RU2617417C1 - Method of collection and fixation of nematodes parasiting in abomasum and in intestines of ruminants - Google Patents

Abstract

FIELD: veterinary medicine.

SUBSTANCE: method for collecting and fixing of parasitic nematodes in the abomasum and intestines of ruminants comprises abomasum and small intestine extraction with contents during autopsy. They are incubated for 1 hour at 4°C. Content and scrapings abomasum and small intestine mucous membrane is placed in a bucket or other container, mixing with water in a ratio of 1:1. They are held for 10-15 minutes, the supernatant is decanted with repetition cycle in 3-5 times, until the supernatant becomes transparent. Further precipitate is fixed with 96% ethanol in a ratio of 1:1. Collected and recorded nematodes have morphological structure that is suitable for microscopy and studies of DNA.

EFFECT: simplification of the process of collecting parasitic nematodes during autopsy of ruminants, reducing losses when collecting the samples, with the exception of mixing samples from different animals, providing the possibility of studying DNA collected nematodes.

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Description

The invention relates to the field of agriculture, in particular veterinary helminthology, and can be used to diagnose ruminant nematodes.

Nematodoses are widespread in ruminants both in Russia and in other countries. Damage from nematodoses consists of a deterioration in feed digestibility, a decrease in the rate of development of young animals, an increase in susceptibility to infectious diseases, and a decrease in productivity [1, 3, 5]. According to modern classification, nematodes parasitizing in the abomasum and small intestine belong mainly to the superfamily Trichostrongyloidea and comprise several dozen species [7]. These species differ both in biological features and in resistance to anthelmintic drugs [6]. The methods of intravital diagnosis of helminthiases (coproovoscopy, coprolarvoscopy) do not accurately determine the taxonomic composition of nematodes parasitizing in ruminants in the abomasum and small intestine. In most cases, with coproovoscopy, the taxonomic affiliation of such nematodes can only be determined to the level of the suborder, and with coprolarvoscopy, to the genus. Post-mortem diagnosis (helminth autopsy) makes it possible to accurately determine the species composition of nematodes, but the existing method for collecting and fixing nematodes has drawbacks.

The most widely used method of collecting and fixing nematodes parasitizing in the abomasum and small intestine is still the method proposed in 1928 [4], or its modification in 1971 [2]. According to this method, for washing from mucus and digestive juices, it is proposed to place the contents of the abomasum and small intestine in a bag of mill gas or gauze (with a mesh of 0.2-0.4 mm ² ), tie and then rinse under running water or rinse in a container with water or a pond. At the same time, helminthological material is often not completely washed from digestive juices, which can cause the destruction of nematodes. In some cases, the bags are torn, which leads to the loss of collected material. A certain number of nematodes get stuck in the folds and seams of the bag, which cannot be determined by visual inspection, but when the bag is reused, such nematodes can get into the material from another animal, which will distort the obtained scientific data. The Barbagallo liquid (3% formalin solution (40% formaldehyde) in a 0.8% sodium chloride solution) used to fix nematodes destroys DNA, which makes it impossible to use nematode samples for molecular diagnostics and molecular taxonomy.

In this regard, the objective of this study was to create a method for collecting and fixing nematodes, providing sufficient washing of nematodes from digestive juices, minimizing loss of samples during collection, preventing mixing of samples from different animals, and also allowing the use of collected nematodes for DNA studies.

The proposed method for collecting and fixing nematodes includes: 1. opening an abomasum and small intestine (without separation); 2. visual inspection of their contents and the extraction of large helminths; 3. transferring the contents into a plastic, metal or glass container with a volume of 5-10 liters; 4. the addition of water in a ratio of 1: 1 by volume; 5. mixing; 6. sedimentation for 10-15 minutes; 7. discharge of the supernatant. Actions 4 to 7 are repeated 3-5 times until the supernatant is clear. After this, the precipitate (matrix) is fixed with 96% ethanol in a ratio of 1: 1.

The proposed method has the following advantages:

1. Ease of use - there is no need to make a bag of gauze or mill gas, a plastic or metal bucket, a glass jar, etc. can be used as a washing tank.

2. Minimized sample loss during collection.

3. Excluded mixing samples from different animals.

4. Fixation of the material in 96% ethanol allows the further use of the detected nematodes for DNA studies.

These positive signs of the proposed method in comparison with the prototype ensure that the technical solution meets the criterion of "novelty" and this allows us to conclude that the technical solution meets the criterion of "significant differences".

The effectiveness of the proposed method is demonstrated by the following examples:

Example 1. Collection and fixation of nematodes during helminthological dissection of abomasum and small intestine of cattle.

After opening the abdominal cavity, ligatures were placed on the abomasum (at the border with the book) and the small intestine (at a distance of about 100 cm from the abomasum) to prevent leakage of contents, then the abomasum and small intestine, without separation from each other, were cut off from other parts of the digestive system. After this, the abomasum and small intestine were kept for 1 hour at a temperature of + 4 ° C and then cut lengthwise with scissors. A visual inspection of the internal contents was carried out for the presence of large helminths, then the contents (the volume of which was about 3 l) were placed in a plastic bucket, 3 l of tap water was poured, mixed. The mucous membrane of the abomasum and small intestine was examined for the presence of nematodes, scraped with a scalpel and washed with water in a bucket of contents. After settling for 10-15 minutes, the supernatant was decanted, 3 L of water was again added to the precipitate, mixed and settled. The washing of the precipitate was repeated 3-5 times until the supernatant became transparent. After this, the precipitate (matrix) was fixed with 96% ethanol in a ratio of 1: 1 by volume and poured into another storage vessel. The tank in which the sediment was washed was thoroughly rinsed with tap water to prevent contamination of the following test samples.

Subsequently, the matrix was examined in a laboratory under a binocular magnifier of the MBI-15 type. For this, 1-2 ml portions of the matrix were placed in a Petri dish, viewed at a 12.5-25-fold increase. The detected nematodes were removed and placed in test tubes or bottles with 96% ethanol. Microscopic examination of the detected nematodes revealed that 96% ethanol does not have a destructive effect on their morphological structures, and various DNA fragments of ribosomal and mitochondrial DNA about 700 nucleotides in size were successfully obtained during DNA studies, which is sufficient for diagnostic and taxonomic studies.

Example 2. Collection and fixation of nematodes during helminthological dissection of the abomasum and small intestine of a sheep.

Analogously to example 1, the abdominal cavity of a sheep was opened, the abomasum and the small intestine (in this case, about 50 cm long) were separated, kept for 1 hour at a temperature of + 4 ° C, cut, and transferred to a plastic bucket. The volume of the contents was about 1 l, 1 l of water was added to it, then the procedure was similar to that described in example 1. The collected and fixed nematodes of the sheep had morphological structures suitable for microscopy, and DNA fragments of these samples were successfully obtained fragments of ribosomal and mitochondrial DNA about 700 nucleotides in size.

Example 3. Collection and fixation of nematodes during helminthological dissection of the abomasum and small intestine of European roe deer.

Similarly to examples 1 and 2, the abdominal cavity of the European roe deer was opened, the abomasum and the small intestine (in this case about 30 cm long) were separated, they were kept for 1 hour at a temperature of + 4 ° C, cut, and transferred to a plastic bucket. The volume of the contents was about 0.5 l, 0.5 l of water was added to it, then the procedure was similar to that described in example 1. The collected and fixed samples of European roe deer nematodes were suitable for microscopy and DNA studies.

The obtained results show that the proposed "Method for collecting and fixing nematodes parasitizing in the abomasum and small intestine of ruminants", compared to the prototype - the method of complete helminthological autopsy, is easier to use, minimizes loss of samples during collection, eliminates mixing of samples from different animals, provides good preservation of the morphological structures of nematodes and allows you to study the DNA of the collected samples.

Literature

1. Akbaev M.Sh., Vodyanov A.A., Kosminkov N.E., A.I. Yatusevich, Pashkin P.I., Vasilevich F.I. Parasitology and invasive animal diseases. - M .: Kolos. - 1998 .-- 743 p.

2. Ivashkin V.M., Kontrimavichus V.N., Nazarova N.S. Methods of collection and study of helminths of terrestrial mammals. - M .: Science. - 1971. - 124 p.

3. Safiullin R.T. Distribution and economic damage from the main helminthiases of ruminants // Veterinary medicine. - 1997. - No. 6. - S. 28-32.

4. Skryabin K.I. The method of complete helminthological dissections of vertebrates, including humans. - M.: Publishing House of Moscow State University, 1928 .-- 45 p.

5. Skryabin K.I., Petrov A.M. Fundamentals of Veterinary Nematology. - M .: Kolos - 1964 .-- 527 p.

6. Cintra M.C.R., Teixeira V.N., Nascimento L.V., Sotomaior C.S. Lack of efficacy of monepantel against Trichostrongylus colubriformis in sheep in Brazil // Veterinary Parasitology. - 2016. - V. 216. - P. 4-6.

7. Durette-Desset M.C., Hugot J.P. A cladistic analysis of the Trichostrongyloidea (Nematoda) // Int. J. Parasitol. - 1999.- V. 29 (7). - P. 1065-1086.

Claims (1)

The method of collection and fixation of nematodes parasitizing in the abomasum and small intestine of ruminants, which consists in removing the abomasum and small intestine with contents during postmortem examination, holding them for 1 hour at a temperature of + 4 ° C, characterized in that the contents and scrapings of the mucous membranes the abomasum and small intestine membranes are placed in a bucket or other container, mixed with water in a ratio of 1: 1, settled for 10-15 minutes, the supernatant is drained with a repeat of the cycle 3-5 times until the supernatant becomes transparent, and Then, the precipitate is fixed with 96% ethanol in a 1: 1 ratio, while the collected and fixed nematodes retain the morphological structures suitable for microscopy and DNA studies.