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Culture method and application of lawsonia intracellularis

A culture method and technology of Lawsonia, applied in the field of culture of Lawsonia intracellularis, can solve the problems of not fully reflecting the infection characteristics of Lawsonia intracellularis, unsuccessful separation and culture, long cell culture cycle, etc. Research, easy practical guidance and data support, the effect of short training period

Pending Publication Date: 2019-11-19
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Due to the special culture conditions, less than 25 strains have been successfully isolated in the world, and fewer strains are preserved. There is no relevant report on successful isolation and culture in China so far.
However, the cells used to culture LI in the current reports are not derived from host cells, and cannot fully reflect the infection characteristics of Lawsonia intracellulare in the pig gastrointestinal tract; in addition, the prior art cell culture cycle is long, and the isolation and detection of the bacteria in the later stage very unfavorable

Method used

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  • Culture method and application of lawsonia intracellularis
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  • Culture method and application of lawsonia intracellularis

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Embodiment 1

[0049] The culture method of embodiment 1 Lawsonia intracellulare comprises the following steps:

[0050] 1) Cell culture: Take out the cells from the liquid nitrogen tank, and quickly shake them in a 37°C water bath to completely melt within 1 min. The thawed cells (cell concentration is about 0.5×10 6 / mL~2×10 8 / mL) into 4mL DMEM complete medium, at 37°C, 5% CO 2 cultured in an incubator. After the cells adhered to the wall, the cells were slowly washed 2 times with PBS. When the confluence of the cells reached 80%, the cells were digested with trypsin, the cell suspension was mixed and spread in a 24-well plate, and 0.1 mL of the cell suspension (concentration 0.5×10 6 / mL) and 0.9mL DMEM complete medium, mix well and culture in the incubator.

[0051] 2) Inoculation and culture of Lawsonia: when the cells were attached to the wall for 6 hours, the positive strains of Lawsonia intracellularis (concentration of 10 4.9 TCID 50 / ml) and cell culture medium were inocula...

Embodiment 2

[0053] The cultivation method of embodiment 2 Lawsonia intracellulare comprises the following steps:

[0054] 1) Cell culture: Take out the porcine intestinal cells from the liquid nitrogen tank, and quickly shake them in a 37°C water bath to completely melt within 1 min. The thawed cells (cell concentration is about 0.5×10 6 / mL~2×10 8 / mL) into 4mL DMEM complete medium, at 37°C, 5% CO 2 cultured in an incubator. After the cells adhered to the wall, the cells were slowly washed 3 times with PBS. When the confluence of the cells reached 85%, the cells were digested with trypsin, the cell suspension was mixed and spread in a 24-well plate, and 0.1 mL of the cell suspension was added to each well (the concentration was 1×10 7 / mL) and 0.9mL DMEM complete medium, mix well and culture in the incubator.

[0055] 2) Inoculation and culture of Lawsonia: when the cells were adhered to the wall for 7 hours, the positive strains of Lawsonia intracellularis (concentration of 10 4.9...

Embodiment 3

[0058] The culture method of Lawsonia intracellulare comprises the following steps:

[0059] 1) Cell culture: Take out the porcine intestinal cells from the liquid nitrogen tank, and quickly shake them in a 37°C water bath to completely melt within 1 min. The thawed cells (cell concentration is about 0.5×10 6 / mL~2×10 8 / mL) into 4mL DMEM complete medium, at 37°C, 5% CO 2 cultured in an incubator. After the cells adhered to the wall, the cells were slowly washed 2 times with PBS. When the confluence of the cells reached 90%, the cells were digested with trypsin, the cell suspension was mixed and spread in a 24-well plate, and 0.1 mL of the cell suspension (concentration 2×10 8 / mL) and 0.9mL DMEM complete medium, mix well and culture in the incubator.

[0060] 2) Inoculation and cultivation of Lawsonia: when the cells were adhered to the wall for 8 hours, the positive strains of Lawsonia intracellularis (concentration of 10 4.9 TCID 50 / ml) and cell culture medium were ...

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Abstract

The invention discloses a culture method of lawsonia intracellularis. The culture method comprises the following steps of step 1, performing adherence culture to obtain pig intestinal tract cells; step 2, after cell adherence culture for 6-8h, inoculating cells with lawsonia intracellularis and cell culture fluid in the volume ratio being (0.01-1) to 1, performing placing in improved micro aerobicenvironment, and performing culture for 1-96h; and step 3, performing trypsinization on infected cells, collecting the infected cells, performing re-suspending on the infected cells with cell frozen-storage fluid, and performing transferring into a liquid nitrogen jar for storage. The invention further discloses an application of the lawsonia intracellularis cultured by the culture method. The lawsonia intracellularis is applied to research of lawsonia intracellularis separated culture and pathogenicity characteristics. The pig intestinal tract cells are used for culturing the lawsonia intracellularis, the culture in vitro environment in the body of a host is strictly simulated, besides, an improved micro aerobic culture box is used for culturing the lawsonia intracellularis, the operation is simple, the culture cycle is short, the cost is low, and the success rate of culturing the lawsonia intracellularis is greatly increased.

Description

technical field [0001] The invention belongs to the field of veterinary biotechnology, and in particular relates to a cultivation method and application of Lawsonia intracellulare. Background technique [0002] Lawsonia intracellularis (Lawsonia Intracellularis, LI) belongs to the genus Desulfovibrio, a gram-negative bacterium that is curved, comma-shaped or S-shaped, with a size of 1.25-1.75 μm×0.25-0.43 μm, obligate Parasitic in the intestinal cells of animals, it can cause porcine proliferative enteritis (PPE) characterized by adenomatous proliferation of immature enterocytes in the ileum and colonic crypts. Although the mortality rate of PPE is not high, the disease can cause clinical symptoms such as diarrhea and slow growth in sick pigs, seriously reducing the feed utilization rate of animals, and causing huge economic losses. According to reports, the annual losses caused by PPE in the United States and the United Kingdom are as high as 20 million US dollars and 4 mi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/20C12N5/071C12R1/01
CPCC12N1/20C12N5/0679
Inventor 谢书宇陈冬梅袁宗辉罗万和武梦茹孟奎宇潘源虎瞿玮程古月黄玲利谢长清王旭陶燕飞刘振利
Owner HUAZHONG AGRI UNIV
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