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Separation and purification method of eimeria tenella sporozoite and inoculation method

A technology of Eimeria and Eimeria balls is applied in the field of separation and purification of sporozoites of Eimeria tenella. Injury, increase the success rate of vaccination, the effect of low degree of dependence

Pending Publication Date: 2021-06-01
SOUTH CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, this method takes a long time and involves more equipment, which brings high instability to obtain cloned oocyst populations
In the prior art, there are also reports of using a microscope to separate monosporangia and establish Eimeria tenella clones, but the success rate is only 17.8%.
It is also reported in the prior art that the monospores are absorbed by microscope operation and injected into the chicken cecum exposed after dissection to realize the isolation and inoculation of the monospores, but the success rate is only 22%, and the operation difficulty factor is large. The demand for precision equipment is high, which brings a lot of inconvenience to the research

Method used

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  • Separation and purification method of eimeria tenella sporozoite and inoculation method
  • Separation and purification method of eimeria tenella sporozoite and inoculation method
  • Separation and purification method of eimeria tenella sporozoite and inoculation method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Propagation, isolation and purification of Eimeria tenella oocysts.

[0041] 1. Eimeria tenella Zengcheng strain (ZC strain) was inoculated into five 1-week-old Qingyuan Ma chickens, and the coccidia were checked by flotation in saturated saline on the 5th day after infection (5days post inoculation, 5d p.i.). , collect 6-10d p.i. containing feces containing oocysts.

[0042] 2. Add 2 times the volume of water to dilute the feces, pass through an 80-mesh metal mesh sieve, knead the feces while sieving, add 2 times the volume of water to the feces residue, mix and pass through an 80-mesh metal mesh sieve to collect the filtrate; pass the filtrate through 100 Mesh metal mesh sieve, knead the feces during sieving, and then pass the collected filtrate through a 300 mesh nylon mesh sieve to collect the filtrate.

[0043] 3. Put the filtrate in a centrifuge tube, centrifuge at 1500rpm for 3min, collect the precipitate, and remove the white urate impurities in the upper layer...

Embodiment 2

[0051] Injection site localization.

[0052] Three 1-week-old Qingyuan Ma chickens were used for clinical trials. First, 3 chicks were taken for abdominal autopsy, and the specific positions of the gizzard and the middle of the small intestine (jejunum and ileum) of the chicks were measured using a vernier caliper, and the length, width, and height of the organs from the tail of the chicken keel were recorded respectively. Two more chicks were taken, and the bromophenol blue indicator was sucked up with a 1mL syringe, and injected into the predicted position of gizzard and small intestine in vivo. Finally, the abdominal cavity of the chick was dissected, and the injection of bromophenol blue in the small intestine, gizzard serosa layer and contents were observed, and the exact location of gizzard and small intestine injection was finally determined.

[0053] Before officially entering the inoculation experiment, first understand the 1-week-old No-Ma chicken ( figure 2 A) Th...

Embodiment 3

[0055] Purification and inoculation of Eimeria tenella sporangia.

[0056] 1. Take the sporulated oocysts purified in Example 1, add sterile glass beads at a volume ratio of 1:1, shake and break for 10 minutes, observe under a microscope until all oocyst walls are broken, repeat the observation 3 times, and wait until all the sporangia have escaped. Stop vibrating when going out. Wash 3 times with sterile PBS solution, centrifuge the washed sporangia liquid at 3600rpm for 5min, and discard the supernatant. The precipitate was mixed evenly with 20mL PBS, filtered through a metal mesh sieve with a pore size of 20μm, centrifuged at 3600rpm for 5min, the supernatant was discarded, and 10mL PBS was added to mix well for later use.

[0057] 2. Inject the purified sporangia into the gizzard and small intestine of 1-week-old Qingyuan No-Ma chickens through a 1mL syringe. The gizzard injection dose is 100,000 sporangia / feather; the control group is set up and an equal volume of PBS is...

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Abstract

The invention provides a separation and purification method of eimeria tenella sporozoite and an inoculation method, and relates to the technical field of biology. The separation and purification method comprises the following steps: taking infected chicken manure, adding water, and filtering; centrifuging, adding a saturated saline solution into the precipitate, centrifuging, and adding water into the supernate to obtain an oocyst diluent; centrifuging, and adding chloramine T solution into precipitate to obtain sporulated oocyst suspension; centrifuging, adding saturated saline solution into the precipitate for resuspending, centrifuging and collecting oocyst precipitate; adding a sodium hypochlorite solution for resuspension, and collecting oocysts; and shaking and crushing, stopping shaking when all sporangia escapes, centrifuging to obtain sporangia, adding pancreatin digestive juice for digestion, filtering, centrifuging, and collecting precipitate to obtain eimeria tenella sporozoites. According to the inoculation method, the sporangiums or sporozoites are accurately injected into the middle sections of muscular stomachs and small intestines of the chickens, and successful infection is achieved. The purification degree of the sporangiums and sporozoites obtained by the purification method is high, the experimental operation is simple and convenient, and the infection success rate after injection inoculation reaches 100%.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a separation and purification method and an inoculation method of Eimeria tenella sporozoites. Background technique [0002] Coccidiosis is considered to be one of the important infectious diseases of poultry, causing economic losses of more than 2 billion pounds to the poultry industry worldwide every year. Eimeria coccidiosis is the main pathogen causing coccidiosis in poultry, which can cause symptoms such as diarrhea, weight loss, reduced egg production, and sometimes even death. In addition, Eimeria infection can facilitate opportunistic infections with other pathogens such as Clostridium perfringens. Also, its oocysts are highly resistant to the environment, which makes control measures rather difficult. Conventional disease control strategies rely primarily on preventive medications, but Eimeria, the cause of coccidiosis in chickens, has developed resistance to most anticocc...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/10C12N1/02A01K67/02C12R1/90
CPCC12N1/10C12N1/02A01K67/02
Inventor 林瑞庆周德荣陆肖蔡晓懿方园婷蒋嘉豪孟甜王瑞珍翁亚彪
Owner SOUTH CHINA AGRI UNIV
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