RU2712892C1 - Method of in-vitro egg collection from trichuris (= trichocephalus) ovis, which causes trichuriasis of domestic and wild ruminant - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention relates to veterinary medicine. Method for in-vitro collection of eggs from females of Trichocephalus ovis, which causes a trichuriasis of domestic and wild ruminants, involves that of colon material of spontaneously infected animals when observing helminthological methods on exposing animals after preliminary washing in isotonic solution of 0.9 % sodium chloride, 250 and more live mature females Trichocephalus ovis are sampled. Five females are placed in separate bacteriological dishes with normal Tyrode solution. Exposure at t=25 °C for 3 hours under conditions of a thermostat to obtain pure collection of mature Trichocephalus ovis eggs.

EFFECT: invention enables to obtain high-quality material for analysis.

1 cl, 2 ex

Description

The invention relates to the field of veterinary medicine, and more specifically to parasitology (= helminthology) to the "Method for collecting eggs in vitro from the causative agent of trichocephalosis of domestic and wild ruminant Trichuris (= Trichocephalus) ovis" and intended for use in helminthological laboratory and / or field studies net collections of mature eggs from living, sexually mature females of Trichuris (= Trichocephalus) ovis, from spontaneously infected with the causative agent of trichocephalosis of domestic and / or wild ruminants, which is necessary in the treatment and prevention of trichocephalosis of domestic and / or di ruminants, screening of drugs (anthelmintics) and genetic research - DNA.

Trichocephalosis is a disease of domestic and wild ruminants, which is registered in many countries of the world and in the Russian Federation. [2, 3, 4, 5, 6, 7, 8, 9] The disease often proceeds in a chronic form in adult domestic ruminants, but can also occur in an acute form, especially in young animals, which can cause the death of the latter if untimely diagnosis and treatment and preventive measures are not carried out. [4, 5] The death of domestic and wild ruminants can cause huge economic losses to zoos, circuses, reserves, hunting and farming enterprises. Therefore, the development of various methods that enable researchers to study in more detail the epizootology, biology and species composition of ruminant trichocephaliasis pathogens will allow the development of more effective measures to combat and prevent this disease.

Known methods for collecting helminth eggs from domestic ruminants [G. Kotelnikov - 1, including from circus animals: V.E. Pasechnik et al., - 5] using coproovoscopy methods (flotation, sedimentation, combined), however, they have the following significant drawback:

With these methods, the type of invasion causative agent is unknown, since according to the literature 11 domestic and wild ruminants of the Russian Federation can parasitize 11 species of trichocephalus of the causative agents of trichocephalosis: Trichuris ovis Abildgaard, 1795; T. globulosa Linstow, 1901; T. skrjabini Baskakov, 1924; T. tarandi Puschmenkov, 1939; T. longispiculus Artjuch 1948; T. dzeirani Artjuch, 1948; T. lani Artjuch, 1948; T. capreoli Artjuch, 1948; T. baskakovi Mizkewitch, 1959; T. massino Mizkewitch, 1959; T.schumakovitschi Savinkova, 1967). Therefore, it is impossible to obtain egg collections by coproovoscopy from a certain type of trichocephalus (in our case, the species Trichuris ovis).

You can use the well-known "Method for intravital differential diagnosis of trichocephalus Trichocephalus ovis, Trichocephalus skrjabini, Trichocephalus capreoli from ruminants" [Pasechnik V.E. - 6] This method is effective in making a lifetime diagnosis, that is, clarifying the type of pathogen: Trichuris ovis, T. skrjabini, T. capreoli and others, if we do not have the opportunity to examine the dead ruminants directly at autopsy.

However, if it is necessary to collect mature eggs from a specific species of whipworm females (in our case, from the species: Trichuris (= Trichocephalus) ovis), this method is unfortunately unsuitable because in this method the eggs trichocephalus (= trichuris) enlighten in a mixture of 98.5% - solution of glycerol with a density of 1,256 and 40% lactic acid, taken in a ratio of 1: 1.

Therefore, trichocephalus eggs of any kind (in our case, species: Trichuris ovis) will be unsuitable for cultivation before the invasive stage, after applying this method.

The well-known "Method for in vivo collection of fertilized eggs (in vitro) from the causative agent of the parasitic zoonosis Trichuris (= Trichocephalus) vulpis" [Pasechnik V.E. - 6], which has disadvantages:

Trichuris vulpis is a parasite typical of domestic and wild predators [Pasechnik V.E. - 2, 4, 5, 7], a Trichuris ovis is a typical helminth for domestic and wild ruminants. [2, 3, 4, 5, 6, 9] Therefore, the conditions in the laboratory environment (used solution, temperature and exposure time) for these whipworm females will differ sharply:

other disadvantages: the need to use test tubes, which significantly complicates the sowing of Trichuris ovis eggs, for cultivation to the invasive stage and lengthens the working time of the experimenters;

the use of an official isotonic solution (0.9%) of sodium chloride for collecting eggs from female parasitic zoonosis of predatory Trichuris vulpis, however, the use of this solution in our method for females of Trichuris ovis ruminant parasite is impractical, since the number of eggs in the above-mentioned known method of collecting eggs significantly inferior to the proposed method;

the temperature regime with this known method for collecting eggs from Trichuris vulpis whipworm females is significantly higher (t = 37.5 ° - 39 ° C), which significantly affects the number of eggs obtained by our method from Trichuris ovis females, ruminant whipworm, so how at t> 30 ° C the number of eggs secreted by a living, sexually mature female Trichuris ovis decreases significantly, which is unacceptable, since very often thousands of eggs are needed for infection of experimental animals;

A known method of collecting eggs [V.E. Pasechnik - 2, 4] for laboratory research, in which researchers to collect eggs from helminths section the genitals of females: vaginas, vulva, and uterus.

This method has the following disadvantages:

1) the resulting material (parasite eggs) is very heavily contaminated with parasite tissue residues, which in the course of the experiment causes the development of bacterial microflora and rotting (= destruction) of helminth eggs;

2) the extraction of eggs from the uterus of the parasite (as described above) additionally contaminates the material with a large number of immature eggs.

3) with this method of collecting eggs, obtaining up to 100% of mature eggs is impossible, since the bulk of mature eggs are in the distal, which does not ripen in the uterus of the parasite) and, in addition, during the experiments, the researchers need to cultivate to an invasive stage in order to study laboratory experiments of biology and epizootology of parasites fees in quantities sometimes numbering thousands of mature eggs.

Obviously, for this reason, it is extremely difficult and almost impossible for a researcher to get large quantities of clean collections of up to 100% mature eggs from Trichuris (= Trichocephalus) ovis for experiments in the laboratory and / or field conditions.

Based on the shortcomings of the above-mentioned known methods for collecting eggs from helminths, we used the well-known physiological saline solution Tyrode M. (according to Sviridov S.V. , 2007) [8]: solution composition (g / l of distilled water): NaCl - 8.00, KCl - 0.20, CaCl ₂ - 0.20, MgCl ₂ - 0.10, NaH ₂ PO ₄ - 0.05, NaHCO ₃ - 1.00, glucose - 1.00

- It is used as a blood substitute in humanitarian medicine, which allowed to obtain good results on the collection of eggs from Trichuris (= Trichocephalus) ovis.

The aim of the invention is the use of our first developed "Method of egg collection in vitro from the causative agent of trichocephalosis of domestic and wild ruminants Trichuris (= Trichocephalus) ovis", to obtain in a natural way up to 100% mature eggs from live, mature females

Trichuris ovis and intended for conducting experiments in laboratory and / or field conditions in the study of therapy and prevention of ruminant trichocephalosis, screening of drugs (anthelmintics) and genetic DNA studies.

This goal is achieved by the fact that when collecting eggs from the causative agents of trichocephalosis, we use the first-developed “Method for collecting eggs in vitro from the causative agent of trichocephaliasis of domestic and wild ruminant Trichuris (= Trichocephalus) ovis”, which consists in the fact that the colon material is spontaneously infected domestic and wild ruminants in the study of helminthological methods at autopsy of animals [according to K.I. Scriabin - 9], 250 or more living sexually mature females of Trichocephalus ovis were selected after preliminary washing in isotonic (0.9%) sodium chloride solution, 5 females were placed in separate bacteriological plates with Tyrode saline solution and exposed at t = 25 ° C for 3 h in a thermostat, after which a clean collection of mature Trichocephalus ovis eggs isolated by live females is obtained.

The experimenter receives a net collection of up to 100% mature eggs from Trichuris (= Trichocephalus) ovis females of ruminant trichocephalosis.

A comparative analysis of the proposed technical solution with the prototype: a known method of collecting eggs by sectioning (= destruction) of the female genital organs of parasites, shows that the claimed method differs from the known one in that:

separate (= individual) freshly harvested ruminants from open organs were collected of live, sexually mature Trichuris females (= Trichocephalus) ovis in each bacteriological cup with Tyrode solution and exposed at 3 ° C for 3 hours under a thermostat.

After this exposure, experimenters obtain, with an efficiency of up to 100%, a clean collection of mature helminth eggs naturally secreted from the genitals (vulva, vagina, uterus) of the females, since they are forced to isolate up to 100% mature, pure into Tyrode solution and therefore into bacteriological plates Trichuris eggs (= Trichocephalus) ovis.

These differences allow us to conclude that the claimed technical solution meets the criterion of "novelty." Signs that distinguish the claimed technical solution from the prototype are not identified in other technical solutions, and therefore they provide the claimed technical solution with the criterion of "significant differences".

Examples of specific performance of the proposed method.

Example 1. In a study in meat processing plants, slaughterhouses in Russia (Moscow region), the Republic of Moldova - of the large intestine and especially the cecum from spontaneously infected trichocephalic domestic ruminants (cattle, sheep), live, mature 500 Trichuris females were collected (= Trichocephalus) ovis, which were used to obtain egg collections in two ways :.

1st (experiment) on the proposed “Method of collecting eggs in vitro from the causative agent of ruminant trichocephaliasis Trichuris (= Trichocephalus) ovis”, which consists in the selection of only live, mature 250 or more Trichuris ovis females after helminthological studies of the colon with an autopsy from spontaneously infected with the pathogen ruminant trichocephalosis with preliminary washing of live, mature Trichuris ovis females from the substrate in isotonic sodium chloride solution (0.9%) and 5 females were placed in each bacteriological plate with Tyrode physiological saline and their exposure t = 25 ° C for 3 hours under conditions of a thermostat.

and the 2nd (control) known “Method for collecting fertilized eggs (in vitro) from the causative agent of the parasitic zoonosis Trichuris (= Trichocephalus) vulpis” [according to B.E. Pasechnik -], which consists in the selection of 250 live, sexually mature Trichuris ovis females, after helminthological studies of the large intestine with an autopsy from ruminant spontaneously infected ruminant trichocephalosis pathogen without preliminary washing of live, mature Trichuris ovis females from the substrate in isotonic solution (0.9%) of chloride sodium in separate tubes with an official isotonic solution (0.9%) of sodium chloride (solutio Natrii chlorati isotonica), and exposure of the tubes at t = 37.5 ° -39 ° C for 5 hours in a thermostat.

After 5 hours, the results of the effectiveness of these two methods of collecting eggs from females of the ruminant trichocephaliasis pathogen were compared: Trichuris (= Trichocephalus) ovis. As a result, it was found that:

by a known method (control) that is, without preliminary washing live, sexually mature Trichuris ovis females from the substrate in isotonic sodium chloride solution (0.9%) and placing them in test tubes with isotonic sodium chloride solution (0.9%) (solution Natrii chlorati isotonica) and exposure of tubes with Trichuris ovis females at t = 37.5 ° C-39 ° C for 5 hours under thermostat conditions, the number of eggs isolated by 250 females in solution:

at t = 37.5 ° -39 ° C it amounted to 759 eggs with an exposure of 5 hours in a thermostat.

And according to the proposed method (1st experiment), 250 live, sexually mature Trichuris ovis females were selected with their preliminary washing from the substrate in an isotonic solution (0.9%) of sodium chloride solution in Tyrode solution and 5 live, sexually mature Trichuris ovis females placed in each bacteriological plate and exposure in a thermostat at t = 25 ° C for 3 and 5 hours:

at t = 25 ° C it amounted to 2957 eggs for 3 hours and 3530 eggs for 5 hours.

Analyzing the results of comparative methods of the known (2nd control) and the proposed (1st experiment), it is clearly seen that the developed method of collecting eggs with the worked out parameters:

preliminary washing of live, sexually mature Trichuris ovis females from the substrate in isotonic sodium chloride solution (0.9%);

collecting live, sexually mature females of Trichuris ovis, not in test tubes, but in bacteriological cups - which allows you to save a larger number of mature eggs and directly in the bacteriological cups carry out the cultivation of T. ovis eggs to the invasive stage;

compliance with the temperature regime (not at t = 37 ° -39 ° C, but at t = 25 ° C);

and the use of another solution (not an official isotonic solution (0.9%) of sodium chloride, but Tyrode saline);

and exposure time (not within 5 hours, but within 3 hours) has a clear advantage over the known method, which was confirmed experimentally.

Therefore, this example convincingly shows the advantage of the proposed "Method of egg collection in vitro from the causative agent of trichocephalosis of domestic and wild ruminants: Trichuris (= Trichocephalus) ovis".

Example 2. In a study in meat processing plants, slaughterhouses in Russia (Moscow region), and the Republic of Moldova — of the large intestine and especially the cecum from spontaneously infected trichocephalic domestic ruminants (sheep and cattle), 500 live, mature Trichuris females (= Trichocephalus) were collected ) ovis.

Collected 500 live and mature Trichuris ovis, were used to obtain egg collections in two ways:

1st (experiment) according to the claimed method of collecting eggs (250 live Trichuris ovis females still mature and sexually matured, previously washed from the substrate in isotonic sodium chloride solution (0.9%), followed by placement of 5 females in each bacteriological plate with Tyrode physiological saline and exposing them at t = 25 ° C for 3 hours under thermostat conditions, where they randomly lay eggs in tubes with Tyrode saline.

and 2nd (control) by a known method of collecting eggs by opening the vulva, vagina, uterus of 250 Trichuris ovis females.

Subsequently, 2500 eggs of Trichuris ovis randomly selected (= in a row) obtained by our proposed method (1st experiment) and by a known method by destruction of the genitals of female helminths (2nd control) were cultivated in an incubator at t = 30 ° C and viewed under a microscope to compare the proposed method for collecting eggs (1st — experience) and the known method — by opening the genitals of females: uterus, vagina, vulva (2nd — control).

As a result, it turned out that only 66% (1650 eggs) developed in the collection of eggs by the known method (2nd — control), and the rest of the eggs began to rot, and with the proposed method (1st — experiment), the eggs were collected up to 100% cases were live and developed without the presence of second microflora.

Thus, the egg cultivation results collected according to the method developed and proposed by us (1st experiment) confirmed the data obtained using the proposed “Method of egg collection in vitro from the causative agent of trichocephalosis of domestic and wild ruminant Trichuris (= Trichocephalus) ovis”, which identified by us for the first time.

Using the proposed “Method of egg collection in vitro from the causative agent of trichocephalosis of domestic and wild ruminants: Trichuris (= Trichocephalus) ovis” provides, compared with existing methods of egg collection: coprooscopy, egg collection by sectioning (= destruction) of the genital organs of female helminths (vulva, vaginas, uterus) the following advantages:

the trichocephalic species is known: Trichuris ovis (in contrast to the known egg collection methods: coprooscopy of sedimentation, flotation, combined, when the trichocephalic species is unknown in domestic and wild ruminants in the Russian Federation, according to published data, 11 trichocephalic species can parasitize: T. ovis; T. globulosa ; T. skrjabini et al., Therefore, the known intravital methods for collecting eggs (flotation, sedimentation, combined) are unsuitable for collecting eggs T. ovis;

reduces time and reduces the complexity of research by eliminating the need to open the genitals of females: vulva, vagina, uterus Trichuris ovis (in contrast to the method of collecting eggs by opening the genitals of female helminths);

eliminates the contamination of the obtained egg collection by scraps of tissue particles of the female genital organs (vulva, vagina, uterus) from Trichuris ovis (in contrast to the method of collecting eggs by opening the genitals of female helminths);

excludes decay and development of a second bacterial infection in the culture of the method of efficient and clean egg collection in vitro obtained by our proposed method of ruminant trichocephalosis: Trichuris ovis (in contrast to the method of collecting eggs by opening the genitals of female parasites);

collecting live, sexually mature Trichuris ovis females, not in test tubes, but in bacteriological plates allows:

a) maintain a larger number of mature eggs,

b) and directly carry out the cultivation and observation of the egg culture of Trichuris ovis (in contrast to the known method of egg collection (in vitro) from the causative agent of parasitic zoonosis Trichuris (= Trichocephalus) vulpis, in which test tubes, rather than bacteriological plates, are used, which significantly complicates sowing eggs Trichuris (= Trichocephalus) ovis, for cultivation to the invasive stage and lengthens the working time of the experimenter [7]);

compliance with a more efficient temperature regime (not at t = 37 ° 39 ° C, but at t = 25 ° C), allows you to get a much larger number of mature eggs (in contrast to the known method of egg collection (in vitro) from the causative agent of parasitic zoonosis Trichuris ( = Trichocephalus) vulpis [7]);

the use of another solution: not an official isotonic solution (0.9%) of sodium chloride, but Tyrode physiological saline has a distinct advantage in contrast to the known method of collecting [7] eggs (in vitro) from the causative agent of parasitic zoonosis Trichuris (= TrichocephaIus) vulpis, which and it was confirmed by us experimentally;

the exposure time (not within 5 hours, but within 3 hours) made it possible to obtain a sufficiently large number of mature eggs necessary for experiments, since when exposed for more than 5 hours, Trichuris ovis females begin to lay in a rather large number of unfertilized eggs from the uterus of females whipworm, and this significantly affects the ability to conduct high-quality experiments, which is another advantage and distinguishability of our proposed method of collecting eggs in vitro from the causative agent of trichocephalosis of domestic and wild ruminants Trichuris (= Trichoceohalus) ovis, by known collection method [7] eggs (in viro) from exciter parasitic zoonosis Trichuris (= Trichocephalus) vulpis (this was also confirmed experimentally);

“An in vitro method for collecting eggs from the causative agent of trichocephalosis of domestic and wild ruminants Trichuris (= Trichocephalus) ovis” is easily practiced in laboratory conditions;

the proposed method provides up to 100% clean egg collection in vitro, without scraps of tissue particles of the genital organs (vulva, vagina, uterus) from living, mature and identified to the species Trichuris (= Trichocephalus) ovis females, from 11 parasitizing trichuris species in ruminants of the Russian Federation , the causative agent of trichocephaliasis from ruminant spontaneously infected trichocephalus in zoos, circuses, and in farms, which is necessary for the treatment and prevention of trichocephalosis of domestic and / or wild ruminants, screening of drugs (anthelmintics) and genetic studies DNA research.

Sources of information taken into account when preparing the application:

1. Kotelnikov G.A. Helminthological studies of animals and the environment. //Directory. - M. - Kolos. - 1983. - 208 p.

2. Pasechnik V.E. Cross infection with trichocephalus of pricked dogs and sheep in Moldova. // Bull. All-Union Institute of Helminthology. - M. - 1984. - Issue. -37. - S. 57.

3. Pasechnik V.E. On the morphology of Trichuris species (= Trichocephalus) ovis Abildgaard, 1795, T. skrjabini Baskakov, 1924 // Proceedings of the All-Russian Research Institute of Helminthology named after K.I. Scriabin. - M. - 1999. - Ed. Russian Agricultural Academy. - Tom. - 35. - S. 85 - 120.

4. Pasechnik V.E. Diss. candidate of veterinary sciences. - M. - All-Russian Research Institute of Helminthology named after K.I. Scriabin (VIGIS) -2000. - S. 124-125.

5. Pasechnik V.E., Uspensky A.V., Mikulets Yu.I., Zhdanova V.Yu. Guidelines for the diagnosis, prevention and control measures for helminthiases of circus animals. // M. - Ed. Russian Agricultural Academy. (RAAS) - 2008 - 50 p.

6. Pasechnik V.E. Method for intravital differential diagnosis of trichocephalus Trichocephalus ovis, Trichocephalus skrjabini and Trichocephalus capreoli ruminants. // Bulletin of the Federal Service for Intellectual Property, Patents and Trademarks of the Russian Federation. - M., 2010 - 22. (Patent for the invention No. 2396069)

7. Pasechnik V.E. The method of in vivo collection of fertilized eggs (in vitro) from the causative agent of the parasitic zoonosis Trichuris (= Trichocephalus) vulpis // Bulletin of the Federal Service for Intellectual Property, Patents and Trademarks of the Russian Federation. - M. - 2015. - No. 21 (Patent for invention No. 2557973).

8. Sviridov S.V. Balanced and special solutions of electrolytes // Difficult patient No. 8. - T. 5. - 2007 - S. 37.

9. Skryabin K.I. The method of complete helminthological dissections of vertebrates, including humans. // Ed. 1st Moscow State University. - M. - 1928. - 45 p.

10. Skryabin K.I., Shikhobalova N.P. and I.V. Orlov. Trichocephalids and capillariids of animals and humans and the diseases caused by them. // Basics of nematology. Ed. USSR Academy of Sciences. - M. - 1957 -T. VI. - 587 p.

Claims (1)

A method of collecting mature eggs in vitro from females of the causative agent of trichocephalosis Trichocephalus ovis, which consists in the fact that from the colon material of spontaneously infected domestic and wild ruminants, when examining animals with helminthological methods, 250 or more living sexually mature females of Trichocephalus ovis are selected after preliminary washing 0.9% sodium chloride solution, placed 5 females in separate bacteriological plates with Tyrode physiological saline, exposed at t = 25 ° C for 3 hours in a thermostat, after which o receive a clean collection of mature Trichocephalus ovis eggs secreted by live females.