CN100366726C - Culture solution and preservation solution for coccidian oocyst - Google Patents
Culture solution and preservation solution for coccidian oocyst Download PDFInfo
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Abstract
The present invention discloses a culture solution and a preservation solution for coccidian oocysts. The culture solution and the preservation solution are chloramine T solutions prepared by fully shaking chloramine T and pure water according to the proportion of (0.5 to 8.0): 100. After fresh coccidian oocysts are separated and purified from chicken excrement containing the oocysts, the precipitates of the oocystes are suspended in the culture solution of the chloramine T solution under the culture temperature of 25 to 30 DEG C, the culture humidity of more than 50%, the oocyst density of no more than 1000 thousand oocysts per ml and the depth of no more than 0.7cm for 24 to 72 hours and the culture solution is slightly stirred for more than three times every day. When more than 80% of oocysts are spored, the oocysts are not cultured, the culture solution of the oocysts is collected and centrifugally concentrated, the concentrated culture solution is added into the preservation solution of the chloramine T solution to be preserved in a refrigerator under 4 to 8 DEG C. The culture solution and the preservation solution has the advantages of high sporing rate for the coccidian oocysts, no heavy metal substance residue in chicken bodies and no environmental pollution.
Description
Technical field:
The invention belongs to a kind of live vaccine pathogen culture liquid and preserve liquid, or rather, the present invention adopts nutrient solution and the preservation liquid of chloramine-T liquid as coccidiosis of chicken vaccine coccidian oocyst.
Background technology:
Coccidiosis of chicken is one of principal disease of harm intensification poultry husbandry, in recent years it is prevented and treated still based on medicine.Yet along with extensively and continuously using of anticoccidial drug, resistance and drug residue two large problems are on the rise, and have caused global vigilance.Along with the arrival of green livestock industry, using coccidiosis vaccine immunoprophylaxis coccidiosis of chicken is trend of the times.Worm strain vaccine (the strong virus live vaccine of coccidian oocyst) arrived the development course of coccidia subunit vaccine and recombiant vaccine again to causing weak worm strain vaccine (coccidian oocyst attenuated live vaccines) a little less than the coccidiosis of chicken vaccine had experienced non-causing.But up to now, the coccidiosis of chicken vaccine that uses in the production mainly still is preceding two kinds of vaccines.The production technique of chicken coccidial oocyst living vaccine is very different with the production of virus, bacteriosis class vaccine, and this is main relevant with the distinctive growth modes of reproduction of chicken coccidia.After the spore coccidian oocyst infected chicken, intracutaneous formed a large amount of egg capsules and excretes with ight soil through 4～6 days growth on intestinal mucosa.The egg capsule of discharging must form sporozoite through a spore process external, just has infectivity again.The coccidian oocyst sporeization needs suitable temperature, humidity and competent oxygen.The desired suitable temp of cultivation coccidian oocyst, humidity condition are to create easily, but the coccidian oocyst that separates from ight soil, collects can have bacterium more or less, consumption oxygen is usually competed in a large amount of breedings of bacterium when cultivating coccidian oocyst, so nutrient solution should add the material of bacteria growing inhibiting.2.5% potassium dichromate solution is a kind of strong oxidizer, and the growth and breeding that it both can provide oxygen also can suppress bacterium is as the coccidian oocyst nutrient solution of routine always and preserve liquid is widely used.But potassium bichromate is a kind of heavy metal compound, and life-time service can be residual in animal body and environment, influences human and animal's health.Along with improving constantly of living standards of the people, the whole society more and more payes attention to meat product edible safety problem and environmental protection problem.Seek a kind of more suitable coccidian oocyst cultivation and preserve and become the important topic that the coccidiosis vaccine investigator presses for solution with solution.
Summary of the invention:
Purpose of the present invention is exactly some shortcomings that overcome prior art, on the coccidiosis vaccine production technique, realize breaking through and innovation, adopt 0.5%～8% chloramine-T (Chloramine T) solution to replace 2.5% conventional potassium bichromate solution and cultivate and preserve and use solution, avoided the residual and environmental pollution of heavy metal substance at the chicken body as coccidian oocyst.
The present invention realizes by following technical proposals: a kind of coccidian oocyst is cultivated and is preserved and use solution, it is characterized in that this nutrient solution and to preserve liquid be to adopt chloramine-T and pure water with (0.5-8.0): 100 ratio is formation chloramine-T solution through fully shaking up preparation.
The described nutrient solution and the cultivation of preserving liquid with preserve using method and be: after the chicken manure that contains egg capsule separates just and is purified into fresh coccidian oocyst, the egg capsule throw out is suspended in the described chloramine-T water culture liquid, 25 ℃～30 ℃ of the culture temperature of nutrient solution, cultivate humidity more than 50%, egg capsule density and be no more than 1,000,000 egg capsule/ml, the nutrient solution degree of depth and be no more than 0.7cm, every day stir culture liquid is more than 3 times gently, incubation time is 24～72 hours; When the egg capsule more than 80% is finished spore, stop to cultivate, results egg capsule nutrient solution adds above-mentioned chloramine-T solution again and preserves to preserve in mid-4 ℃～8 ℃ refrigerators of liquid and get final product after centrifugal the concentrating.
Through the coccidian oocyst of above-mentioned nutrient solution and the processing of preservation liquid, its spore rate is up to more than 80%, and the steriling test result is negative, and it is uninfluenced to preserve 9 months egg capsule vigor down at 4 ℃～8 ℃, and effect is better than 2.5% traditional potassium bichromate solution.
Chloramine-T is a kind of organic chlorine derivative that contains, and molecular formula is C
7H
7NClNO
2SNa3H
2O contains 24% available chlorine.Chloramine-T has been applied in the clinical medicine bacterium, virus, fungi, brood cell all being had killing action as disinfectant for external use.Its mechanism of action is that hypochlorous acid is decomposed in the water-soluble back of chloramine-T, discharges reactive chlorine, makes the oxidized inefficacy of enzyme in the microorganism, and the destroy microorganisms normal metabolic makes bacterium death, thereby reaches germ-resistant purpose.Chloramines itself also has direct disinfection, and its fungicidal activity is stronger in acidity, and because of being difficult for disengaging chlorine, so germicidal action weakens in the alkalescence.Chloramines generates hypochlorous acid, and to generate hypochlorous acid than hypochlorite slow, lasting so disinfection is more weak, and pungency, corrodibility are less.
The present invention is by the more weak and persistent germicidal action of chloramine-T, when egg capsule is cultivated or the bacterium of desire growth when preserving kill, guarantee the required sufficient oxygen of coccidian oocyst existence, can be used as the nutrient solution of coccidian oocyst and preserve liquid.On the other hand, chloramine-T is to cure disinfectant for external use and be found as a kind of people the earliest, and secular application confirms it to animal body nontoxicity and residual, and is free from environmental pollution.The present invention adopts 0.5%～8% chloramine-T solution to replace the improvement of 2.5% conventional potassium bichromate solution as coccidian oocyst nutrient solution and this technology of preservation liquid on the coccidiosis vaccine production technique, still belongs to initiative at home and abroad, and not seeing has similar research report.
Embodiment
The present invention adopts 0.5%～8% chloramine-T solution to replace 2.5% conventional potassium bichromate solution as coccidian oocyst nutrient solution and preservation liquid on the coccidiosis vaccine production technique, significantly improved the spore rate of coccidian oocyst, avoided the residual and environmental pollution of heavy metal substance at the chicken body.
Further specify effect of the present invention with example below:
Example 1 oxygen amine T is to the influence of coccidian oocyst spore development
1 materials and methods
1.1 nutrient solution
Negative control (pure water), positive control (2.5% potassium dichromate solution), 0.5%, 1%, 2%, 4%, 8% chloramine-T liquid.
1.2 the preparation of fresh egg capsule
Coccidia is mixed egg capsule infect 40 14 age in days healthy chicks with 10,000 egg capsules/dosage only, the ight soil of discharging in 6 hours is collected in infection back ovulation in the 7th day capsule peak period, and the production technique isolation and purification for preparing by coccidial vaccine goes out fresh coccidian oocyst.
1.3 test grouping and processing
It is 7 parts that clean coccidian oocyst is divided equally, is suspended in respectively in above-mentioned 7 kinds of designed nutrient solutions, and making culture density is 80,000 egg capsule/ml, puts in 29 ℃ of incubators and cultivates, and stirs once gently every several hrs.Respectively at cultivating back 14h, 21h, 28h and 38h, calculate every group of coccidian oocyst number that can carry out sporeization with blood counting chamber, calculate the spore rate.After the spore end, get the 0.1ml nutrient solution for every group, be inoculated in respectively on the nutrient agar plate, put 37 ℃ and cultivated 3 days, observe the bacterial growth situation.
2 results and conclusion
2.1 coccidian oocyst spore situation
The results are shown in Table 1.As can be seen from Table 1, have 70%～80% during spore when cultivate 24 hours coccidian oocysts at 2.5% potassium dichromate solution, coccidian oocyst in 0.5%, 1%, 2%, 4%, 8% chloramine-T liquid group has 80%～90% complete sporeization, and the latter is higher than the former.As seen, use 0.5%～8% chloramine-T solution as the chicken coccidial oocyst nutrient solution, can significantly improve the spore rate of coccidian oocyst, its spore effect is better than 2.5% conventional potassium bichromate solution.
Table 1 chloramine-T is to the influence test of coccidian oocyst spore rate
| Group | The spore rate (%) of different incubation time coccidian oocysts | |||
| 14h | 21h | 28h | 38h | |
| The pure water control group | 19 | 66 | 82 | 84 |
| 2.5% potassium bichromate group | 31 | 70 | 82 | 86 |
| 0.5% chloramine-T group | 45 | 82 | 88 | 89 |
| 1% chloramine-T group | 48 | 85 | 93 | 94 |
| 2% chloramine-T group | 33 | 80 | 86 | 93 |
| 4% chloramine-T group | 42 | 81 | 85 | 91 |
| 8% chloramine-T group | 43 | 88 | 90 | 93 |
2.2 steriling test result
Each group nutrient solution is inoculated in respectively on the nutrient agar plate, puts 37 ℃ and cultivated 3 days, the result is as follows: pure water control group flat board covers with bacterium colony; 2.5% potassium dichromate solution control group flat board grows 3 bacterium colonies; 0.5%, all aseptic length of being born of 1%, 2%, 4%, 8% chloramine-T liquid group flat board.As seen, chloramine-T liquid can complete bacteria growing inhibiting.
Example 2 coccidian oocysts are preserved the mensuration of its vigor in 0.5%～8% chloramine-T liquid
1 materials and methods
1.1 test chicken and raising thereof
Buy the qualified south of the Five Ridges yellow chicken kind egg from the herding of academy of agricultural sciences, Guangdong Province, hatching is to going out shell, and isolated rearing is in the wire mesh cage that flame disinfection is crossed, and the complete feed of the no anticoccidial medicine of feeding, is drunk through boiling the boiling water of processing through 70 ℃ of baking 12h with preceding.Before the test, check that ight soil non-ball worm egg capsule pollutes.
1.2 coccidiosis of chicken tetravalence living vaccine
Develop by Zhengdian Biological Tech. Co., Ltd., Foshan City, contain Eimeria tenella (Eimeriatenella), poison Eimeria (E.necatrix), Eimeria maxima (E.maxima) and 4 kinds of heap type Eimeria (E.acervulina), be stored in 0.5%～8% chloramine-T liquid present embodiment and adopt in the 1% chloramine-T solution.Product batch number is 20040001,20040002,20040003.
Use the worm strain 1.3 attack
Be the corresponding parnet strain of each vaccine strain, separate, identify and preserve by Zhengdian Biological Tech. Co., Ltd., Foshan City.
1.4 test grouping and processing
After 3 batches of coccidiosis of chicken tetravalence living vaccines of preparation are preserved 0 month, 3 months, 6 months, 9 months respectively at 4 ℃ of refrigerators, oral vaccination immunity 3 Japanese instar chicklings; Other establishes a blank group.Each organizes chicken isolated rearing under the same conditions, and (raising on the bedding and padding, is bedding and padding with the wood chip, 15/m of stocking density
2).Back 28 days of immunity, every chicken was observed 5～7 with the oral attack of strong malicious spore mixing egg capsule (containing 100,000 of heap type Eimerias, each 50,000 of other 3 kinds of coccidias), all chickens are slaughtered cut open inspection, check to suit every chicken (Eimeria tenella is checked caecum at the position; Poison Eimeria and check small intestine middle part and caecum; Eimeria maxima is checked the small intestine middle part; Heap type Eimeria inspection duodenum) pathology is also kept the score.
2 results and conclusion
The results are shown in Table 2.As known from Table 2, in 4 ℃～8 ℃ refrigerators, preserved 0 month, 3 months, 6 months, 9 months coccidiosis of chicken tetravalence living vaccine, all can produce good immunizing power at immune 3 Japanese instar chicklings after 28 days, it is uninfluenced to show that coccidian oocyst is preserved 9 months its vigor under 4 ℃～8 ℃ in 0.5%～8% chloramine-T liquid.
Table 2 coccidian oocyst is preserved its vitality test result in 0.5%～8% chloramine-T liquid
| Group | The vaccine lot number | The vaccine shelf time | The test chicken number | Ight soil is kept the score | Survival rate (%) | Lesion score | ||
| Caecum | Duodenum | The small intestine stage casing | ||||||
| Immune group 1 | 20040001 | 0 month | 20 | 0 | 100 | 0.55 | 0.36 | 1.22 |
| 3 months | 20 | 0 | 100 | 0.57 | 0.33 | 1.17 | ||
| 6 months | 20 | 0 | 100 | 0.65 | 0.46 | 1.24 | ||
| 9 months | 20 | 0 | 100 | 0.74 | 0.58 | 1.36 | ||
| Immune group 2 | 20040002 | 0 month | 20 | 0 | 100 | 0.52 | 0.35 | 1.13 |
| 3 months | 20 | 0 | 100 | 0.55 | 0.38 | 1.14 | ||
| 6 months | 20 | 0 | 100 | 0.69 | 0.47 | 1.21 | ||
| 9 months | 20 | 0 | 100 | 0.83 | 0.75 | 1.39 | ||
| Immune group 3 | 20040003 | 0 month | 20 | 0 | 100 | 0.46 | 0.33 | 1.07 |
| 3 months | 20 | 0 | 100 | 0.42 | 0.35 | 1.03 | ||
| 6 months | 20 | 0 | 100 | 0.75 | 0.36 | 1.22 | ||
| 9 months | 20 | 0 | 100 | 0.86 | 0.65 | 1.35 | ||
| Blank | - | 0 month | 20 | ++++ | 65 | 3.56 | 3.07 | 3.35 |
| 3 months | 20 | ++++ | 65 | 3.59 | 3.02 | 3.26 | ||
| 6 months | 20 | ++++ | 65 | 3.55 | 3.06 | 3.41 | ||
| 9 months | 20 | ++++ | 65 | 3.53 | 3.12 | 3.33 |
Example 3 coccidiosis of chicken tetravalence living vaccines are to the acute toxicity test of mouse
1 materials and methods
1.1 experimental animal
40 of the kunming mices of body weight 18～24g, male and female half and half are available from Zhongshan Medical Univ..Raised 7 days under controlled condition before the test, health condition and physiological situation all show well.
1.2 tried thing
Coccidiosis of chicken tetravalence living vaccine, lot number 20040001 by Zhengdian Biological Tech. Co., Ltd., Foshan City's development, includes 0.5%～8% chloramine-T liquid.
1.3 test grouping and processing
Above-mentioned 40 kunming mices are divided into 4 groups at random, 10 every group (male and female half and half).After stopping eating 4 hours, the stomach approach gives coccidiosis of chicken tetravalence living vaccine (shaking up energetically with preceding) 10 μ l respectively to 1～3 group of every mouse, 50 μ, 1,100 μ are the blank group for 1, the 4 group to irritate.Gavage the back and observed 7 days continuously, toxicity symptom and disease time, death toll and the death time of record test mice.If mouse continues deadly after gavaging 4 days, then prolong observing time to 14 day.
2 results and conclusion
As shown in table 3, when being equivalent to coccidiosis of chicken tetravalence living vaccine immunity chicken respectively, recommends test mice usage quantity (10ml/ bottle, use for 1000 plumage parts, be average every plumage 10 μ 1) during 1 times, 5 times, 10 times the amount of gavaging, mouse does not have any toxicity symptom, 100% survival shows when the chloramine-T low dosage uses the animal body free of toxic effects.
Table 3 coccidiosis of chicken tetravalence living vaccine is to the The acute toxicity tests of mouse
| Group | Gavage dosage (μ l/ only) | The amount of gavaging is equivalent to the multiple of actual amount | The experimental animal number | The dead animal number | Mortality ratio (%) | Survival rate (%) |
| 1 | 10 | 1 | 10 | 0 | 0 | 100 |
| 2 | 50 | 5 | 10 | 0 | 0 | 100 |
| 3 | 100 | 10 | 10 | 0 | 0 | 100 |
| 4 | - | - | 10 | 0 | 0 | 100 |
Claims (2)
1. a coccidian oocyst is cultivated and is preserved and use solution, and it is characterized in that this nutrient solution and to preserve liquid be to adopt chloramine-T and pure water with (0.5-8.0): 100 ratio prepares and the chloramine-T solution of formation through fully shaking up.
2. adopt the described coccidian oocyst of claim 1 to cultivate and preserve the using method of using solution, it is characterized in that, described nutrient solution with the cultivation of preserving liquid with preservation condition is: after the chicken manure that contains egg capsule separates just and is purified into fresh coccidian oocyst, the egg capsule throw out is suspended in the described chloramine-T water culture liquid, 25 ℃～30 ℃ of the culture temperature of nutrient solution, cultivate humidity more than 50%, egg capsule density and be no more than 1,000,000 egg capsule/ml, the nutrient solution degree of depth and be no more than 0.7cm, every day stir culture liquid is more than 3 times gently, incubation time is 24～72 hours; When the egg capsule more than 80% is finished spore, stop to cultivate, results egg capsule nutrient solution adds above-mentioned chloramine-T solution again and preserves to preserve in mid-4 ℃～8 ℃ refrigerators of liquid and get final product after centrifugal the concentrating.
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Families Citing this family (9)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102174408B (en) * | 2011-01-10 | 2012-08-22 | 内蒙古神元生物工程股份有限公司 | Method for batch preparation of chicken Eimeria coccidium oocysts |
| 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 |
| CN103710265A (en) * | 2013-12-23 | 2014-04-09 | 广东省农业科学院动物卫生研究所 | Application of chloramine B solution in chicken coccidiosis oocyst culture and preservation |
| CN106754379B (en) * | 2016-11-25 | 2021-05-25 | 佛山市正典生物技术有限公司 | Middle-size Eimeria acervulina precocious strain for rabbits as well as preparation method and application thereof |
| CN107347873B (en) * | 2017-07-12 | 2021-02-19 | 中国农业大学 | Eimeria coccidium cryopreservation agent and Eimeria coccidium cryopreservation method |
| CN111713488B (en) * | 2020-07-16 | 2022-01-18 | 松山湖材料实验室 | Cryopreservation agent, preparation method thereof and application thereof in coccidian oocysts |
| CN112400865B (en) * | 2020-11-27 | 2022-05-03 | 松山湖材料实验室 | Wall-breaking agent composition, preparation method thereof and application thereof in oocysts |
| CN112369410B (en) * | 2020-11-27 | 2022-05-03 | 松山湖材料实验室 | Oocyst wall breaking agent composition and preparation method and application thereof |
| CN112877215A (en) * | 2021-03-02 | 2021-06-01 | 华南农业大学 | Separation and purification method and inoculation method of Eimeria tenella sporozoites |
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| CN1348490A (en) * | 1999-02-26 | 2002-05-08 | 美国辉瑞有限公司 | Method for the purification, recovery, and sporulation of cysts and oocysts |
| CN1547607A (en) * | 2001-08-30 | 2004-11-17 | Improved methods for producing oocysts | |
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| CN1348490A (en) * | 1999-02-26 | 2002-05-08 | 美国辉瑞有限公司 | Method for the purification, recovery, and sporulation of cysts and oocysts |
| CN1547607A (en) * | 2001-08-30 | 2004-11-17 | Improved methods for producing oocysts | |
| CN1652816A (en) * | 2002-05-21 | 2005-08-10 | 先灵-普劳有限公司 | Methods for the in vitro culture of sporozoea sp. and uses thereof |
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