria of chickens showed 93% IE and against eggs of pork roundworm IE was equal to 93.9%.

Example 4. Study of the ovocidal activity of different concentrations of the agent "Cystodes-ultra" against the eggs of toxocara in dogs in a laboratory experiment. The ovocidal activity of different concentrations of "Cystodes-ultra" and the recommended concentration of phenol was studied in an experiment on the cultivation of eggs Toxocara canis in a thermostat at 26-28 ° C in Petri dishes in a humid chamber for 30 days, 500 specimens were placed in each of them. eggs. The first control chamber, where cultivation took place in saline. In the second, third and fourth chambers were made solutions containing 3; 4 and 5% concentration of the complex agent "Cystodesis-ultra", respectively. A 4% phenol concentration (base preparation) was introduced into the fifth chamber. After a 24-hour exposure, Toxocara canis eggs were washed three times with distilled water, microscoped to reveal changes in the structure, shell integrity, and put on cultivation. During the period of egg cultivation, aeration was carried out once every two days, and embryogenesis was monitored. The viability of Toxocara canis eggs was ascertained by light microscopy and staining.

When viewed under a microscope after a 24-hour exposure of T. canis eggs in various disinfectants, no changes in their structure were revealed before being placed for cultivation. And the eggs of the control group, which were in saline, were at the stage of two blastomeres. Further, when viewed after 2; four; For 6 and 8 days, active development of blastomeres was observed in them, there were from 4 to 32, etc. In the second group, where T. canis eggs were treated with a 3% solution of Cystodes-ultra, at the indicated periods of development of blastomeres, 3-10% of the eggs were observed. Whereas in the third and fourth groups, where the eggs of T. canis were treated with 4 and 5% solutions of "Cystodes-ultra" at all periods of observation of the development of blastomeres and larvae in eggs were not observed. In the fifth group, where the eggs of T. canis were treated with the basic preparation phenol 4%, the development of blastomeres and then larvae at different times was noted in 9-17% of the eggs.

Our observations after 9; eleven; 13 days and further from the beginning of cultivation showed the development of larvae inside the eggs of T. canis, which made active movements until the 24-26-day period, and then they had a rest period and they moved only when heated.

The clearest picture of the development of blastomeres and larvae inside the invasive eggs of T. canis was observed by us in the control group.

In the second and fifth groups, blastomeres and larvae did not develop in all T. canis eggs, which were found and isolated for further observation.

After 30 days of cultivation of T. canis eggs in a thermostat, a final assessment was made, according to which, in the control group, 96% of T. canis eggs had developed larvae inside. In the second group, after treatment with a 3% concentration of Cystodesis-ultra, only 5% of T. canis eggs had developed larvae inside. And in the fifth group, after treatment with a 4% phenol solution, this figure was 12%.

The intensity of the "Cystodes-ultra" agent against T. canis eggs in 3% concentration was 94.8%, in 4 and 5% concentrations -100%. Phenol, taken as a base drug, provided 86.5% intensity.

Differentiation of live eggs from dead eggs was carried out by staining 30 eggs from each group. For coloring eggs T. canis used a solution consisting of methylene blue, lactic acid and caustic alkali in the ratio: 0.05; 0.5 g and 15 ml, respectively. Live eggs from the chambers were not stained, and the embryos of dead eggs were colored blue.

As a result of the research, the active and effective concentration of the complex agent "Cystodesis-ultra" against various invasive objects in laboratory experiments and in a bioassay on different models, which is suitable for disinfestation of objects of the external environment, was determined.

Information sources

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4. Safiullin R.T. Parasitic diseases of birds, means and methods of control. - M, 2019 .-- 260 p.

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Claims (2)

A complex agent for disinfestation against oocysts of parasitic protozoa and eggs of animal helminths, characterized by the fact that the initial components are used in the following ratio, wt%: glutaraldehyde eight alkyldimethylbenzylammonium chloride five isopropyl alcohol 12 polyethylene glycol-400 thirty water 45