CN103710265A - Application of chloramine B solution in chicken coccidiosis oocyst culture and preservation - Google Patents
Application of chloramine B solution in chicken coccidiosis oocyst culture and preservation Download PDFInfo
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- CN103710265A CN103710265A CN201310717171.1A CN201310717171A CN103710265A CN 103710265 A CN103710265 A CN 103710265A CN 201310717171 A CN201310717171 A CN 201310717171A CN 103710265 A CN103710265 A CN 103710265A
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- 241000287828 Gallus gallus Species 0.000 title claims abstract description 51
- QDHHCQZDFGDHMP-UHFFFAOYSA-N Chloramine Chemical compound ClN QDHHCQZDFGDHMP-UHFFFAOYSA-N 0.000 title claims abstract description 44
- 210000003250 oocyst Anatomy 0.000 title claims abstract description 30
- 238000004321 preservation Methods 0.000 title abstract description 25
- 208000003495 Coccidiosis Diseases 0.000 title abstract description 7
- 206010023076 Isosporiasis Diseases 0.000 title abstract description 7
- 239000007864 aqueous solution Substances 0.000 claims abstract description 26
- 239000000243 solution Substances 0.000 claims abstract description 25
- 235000015097 nutrients Nutrition 0.000 claims description 10
- 239000007788 liquid Substances 0.000 claims description 9
- KMUONIBRACKNSN-UHFFFAOYSA-N potassium dichromate Chemical compound [K+].[K+].[O-][Cr](=O)(=O)O[Cr]([O-])(=O)=O KMUONIBRACKNSN-UHFFFAOYSA-N 0.000 abstract description 14
- 241000499563 Eimeria necatrix Species 0.000 abstract description 12
- 241000223934 Eimeria maxima Species 0.000 abstract description 9
- 241000223924 Eimeria Species 0.000 abstract description 8
- 241000223932 Eimeria tenella Species 0.000 abstract description 8
- 230000000694 effects Effects 0.000 abstract description 2
- 230000007918 pathogenicity Effects 0.000 abstract 1
- 239000003761 preservation solution Substances 0.000 abstract 1
- 230000028070 sporulation Effects 0.000 abstract 1
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- 235000013330 chicken meat Nutrition 0.000 description 40
- 238000012360 testing method Methods 0.000 description 15
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- 210000004534 cecum Anatomy 0.000 description 13
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- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 3
- 241000223931 Eimeria acervulina Species 0.000 description 2
- 208000032843 Hemorrhage Diseases 0.000 description 2
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 2
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- 230000017074 necrotic cell death Effects 0.000 description 1
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- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
Abstract
The invention discloses an application of a chloramine B solution in chicken coccidiosis oocyst culture and preservation. According to the invention, study finds out that the sporulation rate of the chicken coccidiosis oocysts can be effectively improved with the chloramine B aqueous solution as a chicken coccidiosis oocyst culture solution. The oocysts of the chicken eimeria tenella, eimeria acevulina, eimeria maxima and eimeria necatrix are not affected in activity if being preserved within 6 months; if being preserved within 8 months, the yield of the oocysts is reduced within 25%; the pathogenicity is not obviously affected. Therefore, the chloramine B aqueous solution can be used as the culture and preservation solution for the chicken coccidiosis oocysts in stead of potassium dichromate.
Description
Technical field
The present invention relates to the application of chloramine B solution in chicken coccidial oocyst is cultivated and preserved.
Background technology
To the cultivation of coccidial vaccine egg capsule and preserve the success or failure of the cost of direct relation vaccine and immunity.In laboratory, operable egg capsule nutrient solution has 0.1% formaldehyde, 1% sulfuric acid and 2.5% potassium bichromate etc. at present, conventionally use 2.5% potassium bichromate solution, but potassium bichromate is a kind of heavy metal compound, life-time service can residue in animal body and environment, affects human and animal healthy.Thereby urgently research and develop a kind of to human body and harmless coccidial vaccine egg capsule nutrient solution and the preservation liquid of animal.
Summary of the invention
Present inventor finds under study for action: chicken coccidial oocyst can be cultivated and long-term preservation in chloramine B solution, chloramine B formal name used at school is benzsulfamide chlorine sodium salt, English name is chloramine B, crystal or powder for white or micro-Huang, soluble in water, good stability, be a kind of sterilizing agent with broad-spectrum bactericidal capacity, be widely used in the sterilization of health care, drinking-water, tableware, vessel, swimming pool etc.The present invention is using it as chicken coccidial oocyst nutrient solution and preserve liquid.
Thereby, the object of the present invention is to provide the application of chloramine B solution in vaccine to chicken coccidiosis is cultivated and preserved.
Described chloramine B solution can be the aqueous solution, and concentration is 0.6～10%, is preferably 1.2%, by mass percentage.
The invention has the beneficial effects as follows:
Utilize chloramine B solution can effectively improve the spore rate of chicken coccidial oocyst as the nutrient solution of chicken coccidial oocyst.And chicken is tender, heap type, that huge and Eimeria Necatrix egg capsule is saved to 6 months vigor in the chloramine B aqueous solution is still unaffected, is saved to 8 months egg capsule output reductions and is no more than 25%, still shows excellent activity.Therefore, chloramine B solution can replace potassium bichromate as the cultivation of chicken coccidial oocyst and preserve liquid.
Embodiment
Below in conjunction with embodiment, the present invention is further illustrated, but be not limited to this.If no special instructions, " % " below all refers to mass percent.
Embodiment
Below we can and preserve liquid as the nutrient solution of chicken coccidial oocyst with the experimental verification chloramine B aqueous solution.
1. test materials
1.1 experimental animals: Lingnan Yellow chick, by animal science institute of Guangdong Academy of Agricultural Sciences, provided, raise in the special-purpose Animal House of having sterilized; Chicken coop and the equal strict sterilization of vessel used, free choice feeding and purified drinking water; Before experiment, observe chick and have or not clinical symptom and continuous 2d to check that ight soil has or not coccidian oocyst, standby.
The 1.2 102 type broiler chicken Liao You Xin Nan of Guangdong Province feed corporation,Ltd customize the material of brooding, containing any anticoccidial drug.
The precocious system of 1.3 Eimeria tenella GD strains, heap type Eimeria GD strain, Eimeria maxima GZ strain, Eimeria Necatrix GD strain precocity are that Guangdong Academy of Agricultural Sciences animal health institute Parasites research department preserves.
1.4 chloramine Bs, potassium bichromate: purchased from lark prestige Science and Technology Ltd..
2. test method
The preparation of the fresh egg capsule of 2.1 chicken coccidia
Egg capsule 5 * 10 mixes in the precocious system of 14 Japanese instar chickling per oral inoculation Eimeria tenella GD strains, heap type Eimeria GD strain, Eimeria maxima GZ strain, the precocious system of Eimeria Necatrix GD strain etc.
4individual (wherein heap type Eimeria GD strain 2 * 10
4, other three kind 1 * 10
4), collection attack after worm 7 discharge feces just, press < < coccidiosis of chicken and learn > > (Suo Xun, Li Guoqing compiles, 1998) method separation and the fresh chicken coccidial oocyst of purifying.
The Sporulated of 2.2 chicken coccidial oocysts in the chloramine B aqueous solution
The potassium dichromate aqueous solution of preparation 2.5% and the chloramine B aqueous solution of 5 kinds of concentration: 0.6%, 1.2%, 2.4%, 4.8%, 10%; The new freshly-slaughtered poultry coccidia of fresh collection is mixed to egg capsule and be divided into 7 groups, every group 2.0 * 10
5individual egg capsule, uses respectively the chloramine B aqueous solution, high purity water and potassium bichromate resuspended, is placed in 28 ℃ of shaking bath concussions and cultivates.Respectively at 15h after cultivating, 20h, 25h, 30h, 40,60h, blood counting chamber method records the quantity of Sporulated Oocysts, calculates spore rate.
2.3 steriling test
After Sporulated finishes, get resuspended liquid 100 μ L for every group, be coated with LB dull and stereotyped, put in 37 ℃ of incubators and cultivate 72h, observe bacterial growth situation.
2.4 chickens are tender, heap type, the research of huge and Eimeria Necatrix egg capsule shelf time in the chloramine B aqueous solution
2.4.1 Eimeria tenella GD strain precocity is the test of egg capsule preservation period
By E.tenella GD strain precocity, be that egg capsule is stored in the chloramine B aqueous solution, be placed in respectively 4 ℃ of refrigerators 0 to 12 months, dosage peroral infection 7 age in days chickens as shown in the table, after observe infecting, the death of test chicken in 1 week, clinical bloody stool, caecum egg capsule are counted, caecum lesion is scored and the relative weight gain situation, the impact of the egg capsule of evaluating different preservation perives on 7 age in days chicken virulencies.
Table 1E.tenella GD strain precocity is egg capsule preservation period test design
2.4.2 Eimeria Necatrix GD strain precocity is the test of egg capsule preservation period
By E.necatrix GD strain precocity, be that egg capsule is stored in the chloramine B aqueous solution, be placed in respectively 4 ℃ of refrigerators 0 to 12 months, dosage peroral infection 7 age in days chickens as shown in table 2, after observe infecting the death of test chicken in 1 week, clinical bloody stool, caecum egg capsule count, small intestine lesion score and the relative weight gain situation, the impact of the egg capsule of evaluating different preservation perives on 7 age in days chicken virulencies.
Table 2 Eimeria Necatrix GD strain precocity is egg capsule preservation period test design
2.4.3 Eimeria maxima GZ strain precocity is the test of egg capsule preservation period
E.maxima GZ strain egg capsule is stored in the chloramine B aqueous solution, be placed in respectively 4 ℃ of refrigerators 0 to 12 months, dosage peroral infection 7 age in days chickens as shown in table 3, the death of test chicken in 1 week after observe infecting, infect after the 8th day egg capsule count, small intestine lesion score and the relative weight gain situation, the impact of the egg capsule of evaluating different preservation perives on 7 age in days chicken virulencies.
Table 3 Eimeria maxima GZ strain precocity is egg capsule preservation period test design
2.4.4 heap type Eimeria GD strain precocity is the test of egg capsule preservation period
E.acervulina GD strain egg capsule is stored in the chloramine B aqueous solution, be placed in respectively 4 ℃ of refrigerators 0 to 12 months, dosage peroral infection 7 age in days chickens as shown in table 4, the death of test chicken in 1 week after observe infecting, infect after the 7th day egg capsule count, duodenum pathology is scored and the relative weight gain situation, the impact of the egg capsule of evaluating different preservation perives on 7 age in days chicken virulencies.
Table 4 heap type Eimeria GD strain precocity is egg capsule preservation period test design
3 results
The Sporulated of 3.1 chicken coccidial oocysts in different nutrient solutions
Result is as shown in table 5, and the most applicable cultivation chicken coccidial oocyst of the chloramine B aqueous solution that concentration is 1.2%, can replace potassium bichromate, improves the spore rate of egg capsule.
The spore rate of table 5 chicken coccidial oocyst in different nutrient solutions
3.2 steriling test
Result shows, except high purity water group flat board covers with bacterium, potassium bichromate and the chloramine B group flat board bacterium that all do not grow, illustrates that the chloramine B aqueous solution can replace potassium bichromate bacteria growing inhibiting.
The shelf time of 3.3 chicken coccidial oocysts in nutrient solution
3.3.1 Eimeria tenella GD strain preservation period test-results
Test-results shows, E.tenella GD strain precocity be egg capsule in the chloramine B aqueous solution, under 4 ℃ of conditions, preserve in 6 months, vigor is not affected, in 8 months, in egg capsule yield reducation 25%, virulence is not all significantly affected, as table 6.
Table 6E.tenella GD strain precocity is the preservation period test-results of egg capsule
3.3.2 Eimeria Necatrix GD strain preservation period test-results
Test-results shows, E.necatrix GD strain precocity be egg capsule in the chloramine B aqueous solution, under 4 ℃ of conditions, preserve in 6 months, vigor is not affected, in 8 months, in egg capsule yield reducation 25%, virulence is not all significantly affected, as table 7.
Table 7E.necatrix GD strain precocity is the preservation period test-results of egg capsule
3.3.3 Eimeria maxima GZ strain preservation period test-results
Test-results demonstration, E.maxima GZ strain egg capsule, in the chloramine B aqueous solution, is preserved in 6 months under 4 ℃ of conditions, and vigor is not affected, and in 8 months, in egg capsule yield reducation 25%, virulence is not all significantly affected, as table 8.
The preservation period test-results of table 8E.maxima GZ strain egg capsule
3.3.4 heap type Eimeria GD strain preservation period test-results
Test-results demonstration, E.acervulina GD strain egg capsule, in the chloramine B aqueous solution, is preserved in 6 months under 4 ℃ of conditions, and vigor is not affected, and in 8 months, in egg capsule yield reducation 25%, virulence is not all significantly affected, as table 9.
The preservation period test-results of table 9E.acervulina GD strain egg capsule
4 conclusions
This research is by the screening to chloramine B concentration of aqueous solution, inquire into the chloramine B aqueous solution to the cultivation of chicken coccidial oocyst and preservation condition, find that chicken coccidial oocyst can improve spore rate in the chloramine B aqueous solution, especially in 1.2% chloramine B aqueous solution miospore rate, can bring up to 93%, and growth that can anti-bacteria; And chicken is tender, heap type, huge and Eimeria Necatrix egg capsule were saved in 6 months, vigor is not affected, and in 8 months, in egg capsule yield reducation 25%, virulence is not all significantly affected.Therefore, the chloramine B aqueous solution can replace potassium bichromate as the cultivation of chicken coccidial oocyst and preserve liquid.
Above embodiment is only for introducing preferred case of the present invention, and to those skilled in the art, any apparent changes and improvements of carrying out in the scope that does not deviate from spirit of the present invention, all should be regarded as a part of the present invention.
Note: 1
Bloody stool is scored: 0 minute, without bloody stool; 1 minute, be less than 25% blood stool; 2 minutes, the blood stool of 25-50%; 3 minutes, the blood stool of 50-75%; 4 minutes, the blood stool more than 75%.
Lesion score standard
After infecting, the pathology that 1.1 Eimeria tenellas cause cuts open inspection caecum (when both sides caecum lesion is inconsistent, being as the criterion with a serious side) between 5th～7 days.
0 minute without the visible pathology of naked eyes.
Within+1 minute, cecal content is normal, and intestines wall mucous membrane face is dispersed in blutpunkte as seen on a small quantity, or blood spots.
+ 2 minutes cecal content band blood, intestines wall mucous membrane face blutpunkte or blood spots quantity are comparatively intensive, intestines wall thickening.
+ 3 minutes caecum silght enlargements, do not have normal caecum ight soil in enteric cavity, intestinal mucosa thickens, and be full of garnet blood or occur caecum core (being the cheesy Banana Type block of canescence) in caecum chamber.
+ 4 minutes caecum heavy swellings, do not have caecum excrement or have been enclosed in intestines core in caecum chamber, be full of downright bad sludged blood and caecum mucous membrane fragment in enteric cavity.The chicken lethal because of this coccidia be designated as+and 4 minutes.
After infecting, the pathology that 1.2 Eimeria Necatrixes cause cuts open inspection small intestine between 5th～7 days.
0 minute without the visible pathology of naked eyes.
Blutpunkte and white point that+1 minute visible tip-like size of small intestine stage casing serosal surface is dispersed in, visible a small amount of orange content in enteric cavity, intestines wall slightly thickens.
+ 2 minutes intestines wall thickenings, serosal surface has a large amount of blutpunktes and white point, and there is slight flatulence in small intestine stage casing, and enteric cavity part is mixed with sludged blood.
Within+3 minutes, serosal surface is covered with red blutpunkte and white point, enteric cavity air-blowing expansion, and intestines wall obviously thickens, and contains blood clot and the downright bad epithelium coming off of volume in whole intestinal contents, and intestinal mucosa face is coarse, there is no normal intestinal contents.
The swelling of+4 minutes small intestine miding level heights, there is atrophy, obviously shorten in intestinal segment, and pathology extends to duodenum and small intestine back segment, contains color of soy sauce or brown mucus in small intestine contents, and intestinal mucosa is hemorrhage, necrosis.The chicken lethal because of this coccidia be designated as+and 4 minutes.
After infecting, the pathology that 1.3 Eimeria maximas cause cuts open inspection small intestine between 5th～7 days.
0 minute without the visible pathology of naked eyes.
, there is a small amount of orange mucus+1 minute visible less blutpunkte of small intestine stage casing mucous membrane face in enteric cavity.
+ 2 minutes there is slight flatulence in small intestine stage casing, and the visible many blutpunktes of serosal surface, extend to duodenum, visible a large amount of orange mucus in enteric cavity.
Dilatation of intestine in+3 minutes, does not have normal intestinal contents, and intestinal contents is mixed with blood clot and orange mucus, the attenuation of intestines wall, and mucous membrane face is coarse.
Intestinal tube swelling in+4 minutes, intestinal contents thickness, is reddish-brown, and is mixed with a large amount of blood clots, is the light meat water colour of washing, and intestinal mucosa is congested, hemorrhage, come off.The chicken lethal because of this coccidia be designated as+and 4 minutes.
After infecting, the pathology that 1.4 heap type Eimerias cause cuts open inspection duodenum between 5th～7 days.
0 minute without the visible pathology of naked eyes.
+ 1 minute duodenal serosa face and mucous membrane face are all dispersed in striation hickie as seen, and transversely arranged, it is trapezoidal that outward appearance is.
Within+2 minutes, pathology is comparatively intensive, but does not merge, and on Duodenal Mucosa, coated with transversely arranged striation hickie, outward appearance is scalariform, and pathology extends to jejunum, and alimentary canal content is thinner, and intestinal mucosa thickens.
+ 3 minutes visible intensive canescence focuses of Duodenal Mucosa face, pathology extends to yolk stalk place, and enteron aisle is pale, has volume moisture in enteric cavity, and content is water sample liquid.
It is light grey that+4 minutes Duodenal Mucosa faces are, and striation hickie merges completely, is full of butteriness exudate in enteric cavity, intestines wall thickening, and the chicken lethal because of this coccidia be designated as+and 4 minutes.
Claims (7)
1. the application of chloramine B solution in chicken coccidial oocyst is cultivated and preserved.
2. application according to claim 1, is characterized in that described chloramine B solution is the chloramine B aqueous solution.
3. application according to claim 1 and 2, is characterized in that, the concentration of described chloramine B solution is 0.6～10%, by mass percentage.
4. application according to claim 3, is characterized in that, the concentration of described chloramine B solution is 1.2%, by mass percentage.
5. application according to claim 1 and 2, is characterized in that, described application refers to chloramine B solution as the nutrient solution of chicken coccidial oocyst or preserves liquid.
6. a chicken coccidial oocyst nutrient solution, it contains 0.6～10% chloramine B, by mass percentage.
7. chicken coccidial oocyst is preserved a liquid, and it contains 0.6～10% chloramine B, by mass percentage.
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| CN107019799A (en) * | 2016-01-30 | 2017-08-08 | 天津赫莱恩特生物科技有限公司 | A kind of application of chicken coccidiasis tetravalence attenuated live vaccines composition in production |
| CN107022488A (en) * | 2016-01-29 | 2017-08-08 | 天津赫莱恩特生物科技有限公司 | A kind of chicken coccidial oocyst protection solution and preparation method thereof |
| CN107022487A (en) * | 2016-01-30 | 2017-08-08 | 天津赫莱恩特生物科技有限公司 | The preparation method and applications of coccidian oocyst activity protecting agent in a kind of chicken manure |
| CN107347873A (en) * | 2017-07-12 | 2017-11-17 | 中国农业大学 | A kind of Eimeria freezes agent and cryopreservation methods |
| CN108085322A (en) * | 2018-01-30 | 2018-05-29 | 广东省农业科学院动物卫生研究所 | A kind of Eimeria species autophagy GAP-associated protein GAP Etatg6 coding gene sequences and its acquisition methods |
| 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 |
| CN108251436A (en) * | 2018-01-30 | 2018-07-06 | 广东省农业科学院动物卫生研究所 | A kind of Eimeria species autophagy GAP-associated protein GAP Etatg8 coding gene sequences and its acquisition methods |
| CN111670898A (en) * | 2020-07-16 | 2020-09-18 | 松山湖材料实验室 | Coccidia oocyst cryopreservation agent and preparation method and application thereof |
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