RU2473899C2 - Method for detection of toxocara eggs in faeces - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: for the purpose of analysis, faeces 3-5 g are sampled and placed in a glass, filled with a small amount of water, thoroughly mixed to produce a homogenous suspension, added with water 50-100 ml and filtered through a metal sieve cloth or gauze, into a clean glass. After 5-minute settlement, a supernatant is poured out. Such amount of the suspension is left on the glass bottom to place goes into a centrifuge test tube. It is followed by 3-minute centrifugation at 3000 g; there after the whole liquid is poured out to the deposition to be added with a flotation fluid consisting of saturated solutions of zinc chloride (1.5 kg of ZnCl₂ per 1 l of water), sodium chloride (0.42 kg of NaCl per 1 l of water) and sugar (1.67 kg of Cl₂H₂₂O₁₁ per 1 l of water) taken in proportions 1.75:1:1. The deposition is shaken to produce a homogenous suspension and centrifuged for 3 minutes at 1500 g. A metal loop is used to skim a surface film to be transferred onto a slide and analysed under microscope.

EFFECT: method is fast and possess high diagnostic effectiveness.

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Description

This invention relates to the field of veterinary medicine. It can be used for coproscopic diagnosis of intestinal carnivore helminthiases.

Intestinal helminthiases of carnivores cause great damage to animal husbandry, which consists of a decrease in productivity, product quality and mortality of animals. Sexually mature helminths of the gastrointestinal tract are localized both in the stomach and in the lumen of the intestine. The larval stages of Toxocara canis, through the arterial system, enter various tissues and are encapsulated while maintaining viability. When pregnancy occurs in bitches, part of the larvae activates and migrates through the placenta, causing intrauterine infection of the fetus. Cannibalism plays an important role in the infection of carnivores, when the encapsulated larvae in the muscles of some serve as a source for the infection of others. Animals affected by intestinal helminthiases secrete a huge number of eggs into the external environment, which are highly resistant to adverse environmental factors.

The main specific prevention of helminthiases in animals is prophylactic deworming, the appropriateness of which is determined by the results of helminthovoscopic studies.

The following methods are used for helminthovoscopic diagnosis of intestinal helminthiases of carnivores:

1. The Fulleborn method. A sample of feces weighing 3-5 g is placed in a glass, poured with a small amount of flotation liquid, stirred thoroughly, then 50-100 ml of this solution is added and filtered through a metal sieve or gauze in one layer in a dry, clean glass. The suspension is left to stand for 40-60 minutes, then, by touching the loop, the surface film is removed, transferred to a microscope slide [1].

The disadvantage of this method is that egg flotation takes a long time (40-60 minutes), and the method is not sufficiently effective.

2. The Kotelnikov-Khrenov method. The technique of this method is similar to the Fulleborn method, but ammonium nitrate (granular ammonium nitrate) is used as a flotation fluid. The surface film can be microscopic after 10 minutes [2].

The disadvantage of this method is the strong saturation of the flotation solution, as a result of which there is a rapid crystallization of droplets on a glass slide, which impedes the study of the material and negatively affects the effectiveness of the studies.

The purpose of the invention is the development of a more effective method for the microscopic diagnosis of carnivore helminthiases in order to determine the true helminthological situation for appropriate treatment and prophylactic measures in the studied objects.

A method for the diagnosis of carnivorous helminthiases is carried out according to the following scheme: first, a combined flotation mixture of three ingredients is prepared:

1) a saturated solution of zinc chloride (2 kg of ZnCl ₂ per 1 liter of water);

2) a saturated solution of sodium chloride (0.42 kg NaCl per 1 liter of water);

3) sucrose (1.67 kg per 1 liter of water).

Next, these three components are mixed in a ratio of 1.75: 1: 1.

A fecal sample weighing 3-5 g is placed in a glass, poured with a small amount of water, thoroughly stirred until a uniform suspension appears, then another 50-100 ml of water is added and filtered through a metal sieve or gauze in a dry, clean glass. After 5 minutes of settling, the supernatant is drained. At the bottom of the glass leave such an amount of suspension that would fit in a centrifuge tube. Then a 3-minute centrifugation is carried out at 3000 g, after which all the liquid is drained to the precipitate, and a three-component flotation solution is added to the precipitate. The precipitate is agitated until a suspension is obtained and centrifuged again for 3 minutes at 1500g. After the metal loop, the surface film is removed, transferred to a glass slide and examined under a microscope.

Example 1. In the Municipal Institution “SOBZh”, Kazan (reception and overexposure station for stray animals), dogs were examined and stool samples were taken (10 adults and 25 puppies) for invasion by intestinal nematodes (Toxocara canis and Toxoascaris leonine).

Helminthovoscopic examination was carried out using the Fulleborn methods and the patented method. The research was carried out on the basis of the Department of Parasitology and Radiobiology of Kazan State Academy of Veterinary Medicine named after N.E.Bauman. The study of feces samples according to the Fulleborn method was carried out as follows: 3 g of feces was poured with a small amount of saturated sodium chloride solution (400 g of salt was dissolved in 1 liter of boiled water), thoroughly mixed, then 50-100 ml of this solution was added. The suspension was filtered through a metal sieve or cheesecloth into a conical flask and left to settle. After 30-45 min, the bulk of the eggs floated to the surface due to the lower specific gravity compared to a saturated solution of sodium chloride.

The patented method was carried out as follows: a fresh sample of feces weighing 3 g was suspended in 50 ml of water, filtered through a metal sieve with a mesh size of 0.5 × 0.5 mm. The resulting filtrate was centrifuged at 3000g for 3 minutes, the resulting precipitate was suspended in a flotation solution and centrifuged again at 1500g for 3 minutes. Helminth pop-up eggs, concentrated in the surface film of the solution, were removed using a standard metal loop and their number was counted in a volume of 0.2 ml of flotation fluid without a cover glass with an eyepiece with a magnification of × 8 and a lens with a magnification of × 20.

Preparation of a flotation solution for a patented method for studying feces samples: a three-ingredient flotation mixture is used, which includes saturated aqueous solutions of zinc chloride (ZnCl ₂ , ρ - 1.82), sodium chloride (NaCl, ρ - 1.19) and sucrose (C ₁₂ H ₂₂ O ₁₁ , ρ - 1.28) in a ratio of 1.75: 1: 1 (v / v / v). The specific gravity of the prepared flotation solutions is determined using a densimeter at t 25 ° C.

As a result of the examination, the patented method made it possible to identify almost an order of magnitude more number of helminth eggs (table 1). The Fulleborn method turned out to be much less effective. The patented method showed 100% invasion of the examined animals. In addition, the patented method revealed unfertilized eggs of Toxoascaris leonine.

Table 1. The study of samples of feces of dogs belonging to MU "SOBZH" Kazan "Peace" in the summer No. p / p Groups of animals The number of eggs in 1 g of feces Fullebourne patentable one 1 group 34 ± 1,5 203 ± 3.5 2 2 group 45 ± 4.1 300 ± 10.5 3 3 group 50 ± 2.5 315 ± 1.51 four 4 group 75 ± 3.8 424 ± 11.7 5 5 group 100 ± 7.55 185 ± 8.68 6 6 group 56 ± 3,5 125 ± 5.2 Note: 1-5 groups - puppies;
6 - group - adult dogs.

Thus, the patented method for diagnosing helminthiases in carnivores has a much more pronounced diagnostic efficiency compared to the analogue used (Fulleborn method).

According to the results of helminthovoscopic examination, deworming of dogs was carried out (azinox plus at a dose of 1 tablet per 10 kg of animal body weight).

Literature

1. Lutfullin M.Kh., Latypov D.G., Kornishina M.D. Veterinary helminthology. Kazan, Idel-Press, 2007, 232 pp.

2. Lutfullin M.Kh., Latypov D.G., Kornishina M.D. Helminth-coproscopic studies of animals. Kazan, 2002, 24 p.

3. Akbaev M.Sh. et al. Parasitology and invasive animal diseases. Moscow, Kolos, 1998, 744 pp.

4. Akbaev M.Sh. et al. Parasitology and invasive animal diseases. Moscow, KolosS, 2002, 743 pp.

Claims (1)

A method for the diagnosis of carnivore helminthiases, which consists in the fact that a fecal sample weighing 3-5 g is placed in a glass, poured with a small amount of water, stirred, add another 50-100 ml of water and filtered through a metal sieve in a clean glass, the suspension is left to stand for 5 minutes, after the supernatant is drained, and the sediment is transferred to a centrifuge tube, then a 3-minute centrifugation is carried out at 3000 g, after all the liquid is drained to the sediment, a three-component flotation solution consisting of saturated solutions of zinc chloride (1.5 kg ZnCl ₂ per 1 l), sodium chloride (0.42 kg NaCl per 1 l) and sugar (1.67 kg C ₁₂ H ₂₂ O ₁₁ per 1 l), taken in a ratio of 1, 75: 1: 1, the precipitate is shaken and centrifuged again for 3 min at 1500 g, then the surface film is removed with a metal loop, transferred to a glass slide and microscopic.