CN113016713B - Method for separating echinococcus granulosus eggs from dog feces and hatching ouncercaria sexually in vitro - Google Patents
Method for separating echinococcus granulosus eggs from dog feces and hatching ouncercaria sexually in vitro Download PDFInfo
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K67/00—Rearing or breeding animals, not otherwise provided for; New breeds of animals
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Abstract
The invention discloses a method for separating Echinococcus granulosus eggs from dog feces and incubating oncophora hexadactyloides in vitro. The invention provides a method for preparing echinococcus granulosus.A technical scheme to be protected is that the method comprises the steps of separating to obtain echinococcus granulosus eggs, hatching the echinococcus granulosus eggs by pepsin, shelling by a shelling solution, and purifying by a Percoll culture solution to obtain the echinococcus granulosus.A technical scheme to be protected by the invention is a method for preparing the echinococcus granulosus.A. The Percoll culture solution is prepared by mixing 10 XNCTC 135, a glutamine solution, gentamicin and Percoll. Experiments prove that the obtained oncosphere can be hatched to obtain the oncosphere with less impurities, high purification degree and high survival rate, and the obtained oncosphere can obtain a primary infection model of echinococcosis hepatica after infecting a mouse by the hepatic portal vein, thereby providing a model and research foundation for the subsequent fields relating to echinococcosis pathogenic mechanism, medicine research and development and the like.
Description
Technical Field
The invention relates to the technical field of medicine, in particular to a method for separating Echinococcus granulosus eggs from dog feces and incubating oncophora elaterium in vitro.
Background
Echinococcosis, commonly known as Echinococcosis (Echinococcosis), is a highly dangerous zoonosis caused by echinococcus larvae (Echinococcosis). Echinococcus echinococcus requires two mammals to complete their life history, and the adult, i.e., tapeworm, is parasitic in the small intestine of the terminal host canine; cyst is the larval stage of Echinococcus, which is parasitic in parenchymal organs of humans, herbivorous livestock or rodents (intermediate hosts), mainly the liver and lungs. Echinococcosis, which causes infection in humans, is mainly of two types: cystic Echinococcosis (CE) caused by Echinococcus granulosus (Eg) and Alveolar Echinococcosis (AE) caused by Echinococcus multilocularis (e.m). There are four stages of marked development of echinococcus granulosus and echinococcus multilocularis: both adult (in the terminal host), egg (in the terminal host, mature adult egg after 45 days of infection)/oncosphere (in the intermediate host), cyst larva (in the intermediate host) and metacercaria (in the intermediate host).
The dogs are terminal hosts, the sheep and the cattle are intermediate hosts, people infect the echinococcosis cysticercosis due to the miseating of echinococcus granulosus eggs discharged by the dogs, echinococcus growth capacity is high, the increase of cyst volume can press surrounding tissues to atrophy and dysfunction, secondary infection is easily caused, and the rupture of cysts can cause anaphylactic reaction and even death, so that the health of people is greatly damaged. Echinococcosis is very prevalent, specifically, it is widely prevalent, and is prevalent almost all over the world. The serological positive rate of the population in different popular areas is 5-60%, 300 ten thousand patients with cyst more than 2cm are diagnosed by ultrasonic (the selection of the hazard and control strategy of the echinococcosis in Zhang wenbao, guo gang, lijun.) [ J ]. Chinese animal health care, 2017,19 (007): 4-7 ]. The worldwide adjustment life of cystic echinococcosis for disability is estimated to be 100 to 300 million people years (DALYs).
The current treatment for hepatocystic echinococcosis relies mainly on surgery and there is a risk of recurrence. Clinical drug therapy currently mainly uses benzdanazol and albendazole, but the cure rate for patients is not high. In order to understand the pathogenesis of a disease and to ultimately treat the disease, a great deal of preliminary research work on drugs and pathogenesis in experimental animals is urgently needed. However, the current model of hepatocysticercosis is usually to use the metacercaria or cyst to perform liver puncture or hepatic portal vein injection on the experimental animal to establish a secondary infection model, which is different from the primary infection actually existing in nature, so that the experimental data obtained on the model is still different from the application of the model to human.
Disclosure of Invention
The technical problem to be solved by the invention is how to separate and purify the echinococcus ova and/or how to separate and obtain the echinococcus hexadactylosis.
In order to solve the technical problems, the invention firstly provides a method for preparing echinococcus granulosus oncosphere. The method comprises the following steps: isolating Echinococcus granulosus eggs from Echinococcus granulosus-infected stool; incubating the Echinococcus granulosus eggs using pepsin; shelling by using a shelling liquid; the Echinococcus granulosus oncosphere was obtained by purification using Percoll medium.
The Percoll culture solution consists of solute and solvent. The solvent is Percoll. The solutes may be 10 × NCTC135 liquid medium, glutamine and gentamicin. In the Percoll culture solution, the volume percentage of the 10 XNCTC 135 liquid culture medium can be 10 percent, the content of glutamine is 2M glutamine, and the mass percentage of gentamicin is 5 percent.
Isolating Echinococcus granulosus eggs as described above, comprising the steps of:
a1 Taking the feces of echinococcus granulosus infected dogs, diluting the feces with 0.85% NaCl solution, filtering, collecting filtrate, centrifuging the filtrate, collecting precipitate, and resuspending the precipitate with 0.85% NaCl solution to obtain a liquid which is a host feces-like suspension containing the echinococcus granulosus eggs.
A2 Adding the host manure-like suspension containing the echinococcus granulosus eggs into the upper layer of the saturated sucrose solution, and carrying out purification egg treatment to obtain purified echinococcus granulosus eggs.
The treatment of purified eggs described above in step A2) comprises the following steps: and adding the host manure-like suspension containing the Echinococcus granulosus eggs into the upper layer of the saturated sucrose solution, and then stirring and diffusing the upper layer of the saturated sucrose solution. The stirring diffusion treatment is to use a stirring rod to rotate and stir at a constant speed of 80-90 circles/min for 60 circles to layer the liquid, absorb the upper layer liquid, dilute the upper layer liquid with 0.85% NaCl solution, and then centrifugally collect the precipitate.
The stirring diffusion treatment time is less than 1 minute.
In the above step A2), the volume ratio of the saturated sucrose solution to the host fecal suspension containing echinococcus granulosus eggs may be 9. The time of the host feces-like suspension containing Echinococcus granulosus eggs in the saturated sucrose solution is less than 30 minutes.
In order to solve the technical problems, the invention also provides a method for separating echinococcus granulosus eggs, which is characterized by comprising the following steps: the method comprises separating Echinococcus granulosus eggs from Echinococcus granulosus-infected faeces, the separation of Echinococcus granulosus eggs, comprising the steps of:
a1 Taking the feces of echinococcus granulosus infected dogs, diluting the feces with 0.85% NaCl solution, filtering, collecting filtrate, centrifuging the filtrate, collecting precipitate, and resuspending the precipitate with 0.85% NaCl solution to obtain a liquid which is a host feces-like suspension containing the echinococcus granulosus eggs.
A2 Adding the host fecal-like suspension containing the echinococcus granulosus eggs to the upper layer of the saturated sucrose solution, and performing purification treatment on the eggs to obtain purified echinococcus granulosus eggs.
The treatment of purified eggs described in A2) above comprises the following steps: and adding the host manure-like suspension containing the Echinococcus granulosus eggs into the upper layer of the saturated sucrose solution, and then stirring and diffusing the upper layer of the saturated sucrose solution. The stirring diffusion treatment is to use a stirring rod to rotate and stir at a constant speed of 80-90 circles/min for 60 circles to layer the liquid, absorb the upper layer liquid, dilute the upper layer liquid with 0.85% NaCl solution, and then centrifugally collect the precipitate.
The volume ratio of the saturated sucrose solution to the host fecal suspension containing Echinococcus granulosus eggs described above may be 9.
In order to solve the above technical problem, the present invention further provides an application, wherein the application is any one of the following:
p1, use of any of the methods described above for the construction of a model of primary infectious echinococcosis;
p2, application of the method for separating the echinococcus granulosus eggs in the separation of active parasite eggs.
The active parasite egg may be taenidae eggs. The Taenia solium may be Echinococcus granulosus (Echinococcus grandis), echinococcus multocida (Echinococcus multilocularis), cestode in vivo (Multiceps), taenia vesiculosus (Taenia. Hydatigera), cestode canis (Dipylidium caninum), taenia bovis (Taenia saginata), or Taenia suis (Taenia solium).
The invention discloses a method for enriching, separating and purifying clean eggs from dog feces and hatching oncosphere. Experiments prove that the method for separating the echinococcus granulosus eggs and the echinococcus granulosus oncosphere larvae prepared by the method have high purification degree, and can be applied to the fields of subsequent echinococcus disease pathogenesis, drug research and development and the like.
Drawings
FIG. 1 shows the isolation of Echinococcus granulosus eggs from a saturated sucrose solution. A. Stirring to fully disperse the dung sample of the dog containing the worm eggs, wherein the arrow indicates that the dung sample is 2-3cm below the liquid level; B. separating the feces sample of the dog containing the worm eggs after centrifugation, wherein an arrow indicates a sampling worm egg layer; C. an egg enrichment zone appears after repeated stirring and centrifugation, and an arrow mark shows the egg enrichment zone.
FIG. 2 shows the effect of different stirring diffusion treatments on the later purification and separation of eggs. A. Stirring for 20 circles; B. stirring for 40 circles; C. stirring for 60 circles; arrows indicate isolated worm eggs. Panel A is at 50 times magnification, and panels B and C are at 100 times magnification.
FIG. 3 shows the process of shelling eggs treated with enzyme. Arrows indicate the unshelled eggs. At 100 times magnification.
FIG. 4 shows the microscopic examination of the isolated oncophora by Percoll centrifugation, in which black arrows indicate the deshelled oncophora; unshelled worm eggs are shown by white arrows. At 100 times magnification.
Detailed Description
The present invention is described in further detail below with reference to specific embodiments, and the examples are given only for illustrating the present invention and not for limiting the scope of the present invention. The examples provided below serve as a guide for further modifications by a person skilled in the art and do not constitute a limitation of the invention in any way.
The experimental procedures in the following examples, unless otherwise indicated, are conventional and are carried out according to the techniques or conditions described in the literature in the field or according to the instructions of the products. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
The sources of reagents and consumables in the following examples are as follows:
15mL sterile centrifuge tube (Corning, USA, cat number 430791).
A3 mL disposable plastic pipette (Shanghai Jing Ansheng Biotech Co., ltd., product number: J00082).
Penicillin-streptomycin mixed solution (100X, hyclone, cat # SV 30010).
10 XNCTC 135 liquid medium (Seebio, cat # N3262).
Percoll (density 1.130 g/mL) (GE, cat # 17089102).
RPMI1640 cell culture solution (Hyclone, cat # SH30809.01-500 mL).
200mM glutamine (Beijing Sorbao science and technology Co., ltd., cat # G0200).
Gentamicin (Beijing Solebao science and technology Co., ltd., product number: G8170).
Sterile D-Hanks solution (Beijing Solebao science and technology Co., ltd., cat. No.: H1040).
Nystatin solution (Nystatin 10mg/mL, as in Gibbelofop, cat # SJH 0025).
Trypsin (1.
Pepsin (. Gtoreq.250 units/mg, sigma, cat # P7000-100G).
Example 1 enrichment and isolation purification of Echinococcus caninum eggs
The experiment was set up in triplicate.
1. The echinococcus granulosus protocoid is precipitated (stored in the laboratory, protocoid is separated from naturally infected liver echinococcosis sheep liver, and sick liver entrusted workers collect from Xinjiang Ulluqi Hualing cattle and sheep slaughter house), 1.2mL protocoid is orally infected with beagle dogs, dog anus is adhered by using sticky tapes every day after 45 days of infection, and after the appearance of insect eggs is discovered by microscopic examination, the insect eggs are separated from dog feces.
2. And (3) putting a certain amount of dog dung into a measuring cylinder, adding a sterile 0.85% NaCl solution, and stirring and diluting by using a stirring rod.
The lower part of a coarse screen of 3.100-120 meshes is connected with a barrel, diluted excrement is put into the screen, excrement particles are ground into homogenate by a stirring rod, and the homogenate is washed and sieved by a sterile 0.85% NaCl solution.
4. The filtrate was centrifuged at 1000g for 5min, the precipitate was collected and the supernatant discarded. The precipitate is a suspended fecal-like precipitate.
5. The fecal pellet was suspended with sterile 0.85% NaCl solution, placed on a 200 mesh sieve, and filtered by rinsing with sterile 0.85% NaCl solution.
6. And (4) repeating the step.
7. Adding a sterile 0.85% NaCl solution of two sediment volumes to suspension filter the fecal pellet to obtain a host fecal sample suspension containing Echinococcus granulosus eggs.
8. 9mL of a saturated sucrose solution (67.1% sucrose in water) was added to a 15mL sterile centrifuge tube, and 3mL of a host fecal suspension containing Echinococcus granulosus eggs was added to the supernatant using a 3mL disposable plastic pipette. And (2) extending the suction head into the liquid level of the sample, penetrating the suction head through the excrement layer until the distance between the sample and the sucrose solution is 2-3cm (shown as A in figure 1), and continuously rotating and stirring at a constant speed along the pipe wall to fully diffuse the host excrement suspension containing the echinococcus granulosus eggs in the saturated sucrose solution. The stirring diffusion (namely the treatment of uniform-speed rotation stirring, namely the treatment of continuously floating worm eggs in saturated sucrose solution) is provided with five treatments: 20 turns, 40 turns, 60 turns, 80 turns, 100 turns. The speed is 80-90 circles/minute.
9. Fully diffusing the feces-like suspension in a saturated sucrose solution under five stirring diffusion treatments, centrifuging for 5min at 1000g to layer the feces to obtain a sampling worm egg layer (shown as B in figure 1), sucking the feces-like suspension containing the worm eggs by using a suction pipe in the sampling worm egg layer (taking care not to suck the feces adhered to the pipe wall as much as possible, otherwise, easily separating the later-stage worm eggs to be impure), adding sterile 0.85 percent NaCl solution with 3 times volume, centrifuging for 5min at 1000g after uniform stirring, discarding supernatant and retaining precipitate, wherein the precipitate is echinococcus canis fine-grained eggs. The steps 8-9 can be repeated according to the cleanliness of the eggs (preferably, the time for continuously floating the eggs by saturated cane sugar is not more than 30 minutes, otherwise, the eggs are easy to deform, crack and inactivate). Then, the precipitate was replaced with sterile 0.85% NaCl solution to obtain five kinds of worm egg suspensions purified under the conditions of stirring and diffusion treatment.
And (3) repeating the step (8) on the obtained 3mL worm egg suspension, centrifuging for 5min at 1000g, wherein different layered color bands (shown as C in figure 1) can be seen, the light color band on the uppermost layer is a worm egg concentrated band, and the darker color bands on the middle and lower layers are feces fragment bands containing a small amount of worm eggs. The upper layer of the worm egg zone is the final purified worm egg layer.
10. Observing the eggs in the finally purified egg layer under a microscope, and comparing the purification degrees of the obtained eggs under the five stirring diffusion treatment conditions. The separation and purification results of different stirring diffusion treatments (i.e. 80-90 circles/minute of uniform stirring turns) are shown in FIG. 2 (in FIG. 2, A is 20 circles, B is 40 circles, and C is 60 circles). The result shows that the dog dung impurities are gradually reduced along with the increase of the number of stirring turns (the dog dung impurities can influence the primary infection of mice by the ancylophorus hookeri, so that the death rate is obviously increased and the immune state is changed), the egg impurities separated when the uniform stirring is over 60 turns are greatly reduced (shown as C in figure 2), and the purification degree is high; but the effect of separating and purifying the worm eggs is not different when the number of stirring circles at constant speed exceeds 60 circles (80 circles and 100 circles). Therefore, 60 circles are the optimal stirring times, and the time of stirring 60 circles at a constant speed of 80-90 circles/minute is the optimal stirring diffusion treatment. And (3) separating and purifying for three times while stirring for 60 circles, wherein the average number of eggs separated from each gram of dog dung is 5605, 6302 and 5900.
Sucking the egg layer which is preliminarily removed with dog feces impurities to purify and clean the eggs, adding 10uL of 100 Xdouble antibody (penicillin-streptomycin mixed solution) into each 1mL of suspension until the final concentration is 1% and 10uL of nystatin solution with the concentration of 10mg/mL until the final concentration is 10mg/L to obtain the purified egg suspension, and refrigerating and storing at 4 ℃ for 1-2 years.
The results of separation and enrichment of the echinococcus granulosus infected dog feces by ova show that the separation and purification method of the invention can obtain the echinococcus granulosus ova with less impurities and high purification degree.
Example 2 incubation and Collection of Oncomelania littoralis
The experiments of this example were all set up in triplicate.
The egg shelling liquid consists of solute and solvent, the solute is trypsin and NaHCO 3 And sterile sheep bile, and the solvent is RPMI1640 cell culture solution. In the egg shelling liquid, the content of trypsin is 1 percent (mass percentage content), and NaHCO is used 3 The content of (A) is 1% (mass percentage content), and the content of the aseptic sheep bile is 5% (volume percentage content).
The Percoll culture solution in the following embodiment consists of a solute and a solvent, wherein the solvent is Percoll, the solute is 10 XNCTC 135 culture medium, glutamine and gentamicin, and in the Percoll culture solution, the volume percentage content of the 10 XNCTC 135 culture medium is 10%, the content of the glutamine is 2M glutamine, and the mass content of the gentamicin is 5%.
The preparation method of 1L Percoll culture solution is as follows: 100% Percoll of 100mL 10 XNCTC 135, 10mL 200mM glutamine solution, 50mg gentamycin, 890 mL.
The four purified egg suspensions obtained in example 1 after stirring and diffusion treatment (80-90 circles/min, 20 circles, 40 circles, 60 circles and 80 circles with uniform rotation) were respectively diluted with 0.85% NaCl solution to reach an egg density of 10 5 Count per mL (egg density counting method: diluting the egg precipitate with 0.85% NaCl solution, randomly taking 2uL egg suspension, observing under microscope, and counting up to 200, the concentration of the egg diluted suspension is 10 5 one/mL), then, a 1% pepsin solution (a solution obtained by diluting the solution with sterile D-Hanks until the content of pepsin is 1% (by mass) and adjusting the pH to 2.0 with concentrated hydrochloric acid) in an approximately 10-fold volume was added, and the mixture was incubated at 37 ℃ for 45min with shaking several times. Centrifuging at 1500g for 5min, removing supernatant, adding egg shelling solution, incubating at 37 deg.C for 40min, taking out the test tube every 5min, shaking, and observing egg shelling condition by microscopic examination. After incubation for about 1h, most of the oncophores are hulled (FIG. 3), and the supernatant is discarded after centrifugation at 1500g for 2 min.
Washing the oncosphere precipitate with sterile normal saline, centrifuging for 10min at 4 ℃ and 500g, further purifying to remove egg shells and residual impurities, specifically, adding 5mL of Percoll culture solution at the bottom of a 15mL sterile centrifuge tube, sucking 1mL of suspension (containing 10 ten thousand oncospheres) and adding the suspension to the surface of the centrifuge tube containing 5mL of Percoll culture solution, and centrifuging for 30min at 1500g and 5 ℃. The oncosphere is in a thin mist shape and exists in a gradient layered interface. Transferring the oncorhynchophorus (the extraction of percoll is reduced as much as possible, otherwise, the loss of the oncorhynchophorus in the subsequent washing and centrifugation process is easily caused), adding 10mL of sterile physiological saline at 4 ℃ into a 15mL centrifuge tube, carrying out centrifugation twice at 500g for 10min each time, and finally suspending the oncorhynchophorus sediment by using 1mL of RPMI1640 to obtain oncorhynchophorus suspension.
Performing microscopic examination on 10uL of the oncosphere suspension (see table 1 and figure 4 for details), and performing stirring diffusion treatment and separation purification on the oncosphere and the uncoated worm eggs by rotating at a constant speed for 60 circles at 80-90 circles/min to obtain the total density of 93 oncosphere and uncoated worm eggs, wherein the ratio of the uncoated oncosphere to the uncoated oncosphere can reach 80 +/-2%; the total density of the shelled oncophora and unshelled egg obtained by 80-circle stirring, diffusion and purification is 78 +/-4/uL, wherein the shelled oncophora can reach 61 +/-4%/uL; the purified samples after 20 and 40 circles of stirring still have relatively more impurities, wherein the total density of the shelled and unshelled larvae obtained after 20 circles of stirring and subsequent purification is 34 +/-3/uL, and the shelled and unshelled larvae can reach 61 +/-4%/uL; the total density of the unshelled coenuridae and unshelled worm eggs obtained by stirring for 40 circles and subsequent purification is 49 +/-3/uL, wherein the unshelled coenuridae can reach 68 +/-6%/uL.
In the process of example 1, the host manure-like suspension containing Echinococcus granulosus eggs was placed in a saturated sucrose solution, and the total count of unshelled and unshelled eggs in the diffusion treatment group was statistically different by a formula differential analysis system, wherein the total count was sufficiently diffused by stirring at a constant speed of 80-90 cycles/min for different cycles of stirring (F =60.9, P)<0.01 And the 60-circle stirring diffusion treatment group and the 80-circle stirring diffusion treatment group have no statistical difference, but are both obviously higher than the groups stirred for 20 times and 40 times. The ratio of the components of the shelled and hatched oncosphere is that the statistical difference is not seen in the four groups of different stirring and diffusion treatment modes (X) 2 ＝9.2，P＝0.02>0.01)。
TABLE 1 influence of different stirring frequencies on the subsequent shelling of egg suspensions
The four oncophores obtained by the stirring diffusion treatment, separation and purification are prepared into the oncophora elata with the density of 2 x 10 by using RPMI1640 4 Diluting the oncosphere with RPMI1640 solution, observing 2uL oncosphere suspension at random under microscope, and counting up to 40 eggs, wherein the concentration of the egg diluted suspension is 2 × 10 4 mL), adding 10uL of 100 Xdouble antibody (mixed solution of penicillin-streptomycin) to a final concentration of 1% and 10uL of nystatin solution with a concentration of 10mg/mL to a final concentration of 10mg/L to obtain a suspension of the ancylostoma, and refrigerating the suspension at 4 ℃.
The present invention has been described in detail above. It will be apparent to those skilled in the art that the invention can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While the invention has been described with reference to specific embodiments, it will be appreciated that the invention can be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains. The use of some of the essential features is possible within the scope of the claims attached below.
Claims (8)
1. A method for producing echinococcus granulosus oncosphere comprising: separating Echinococcus granulosus eggs from Echinococcus granulosus-infected feces, incubating the Echinococcus granulosus eggs with pepsin, removing shells with a shelling solution, and purifying with a Percoll culture solution to obtain Echinococcus granulosus hexachelencis;
the Percoll culture solution consists of a solute and a solvent, wherein the solvent is Percoll, and the solute is a 10 XNCTC 135 liquid culture medium, glutamine and gentamicin; in the Percoll culture solution, the volume percentage content of a 10 XNCTC 135 liquid culture medium is 10 percent, the content of glutamine is 2M glutamine, and the mass content of gentamicin is 5 percent;
the separation of the echinococcus granulosus eggs comprises the following steps:
a1 Taking feces of echinococcus granulosus infected dogs, diluting the feces with a 0.85% NaCl solution, filtering, collecting a filtrate, centrifuging the filtrate, collecting a precipitate, and resuspending the precipitate by using a 0.85% NaCl solution to obtain a liquid which is a host feces-like suspension containing echinococcus granulosus eggs;
a2 Adding the host fecal-like suspension containing the echinococcus granulosus eggs to the upper layer of the saturated sucrose solution, and performing purification treatment on the eggs to obtain purified echinococcus granulosus eggs.
2. The method of claim 1, wherein: a2 The treatment of the purified eggs in) comprises the following steps: adding the host manure sample suspension containing the echinococcus granulosus eggs to the upper layer of the saturated sucrose solution, and then stirring and diffusing the upper layer of the saturated sucrose solution; the stirring diffusion treatment is to use a stirring rod to rotate and stir at a constant speed of 80-90 circles/min for 60 circles to layer the liquid, absorb the upper layer liquid, dilute the upper layer liquid with 0.85% NaCl solution, and then centrifugally collect the precipitate.
3. The method according to claim 1 or 2, characterized in that: a2 In) the volume ratio of the saturated sucrose solution to the host fecal-like suspension containing Echinococcus granulosus eggs is 9.
4. A method for separating Echinococcus granulosus eggs, which is characterized by comprising the following steps: the method comprises separating Echinococcus granulosus eggs from Echinococcus granulosus-infected faeces, the separation of Echinococcus granulosus eggs, comprising the steps of:
a1 Taking feces of echinococcus granulosus infected dogs, diluting the feces with 0.85% NaCl solution, filtering, collecting filtrate, centrifuging the filtrate, collecting precipitate, and resuspending the precipitate with 0.85% NaCl solution to obtain a liquid which is a host feces-like suspension containing echinococcus granulosus eggs;
a2 Adding the host manure-like suspension containing the echinococcus granulosus eggs into the upper layer of the saturated sucrose solution, and carrying out purification egg treatment to obtain purified echinococcus granulosus eggs.
5. The method of claim 4, wherein: a2 The treatment of the purified eggs in) comprises the following steps: adding the host manure-like suspension containing the Echinococcus granulosus eggs into the upper layer of the saturated sucrose solution, and then stirring and diffusing the upper layer of the saturated sucrose solution; the stirring diffusion treatment is to use a stirring rod to rotate and stir at a constant speed of 80-90 circles/min for 60 circles to layer the liquid, absorb the upper layer liquid, dilute the upper layer liquid with 0.85% NaCl solution, and then centrifugally collect the precipitate.
6. The method of claim 5, wherein: a2 In) the volume ratio of the saturated sucrose solution to the host fecal-like suspension containing Echinococcus granulosus eggs is 9.
7. Use of the method according to any one of claims 1 to 6 for the construction of a model of primary infectious echinococcosis.
8. Use of a method according to any one of claims 4 to 6 for the isolation of active parasite eggs.
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