RU2802317C1 - Disinfestation agent against parasitic protozoa in young cattle - Google Patents

Abstract

FIELD: veterinary parasitology.

SUBSTANCE: invention relates to an agent of disinvasion against parasitic protozoa of young cattle. Disinfestation agent against parasitic protozoa of young cattle, includes the following components: 6.0 wt.% of glutaraldehyde, 7.0 wt.% of alkyldimethylbenzylammonium chloride, 27.0 wt.% of polyethylene glycol-400, 25.0 wt.% of surfactants, water — up to 100.

EFFECT: above invention is an affordable agent suitable for disinfestation of premises for keeping young cattle and technological equipment in preparation for settling with young animals.

1 cl, 2 tbl, 2 ex

Description

The invention relates to the field of veterinary parasitology and can be used in livestock farms and in the personal farmstead of citizens against exogenous stages of development of parasitic protozoa in young cattle.

Cattle breeding is one of the main branches of animal husbandry in Russia and plays an important role in providing the population with such important food products as meat and milk. And the resulting by-product - manure, is the most valuable raw material for the preparation of organic fertilizers, which help improve soil fertility and increase the yield of grain and fodder crops.

It is known from the experience of the development of cattle breeding in a planned economy that various parasitic diseases often prevent the increase in livestock and increase in productivity of animals, among them parasitic protozoa, helminths and ectoparasites occupy a special place in cattle, which are quite widespread [1, 5].

Of the parasitic protozoa, Eimeria, Cryptosporidium and Buxtonella are the most common, which infect animals of different ages, but they have the greatest negative effect on the body in young animals. Calves of the milk period become infected in the first days of life after birth and are seriously ill with cryptosporidiosis, calves of 2-6 months of age and older are most often ill with eimeriosis, and calves from 6 months of age and older are most often affected by buxstonellosis.

In cattle, 15 species of eimeria have been described to date, causing eimeriasis, which belong to the Apicomplexa type, Sporozoa class, Coccidia order, Eimeriidae family, Eimeriinae subfamily, Eimeria genus. The species Eimeria bovis, E.zuernii, E.ellipsoidalis, E.bukidnonensis are of the greatest importance for young cattle. Studies have shown that calves are most often parasitized by Cryptosporidium species Criptosporidium parvum and C.muris. Cattle are parasitized by one species of Buxtonella Bwctonella sulcata [2-4].

The pathogenic effect of cryptosporidium and eimeria is due to the massive death of invaded epithelial cells and villi, inflammation of the intestinal wall, and impaired absorption of nutrients from the intestine. Animals of older age groups suffer from mild eimeriosis and remain carriers of pathogens. The pathogenic effect of Buxtonella is due to a violation of the integrity of the mucous membrane of the caecum, the absorption capacity of the intestine at their location. In the course of their life, these parasites release toxins that lead to dysfunction of the affected areas of the intestine.

Studies have established that animals infected with parasitic protozoa daily excrete a large number of oocysts and cysts with feces, which pollute environmental objects with invasive elements that are very stable in the environment and retain their viability for a long time.

Our studies have shown that the most polluted by invasive elements of parasitic protozoa and helminths are the objects of the external environment in the premises for keeping animals are the floor of the machines and passages. Walls of pens and animal feeders are less contaminated with invasive elements.

The wide distribution of parasitic protozoa is facilitated by various violations of the technology of growing young cattle: increased air humidity, crowding in rooms, inadequate feeding and violations of veterinary and sanitary rules.

To achieve sustainable results in the fight against parasitic protozoa in young cattle, scientists and practitioners have proposed a number of agents and preparations against both endogenous and exogenous stages. The problem of parasitic protozoa in young cattle continues to pose challenges for researchers to improve measures to combat the exogenous stage of development of parasites.

Of the modern drugs used against exogenous stages of development of parasitic protozoa in young cattle, it should be noted drugs based on chlorine, phenol, iodine, caustic alkalis and others, which are mostly monovalent and do not meet the requirements of veterinary practice in terms of efficiency.

Based on the above, for the disinfestation of environmental objects against oocysts of parasitic protozoa of young cattle, a complex agent "Cystodes-2" has been proposed, which has the following composition: active ingredients (DA) - glutaraldehyde 6.0%, alkyldimethylbenzylammonium chloride 7.0% and auxiliary components - polyethylene glycol 400-27.0%, surfactants - 25.0% and water up to 100% when used together in optimal concentrations in the ratio of mass,% - 6.0; 7.0; 27.0; 25.0.

When considering the active substances, it should be noted that glutaraldehyde is a well-known disinfectant for external use and is active against oocysts and cysts of parasitic protozoa. The second active ingredient, alkyldimethylbenzylammonium chloride, is a reliable antiseptic, antiprotozoal and antifungal agent and is known as an effective disinfectant. Based on the noted advantages of the active substances, when used together against oocysts and cysts of parasitic protozoa in young cattle, they can have a synergistic effect on the parasitic object, and the auxiliary substances used help to ensure better cleaning of the floor, walls and process equipment from biocontamination and more evenly distribute the DI over object surface.

Used as an auxiliary agent, polyethylene glycol-400 (PEG-400) has high emulsifying properties, which ultimately has an effective softening effect on oocysts and cysts of parasitic protozoa. Later, under the action of polyethylene glycol, the cell membrane of the shell of oocysts and cysts of parasitic protozoa partially dissolves, opening access for the penetration of the oocysts and cysts of the disinfectant. In addition, among the auxiliary components used, surfactants should be noted, which provide better cleaning of the floor, walls and process equipment from biological contaminants and evenly distribute the active ingredient over the surface, contributing to an increase in efficiency.

The task of our research was to develop a means of disinfestation against oocysts and cysts of parasitic protozoa in young cattle using available means, suitable for use both manually and mechanically, without corrosive effect on the technological equipment in livestock buildings.

The solution of this problem is achieved by the fact that as a means of disinfestation against oocysts and cysts of parasitic protozoa of young cattle, the complex agent "Cistodes-2" compiled by us was used, which includes: active ingredients - glutaraldehyde 6%, alkyldimethylbenzylammonium chloride 7, 0% and auxiliary components - polyethylene glycol-400 - 27.0%, surfactants - 25.0% and water up to 100%, respectively, in the ratio of mass, %: 6.0; 7.0; 27.0; 25,.

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

The Applicant also does not know the means of a similar purpose, in which the distinctive features of the claimed invention were disclosed with the receipt from its use in any set of features of the specified technical result. Therefore, the claimed invention meets the condition of patentability - inventive step.

Evidence of the industrial applicability of the claimed is given in the following examples.

Examples of specific performance.

Example 1. Preparation of working solutions, cultures of oocysts of eimeria and cryptosporidium of cattle and carrying out a lysis test with different concentrations of a disinfectant.

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

Initially, a complex disinfectant against parasitic protozoa of young cattle was prepared according to the above recipe.

Working solutions with different concentrations of the Cystodez-2 complex disinfectant were prepared as follows. 750 ml of distilled water were poured into the prepared four glass flasks per 1 liter, and 30 ml were added to the first three of them; 40 and 50 ml of Cystodez-2 were thoroughly mixed for seven minutes, then the volume was adjusted to a liter, stirred again for seven minutes and the solution was ready for use. In the fourth flask, 40 g of crystalline phenol was added to 750 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 cultures of Eimeria spp. and Criptosporidium spp. in young cattle. To collect oocysts of eimeria and cryptosporidium, feces of young cattle spontaneously infected with eimeria and cryptosporidium were used, in the conditions of a livestock farm in the Moscow region. Material received from the farm from infected calves was examined by the Fülleborn flotation method using a saturated sodium chloride solution. The surface film from the glasses was removed using a parasitological loop and collected in a Petri dish with dichlorinated water, the oocysts were washed three times from salt, their number was counted in 1 μl and in 1 ml, mixed with a preservative layer - 2.0% potassium dichromate solution and used in further work, when cultivating them in a thermostat at a temperature of +26°C in laboratory conditions. When cultivating for seven days, the culture was aerated daily (40 minutes), and from the fourth day, the oocyst culture was examined and evaluated under a microscope. After seven days of cultivation, the final evaluation of the culture was carried out, the percentage of sporulation was determined, which was 97.5% in the culture of Eimeria spp. and 98.0% in the culture of Criptosporidium spp. SWH buffer was used to dilute the prepared culture. When preparing the buffer, 17.5 ml of 10.0% calcium chloride solution and 5 ml of 10.0% magnesium sulfate solution were added to 3300 ml of distilled water, autoclaved for 20 minutes at 120°C. The concentration of hydrogen ions in the solution was determined using an Akvilon-410 instrument.

Lysis test of Eimeria spp. and Criptosporidium spp. oocyst cultures. with the prepared concentrations of the complex agent (3; 4 and 5.0% solutions of the agent and 4.0% phenol) were carried out in the laboratory. To do this, 50 ml of the prepared agent, phenol, and SWH buffer were placed separately in a 250 ml Erlenmeyer flask, and 50 ml of a solution with Eimeria and Cryptosporidium oocysts at a concentration of 2000 and 3000 oocysts/ml, respectively, was added. Then the flasks with contents were placed on a vibrating table at a speed of 100 rpm for 2 hours.

After that, the contents from the flask were poured into a 1500 ml plastic bottle with a screw cap, the flask with the rest of the solution was rinsed several times and poured into a plastic bottle, and the volume was adjusted to 1500 ml. The contents of the bottle were stirred by inverting (three times) and left at room temperature (22°C) for 24 hours. At the end of the time, the solution was drained to the 30 ml mark, and the precipitate was poured into a new container with a volume of 100 ml, the plastic bottle was rinsed several times with buffer, bringing the volume to 50 ml, and used in further work.

Example 2. Bioassay for experimental infection of calves to determine the effectiveness of a complex tool for disinfestation of machine tools.

To determine the effectiveness of different concentrations of a complex agent for disinfestation in the conditions of the cattle breeding economy of the Moscow region, a bioassay was carried out on experimental infection of 36 calves 10-14 days old with a pre-prepared culture of cryptosporidium and 36 calves of 3 months of age with a culture of eimeria, which were free from cryptosporidium and eimeria and their feed did not contain anti-coccidial drugs. Experimental calves of different ages were subjected to a clinical examination, numbered and, according to the principle of analogues, were divided into 6 groups of 6 animals each.

Calves of 10-14 days of age were kept in cages, and calves of 3 months of age were kept in pens for 6 heads. To control the concentration of oocysts of eimeria (2000 ind./ml) and cryptosporidium (3000 ind./ml), a MacMaster camera and an MBS microscope were used. To dilute the culture, SWH buffer was used so that it was possible to give 5 ml of suspension to each calf with a total of 10,000 Eimmeria oocysts/head and 15,000 Cryptosporidium oocysts/head.

Calves of groups 1, 2 and 3 of each age group were given gradually inside using a syringe with a rubber tube at the end of 5 ml of a suspension of oocysts of Eimeria and Cryptosporidium, treated with 3; 4 and 5.0% solutions of the complex agent. Calves of the 4th group - 5 ml of a suspension of oocysts of eymeria and cryptosporidium, treated with a 4.0% phenol solution. Calves of the 5th group were given orally 5 ml of a suspension of oocysts of eimeria and cryptosporidium diluted with buffer, which served as an infected control. Calves of the 6th group received 5 ml of a buffer solution, which served as an uninfected control. During the test, the calves of all six groups of each age group were in similar conditions and had the same diet. The experimental animals were subjected to daily clinical observations, paying attention to their general condition, their behavior, food and water intake, and visible physiological changes.

To establish the isolation of oocysts of eimeria and cryptosporidium in the faeces of calves of different age groups, fresh feces were collected daily from 15 to 21 days after the introduction of the invasive material, weighed, water was added to a mass of 3000 g, mixed with a mixer for five minutes. Samples for further studies were taken from each group in the amount of 30 g, preserved with a 4.0% potassium bichromate solution, brought to a homogeneous mass and stored in a refrigerator at +4°C.

The presence of oocysts in the feces of calves was determined by the Fülleborn flotation method using a saturated sodium chloride solution with a density of 1.18 g/cm ³ and the number of oocysts/g of faeces was counted using a McMaster chamber.

The effectiveness of different concentrations of the complex agent and 4.0% concentration of phenol was established based on the percentage reduction in oocysts of eimeria and cryptosporidium after exposure to the marked concentrations of the complex agent of disinfestation, compared with infected control calves.

Conducted studies of experimental calves of the first group of 3 months of age, which were given a suspension of eimerium oocysts treated with a 3.0% concentration of a complex agent, oocysts of eimerium in feces were found after three, four and seven days, the average in one chamber over the period of research was 4 .71. In g/faeces of calves of the first group, 942 specimens were found. oocysts, which is 15.6% of the control (Table 1). The intensity of the complex agent in a 3.0% concentration or the percentage of reduction in the release of oocysts after exposure to the agent was:

In the calves of the second and third groups, which were prescribed a suspension of oocysts treated with 4 and 5.0% concentrations of the complex agent, in the study of faecal samples, oocysts of eimerium were not found in any case, which indicates a 100% effectiveness of the complex agent in the tested concentrations against oocysts of Eimeria calves.

Calves of the fourth group, who were prescribed a suspension of eimerium oocysts treated with a 4.0% concentration of phenol (base preparation), oocysts of eimerium in the faeces were found in all periods of research in the amount of 8.3 to 12.3 in the chamber, the average in the chamber for study period 6.14. The number of eimeria in g/faeces was 1228 or 20.3% of the control. The intensity of phenol 4.0% against oocysts of Eimeria calves was 79.7%.

The calves of the fifth control group, which received 2000 oocysts/ml at all times of the study with faeces, excreted eimerium oocysts in the amount from 10.2 to 45.4 in the chamber, the average for the study period was 30.2. In the calves of the control group in g / feces during the study, 6040 copies were found. Eimeria oocysts and this indicator was used by us as a starting point when calculating the intensity of effectiveness of the tested complex agent and the base preparation.

Eimeria oocysts were not found in calves of the sixth control group at all periods of the study.

Our studies have shown that the intensity of the complex agent at a 3.0% concentration against calf eimerium oocysts was 84.4%, at concentrations of 4 and 5.0%, the tested agent showed 100% efficiency, and the base drug phenol 4, 0% provided 79.7% intensity efficiency.

In the study of experimental calves 10-14 days old in animals of the first group, which received a suspension of cryptosporidium oocysts treated with a 3.0% concentration of complex agents, cryptosporidium oocysts were found in feces after two, three, five and six days, the average one chamber per study period 6.83. In g/faeces of calves of the first group, 1366 specimens were found. oocysts, which is 12.84 as a percentage of the control (Table 2). The intensity of the complex agent at a 3.0% concentration, after exposure to cryptosporidium oocysts, was:

The calves of the second and third groups, which were given a suspension of oocysts treated with 4 and 5.0% concentration of the complex agent, were free from oocysts during the noted periods, which indicates a 100% effectiveness of the complex agent against cryptosporidium oocysts.

In calves of the fourth group, which received a suspension of cryptosporidium oocysts treated with a 4.0% concentration of phenol, oocysts in the feces were found in all periods of research in an amount from 1.3 to 23.1 in the chamber, the average in the chamber for the period of research was 11, 35. The number of oocysts in g/feces was 2270 or 21.3% of the control. The intensity of phenol 4.0% against the oocysts of cryptosporidium calves was 78.7%.

The calves of the fifth control group, which received 3000 oocysts/ml at all times of the study with faeces, isolated cryptosporidium oocysts in the amount from 18.4 to 76.5 in the chamber, the average for the study period was 53.2. The number of oocysts in 1 g of feces was 10640 ind.

The calves of the sixth control group were free from cryptosporidium oocysts at all times of the study.

The results of the conducted studies give us reason to recommend a tested complex agent in a 4.0% concentration in livestock farms for the disinfestation of premises for keeping young cattle and technological equipment in order to combat the exogenous stage of development of eimeria and cryptosporidium in their preparation for settling with young cattle.

Information sources:

1. Akbaev M.Sh., Vasilevich F.I., Akbaev R.M. and others. Parasitology and invasion diseases of animals - M., 2008.-776 p.

2. Vershinin, I.I. Animal coccidiosis and their differential diagnosis / I.I. Vershinin. - Yekaterinburg, 1996. - 264 p.

3. Krylov M.V. Key to parasitic protozoa. - St. Peter., 1996.-602 p.

4. Rules for disinfection and disinfestation of objects of state veterinary supervision. No. 13-5-02/0522 dated 15.07.2002.-76 p.

5. Safiullin R.T., Shibitov S.K. Parasitosis of young cattle in the farms of the Central and Ural regions of Russia // Proceedings of the scientific conference "Theory and practice of combating parasitic diseases". M., 2019. Issue. 20. S. 264-269.

APPLICATION

Claims (2)

Disinfestation agent against parasitic protozoa of young cattle, including components, wt.%: glutaraldehyde 6.0 alkyldimethylbenzylammonium chloride 7.0 polyethylene glycol-400 27.0 surfactants 25.0 water up to 100