Detection method for viability of Eimeria tenella oocysts

Abstract

The present invention provides a method for detecting the viability of chicken coccidian oocysts, comprising the following steps: respectively obtaining the sporozoite escape rate of a chicken coccidian oocyst sample to be tested and the sporozoite escape rate of a control chicken coccidian oocyst sample, analyzing the sporozoite escape rate and the sporozoite escape rate, and detecting the viability of the chicken coccidian oocyst sample to be tested according to the obtained analysis result. The present invention is suitable for detecting the viability of chicken coccidian oocysts and chicken coccidian oocyst vaccines, has simple operation and high repeatability, and is applicable to clinical production and laboratory promotion and detection.

Description

Technical Field

[0001] The present invention relates to the technical field of chicken coccidia, and particularly to a method for detecting the viability of chicken coccidian oocysts and its application. Background Art

[0002] The prevention of chicken coccidiosis mainly relies on drugs and vaccines. In the breeding industry, chicken coccidiosis is the most harmful, greatly affecting the breeding efficiency and being one of the main diseases restricting the development of large-scale chicken farming. Chicken coccidiosis caused by 7 species of coccidia represented by Eimeria tenella is an important intestinal parasitic disease that seriously endangers the intestinal health of chicken flocks, causing huge economic losses to the chicken farming industry every year. It is estimated that the annual global cost for the prevention and treatment of chicken coccidiosis alone exceeds $3 billion.

[0003] Chicken coccidiosis is a protozoal disease that parasitizes in the intestinal epithelial cells of chickens. In the clinical onset characteristics, farm animals infected with Eimeria spp., especially chicks, often show listlessness, accompanied by diarrhea, and even bloody stools. At the same time, the production performance is significantly reduced, and the slaughter volume is also severely affected. Therefore, this disease causes huge economic losses to the poultry industry in China and even the world.

[0004] Chicken coccidian oocysts are endoparasitic protozoa with a complete oocyst wall. A sporulated and viable chicken coccidian oocyst contains 4 sporocysts with complete structures, and each sporocyst structure contains two infective and viable sporozoites. Because of the oocyst wall and sporocyst wall, it can well protect the sporozoites and prevent damage to the oocysts by the external environment. The life cycle of chicken coccidian oocysts includes two stages: in vivo infection and in vitro sporulation.

[0005] At present, the control of chicken coccidiosis more often adopts the method of immunization with live vaccines of chicken coccidian oocysts. However, there are also potential safety hazards of virulence reversion in the use of live vaccines of chicken coccidian oocysts, and live vaccines of chicken coccidian oocysts also have limitations such as short storage time and strict storage methods. Therefore, it is of great significance for the comprehensive prevention and control of chicken coccidiosis to be able to evaluate the viability of live chicken coccidian oocyst vaccines in a timely and effective manner and ensure the effectiveness and safety of each batch of live chicken coccidiosis vaccines. However, based on the traditional in vivo animal test evaluation standard, there are limitations such as time-consuming, laborious, and the need for an evaluation site. Therefore, establishing a set of safe, simple, fast, and laboratory-operable methods for evaluating the viability of live chicken coccidian oocyst vaccines has become an urgent problem to be solved in chicken production. Summary of the Invention

[0006] Based on this, it is necessary to provide a method for detecting the viability of chicken coccidian oocysts that is safe, simple, fast, and laboratory-operable, which helps to quickly and timely provide the results of evaluating the viability of chicken coccidian oocyst vaccines clinically.

[0007] One aspect of the present invention provides a method for detecting the viability of chicken coccidian oocysts.

[0008] The technical solution of the present invention includes:

[0009] A method for detecting the viability of chicken coccidian oocysts, comprising the following steps:

[0010] Respectively obtain the sporozoite emergence rate Y of the chicken coccidian oocyst sample to be tested and the sporozoite emergence rate Y' of the control chicken coccidian oocyst sample, analyze the sporozoite emergence rate Y and the sporozoite emergence rate Y', and detect the viability of the chicken coccidian oocyst sample to be tested according to the obtained analysis results;

[0011] Among them, the method for obtaining the sporozoite emergence rate Y includes the following steps:

[0012] Provide a chicken coccidian oocyst sample to be tested and prepare an oocyst suspension;

[0013] Physically break the oocyst suspension to prepare a sporocyst suspension, and record the amount of sporocysts in the sporocyst suspension as A;

[0014] Mix the sporocyst suspension, bile and trypsin, and carry out digestion to prepare a sporozoite suspension, and record the amount of sporozoites in the sporozoite suspension as B and the dilution factor of the sporocyst suspension as C;

[0015] Calculate the sporozoite emergence rate Y according to the A, B and C;

[0016] Obtain the sporozoite emergence rate Y' by referring to the method for obtaining the sporozoite emergence rate Y; the control chicken coccidian oocyst sample has the same batch as the chicken coccidian oocyst sample to be tested and the viability result verified by animal experiments is qualified.

[0017] In one embodiment, detecting the viability of the chicken coccidian oocyst sample to be tested according to the obtained analysis results includes:

[0018] Under the condition that the deviation of the sporozoite emergence rate Y relative to the sporozoite emergence rate Y' is less than 10%, the viability result verified by animal experiments corresponding to the chicken coccidian oocyst sample to be tested is qualified.

[0019] In one embodiment, the step of physical breaking includes: mixing the oocyst suspension and glass beads and shaking.

[0020] In one embodiment, the shaking conditions include: the shaking speed is 2900 rpm to 3100 rpm, and the shaking time is 2.5 min to 3.5 min.

[0021] In one embodiment, the mass of the glass beads corresponding to each 0.5 mL of the oocyst suspension is 200 mg.

[0022] In one embodiment, the diameter of the glass beads is 0.8 mm to 2 mm.

[0023] In one embodiment, the digestion conditions include: a digestion temperature of 39°C to 41°C and a digestion time of 48 min to 52 min.

[0024] In one embodiment, the dosage of the bile corresponding to every 1 mL of the sporocyst suspension is 0.04 mL to 0.05 mL.

[0025] In one embodiment, the dosage of the pancreatin corresponding to every 1 mL of the sporocyst suspension is 0.008 g to 0.01 g.

[0026] In one embodiment, the calculation formula for the sporozoite escape rate Y is: sporozoite escape rate Y = B × C / (2 × A) × 100%.

[0027] In one embodiment, the oocyst content in the oocyst suspension is 100,000 to 500,000 per mL.

[0028] In one embodiment, the chicken coccidia oocyst is an oocyst vaccine.

[0029] Compared with the traditional scheme, the present invention has the following beneficial effects:

[0030] The method for detecting the viability of chicken coccidia oocysts of the present invention is simple in the instruments used, easy to prepare the reagents, and easy to operate and interpret when detecting the viability of chicken coccidia oocysts. Compared with the traditional in-vivo detection method for the viability of chicken coccidia vaccine oocysts, the technical solution of the present invention for detecting the viability of chicken coccidia oocyst vaccine helps to quickly and timely provide the evaluation results of the viability of chicken coccidia oocyst vaccine, and is more suitable for clinical production and laboratory promotion and detection. Detailed implementation manners

[0031] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the technical field to which this invention belongs. The terms used in the specification of this invention herein are only for the purpose of describing specific embodiments, and are not intended to limit the present invention. The term "and / or" used herein includes any and all combinations of one or more of the related listed items.

[0032] Terms

[0033] Unless otherwise stated or there is a contradiction, the terms or phrases used herein have the following meanings:

[0034] In the present invention, terms such as "multiple", "diverse", "multiple times", "pluralistic", etc., unless otherwise specifically defined, mean greater than or equal to 2 in quantity. For example, "one or more" means one or greater than or equal to two.

[0035] In the present invention, terms such as "preferred", "better", "more preferable", "advisable" are only used to describe embodiments or examples with better effects, and it should be understood that they do not constitute a limitation on the protection scope of the present invention.

[0036] In the present invention, terms such as "further", "even further", "especially", etc. are used for descriptive purposes, indicating differences in content, but should not be construed as a limitation on the protection scope of the present invention.

[0037] In the present invention, the terms "optionally", "optional", "option" mean having or not having, that is, either of the two parallel options of "having" or "not having". If the term "optional" appears multiple times in a technical solution, unless otherwise specifically stated and there are no contradictions or mutual restrictions, each "optional" is independent.

[0038] In the present invention, in terms such as "the first aspect", "the second aspect", "the third aspect", "the fourth aspect", etc., the terms "first", "second", "third", "fourth", etc. are only used for descriptive purposes, and cannot be understood as indicating or implying relative importance or quantity, nor can they be understood as implicitly indicating the importance or quantity of the indicated technical features. Moreover, "first", "second", "third", "fourth", etc. only serve the purpose of non-exhaustive listing and description, and it should be understood that they do not constitute a closed limitation on quantity.

[0039] In the present invention, for the technical features described in an open-ended manner, it includes both the closed technical solutions composed of the listed features and the open technical solutions containing the listed features.

[0040] In the present invention, regarding numerical intervals (i.e., numerical ranges), unless otherwise specifically stated, the selectable numerical values are considered continuous within the above numerical intervals, and include the two numerical endpoints of this numerical range (i.e., the minimum value and the maximum value), as well as each numerical value between these two numerical endpoints. Unless otherwise specifically stated, when the numerical interval only refers to the integers within this numerical interval, it includes the two endpoint integers of this numerical range, as well as each integer between the two endpoints. In this article, it is equivalent to directly listing each integer. For example, t is an integer selected from 1 to 10, which means t is any integer selected from the integer group composed of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10. In addition, when multiple ranges are provided to describe features or characteristics, these ranges can be combined. In other words, unless otherwise specified, the ranges disclosed herein should be understood to include any and all sub-ranges subsumed therein.

[0041] The temperature parameters in the present invention, unless otherwise specified, are allowed to be constant temperature treatment or allowed to vary within a certain temperature range. It should be understood that the constant temperature treatment allows the temperature to fluctuate within the accuracy range controlled by the instrument. It is allowed to fluctuate within a range such as ±5°C, ±4°C, ±3°C, ±2°C, ±1°C.

[0042] In the present invention, regarding the percentage content, unless otherwise specified, for solid-liquid mixing and solid-solid mixing, it refers to the mass percentage, and for liquid-liquid mixing, it refers to the volume percentage.

[0043] In the present invention, regarding the percentage concentration, unless otherwise specified, it refers to the final concentration. The final concentration refers to the proportion of the added component in the system after adding the component.

[0044] All reagents and water in the present invention, unless otherwise specified, only use analytical pure reagents and comply with the regulations of grade three water in GB / T 6682. For standard titration solutions, preparations and products required in the experiment, unless otherwise specified, they are prepared according to the regulations of GB / T601 and GB / T 603.

[0045] Currently, for the control of chicken coccidiosis, the method of immunizing with live chicken coccidia oocyst vaccines is more commonly used. However, there are also potential safety hazards of virulence reversion during the use of live chicken coccidia oocyst vaccines. Live chicken coccidia oocyst vaccines also have limitations such as short storage time and strict storage methods. Therefore, being able to timely and effectively evaluate the viability of live chicken coccidia oocyst vaccines and ensure the effectiveness and safety of each batch of live chicken coccidiosis vaccines is of great significance for the comprehensive prevention and control of chicken coccidiosis. However, based on the traditional in vivo animal test evaluation standards, there are limitations such as time-consuming, laborious, and the need for evaluation sites. Therefore, establishing a safe, simple, fast, and laboratory-operable method for evaluating the viability of live chicken coccidia oocyst vaccines has become an urgent problem to be solved in chicken farming production.

[0046] To solve the above problems, the present invention provides a method for detecting the viability of chicken coccidia oocysts, including the following steps:

[0047] One aspect of the present invention provides a method for detecting the viability of chicken coccidia oocysts.

[0048] The technical solution of the present invention includes:

[0049] A method for detecting the viability of chicken coccidia oocysts, including the following steps:

[0050] Respectively obtain the sporozoite escape rate Y of the chicken coccidia oocyst sample to be tested and the sporozoite escape rate Y' of the control chicken coccidia oocyst sample, analyze the sporozoite escape rate Y and the sporozoite escape rate Y', and detect the viability of the chicken coccidia oocyst sample to be tested according to the obtained analysis results;

[0051] Among them, the method for obtaining the sporozoite escape rate Y includes the following steps:

[0052] Provide a chicken coccidia oocyst sample to be tested and prepare an oocyst suspension;

[0053] Physically break the wall of the oocyst suspension to prepare a sporocyst suspension, and record the amount of sporocysts in the sporocyst suspension as A;

[0054] Mix the sporocyst suspension, bile and trypsin, and carry out digestion to prepare a sporozoite suspension. Record the amount of sporozoites in the sporozoite suspension as B, and record the dilution factor of the sporocyst suspension as C;

[0055] Calculate the sporozoite escape rate Y according to the A, B and C;

[0056] Obtain the sporozoite escape rate Y' by referring to the method for obtaining the sporozoite escape rate Y; the control chicken coccidia oocyst sample has the same batch as the chicken coccidia oocyst sample to be tested and the vitality result verified by animal experiments is qualified.

[0057] Optionally, detecting the vitality of the chicken coccidia oocyst sample to be tested according to the obtained analysis result includes:

[0058] Under the condition that the deviation of the sporozoite escape rate Y from the sporozoite escape rate Y' is less than 10%, the vitality result verified by animal experiments corresponding to the chicken coccidia oocyst sample to be tested is qualified.

[0059] Optionally, the step of physical wall breaking includes: mixing the oocyst suspension and glass beads and shaking.

[0060] Optionally, the conditions for shaking include: the shaking speed is 2900 rpm to 3100 rpm, and the shaking time is 2.5 min to 3.5 min.

[0061] Further, the shaking speed is 2900 rpm, 2950 rpm, 3000 rpm, 3050 rpm, 3100 rpm.

[0062] Further, the shaking time is 2.5 min, 2.6 min, 2.7 min, 2.8 min, 2.9 min, 3 min, 3.1 min, 3.2 min, 3.3 min, 3.4 min, 3.5 min.

[0063] Optionally, the mass of the glass beads corresponding to every 0.5 mL of the oocyst suspension is 200 mg.

[0064] Optionally, the diameter of the glass beads is 0.8 mm to 2 mm.

[0065] Preferably, the diameter of the glass beads is 1 mm.

[0066] Optionally, the digestion conditions include: the digestion temperature is 39°C to 41°C, and the digestion time is 48 min to 52 min.

[0067] Furthermore, the digestion temperature is 39°C, 40°C, 41°C.

[0068] Furthermore, the digestion time is 48 min, 49 min, 50 min, 51 min, 52 min.

[0069] Optionally, the dosage of the bile corresponding to every 1 mL of the sporocyst suspension is 0.04 mL to 0.05 mL.

[0070] Optionally, the dosage of the pancreatin corresponding to every 1 mL of the sporocyst suspension is 0.008 g to 0.01 g.

[0071] Furthermore, the dosage of the bile corresponding to every 1 mL of the sporocyst suspension is 0.04 mL, 0.045 mL, 0.05 mL.

[0072] Furthermore, the dosage of the pancreatin corresponding to every 1 mL of the sporocyst suspension is 0.008 g, 0.009 g, 0.01 g.

[0073] Preferably, the calculation formula for the sporozoite escape rate Y is: sporozoite escape rate Y = B × C / (2 × A) × 100%.

[0074] Optionally, the oocyst content in the oocyst suspension is 100,000 / mL - 500,000 / mL.

[0075] Furthermore, the oocyst content is 100,000 / mL, 110,000 / mL, 120,000 / mL, 130,000 / mL, 140,000 / mL, 150,000 / mL, 160,000 / mL, 170,000 / mL, 180,000 / mL, 190,000 / mL, 200,000 / mL, 210,000 / mL, 220,000 / mL, 230,000 / mL, 240,000 / mL, 250,000 / mL, 260,000 / mL, 270,000 / mL, 280,000 / mL, 290,000 / mL, 300,000 / mL, 310,000 / mL, 320,000 / mL, 330,000 / mL, 340,000 / mL, 350,000 / mL, 360,000 / mL, 370,000 / mL, 380,000 / mL, 390,000 / mL, 400,000 / mL, 410,000 / mL, 420,000 / mL, 430,000 / mL, 440,000 / mL, 450,000 / mL, 460,000 / mL, 470,000 / mL, 480,000 / mL, 490,000 / mL, 500,000 / mL.

[0076] Preferably, the chicken coccidia oocyst is a chicken coccidia oocyst vaccine.

[0077] Optionally, before preparing the sporozoite suspension, a step of terminating digestion is further included.

[0078] Optionally, the temperature during the termination of digestion is 3°C to 5°C.

[0079] Preferably, the temperature during the termination of digestion is 3°C, 4°C, or 5°C.

[0080] Optionally, a hemocytometer is used to count the content of sporocysts and the content of sporozoites.

[0081] Optionally, the coccidia in the to-be-detected sample of chicken coccidian oocysts are selected from one of Eimeria tenella, Eimeria acervulina, Eimeria maxima, Eimeria necatrix, and Eimeria brunetti.

[0082] The following is further described in conjunction with specific embodiments.

[0083] Example 1

[0084] 1. Test drug

[0085] A certain domestic chicken coccidian oocyst vaccine brand 1 to be evaluated and detected; the chicken coccidian oocyst vaccines that have been detected as qualified and unqualified in the same batch are used for comparison.

[0086] 2. Test reagents

[0087] In this test, all reagents and water, unless otherwise specified, only analytical pure reagents and water meeting the requirements of grade 3 water in GB / T 6682 are used. For the standard titration solutions, preparations, and products required in the test, unless otherwise specified, they are prepared according to the provisions of GB / T 601 and GB / T 603.

[0088] 3. Preparation of solutions

[0089] Prepare phosphate buffer solution (PBS): 8 g of sodium chloride, 0.2 g of potassium chloride, 3.88 g of disodium hydrogen phosphate, 0.24 g of potassium dihydrogen phosphate, 1 L of double-distilled water, autoclaved, and stored at 4°C for standby;

[0090] Prepare 1% bile pancreatin digestive solution: 5 mL of fresh bile, 1 g of pancreatin, 95 mL of PBS, dissolved by aseptic operation and then aliquoted into 5 mL / tube, stored at -20°C, and thawed immediately before use.

[0091] 4. Test instruments and consumables

[0092] a) Electronic balance; b) Low-speed centrifuge; c) Stirrer; d) Optical microscope; e) Counter; f) Hemocytometer; g) Water bath; h) Low-speed shaker; i) Pipette; j) 1.5 mL EP tubes, 5 mL EP tubes; k) 20 μL pipette tips, 1000 μL pipette tips.

[0093] 5. Method for detecting the viability of oocysts of chicken coccidiosis vaccine

[0094] 5.1 Oocyst counting: Mix the oocyst samples of chicken coccidiosis vaccine to be detected, and perform preliminary counting using a hemocytometer.

[0095] 5.2 Oocyst concentration treatment: Mix the vaccine oocysts evenly, draw a certain amount of oocyst fluid into a 1.5 mL EP tube for concentration, centrifuge at 10000 rpm / min for 2 min, discard the supernatant, and wash and preserve the solution. Take the precipitate, suspend it with 1 mL of PBS and collect it into one tube to prepare an oocyst suspension, and store it at 4°C for standby (the content in the oocyst suspension is recommended to be 100,000 / mL - 500,000 / mL).

[0096] 5.3 Oocyst wall breaking with glass beads and sporocyst counting: Mix 0.5 mL of the purified oocyst suspension with 200 mg of glass beads, shake at 3000 rpm on a low-speed shaker for 3 min (at this time, the sporocyst release rate reaches more than 95%), use a pipette to aspirate the oocyst suspension after wall breaking and accurately count the sporocyst content A (in ten thousands / mL) using a hemocytometer, and store it at 4°C for standby. (If there are many impurities in the sample and sporocyst counting cannot be performed, it is recommended to dilute it reasonably and then perform the counting.)

[0097] 5.4 Digestion treatment with 1% bile trypsin digestion solution and sporozoite counting: Take 100 μL of the accurately counted sporocysts, add 900 μL of the pre-prepared bile trypsin digestion solution (at this time, the dilution factor C of the sporocyst suspension is 10), digest in a 41°C water bath for 50 min, take it out and place it at 4°C to terminate digestion, aspirate the digested sporocyst fluid for sporozoite counting, accurately count the sporozoite content as B (in ten thousands / mL), and according to the calculation formula: sporozoite escape rate Y = B×C / (2×A)×100%, calculate that the sporozoite escape rate Y at this time is 5×B / A×100%.

[0098] 5.5 Take the oocysts of the vaccine that have passed the animal experiment of the same brand and batch and perform the same operations (such as steps 5.1 - 5.4) for treatment, calculate the sporozoite escape rate as Y', and use it as a parallel reference.

[0099] 6. Result determination

[0100] Take the oocysts of the vaccines that have passed and failed the animal experiments of the same brand and batch, and perform the same operations (Steps 5.1 - 5.4) to calculate the sporozoite escape rate as a parallel reference. Compare the sporozoite escape rate results of the test samples with those of the qualified batches, and a deviation less than 10% is within the credible range.

[0101] The experimental data of the test samples and control samples of a domestic chicken coccidia oocyst vaccine brand 1 are recorded in Table 1.

[0102] Table 1 Sporozoite escape rate and verification results of a domestic chicken coccidia brand 1

[0103]

[0104] Example 2

[0105] 1. Test drug

[0106] A domestic chicken coccidia oocyst vaccine brand 2 to be evaluated and tested; qualified and unqualified chicken coccidia oocyst vaccines of the same batch for control comparison.

[0107] 2. Test reagents

[0108] In this test, unless otherwise specified, only analytical pure reagents and water meeting the requirements of Grade 3 water in GB / T 6682 are used. Standard titration solutions, preparations and products required in the test are prepared in accordance with the provisions of GB / T 601 and GB / T 603 when no other requirements are specified.

[0109] 3. Preparation of solutions

[0110] Prepare phosphate buffer solution (PBS): 8 g of sodium chloride, 0.2 g of potassium chloride, 3.88 g of disodium hydrogen phosphate, 0.24 g of potassium dihydrogen phosphate, 1 L of double-distilled water, autoclave and store at 4°C for later use;

[0111] Prepare 1% bile pancreatin digestive solution: 5 mL of fresh bile, 1 g of pancreatin, 95 mL of PBS, dissolve by aseptic operation and dispense into 5 mL / tube, store at -20°C, and thaw immediately before use.

[0112] 4. Test instruments and consumables

[0113] a) Electronic balance; b) Low-speed centrifuge; c) Stirrer; d) Optical microscope; e) Counter; f) Hemocytometer; g) Water bath; h) Low-speed oscillator; i) Pipette; j) 1.5 mL EP tube, 5 mL EP tube; k) 20 μL pipette tip, 1000 μL pipette tip.

[0114] 5. Method for detecting the viability of chicken coccidia vaccine oocysts

[0115] 5.1 Oocyst count: Mix the chicken coccidiosis vaccine oocyst sample to be tested and perform a preliminary count using a hemocytometer.

[0116] 5.2 Oocyst concentration treatment: Mix the vaccine oocysts evenly, extract a certain amount of oocyst fluid into a 1.5mL EP tube for concentration, centrifuge at 10000rpm / min for 2min, discard the supernatant, and elute the preservation solution. Take the precipitate and suspend it in 1mL PBS to collect it into a tube to prepare the oocyst suspension, and store it at 4℃ for later use (the recommended oocyst content in the oocyst suspension is 100,000 / mL-500,000 / mL).

[0117] 5.3 Oocyst wall breaking with glass beads, sporangium counting: Take 0.5 ml of purified oocyst suspension and mix with 200 mg of glass beads, shake on a low-speed oscillator at 3000 rpm for 3 min (the sporangium release rate is over 95% at this time), use a pipette to draw the broken oocyst suspension, use a blood cell counting plate to accurately count the sporangium content A (10,000 / mL), and store at 4°C for later use. (Note: If the sample has too many impurities and sporangium counting cannot be performed, it is recommended to dilute it reasonably before counting.)

[0118] 5.4 1% bile pancreatic digestion solution digestion, sporozoite count: take 100uL of the sporangium after accurate counting, add 900uL of the prepared bile pancreatic digestion solution (the dilution factor C of the sporangium suspension is 10 at this time), digest in a 41°C water bath for 50min, take out and place at 4°C to terminate digestion, aspirate the digested sporangium fluid for sporozoite count, accurately count the sporozoite content B (10,000 / mL), according to the calculation formula: sporozoite escape rate = B×C / (2×A)×100%, the sporozoite escape rate at this time is calculated to be 5×B / A×100%.

[0119] 5.5 Take the vaccine oocysts of the same brand and batch that have passed the animal experiment and perform the same operation (steps 5.1-5.4) to calculate the sporozoite escape rate as a parallel reference.

[0120] 6. Result determination

[0121] Take the same brand and batch of vaccine oocysts that have passed and failed animal experiments and perform the same operation (steps 5.1-5.4) to calculate the sporozoite escape rate as a parallel reference. Interpret the sporozoite escape rate results of the test samples and compare them with the qualified batches that have been tested. The deviation is less than 10% in the credible range.

[0122] The experimental data of the test and control products of a domestic chicken coccidia oocyst vaccine brand 2 are recorded in Table 2.

[0123] Table 2 Sporozoite escape rate and verification results of a domestic chicken coccidia brand 2

[0124]

[0125] The technical features of the above-described embodiments can be combined arbitrarily. For the sake of brevity of description, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, it should be considered as falling within the scope described in this specification.

[0126] The above-described embodiments merely represent several implementation manners of the present invention. The description is relatively specific and detailed, but it should not be construed as a limitation on the scope of the invention patent. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can still be made, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the present invention patent shall be subject to the appended claims.

Claims

1. A method for detecting the viability of Eimeria tenella oocysts, characterized in that, The method includes the following steps: Obtain the sporozoite emergence rate Y of the chicken coccidia oocyst sample to be tested and the sporozoite emergence rate Y' of the control chicken coccidia oocyst sample respectively, analyze the sporozoite emergence rate Y and the sporozoite emergence rate Y', and detect the viability of the chicken coccidia oocyst sample to be tested according to the obtained analysis result; Among them, the method for obtaining the sporozoite emergence rate Y includes the following steps: Provide the chicken coccidia oocyst sample to be tested and prepare an oocyst suspension; Perform physical wall-breaking on the oocyst suspension to prepare a sporocyst suspension, and record the sporocyst content in the sporocyst suspension as A; Mix the sporocyst suspension and the bile-trypsin digestive solution for digestion. The dilution factor of the sporocyst suspension is recorded as C, prepare a sporozoite suspension, and record the sporozoite content in the sporozoite suspension as B; Calculate the sporozoite emergence rate Y according to the A, B, and C; The calculation formula for the sporozoite emergence rate Y is: sporozoite emergence rate Y = B×C / (2×A)×100%; Obtain the sporozoite emergence rate Y' of the control chicken coccidia oocyst sample by referring to the method for obtaining the sporozoite emergence rate Y; the control chicken coccidia oocyst sample has the same batch as the chicken coccidia oocyst sample to be tested and the viability result verified by animal experiments is qualified; Detecting the viability of the chicken coccidia oocyst sample to be tested according to the obtained analysis result includes: Under the condition that the deviation of the sporozoite emergence rate Y from the sporozoite emergence rate Y' is less than 10%, the viability result verified by animal experiments corresponding to the chicken coccidia oocyst sample to be tested is qualified.

2. The method for detecting the viability of Eimeria tenella oocysts according to claim 1, characterized in that, The step of physical wall-breaking includes: mixing the oocyst suspension and glass beads and shaking.

3. The detection method of the viability of Eimeria tenella oocysts according to claim 2, characterized in that, The conditions for shaking include: the shaking speed is 2900 rpm to 3100 rpm, and the shaking time is 2.5 min to 3.5 min.

4. The method for detecting the viability of chicken coccidia oocysts according to claim 2, wherein the mass of the glass beads corresponding to every 0.5 mL of the oocyst suspension is 200 mg; or / and, the diameter of the glass beads is 0.8 mm to 2 mm.

5. The method for detecting the viability of Eimeria tenella oocysts according to any one of claims 1 to 4, characterized in that, The conditions for digestion include: the digestion temperature is 39°C to 41°C, and the digestion time is 48 min to 52 min.

6. The detection method for the viability of Eimeria tenella oocysts according to any one of claims 1 to 4, characterized in that, The dosage of the bile corresponding to every 1 mL of the sporocyst suspension is 0.04 mL to 0.05 mL and the dosage of trypsin is 0.008 g to 0.01 g.

7. The method for detecting the viability of Eimeria tenella oocysts according to any one of claims 1 to 4, characterized in that, The oocyst content in the oocyst suspension is 100,000 / mL - 500,000 / mL.

8. The method for detecting the viability of Eimeria tenella oocysts according to any one of claims 1 to 4, characterized in that, The chicken coccidia oocyst is a chicken coccidia oocyst vaccine.

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