CN102174408B - Method for batch preparation of chicken Eimeria coccidium oocysts - Google Patents
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Abstract
The invention discloses a method for batch preparation of chicken Eimeria coccidium oocysts, and particularly relates to a separation and purification method for producing oocysts (antigens) of chicken Eimeria coccidium vaccines. The method for preparing the chicken Eimeria coccidium oocysts comprises the following process steps: fecal sample processing, continuous screening, saturated brine float centrifuging, oocyst centrifugal depositing and oocyst purifying. According to the method, the industrial preparation of the chicken Eimeria coccidium oocysts can be realized, the production efficiency can be improved, and the cost can be reduced.
Description
Technical field
A kind of chicken Eimeria egg capsule batch preparation relates to the separation purification method of the egg capsule (antigen) of producing chicken Eimeria vaccine.
Background technology
Sick about the chicken Eimeria
Eimeria (Eimeria) coccidia (Coccidium) is the one type of obligatory parasitism protozoon that parasitizes the chicken enteron aisle, causes coccidiosis of chicken (Coccidiosis).According to statistics; The chicken coccidia that the whole world has been reported has 15 kinds; Wherein, Have 5 kinds to have significantly pathogenicly, they are: Eimeria tenella (E.tenella), poison Eimeria (E.necatrix), Eimeria maxima (E.maxima), heap shape Eimeria (E.acervulina) and gentle Eimeria (E.mitis).In the pathogenic coccidia of 5 kinds of tools, there are 3 kinds to chick tool hazardness, they are: Eimeria tenella, Eimeria maxima and heap shape Eimeria.
Coccidiosis is a kind of ubiquitous infectious eqpidemic disease, and every in the world have the place of chicken just to have the chicken coccidia, and the generation of coccidiosis is just arranged, and the outburst of this disease usually appears in the mass-producing chicken house incident suitable place that is coccidiosis of chicken.Coccidiosis of chicken is that a kind of infection is rapid, propagates soon and extensively, and the high lytic infection of pathogenicity rate, lethality rate is sick, is preventing and treating improperly under the situation, and infection rate and mortality ratio all can be up to more than 50%.Therefore, coccidiosis of chicken causes serious economy loss to aviculture, and the annual loss that causes because of this disease in the whole world is up to tens billion of yuans, and the financial loss that China causes because of this disease every year is no less than 10,000,000,000 yuan.In today that poultry husbandry intensification degree improves constantly, coccidiosis of chicken is an indispensable link in the prevention and control of poultry husbandry eqpidemic disease.
The control of coccidiosis of chicken
From the eighties in last century, the prevention and control of coccidiosis of chicken have obtained major progress, mainly have benefited from the appearance of a large amount of anticoccidial drugs, and obtain gratifying effect.Yet; Mostly a large amount of anticoccidial drugs is chemical classes product (like Robenidine, nicarbazine, dinitolmide, diclazuril, nicarbazine and sulfa drugs) and antibiotics product (like monensin, Salinomycin., draw husky rhzomorph, that draws rhzomorph, Maduramicin, Hainan mycin), and the life-time service of these products must cause the appearance of resistance and drug residue phenomenon.And the use of various anticoccidial drugs is the administration of companion material, in fact the nineties in last century anti-medicine phenomenon very general, the development pressure pcl of new drug increases in this case, the prevention and control difficulty of the enterprise of raising chickens strengthens day by day.
Got into since 21 century; Pharmaceutical chemicals and antibiotic use are restricted in the global range; The food safety system receives increasing concern; Developed country has forbidden the use of chemical anticoccidial drug basically, and the use of antibiotics anticoccidial drug also is subject to significant restrictions, and has prohibited this series products in the U.S., Japan and the European Community and has appeared in the feed.The application that does not add restriction in China owing to various anticoccidial drugs, resistance generally produces, and the product that has (like monensin) has not had clinical use value.The food safety policy of China comes into effect in recent years; Human consumer's self health perception strengthens day by day simultaneously; The atrophy day by day of the market of anticoccidial drug; And China also will implement forbid or limit the marketing policy of using anticoccidial drug in the recent period, and coccidiosis of chicken prevents and treats situation anxiety suddenly like this.
For effective control coccidiosis of chicken, guarantee the sound development of poultry husbandry, press for the new method of preventing and treating of seeking, this wherein the vaccine inoculation prevention be one of approach that has future.Yet traditional coccidian oocyst stripping technique is not suitable for industrialization production, need be applicable to the technical matters of large-scale production in a hurry.
Existing egg capsule stripping technique
Chicken coccidial oocyst must separate just from chicken manure; Existing stripping technique has multiple, but can reduce 2 types, and one type is floating method; Another kind of is density gradient centrifugation, wherein floating saturated brine floating method, sucrose solution floating method, saturated sal epsom floating method and the saturated chromatosulfuric acid floating method of comprising; It is sucrose density gradient centrifugation that gradient centrifugation has only a kind of.
Floating method
Because the density (1.103～1.064) of coccidian oocyst is between the density of water and saturated brine (sucrose solution, saturated sal epsom and saturated chromatosulfuric acid); When the egg capsule solution that will contain impurity was placed on that whizzer is centrifugal in the water, egg capsule and a spot of material sank than water-mass density is big together, because buoyancy; Than the little impurity come-up of water-mass density; After centrifugal, abandon supernatant, removed the impurity littler than water-mass density; When the egg capsule solution that will contain impurity is placed in the saturated brine (sucrose solution, saturated sal epsom and saturated chromatosulfuric acid) when centrifugal; Because buoyancy, egg capsule and a spot of material be than the little come-up together of saturated brine density, centrifugal after; Abandon deposition, removed the impurity bigger than saturated brine density.Subsequently, isolating egg capsule is handled with Youxiaolin, removes the bur on bacterium and egg capsule surface.
Saturated brine floating method operation steps:
The 1st step: get infected chicken fresh excreta 10g, add water and be mixed into liquid manure for 10 times;
The 2nd step: liquid manure filters through 60 order sub-sieves, keeps filtrating;
The 3rd step: filtrating is supplied the water yield and is filtered through 200 order sub-sieves, keeps filtrating;
The 4th step: it is centrifugal through 2000rpm * 10min to filtrate, and abandons supernatant;
The 5th step: throw out adds saturated brine, and 2000rpm * 10min is centrifugal;
The 6th step: repeated for the 5th step;
The 7th step: get supernatant liquid and add water, 3000rpm * 15min is centrifugal, and throw out is egg capsule;
The 8th step: throw out adds water, the centrifugal flush away salinity of 3000rpm * 15min.
Sucrose solution floating method operation steps:
The 1st step: get infected chicken fresh excreta 10g, add water and be mixed into liquid manure for 2～5 times;
The 2nd step: liquid manure filters through 60 order sub-sieves, keeps filtrating;
The 3rd step: filtrating is supplied the water yield and is filtered through 100 or 200 order sub-sieves, keeps filtrating;
The 4th step: filtrating adds equivalent sucrose solution (sheatherShi liquid) and mixes, and is centrifugal through 3000rpm * 3min;
The 5th step: fish for above-mentioned centrifuge tube surface supernatant liquid in another tool water receptacle with 20～25 order dredgers;
The 6th step: 3000rpm * 3min is centrifugal, the flush away sugar soln;
The 7th step: repeated for the 6th step, throw out is egg capsule.
Saturated sal epsom floating method operation steps:
The 1st step: get infected chicken fresh excreta 10g, add water and be mixed into liquid manure for 2～5 times;
The 2nd step: liquid manure filters through 60 order sub-sieves, keeps filtrating;
The 3rd step: filtrating is supplied the water yield and is filtered through 100 or 200 order sub-sieves, keeps filtrating;
The 4th step: filtrating adds the saturated Adlerika of equivalent and mixes, and is centrifugal through 3000rpm * 3min;
The 5th step: fish for above-mentioned centrifuge tube surface supernatant liquid in another tool water receptacle with 20～25 order dredgers;
The 6th step: 3000rpm * 3min is centrifugal, the saturated Adlerika of flush away;
The 7th step: repeated for the 6th step, throw out is egg capsule.
Saturated chromatosulfuric acid floating method operation steps:
The 1st step: get infected chicken fresh excreta 10g, add water and be mixed into liquid manure for 10 times;
The 2nd step: 1500rpm * 5min is centrifugal, keeps throw out;
The 3rd step: add 4～5 times of amount chromatosulfuric acid liquid in the throw out, abundant mixing under the condition of ice bath, it is centrifugal to carry out 1500rpm * 5min at once;
The 4th step: with suction pipe sucking-off supernatant, add 20 times of frozen water, 1500rpm * 5min is centrifugal again;
The 5th step: repeat centrifugal 2 times of 1500rpm * 5min, throw out is egg capsule.
Sucrose density gradient centrifugation
Ultimate principle
Between 1.103～1.064, process density gradient according to the density of coccidian oocyst, add,, collect the egg capsule layer between two gradients through centrifugal with the isolating egg capsule of saturated brine floating method with sucrose, with density greatly and the little impurity of density separate with egg capsule.
Operation steps:
The 1st step: press the saturated brine floating method and handle ight soil, separate worm's ovum;
The 2nd step: sucrose solution gradient preparation (pressing Suo Xun, Yang Xiaoye, 2005);
The 3rd step: get the isolating concentrated egg capsule liquid 5ml of saturated brine floating method, place on the liquid level the centrifugal 30min of 1500g;
The 4th step: collect each gradient layer of inspection and deposition respectively, have each layer of egg capsule to collect respectively, with saline water or tap water centrifuge washing, collecting precipitation thing;
The 5th step: throw out is resuspended in the 5ml water, once more gradient centrifugation, repeated collection and washing process.
The analysis of existing manufacturing technology:
The saturated brine floating method is the most frequently used coccidian oocyst stripping technique in laboratory, and this technology is simple relatively, is easy to grasp favorable repeatability, reliable results.But be used for industrialization production major defect is arranged.To handle the 300kg chicken manure day is that the industrialization production of split run production capacity is example, and the liquid manure amount after the processing can reach 3300 liters, and after 60 orders and 200 mesh sieves divided, 90% liquid manure became filtrating (excrement slag＜10%), and whole filtrating reaches 2970 liters.
The whizzer of present domestic maximum capacity is 4 * 500ml (rotating speed is not less than 3000rpm), once centrifugally can only handle 2000ml, handle 2970 liters of filtratings, and a bench centrifuge needs non-stop run 1485 times; Like 1 bench centrifuge darg 10 times; Then need 148 super-magnum centrifuges, this does not also comprise follow-up a large amount of spinning and purifying, therefore; This technology obviously can not be used for large-scale production, and the vaccine of producing through this kind method uses owing to too high also being inappropriate in the production of cost.
With chicken coccidia pentavalent vaccine is example, and 3 fryer day in age week defecation are by 100 grams, and daily output 300kg ight soil needs 3000 chickens; Each plants 600, and every chicken (by 7 days) in one-period ovulates, and capsule is about can to prepare 10000 plumage vaccines, and each cycle can be produced 6,000,000 plumage vaccines; Annual by production in 300 days; Totally 42 production cycles, total can be produced vaccine 2.52 hundred million plumages, and this is the output of a medium-scale vaccine factory.
In view of this, the industrialized manufacturing technique of vaccine to chicken coccidiosis can not prolong uses conventional art, and formulating new technical process is the only way of industrialization.
The defective of sucrose solution floating method though the liquid manure amount reduces, needs the sucrose solution of adding and filtrating equivalent basically with the saturated salt water law; Still need a large amount of centrifugal treating repeatedly; Not only need a large amount of whizzers, and centrifugal treating is time-consuming too much, this method can not be used for industrialization production.
Saturated sal epsom floating method is except that having above-mentioned defective; This fado is used for the laboratory and separates Tyzzeria perniciosa (Tyzzeria perniciosa) and Wenyonella philiplevinei (Wenyonella philiplevine) egg capsule, seldom is used for the separation of chicken pathogenic Eimeria egg capsule.
Saturated chromatosulfuric acid floating method not only needs a large amount of centrifugal treating, also needs condition of ice bath, and this does not obviously meet the working condition of vaccine to chicken coccidiosis, and cost is too high.
The sucrose density gradient centrifugation process is complicated, condition preparation difficulty, and sepn process has comprised the saturated brine floating method, and weak point is no longer tired to be stated.
Summary of the invention
The purpose of this invention is to provide the method that industrialization prepares chicken Eimeria egg capsule.
Method of the present invention is:
A, excrement appearance are handled: get the infected chicken fresh excreta; Add the water dilution mixing of 3 times of its weight, behind the adding stomach en-thorough mixing, leave standstill the water that adds 7 times of excrement weight after 10 minutes again; Mixing makes into liquid manure, and wherein pepsic add-on is a 0.05mg/ gram ight soil;
B, screening processing continuously: liquid manure is handled through multi-layer type vibrosieve filter, promptly at the uniform velocity adds liquid manure from charging opening, is filtered by the 1st layer to the 4th layer continuous sub-sieve; Add the water flushing simultaneously; After take off the 4th layer of screen cloth, water washes out all trapped substances, to the graduated containers for future use of tool;
Multi-layer type vibrosieve filter: or claim the multi-layer type vibrating screen classifier, establish 4 layers altogether, the 1st layer of 60 order, the 2nd layer of 200 order, the 3rd layer of 325 order, the 4th layer of 1250 order;
C, saturated brine are floating centrifugal: it is floating that trapped substance adds the abundant mixing of saturated brine of 5 times of volumes, and 2500rpm * 10min is centrifugal;
D, egg capsule centrifugation: the swimmer on sucking-off centrifuge tube surface adds to and is less than 10 times of water mixings of its volume, and 2500rpm * 10min is centrifugal;
E, egg capsule purify: remove supernatant, add water to centrifuge tube and be full of mixing, 2500rpm * 10min is centrifugal, and throw out is egg capsule.
The characteristics of present technique:
(1) simplified technology: present technique comes down to the technique improvement to the saturated salt water law, and whole egg capsule sepn process has reduced by three unit, the simplified working procedures of Technology 37.5%.
(2) be applicable to industrialization production: with the 300kg chicken manure is example; 3300 liters of liquid manures are through screening processing continuously; 60 orders, 200 orders and 325 mesh sieves have been held back the excrement slag bigger than egg capsule, and all flow through 1250 mesh sieves less than the excrement slag and the water of egg capsule, the trapped substance that resides in 1250 mesh sieves have only egg capsule with the identical impurity of egg capsule size; Its total amount is about 2% of liquid manure total amount, promptly 66 liters.With above-mentioned large vol whizzer centrifugal treating, 1 bench centrifuge needs 33 times, if use 6 bench centrifuges, then the work of every machine split run is 5.5 times.Meet the matched and the post setting of medium-sized vaccine factory, if will enlarge output, only needing increases by 6 bench centrifuges, can reach and produce the above pentavalent coccidial vaccine of 500,000,000 plumages per year, as the production trivalent vaccine then output can reach more than 700,000,000 plumages.
(3) excrement appearance adds a small amount of stomach en-(0.05mg/ restrains ight soil) in handling, and its role is to destroy intestinal tissue, foreign organic matter in the ight soil, helps screening and filters, and simultaneously, also can remove the adhesion that reaches egg capsule and foreign material between the egg capsule.
(4) shortened egg capsule and separated the time spent, compared, saved disengaging time more than 50% with conventional art.Reduced centrifugal number of times, traditional stripping technique is after filtering through 200 mesh sieves, and egg capsule and water and bulky stool appearance impurity concrete must pass through centrifugal treating and just can make egg capsule and water sepn together, and then it is floating to carry out salt solution.And new technology has been saved centrifugal before floating, and reason is directly to have accomplished 3 separation: 1. big than egg capsule foreign material; 2. than little foreign material of egg capsule and 3. water.
For industrialization production, as continue to use traditional technology, the spinning of this operation needs equally a large amount of repeatedly centrifugal repeatedly, is impracticable, and new technology has been eliminated " bottleneck " of original technology fully.
(5) significantly reduce the usage quantity of saturated brine, reduced cost.Because sieve apparatus has increased by 325 order removal of impurities continuously, has reduced the swimmer total amount, can reduce the usage quantity about 30% of saturated brine.
(6) improved the separation rate of egg capsule,, increased contacting of filtrate and screen cloth because sieve apparatus can produce the ternary motion continuously, can more effective filtration egg capsule, make the separation rate raising 10% of egg capsule, finally can increase the output 10% of vaccine.
Embodiment
Get infected chicken fresh excreta 20kg, add 60 liters of abundant mixings of water, add stomach en-1000mg, mixing leaves standstill 10min; Add 140 premium on currency afterwards again, be mixed into liquid manure; About 220 liters of liquid manure total amount; Start continuous sieve apparatus; Liquid manure from charging opening at the uniform velocity (10-15 liter/minute) add, add 20 liters of entry after liquid manure adds again, keep the sieve apparatus running simultaneously; To wash the not filtration egg capsule that is retained in the 1st, 2,3 layer of screen cloth as far as possible; Take off the 4th layer of sub-sieve afterwards, water flushing trapped substance is in heaps, is collected in the graduated container of tool subsequent use; (V: V) the saturated brine mixing is floating, is sub-packed in the 500ml centrifuge tube, and 2500rpm * 10min is centrifugal to add 5 times by the trapped substance amount; The swimmer on sucking-off centrifuge tube surface adds at least 10 times of water mixings, and 2500rpm * 10min is centrifugal; It is centrifugal that taking precipitate adds enough water (filling it up with centrifuge tube) 2500rpm * 10min, and throw out is egg capsule.
Claims (1)
1. a chicken Eimeria (Eimeria coccidia) egg capsule batch preparation is characterized in that:
A, excrement appearance are handled: get the infected chicken fresh excreta; Add the water dilution mixing of 3 times of its weight, behind the adding stomach en-thorough mixing, leave standstill the water that adds 7 times of excrement weight after 10 minutes again; Mixing makes into liquid manure, and wherein pepsic add-on is a 0.05mg/ gram ight soil;
B, screening processing continuously: liquid manure is handled through multi-layer type vibrosieve filter, promptly at the uniform velocity adds liquid manure from charging opening, is filtered by the 1st layer to the 4th layer continuous sub-sieve; Add the water flushing simultaneously; After take off the 4th layer of screen cloth, water washes out all trapped substances, to the graduated containers for future use of tool;
Multi-layer type vibrosieve filter: or claim the multi-layer type vibrating screen classifier, establish 4 layers altogether, the 1st layer of 60 order, the 2nd layer of 200 order, the 3rd layer of 325 order, the 4th layer of 1250 order;
C, saturated brine are floating centrifugal: it is floating that trapped substance adds the abundant mixing of saturated brine of 5 times of volumes, and 2500rpm * 10min is centrifugal;
D, egg capsule centrifugation: the swimmer on sucking-off centrifuge tube surface adds to and is less than 10 times of water mixings of its volume, and 2500rpm * 10min is centrifugal;
E, egg capsule purify: remove supernatant, add water to centrifuge tube and be full of mixing, 2500rpm * 10min is centrifugal, and throw out is egg capsule.
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Families Citing this family (8)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102726617A (en) * | 2012-07-05 | 2012-10-17 | 烟台青大生物制药有限公司 | Feed additive composed of cellulose and sodium bicarbonate, feed and applications of feed additive and feed, as well as chicken coccidiosis live oocyst 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 |
| CN103881913A (en) * | 2014-03-07 | 2014-06-25 | 广东省农业科学院动物卫生研究所 | Industria production method for separating and purifying chicken eimeria coccidium oocyst |
| CN111154653B (en) * | 2020-01-16 | 2020-11-20 | 中国农业科学院兰州兽医研究所 | Method for purifying insect eggs through iodixanol density gradient centrifugation |
| CN111733079B (en) * | 2020-07-08 | 2021-09-07 | 佛山市正典生物技术有限公司 | Separation composition, preparation method thereof and chicken coccidian oocyst separation method using same |
| 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 |
| CN114381375A (en) * | 2021-12-21 | 2022-04-22 | 佛山市正典生物技术有限公司 | Treatment method and application of chicken Eimeria maxima excrement mucus |
| CN114380474B (en) * | 2021-12-21 | 2022-10-21 | 佛山市正典生物技术有限公司 | Excrement treating agent and method and application thereof for treating excrement containing chicken cecum core |
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