CN112877215A

CN112877215A - Separation and purification method and inoculation method of Eimeria tenella sporozoites

Info

Publication number: CN112877215A
Application number: CN202110227960.1A
Authority: CN
Inventors: 林瑞庆; 周德荣; 陆肖; 蔡晓懿; 方园婷; 蒋嘉豪; 孟甜; 王瑞珍; 翁亚彪
Original assignee: South China Agricultural University
Current assignee: South China Agricultural University
Priority date: 2021-03-02
Filing date: 2021-03-02
Publication date: 2021-06-01

Abstract

The invention provides a separation and purification method and an inoculation method of Eimeria tenella sporozoites, and relates to the technical field of biology. The separation and purification method comprises the following steps: adding water into the infected chicken manure, and filtering; centrifuging, adding saturated saline solution into the precipitate, centrifuging, and adding water into the supernatant to obtain oocyst diluent; centrifuging, adding chloramine T solution into the precipitate to obtain sporulated oocyst suspension; centrifuging, adding saturated salt solution into the precipitate for heavy suspension, centrifuging and collecting the oocyst precipitate; adding sodium hypochlorite solution for resuspension, and collecting oocysts; and (3) oscillating and breaking, stopping oscillating when the sporangium is completely escaped, centrifuging to obtain the sporangium, adding pancreatin digestive juice for digestion, filtering, centrifuging and collecting precipitates to obtain the eimeria tenella sporozoites. The inoculation method of the invention is to accurately inject the sporangium or the sporozoite into the middle section of the chicken muscle stomach and the small intestine to realize successful infection. The purifying method of the invention has the advantages of high purifying degree of the sporangium and the sporozoite, simple and convenient experimental operation and 100 percent of infection success rate after injection and inoculation.

Description

Separation and purification method and inoculation method of Eimeria tenella sporozoites

Technical Field

The invention relates to the technical field of biology, in particular to a separation and purification method and an inoculation method of Eimeria tenella sporozoites.

Background

Coccidiosis is considered one of the important infectious diseases of poultry, causing economic losses of over 20 billion pounds each year to the poultry industry worldwide. Eimeria is a major pathogen causing coccidiosis in poultry, and can cause symptoms such as diarrhea, weight loss, decreased egg production, and the like, and sometimes even death. In addition, infection with eimeria can promote opportunistic infections with other pathogens, such as clostridium perfringens. Moreover, their oocysts are very resistant to the environment, which makes control measures quite difficult. Conventional disease management strategies have relied primarily on prophylactic administration, but the chicken coccidiosis causing Eimeria have developed resistance to most anticoccidial drugs. Therefore, in recent years, researchers at home and abroad have conducted a great deal of research on the identification, gene editing, genetic stability, vaccine development and the like of coccidian species of chicken.

The chicken coccidiosis oocysts contain 4 sporangia, and genetic genes among the sporangia are inconsistent, but each sporangia contains 2 sporozoites with identical genomes. The populations used for molecular genetic studies must be completely genomically identical, and the products obtained by traditional single oocyst isolation techniques present the possibility of genomic impurities, and therefore, the manner in which monosporal and monosporal inoculations obtain cloned populations appears to be of great importance. Research reports that monosporangies are separated from an oocyst population with early maturity and drug resistance, and molecular genetic identification shows that the offspring inoculated by oral administration is only from 2 maternal oocysts, which indicates that the monosporangic inoculated population is consistent in genetic genes, namely a true cloned population.

At present, the prior art has enabled the isolation and purification of oocysts, sporocysts and sporozoites, for example, by purifying coccidia sporozoites by DE-52 anion exchange chromatography. However, this method is time consuming, involves more equipment, and results in higher instability for obtaining a colony of cloned oocysts. There are also reports in the prior art of using a microscope to isolate monosporangium and establish clones of Eimeria tenella, but the success rate is only 17.8%. In the prior art, the monospore is absorbed by using microscope operation and injected into the cecum exposed after dissection, so that the separation and inoculation of the monospore are realized, but the success rate is only 22%, the operation difficulty coefficient is high, the requirement degree of precision equipment is high, and much inconvenience is brought to research.

Disclosure of Invention

Therefore, there is a need to provide a method for separating and purifying sporozoites of Eimeria tenella, which can obtain high purification degree of sporangium and sporozoites and greatly improve the infection success rate after inoculation.

A method for separating and purifying Eimeria tenella sporozoites comprises the following steps:

s1, taking the excrement of the chicken infected with Eimeria tenella, adding water for dilution, sieving the mixture, and keeping the filtrate;

s2, centrifuging the filtrate, collecting the precipitate, adding saturated saline solution for resuspension, centrifuging, collecting the supernatant, and adding water for dilution to obtain an oocyst diluent;

s3, centrifuging the oocyst diluent, collecting precipitates, adding a chloramine T solution, and obtaining sporulated oocyst suspension after the oocysts are sporulated;

s4, centrifuging the sporulated oocyst suspension, collecting precipitates, adding saturated saline solution for resuspension, centrifuging, and collecting oocysts;

s5, adding a sodium hypochlorite solution into the oocyst sediment for resuspension, centrifuging, and collecting oocysts on the upper layer;

s6, preparing oocysts into suspension, oscillating and crushing, stopping oscillating when all the sporangiums escape, centrifuging, and collecting precipitates to obtain the sporangiums;

s7, preparing sporangium into suspension, centrifuging, collecting precipitate, adding pancreatin digestive juice, digesting until all sporozoites are released, stopping digesting, centrifuging, collecting precipitate, preparing into suspension, and filtering to obtain precipitate, i.e. Eimeria tenella sporozoites.

The separation and purification method provided by the invention is improved by adopting a conventional separation and purification method, adopting chloramine T as an inducer, using glass beads to shake and break (8-10min), prolonging the digestion time (60-90min), digesting by using an optimized pancreatin digestive juice, enabling sporocysts to completely escape, then enabling sporozoites to completely escape through digestion, and then filtering the sporozoites by using a G3 sand core funnel, thereby facilitating the purification of the sporocysts and the sporozoites and improving the extraction rate of the sporozoites. Moreover, the separation and purification method has low dependence on precision equipment or instruments, and can realize the high-efficiency purification of the Eimeria tenella sporangia and sporozoites by adopting conventional reagents and equipment in a laboratory.

In one embodiment, the step S1 specifically includes: inoculating Eimeria tenella to 1-2 weeks old partridge, detecting coccidia 5-6 days after infection, collecting 6-10 days of feces containing Eimeria tenella oocysts; adding 2-3 times of water to dilute the feces, sequentially sieving with 80 mesh, 100 mesh and 300 mesh sieves, and retaining the filtrate.

In one embodiment, the step S2 specifically includes: centrifuging the filtrate at 1400-1600rpm for 3-4min, collecting precipitate, adding 1-3 times of saline solution, centrifuging at 2700-2900rpm for 3-4min, collecting supernatant, and adding 4-6 times of water for dilution to obtain oocyst diluent.

In one embodiment, the step S3 specifically includes: centrifuging the oocyst diluent at 3200-3400rpm for 1-3min, collecting the precipitate, adding water for washing, centrifuging, retaining the precipitate, adding a chloramine T solution with the concentration of 1.0 w/v%, culturing at 28 ℃, and obtaining sporulated oocyst suspension after the oocysts are sporulated.

In one embodiment, the step S4 specifically includes: centrifuging the sporulated oocyst suspension at 3500-3700rpm for 4-6min, collecting the precipitate, adding water for washing, centrifuging at 3500-3700rpm for 4-6min, retaining the precipitate, adding saturated saline solution for resuspension, collecting the upper oocysts, adding 4-6 times of water for washing, centrifuging at 3500-3700rpm for 4-6min, and collecting the oocyst precipitate.

In one embodiment, the step S5 specifically includes: adding 4-6 times of sodium hypochlorite solution with concentration of 30 w/v% into the oocyst sediment for resuspension, standing at 0 ℃ for 5-7min, centrifuging at 3500-.

In one embodiment, the step S6 specifically includes: taking oocysts to prepare suspension, adding glass beads with the volume of 1-2 times of the oocysts, carrying out oscillation crushing for 8-10min, stopping oscillation when all the sporangiums escape, centrifuging at 3500-3700rpm for 4-6min, and collecting precipitates to obtain the sporangiums.

In one embodiment, the step S7 specifically includes: taking sporangium to prepare suspension, adding pancreatin digestive juice, performing shock digestion at the temperature of 40-42 ℃ and the rotational speed of 160rpm for 60-90min, stopping digestion until all sporozoites are released, centrifuging at the speed of 3500 rpm and 4500rpm for 8-12min, taking precipitate, adding PBS, mixing uniformly, filtering by a G3 sand core funnel by using a mechanical vacuum pump, and centrifuging at the speed of 3500 rpm and 4500rpm for 8-12min, wherein the precipitate is the eimeria tenella sporozoites.

In one embodiment, the pancreatin digest is a PBS buffer solution containing 0.5 w/v% trypsin and 5 w/v% sodium taurodeoxycholate.

The invention also provides an inoculation method of the Eimeria tenella sporozoites, which comprises the steps of taking the Eimeria tenella sporozoites obtained by the separation and purification method, carrying out heavy suspension by using PBS buffer solution, and injecting the Eimeria tenella sporozoites into the small intestine and/or the vein of the chicken.

The invention also provides an inoculation method of the Eimeria tenella sporangia, the Eimeria tenella sporangia obtained by the separation and purification method is taken, resuspended by PBS buffer solution and then injected into the muscular stomach and/or small intestine of the chicken.

According to the inoculation method, the sporangium or the sporozoite is accurately injected into the muscular stomach and/or the small intestine of the chicken, so that mechanical damage caused by passing through the crop and the muscular stomach is avoided, the inoculation success rate of the sporangium and the sporozoite is favorably improved, and a foundation is laid for the injection inoculation of the monosporangium and the monospora and the acquisition of cloned oocyst groups.

Compared with the prior art, the invention has the following beneficial effects:

the separation and purification method provided by the invention is improved by adopting a conventional separation and purification method, adopting chloramine T as an inducer, using glass beads to shake and break (8-10min), prolonging the digestion time (60-90min), adopting optimized pancreatin digestive juice to digest, enabling sporocysts to completely escape, then enabling sporozoites to completely escape through digestion, and then filtering the sporozoites by using a G3 sand core funnel, thereby being beneficial to purification of the sporocysts and the sporozoites and improving the extraction rate of the sporozoites. Moreover, the separation and purification method has low dependence on precision equipment or instruments, and can realize the high-efficiency purification of the Eimeria tenella sporangia and sporozoites by adopting conventional reagents and equipment in a laboratory.

According to the inoculation method, the sporangium or the sporozoite is accurately injected into the muscular stomach and/or the small intestine of the chicken, mechanical damage caused by passing through the craw and the muscular stomach is avoided, the inoculation success rate of the sporangium and the sporozoite is favorably improved, experiments show that the inoculation success rate of the sporangium and the sporozoite purified by the method reaches 100%, and a foundation is laid for the injection inoculation of the monosporangium and the monospora and the acquisition of cloned oocyst groups.

Drawings

FIG. 1 is a morphogram of Eimeria tenella oocysts, sporangia and sporozoites purified in the examples;

wherein A is purified sporulated oocyst, B is purified sporangium, C is purified sporozoite;

FIG. 2 is a diagram of the injection site of the myostomach and small intestine in practice;

wherein, A is 1-week-old Qingyuan partridge, B is the position mark of the muscular stomach and the small intestine, C is the successful injection of bromophenol blue into the muscular stomach, and D is the successful injection of bromophenol blue into the small intestine;

FIG. 3 is a graph of ovulation capsules of intramyogastric inoculation of sporangium in the example;

FIG. 4 is a graph of the ovulation sac of the small intestine inoculated with sporangia in the example;

FIG. 5 is a graph of the ovulation capsule of the small intestine inoculated with sporozoites;

FIG. 6 is a graph of ovulatory capsules inoculated intravenously with seed spores.

Detailed Description

To facilitate an understanding of the invention, a more complete description of the invention will be given below in terms of preferred embodiments. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items.

Example 1

And (3) propagation, separation and purification of Eimeria tenella oocysts.

1. E.tenella Zengcheng strain (ZC strain) is inoculated to 5 Qingyuan partridge chickens of 1 week old, and coccidian investigation is carried out on the 5 th day after infection (5days post infection, 5d p.i.) by a saturated saline floating method to collect 6-10d p.i. oocyst-containing excrement.

2. Diluting feces with 2 times of water, sieving with 80 mesh metal screen, twisting feces, adding 2 times of water into residue, mixing, sieving with 80 mesh metal screen, and collecting filtrate; sieving the filtrate with 100 mesh metal screen, twisting feces, collecting filtrate, sieving with 300 mesh nylon screen, and collecting filtrate.

3. Putting the filtrate in a centrifuge tube, centrifuging at 1500rpm for 3min, collecting precipitate, and removing the white urate impurity on the upper layer. And washing the feces with clear water until the supernatant is clear, and finally combining the precipitates.

4. Wiping water drops on the inner wall of the centrifugal tube, adding 2 times of saturated salt water into the sediment, centrifuging at 2800rpm for 3min, collecting the supernatant, and adding 5 times of clear water for dilution. Adding 2 times volume of saturated saline solution into the precipitate, centrifuging at 2800rpm for 3min, collecting supernatant, and mixing the supernatants to obtain oocyst dilution.

5. The oocyst dilutions were centrifuged at 3300rpm for 2min and resuspended by washing with clear water. Cell counts were performed.

6. Centrifuging, adding 1 w/v% chloramine T solution into the precipitate, culturing for 24h at 28 ℃ under aerobic shaking with the concentration of oocysts of 10-20 ten thousand oocysts/mL, obtaining a fresh sporulated oocyst suspension, centrifuging at 3600rpm for 5min (the following centrifugation steps are all rotating speeds), discarding the supernatant, adding water, washing for 3 times, and washing off chloramine T.

7. Adding saturated saline water into the precipitate for resuspension, centrifuging, collecting the uppermost oocyst, adding 5 times volume of water for resuspension, centrifuging, and collecting the oocyst precipitate.

8. Adding 5 times volume of 30 w/v% sodium hypochlorite solution into the oocyst precipitate, resuspending, standing in ice bath for 6min, centrifuging, collecting the uppermost oocyst layer, adding 5 times volume of water, mixing, centrifuging, washing with water for 3 times, and washing off sodium hypochlorite.

9. To the oocyst pellet was added an appropriate amount of 1 w/v% chloramine T solution and stored at 4 ℃ until use.

Example 2

The injection site is located.

3 Qingyuan partridge chickens of 1 week old were taken for clinical trials. Firstly, 3 chickens are taken to be subjected to abdominal cavity dissection, the specific positions of the muscular stomach and the middle section (jejunum and ileum) of the small intestine of the chickens are obtained by measuring with a vernier caliper, and the length, width and height of the organs from the tail end of the chicken keel process are respectively recorded. Two chickens were then selected and the bromophenol blue indicator was aspirated by a 1mL syringe and injected into the predicted locations of the myostomach and small intestine. And finally, cutting open the abdominal cavity of the chicken, observing the injection condition of bromophenol blue in the small intestine, the serosal layer of the muscular stomach and the content, and finally determining the exact injection position of the muscular stomach and the small intestine.

Prior to formal entry into the vaccination experiment, the specific location of the myostomach and small intestine of 1 week old partridge chickens (fig. 2A) was known. After the abdominal cavity of the chicken is dissected, the tail end of the chicken sternum is taken as the center, and the measurement is carried out by a vernier caliper, so as to obtain the positions of the middle sections of the stomach and the small intestine: the length is 8.0 +/-1 mm, the width is 9.4 +/-1 mm, and the depth is 15 +/-1 mm (figure 2B). Using bromophenol blue as an indicator, performing fixed-point injection on the predicted part, wherein the result is as shown in the figure, the muscular stomach is positioned at the right side of the abdominal cavity, the position of the muscular stomach can be easily sensed by touching, and the indicator is successfully injected into the muscular stomach (figure 2C); the middle small intestine (jejunum and ileum) was located on the left side of the abdominal cavity, and the indicator was successfully injected into the small intestine (fig. 2D). In conclusion, experiments show that the myogastric and small intestine are accurately positioned, and the indicator is successfully and accurately injected.

Example 3

Purifying and inoculating the Eimeria tenella sporangium.

1. Taking the sporulated oocysts purified in example 1, adding sterile glass beads in a volume ratio of 1:1, carrying out shaking and crushing for 10min, observing under a microscope until all the oocyst walls are crushed, repeatedly observing for 3 times, and stopping shaking when all the sporangiums escape. Washed 3 times with sterile PBS solution, the washed cyst fluid was centrifuged at 3600rpm for 5min, and the supernatant was discarded. The precipitate was mixed with 20mL PBS, filtered through a 20 μm pore size wire mesh screen, centrifuged at 3600rpm for 5min, the supernatant was discarded, and 10mL PBS was added and mixed for further use.

2. Injecting the purified sporangia into the muscular stomach and small intestine of Qingyuan partridge chickens of 1 week old by a 1mL injector, wherein the injection dosage of the muscular stomach group is 10 ten thousand sporangia/feather; the control group was set and injected simultaneously with an equal volume of PBS. The injection dosage of the small intestine group is 10 ten thousand sporangium/feather; the control group was set and injected simultaneously with an equal volume of PBS. The experimental group and the control group were 5 chicks.

Example 4

Purifying and inoculating Eimeria tenella sporozoites.

1. Adding sterile glass beads into the sporulated oocysts purified in example 1 in a volume ratio of 1:1, carrying out shaking and crushing for 10min, and observing under a microscope that all oocyst walls are crushed, the sporangium escapes, and stopping shaking. Sucking the cyst fluid out, washing with PBS 3 times, centrifuging the washed PBS and cyst fluid at 3500-. Adding pancreatin digestive juice (PBS buffer solution containing 0.75 w/v% trypsin and 10 w/v% sodium taurodeoxycholate, pH7.6), shaking at constant temperature of 41 deg.C and 150rpm, digesting for about 60min, and examining sporozoite overflow under microscope until all sporozoites are released, and stopping digestion. And (3) sucking the digested sporozoite mixed solution into a centrifuge tube, centrifuging for 10min at the temperature of 4 ℃ at 4000r/min, discarding the supernatant, washing for 3 times by using PBS, repeatedly centrifuging, and discarding the supernatant. Adding a small amount of PBS into the precipitate, mixing, filtering with a G3 sand core funnel by using a mechanical vacuum pump, counting sporozoites by using a blood counting chamber, and storing at 4 ℃ for later use.

2. Injecting the purified sporozoites into the infrawinged vein and small intestine of the Qingyuan partridge chickens through a 1mL injector, and injecting 1-week-old partridge chickens into the small intestine group, wherein the dosage is 5 ten thousand sporozoites/feathers; the control group was set and injected simultaneously with an equal volume of PBS. Injecting 4 weeks old partridge into the intravenous group, wherein the dosage is 2 ten thousand sporozoites/feathers; the control group was set and injected simultaneously with an equal volume of PBS. The experimental group and the control group were 5 chicks.

Example 5

And observing the morphology of the oocysts, sporangiums and sporozoites of the purified Eimeria tenella.

Morphological observation was conducted on the isolated and purified Eimeria tenella sporulated oocysts, sporangia and sporozoites in the above examples to observe, identify the biological structure thereof and confirm the purity of the coccidia purification. The results show that sporulated oocysts (FIG. 1A) are in the shape of a wide oval, approximately the size: 24 μm × 19 μm; sporangia (FIG. 1B) are oval and about 15 μm by 6 μm in size; the sporozoites (FIG. 1C) were crescent shaped and approximately 10 μm by 2 μm in size. Moreover, through microscopic observation, the purity of each of the oocysts, the sporocysts and the sporozoites is high, and basically no impurities exist.

Example 6

Oocyst shedding dynamic monitoring.

1. Oocyst number detection method

The myxogastric group and small intestine group inoculated with sporangium, and the venous group and small intestine group inoculated with sporozoite, 4-13d p.i. the coccidium content (OPG) in each gram of feces was measured with a Mycoplasma counting plate. Taking 2g of excrement, putting the excrement into a beaker, adding 5mL of water, stirring and uniformly mixing, adding saturated saline to 60mL, filtering the mixture by using a 60-mesh copper screen after uniformly mixing, sucking the excrement liquid, injecting the excrement liquid into a counting chamber of a Macmester counting plate, standing for 5 minutes, and counting Oocysts (OPG) in two scale chambers under a microscope. The average value A of the egg numbers of the two counting chambers is multiplied by 200, and the egg sac number per gram of excrement is obtained. The number of oocysts OPG per gram of feces was calculated according to the following formula:

OPG＝(n1+n2)/2÷0.15×60÷2＝A×200

wherein (n1+ n2)/2 is the average number A of oocysts in each counting chamber, 0.15 is the effective volume of each counting chamber and is 0.15mL, 60 is the total volume of the liquid dung and is 60mL, and 2 is the gram number of the used excrement and is 2 g.

2. Ovulatory cyst stage after sporangial inoculation

2.1 ovulation phase after intragastric inoculation of sporangium

The purified sporangia (10 ten thousand per feather) is accurately injected into the muscular stomach of a 1-week-old partridge chicken, and 5 chickens are obtained in total; the negative control group was injected simultaneously with the same volume of PBS for a total of 5 chickens. OPG was assayed at 4-13d p.i., total daily stool weight was weighed for each group, daily oocyst production was calculated for each chicken, and a intramyogastric sporocyst exclusion oocyst curve was prepared (FIG. 3). The results show that the control group is negative, the ovulation period in the experimental group is 6-12d p.i., the ovulation peak is 7d p.i., and the yield/feather of the oocysts is 121.07 multiplied by 10⁵And (4) respectively.

2.2 sporangium inoculation in the post-oocyst stage of small intestine

Injecting purified sporangia (10 ten thousand per feather) into small intestines of 1-week-old partridge chickens at 3 points, wherein the total number of the chickens is 5; the negative control group was injected simultaneously with the same volume of PBS for a total of 5 chickens. OPG was detected at 4-13d p.i., total daily feces weight of each group was weighed, daily oocyst production per chicken was calculated, and a plot of sporangium-discharging oocysts inoculated in the middle section of small intestine was prepared (FIG. 4). The results show that the control group is negative, the oocyst discharging period in the experimental group is 7-12d p.i., the ovulation peak is 9d p.i., and the oocyst yield/feather is 114.99 multiplied by 10⁴And (4) respectively.

3. Ovulatory cyst stage after sporozoite inoculation

3.1, in the post-oocyst stage of sporozoite inoculation on the small intestine

Injecting purified sporozoites (5 ten thousand per feather) into the small intestine of 1 week old partridge chicken at 3 points, wherein the total number of the chickens is 5; the negative control group was injected simultaneously with the same volume of PBS for a total of 5 chickens. OPG was detected at 4-13d p.i., total daily feces weight of each group was weighed, daily oocyst yield of each chicken was calculated, and a midgut seeded sporulation oocyst ejection curve was prepared (FIG. 5). The results show that the control group is negative, the ovulation period in the experimental group is 6-11 dp.i., the ovulation peak is 8 dp.i., and the oocyst yield/feather is 56.32 multiplied by 10⁴And (4) respectively.

3.2 post-oocyst ejection phase of sporozoite intravenous injection

Injecting purified sporozoites (2 ten thousand per feather) into the lower part of the wing of the 4-week-old partridge chicken by intravenous injection, and 5 chickens in total; the negative control group was injected simultaneously with the same volume of PBS for a total of 5 chickens. OPG was assayed at 4-13d p.i., total daily stool weight was weighed for each group, daily oocyst production per chicken was calculated, and a sporozoite injection intravenous oocyst curve was prepared (FIG. 6). The results showed that both the control and experimental groups were negative and no oocysts were produced at 4-13d p.i.

Example 7

Inoculation success/infection success detection.

In the experiment, under the condition that the sporozoites and the sporangiums of the Eimeria tenella are injected and inoculated to the muscular stomach and/or the middle section of the small intestine of 1-week-old partridges, all partridges (20 partridges) in the experimental group are subjected to excrement detection at 6-7 dp.i., and the inoculation success rate reaches 100%.

Comparative example 1

The method for purifying Eimeria tenella sporulated oocysts is mainly different from the purification method of example 1 in the solution for inducing oocyst sporulation and the induction conditions, and the preservative for preserving oocysts.

The specific method comprises the following steps:

(1) adding tap water with the volume of 5-10 times of that of the excrement into the excrement, uniformly stirring the mixture, and then filtering the mixture by using a 80-mesh sieve; in the process, a plastic soft shovel is required to be used for stirring continuously, so that most of the water solution is filtered through a screen and collected into a plastic basin; and placing the filtered excrement residues in another plastic basin, adding tap water into the excrement residues, further filtering, and finally discarding the excrement residues.

(2) Filtering all the filtrate for the second time by using a 160-mesh sieve, and collecting the filtrate into a plastic basin; standing the filtrate for 2h, and quickly pouring off the supernatant; centrifuging the obtained suspension at 3600rpm for 5min (the setting is adopted in the following centrifugation), and collecting the deposited precipitate;

(3) stirring with glass rod, precipitating, adding 2 times volume of saturated saline, stirring, and centrifuging to float oocysts; pouring the obtained oocyst-containing supernatant into a large beaker, uniformly distributing the oocyst-containing supernatant into a centrifugal tank, adding tap water with the volume at least 5 times that of the supernatant, and then centrifuging and precipitating the oocysts;

(4) the obtained oocyst sediment is resuspended by potassium dichromate solution and transferred into a small conical flask, the volume of the potassium dichromate is determined according to the number of the oocysts, and each milliliter contains no more than 100 ten thousand of the oocysts;

(5) inoculating the collected oocysts with an air blowing device, placing in a 28 ℃ incubator, ventilating and sporulating for 48 hours (adding water when paying attention to 24 hours, compensating for water evaporated by air blowing incubation); after sporulation, the oocysts were labeled and stored at 4 ℃.

The inducer and the preservative in the comparative example are potassium dichromate, belong to highly toxic and explosive products, and bring potential safety hazards to experimental processes and scientific researchers. Therefore, the invention adopts 1% chloramine T as an inducer and a preservative of sporulated oocysts, belongs to an external disinfectant, and has low toxicity, safety and stability.

Comparative example 2

The purification method of Eimeria tenella sporozoites is different from the purification method of example 4 mainly in the crushing mode of oocysts, the type of digestive juice and the purification mode of sporozoites.

The specific method comprises the following steps:

(1) taking a certain amount of sporulated oocysts treated with sodium hypochlorite according to the experiment needs, and washing 3 times (3000rpm for 3min) with PBS; adding 1-2 times volume of PBS into the oocyst sediment, mixing uniformly, adding into a grinder for grinding, stopping when about 90% of oocysts are broken through microscopic examination (taking 1 microliter of ground material from the grinder for microscopic examination, only when the oocysts are rarely seen in visual field; transferring sporangium suspension (containing a small amount of oocysts and oocyst walls) from the grinder to a centrifuge tube, washing twice (3000rpm3min) with PBS, re-suspending the sporangium sediment with a de-cyst buffer (0.75% trypsin and 10% chicken bile), incubating in water bath at 41-42 ℃ for about 40 min, and reversing and mixing uniformly every 5 min;

(2) performing microscopic examination on the cyst shedding condition of sporozoites at 30min, and stopping incubation when the sporozoites are rarely seen in a visual field generally; centrifuging to remove the de-encapsulation buffer solution, resuspending the precipitate with glycine eluent preheated to 42 ℃ and centrifuging and washing for 3 times;

(3) column purification of sporozoites: resuspending all the precipitates with about 1mL of glycine eluent, adding the resuspension solution into the column when the cellulose is basically settled, starting elution (the flow rate is 3-4 of 10 × shelves), collecting the eluent by using a 50mL centrifuge tube, wherein the process lasts for 20min to 1h, and determining the stop time according to the result of microsporoscopy in the eluent; in the process, the upper layer of the cellulose is gently stirred to the middle part by a glass rod at intervals, and the bottom part of the cellulose is not stirred to prevent oocysts or oocyst walls and the like from directly entering a collecting pipe; if the room temperature is low, an infrared lamp is arranged before elution, and the chromatographic column is heated and insulated; and centrifuging the collected eluent at 3000rpm for 5min to obtain a precipitate, namely the purified sporozoite, resuspending the sporozoite by using a complete Cytomix solution, and transferring the sporozoite to a 1.5mL centrifuge tube for later use.

The purification method of Eimeria tenella sporangia and sporozoite adopted in the comparative example has the defects of complicated process and long time consumption, and the main reasons are as follows: the core of the method is the preparation of a DE-52 cellulose chromatographic column, and sporozoite purification is carried out by utilizing the chromatographic column; the preparation of the chromatographic column requires 1 day and the experimental process is complicated; cellulose chromatography columns are easily clogged by the oocyst wall when the sporozoites are overloaded for purification.

The technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.

The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.

Claims

1. A method for separating and purifying Eimeria tenella sporozoites is characterized by comprising the following steps:

s6, preparing oocysts into suspension, oscillating and crushing, stopping oscillating when the sporangiums escape, centrifuging, and collecting precipitates to obtain the sporangiums;

s7, preparing sporangium into suspension, centrifuging, collecting precipitate, adding pancreatin digestive juice, digesting until all sporozoites are released, stopping digesting, centrifuging, collecting precipitate, preparing into suspension, filtering, centrifuging, and obtaining precipitate which is the eimeria tenella sporozoites.

2. The separation and purification method according to claim 1, wherein the step S1 specifically comprises: inoculating Eimeria tenella to 1-2 weeks old partridge, detecting coccidia 5-6 days after infection, collecting 6-10 days of feces containing Eimeria tenella oocysts; adding 2-3 times of water to dilute the feces, sequentially sieving with 80 mesh, 100 mesh and 300 mesh sieves, and retaining the filtrate.

3. The separation and purification method according to claim 1, wherein the step S2 specifically comprises: centrifuging the filtrate at 1400-1600rpm for 3-4min, collecting precipitate, adding 1-3 times of saline solution, centrifuging at 2700-2900rpm for 3-4min, collecting supernatant, and adding 4-6 times of water for dilution to obtain oocyst diluent.

4. The separation and purification method according to claim 1, wherein the step S3 specifically comprises: centrifuging the oocyst diluent at 3200-3400rpm for 1-3min, collecting the precipitate, adding water for washing, centrifuging, retaining the precipitate, adding a chloramine T solution with the concentration of 1.0 w/v%, culturing at 28 ℃, and obtaining sporulated oocyst suspension after the oocysts are sporulated.

5. The separation and purification method according to claim 1, wherein the step S4 specifically comprises: centrifuging the sporulated oocyst suspension at 3500-3700rpm for 4-6min, collecting the precipitate, adding water for washing, centrifuging at 3500-3700rpm for 4-6min, retaining the precipitate, adding saturated saline solution for resuspension, collecting the upper oocysts, adding 4-6 times of water for washing, centrifuging at 3500-3700rpm for 4-6min, and collecting the oocyst precipitate.

6. The separation and purification method according to claim 1, wherein the step S5 specifically comprises: adding 4-6 times of sodium hypochlorite solution with concentration of 30 w/v% into the oocyst sediment for resuspension, standing at 0 ℃ for 5-7min, centrifuging at 3500-.

7. The separation and purification method according to claim 1, wherein the step S6 specifically comprises: preparing oocysts into suspension, adding 1-2 times of glass beads, performing oscillation crushing for 8-10min, stopping oscillation when all the sporangia escape, centrifuging at 3500-3700rpm for 4-6min, and collecting precipitate to obtain sporangia.

8. The separation and purification method according to any one of claims 1 to 7, wherein the step S7 is specifically: taking sporangium to prepare suspension, adding pancreatin digestive juice, performing shock digestion at the temperature of 40-42 ℃ and the rotational speed of 160rpm for 60-90min, stopping digestion until all sporozoites are released, centrifuging at the speed of 3500 rpm and 4500rpm for 8-12min, taking precipitate, adding PBS, mixing uniformly, filtering by a G3 sand core funnel by using a mechanical vacuum pump, and centrifuging at the speed of 3500 rpm and 4500rpm for 8-12min, wherein the precipitate is the eimeria tenella sporozoites.

9. The method for separation and purification according to claim 8, wherein the pancreatin digest is a PBS buffer solution containing 0.5 w/v% trypsin and 5.0 w/v% sodium taurodeoxycholate.

10. A method for inoculating Eimeria tenella sporozoites, which comprises the steps of taking Eimeria tenella sporocysts or sporozoites obtained by the purification method of any one of claims 1-9, re-suspending with PBS buffer solution, and injecting into the muscular stomach and/or small intestine of chicken.