RU2567845C1 - METHOD OF SEPARATION OF LARVAE T.canis BY METHOD OF DIGESTING LUNG AND LIVER PARENCHYMA OF CARNIVORES - Google Patents

Abstract

FIELD: veterinary medicine.

SUBSTANCE: method comprises sampling a mass of 50.0 g. The sample is ground in a meat grinder with a grating diameter of 3-4 mm. The artificial gastric juice is prepared according to the following recipe: 11.0 cm³ of concentrated hydrochloric acid (specific gravity 1.2) is brought to 1000.0 cm³ with tap water heated to 41-42°C, 7.0 g of food porcine pepsin is added. The artificial gastric juice is poured into the reactor of the apparatus "Gastros" and heated to 41-42°C. The cup with the ground sample is placed, and the apparatus is set in the digestion mode of 50 min with active stirring and settling time of overcook of 10 min. The precipitate is poured in a volume of 20.0 cm³. The presence of larvae is determined under a magnifying glass. For concentration the method of centrifugation at 5000 rev/min for 10 min is used. Then from the test-tube 10.0 cm³ of the upper layer of the liquid is aspirated. The remaining precipitate with the larvae is kept for further study by freezing at -20°C.

EFFECT: method enables to detect effectively the pathogen of toxocarosis at post-mortem diagnostics of this disease.

1 ex

Description

The invention relates to the field of veterinary medicine (parasitology) and can be used for the posthumous diagnosis of toxocariasis of carnivores and the isolation of migrating second-stage toxocari larvae from lung or liver parenchyma in order to obtain parasitic proteins with antigenic properties.

The application of this method in practical veterinary medicine will significantly clarify the epidemiological situation of carnivores toxocariasis and assess the risk of people becoming infected with these helminths, justify the need for control over the development of the parasite population in cities, and obtain specific material for creating test systems or vaccines.

Toxocariasis is an invasive zoonotic disease that has important epidemiological and epizootic and great social significance, the causative agent of which is the nematode of the family Anisakidae, genus Toxocara, a species of Toxocara canis (Werner, 1782) - canine parasite. A significant accumulation of dogs in urban settlements, their frequent invasion by helminths, and the lack of stone disinfestation agents creates a high level of soil contamination with nematode eggs, which makes them one of the important epidemiological components of the urban environment. Defeats of a person under these conditions by migrating toxocar larvae (the so-called “larva migrans”) are not uncommon, and for some groups of the population, for example children, pose a significant threat to health.

Young dogs are most infected, and puppies from 90 to 100% are infected with toxocaras from birth to 30 days, due to the ability of the larvae to resume their activity and continue migration during lactation during lactation, while they enter the organs and tissues fetus. Most of the larvae that emerge from the eggs in the gastrointestinal tract complete a hepatopulmonary migration and reach a mature stage in the intestine. Subsequently, the release of toxocar eggs into the external environment begins. One female toxocara is capable of secreting 200,000 eggs per day with feces, so environmental contamination is growing very quickly. In dogs older than one year, larvae usually accumulate in somatic tissues and remain viable for 2–3 years [1]. A high percentage of dogs infected with toxocaras is the result of lifelong asymptomatic carriage of larvae [2].

In this regard, it is urgent to develop and improve diagnostic methods to increase the efficiency of detection of tissue, migrating toxocar larvae before helminths enter the intestines and reach puberty (no invasive elements are released into the external environment). On the other hand, the presented method allows us to clarify the diagnosis in cases of sudden death of puppies with a low degree of invasion or with pathology of an unclear etiology.

A known method of obtaining a culture of larvae of Toxocara canis, developed P.A. Solopov (2009) [2] from fertilized eggs. The toxocar egg suspension is kept in the laboratory at a temperature of + 22 ° C, relative humidity of 85% and natural light. The cultivation of toxocar eggs is performed in an environment containing 1% glutamine. When cultured in saline after ten days from the start of the experiment, 97.5% of the eggs were destroyed. Artificial gastric juice consisting of pancreatin, pork bile and 1% hydrochloric acid was added to the ripened eggs. Processing of toxocar eggs with artificial gastric juice was carried out in a thermostat at a temperature of 37.5 ° C for 8-10 hours. After liberation of the nematode larvae from the egg membranes, they were separated from the liquid by centrifugation at 2500 rpm for 15 minutes. Microscopic examination after centrifugation in the sediment contained mobile second stage toxocar larvae.

The author concludes that the optimal parameters for the cultivation of Toxocara canis eggs are: temperature + 23-25 ° C, humidity 82-85%. The completion of larval formation and manifestation of activity is observed after 25-30 days during the cultivation of eggs isolated from the uterus of toxocar in a 1% glutamine solution with the addition of streptomycin and nystatin [2].

A method for obtaining a culture of T.canis larvae of the 2nd stage P.A. Solopova requires a long time period (30 days) and is time consuming. Also, to carry out this work, it is necessary to have sexually mature females of T. canis, isolate the culture of fertilized eggs from the uterus, prepare a nutrient medium and select optimal cultivation conditions. It should be noted that the obtained larvae did not migrate in the host organism and may not have the necessary antigenic proteins and penetration enzymes in their protein composition. In addition, the method proposed by P.A. Solopov, cannot be a tool for the posthumous diagnosis of toxocariasis in the absence of helminths in the intestines of the animal, and the researcher has constant contact with invasive material with hypeallergenic properties.

Attempts to develop ways to digest muscle tissue were made more than 100 years ago. To study the pathology caused by Trichinella and the isolation of larvae, Ströbel (1911) [3] first used the digestion of muscle tissue in artificial gastric juice (IZHS) with a content of 0.5% hydrochloric acid and 0.7% pepsin (5, 0 ml of concentrated HCl and 7.0 g of pepsin per 1 liter of solution). Digestion was carried out at 38 ° C. This method of artificial muscle digestion was widespread and constantly improved [4, 5].

For the first time, Ransom (1916) [6] was used for the diagnosis of pig trichinosis with the use of muscle digestion method, which filled 50.0 g of crushed muscles with 600.0 ml of gastric juice (1% hydrochloric acid and 0.25% pepsin or 10.0 ml and 2.5 g per 1 liter of solution, respectively), and the mixture was incubated in a beaker at 37-40 ° C for 18-24 hours.

The original technique of accelerated muscle digestion, which has become classical and allows you to get the result of the study after 3.5-4.5 hours, was developed by P.A. Vladimirova (1963, 1965, 1968, 1969, 1972) [7, 8, 9, 10, 11]. She introduced 10.0 g of crushed muscles into a flask and poured 15-20 times (by weight of minced meat) the amount of IZHS, calculated on the basis of 1% hydrochloric acid and 3% pepsin (10.0 ml of HCl and 30.0 g of pepsin per 1 liter of solution). Incubation was carried out at a temperature of 37-42 ° C with periodic stirring, the precipitate after digestion was studied in a Petri dish.

These methods were used to study muscle tissue, mainly for the diagnosis of trichinosis.

Closest to the claimed method is a method for detecting trophozoites of sarcocysts [12]. A method for detecting trophozoites of a sarcocyst, including sampling muscle tissue, grinding, laying in flasks, introducing artificial gastric juice, incubation, centrifugation, sediment testing, characterized in that the artificial gastric juice is made up according to the recipe: distilled water at a temperature of 41-42 ° C to 1000 , 0 ml, concentrated hydrochloric acid with a specific gravity of 1.2 12.0-15.0 ml, food pork pepsin 20.0-30.0 g, and incubation is carried out in the Gastros apparatus at a temperature of 41-42 ° C, digest defend, after which the precipitate is subjected centrifugation, 8.0 ml of the upper liquid layer is aspirated from the test tube, a drop is taken from the lower layer and a smear is made on a glass slide, and the remainder is pipetted onto a glass slide and dried, then the prepared preparations are stained according to Romanovsky-Giemsa for 10 minutes and examined under a microscope at magnification × 400 and × 1250 with immersion.

This method is used to diagnose tissue forms of protozoa in the muscle tissue of cattle.

The technical task of the invention is the development of a method for the posthumous diagnosis of toxocariasis of carnivores and the isolation of migrating second-stage toxocari larvae from the lung and / or liver parenchyma.

The technical result is solved by the fact that to isolate toxocar larvae, a method of digestion of the parenchyma of the lungs and liver of carnivores in IHL was used. For this, samples of lung tissue and liver tissue were taken, crushed, artificial gastric juice was prepared according to the following recipe: concentrated hydrochloric acid with a specific gravity of 1.2 11.0 cm ^{3 was} brought with water of tap temperature 41-42 ° C to 1000.0 cm ³ and added food pork pepsin 7.0 g. The ground IHL sample was placed in the reactor of the Gastros apparatus, incubated for 50 minutes, the digest settled for 10 minutes, and the sediment was examined in a volume of 20.0 cm ³ . To concentrate and preserve the larvae, the obtained material was centrifuged at 5000 rpm for 10 min, then 10.0 cm ^{3 of the} upper liquid layer was aspirated from the tube, the remaining sediment with larvae was stored for further studies by freezing at -20 ° C.

The advantage of the artificial digestion method is that with this method you can immediately examine a large mass of samples and detect a very weak infection. The device "Gastros" is designed by the developer to isolate the larvae of worms (trichinella), which are localized in the striated musculature of the pig. We have developed a method using the Gastros apparatus to isolate toxocari larvae, which are localized in the parenchyma of the lungs and liver of carnivores during migration.

Known methods are aimed at the digestion of muscle tissue, our goal was to digest the lung and liver tissue, which requires an increase in the concentration of acid and pepsin to accelerate and better digest.

To isolate the larvae, we proposed a recipe for artificial gastric juice: concentrated hydrochloric acid (specific mass 1.2) 11.0 cm ^3, bring to 1000.0 cm ³ with tap water at 41-42 ° C and add food pork pepsin 7.0 g .

The proposed method allows you to effectively detect the causative agent of toxocariasis in the posthumous diagnosis of this disease, in cases where the invasion is insignificant and when there are no sexually mature helminths in the intestine, on the other hand, in this way a pure culture of larvae can be obtained, which will serve as material for further genetic studies, and also for the production of proteins with good diagnostic or protective properties. The results of the research will allow us to develop and improve methods for the diagnosis of carnivore toxocariasis at the stage of parasite migration and more effectively carry out anti-epizootic measures aimed at combating zoonoses.

The method is as follows.

The selected samples of lung and / or liver parenchyma weighing 50.0 g are carefully ground in a meat grinder with a grid diameter of 3-4 mm. Artificial gastric juice is prepared previously separately according to the following recipe: 11.0 cm ^{3 of} concentrated hydrochloric acid (specific gravity 1.2) is adjusted to 1000.0 cm ^{3 with} tap water, heated to 41-42 ° C, 7.0 g of edible pork is added pepsin. IZHS are poured into the reactor of the Gastros apparatus of the NPO Petrolizer (St. Petersburg), after warming up to 41-42 ° C, a glass with crushed sample is placed in it, and the apparatus is set into operation for 60 minutes at 41-42 ° C ( the digestion time is 50 minutes, the digestion time is 10 minutes), while the sample is automatically mixed and the selected temperature is maintained. After settling out of the reactor, the precipitate is drained in a volume of 20.0 cm ³ . The presence of larvae is determined under a magnifying glass. For concentration, the centrifugation method is used at 5000 rpm for 10 min, then 10.0 cm ^{3 of the} upper liquid layer is aspirated from the tube, the remaining sediment with larvae is stored for further studies by freezing at -20 ° C.

The method of isolation of T.canis larvae by digesting the parenchyma of the lungs and liver of carnivores is illustrated by the following example.

Example.

Three corpses of an outbred street puppy, 1.5 months of age, who died suddenly without pronounced clinical signs, were delivered to the clinic. A total of 8 puppies in the litter, 5 others remained clinically healthy, the dead were weakened and less developed. According to the results of an autopsy, helminths were not detected in the intestine.

Puppy samples of lung parenchyma and liver were collected from puppies. A sample weighing 50 g was ground in a meat grinder with a grid diameter of 3-4 mm. IZHS were prepared separately: 11.0 cm ^{3 of} concentrated hydrochloric acid (specific gravity 1.2) was brought to 1000.0 cm ^{3 with} tap water heated to 41-42 ° C, and 7.0 g of pork pepsin was added. The resulting mixture was thoroughly mixed and poured into the Gastros apparatus for digestion, and a beaker with ground sample was placed in the reactor. Cultivation parameters: temperature 41-42 ° C, digestion time 50 minutes, digestion time 10 minutes. The degree of digestion was determined visually. The resulting digest is a muddy broth without large framed tissue particles; a thin brownish precipitate remains at the bottom of the reactor.

After that, 20.0 cm ^{3 of} sediment was poured into a Petri dish and the presence of larvae was determined under a magnifying glass, then it was poured into a centrifuge tube and centrifuged at 5000 rpm for 10 minutes, then 10.0 cm ^{3 of the} upper liquid layer was aspirated, the remaining sediment with larvae were kept for further studies by freezing at -20 ° C.

As a result, an invasion of stage II toxocar larvae was detected by the method of digesting lung and liver tissue in two of the three dead puppies.

findings

1. It has been established that the developed method for isolating T.canis larvae by digesting the parenchyma of the lungs and liver of carnivores is easily feasible, highly effective, and has no analogues.

2. The time of isolation of toxocar larvae by digestion in IZHS, including sample preparation and evaluation of the result, 80 minutes.

3. It has been established that in the proposed modes, a pure culture of organ parenchyma of impurities of toxocar larvae of the second stage is released.

Bibliography

1. Tumolskaya N.I., Sergiev V.P., Lebedeva M.N., Mazmanyan M.V., Poletaeva O.G. and other Toxocariasis. Clinic. Diagnostics. Treatment. Prevention - Novosibirsk, 2004 .-- 48 p.

2. Solopov P.A. Enzyme-linked immunosorbent assay for the diagnosis of dog toxocariasis, seroepizootological monitoring and therapy. Diss. Cand. vet. sciences. Ryazan, 2009 .-- 114 p.

3. Ströbel N., 1911. - "Münchener med. Wochenschr.", 58, 6, S. 121-125.

4. Bessonov AS, 1970. Epizootology (epidemiology), diagnosis and prevention of trichinosis (monographic analysis and own research). Doctoral diss., Moscow.

5. Bessonov A.S. Diagnosis of trichinosis (part two). Vilnius, Mintis, 1975, 383 pp.

6. Ransom V.N., 1916. - "J. Agric. Res.", 5, 18, p. 819-854.

7. Vladimirova P.A., 1963. Mat. reports of the All-Union Scientific Conference dedicated to the 90th anniversary of the Kazan Veterinary Institute. Kazan, s. 134-135.

8. Vladimirova PA, 1965. The influence of various factors on accelerating the process of digestion of muscles in artificial gastric juice to identify trichinella larvae in pork. - Candidate diss., Moscow.

9. Vladimirova PA, 1968. The work of young scientists. Livestock and veterinary medicine. - In the book: mat. conferences held in 1965 and 1966 M., p. 337-340.

10. Vladimirova R.A., 1969. - In: II International Conference on Trichinellosis, Wroclaw, June 26-29, 1969. Abstract of Papers. Wroclaw, p. 173-174.

11. Vladimirova P.A. Prospects for the use in practice of the method of digesting muscle samples in artificial gastric juice for the diagnosis of trichinosis. // Mat. doc. All conf. on the problem of trichinosis of humans and animals. - Vilnius. - 1972, - p. 124.

12. Usha B.V., Glamazdin I.G., Davydov E.V. A method for detecting trophozoites of a sarcocyst. RF patent 2415416 C1, 08/11/2009.

Claims (1)

A method for detecting T.canis larvae by digesting the parenchyma of the lungs and liver of carnivores, including sampling weighing 50.0 g, grinding in a meat grinder with a grate diameter of 3-4 mm; the preparation of artificial gastric juice according to the following recipe: 11.0 cm ^{3 of} concentrated hydrochloric acid (specific gravity 1.2) is adjusted to 1000.0 cm ^{3 with} tap water heated to 41-42 ° C, 7.0 g of pork pepsin IZHS is added , poured into the reactor of the Gastros apparatus, heated to 41-42 ° C, placed a glass with crushed sample and the apparatus was set in the digestion mode for 50 minutes with active stirring, and the digest settling time was 10 minutes, the precipitate was drained in a volume of 20.0 cm ³ , the presence of larvae is determined under a magnifying glass, the method is used for concentration centrifugation at 5000 rpm for 10 min, then 10.0 cm ^{3 of the} upper liquid layer is aspirated from the tube, the remaining sediment with larvae is stored for further studies by freezing at -20 ° C.