CN101045159A - Method for preparing vaccine to chicken coccidiosis - Google Patents
Method for preparing vaccine to chicken coccidiosis Download PDFInfo
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- CN101045159A CN101045159A CN 200710055622 CN200710055622A CN101045159A CN 101045159 A CN101045159 A CN 101045159A CN 200710055622 CN200710055622 CN 200710055622 CN 200710055622 A CN200710055622 A CN 200710055622A CN 101045159 A CN101045159 A CN 101045159A
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Abstract
A process for preparing the vaccine of chicken coccidiosis includes such steps as separating the oogonia from chicken coccidian, culturing in the solution of potassium bichromate until more than 90% of oogonia become spores, storing in refrigerator at 4 deg.C, adding distilled water, centrifugal removal of potassium bichromate solution, counting, ice bath, and ultrasonic weakening.
Description
Technical field
The present invention relates to a kind of preparation method of vaccine to chicken coccidiosis, is the method that a kind of ultrasound wave causes weak strong malicious Eimeria Tenella egg capsule.
Background technology
Chicken coccidiosis is that a kind of distribution is very wide, the intestinal parasitic protozoa disease that hazardness is very big, and it mainly is that it is caused by nine kinds of coccidiosiss of Eimeria because Eimeria parasitizes the disease that enterocyte causes.Cause of disease is subordinate to the multiple door in top, the Eimeria protozoon of Eucoccida, its life cycle are divided into schizogony, gametogonia and Sporogony three phases, and preceding two stages carry out in the chicken body, the latter half carries out external, but the egg capsule peroral infection of sporeization therebetween.Aborning, chicken coccidiosis not only causes the large quantities of death of chickling, and even more serious is obviously to reduce the fertility performance of chicken, causes great economic loss, especially all the more so to the intensification poultry husbandry.This disease is a feature with hemorrhagic enteritis, dysentery, chickling high incidence and mortality rate, and the chickling infection rate is up to more than 80% sometimes, and mortality rate can reach 20%-50%, becomes one of important diseases of harm poultry husbandry.
At present, the control of this disease is mainly contained drug preventing and controlling method and vaccine control method, wherein the medical treatment coccidiosis mainly is by mix the effect that the anticoccidial drug that mixes prevention or therapeutic dose reaches the control coccidiosis in feedstuff or drinking-water.Coccidiosis medicine commonly used has: sulfaquinoxaline (SQ4), sulfadimidine (SM2), ammonia third beautiful jade, the spirit of ball dysentery, Robenidine, Diverdine, nicarbazine, the spirit of ball dysentery, coccidiosis powder, Australia's chlorine halofuginone hydrobromide and quinolones etc., but the life-time service medical treatment can cause drug resistance strain to produce, and control costs an arm and a leg.Along with progress of science and technology, use vaccine to prevent the generation of chicken coccidiosis just to become not only science but also the economic method of preventing and treating, have bright development prospect.The vaccine to chicken coccidiosis of having developed both at home and abroad has virulent strain worm Seedling, low virulent strain worm Seedling and coccidiosis gene engineering vaccine etc. at present.Wherein, virulent strain worm Seedling is that the mixing egg capsule coccidial vaccine Coccivac of dMitChel animal health company is representative with U.S. Dorn, it contains the mixing egg capsule of 8 kinds of coccidiosiss for chicken inoculation at the 5-10 age in days by drinking-water or feedstuff on a small quantity, breed the back by the chicken group filial generation egg capsule is sowed on the bedding and padding, make chicken group repeated infection and continuous enhancing immunity.But this strong poisonous insect Seedling comes out and is not widely used over more than 40 year, and main cause is that it exists two big defectives: the one, and the various coccidiosis strains that are present in the worm Seedling all are fully pathogenic; The 2nd, the egg capsule skewness owing to sneaking in drinking-water or the feedstuff causes the chicken group shot to go into the inconsistent of egg capsule amount, and the chicken that the result has infects too light, is not enough to produce immunity, and the chicken that has is taken in egg capsule and too much causes morbidity.
Because the virulent strain worm Seedling of living without a little less than causing, if the immunity amount is inaccurate or the quantity of immunity inoculation egg capsule after chicken is gone down to posterity is more and more, might cause the outburst of coccidiosis, so attenuated vaccine arises at the historic moment.The weak method that causes to the virulent strain coccidiosis at present mainly contains three kinds: physics and chemistry causes weak method, Embryo Gallus domesticus goes down to posterity and causes weak method and precocious egg capsule selection-breeding method.
Physics and chemistry cause weak method be by egg capsule is cooled off, physical treatment such as heating, ultrasonic, ionizing radiation, and make it attenuation.
Embryo Gallus domesticus goes down to posterity and causes weak method is the coccidiosis zygoblast to be inoculated on the chick chorioallantoic membrane cultivate; continuous passage repeatedly back obtains low to chicken pathogenic; but still keep well immunogenic " Embryo Gallus domesticus is suitable for strain "; its pathogenicity weaken be since the 2nd generation schizont from original proper mucous membrane then all colonize in the epithelial cause of enteraden; do not cause bleeding; little to tissue injury, with the chicken of weak worm strain immunity with greetings the attack of virulent strain there is good protective action.But Embryo Gallus domesticus goes down to posterity and causes weak method four big shortcomings are arranged: 1. attenuated character can be unstable relatively, and virulence is returned strong fast when transferring chicken to and go down to posterity; 2. the egg capsule of worm strain is relatively more difficult a little less than causing by Embryo Gallus domesticus production shiploads of merchandiseization; 3. increase with the Embryo Gallus domesticus passage number, the immunogenicity of polypide will reduce or lose, and in fact is difficult to obtain the immunity that both keeps good, makes the more weak best of breed of virulence again; Though 4. topmost limitation is the widest popular huge, heap type and precocious Eimeria and can grows in Embryo Gallus domesticus, the egg capsule undergrowth, therefore by Embryo Gallus domesticus go down to posterity productions " full price " cause a little less than the worm Seedling just be restricted.
Precocious egg capsule selection-breeding method is to cause weak worm strain according to Eimeria tenella, weakening of its pathogenicity is because the 2nd not differentiation or become very little mechanism when maturation fully of generation schizont, the worm strain is made coccidial vaccine and can be induced chicken body generation immunity a little less than utilizing these to cause, and simultaneously chicken is kept harmless.This worm Seedling can prevent morbidity, rather than the opposing infection, although still have egg capsule to discharge with chicken group behind the Seedling, chicken group's coccidiosis obtains prevention and control really.Oral coccidial vaccine as being developed and put into production by professor Duan Jiashu of Beijing Agricultural College adopts the method to make.In addition, people are making great efforts exploitation coccidiosis gene vaccine at present, attempt coming immune chicken in the hope of protection with the antigen of purification.Use monoclonal antibody that hybridoma technology produces anticoccidial as Garker (1982) and identify that coccidiosis antigen achieves success, developed the subunit seedling of control coccidiosis of domestic fowls; Danforth (1986) succeeds in developing coccidiosis reorganization Seedling first, promptly from escherichia coli, extracted the antigen (called after 5401) of Eimeria tenella, with attacking with egg capsule of the same race behind the 5401 immune chickens, be better than matched group significantly at aspects such as body weight gains, feed conversion rates, with heap type Eimeria no cross reaction.Utilize technique for gene engineering that the understanding of fundamental property of coccidium infection and immunity has been obtained progress, but far away, change the practical stage over to need time from the target of making gene engineering vaccine.
Summary of the invention
The object of the present invention is to provide a kind of preparation method of vaccine to chicken coccidiosis, for overcoming the technological deficiency that existing virulent strain chicken coccidiosis causes weak method, its safe in utilization, easy and simple to handle, effective chicken coccidiosis ultrasound wave causes weak method.
Technical scheme of the present invention is achieved in that a kind of preparation method of vaccine to chicken coccidiosis, it is characterized in that: be to adopt ultrasound wave to cause weak method, its concrete step is: (1) at first carries out the egg capsule sporeization, the amplification chicken coccidial oocyst, isolated egg capsule is put into the culture dish of 2.5% the potassium bichromate solution that is equipped with 10 times of volumes, the about 0.6cm of solution deep, putting into temperature is 28～30 ℃ of calorstats, cultivates 5 days; Gently stir 4～5 time culture fluid every day between culture period, when the egg capsule more than 90% is finished spore, stops to cultivate, and places 4 ℃ of refrigerator storage standby; (2) secondly carry out ultrasound wave and cause weak egg capsule, with 3000-5000 rev/min of the spore egg capsule adding distil water of above collection is centrifugal remove potassium bichromate solution 2-3 time after, place the 100ml small beaker, after again small beaker being put into the 1000ml large beaker that rubble ice is housed behind the counting and being carried out ice bath, place on the ultrasonoscope sound proof box lifting platform, after making lifting platform rise to horn immersion solution 10mm, power is transferred to 200 watts of minimum positions, adjusting ultrasonic time is 3 seconds, the adjusting play time is that ultrasonic time is 8-12 minute a little less than causing in 3 seconds.(3) after the ultrasonic end,, 100ml coccidian oocyst liquid is added 2% potassium bichromate solution 100ml if do not use immediately, 1% sodium azide solution 1.5ml, it is standby to place 4 ℃ of refrigerators; The distilled water that before using coccidian oocyst solution is added 10 times of volumes, with 4000 rev/mins centrifugal 3 times, each 3 minutes, after removing potassium dichromate, sodium azide solution, make oral vaccine.Carry out the egg capsule counting with the blood cell counting plate method, feed to chickling in the drip mode by 4000 spore egg capsules of every chicken, first immunisation 7 ages in days, secondary immunity are 14 ages in days.Can not feed anticoccidial drug to chicken in 2 days before and after the oral immunity.
Good effect of the present invention is: cause the weak equal no pathogenicity of coccidiosis, the inoculation chickling is safe and reliable, and has good immunogenicity; Cause weak method and be directly, in official hour, can cause weak multiple chicken coccidiosis simultaneously,, and keep good immunogenicity as tender, heap type, huge, murder by poisoning and precocious Eimeria etc.; The simple and easy practicality of its method is convenient to the rural area and is applied; And reduced use cost.
The present invention will be further described below in conjunction with the test example:
Strong malicious coccidiosis causes weak test:
Chicken coccidial oocyst cause weak process: gather the coccidiosis chicken manure just → collect mix egg capsule → propagation Eimeria egg capsule → Eimeria egg capsule to place ultrasonoscope to cause weak → count egg capsule → oral vaccination chickling.
The operating procedure of immunity inoculation: the potassium dichromate that will contain spore coccidian oocyst 50ml, sodium azide solution adds centrifugal the removing of distilled water of 10 times of volumes, centrifugal 3 times, each centrifugal 3 minutes, revolution is 4000 rev/mins, supernatant after centrifugal is outwelled, to remove potassium dichromate, sodium azide solution, with the precipitation of the egg capsule after centrifugal adding distil water 50ml, even with the suction pipe pressure-vaccum, carry out the egg capsule counting with the blood cell counting plate method, use 1000 μ l micropipettors to give the oral instillation of chicken, feed to chickling in the drip mode by 4000 spore egg capsules of every chicken, first immunisation 7 ages in days, secondary immunity are 14 ages in days.Can not feed anticoccidial drug to chicken in 2 days before and after the oral immunity.
Be the immunoprophylaxis test below:
Immune protective test is divided into 130 7 aa broiler chicken and causes 90 of weak immune group, 30 of unlikely weak immune group, 10 3 groups of not immune matched group.Cause weak immune group: 90 7 aa broiler chicken young birds are divided into 3 groups, and every group of 30 chickens are divided into and ultrasonicly cause weak 5 minutes groups, ultrasonicly cause weak 10 minutes groups, ultrasonicly cause weak 15 minutes groups, cause weak Eimeria tenella egg capsule oral immunity with ultrasound wave.Ultrasonic cause weak 5 minutes the group 30 chickens be divided into 3 groups again, every group of 10 chickens, every group of immunizing dose is respectively 4 * 10
3, 8 * 10
3, 16 * 10
3Individual/as only, to carry out 2 immunity altogether, head exempts from 7 ages in days, and two exempt from 14 ages in days, attack worm with street strain's egg capsule during 21 ages in days, and attacking worm dosage is 5 * 10
4Individual/only.Ultrasonic weak 10 minutes groups, the ultrasonic grouping that causes weak 15 minutes groups and the immunization method of causing causes weak 5 minutes groups with ultrasonic.Unlikely weak immune group: use without ultrasound wave and cause weak 30 7 age in days AA broiler chicken of Eimeria tenella egg capsule immunity, 10 every group, immunizing dose is respectively 4 * 10
3, 8 * 10
3, 16 * 10
3Individual/only, head exempts from 7 ages in days, and two exempt from 14 ages in days, attack worm with street strain's egg capsule during 21 ages in days, and attacking worm dosage is 5 * 10
4Individual/only.Not immune matched group: attack worm with street strain's egg capsule during 21 ages in days, attacking worm dosage is 5 * 10
4Individual/only.Experimental session is measured the body weight change of respectively organizing chicken respectively, attacks behind the worm the 7th day with its immune performances of index determining such as cecal lesion score, number of eggs ovulated, feces score, and calculates and respectively organize immune protective rate.The results are shown in Table-1.
Table-1 ultrasound wave causes weak Eimeria tenella immune protective
| 7,14 ages in days immunity Immunization the 7th, 14th day | 21 ages in days are attacked worm Attack on the 21th day | ||||||||
| Cause weak time Attenuate time | Group | Immunizing dose 1 * 10 3?Dose ? | Chicken is counted Numbe r of chick en | Dosage 1 * 10 4?Dose ? ? | Cecal lesion score Scores of caecum | Egg capsule counting 1 * 10 4?OPG ? | Feces score Scores of feces | Average weight gain g/ Average weight gaining | Protective rate/Rate protection |
| Cause weak 5 minutes Attenuate 5minute | ?1 ?2 ? ?3 | ?4 ?8 ? ?16 | ?10 ?10 ? ?10 | ?5 ?5 ? ?5 | ?2 ?2 ? ?1 | ?2 ?1 ? ?0.4 | ?2 ?1 ? ?0 | ?144 ?165 ? ?155 | ?90 ?70 ? ?70 |
| Cause weak 10 minutes Attenuate 10 minute | ?4 ?5 ?6 | ?4 ?8 ?16 | ?10 ?10 ?10 | ?5 ?5 ?5 | ?2 ?2 ?2 | ?0.7 ?0.5 ?0.2 | ?1 ?1 ?1 | ?180 ?170 ?168 | ?100 ?100 ?100 |
| Cause weak 15 minutes Attenuate 15 minute | ?7 ?8 ?9 | ?4 ?8 ?16 | ?10 ?10 ?10 | ?5 ?5 ?5 | ?3 ?2 ?3 | ?1.7 ?0.7 ?0.4 | ?3 ?2 ?2 | ?140 ?165 ?167 | ?100 ?100 ?100 |
| Unlikely weak F2 Not Attenuate | ?10 ?11 ?12 | ?4 ?8 ?16 | ?10 ?10 ?10 | ?5 ?5 ?5 | ?2 ?2 ?1 | ?0.9 ?0.7 ?0.4 | ?2 ?1 ?0 | ?135 ?130 ?140 | ?80 ?70 ?60 |
| Matched group (not immune) Control (Nonimmunization) | ?13 ? | ?10 ? | ?5 ? | ?4 ? | ?14.5 ? | ?4 ? | ?98 ? | ?60 ? | |
The result
Cecal lesion score: after attacking worm, cutd open in the 7th day and test chicken extremely, get its caecum, observe the caecum lesion degree.When the both sides caecum lesion is inconsistent, be as the criterion with a serious side, the results are shown in Table-2.
Above-mentioned each test group and matched group are carried out statistics biology, and the result shows: cause weak 5 minutes, 10 minutes and unlikely weak Eimeria tenella egg capsule test group and matched group significant difference (p＜0.05).Cause weak 15 minutes the group in 4 * 10
3, 16 * 10
3An individual/test group and matched group difference are not remarkable, and cecal lesion score is 3 minutes, and caecum lesion is comparatively serious, and 8 * 10
3The group cecal lesion score is 2 minutes, and the caecum infringement is medium.Cause weak 10 minutes group cecal lesion scores and be 2 fens, the caecum infringement is medium.Cause weak 5 minutes identical with the cecal lesion score of unlikely weak F2 group, immunizing dose is 4 * 10
3, 8 * 10
3, 16 * 10
3Individual/cecal lesion score only is 2 minutes, 2 minutes, 1 minute.Not immune matched group cecal lesion score is 4 minutes, and caecum lesion is the most serious.
Attack behind the worm the 7th day egg sac number in every gram feces
For the ease of observing, adopt 5g feces to measure wherein egg sac number in the test, then with the result divided by 5 egg sac numbers of counting every gram feces, after attacking worm, collected the feces on the same day on the 7th day and observe egg sac number, the results are shown in Table-2.
Statistic analysis result shows: each organizes egg sac number in the feces and matched group difference extremely significantly (p＜0.01).Cause in each test group of weak egg capsule immunity, so that weak 10 minutes group discharge egg capsule sums are minimum, OPG is 1.4 * 10
4, causing weak 5 minutes groups and discharge the egg capsule sum at most, OPG is 3.4 * 10
4
The feces score
The 7th day the feces of worm of attacking against each other is scored and shown: cause weak 5 minutes, cause weak 10 minutes, feces score significant difference (p＜0.05) between unlikely weak F2 group and the matched group, it is not remarkable to cause between weak 15 minutes groups and the matched group difference.
Body weight change result
The weightening finish significant difference of weightening finish of test group chicken and matched group (p＜0.05) wherein causes weak 10 minutes groups 4 * 10
3Individual/egg capsule immune group weightening finish at most, average weight gain reaches 180 grams/only.
Protective rate
Caused weak 10 minutes and the protective rate of 15 minutes test group is 100%, caused weak 5 minutes and the protective rate of unlikely weak F2 group is 60 ~ 90%, the protective rate of immune matched group is not 60%.
Discuss
From above result of the test as can be seen, aspect immune protective rate, the immune protective rate that causes weak egg capsule immune group all is higher than unlikely weak immune group and matched group.Cause weak 10 minutes, cause weak 15 minutes protective rates of 2 groups and all reach 100%; cause weak 5 minutes protective rate and be respectively 90%, 70%, 70%; the protective rate 80%, 70%, 60% that all is higher than unlikely weak group also is higher than the not protective rate 60% of immune matched group simultaneously.
Aspect the broiler chicken average weight gain, after causing 3 immunizing dose immunity in the group in weak 10 minutes, the broiler chicken average weight gain is respectively 180,170,168g/ only, all be higher than the average weight gain that causes weak 5 minutes groups, 15 minutes groups, unlikely weak group and matched groups, explanation is aspect weightening finish, and the immune effect that causes weak 10 minutes groups is best.
Aspect the feces score, cause the feces score after 3 immunizing dose immunity in the group in weak 10 minutes and be 1 fen, causing weak 5 minutes groups and the unlikely weak feces of organizing was respectively 2 minutes, 1 minute, 0 minute, and the feces score that causes weak 15 minutes groups is 3 minutes, 2 minutes, 2 minutes, the score of matched group feces is 4 minutes, can illustrate that the feces score that causes weak 10 minutes groups is minimum, immune effect is best.
Attacking the egg capsule of worm after 7 days aspect output, causing that egg capsule counting OPG is respectively 0.7 * 10 in weak 10 minutes groups
4, 0.5 * 10
4, 0.2 * 10
4, all be lower than other and cause weak immune group, unlikely weak immune group and not immune matched group.
Aspect cecal lesion score, the cecal lesion score that causes after weak 10 minutes 3 immunizing doses in the group are exempted from is 2 fens, with cause weak 5 minutes and unlikely weak group remains basically stable, be lower than cause weak 15 minutes the group and not immune matched group, illustrate that thus the cecal lesion score that causes after weak 10 minutes 3 immunizing doses in the group are exempted from is less, immune effect is better.
In sum, adopting supercritical ultrasonics technology to cause weak Eimeria tenella egg capsule than cause weak egg capsule without ultrasound wave is safely and effectively to the prevention chicken coccidiosis, and the protective rate of broiler chicken obviously improves, and weightening finish significantly.Especially cause weak 10 minutes groups, head exempts from 7 ages in days, and two exempt from 14 ages in days, and immunizing dose is 4 * 10
3The group immune effect is ideal.
The specific embodiment:
The present invention will be further described below in conjunction with embodiment:
Embodiment 1:
At first carry out the egg capsule sporeization, the amplification chicken coccidial oocyst is put into the culture dish of 2.5% the potassium bichromate solution that is equipped with 10 times of volumes with isolated egg capsule, the about 0.6cm of solution deep, and putting into temperature is 28～30 ℃ of calorstats, cultivates 5 days; Gently stir 4～5 time culture fluid every day between culture period, when the egg capsule more than 90% is finished spore, stops to cultivate, and places 4 ℃ of refrigerator storage standby; Next carries out ultrasound wave and causes weak egg capsule, with 3000 rev/mins of the spore egg capsule adding distil waters of above collection are centrifugal remove potassium bichromate solution 2 times after, place the 100ml small beaker, after again small beaker being put into the 1000ml large beaker that rubble ice is housed behind the counting and being carried out ice bath, place on the ultrasonoscope sound proof box lifting platform, after making lifting platform rise to horn immersion solution 10mm, power is transferred to 200 watts of minimum positions, adjusting ultrasonic time is 3 seconds, the adjusting play time is that ultrasonic time is 8 minutes a little less than causing in 3 seconds.After the ultrasonic end,, 100ml coccidian oocyst liquid is added 2% potassium bichromate solution 100ml if do not use immediately, 1% sodium azide solution 1.5ml, it is standby to place 4 ℃ of refrigerators; The distilled water that before using coccidian oocyst solution is added 10 times of volumes, with 4000 rev/mins centrifugal 3 times, each 3 minutes, after removing potassium dichromate, sodium azide solution, make oral vaccine.Carry out the egg capsule counting with the microscope slide counting method, promptly draw 1ml coccidian oocyst liquid, be dissolved in and make 10 times diluent in the 10ml water with suction pipe, after stirring, taking-up 0.05ml places on the microscope slide, covers to add coverslip again, counts the egg sac number in the whole coverslip.Its computing formula is egg sac number * 200 in egg sac number=coverslip in every milliliter of coccidian oocyst liquid; Feed to chickling in the drip mode by 4000 spore egg capsules of every chicken, first immunisation 7 ages in days, secondary immunity are 14 ages in days.Can not feed anticoccidial drug to chicken in 2 days before and after the oral immunity.
Embodiment 2:
At first carry out the egg capsule sporeization, the amplification chicken coccidial oocyst is put into the culture dish of 2.5% the potassium bichromate solution that is equipped with 10 times of volumes with isolated egg capsule, the about 0.6cm of solution deep, and putting into temperature is 28～30 ℃ of calorstats, cultivates 5 days; Gently stir 5 time culture fluid every day between culture period, when the egg capsule more than 90% is finished spore, stops to cultivate, and places 4 ℃ of refrigerator storage standby; Next carries out ultrasound wave and causes weak egg capsule, with 4000 rev/mins of the spore egg capsule adding distil waters of above collection are centrifugal remove potassium bichromate solution 3 times after, place the 100ml small beaker, after again small beaker being put into the 1000ml large beaker that rubble ice is housed and being carried out ice bath, place on the ultrasonoscope sound proof box lifting platform, after making lifting platform rise to horn immersion solution 10mm, power is transferred to 200 watts of minimum positions, adjusting ultrasonic time is 3 seconds, the adjusting play time is that ultrasonic time is 10 minutes a little less than causing in 3 seconds.After the ultrasonic end,, 100ml coccidian oocyst liquid is added 2% potassium bichromate solution 100ml if do not use immediately, 1% sodium azide solution 1.5ml, it is standby to place 4 ℃ of refrigerators; The distilled water that before using coccidian oocyst solution is added 10 times of volumes, with 4000 rev/mins centrifugal 3 times, each 3 minutes, after removing potassium dichromate, sodium azide solution, make oral vaccine.Carry out the egg capsule counting with the microscope slide counting method, feed to chickling in the drip mode by 4000 spore egg capsules of every chicken, first immunisation 7 ages in days, secondary immunity are 14 ages in days.Can not feed anticoccidial drug to chicken in 2 days before and after the oral immunity.
Embodiment 3:
At first carry out the egg capsule sporeization, the amplification chicken coccidial oocyst is put into the culture dish of 2.5% the potassium bichromate solution that is equipped with 10 times of volumes with isolated egg capsule, the about 0.6cm of solution deep, and putting into temperature is 28 ~ 30 ℃ of calorstats, cultivates 5 days; Gently stir 4 ~ 5 time culture fluid every day between culture period, when the egg capsule more than 90% is finished spore, stops to cultivate, and places 4 ℃ of refrigerator storage standby; Next carries out ultrasound wave and causes weak egg capsule, with 5000 rev/mins of the spore egg capsule adding distil waters of above collection are centrifugal remove potassium bichromate solution 3 times after, place the 100ml small beaker, after again small beaker being put into the 1000ml large beaker that rubble ice is housed and being carried out ice bath, place on the ultrasonoscope sound proof box lifting platform, after making lifting platform rise to horn immersion solution 10mm, power is transferred to 200 watts of minimum positions, adjusting ultrasonic time is 3 seconds, the adjusting play time is that ultrasonic time is 12 minutes a little less than causing in 3 seconds.After the ultrasonic end,, 100ml coccidian oocyst liquid is added 2% potassium bichromate solution 100ml if do not use immediately, 1% sodium azide solution 1.5ml, it is standby to place 4 ℃ of refrigerators; The distilled water that before using coccidian oocyst solution is added 10 times of volumes, with 4000 rev/mins centrifugal 3 times, each 3 minutes, after removing potassium dichromate, sodium azide solution, make oral vaccine.Carry out the egg capsule counting with the microscope slide counting method, feed to chickling in the drip mode by 4000 spore egg capsules of every chicken, first immunisation 7 ages in days, secondary immunity are 14 ages in days.Can not feed anticoccidial drug to chicken in 2 days before and after the oral immunity.
Claims (1)
1, a kind of preparation method of vaccine to chicken coccidiosis, it is characterized in that: be to adopt ultrasound wave to cause weak method, its concrete step is: (1) at first carries out the egg capsule sporeization, the amplification chicken coccidial oocyst, isolated egg capsule is put into the culture dish of 2.5% the potassium bichromate solution that is equipped with 10 times of volumes, the about 0.6cm of solution deep, putting into temperature is 28～30 ℃ of calorstats, cultivates 5 days; Gently stir 4～5 time culture fluid every day between culture period, when the egg capsule more than 90% is finished spore, stops to cultivate, and places 4 ℃ of refrigerator storage standby; (2) secondly carry out ultrasound wave and cause weak egg capsule, with 3000-5000 rev/min of the spore egg capsule adding distil water of above collection is centrifugal remove potassium bichromate solution 2-3 time after, place the 100ml small beaker, after again small beaker being put into the 1000ml large beaker that rubble ice is housed behind the counting and being carried out ice bath, place on the ultrasonoscope sound proof box lifting platform, after making lifting platform rise to horn immersion solution 10mm, power is transferred to 200 watts of minimum positions, adjusting ultrasonic time is 3 seconds, the adjusting play time is that ultrasonic time is 8-12 minute a little less than causing in 3 seconds.(3) after the ultrasonic end,, 100ml coccidian oocyst liquid is added 2% potassium bichromate solution 100ml if do not use immediately, 1% sodium azide solution 1.5ml, it is standby to place 4 ℃ of refrigerators; The distilled water that before using coccidian oocyst solution is added 10 times of volumes, with 4000 rev/mins centrifugal 3 times, each 3 minutes, after removing potassium dichromate, sodium azide solution, make oral vaccine.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103251938A (en) * | 2013-04-15 | 2013-08-21 | 内蒙古神元生物工程股份有限公司 | Industrial production process of eimeria coccidiosis attenuated vaccine |
| CN103374526A (en) * | 2012-04-18 | 2013-10-30 | 上海市农业科学院 | Coccidian oocyst culture preservative fluid and application method thereof |
| CN107904247A (en) * | 2017-12-26 | 2018-04-13 | 吉林大学 | One kind restructuring Eimeria species Alphavirus particle vaccines and preparation method |
| CN108102919A (en) * | 2017-12-31 | 2018-06-01 | 天津赫莱恩特生物科技有限公司 | A kind of method and its application for improving chicken eimeria tenella egg capsule Sporulated efficiency |
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2007
- 2007-04-30 CN CN200710055622A patent/CN100584377C/en not_active Expired - Fee Related
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103374526A (en) * | 2012-04-18 | 2013-10-30 | 上海市农业科学院 | Coccidian oocyst culture preservative fluid and application method thereof |
| CN103251938A (en) * | 2013-04-15 | 2013-08-21 | 内蒙古神元生物工程股份有限公司 | Industrial production process of eimeria coccidiosis attenuated vaccine |
| CN103251938B (en) * | 2013-04-15 | 2015-07-15 | 内蒙古神元生物工程股份有限公司 | Industrial production process of eimeria coccidiosis attenuated vaccine |
| CN107904247A (en) * | 2017-12-26 | 2018-04-13 | 吉林大学 | One kind restructuring Eimeria species Alphavirus particle vaccines and preparation method |
| CN108102919A (en) * | 2017-12-31 | 2018-06-01 | 天津赫莱恩特生物科技有限公司 | A kind of method and its application for improving chicken eimeria tenella egg capsule Sporulated efficiency |
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