CN106754379A - A kind of medium-sized Eimeria Precocious Strains of rabbit and preparation method thereof, application - Google Patents
A kind of medium-sized Eimeria Precocious Strains of rabbit and preparation method thereof, application Download PDFInfo
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Abstract
The invention discloses medium-sized Eimeria Precocious Strains of a kind of rabbit and preparation method thereof, application, methods described includes：By rabbit feces collection and isolate and purify and obtain the medium-sized Eimeria velogen strain Sporulated Oocysts of rabbit, by the medium-sized Eimeria velogen strain Sporulated Oocysts infection weaned newborn rabbit of the rabbit, the medium-sized Eimeria egg capsule of N-Generation rabbit by obtaining latent period shortening after repeatedly passage, the i.e. medium-sized Eimeria Precocious Strains of rabbit；For the medium-sized Eimeria field epidemic strain of rabbit, the medium-sized Eimeria Precocious Strains of rabbit prepared by the present invention are pure and pathogenicity is weaker, can be directly used for preparing vaccine；And the medium-sized Eimeria Precocious Strains preparation method of rabbit disclosed by the invention is simply easily realized.
Description
Technical field
The present invention relates to Eimeria application field, more particularly to a kind of medium-sized Eimeria Precocious Strains of rabbit and
Its preparation method, application.
Background technology
Coccidiosis of rabbit is posted by one or more Eimerias of sporozoa Eucoccida Amy otology Eimeria
A kind of parasitic protozoa disease for causing is born in rabbit hepatic duct and enteron aisle.The disease brings greatly harm to rabbit keeping, seriously
Hinder the development of rabbit keeping, China is classified as it two class animal epidemics, with rabbit pest, rabbit brucellosis, rabbit psoroptic mange together by
Referred to as four big rabbits.It is reported that the rabbit that China there are about 41.9% has infected coccidia, in NORTHWEST CHINA, northeast and southwestern infection rate
Unexpectedly more than 60%.The rabbit of all kinds is all susceptible to the disease, wherein with after wean to 3 monthly ages children rabbit infection and morbidity the most
Seriously, the death rate is up to 40%~70%, is to support disease common during rabbit produces.Medium-sized Eimeria (Eimeria
Media) planted as rabbit coccidia is one of, it is popular fairly common in rabbit cultivation farm, it is the main worm for causing coccidiosis of rabbit
Kind.
The current method for preventing and treating rabbit coccidia is mainly carries out medical treatment using anticoccidial drug, and current anticoccidial drug species is many
Many, prevention effect is uneven, tends to cause the serious consequences such as drug resistance and food-safety problem.No matter is research discovery
It is the poor raiser of condition or management preferably intensive warren, it is ineffective without anticoccidial drug, can be detected in rabbit group
To rabbit coccidia.Developing rabbit coccidial vaccine not only can effectively control coccidiosis of rabbit, and will not produce drug resistance, not have food to pacify
Problem in all directions.At present, forbid being added in feed the cry also more and more higher of coccidiostat, entered using rabbit coccidial vaccine
Row immunoprophylaxis will substitute existing medical treatment, occupy leading position.Illustrate that there is rabbit coccidial vaccine wide market to answer
Use prospect.Rabbit coccidia is obligatory parasitism protozoon, only rabbit is produced pathogenic thus fool proof；The use of the pathogen
Relatively simple, production cost is relatively low, has competitive advantage in market.
But, the pathogenicity of the field epidemic strain of rabbit coccidia is general relatively strong, is not suitable for being directly used in and prepares vaccine, therefore right
It is very necessary to reduce the virulence of rabbit coccidia that it carries out precocious seed selection, and the seed selection of rabbit Eimeria Precocious Strains is rabbit coccidia work epidemic disease
The development of seedling lays the foundation.
Therefore, prior art has yet to be improved and developed.
The content of the invention
In view of above-mentioned the deficiencies in the prior art, it is an object of the invention to provide a kind of medium-sized Eimeria Precocious Strains of rabbit
And preparation method thereof, application, it is intended to the field epidemic strain pathogenicity for solving rabbit coccidia is stronger, is not suitable for being directly used in and prepares vaccine
Problem.
Technical scheme is as follows：
A kind of preparation method of the medium-sized Eimeria Precocious Strains of rabbit, wherein, including step：
A, by coccidia separation method obtain rabbit excrement in hybrid encephalitozoon cuniculi；
B, hybrid encephalitozoon cuniculi is transferred in the plate equipped with nutrient solution, after being incubated 20~36h, obtains hybrid
Rabbit coccidia Sporulated Oocysts；
C, coccidia purification process is carried out to hybrid rabbit coccidia Sporulated Oocysts, obtain the medium-sized Eimeria of rabbit malicious by force
Strain, the medium-sized Eimeria velogen strain of rabbit is put into after being incubated the scheduled time in nutrient solution, obtains the medium-sized Eimeria of rabbit strong
Strain Sporulated Oocysts；
D, by the medium-sized Eimeria velogen strain Sporulated Oocysts peroral infection weaned newborn rabbit of the rabbit of scheduled volume；
E, per collecting once the excrement of the weaned newborn rabbit every other hour, and check in the excrement whether there is the medium-sized Chinese mugwort of rabbit
U.S. ear coccidian oocyst, checking the medium-sized Eimeria of rabbit in the excrement of weaned newborn rabbit to first time from infection weaned newborn rabbit
The time interval of egg capsule is designated as the first time latent period of the medium-sized Eimeria egg capsule of the rabbit；
F, by from the excrement of weaned newborn rabbit collect the medium-sized Eimeria egg capsule of rabbit be trained the medium-sized Amy ear ball of rabbit
Worm Sporulated Oocysts, and by the new weaned newborn rabbit of the medium-sized Eimeria Sporulated Oocysts peroral infection of the rabbit；
G, per collecting once the excrement of the new weaned newborn rabbit every other hour, and check in the excrement and whether have a rabbit
Type Eimeria egg capsule, in rabbit is checked in the excrement of new weaned newborn rabbit to first time from the new weaned newborn rabbit of infection
The time interval of type Eimeria egg capsule is designated as second latent period of the medium-sized Eimeria egg capsule of the rabbit；
H, the above-mentioned succeeding generations of repetition, until the n-th latent period time of the medium-sized Eimeria egg capsule of rabbit is than for the first time
When the latent period time shortens 20~26h, then using the medium-sized Eimeria egg capsule of the rabbit of n-th latent period as the medium-sized Amy ear of rabbit
Coccidia Precocious Strains.
It is preferred that the preparation method of the medium-sized Eimeria Precocious Strains of described rabbit, wherein, the step A is specifically included：
A1, the graininess rabbit excrement of collection is smashed to pieces, and the mixing filtering that adds water, obtain filtrate；
A2, the filtrate is carried out centrifugal treating, abandoning supernatant retains precipitation；
A3, centrifugal treating that the precipitation repeatedly adds water are until supernatant clarification, the precipitation saturated aqueous common salt that will be obtained
Dissolving is mixed, and is centrifuged again, collects supernatant；
A4, by the supernatant dilute with water, be centrifuged again, collect precipitation, it is described precipitation be hybrid rabbit coccidia ovum
Capsule.
It is preferred that the preparation method of the medium-sized Eimeria Precocious Strains of described rabbit, wherein, centrifugal speed is 2000~
3000rpm/min。
It is preferred that the preparation method of the medium-sized Eimeria Precocious Strains of described rabbit, wherein, the nutrient solution is 0.5~
3% potassium bichromate solution or 0.5~3% chlorine ammonia T solution.
It is preferred that the preparation method of the medium-sized Eimeria Precocious Strains of described rabbit, wherein, incubation temperature is 25~30
℃。
It is preferred that the preparation method of the medium-sized Eimeria Precocious Strains of described rabbit, wherein, scheduled volume in the step D
It is 5000~100000.
A kind of medium-sized Eimeria Precocious Strains of rabbit, wherein, it is precocious using the medium-sized Eimeria of above-mentioned any one rabbit
The preparation method of strain is prepared from.
A kind of application of the medium-sized Eimeria Precocious Strains of rabbit, wherein, the medium-sized Eimeria Precocious Strains of above-mentioned rabbit are used
In preparing vaccine.
Beneficial effect：The present invention provides a kind of medium-sized Eimeria Precocious Strains of rabbit and preparation method thereof, application, the side
Method includes：By rabbit feces collection and isolate and purify and obtain the medium-sized Eimeria velogen strain Sporulated Oocysts of rabbit, by the rabbit
Medium-sized Eimeria velogen strain Sporulated Oocysts infect weaned newborn rabbit, the N by obtaining latent period shortening after repeatedly passage
For the medium-sized Eimeria egg capsule of rabbit, the i.e. medium-sized Eimeria Precocious Strains of rabbit；Flowed relative to the medium-sized Eimeria field of rabbit
For row strain, the medium-sized Eimeria Precocious Strains of rabbit prepared by the present invention are pure and pathogenicity is weaker, can be directly used for preparing epidemic disease
Seedling；And the medium-sized Eimeria Precocious Strains preparation method of rabbit disclosed by the invention is simply easily realized.
Brief description of the drawings
Fig. 1 is a kind of flow chart of the medium-sized good embodiment of Eimeria Precocious Strains preparation method of rabbit of the invention.
Specific embodiment
The present invention provides a kind of medium-sized Eimeria Precocious Strains of rabbit and preparation method thereof, application, to make mesh of the invention
, technical scheme and effect it is clearer, clear and definite, referring to the drawings and give an actual example that the present invention is described in more detail.Should
Work as understanding, the specific embodiments described herein are merely illustrative of the present invention, be not intended to limit the present invention.
Fig. 1 is referred to, Fig. 1 is a kind of medium-sized Eimeria Precocious Strains of rabbit of the invention and preparation method thereof preferred embodiment
Flow chart, as illustrated, it includes step：
S100, by coccidia separation method obtain rabbit excrement in hybrid encephalitozoon cuniculi；
Specifically, the step S100 is specifically included：
S110, the graininess rabbit excrement of collection is smashed to pieces, and the mixing filtering that adds water, obtain filtrate；
Specifically, the rabbit excrement of the mixing street strain containing the medium-sized Eimeria of rabbit that will be collected from field is smashed to pieces, and
Plus appropriate distilled water or purified water, mix；Then with 100 mesh, the screen filtration of 300 mesh, excrement slag is reclaimed, then add suitable quantity of water to mix
Even filtering, until the most of egg capsule in excrement slag has all been filtered out；
S120, the filtrate is carried out centrifugal treating, abandoning supernatant retains precipitation；
Specifically, the filtrate that will have been filtered is centrifuged under the rotating speed of 2000~3000rpm/min, and abandoning supernatant is obtained
Precipitation containing coccidian oocyst；
S130, centrifugal treating that the precipitation repeatedly adds water are until supernatant clarification, the precipitation saturated common salt that will be obtained
Water dissolves are mixed, and are centrifuged again, collect supernatant；
Specifically, repeated centrifugation step, until the supernatant clarification after centrifugation；The saline solution of the precipitation saturation that will be obtained
(or Adlerika of equal buoyancy) dissolving is mixed, is centrifuged by 2000~3000rpm/min, collects supernatant, until
Most coccidian oocyst has all floated collection in precipitation；
S140, by the supernatant dilute with water, be centrifuged again, collect precipitation, it is described precipitation be hybrid rabbit coccidia
Egg capsule.
Specifically, add 4 in 1 part of saturated aqueous common salt~ratio of 10 parts of distilled water or purified water, coccidia is contained described in dilution
The supernatant of egg capsule, is centrifuged through 2000~3000rpm/min, collects precipitation, and the precipitation is hybrid encephalitozoon cuniculi.
S200, hybrid encephalitozoon cuniculi is transferred in the plate equipped with nutrient solution, after being incubated 20~36h, is mixed
Close kind of a rabbit coccidia Sporulated Oocysts；
Specifically, the nutrient solution is 0.5~3% potassium bichromate solution or 0.5~3% chlorine ammonia T solution, is incubated temperature
Spend is 25~30 DEG C；It is preferred that hybrid encephalitozoon cuniculi is transferred in the plate equipped with nutrient solution, incubation temperature is 27
DEG C, incubation time is 28h, obtains the stronger hybrid rabbit coccidia Sporulated Oocysts of activity.
S300, coccidia purification process is carried out to hybrid rabbit coccidia Sporulated Oocysts, obtain the medium-sized Eimeria of rabbit strong
Strain, the medium-sized Eimeria velogen strain of rabbit is put into after being incubated the scheduled time in nutrient solution, obtains the medium-sized Eimeria of rabbit
Velogen strain Sporulated Oocysts；
Specifically, that single rabbit is isolated from the hybrid encephalitozoon cuniculi of Sporulated by single oocyst isolation technique is medium-sized
Eimeria velogen strain egg capsule, each egg capsule oral administration is fed for 1 rabbit, and each rabbit needs strict isolated rearing, and institute is useful
Tool, feed, equal strict sterilization of drinking water；The 2nd day after infection, daily excrement inspection is multiple, to decontaminate；Opened after collecting 96 hours respectively
The excrement of beginning discharge egg capsule, separates egg capsule, and expanding propagation once, obtains the medium-sized Eimeria of purebred rabbit after Sporulated, is
Medium-sized Eimeria velogen strain, Sporulated maturation is allowed to through culture, obtains the medium-sized Eimeria velogen strain Sporulated ovum of rabbit
Capsule.
S400, by the medium-sized Eimeria velogen strain Sporulated Oocysts peroral infection weaned newborn rabbit of the rabbit of scheduled volume；
Specifically, 5000~100000 medium-sized Eimeria velogen strain Sporulated Oocysts peroral infections of rabbit can be broken
Milk son rabbit；
S500, per collecting once the excrement of the weaned newborn rabbit every other hour, and check in the excrement and whether have a rabbit
Type Eimeria egg capsule, checking the medium-sized Amy ear of rabbit in the excrement of weaned newborn rabbit to first time from infection weaned newborn rabbit
The time interval of coccidian oocyst is designated as the first time latent period of the medium-sized Eimeria egg capsule of the rabbit；
It is preferred that understanding that the first time latent period of the medium-sized Eimeria egg capsule of rabbit is 94~98h according to experiment；In order to carry
High workload efficiency, it is preferable that the present invention can start to receive within preceding 5 hours that the medium-sized Eimeria egg capsule of rabbit reaches latent period
Collect the excrement of weaned newborn rabbit, while whether having the medium-sized Eimeria egg capsule of rabbit, the rabbit in the excrement in checking the excrement
Medium-sized Eimeria egg capsule is the medium-sized Eimeria egg capsule of generation rabbit；Young in wean from infection weaned newborn rabbit to first time
The time interval that the medium-sized Eimeria egg capsule of rabbit is checked in the excrement of rabbit is designated as the medium-sized Eimeria egg capsule of the rabbit
First time latent period；
S600, by from the excrement of weaned newborn rabbit collect the medium-sized Eimeria egg capsule of rabbit be trained the medium-sized Amy ear of rabbit
Coccidia Sporulated Oocysts, and by the new weaned newborn rabbit of the medium-sized Eimeria Sporulated Oocysts peroral infection of the rabbit；
Specifically, the medium-sized Eimeria egg capsule of generation rabbit is cultivated in nutrient solution, in obtaining the generation rabbit of Sporulated
Type Eimeria egg capsule, using the medium-sized Eimeria egg capsule of the generation rabbit of 2~5 times of first amount of subinfection weaned newborn rabbit
The weaned newborn rabbit for going infection new；The amount of the medium-sized Eimeria egg capsule of generation rabbit that each subinfection weaned newborn rabbit is used afterwards
It is last 2~5 times；The medium-sized Eimeria velogen strain Sporulated ovum of rabbit that for example the first subinfection weaned newborn rabbit is used
The amount of capsule is 5000~100000, then the medium-sized Eimeria egg capsule of generation rabbit that the new weaned newborn rabbit of the second subinfection is used
Amount be 10000~500000, the like.
S700, per collecting once the excrement of the new weaned newborn rabbit every other hour, and check in the excrement whether have
The medium-sized Eimeria egg capsule of rabbit, being checked in the excrement of new weaned newborn rabbit to first time from infecting new weaned newborn rabbit
The time interval of the medium-sized Eimeria egg capsule of rabbit is designated as second latent period of the medium-sized Eimeria egg capsule of the rabbit；
S800, the above-mentioned succeeding generations of repetition, first is compared until the n-th latent period time of the medium-sized Eimeria egg capsule of rabbit
When the secondary latent period time shortens 20~26h, then using the medium-sized Eimeria egg capsule of the rabbit of n-th latent period as the medium-sized Amy of rabbit
Ear coccidia Precocious Strains.
Specifically, the present invention passes through rabbit feces collection and isolates and purifies to obtain the medium-sized Eimeria velogen strain Sporulated of rabbit
Egg capsule, the medium-sized Eimeria velogen strain Sporulated Oocysts infection weaned newborn rabbit of the rabbit is dived by after repeatedly passage
The medium-sized Eimeria egg capsule of N-Generation rabbit that the hidden phase shortens, the i.e. medium-sized Eimeria Precocious Strains of rabbit；Relative to the medium-sized Amy of rabbit
For the epidemic strain of ear coccidia field, the medium-sized Eimeria Precocious Strains of rabbit prepared by the present invention are pure and pathogenicity is weaker, can be straight
Connect for preparing vaccine；And the medium-sized Eimeria Precocious Strains preparation method of rabbit disclosed by the invention is simply easily realized.
Based on the above method, a kind of medium-sized Eimeria Precocious Strains of rabbit of the invention, wherein, using above-mentioned any one rabbit
The preparation method of medium-sized Eimeria Precocious Strains is prepared from.
Based on a kind of above-mentioned medium-sized Eimeria Precocious Strains of rabbit, the present invention also provides a kind of rabbit medium-sized Eimeria morning
The application of ripe strain, wherein, the medium-sized Eimeria Precocious Strains of above-mentioned rabbit are used to prepare vaccine.
Further, rabbit medium-sized Eimeria Precocious Strains of the present invention also to preparing have done some biological natures and safety
The detection of type, stability, it is specific as follows：
1st, the detection of biological property：
The medium-sized Eimeria Precocious Strains main parasitic of rabbit prepared by the present invention may extend to small in duodenum, when serious
Intestines posterior segment, egg capsule is in oval long, and mean size is 30.30~30.41 × 18.12~18.21 μm, and shape index is 1.62
~1.70, latent period is 72h.
2nd, safety detection：
Method：40~55 close age in days SPF rabbits 20 of body weight are taken, 4 groups are randomly divided into, every group 5, is claimed before inoculation
Weight；1st (single dose infected group), 2 (single dose superinfection groups), 3 (10 multiple dose infected groups) group distinguishes oral infection per rabbit
Medium-sized Eimeria Precocious Strains Sporulated Oocysts 1 × 103, 1 × 103, 10 × 103It is individual；4th group is blank control group.After inoculation
Within two weeks, the Gain weight of observed and recorded each group rabbit, excrement situation and death condition；After observing two weeks, the 2nd group with phase
With method and dosage superinfection once；Continue the above-mentioned project of observed and recorded two weeks after superinfection.
As a result：Safety detection the results are shown in Table 1 and table 2.Result shows：Production is main with medium-sized Eimeria Precocious Strains
Pathogenicity index is relatively low, with preferable security.
The medium-sized Eimeria Precocious Strains safety detection result of table 1
The medium-sized Eimeria Precocious Strains safety detection superinfection result of table 2
3rd, immunogenicity detection：
Method：40~55 age in days SPF rabbits 30 are taken, is divided into 6 groups, every group 5, wherein the 1st, 2,3,4 group is immune group, often
Group rabbit distinguishes the Sporulated Oocysts 500,1000,2000 and 4000 of the medium-sized Eimeria Precocious Strains of per oral inoculation, and the 5th group is
Positive controls (are not immunized, attack worm)；6th group is negative control group (not being immunized, do not attack worm).By 6 groups of rabbits respectively in identical ring
Isolated rearing under the conditions of border.Weighed after 13 days, medium-sized Eimeria velogen strain Sporulated Oocysts 1 × 10 are orally attacked per rabbit5It is individual,
Attack after worm and observe 5~7, record its excrement situation, and collect respectively, calculate and attack after worm the 6th day positive controls and immune
Group each group rabbit gram excrement egg sac number.All rabbits were weighed in 7th, count each group rabbit body weight and calculate the relative weight gain, and relatively
Egg capsule slip, body weight increase rate and excrement situation are used as evaluation index.
As a result：The medium-sized Eimeria Precocious Strains immunogenicity testing result of production as shown in table 3, as a result shows：It is raw
Produce and be resistant to its parent's velogen strain 10 × 10 with four different immunizing dose groups experiment rabbits of medium-sized Eimeria Precocious Strains4It is individual
The attack of Sporulated Oocysts, illustrates that production has good immunogenicity with medium-sized Eimeria Precocious Strains.
Table 3 is produced with medium-sized Eimeria Precocious Strains immunogenicity testing result
4th, Detection of Stability：
4th, 1 medium-sized Eimeria Precocious Strains difference storage life content assaying method
Medium-sized Eimeria Precocious Strains Sporulated Oocysts are placed in 2~8 DEG C of preservations, respectively at preserve 0 month, 3 months,
6 months, after 9 months, with Cecil McMaster tally survey Sporulated Oocysts content；Average every milliliter Sporulated Oocysts amount should be
50.0×104± 10%；
4th, 2 medium-sized Eimeria Precocious Strains difference storage life immunogenicity detection method
Taking the medium-sized Eimeria Precocious Strains egg capsule being placed under the conditions of 2~8 DEG C after preserving 0,3,6,9 months respectively is carried out
Immunogenicity is detected.15 40~55 age in days SPF rabbits are randomly divided into 3 groups, every group 5, each group rabbit body weight is recorded.Wherein
1 group is immune group, and 1000 Precocious Strains Sporulated Oocysts of oral vaccination are distinguished per rabbit；2nd group be positive controls (be not immunized,
Attack worm), the 3rd group is negative control group (not being immunized, do not attack worm).By the isolated rearing under the same conditions respectively of 3 groups of rabbits.After immune
13rd, weigh, every rabbit orally attacks medium-sized Eimeria parent velogen strain 1 × 105Individual Sporulated Oocysts.Seen after attacking poison
Examine 5~7, record its excrement situation, and collect respectively, calculate and attack the 6th day positive controls and immune group each group rabbit after worm
Gram excrement egg sac number.All rabbits were weighed in 7th, statistics each group rabbit body weight simultaneously calculate the relative weight gain, with relative egg capsule slip,
Body weight increase rate and excrement situation are used as evaluation index
4th, 3 medium-sized Eimeria Precocious Strains difference storage life assay result
Medium-sized Eimeria Precocious Strains difference storage life Content measurement result is shown in Table 4.Knowable to the measurement result of table 4
Medium-sized Eimeria Precocious Strains are preserved under the conditions of 2~8 DEG C, and respectively at behind 0 month, 3 months, 6 months, 9 months, sampling is counted
Number, it is 50.0 × 10 that egg capsule content meets average every milliliter Sporulated Oocysts amount4The requirement of ± 10%.
The stability test egg capsule assay result of table 4
4th, 4 medium-sized Eimeria Guangxi Strain Precocious Strains difference storage life immunogenicity determinations：
Medium-sized Eimeria Guangxi Strain Precocious Strains difference storage life immunogenicity determinations such as 5~1 to table of table 5~4
It is shown：
The medium-sized Eimeria Guangxi Strain Precocious Strains of table 5~1 preserve 0 month Study On Immunogenicity result
The medium-sized Eimeria Guangxi Strain Precocious Strains of table 5~2 preserve 3 months Study On Immunogenicity results
The medium-sized Eimeria Guangxi Strain Precocious Strains of table 5~3 preserve 6 months Study On Immunogenicity results
The medium-sized Eimeria Guangxi Strain Precocious Strains of table 5~4 preserve 9 months Study On Immunogenicity results
Result shows：By the strain of the result of table 5~1~5~4 visible worm preserved at 2~8 DEG C 3 months, 6 months, after 9 months,
With 0 month worm strain immunogenicity of preservation without significant difference, good immunogenicity is respectively provided with.In sum, medium-sized Amy is determined
The storage life of ear coccidia Guangxi Strain Precocious Strains is 9 months.
In sum, the present invention provides a kind of medium-sized Eimeria Precocious Strains of rabbit and preparation method thereof, application, the side
Method includes：By rabbit feces collection and isolate and purify and obtain the medium-sized Eimeria velogen strain Sporulated Oocysts of rabbit, by the rabbit
Medium-sized Eimeria velogen strain Sporulated Oocysts infect weaned newborn rabbit, the N by obtaining latent period shortening after repeatedly passage
For the medium-sized Eimeria egg capsule of rabbit, the i.e. medium-sized Eimeria Precocious Strains of rabbit；Flowed relative to the medium-sized Eimeria field of rabbit
For row strain, the medium-sized Eimeria Precocious Strains of rabbit prepared by the present invention are pure and pathogenicity is weaker, can be directly used for preparing epidemic disease
Seedling；And the medium-sized Eimeria Precocious Strains preparation method of rabbit disclosed by the invention is simply easily realized.
It should be appreciated that application of the invention is not limited to above-mentioned citing, and for those of ordinary skills, can
To be improved according to the above description or converted, for example, all these modifications and variations should all belong to right appended by the present invention wanting
The protection domain asked.
Claims (8)
1. the preparation method of the medium-sized Eimeria Precocious Strains of a kind of rabbit, it is characterised in that including step：
A, by coccidia separation method obtain rabbit excrement in hybrid encephalitozoon cuniculi；
B, hybrid encephalitozoon cuniculi is transferred in the plate equipped with nutrient solution, after being incubated 20~36h, obtains hybrid rabbit ball
Worm Sporulated Oocysts；
C, coccidia purification process is carried out to hybrid rabbit coccidia Sporulated Oocysts, obtain the medium-sized Eimeria velogen strain of rabbit, will
The medium-sized Eimeria velogen strain of rabbit is put into after being incubated the scheduled time in nutrient solution, obtains the medium-sized Eimeria velogen strain spore of rabbit
Sonization egg capsule；
D, by the medium-sized Eimeria velogen strain Sporulated Oocysts peroral infection weaned newborn rabbit of the rabbit of scheduled volume；
E, per collecting once the excrement of the weaned newborn rabbit every other hour, and check in the excrement whether there is the medium-sized Amy ear of rabbit
Coccidian oocyst, checking the medium-sized Eimeria egg capsule of rabbit in the excrement of weaned newborn rabbit to first time from infection weaned newborn rabbit
Time interval be designated as the first time latent period of the medium-sized Eimeria egg capsule of the rabbit；
F, by from the excrement of weaned newborn rabbit collect the medium-sized Eimeria egg capsule of rabbit be trained the medium-sized Eimeria spore of rabbit
Sonization egg capsule, and by the new weaned newborn rabbit of the medium-sized Eimeria Sporulated Oocysts peroral infection of the rabbit；
G, per collecting once the excrement of the new weaned newborn rabbit every other hour, and check in the excrement whether there is the medium-sized Chinese mugwort of rabbit
U.S. ear coccidian oocyst, checking the medium-sized Chinese mugwort of rabbit in the excrement of new weaned newborn rabbit to first time from infecting new weaned newborn rabbit
The time interval of U.S. ear coccidian oocyst is designated as second latent period of the medium-sized Eimeria egg capsule of the rabbit；
H, the above-mentioned succeeding generations of repetition, until the n-th latent period time of the medium-sized Eimeria egg capsule of rabbit than hiding for the first time
When time phase shortens 20~26h, then using the medium-sized Eimeria egg capsule of the rabbit of n-th latent period as the medium-sized Eimeria of rabbit
Precocious Strains.
2. the preparation method of the medium-sized Eimeria Precocious Strains of rabbit according to claim 1, it is characterised in that the step
A is specifically included：
A1, the graininess rabbit excrement of collection is smashed to pieces, and the mixing filtering that adds water, obtain filtrate；
A2, the filtrate is carried out centrifugal treating, abandoning supernatant retains precipitation；
A3, centrifugal treating that the precipitation repeatedly adds water are until supernatant clarification, the precipitation that will be obtained saturated common salt water dissolves
Mix, be centrifuged again, collect supernatant；
A4, by the supernatant dilute with water, be centrifuged again, collect precipitation, it is described precipitation be hybrid encephalitozoon cuniculi.
3. the preparation method of the medium-sized Eimeria Precocious Strains of rabbit according to claim 2, it is characterised in that centrifugal speed
It is 2000~3000rpm/min.
4. the preparation method of the medium-sized Eimeria Precocious Strains of rabbit according to claim 1, it is characterised in that the culture
Liquid is 0.5~3% potassium bichromate solution or 0.5~3% chlorine ammonia T solution.
5. the preparation method of the medium-sized Eimeria Precocious Strains of rabbit according to claim 1, it is characterised in that incubation temperature
It is 25~30 DEG C.
6. the preparation method of the medium-sized Eimeria Precocious Strains of rabbit according to claim 1, it is characterised in that described predetermined
Measure is 5000~100000.
7. medium-sized Eimeria Precocious Strains of a kind of rabbit, it is characterised in that prepared using described in 1~6 any one claim
Method is prepared from.
8. the application of the medium-sized Eimeria Precocious Strains of a kind of rabbit, it is characterised in that by the medium-sized Amy of rabbit described in claim 7
Ear coccidia Precocious Strains are used to prepare vaccine.
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| 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 |
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| CN113528347A (en) * | 2021-06-28 | 2021-10-22 | 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 |
| 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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