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Construction method and application of diarrhetic escherichia coli infected animal model

A technology for Escherichia coli and animal infection, which is applied in the field of constructing an animal model of diarrheal Escherichia coli infection, and can solve the problems of unclear pathogenic mechanism and host immune regulation mechanism.

Active Publication Date: 2022-07-15
CHINA AGRI UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Cattle with Escherichia coli diarrhea are often accompanied by severe colonic inflammation, which demonstrates the important role of colonic inflammation in the occurrence and development of Escherichia coli diarrhea in dairy cattle, but the pathogenic mechanism of DEC and the host immune regulation mechanism still unclear

Method used

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  • Construction method and application of diarrhetic escherichia coli infected animal model
  • Construction method and application of diarrhetic escherichia coli infected animal model
  • Construction method and application of diarrhetic escherichia coli infected animal model

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Example 1 Isolation and purification of DEC strains

[0034] The DEC strain (Escherichia coli 1587) was isolated from calves clinically suffering from Escherichia coli diarrhea, which can cause acute diarrhea in dairy cows. Through 16S rDNA sequencing (SEQ ID NO: 1), according to the sequencing results and the above-mentioned microbiological characteristics and physiological and biochemical characteristics, the bacteria was identified as diarrhea Escherichia coli (Diarrheagenic Escherichia coli), the serotype was O101:H9, and the bacteria had Typical cell adhesion characteristics, carrying the astA cell adhesion virulence gene, belonging to multidrug-resistant CTX-M-15-positive extended-spectrum β-lactamase-producing Escherichia coli.

Embodiment 2

[0035] Example 2 DEC strain has good colon cell adhesion

[0036] Caco-2 cells were grown in MEM medium containing 10% fetal bovine serum at 2 x 10 6 Cells / well were seeded into 6-well cell culture plates, 37°C, 5% CO 2 Cells were cultured in a cell incubator for 12 h to allow the cells to adhere completely. After the cells adhered, the original medium was aspirated and washed twice with MEM complete medium. Prepare the MEM suspension containing DEC, dilute it by 10 times, drop the dilutions of different dilutions on the MacConkey solid medium, invert the culture in a 37°C incubator for 16 hours, and count the bacterial colonies to obtain the total bacterial colonies. number. According to the infection dose of MOI=100, 1 mL of MEM complete medium containing DEC was added to the experimental group, and 1 mL of MEM complete medium containing the same amount of Escherichia coli K12 strain was added to the control group, and the culture was continued. Cells were harvested 1 to...

Embodiment 3

[0037] Example 3 Construction of a mouse model of diarrheal Escherichia coli infection

[0038] This embodiment adopts the figure 2 The modeling process shown includes the following steps:

[0039] Cultivation and counting of DEC: Bacteria were passaged in broth (LB) medium, colonies were counted in MacConkey agar medium, single colonies were picked and inoculated in LB medium, and cultured with shaking at 37°C and 200rpm for 16h. When counting bacteria, the bacterial solution was diluted 10-fold with PBS, and 10 μL of the dilution solution corresponding to each dilution was drawn into the MacConkey agar medium, and three replicates were set for each dilution. Inverted culture was continued for 16h, and the colony was counted to obtain the bacterial CFU value.

[0040] Under constant temperature, ventilation and dry conditions, 6-week-old female specific pathogen-free (SPF) CD-1 female mice were grouped and housed in separate cages, with free access to food and water, and t...

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Abstract

The invention provides a construction method and application of a diarrhetic escherichia coli infected animal model. According to the construction method, experimental animal (mouse) infection is carried out in a manner of intraperitoneal injection of diarrhetic escherichia coli bacterial suspension. The mouse diarrhea model constructed by utilizing the diarrhetic Escherichia coli can be widely applied to the related fields of prevention, control and treatment of dairy cow diarrhea induced by diarrhetic Escherichia coli infection, dairy cow intestinal health research and the like, and a proper experimental animal model is provided for mechanism research of dairy cow escherichia coli diarrhea.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for constructing an animal model of diarrheal Escherichia coli infection and its application. Background technique [0002] Diarrhea is a common physiological or pathological reaction of dairy cows, with watery feces, rapid dehydration and autotoxicity as the main symptoms. It is a serious clinical disease. The causes of diarrhea in dairy cows mainly include pathogenic microorganisms such as Escherichia coli, rotavirus, coronavirus and Cryptosporidium, as well as external factors such as nutrition, environment and feeding management. Newborn calves have an immature rumen and immature immune system, making them susceptible to diarrhea in dairy cows. After statistics on the incidence of 450,000 heifers in my country, it was found that the incidence of lactating calves accounted for 51.4%, of which diarrhea accounted for 72.8%. [0003] Studies have shown that the morbidity a...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A01K67/02C12N1/20A61K49/00C12R1/19
CPCA01K67/02A61K49/0008Y02A50/30
Inventor 曹志军何至远马玉林
Owner CHINA AGRI UNIV
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