CN106754379B - Middle-size Eimeria acervulina precocious strain for rabbits as well as preparation method and application thereof - Google Patents
Middle-size Eimeria acervulina precocious strain for rabbits as well as preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses a medium Eimeria acervulina precocious strain in a rabbit and a preparation method and application thereof, wherein the method comprises the following steps: collecting, separating and purifying rabbit feces to obtain sporulated oocysts of a rabbit medium Eimeria strain, infecting the sporulated oocysts of the rabbit medium Eimeria strain with weaned rabbits, and carrying out multiple passages to obtain the rabbit medium Eimeria oocysts with shortened latent period in the Nth generation, namely the rabbit medium Eimeria strain early; compared with the wild epidemic strain of the middle Eimeria tenella, the early-maturing strain of the middle Eimeria tenella prepared by the method is pure and weak in pathogenicity, and can be directly used for preparing vaccines; the preparation method of the middle Eimeria tenella precocious strain in rabbits, disclosed by the invention, is simple and easy to realize.
Description
Technical Field
The invention relates to the field of Eimeria coccidiosis application, in particular to a medium Eimeria coccidiosis precocious strain for rabbits, and a preparation method and application thereof.
Background
The rabbit coccidiosis is a parasitic protozoal disease caused by one or more Eimeria species of Eimeria of Eimeriaceae of Eimeria of Coccidia of Eimeria in Hemophores of Rabbit and intestinal tract. The disease brings great harm to the rabbit industry, seriously hinders the development of the rabbit industry, is classified as two animal epidemic diseases in China, and is called as four-big rabbit disease together with rabbit plague, rabbit brucellosis and rabbit sarcoptidosis. It is reported that about 41.9% of rabbits in China are infected with coccidia, and the infection rate in the northwest, northeast and southwest of China is over 60%. Rabbits of all varieties are susceptible to the disease, wherein the infection and the morbidity of young rabbits from weaning to 3 months are the most serious, the mortality rate is up to 40-70%, and the diseases are common diseases in rabbit breeding production. Eimeria medians (Eimeria media), one of the species of rabbit coccidia, is prevalent in rabbit farms and is the major species responsible for rabbit coccidiosis.
The existing method for preventing and treating rabbit coccidiosis mainly adopts anticoccidial drugs for drug prevention and treatment, and the existing anticoccidial drugs have various types, have uneven prevention and treatment effects, and are easy to cause serious consequences such as drug resistance, food safety problems and the like. The research shows that the rabbit coccidia can be detected in the rabbit group no matter in poor-condition farmers or better-managed intensive rabbit farms, no matter the rabbit groups use the anticoccidial drugs. The development of the rabbit coccidian vaccine can effectively control the rabbit coccidiosis, and cannot generate drug resistance and have no problem in food safety. At present, the call for banning the addition of anticoccidial drugs in feeds is higher and higher, and the rabbit coccidian vaccine for immune prevention is bound to replace the existing drug control, and has a leading position. Thus, the rabbit coccidian vaccine has wide market application prospect. The rabbit coccidiosis is an obligate parasitic protozoa and only produces pathogenicity to rabbits, so the rabbit coccidiosis is very safe; the pathogen is simple to use, low in production cost and competitive in the market.
However, the pathogenicity of wild epidemic strains of the rabbit coccidia is generally stronger, and the strains are not suitable for being directly used for preparing vaccines, so that the early breeding of the strains to reduce the virulence of the rabbit coccidia is necessary, and the breeding of the early-maturing strains of the rabbit eimeria coccidia lays a foundation for the development of live vaccines of the rabbit coccidia.
Accordingly, the prior art is yet to be improved and developed.
Disclosure of Invention
In view of the defects of the prior art, the invention aims to provide a medium-sized Eimeria tenella precocious strain for rabbits, a preparation method and application thereof, and aims to solve the problems that the wild epidemic strain of the Eimeria tenella is strong in pathogenicity and is not suitable for being directly used for preparing vaccines.
The technical scheme of the invention is as follows:
a preparation method of a medium Eimeria tenella precocious strain in a rabbit comprises the following steps:
A. obtaining mixed rabbit coccidium oocysts in rabbit manure by a coccidium separation method;
B. transferring the mixed rabbit coccidian oocysts to a plate filled with a culture solution, and incubating for 20-36 h to obtain the mixed rabbit coccidian sporulated oocysts;
C. carrying out coccidium purification treatment on the sporulated oocysts of the mixed species of rabbit coccidia to obtain a rabbit medium-sized eimeria coccidia virulent strain, and putting the rabbit medium-sized eimeria coccidia virulent strain into a culture solution to incubate for a preset time to obtain sporulated oocysts of the rabbit medium-sized eimeria coccidia virulent strain;
D. orally infecting a predetermined amount of sporozoized oocysts of a virulent strain of eimeria maxima in a rabbit to weaned young rabbits;
E. collecting the excrement of the weaned newborn rabbits every hour, checking whether rabbit medium Eimeria oocysts exist in the excrement, and recording the time interval from the infection of the weaned newborn rabbits to the first detection of the rabbit medium Eimeria oocysts in the excrement of the weaned newborn rabbits as the first prepatent period of the rabbit medium Eimeria oocysts;
F. culturing rabbit eimeria necatrix oocysts collected from stool of weaned young rabbits into rabbit eimeria necatrix sporulated oocysts, and orally infecting new weaned young rabbits with the rabbit eimeria necatrix sporulated oocysts;
G. collecting feces of the new weaned newborn rabbit every hour, checking whether rabbit medium Eimeria oocysts exist in the feces, and recording a time interval from infection of the new weaned newborn rabbit to first checking of the rabbit medium Eimeria oocysts in the feces of the new weaned newborn rabbit as a second prepatent period of the rabbit medium Eimeria oocysts;
H. and repeating the passage process until the N-time latent period time of the rabbit medium-sized Eimeria oocysts is shortened by 20-26 h compared with the first latent period time, and taking the rabbit medium-sized Eimeria oocysts in the N-time latent period as the rabbit medium-sized Eimeria precocious strain.
Preferably, the method for preparing the medium Eimeria tenella precocious strain in the rabbit specifically comprises the following steps:
a1, mashing the collected granular rabbit dung, adding water, mixing uniformly, and filtering to obtain filtrate;
a2, carrying out centrifugal treatment on the filtrate, discarding the supernatant, and keeping the precipitate;
a3, adding water into the precipitate for multiple times, centrifuging until the supernatant is clear, dissolving the obtained precipitate with saturated salt solution, mixing uniformly, centrifuging again, and collecting the supernatant;
and A4, diluting the supernatant with water, centrifuging again, and collecting a precipitate, wherein the precipitate is the mixed rabbit coccidian oocysts.
Preferably, the preparation method of the middle Eimeria tenella precocious strain in the rabbit is characterized in that the centrifugation speed is 2000-3000 rpm/min.
Preferably, the preparation method of the rabbit middle Eimeria tenella precocious strain comprises the step of preparing a culture solution from 0.5-3% of potassium dichromate solution or 0.5-3% of chloroammonia T solution.
Preferably, the preparation method of the middle Eimeria tenella precocious strain in the rabbit comprises the step of incubating at the temperature of 25-30 ℃.
Preferably, the preparation method of the rabbit middle Eimeria tenella precocious strain is characterized in that the predetermined amount in the step D is 5000-100000.
The middle Eimeria acervulina precocious strain for the rabbits is prepared by adopting a preparation method of any one of the middle Eimeria acervulina precocious strains for the rabbits.
The application of the middle Eimeria tenella precocious strain of the rabbit is to prepare a vaccine.
Has the advantages that: the invention provides a medium Eimeria acervulina precocious strain in a rabbit and a preparation method and application thereof, wherein the method comprises the following steps: collecting, separating and purifying rabbit feces to obtain sporulated oocysts of a rabbit medium Eimeria strain, infecting the sporulated oocysts of the rabbit medium Eimeria strain with weaned rabbits, and carrying out multiple passages to obtain the rabbit medium Eimeria oocysts with shortened latent period in the Nth generation, namely the rabbit medium Eimeria strain early; compared with the wild epidemic strain of the middle Eimeria tenella, the early-maturing strain of the middle Eimeria tenella prepared by the method is pure and weak in pathogenicity, and can be directly used for preparing vaccines; the preparation method of the middle Eimeria tenella precocious strain in rabbits, disclosed by the invention, is simple and easy to realize.
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FIG. 1 is a flow chart of a preferred embodiment of a method for preparing a medium Eimeria tenella precocious strain in rabbits in accordance with the present invention.
Detailed Description
The invention provides a medium Eimeria tenella precocious strain in rabbits, and a preparation method and application thereof, and in order to make the purpose, technical scheme and effect of the invention clearer and clearer, the invention is further described in detail by referring to the attached drawings and taking examples. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
Referring to FIG. 1, FIG. 1 is a flow chart of a preferred embodiment of a medium Eimeria tenella strain in rabbits and a method for preparing the same, as shown in the figure, comprising the steps of:
s100, obtaining mixed rabbit coccidium oocysts in rabbit manure by a coccidium separation method;
specifically, the step S100 specifically includes:
s110, mashing the collected granular rabbit manure, adding water, uniformly mixing and filtering to obtain a filtrate;
specifically, crushing rabbit manure which is collected from the field and contains a mixed wild strain of Eimeria necatrix in rabbits, adding a proper amount of distilled water or purified water, and uniformly mixing; then filtering with 100-mesh and 300-mesh screens, recovering the feces, adding a proper amount of water, mixing uniformly and filtering until most of oocysts in the feces are filtered out;
s120, centrifuging the filtrate, discarding the supernatant, and keeping the precipitate;
specifically, centrifuging the filtered filtrate at a rotating speed of 2000-3000 rpm/min, and discarding the supernatant to obtain a precipitate containing coccidian oocysts;
s130, adding water into the precipitate for multiple times, centrifuging until the supernatant is clear, dissolving the obtained precipitate with saturated salt solution, uniformly mixing, centrifuging again, and collecting the supernatant;
specifically, the centrifugation step is repeated until the centrifuged supernatant is clear; dissolving and uniformly mixing the obtained precipitate with saturated salt water (or magnesium sulfate solution with the same buoyancy), centrifuging at 2000-3000 rpm/min, and collecting supernatant until most coccidian oocysts in the precipitate are collected in a floating manner;
s140, diluting the supernatant with water, centrifuging again, and collecting a precipitate, wherein the precipitate is the mixed rabbit coccidia oocysts.
Specifically, diluting the supernatant containing the coccidian oocysts according to the proportion of 1 part of saturated saline solution and 4-10 parts of distilled water or purified water, centrifuging at 2000-3000 rpm/min, and collecting precipitates, wherein the precipitates are the mixed rabbit coccidian oocysts.
S200, transferring the mixed rabbit coccidium oocysts to a plate filled with a culture solution, and incubating for 20-36 hours to obtain the mixed rabbit coccidium sporulated oocysts;
specifically, the culture solution is 0.5-3% of potassium dichromate solution or 0.5-3% of chloroammoniate T solution, and the incubation temperature is 25-30 ℃; preferably, the mixed rabbit coccidian oocysts are transferred to a plate filled with a culture solution, the incubation temperature is 27 ℃, and the incubation time is 28 hours, so that the mixed rabbit coccidian sporulated oocysts with strong activity are obtained.
S300, carrying out coccidium purification treatment on the mixed coccidium sporulated oocysts of the rabbits to obtain medium Eimeria necatrix virulent strains of the rabbits, and putting the medium Eimeria necatrix virulent strains of the rabbits into a culture solution to incubate for a preset time to obtain sporulated oocysts of the medium Eimeria necatrix virulent strains of the rabbits;
specifically, single rabbit medium-sized Eimeria coccidium virulent strain oocysts are separated from sporulated mixed rabbit coccidium oocysts by a single oocyst separation technology, each oocyst is fed to 1 rabbit through mouth, each rabbit needs to be strictly isolated and raised, and all appliances, feeds and drinking water are strictly disinfected; feces are examined for many times every day on the 2 nd day after infection to eliminate pollution; and respectively collecting feces which begin to discharge oocysts after 96 hours, separating the oocysts, carrying out sporulation and then carrying out amplification propagation once to obtain pure rabbit medium-sized Eimeria coccidia which is a medium-sized Eimeria coccidia virulent strain, and culturing to ensure that the medium-sized Eimeria coccidia is sporulated and matured to obtain the sporulated oocysts of the rabbit medium-sized Eimeria coccidia virulent strain.
S400, orally infecting weaned young rabbits with a predetermined amount of sporulated oocysts of a virulent strain of Eimeria mitis in the rabbits;
specifically, 5000-100000 medium-sized Eimeria coccidium virulent strain sporulated oocysts of rabbits can be orally infected into weaned young rabbits;
s500, collecting excrement of the weaned young rabbit every hour, checking whether rabbit medium-sized Eimeria oocysts exist in the excrement, and recording the time interval from infection of the weaned young rabbit to the first time of checking the rabbit medium-sized Eimeria oocysts in the excrement of the weaned young rabbit as a first prepatent period of the rabbit medium-sized Eimeria oocysts;
preferably, the first prepatent period of the medium Eimeria oocysts in rabbits is 94-98 hours according to experiments; in order to improve the working efficiency, preferably, the invention can collect the excrement of the weaned young rabbit within the first 5 hours when the oocysts of the rabbit Eimeria necatrix reach the latent stage and check whether the excrement contains the oocysts of the rabbit Eimeria necatrix, wherein the oocysts of the rabbit Eimeria necatrix in the excrement are the oocysts of the first generation of the rabbit Eimeria necatrix; recording the time interval from the infection of the weaned newborn rabbit to the first detection of Eimeria necatrix in the feces of the weaned newborn rabbit as the first prepatent period of Eimeria necatrix in the rabbit;
s600, culturing the middle Eimeria necatrix oocysts of the rabbits collected from the feces of weaned newborn rabbits into middle Eimeria sporozoized oocysts of the rabbits, and orally infecting new weaned newborn rabbits with the middle Eimeria sporozoized oocysts of the rabbits;
specifically, the first generation of middle Eimeria oocysts of rabbits are cultured in a culture solution to obtain sporulated first generation of middle Eimeria oocysts of rabbits, and the new weaning rabbits are infected with the middle Eimeria oocysts of the rabbits in an amount which is 2-5 times that of the first weaning rabbits; the amount of the Eimeria oocysts in the first generation of rabbits adopted by the weaned newborn rabbit infected every time is 2-5 times of that of the previous time; for example, the amount of sporulated oocysts of the virulent strain of Eimeria acervulina used by the rabbit infected with the weaned newborn rabbit for the first time is 5000-100000, the amount of the oocysts of the Eimeria acervulina used by the first generation of rabbit infected with the new weaned newborn rabbit for the second time is 10000-500000, and the like.
S700, collecting excrement of the new weaned newborn rabbit once every hour, checking whether rabbit medium Eimeria oocysts exist in the excrement, and recording the time interval from infection of the new weaned newborn rabbit to the first time of checking the rabbit medium Eimeria oocysts in the excrement of the new weaned newborn rabbit as a second occult period of the rabbit medium Eimeria oocysts;
and S800, repeating the passage process until the N-time prepatent time of the middle Eimeria oocysts in the rabbit is shortened by 20-26 h compared with the first prepatent time, and taking the middle Eimeria oocysts in the rabbit in the N-time prepatent time as the early-maturing strains of the middle Eimeria.
Specifically, the sporulated oocysts of the rabbit medium-sized eimeria coccidiosis virulent strain are obtained by collecting, separating and purifying rabbit feces, the sporulated oocysts of the rabbit medium-sized eimeria coccidiosis virulent strain infect weaned rabbits, and after multiple passages, the rabbit medium-sized eimeria coccidiosis oocysts with shortened prepatent period are obtained, namely the rabbit medium-sized eimeria coccidiosis precocious strain; compared with the wild epidemic strain of the middle Eimeria tenella, the early-maturing strain of the middle Eimeria tenella prepared by the method is pure and weak in pathogenicity, and can be directly used for preparing vaccines; the preparation method of the middle Eimeria tenella precocious strain in rabbits, disclosed by the invention, is simple and easy to realize.
Based on the method, the medium Eimeria tenella precocious strain for the rabbits is prepared by adopting the preparation method of any one of the medium Eimeria tenella precocious strains for the rabbits.
Based on the medium Eimeria tenella precocious strain in the rabbit, the invention also provides application of the medium Eimeria tenella precocious strain in the rabbit, wherein the medium Eimeria tenella precocious strain in the rabbit is used for preparing the vaccine.
Furthermore, the invention also detects some biological characteristics, safety and stability of the prepared middle Eimeria tenella precocious strain of the rabbit, and the detection method specifically comprises the following steps:
1. detection of biological characteristics:
the rabbit middle Eimeria tenella precocious strain prepared by the invention mainly parasitizes in duodenum, can extend to the middle and rear section of small intestine when serious, and has oocysts in a long oval shape, the average size of 30.30-30.41 multiplied by 18.12-18.21 mu m, the shape index of 1.62-1.70 and the latent period of 72 hours.
2. And (3) safety detection:
the method comprises the following steps: taking 20 SPF rabbits (40-55 days old) with similar body weight, randomly dividing the SPF rabbits into 4 groups, and weighing the SPF rabbits before inoculation, wherein each group comprises 5 SPF rabbits; group 1 (single-dose infected group), group 2 (single-dose re-infected group), and group 3 (10-fold dose infected group) were orally infected with 1X 10 sporulated oocysts of the precocious strain of Eimeria intermedia, respectively, per rabbit3，1×103，10×103A plurality of; group 4 is emptyWhite control group. Within two weeks after inoculation, the weight increment condition, the excrement condition and the death condition of the rabbits of each group are observed and recorded; two weeks after observation, group 2 was re-infected once in the same way and dose; the above items were recorded with continued observation for two weeks after repeated infection.
As a result: the results of the safety tests are shown in tables 1 and 2. The results show that: the main pathogenicity indexes of the medium Eimeria acervulina precocious strain for production are lower, and the safety is better.
Table 1 Medium Eimeria tenella precocious strain safety test results
TABLE 2 safety test for the intermediate Eimeria precocious strain
3. And (3) detecting immunogenicity:
the method comprises the following steps: dividing 30 SPF rabbits with the age of 40-55 days into 6 groups, 5 rabbits in each group, wherein the 1 st, 2 nd, 3 th and 4 th groups are immunization groups, the rabbits in each group are respectively inoculated with 500, 1000 th, 2000 th and 4000 sporulation oocysts of the medium Eimeria tenella precocious strain by mouth, and the 5 th group is a positive control group (non-immune and insect-attacking); group 6 was a negative control group (no immunization, no attack on insects). Groups 6 of rabbits were separately housed under the same environmental conditions. Weighing after 13 days, and orally attacking 1X 10 sporulated oocysts of a virulent strain of Eimeria media by each rabbit5And (3) observing for 5-7 days after insect attack, recording the excrement condition, and respectively collecting and calculating the number of rabbit gram excrement oocysts of each group of the positive control group and the immune group on the 6 th day after insect attack. All rabbits were weighed on day 7, and the weights of the rabbits in each group were counted and the relative weight gain was calculated, as well as the relative oocyst reduction rate, the relative weight gain rate, and the stool condition as evaluation indexes.
As a result: the immunogenicity test results of the middle Eimeria acervulina precocious strain for production are shown in Table 3, and the results show that: middle Eimeria acervulina for productionMature strain four different immunity dose groups test rabbit can tolerate its parent virulent strain 10X 104The sporulated oocyst challenge indicates that the intermediate E.eimeria precocious strain used for production has good immunogenicity.
TABLE 3 immunogenicity test results for the intermediate E.Eimeria precocious strain used in production
4. And (3) stability detection:
4. method for measuring content of 1-type Eimeria acervulina precocious strain with different storage periods
Storing the sporulated oocysts of the medium Eimeria coccidia precocious strain at 2-8 ℃, and measuring the content of the sporulated oocysts by using a Macmester counting plate after storing for 0 month, 3 months, 6 months and 9 months respectively; the average sporulated oocyst amount per ml should be 50.0X 104Plus or minus 10 percent;
4. immunogenicity detection method for different storage periods of medium Eimeria tenella precocious strains
And (3) performing immunogenicity detection on the oocysts of the medium Eimeria tenella precocious strain which are respectively stored at the temperature of 2-8 ℃ for 0, 3, 6 and 9 months. Randomly dividing 15 SPF rabbits with the age of 40-55 days into 3 groups of 5 rabbits, and recording the weight of each group of rabbits. Wherein the group 1 is an immunization group, and 1000 early-maturing strain sporulated oocysts are respectively inoculated to each rabbit orally; group 2 was a positive control group (no immunization, no attack on insects) and group 3 was a negative control group (no immunization, no attack on insects). Groups of 3 rabbits were separately housed under the same conditions. On day 13 after immunization, weighing, and orally attacking the middle Eimeria species virulent strain 1X 10 by each rabbit5Sporulated oocysts. And observing for 5-7 days after the challenge, recording the fecal condition, and respectively collecting and calculating the number of oocysts of rabbit gram feces of each group of the positive control group and the immune group on the 6 th day after the challenge. Weighing all rabbits on day 7, and counting rabbit bodies of each groupWeighing and calculating relative weight gain, and taking relative oocyst reduction rate, relative weight gain rate and feces condition as evaluation indexes
4. Measurement results of different storage periods of medium Eimeria acervulina precocious strain
The content measurement results of different retention periods of the medium Eimeria precocious strain are shown in Table 4. From the determination results in Table 4, it can be seen that the middle Eimeria tenella precocious strain is stored at 2-8 deg.C, and after 0 month, 3 months, 6 months and 9 months, respectively, sampling and counting are performed, and the oocyst content is equal to the sporulated oocyst content of 50.0 × 10 per ml4Requirement of +/-10%.
TABLE 4 stability test oocyst content determination results
4. 4, determination results of immunogenicity of different storage periods of the precocious strain of the Guangxi strain of the medium Eimeria:
the immunogenicity measurement results of different retention periods of the precocious strain of the medium Eimeria guangxi strain are shown in tables 5-1 to 5-4:
table 5-1 results of immunogenicity test on early-maturing strain of Eimeria species in Guangxi after 0-month preservation
Table 5-2 results of immunogenicity test on early-maturing strain of Eimeria species Coccida in Guangxi after 3 months of preservation
Table 5-3 results of immunogenicity test on early-maturing strain of Eimeria species Coccidia in Guangxi strain stored for 6 months
Table 5-4 results of immunogenicity test on early-maturing strain of Eimeria species Coccidia in Guangxi strain stored for 9 months
The results show that: the results in tables 5-1-5-4 show that the insect strain has no obvious difference in immunogenicity from the insect strain stored for 0 month after being stored for 3 months, 6 months and 9 months at the temperature of 2-8 ℃, and has good immunogenicity. In conclusion, the shelf life of the precocious strain of Eimeria medicinalis was determined to be 9 months.
In summary, the invention provides a medium Eimeria tenella precocious strain in rabbits, a preparation method and an application thereof, wherein the method comprises the following steps: collecting, separating and purifying rabbit feces to obtain sporulated oocysts of a rabbit medium Eimeria strain, infecting the sporulated oocysts of the rabbit medium Eimeria strain with weaned rabbits, and carrying out multiple passages to obtain the rabbit medium Eimeria oocysts with shortened latent period in the Nth generation, namely the rabbit medium Eimeria strain early; compared with the wild epidemic strain of the middle Eimeria tenella, the early-maturing strain of the middle Eimeria tenella prepared by the method is pure and weak in pathogenicity, and can be directly used for preparing vaccines; the preparation method of the middle Eimeria tenella precocious strain in rabbits, disclosed by the invention, is simple and easy to realize.
It is to be understood that the invention is not limited to the examples described above, but that modifications and variations are possible to those skilled in the art in light of the above teachings, and that, for example, all such modifications and variations are intended to be included within the scope of the invention as defined in the appended claims.
Claims (4)
1. A preparation method of a medium Eimeria tenella precocious strain in a rabbit is characterized by comprising the following steps:
A. obtaining mixed rabbit coccidium oocysts in rabbit manure by a coccidium separation method;
B. transferring the mixed rabbit coccidian oocysts to a plate filled with a culture solution, and incubating for 20-36 h to obtain the mixed rabbit coccidian sporulated oocysts;
C. carrying out coccidium purification treatment on the sporulated oocysts of the mixed species of rabbit coccidia to obtain a rabbit medium-sized eimeria coccidia virulent strain, and putting the rabbit medium-sized eimeria coccidia virulent strain into a culture solution to incubate for a preset time to obtain sporulated oocysts of the rabbit medium-sized eimeria coccidia virulent strain;
D. orally infecting a predetermined amount of sporozoized oocysts of a virulent strain of eimeria maxima in a rabbit to weaned young rabbits;
E. collecting the excrement of the weaned newborn rabbits every hour, checking whether rabbit medium Eimeria oocysts exist in the excrement, and recording the time interval from the infection of the weaned newborn rabbits to the first detection of the rabbit medium Eimeria oocysts in the excrement of the weaned newborn rabbits as the first prepatent period of the rabbit medium Eimeria oocysts;
F. culturing the medium eimeria coccidia oocysts collected from the feces of the weaned young rabbits into medium eimeria coccidia sporulated oocysts, infecting the medium eimeria coccidia sporulated oocysts of the rabbits with new weaned young rabbits through mouth, and infecting the new weaned young rabbits with the medium eimeria oocysts of one generation of rabbits, the amount of which is 2-5 times that of the first infected weaned young rabbits; the amount of the Eimeria oocysts in the first generation of rabbits adopted by the weaned newborn rabbit infected every time is 2-5 times of that of the previous time;
G. collecting feces of the new weaned newborn rabbit every hour, checking whether rabbit medium Eimeria oocysts exist in the feces, and recording a time interval from infection of the new weaned newborn rabbit to first checking of the rabbit medium Eimeria oocysts in the feces of the new weaned newborn rabbit as a second prepatent period of the rabbit medium Eimeria oocysts;
H. repeating the passage process until the N-time latent period time of the rabbit medium-sized Eimeria oocysts is shortened by 20-26 h than the first-time latent period time, and taking the rabbit medium-sized Eimeria oocysts in the N-time latent period as a rabbit medium-sized Eimeria precocious strain;
the coccidian purification treatment in the step C comprises the following steps: separating single rabbit medium-sized Eimeria strain oocysts from sporulated mixed rabbit coccidian oocysts by a single oocyst separation technology, feeding 1 rabbit with each oocyst through mouth, collecting excrement discharged from the oocysts of infected rabbits, separating the oocysts to obtain pure medium-sized Eimeria strain;
the step A specifically comprises the following steps:
a1, mashing the collected granular rabbit dung, adding water, mixing uniformly, and filtering to obtain filtrate;
a2, carrying out centrifugal treatment on the filtrate, discarding the supernatant, and keeping the precipitate;
a3, adding water into the precipitate for multiple times, centrifuging until the supernatant is clear, dissolving the obtained precipitate with saturated salt solution, mixing uniformly, centrifuging again, and collecting the supernatant;
a4, diluting the supernatant with water, centrifuging again, and collecting a precipitate, wherein the precipitate is the mixed rabbit coccidian oocysts;
collecting the excrement of the weaned newborn rabbits every other hour, specifically collecting the excrement of the weaned newborn rabbits every other hour within 5 hours before reaching a stealth period;
the predetermined amount is 5000 to 100000 pieces.
2. The method for preparing the precocious rabbit Eimeria strain according to claim 1, wherein the centrifugation speed is 2000-3000 rpm/min.
3. The method for preparing the precocious Eimeria rabbit as claimed in claim 1, wherein the culture solution is 0.5-3% potassium dichromate solution or 0.5-3% chlorammonia T solution.
4. The method for preparing the precocious rabbit Eimeria strain according to claim 1, wherein the incubation temperature is 25-30 ℃.
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| CN108619498A (en) * | 2018-05-18 | 2018-10-09 | 佛山市正典生物技术有限公司 | A kind of coccidiosis of rabbit vaccine and its application |
| CN109646674B (en) * | 2019-01-25 | 2022-08-09 | 中国农业大学 | Coccidiosis live vaccine and application thereof |
| CN110218809B (en) * | 2019-07-08 | 2022-04-22 | 西北农林科技大学 | Specific amplification primer SP4124 for sporulated oocysts of Eimeria necatrix and PCR (polymerase chain reaction) detection method of specific amplification primer SP4124 |
| CN110780068B (en) * | 2019-11-18 | 2023-07-04 | 四川农业大学 | Application of Eimeria stichopus MIC3 protein |
| CN113528347B (en) * | 2021-06-28 | 2023-04-18 | 佛山市正典生物技术有限公司 | Method for improving sorting success rate of fluorescent Eimeria oocysts and application |
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