Method for detecting activity of coccidian oocysts and judging quality grade of coccidian oocysts

Abstract

The invention provides a method for detecting the activity of coccidian oocysts and judging the quality grade of the coccidian oocysts, and the method for detecting the activity of the coccidian oocysts comprises the following steps: obtaining the sporozoite escape rate M of a to-be-detected coccidian oocyst sample, and determining whether the activity of the coccidian oocysts is qualified or not according to the sporozoite escape rate M, the method for obtaining the sporozoite escape rate M comprises the following steps: (1) preparing a coccidian oocyst suspension of a coccidian oocyst sample; (2) carrying out wall breaking on oocysts in the coccidium oocyst suspension to enable the oocysts to release sporangiums so as to obtain a sporangium suspension; (3) digesting the obtained sporangium suspension by using digestive juice, so that sporangium releases sporozoites to obtain a sporozoite suspension, counting the sporozoites and sporangium in the sporozoite suspension, and marking the number of the sporozoites and sporangium as B and C respectively, and (4) calculating the sporozoite escape rate M: when the sporozoite escape rate M is greater than or equal to 70%, determining that the activity of the coccidium oocysts is qualified.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of vaccine quality detection, and more particularly relates to a method for detecting the activity of coccidian oocysts and determining the quality grade thereof. BACKGROUND

[0002] Coccidiosis is a common intestinal parasitic disease in breeding production. For example, chicken coccidia can infect chickens of various breeds, genders and age groups, and the harm to chicks is the greatest. Once the chicken population is infected with chicken coccidia wild strains, it is difficult to completely eliminate them from the environment, which brings great economic losses to the chicken production. At present, the use of vaccines for prevention is a common measure to cope with chicken coccidiosis in China, and is also the main method for replacing anticoccidial drugs to prevent and control chicken coccidiosis.

[0003] After chickens are infected with coccidiosis, coccidia reproduce in the chicken body, and coccidian oocysts are excreted with chicken feces into the external environment. At this time, the oocysts are called unsporulated oocysts (see Figure 1 ), which do not have the ability to infect chickens. Unsporulated oocysts form sporulated oocysts through sporogenesis under appropriate temperature, humidity and oxygen conditions. Each sporulated oocyst contains four sporocysts (see Figure 2 ), and each sporocyst contains two sporozoites. At this time, the oocysts have the ability to infect chickens and are called sporulated oocysts (see Figure 3 ). The optimum temperature for chicken coccidian oocyst sporulation is 28℃±1℃. The storage standard of chicken coccidiosis vaccine stipulates that the suitable storage temperature of chicken coccidiosis vaccine is 2-8℃. If the temperature of the vaccine is once lower than 0℃ during storage, the vaccine will freeze, which will greatly reduce or completely inactivate the activity of the oocysts. For coccidiosis vaccines, only when the activity of the oocysts is good (the quality of the coccidiosis vaccine is qualified), can the vaccine successfully infect susceptible hosts and develop and reproduce in the host body to establish an immune response. When the activity of the oocysts of the coccidiosis vaccine decreases or is inactivated (the quality of the coccidiosis vaccine is unqualified or invalid), it will lead to the state that the vaccine cannot develop and reproduce normally in the host body after infecting the host, thereby causing vaccine immunization failure. Therefore, the key point of the transportation and storage process of chicken coccidiosis vaccine is to avoid freezing. Moreover, chicken coccidian sporulated oocysts do not change in morphology after experiencing low temperature freezing (-18℃) or high temperature (more than 35℃) extreme conditions (see Figure 4 and Figure 5 respectively), but the activity detection shows that the activity of the coccidian oocysts has been significantly reduced or completely inactivated at this time.

[0004] The quality control of chicken coccidiosis vaccine runs through the whole chain of production and manufacturing, quality testing, finished product storage, delivery and transportation, and customer storage. Each link needs to observe the release of oocysts and sporozoites in the vaccine regularly, and detect the viability to ensure the quality of the vaccine (i.e. to ensure the viability of the oocysts). The quality detection of chicken coccidiosis vaccine is actually the viability detection of the oocysts. At present, people generally infect animals with chicken coccidiosis vaccine and then detect the reproduction amount of oocysts in the chicken body to determine it. This detection method takes at least 7 days, is time-consuming and labor-intensive, and has high cost.

[0005] The prior art CN115491406A discloses a detection method for the viability of chicken coccidiosis oocysts, which comprises obtaining the sporozoite release rate of a sample of chicken coccidiosis oocysts to be tested and the sporozoite release rate of a sample of chicken coccidiosis oocysts for comparison. If the deviation of the sporozoite release rate of the sample of chicken coccidiosis oocysts to be tested from the sporozoite release rate of the sample of chicken coccidiosis oocysts for comparison is less than 10%, the viability of the sample of chicken coccidiosis oocysts to be tested is qualified. The calculation formula of the sporozoite release rate (Y) disclosed by the prior art is Y = B x C / (2 x A), wherein A is the content of sporocysts in the sporocyst suspension, B is the content of sporozoites in the sporozoite suspension, and C is the dilution multiple of the sporocyst suspension. Although the detection method is simple and has good repeatability compared with the method of first infecting animals with vaccine and then detecting the reproduction amount of oocysts in the chicken body to determine it, the content A of sporocysts and the content B of sporozoites in the calculation formula of the sporozoite release rate (Y) are the contents of two different steps. When there is an operation error in the process of diluting the sporocyst suspension, the calculation result will be inaccurate, and the counting result will fluctuate in a large range. The detection method for the viability of chicken coccidiosis oocysts of the prior art needs to compare the sporozoite release rate (Y) of the sample with the sporozoite release rate (Y) of the control product, and the deviation of less than 10% is the reliable range. In this case, the fluctuation of the Y value directly affects the detection result. In addition, the control product has problems, which also easily leads to inaccurate detection results. In fact, the sporozoite release rate (Y) mentioned in the prior art is a relative value, which easily leads to a large error in the final result, and the accuracy of determining the viability of coccidiosis oocysts is low.

[0006] Therefore, there is a need for a method for reliably and accurately detecting the viability of coccidiosis oocysts. SUMMARY

[0007] In view of the deficiencies in the prior art, the present application provides a method for detecting the viability of coccidiosis oocysts, which is reliable and has good accuracy.

[0008] The method for detecting the viability of coccidiosis oocysts of the present application comprises the steps of obtaining the sporozoite release rate M of a sample of coccidiosis oocysts to be tested, determining whether the viability of the coccidiosis oocysts is qualified according to the sporozoite release rate M,

[0009] wherein the method for obtaining the subsporozoite release rate M comprises the following steps:

[0010] (1) preparing an oocyst suspension of a coccidian oocyst sample;

[0011] (2) breaking the oocysts in the oocyst suspension to release sporocysts from the oocysts, thereby obtaining a sporocyst suspension;

[0012] (3) digesting the obtained sporocyst suspension with a digestive solution to release subsporozoites from the sporocysts, thereby obtaining a subsporozoite suspension, and counting the subsporozoites and sporocysts in the subsporozoite suspension, the number of the subsporozoites and sporocysts being denoted as B and C, respectively;

[0013] (4) calculating the subsporozoite release rate M: and

[0014]

[0015] when the subsporozoite release rate M is ≥ 70%, the coccidian oocyst is qualified,

[0016] when the subsporozoite release rate M is < 70%, the coccidian oocyst is unqualified.

[0017] In another preferred embodiment, when the subsporozoite release rate 70%≤ M≤ 100%, the coccidian oocyst is qualified. In another preferred embodiment, when the subsporozoite release rate 0 < M < 70%, the coccidian oocyst is unqualified.

[0018] In another preferred embodiment, the method further comprises:

[0019] counting the sporocysts in the sporocyst suspension of step (2), the number of the sporocysts being denoted as A,

[0020] denoting the ratio of the volume of the subsporozoite suspension to the volume of the digested sporocyst suspension as the dilution factor D, and

[0021] calculating the subsporozoite recovery rate N of the coccidian oocyst sample,

[0022]

[0023] the subsporozoite recovery rate N and the subsporozoite release rate M are combined to determine whether the coccidian oocyst is qualified, when the subsporozoite release rate M is ≥ 70% and the subsporozoite recovery rate N is ≥ 50%, the coccidian oocyst is qualified, and when the subsporozoite release rate M is < 70% and the subsporozoite recovery rate N is < 50%, the coccidian oocyst is unqualified.

[0024] In another preferred embodiment, the sub-oocyst recovery rate N and the sub-oocyst escape rate M are combined to determine whether the coccidian oocyst viability is qualified, when the sub-oocyst escape rate is 70%≤M≤100% and the sub-oocyst recovery rate is 50%≤N≤115%, the coccidian oocyst viability is qualified, when the sub-oocyst escape rate is 0

[0025] In another preferred embodiment, the step (1) comprises the following steps:

[0026] (1-a) centrifuging the coccidian oocyst sample after shaking well;

[0027] (1-b) adding water or PBS buffer to the precipitate obtained by centrifugation and centrifuging, repeating the operation 2-3 times;

[0028] (1-c) resuspending the precipitate obtained in step (1-b) with PBS buffer to obtain an oocyst suspension.

[0029] The number of oocysts in the oocyst suspension prepared in step (1) is 100,000-500,000 / ml.

[0030] In another preferred embodiment, the PBS buffer is prepared by mixing 8.0 g of sodium chloride, 0.2 g of potassium chloride, 1.44 g of sodium hydrogen phosphate (Na2HPO4) and 0.24 g of potassium dihydrogen phosphate (KH2PO4) together and then adding deionized water or distilled water to a volume of 1000 ml.

[0031] In another preferred embodiment, the wall breaking in step (2) comprises the following steps: adding glass beads to the oocyst suspension and performing shock treatment to break the oocyst wall and release the sporocysts to obtain a sporocyst suspension.

[0032] In another preferred embodiment, the shock treatment in the wall breaking process is performed by using a vortex shaker at 2900-3100 rpm for 50-90 seconds.

[0033] In another preferred embodiment, the diameter of the glass beads is 0.8-1.0 mm.

[0034] In another preferred embodiment, the weight-to-volume ratio of the glass beads to the oocyst suspension is 0.5-3 g / ml, more preferably 1 g / ml.

[0035] In another preferred embodiment, in step (3), the digestion solution comprises sodium taurodeoxycholate, trypsin and PBS buffer. In another preferred embodiment, the preparation method of the digestion solution for digestion is as follows: sodium taurodeoxycholate hydrate and trypsin are added to PBS buffer in an amount of 0.015 g / ml and 0.01 g / ml, respectively.

[0036] In another preferred embodiment, in step (3), the digestive solution comprises trypsin, chicken bile and PBS buffer. In another preferred embodiment, the preparation method of the digestive solution used for digestion is as follows: trypsin and fresh chicken bile are added into PBS buffer respectively, and mixed uniformly, wherein the volume ratio of PBS buffer to fresh chicken bile is 19:1, and the weight volume ratio of trypsin to the total volume of PBS buffer and fresh chicken bile is 0.01 g / ml.

[0037] In another preferred embodiment, the temperature for digestion is 41±3℃, and the time is 40-70 minutes. In another more preferred embodiment, the temperature for digestion is 41±1℃, and the time is 55-65 minutes.

[0038] In another preferred embodiment, the volume ratio of the digestive solution to the sporocyst suspension is 1:7-11, more preferably 1:9.

[0039] In another preferred embodiment, the number A of sporocysts, the number B of microsporocysts and the number C of sporocysts are all the average numbers counted by sampling 2-4 times.

[0040] In another more preferred embodiment, the number A of sporocysts, the number B of microsporocysts and the number C of sporocysts are all the average numbers counted by sampling 3 times.

[0041] The present application also provides a method for determining the quality grade of coccidian oocysts, comprising the steps of: obtaining the microsporocyst release rate M of a sample of coccidian oocysts to be tested, and determining the quality of the coccidian oocysts according to the microsporocyst release rate M,

[0042] The method for obtaining the microsporocyst release rate M comprises the following steps:

[0043] (1) preparing an oocyst suspension of the sample of coccidian oocysts;

[0044] (2) breaking the wall of the oocysts in the oocyst suspension to make the oocysts release sporocysts, thereby obtaining a sporocyst suspension;

[0045] (3) digesting the obtained sporocyst suspension with a digestive solution to make the sporocysts release microsporocysts, thereby obtaining a microsporocyst suspension, and counting the microsporocysts and sporocysts in the suspension, wherein the number of microsporocysts and sporocysts is recorded as B and C respectively; and

[0046] (4) calculating the microsporocyst release rate M:

[0047]

[0048] When the microsporocyst release rate M is 70-79%, the quality of the coccidian oocysts is generally qualified,

[0049] When the oocyst quality is good, the oocyst quality is good when the merzoite release rate M = 80-89%,

[0050] When the oocyst quality is excellent, the oocyst quality is excellent when the merzoite release rate M = 90-100%,

[0051] When the oocyst quality is unqualified, the oocyst quality is unqualified when the merzoite release rate M < 70%.

[0052] In another preferred embodiment, the method for determining the quality grade of coccidian oocysts further comprises:

[0053] counting the number of sporocysts in the sporocyst suspension of step (2), the number of sporocysts being denoted as A,

[0054] calculating the ratio of the volume of the merzoite suspension to the volume of the digested sporocyst suspension, the ratio of the volume of the merzoite suspension to the volume of the digested sporocyst suspension being denoted as dilution factor D, and

[0055] calculating the merzoite recovery rate N of the coccidian oocyst sample,

[0056]

[0057] combining the merzoite recovery rate N and the merzoite release rate M to determine the quality grade of the coccidian oocysts,

[0058] When the oocyst quality is good, the oocyst quality is good when the merzoite release rate M = 80-89% and the merzoite recovery rate N ≥ 50%,

[0059] When the oocyst quality is good, the oocyst quality is good when the merzoite release rate M = 80-89% and the merzoite recovery rate N ≥ 50%,

[0060] When the oocyst quality is excellent, the oocyst quality is excellent when the merzoite release rate M = 90-100% and the merzoite recovery rate N ≥ 50%,

[0061] When the oocyst quality is unqualified, the oocyst quality is unqualified when the merzoite release rate M < 70% and the merzoite recovery rate N < 50%.

[0062] In another preferred embodiment, the oocysts quality is determined by the combination of the oocysts recovery rate N and the oocysts escape rate M, when the oocysts escape rate M = 70-79% and the oocysts recovery rate 50% < N < 115%, the oocysts quality is general; when the oocysts escape rate M = 80-89% and the oocysts recovery rate 50% < N < 115%, the oocysts quality is better; when the oocysts escape rate M = 90-100% and the oocysts recovery rate 50% < N < 115%, the oocysts quality is excellent. In another preferred embodiment, when the oocysts escape rate 0 < M < 70% and the oocysts recovery rate 0 < N < 50%, the oocysts quality is unqualified.

[0063] In another preferred embodiment, the oocysts are chicken oocysts.

[0064] In another preferred embodiment, the oocysts sample is a chicken oocysts vaccine sample. BRIEF DESCRIPTION OF DRAWINGS

[0065] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present application. The application can be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments.

[0066] Figure 1 Microscope picture of unsporulated chicken oocysts;

[0067] Figure 2 Microscope picture of sporulated chicken oocysts, which are differentiated into 4 sporocysts;

[0068] Figure 3 Microscope picture of sporulated chicken oocysts, which are differentiated into 4 sporocysts, each sporocyst contains 2 sporozoites;

[0069] Figure 4 Microscope picture of sporulated chicken oocysts, which are stored at -18°C;

[0070] Figure 5 Microscope picture of sporulated chicken oocysts, which are stored at 35°C;

[0071] Figure 6 Microscope picture of ground and shaken chicken oocysts, in which the oocysts are completely broken and sporocysts are released;

[0072] Figure 7 Microscope picture of ground and shaken chicken oocysts, in which the oocysts are not completely broken and more oocysts can be seen under microscope;

[0073] Figure 8 Microscope picture of sporozoite suspension after digestion of sporocyst suspension, in which the arrows point to the sporozoites;

[0074] Figure 9 A microscope image of the oocysts suspension after digestion, wherein the arrow points to the sporocysts;

[0075] Figure 10 A counting chamber of a hemocytometer. DETAILED DESCRIPTION

[0076] In the process of researching the quality of chicken coccidiosis vaccine, the inventors of the present application found that although the existing technology CN115491406A uses the sporocyst content A in the sporocyst suspension and the sporozoite content B in the sporozoite suspension to calculate the sporozoite escape rate Y to determine the viability of chicken coccidiosis oocysts, since the sporocyst content A and the sporozoite content B come from different suspensions, the error between the actual dilution multiple and the predetermined dilution multiple in the process of diluting the sporocyst suspension leads to inaccurate values of the sporozoite escape rate Y, and the fluctuation range is relatively large (see Example 3 below). Moreover, the detection method of the viability of chicken coccidiosis oocysts in the existing technology is to compare the sporozoite escape rate Y of the sample to be tested with the sporozoite escape rate Y' of the control, and when the deviation of the sporozoite escape rate Y relative to the sporozoite escape rate Y' is less than 10%, the viability of the chicken coccidiosis oocysts is determined to be qualified. The detection method of the existing technology is based on a relative value, and the sporozoite escape rate Y itself has a relatively large fluctuation range, leading to inaccurate detection results. Moreover, the problem of the control itself also leads to inaccurate detection results, so the detection method of the viability of chicken coccidiosis oocysts in the existing technology is not very reliable and accurate.

[0077] In addition, the method of the technology CN115491406A can only determine whether the viability of chicken coccidiosis oocysts is qualified, and cannot classify the chicken coccidiosis oocyst samples according to their viability. In the actual production process, under certain circumstances, it is necessary to classify the qualified oocyst samples (i.e., the oocyst samples that can be artificially infected with coccidia or vaccinated with coccidia vaccine), for example, the sample with excellent coccidia oocyst viability is represented by A, the sample with good coccidia oocyst viability is represented by B, and the sample with general coccidia oocyst viability (such sample can also successfully infect animals, but a considerable part of the oocysts have been inactivated) is represented by C. Therefore, the inventors of the present application further researched the method for detecting the viability of coccidia oocysts, and found that the sporozoite escape rate M calculated using the sporozoite content B and the sporocyst content C in the sporozoite suspension can accurately reflect the viability of coccidia oocysts, and since the sporozoite content B and the sporocyst content C come from the same suspension, the sporozoite escape rate M obtained is not affected by the error between the actual dilution multiple and the predetermined dilution multiple, and does not need to be compared with the control, and is not affected by the quality of the control, so the detection result is more accurate than the existing technology CN115491406A.

[0078] And the present application inventors found that the oocyst viability of chicken coccidium can be reflected by the recovery rate N of sporozoites calculated by the number A of sporocysts from the sporocyst suspension, the number B of sporozoites and the number C of sporocysts from the sporozoite suspension. The sporozoite escape rate M combined with the sporozoite recovery rate N can more reliably and accurately determine the coccidium oocyst viability. On this basis, the present application is completed.

[0079] The method for detecting the coccidium oocyst viability provided by the present application comprises the steps of: obtaining the sporozoite escape rate M of the coccidium oocyst sample to be detected, determining whether the coccidium oocyst viability is qualified according to the sporozoite escape rate M,

[0080] The method for obtaining the sporozoite escape rate M comprises the following steps:

[0081] (1) preparing an oocyst suspension of the coccidium oocyst sample;

[0082] (2) breaking the wall of the oocysts in the oocyst suspension to make the oocysts release sporocysts, obtaining a sporocyst suspension;

[0083] (3) using a digestive solution to digest the obtained sporocyst suspension to make the sporocysts release sporozoites, obtaining a sporozoite suspension, counting the sporozoites and sporocysts in the sporozoite suspension, and the number of the sporozoites and the sporocysts are respectively recorded as B and C; and

[0084] (4) calculating the sporozoite escape rate M:

[0085]

[0086] When the sporozoite escape rate M is ≥70%, the coccidium oocyst viability is qualified, and when the sporozoite escape rate M is <70%, the coccidium oocyst viability is unqualified.

[0087] Further, counting the sporocysts in the sporocyst suspension of step (2), recording the number of sporocysts as A, recording the ratio of the volume of the sporozoite suspension to the volume of the digested sporocyst suspension as the dilution multiple D, and calculating the sporozoite recovery rate N of the coccidium oocyst sample to be detected,

[0088]

[0089] The sporozoite recovery rate N combined with the sporozoite escape rate M can be used as a more effective evaluation method for the coccidium oocyst viability.

[0090] When the subsporozoite release rate M≥70% and the subsporozoite recovery rate N≥50%, the coccidian oocyst viability is qualified; when the subsporozoite release rate M<70% and the subsporozoite recovery rate N<50%, the coccidian oocyst viability is unqualified.

[0091] In the calculation formula of the subsporozoite release rate M, both the number of subsporozoite cysts B and the number of sporozoite cysts C are from the subsporozoite suspension obtained after the final digestion, and are not affected by the error between the actual value and the predetermined value of the ratio of the volume of the subsporozoite suspension to the volume of the sporozoite cyst suspension, so that the numerical range of the obtained subsporozoite release rate M has small fluctuations.

[0092] The subsporozoite release rate M obtained in the method for detecting the coccidian oocyst viability of the application can also directly reflect the quality grade of the coccidian oocyst. According to the actual situation of the coccidian oocyst samples verified by animal experiments, the better the effect of the coccidian oocyst sample on animal infection, the greater the subsporozoite release rate M, which is closer to 100%. The worse the effect of the coccidian oocyst sample on animal infection, the smaller the subsporozoite release rate M, which is generally less than 70%. This may be related to the fact that the better the coccidian oocyst activity, the more subsporozoites released after digestion (see Application Example 1).

[0093] According to the subsporozoite release rate of the coccidian oocyst samples verified by animal experiments, the inventors of the present application have found that: when the subsporozoite release rate M is 70-79%, it can be determined that the coccidian oocyst viability is general, and such a sample can be used for coccidian artificial infection or vaccine immunization, but the infection effect is slightly worse than the expected result; when the subsporozoite release rate M is 80-89%, it can be determined that the coccidian oocyst viability is better, and such a sample can be used for coccidian artificial infection or vaccine immunization, and the infection effect is better, which can achieve the expected result; when the subsporozoite release rate M is 90-100%, it can be determined that the coccidian oocyst viability is excellent, and such a sample can be used for coccidian artificial infection or vaccine immunization, and the infection effect is very good.

[0094] In the calculation formula of the subsporozoite recovery rate N, the ratio of the volume of the subsporozoite suspension to the volume of the sporozoite cyst suspension is denoted as the dilution multiple D, wherein the “volume of the subsporozoite suspension” refers to the volume of the subsporozoite suspension subjected to the final subsporozoite counting. Under normal circumstances, the volume of the subsporozoite suspension directly obtained by digesting the sporozoite cyst suspension is large, which is not convenient for subsporozoite counting, and needs to be concentrated. After concentration, the subsporozoite counting is performed again, and the subsporozoite suspension subjected to counting after concentration is the subsporozoite suspension subjected to the final subsporozoite counting.

[0095] The formula for calculating the recovery rate of sporozoites N, the number of sporocysts A comes from the sporocyst suspension, each sporocyst has 2 sporozoites, so the denominator is the number of sporocysts calculated in the sporocyst suspension A x 2 (i.e. the theoretical value of the recoverable sporozoites). The formula also includes the number of sporozoites in the sporozoite suspension obtained by digestion and the number of sporocysts that are not digested, therefore, the recovery rate of sporozoites N involves values from two steps (the step of obtaining the sporocyst suspension and the step of obtaining the sporozoite suspension), which can be used as a correction reference value for the sporozoite escape rate M, so that the method for detecting the activity of coccidian oocysts using the sporozoite escape rate M is more accurate, because the formula for calculating the sporozoite escape rate M involves values only from the sporozoite suspension, although the calculation result has small fluctuations, but cannot reflect the number of sporocysts in the sporocyst suspension.

[0096] In the case of the operation specification, the oocyst recovery rate N is mainly affected by the quality of the coccidian oocysts, like the oocyst escape rate M. The higher the activity of the coccidian oocysts, the closer the oocyst recovery rate N to 100% (theoretically, the maximum value of the oocyst recovery rate N is 100%). On the contrary, the lower the activity of the coccidian oocysts, the smaller the oocyst recovery rate N (usually, the oocyst escape rate M is greater than 0). Therefore, the oocyst recovery rate N can also be used as a criterion for measuring the activity of the coccidian oocysts. The lower the activity of the coccidian oocysts, the lower the value of the oocysts after digestion and the oocyst recovery rate. The operation error of the operator can easily lead to the fluctuation of the oocyst recovery rate N. For example, 100 μL of sporocyst suspension and 900 μL of digestion solution are mixed to obtain 1000 μL of oocyst suspension. After centrifugal precipitation of the oocyst suspension, 200 μL of the lower precipitate is reserved as the final oocyst suspension (the volume ratio of the final 200 μL of oocyst suspension to 100 μL of sporocyst suspension is the dilution multiple D, which is 2). However, due to the operation error of the operator, the actual lower precipitate is more or less than the predetermined volume of 200 μL (i.e., the actual dilution multiple D is not 2). In this case, the calculation is still based on D = 2, and the oocyst count result in the actual oocyst suspension is larger or smaller, which leads to the error (larger or smaller) of the oocyst recovery rate N. For example, when the actual lower precipitate is less than the predetermined lower precipitate, the numerator value in the calculation formula of the oocyst recovery rate N is larger, which leads to the oocyst recovery rate N ≥ 100%. For example, the oocyst recovery rate N is 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 110%, 111%, 112%, 113%, 114%, 115%, etc. On the contrary, when the actual lower precipitate is more than the predetermined lower precipitate, the numerator value in the calculation formula of the oocyst recovery rate N is smaller, which leads to the oocyst recovery rate being smaller than the actual value, and the oocyst recovery rate N < 100%. For example, the oocyst recovery rate is 5%, 7%, 8%, 10%, 12%, 14%, 15%, 18%, 20% smaller than the actual value, etc. Usually, N ≥ 100% is caused by human operation, mainly due to the fact that the lower precipitate of the actual oocyst suspension is less than the predetermined volume, but this does not affect the value of the oocyst escape rate M, because the parameters of the oocyst escape rate M, i.e., the oocyst number B and the sporocyst number C, come from the same oocyst suspension and do not involve the dilution multiple D. When the oocyst recovery rate N is too small, e.g., ≤ 50%, it is likely to be caused by the low activity of the coccidian oocysts. The error of N caused by human operation is usually about 15%, and can also be about 20%. In actual operation, the maximum value of the oocyst recovery rate N obtained by the inventors can also be about 120%.

[0097] In the description of the present application, when referring to a numerical interval (i.e. a numerical range), unless otherwise specified, the optional numerical values are considered to be continuous within the numerical interval, and include each and every numerical value within the numerical range, including the two numerical endpoints (i.e. the minimum and maximum values) of the numerical range. When referring to a numerical interval, unless otherwise specified, when the numerical interval refers to integers within the numerical interval, the two numerical endpoints are included, and each and every integer between the two numerical endpoints is included. Furthermore, when multiple ranges are provided to describe a feature or characteristic, the ranges can be combined. In other words, unless otherwise specified, ranges disclosed herein are to be understood to include any and all sub-ranges subsumed therein.

[0098] In the description of the present application, unless otherwise specified, a temperature parameter allows for constant temperature treatment, and also allows for variation within a certain temperature interval. It should be understood that the constant temperature treatment allows for fluctuations within the accuracy range of instrument control. Fluctuations within a range such as ±5℃, ±4℃, ±3℃, ±2℃, ±1℃ are allowed.

[0099] In the description of the present application, unless otherwise specified, percentages refer to mass percentages for solid-liquid mixing and solid-solid mixing, and refer to volume percentages for liquid-liquid mixing.

[0100] In the description of the present application, unless otherwise specified, percentages refer to final concentrations. The final concentration refers to the proportion of the added component in the system after the component is added.

[0101] In the description of the present application, unless otherwise specified, only analytically pure reagents and water meeting the third level water requirements of GB / T6682 are used. Standard titration solutions, preparations and products required in the test are prepared in accordance with the requirements of GB / T601 and GB / T603, unless otherwise specified.

[0102] Compared with the prior art, the main advantageous effects of the method for detecting the activity of chicken coccidiosis oocysts vaccine of the present application are:

[0103] 1. The numerical fluctuation range of the oocyst escape rate M is relatively small, and no control product is required, and the accuracy of the detection result is good.

[0104] 2. The calculation formula of the oocyst recovery rate N includes the number of sporocysts in the sporocyst suspension, the number of sporozoites and the number of sporocysts in the sporozoite suspension, and can more comprehensively reflect the activity of the coccidiosis oocysts obtained in the experimental process. When detecting the activity of coccidiosis oocysts, the oocyst escape rate M combined with the oocyst recovery rate N can more scientifically and accurately determine the activity of coccidiosis oocysts, and ensure that the result is true and reliable.

[0105] 3. The size of the M value of the oocyst escape rate can directly reflect the quality grade of the coccidian oocyst, and the coccidian oocyst sample can be classified into general (C level), better (B level) and excellent (A level) according to the quality. This quality classification can be used by production technicians (as an internal control detection standard for coccidian oocyst vaccine products, including online sampling detection during vaccine production, quality (viability) evaluation of vaccine shelf life) and coccidian oocyst vaccine users (for example, to provide auxiliary detection methods, such as evaluation of vaccines when logistics transportation is overdue, evaluation of vaccines when storage temperature is abnormal, and pre-warehouse acceptance detection).

[0106] The application will be further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the application and not to limit the scope of the application. The experimental methods in the following examples are generally carried out under conventional conditions unless otherwise specified. Unless otherwise specified, percentages and parts are weight percentages and weight parts.

[0107] Example 1

[0108] 1.1 Instruments and equipment used are as follows:

[0109] Centrifuge, vortex shaker (IKA VORTEX2), constant temperature water bath, microscope, 1.5 ml conical centrifuge tube, 50 ml centrifuge tube, pipette (1000 μL, 20-200 μL, 2-20 μL), counter, 1 ml syringe, hemocytometer, glass beads (diameter 0.8-1.0 mm, purchased from Yingxu Chemical Machinery (Shanghai) Co., Ltd.).

[0110] The preparation method of the PBS buffer used is as follows: sodium chloride (NaCl) 8.0 g, potassium chloride (KCl) 0.2 g, disodium hydrogen phosphate (Na2HPO4) 1.44 g, potassium dihydrogen phosphate (KH2PO4) 0.24 g, deionized water or distilled water to 1000 ml.

[0111] 1.2 The composition and preparation method of the digestion solution used are as follows:

[0112] The digestion solution (1) tauroursodeoxycholic acid sodium hydrate is selected from the products of Aladdin Company

[0113] The preparation method of the digestion solution 1 is as follows: 0.4 g of trypsin and 0.3 g of tauroursodeoxycholic acid sodium hydrate are added one by one into 20 ml of PBS buffer, and shaken well, for fresh preparation and immediate use.

[0114] The preparation method of the used digestive solution 2 is as follows: fresh bile is collected by dissecting a chicken, 0.2 g of trypsin (a product of Biosharp or Biofroxx Company) and 1 ml of fresh bile are added into 19 ml of PBS buffer solution respectively, and the prepared solution is divided into small portions and stored at -20°C for use.

[0115] 1.3 The method for detecting the activity of chicken coccidial oocysts specifically comprises the following steps:

[0116] (1) Preparation of oocyst suspension

[0117] 1.1 Centrifugal precipitation: 1 bottle of chicken coccidiosis vaccine is evenly shaken, and then evenly divided into 4 1.5 ml conical centrifuge tubes. The centrifuge is operated at 10000-12000 rpm for 3-5 min. The supernatant is discarded, and 0.1 ml of the precipitate is reserved. 2.5 μl of the supernatant is taken and placed on a glass slide. The effect of centrifugal precipitation of oocysts in the supernatant is observed under a microscope. The number of oocysts in the supernatant under the microscope should not exceed 20 per field. If the number of oocysts in the supernatant exceeds 20 per field, continue to centrifugal precipitate. If the number of oocysts in the supernatant is less than 20 per field, proceed to the next step.

[0118] 1.2 Centrifugal washing: the precipitates in the 4 tubes are combined into one tube, and 1000 μL of PBS buffer solution is added for centrifugal washing. The centrifuge is operated at 3-5 min, and the supernatant is discarded. The precipitate is reserved at 0.1 ml. The centrifugal washing operation is repeated for 2 times.

[0119] 1.3 Resuspension: 500 μL of PBS buffer solution is added to the oocyst precipitate obtained in step 1.2 to resuspend, and an oocyst suspension is obtained, in which the number of oocysts is 100-500 thousand per ml.

[0120] (2) Preparation of sporocyst suspension

[0121] 500 μL of the oocyst suspension in step (1) is taken, 0.5 g of glass beads is added, and then the mixture is placed in a vortex shaker and treated at 3000 rpm for 50-90 seconds to break the oocyst wall and release sporocysts. The sporulation of oocysts is checked under a microscope. When more than 95% of the oocysts are broken (see Figure 6 ), the grinding and shaking treatment is terminated, and a sporocyst suspension is obtained.

[0122] The sporocysts in the sporocyst suspension are counted. The specific process is as follows: the sporocyst suspension after grinding and shaking treatment is blown evenly, and 10-11 μL of the sample is taken and injected into the counting chamber of a blood cell counting plate (see Figure 10, the four corner squares are used for counting, the upper left square is No. 1 square, the upper right square is No. 2 square, the lower left square is No. 3 square, and the lower right square is No. 4 square, the total number of the four corner squares is divided by 4 to obtain the average value, the average of the values of three repeated samplings and counting is counted as the number of sporangia A (ten thousand / ml) ;

[0123] (3) Digestion with the digestive solution to obtain the sporozoite suspension

[0124] 100 μL of the sporangia suspension obtained in step (2) is placed in a glass bottle, 900 μL of digestive solution 1 or digestive solution 2 (the digestive solution is preheated at 41°C for 10 min) is added thereto and mixed, the sporangia are digested in a water bath at 41°C to release sporozoites, the glass bottle is shaken every 10 min during the process, and after 60 min, the sporangia are basically digested, the digestion is terminated in a refrigerator at 4°C to obtain the sporozoite suspension.

[0125] During the digestion of the sporangia, samples are taken every certain period of time, the samples are blown and then sucked by a pipette to be injected into a blood cell counting chamber to stand for 2 min, sporozoites are observed under a microscope, and the counting is performed by using a blood cell counting plate. If the number of sporozoites in 16 small squares of the counting plate is not less than 20, the digestion can be stopped (at this time, the sporangia are basically digested, see Figure 8 and Figure 9 ).

[0126] In order to obtain a sporozoite suspension with moderate concentration, the sporozoite suspension obtained by digestion is centrifuged and precipitated, and the process is as follows: the sporozoite suspension obtained by digestion is centrifuged at 10,000-12,000 rpm for 3 min, 800 μL of supernatant is discarded, and the remaining 200 μL of lower precipitate is used as the final sporozoite suspension, which is then placed in a refrigerator at 4°C to terminate the digestion.

[0127] The counting process of the sporozoite suspension is as follows: the final sporozoite suspension is placed in a sample tube and blown, 10-11 μL of the sample is sucked by a pipette and injected into a counting chamber of a blood cell counting plate to count sporozoites and sporangia. The average of the values of three repeated samplings and counting is counted as the number of sporozoites B (ten thousand / ml) and the number of sporangia C (ten thousand / ml). The ratio of the volume of the final sporozoite suspension (200 μL) to the volume of the sporangia suspension (100 μL) is recorded as the dilution multiple D.

[0128] (4) The sporozoite release rate M and the sporozoite recovery rate N are calculated by the following formula,

[0129]

[0130] The dilution multiple D is 2.

[0131] Key control points of each operation step:

[0132] 1. Centrifugal precipitation of oocysts: centrifugal precipitation of supernatant to be discarded, and the centrifugal precipitation effect of oocysts must be observed under a microscope. The specific process is as follows: transfer the supernatant into a 5-ml centrifuge tube, mix and take 2.5 μL of the supernatant for microscopic examination, and the number of oocysts should not exceed 20 per field of view, and less than 10 is the best.

[0133] 2. The vortex speed of the vortex shaker can be calculated according to the specific model, and the strength should not be too large; the vortex process needs to be observed under a microscope, and the wall of the oocyst should be broken and the sporozoites should be prevented from being released. It is recommended to shake for 60 seconds for the first time, then take 10 μL of the liquid and inject it into a hemocytometer plate under a microscope to observe the oocyst wall breaking condition, and if the sporocyst ratio reaches 95%, stop the oocyst wall breaking. If not, increase the vortex treatment time by 10 seconds, and observe again under a microscope until the sporocyst ratio reaches more than 95%.

[0134] 3. Digestion solution preparation: taurine deoxycholate sodium hydrate is recommended to be prepared and used immediately as the digestion solution; chicken bile should be collected to prevent blood from mixing; the digestion solution is recommended to be prepared and used immediately, and the effective period should not exceed 14 days

[0135] 4. Monitor the temperature of the water bath: continuously monitor the temperature of the water bath during the digestion process to keep it within the range of 41±1℃, and stop the digestion after 60 minutes.

[0136] 5. Operation method and counting method of oocyst counting with a hemocytometer plate: after the coccidial oocyst suspension to be detected is fully stirred and mixed, use a sample dropper to drop the sample into a hemocytometer plate, and count the total number of sporulated oocysts in 4 corner squares under a 10x10 microscope, and multiply by 2.5x10 3 , which is the number of sporulated oocysts in 1 ml of oocyst suspension.

[0137] Application Example 1

[0138] The chicken coccidial oocyst control sample and samples 1-5 used in this example are the same batch of chicken coccidiosis tetravalent live vaccine produced by Foshan Zhengdian Biological Technology Co., Ltd., which contains four kinds of coccidial oocysts of Eimeria tenella, Eimeria necatrix, Eimeria maxima and Eimeria acervulina. The control sample is stored in the vaccine storage environment for 7 days; sample 1 is stored in a constant temperature control box at 16-18℃ for 7 days; sample 2 is stored in a constant temperature control box at 28-30℃ for 7 days; sample 3 is stored in a constant temperature control box at 31-33℃ for 7 days; sample 4 is stored in a constant temperature control box at 34-36℃ for 7 days; and sample 5 is inactivated by 100℃ high temperature for 30 minutes.

[0139] The quality of chicken coccidiosis vaccine of the control, sample 1, sample 2, sample 3, sample 4 and sample 5 was obtained through animal experiment. The specific process of the animal experiment was as follows: 60 ephedrine chickens with similar weight of 3 days old were selected and evenly divided into 6 groups, 10 chickens in each group. The chickens in each group were raised in the same conditions in the isolated cages, the ephedrine chickens in the control group were infected with the control, the ephedrine chickens in the first group were infected with sample 1, the ephedrine chickens in the second group were infected with sample 2, the ephedrine chickens in the third group were infected with sample 3, the ephedrine chickens in the fourth group were infected with sample 4, and the ephedrine chickens in the fifth group were infected with sample 5, 1 dose of vaccine per chicken. The feces of each group 5-7 days after inoculation were collected every 24 hours, weighed and subjected to oocyst counting per gram of feces, the total oocyst discharge of each chicken was calculated and the relative oocyst discharge of each group (S, the total oocyst discharge of the control group was recorded as 100%) was calculated. The relative oocyst yield was used as an index for determining whether the vaccine met the requirements, S≥80% indicated that the activity of the vaccine was not significantly affected, that is, the oocyst yield of the vaccine in the animal body met the requirements (qualified), and the vaccine could be used for artificial infection or vaccine immunization of chicken coccidiosis. The grouping and treatment of ephedrine chickens are specifically shown in Table 1, and the oocyst discharge results of ephedrine chicken coccidiosis (total oocyst discharge of each chicken and relative oocyst discharge of each group) are shown in Table 2.

[0140] The formula is converted as follows:

[0141] The counting chamber volume of the McMaster counting plate is 1 cm x 1 cm x 0.15 cm = 0.15 ml,

[0142] The feces content in each 0.15 ml counting chamber is 0.01 g

[0143] Oocysts per gram of feces (OPG) = total number of coccidial oocysts in each counting chamber (a) x 100; a is the number of oocysts counted under a microscope.

[0144] Average oocyst discharge per chicken = OPG x feces weight ÷ number of chickens.

[0145] Relative oocyst discharge (S) = (average oocyst discharge per chicken of the test group ÷ average oocyst discharge per chicken of the control group) x 100%.

[0146] Table 1 Grouping and treatment of ephedrine chickens

[0147]

[0148] Table 2 Oocyst discharge results of ephedrine chickens

[0149]

[0150]

[0151] Note: In the animal test, the relative oocyst discharge of the test groups (Group 1, Group 2, Group 3, Group 4, Group 5) greater than 80% indicates that the sample meets the requirements (qualified), otherwise it does not meet the requirements (unqualified).

[0152] As can be seen from Table 2, the total oocyst discharge of the control group of ephedrine chickens infected with the control sample is the largest, followed by Group 1, Group 2, Group 3, Group 4 and Group 5 in turn. The oocyst activity of the control sample is the best, and the total oocyst discharge of the control group infected with the control sample is the largest; sample 1 is stored in a constant temperature control box at 16-18°C for 7 days, the relative oocyst discharge of Group 1 is 91.4%, indicating that sample 1 meets the requirements; sample 2 is stored in a constant temperature control box at 28-30°C for 7 days, the relative oocyst discharge of Group 2 is 86.5%, indicating that sample 2 meets the requirements; sample 3 is stored in a constant temperature control box at 31-33°C for 7 days, the relative oocyst discharge of Group 3 is 80.0%, indicating that sample 3 meets the requirements; sample 4 is stored in a constant temperature control box at 34-36°C for 7 days, the relative oocyst discharge of Group 4 is 65.0%, which is less than 80.0%, indicating that sample 4 does not meet the requirements; sample 5 is placed at 100°C for 30 minutes, the relative oocyst discharge of Group 5 is 21.5%, indicating that sample 5 has been basically inactivated at 100°C for 30 minutes.

[0153] The number of sporocysts (A, also referred to as "average sporocyst value before digestion") in the sporocyst suspension, the number of sporozoites (B, also referred to as "average sporozoite value after digestion") in the sporozoite suspension, and the number of sporocysts (C, also referred to as "average sporocyst value after digestion") in the sporozoite suspension of the above-mentioned control sample and sample 1-5 were obtained by using the method for detecting the oocyst activity of chicken coccidia in Example 1, and the sporozoite escape rate M and the sporozoite recovery rate N were calculated, and the results are shown in Table 3 below.

[0154] Table 3

[0155]

[0156] Note: Compared with the control sample, samples 1-5 mainly due to the long-term storage of coccidian oocysts in a high-temperature environment, and the oocysts may not withstand mechanical force to break the wall damage, so the number of sporocysts in the sporocyst suspension before digestion is significantly reduced. Therefore, although the initial oocyst amount of the sample and sample 1-5 is consistent (all 1.1 million tetravalent vaccine per bottle), there is a significant difference in the number of sporocysts in the sporocyst suspension after breaking the wall.

[0157] From the results of Table 3, it can be seen that the average sporocyst before digestion, the average sporozoite after digestion and the sporozoite release rate M obtained by the method for detecting the activity of coccidial oocysts according to the present application are consistent with the quality of the samples obtained from the animal experiment, and the better the quality (the better the oocyst activity) of the sample, the greater the average sporocyst before digestion, the average sporozoite after digestion and the sporozoite release rate M. The value of the sporocyst number C after digestion fluctuates, which is mainly affected by the digestion time and the sampling operation of the personnel, but the value of the sporocyst C after digestion of the sample with no active oocysts is relatively small. The sporozoite value after digestion of Sample 2, Sample 3 and Sample 4 decreases with the increase of the storage temperature, and it can be seen that the activity (activity) of the oocyst sporozoite is affected by the storage temperature and decreases with the decrease of the storage temperature.

[0158] From the results of Tables 2 and 3, it can be seen that the relative oocyst yield of the control group is 100%, the sporozoite release rate of the control sample is 92.2%; the relative oocyst yield of the first group is 91.4%, the sporozoite release rate of Sample 1 is 91.2%; the relative oocyst yield of the second group is 86.5%, the sporozoite release rate of Sample 2 is 83.0%; the relative oocyst yield of the third group is 80.0%, the sporozoite release rate of Sample 3 is 70.0%, the relative oocyst yield of the fourth group is 65.0%, the sporozoite release rate of Sample 4 is 68.5%; the relative oocyst yield of the fifth group is 21.5%, the sporozoite release rate of Sample 5 is 36.3%. From the results of Tables 2 and 3, it can be seen that the sporozoite recovery rate of the control sample and the sample in conformity with the provisions is greater than 50%, and the greater the relative oocyst yield of the chicken group infected, the greater the sporozoite recovery rate.

[0159] According to the actual use of the chicken coccidiosis vaccine and the results of Table 3, it can be determined that when the sporozoite release rate M is greater than or equal to 70%, the chicken coccidial oocyst activity is determined to be qualified, and the greater the sporozoite release rate M, the better the chicken coccidial oocyst activity and the better the immune effect. Therefore, the chicken coccidial oocyst activity can be classified according to the sporozoite release rate M, which is divided into A (excellent), B (good) and C (general) (see Table 4 below for the classification criteria).

[0160] Table 4

[0161] Rank Criteria A Oocysts of chicken coccidia samples with a rate of sporozoite release M = 90-100% B Oocysts of chicken coccidia samples with a rate of sporozoite release M = 80-89% C Oocysts of chicken coccidia samples with a rate of sporozoite release M = 70-79%

[0162] Application Example 2:

[0163] According to the method for detecting the activity of chicken coccidial oocysts in Example 1 above, the sporozoite release rate M of the domestic similar chicken coccidiosis vaccine is obtained, and the oocyst activity is determined. The chicken coccidiosis vaccine to be detected is as follows:

[0164] The chicken coccidiosis tetravalent live vaccine (referred to as SCOCVAC-4) of Foshan Zhengdian Biotechnology Co., Ltd. contains four kinds of coccidium oocysts of Eimeria tenella, Eimeria necatrix, Eimeria maxima and Eimeria acervulina.

[0165] The chicken coccidiosis tetravalent live vaccine (referred to as QL tetravalent vaccine) of a well-known brand in the domestic market contains four kinds of coccidium oocysts of Eimeria tenella, Eimeria necatrix, Eimeria maxima and Eimeria acervulina.

[0166] The chicken coccidiosis tetravalent live vaccine (referred to as QL tetravalent vaccine) of a well-known brand in the domestic market contains four kinds of coccidium oocysts of Eimeria tenella, Eimeria necatrix, Eimeria maxima and Eimeria acervulina.

[0167] Table 5

[0168]

[0169] As can be seen from Table 5, there are differences in the quality (i.e., oocyst viability) of chicken coccidiosis vaccines produced by different manufacturers. Moreover, according to the grade determination standard of Table 4 and the test results obtained from Table 5, the SCOCVAC-4 meront escape rate is 94.8%, and the oocyst quality grade is determined as A level; the QL tetravalent vaccine meront escape rate is 86.8%, and the oocyst quality grade is determined as B level; and the T trivalent vaccine meront escape rate is 83.6%, and the oocyst quality grade is determined as B level.

[0170] The meront escape rate M obtained by using the present application can not only accurately determine whether the chicken coccidium oocyst viability meets the requirements, but also determine the level of the chicken coccidium oocyst viability.

[0171] Application Example 3

[0172] Take the same batch of chicken coccidiosis tetravalent live vaccine (referred to as SCOCVAC-4) of Foshan Zhengdian Biotechnology Co., Ltd., which contains four kinds of coccidium oocysts of Eimeria tenella, Eimeria necatrix, Eimeria maxima and Eimeria acervulina. Samples 1-3 are vaccines stored in a vaccine storage environment for 1 month, which have been tested by animal experiments and meet the requirements (quality is qualified). Samples 4-5 are vaccines stored in a vaccine storage environment for 1 month, which are taken out from the storage environment, boiled at 100°C for 30 minutes, and have been tested by animal experiments and do not meet the requirements (quality is unqualified). Respectively, according to the method for detecting coccidium oocyst viability of the present application and the chicken coccidium oocyst viability detection method disclosed in the prior art CN115491406A, the meront escape rates M and Y are obtained, respectively, and the results are shown in Table 6 below. The deviation between the obtained meront escape rate M and the meront escape rate Y is less than 10%, which is within the acceptable range.

[0173] Table 6

[0174]

[0175] Note: In Table 1, according to the prior art CN115491406A, the calculation formula of the sporozoite release rate (Y) = 5*B / A*100%.

[0176] As can be seen from Table 1, the sporozoite release rates M of samples 1-3 calculated by the sporozoite release rate M calculation formula of the present application are all greater than 90%, and the counting results of the three samples have a smaller fluctuation range (the maximum deviation is 3.1%), and the deviation is much smaller than 10%. The sporozoite release rates M of samples 4-5 are all much less than 70%, which is consistent with the determination result of the animal experiment. The counting results of the sporozoite release rates Y of samples 1-3 calculated by the sporozoite release rate Y calculation formula of the prior art CN115491406A have a larger fluctuation range, especially the deviations of samples 2 and 3 are much greater than 10% (up to 13.9%, which is determined to be in an unacceptable range). In this case, according to the determination method disclosed in the prior art (under the condition that the deviation of the sporozoite release rate of the sample to be tested relative to the sporozoite release rate of the control chicken coccidian oocyst sample is less than 10%, the activity of the sample to be tested is qualified), the activity of the chicken coccidian oocyst to be tested cannot be accurately determined.

[0177] The reason why the numerical range calculated by the sporozoite release rate M calculation formula of the present application has a small fluctuation is that B and C in the formula are both from the sporozoite suspension, and are not affected by the error between the actual dilution multiple and the predetermined dilution multiple. The reason why the numerical range calculated by the sporozoite release rate Y calculation formula of the prior art CN115491406A has a large fluctuation is that B and A in the formula are from different suspensions, and are affected by the error between the actual dilution multiple and the predetermined dilution multiple. The greater the error, the greater the deviation of the obtained result, resulting in low accuracy in determining the activity of coccidian oocysts. Therefore, the data in Table 1 show that the sporozoite release rate M calculated by the method for detecting the activity of chicken coccidiosis vaccine oocysts of the present application has a small deviation, and can more accurately detect whether the activity of chicken coccidian oocysts meets the requirements (qualified).

[0178] Application Example 4:

[0179] This application example compares the application effects of digestive solution 1 and digestive solution 2 in coccidian oocyst activity detection, broadens the coccidian oocyst activity detection method and provides a reference basis therefor.

[0180] The chicken coccidiosis live vaccine used in this example is a chicken coccidiosis tetravalent live vaccine (abbreviation: SCOCVAC-4, containing four kinds of coccidium oocysts of Eimeria tenella, Eimeria necatrix, Eimeria maxima and Eimeria acervulina) from Foshan Zhengdian Biotechnology Co., Ltd. The production batch number is 20250602. The same batch of test samples were taken, and in the step of digesting sporocysts, digestion solution 1 and digestion solution 2 were used respectively. The results of the subsporogonial escape rate M of the chicken coccidiosis live vaccine test samples are shown in Table 7.

[0181]

[0182] As can be seen from Table 7, for the same batch of test samples, digestion solution 1 and digestion solution 2 were used as the digestion solution in the step of digesting sporocysts. After digestion with digestion solution 1, the number of subsporogonia was 2.35 million / ml, and the subsporogonial escape rate was 95.1%. After digestion with digestion solution 2, the number of subsporogonia was 2.106 million / ml, and the subsporogonial escape rate was 96.2%. It can be seen that digestion agent 1 and digestion agent 2 both have good digestion characteristics.

[0183] Application Example 5:

[0184] This application example studies the effects of different storage temperatures and times on the oocyst activity of the chicken coccidiosis vaccine, providing a reference basis for the temperature range of coccidiosis vaccine transportation.

[0185] The chicken coccidiosis vaccine used in this example is a chicken coccidiosis tetravalent live vaccine (abbreviation: SCOCVAC-4, containing four kinds of coccidium oocysts of Eimeria tenella, Eimeria necatrix, Eimeria maxima and Eimeria acervulina) from Foshan Zhengdian Biotechnology Co., Ltd.

[0186] Six samples of the same batch of SCOCVAC-4 were taken as control and test samples 1-5. The control was stored in an environment of 2-8°C, and test samples 1-4 were stored in temperature-controlled boxes at different temperatures (8-10°C, 19-21°C, 24-26°C, 31-33°C). Four gradients of duration time were set, i.e. 1 day, 3 days, 5 days and 7 days, and samples were taken at the corresponding time points, with 2 bottles of samples taken each time. The storage temperature and duration time are shown in Table 8. Test sample 5 was inactivated by heating at 100°C for 30 min.

[0187] According to the results of the subsporogonial escape rate M and the subsporogonial recovery rate N of the chicken coccidiosis vaccine of the control and test samples 1-5 obtained in Example 1, see Table 9.

[0188] Table 8

[0189] Group Storage temperature Duration 1 Duration 2 Duration 3 Duration 4 Control 2～8℃ 1 day 3 days 5 days 7 days Test sample 1 8～10℃ 1 day 3 days 5 days 7 days Test sample 2 19～21℃ 1 day 3 days 5 days 7 days Test sample 3 24～26℃ 1 day 3 days 5 days 7 days Test sample 4 31～33℃ 1 day 3 days 5 days 7 days

[0190] Table 9

[0191]

[0192] The following conclusions can be drawn from Table 8:

[0193] (1) From the results of the oocyst release rate M, it can be seen that, compared with the control samples stored at 2-8℃, the oocyst release rate M of the test samples 1-4 stored at 8-10℃, 19-21℃, 24-26℃ and 31-33℃ for 7 days showed a downward trend as a whole, but were all greater than 70%, indicating that the oocyst activity of the test samples was at a stable level, the oocyst activity of the coccidiosis vaccine was not significantly affected, and the test samples met the requirements (quality qualified).

[0194] The oocyst recovery rate of the test samples 1-4 fluctuated greatly. The oocyst recovery rate of the control sample stored for 5 days was 106.7%, and the oocyst recovery rate N was greater than 100%. The oocyst recovery rate N of the control sample stored for 1 day, 3 days and 7 days was 93.6%, 87.8% and 94.3% respectively, and the oocyst recovery rate N was less than 100%. The main reason for the large fluctuation in the oocyst recovery rate was that, after centrifugation of the oocyst suspension, the volume of the remaining lower sediment was less than or greater than the predetermined volume. For example, when the predetermined volume of the remaining lower sediment was 200 μL, and the actual volume of the remaining lower sediment was less than 200 μL, for example, about 180 μL, the number of oocysts B and sporocysts C obtained by microscopic examination were larger, the dilution factor D was still calculated as 2, the numerator value in the oocyst recovery rate formula was larger, and the calculated oocyst recovery rate N was larger, which might be greater than 100%. Conversely, when the predetermined volume of the remaining lower sediment was 200 μL, and the actual volume of the remaining lower sediment was greater than 200 μL, the calculated oocyst recovery rate N was smaller. That is, when the oocyst recovery rate N≥100%, it can be judged that the personnel operation error. However, in this case, the oocyst release rate M is not affected, because the parameters of the number of oocysts B and the number of sporocysts C used in the formula are the same suspension. That is, the human error of the actual volume of the remaining lower sediment being less than the predetermined volume does not affect the oocyst release rate M. However, in the case of poor quality of the coccidiosis vaccine itself, the number of oocysts in the oocyst suspension obtained after digestion will be small, and the calculated oocyst release rate M and oocyst recovery rate N will necessarily be smaller, and the values of the oocyst release rate M and the oocyst recovery rate N will be smaller (the former is less than 70%, and the latter is less than 50%), which can be judged that the coccidiosis vaccine (oocyst sample) is unqualified. The oocyst recovery rate N includes the number of sporocysts in the sporocyst suspension and the number of oocysts and sporocysts in the oocyst suspension, and can more accurately reflect the number of oocysts obtained in the detection process, and therefore can be used as an auxiliary judgment parameter for the oocyst release rate.

[0195] (2) The sporozoite release rate M of the experimental sample 4 is greater than 80% when stored at 31-33°C for 1 day, 3 days and 5 days. The sporozoite release rate M is 70.0% when stored at 31-33°C for 7 days, the sporozoite release quantity is reduced to 0.44 million / ml, the sporozoite recovery rate is 50.0%, and both the sporozoite release rate M and the sporozoite recovery rate N are low. It is shown that the activity of the coccidiosis vaccine is obviously decreased and the quality of the coccidiosis vaccine is general when the coccidiosis vaccine is stored at 31-33°C for 7 days.

[0196] (3) The experimental sample 5 is the coccidiosis vaccine boiled at 100°C for 30 minutes, and the coccidiosis vaccine is usually inactivated. The quantity of sporozoites in the sporozoite suspension obtained by the method and technology of the application is obviously less than that of the experimental samples 1-4, the sporozoite release rate M is 36.3%, the sporozoite recovery rate N is 24.0%, both the sporozoite release rate M and the sporozoite recovery rate N are low, and it is determined that the quality of the coccidiosis vaccine is not qualified (i.e. the quality of the coccidiosis vaccine is unqualified).

[0197] (4) It can be seen from the data of the sample 4 and the sample 5 that if the activity of the oocysts is weakened, the quantity of sporozoites released after digestion is obviously reduced, and the sporozoite recovery rate is low, and both are positively correlated.

[0198] (5) It can also be seen from Table 8 that the quantity of sporozoites of the inactivated coccidiosis oocysts is the smallest. The quantity of sporozoites obtained after the oocysts of the experimental sample 4 are broken and digested is in a downward trend with the extension of the storage time when stored at 31-33°C for 1 day, 3 days, 5 days and 7 days, which shows that the quality of the sample is slowly decreased. The quantity of sporozoites obtained after the oocysts of the control sample and the experimental samples 1, 2 and 3 are broken and digested can be maintained at a certain high level, which shows that the qualities of these samples are qualified. It can be seen that the quality determination method of the coccidiosis vaccine of the application can not only determine the activity of the coccidiosis oocysts reliably and accurately, but also grade the quality of the coccidiosis vaccine.

[0199] According to the above application examples, in actual operation, the skilled person can use the method for detecting the activity of coccidian oocysts and determining the quality grade thereof to determine that the activity of coccidian oocysts is qualified when the calculated sporozoite release rate M of the chicken coccidian oocyst sample is greater than about 70% and the sporozoite recovery rate N is greater than about 50%; the activity of coccidian oocysts is unqualified when the calculated sporozoite release rate M of the chicken coccidian oocyst sample is less than about 70% and the sporozoite recovery rate N is less than about 50%; the activity of coccidian oocysts needs to be re-detected when the calculated sporozoite release rate M of the chicken coccidian oocyst sample is greater than about 70% and the sporozoite recovery rate N is less than about 50%; and the activity of coccidian oocysts needs to be re-detected when the calculated sporozoite release rate M of the chicken coccidian oocyst sample is less than about 70% and the sporozoite recovery rate N is greater than about 50%.

[0200] Therefore, the method for detecting the activity of coccidian oocysts and determining the quality grade thereof of the present application ensures the accuracy of detection of coccidian oocyst samples and the effectiveness of single detection. Moreover, in the case of determining that the chicken coccidian oocyst sample is qualified, the skilled person can directly determine the quality grade thereof according to the size of the sporozoite release rate M.

[0201] All the documents mentioned in the present application are incorporated by reference in the present application as if each document was individually incorporated by reference. In addition, it should be understood that various modifications and changes can be made to the present application by those skilled in the art upon reading the above description of the present application, and such equivalent forms are also within the scope of the appended claims of the present application.

Claims

1. A method for detecting the viability of coccidia oocysts, characterized in that, The method includes the following steps: obtaining the sporozoite escape rate M of the coccidia oocyst sample to be tested, and determining whether the coccidia oocyst viability is qualified based on the sporozoite escape rate M. The method for obtaining the sporozoite escape rate M includes the following steps: (1) Preparation of oocyst suspension of coccidia oocyst samples; (2) The oocysts in the oocyst suspension are broken up to release sporangia, thus obtaining a sporangia suspension. (3) The obtained sporangium suspension is digested with a digestive solution to release sporozoites from the sporangia, resulting in a sporozoite suspension. The number of sporozoites and sporangia in the sporozoite suspension is counted, and the number of sporozoites and sporangia is denoted as B and C, respectively; and (4) Calculate the sporozoite escape rate M: When the sporozoite escape rate M ≥ 70%, the coccidia oocyst viability is qualified. When the sporozoite escape rate M < 70%, the coccidia oocyst viability is unqualified.

2. The method according to claim 1, characterized in that, The method further includes: Count the sporangia in the sporangium suspension from step (2), and record the number of sporangia as A. The ratio of the volume of the sporozoite suspension to the volume of the digested sporangium suspension is denoted as the dilution factor D. Calculate the sporozoite recovery rate N of coccidia oocyst samples: The viability of coccidia oocysts is determined by combining the sporozoite recovery rate N with the sporozoite escape rate M. When the sporozoite escape rate M ≥ 70% and the sporozoite recovery rate N ≥ 50%, the coccidia oocysts are considered to be viable. When the sporozoite escape rate M < 70% and the sporozoite recovery rate N < 50%, the coccidia oocysts are considered to be unviable.

3. The method according to claim 1 or 2, characterized in that, Step (1) includes the following steps: (1-a) After thoroughly shaking the coccidia oocyst sample, centrifuge to precipitate; (1-b) Add water or PBS buffer to the precipitate obtained by centrifugation and centrifuge. Repeat this operation 2 to 3 times. (1-c) Resuspend the precipitate obtained in step (1-b) using PBS buffer to obtain an oocyst suspension. Preferably, the number of oocysts in the oocyst suspension is 100,000 to 500,000 per ml.

4. The method according to claim 3, characterized in that, The PBS buffer used in steps (1-b) and (1-c) is prepared as follows: Mix 8.0g of sodium chloride (NaCl), 0.2g of potassium chloride (KCl), 1.44g of disodium hydrogen phosphate (Na2HPO4) and 0.24g of potassium dihydrogen phosphate (KH2PO4) together, and bring the volume to 1000ml with deionized water or distilled water.

5. The method according to claim 1 or 2, characterized in that, In step (2), the cell wall disruption includes the following steps: adding glass beads to the oocyst suspension and shaking the mixture to rupture the oocyst wall, releasing the sporangia and obtaining a sporangia suspension. More preferably, during the cell wall breaking process, the oscillation is performed using a vortex oscillator at 2900–3100 rpm for 50–90 seconds, and / or More preferably, the diameter of the glass beads is 0.8 mm to 1.0 mm, and the weight-to-volume ratio of the glass beads to the egg sac suspension is 0.5 to 3 g / ml, more preferably 1 g / ml.

6. The method according to claim 1 or 2, characterized in that, In step (3), the digestive solution contains sodium taurodeoxycholate, trypsin, and PBS buffer, and / or The digestive fluid contains trypsin, chicken bile, and PBS buffer, and / or The volume ratio of sporangium suspension to digestion fluid is 1:7 to 11, more preferably 1:9, and / or The digestion temperature is 41±3℃ and the time is 40 to 70 minutes; more preferably, the digestion temperature is 41±1℃ and the time is 55 to 65 minutes.

7. The method according to claim 1 or 2, characterized in that, The number of sporangia (A), sporozoites (B), and sporangia (C) are the average counts from 2 to 4 samplings.

8. A method for determining the quality grade of coccidia oocysts, characterized in that, The method includes the following steps: obtaining the sporozoite escape rate M of the coccidia oocyst sample to be tested, and determining the quality grade of the coccidia oocysts based on the sporozoite escape rate M. The method for obtaining the sporozoite escape rate M includes the following steps: (1) Preparation of oocyst suspension of coccidia oocyst samples; (2) The oocysts in the oocyst suspension are broken up to release sporangia, thus obtaining a sporangia suspension. (3) The obtained sporangium suspension is digested with a digestive solution to release sporozoites from the sporangia, resulting in a sporozoite suspension. The number of sporozoites and sporangia in the suspension is counted, and the number of sporozoites and sporangia is denoted as B and C, respectively; and (4) Calculate the sporozoite escape rate M: When the sporozoite escape rate M = 70-79%, the quality of coccidia oocysts is generally poor. When the sporozoite escape rate M = 80-89%, the quality of the coccidia oocysts is relatively good. When the sporozoite escape rate M = 90-100%, the quality of the coccidia oocysts is excellent. When the sporozoite escape rate M < 70%, the coccidia oocysts are of substandard quality.

9. The method according to claim 8, characterized in that, The method further includes: Count the sporangia in the sporangium suspension from step (2), and record the number of sporangia as A. The ratio of the volume of the sporozoite suspension to the volume of the digested sporangium suspension is denoted as the dilution factor D. Calculate the sporozoite recovery rate N of the chicken coccidiosis vaccine to be tested. The quality grade of coccidia oocysts was determined by combining the sporozoite recovery rate (N) and the sporozoite escape rate (M). When the sporozoite escape rate M = 70-79% and the sporozoite recovery rate N ≥ 50%, the quality of coccidia oocysts is generally poor. When the sporozoite escape rate M = 80-79% and the sporozoite recovery rate N ≥ 50%, the quality of coccidia oocysts is relatively good. When the sporozoite escape rate M = 90-100% and the sporozoite recovery rate N ≥ 50%, the quality of the coccidia oocysts is excellent. When the sporozoite escape rate M < 70% and the sporozoite recovery rate N < 50%, the coccidia oocysts are of substandard quality.

10. The method for detecting coccidial oocyst viability according to any one of claims 1 to 8 or the method for determining the quality grade of coccidial oocysts according to any one of claims 9 to 10, characterized in that, The coccidia oocysts are chicken coccidia oocysts.

Images