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Swine Escherichia coli non-toxic isolate capable of simultaneously expressing F4 and F18 pili and application of swine Escherichia coli non-toxic isolate

A technology of Escherichia coli and F418, which is applied in the field of biomedicine, can solve problems such as instability, weak colonization ability, and strong virulence, and achieve the effect of exerting immune efficacy and ensuring safety

Active Publication Date: 2022-07-15
YANGZHOU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, it has been reported to use wild strains to knock out the enterotoxin gene to construct attenuated strains, but the gene-modified deletion strains have weak colonization ability, are unstable in the body, and are easy to be cleared. Although relatively safe, they still have certain toxicity
Considering that the LT and STb genes are encoded by plasmids, the number of copies of the plasmid is large, it is difficult to artificially knock out multiple copies of the gene and the precise identification of the knockout, and there is a possibility of recombination after the knockout gene, which will lead to stronger virulence. The safety of gene deletion strains needs long-term evaluation

Method used

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  • Swine Escherichia coli non-toxic isolate capable of simultaneously expressing F4 and F18 pili and application of swine Escherichia coli non-toxic isolate
  • Swine Escherichia coli non-toxic isolate capable of simultaneously expressing F4 and F18 pili and application of swine Escherichia coli non-toxic isolate
  • Swine Escherichia coli non-toxic isolate capable of simultaneously expressing F4 and F18 pili and application of swine Escherichia coli non-toxic isolate

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028]Example 1 Isolation and identification of porcine Escherichia coli avirulent isolate F418

[0029] On April 16, 2019, 30 fecal samples of 45-day-old weaned piglets were collected from Liu Jiaqi, and bacteriological detection was carried out according to the national standard method (GB 4789.6-2016). The samples were retained in the laboratory, and about 25 g of the samples were taken and placed in a sterile beaker containing 225 mL of sterile liquid LB medium, fully stirred and then placed in a sterile conical flask for 6 hours with shaking at 180 r / min. Take 10 μL of the culture and inoculate it into 30 mL of intestinal bacteria enrichment medium (the ingredients are 1 g of gelatin trypsin hydrolyzate per 100 mL of water, 0.8 g of disodium hydrogen phosphate dihydrate, 0.2 g of bovine bile salt, 0.0015 g of bright green, and 0.5 g of glucose. g, potassium dihydrogen phosphate 0.2g, pH 7.2±0.2) was placed at 42°C for 18h enrichment. The enrichment solution was streaked ...

Embodiment 2

[0044] Example 2 Biological study of pig-derived Escherichia coli avirulent isolate F418

[0045] Single colonies of the above-mentioned isolates F418, F4 virulent standard strain C83902 and F18ab standard strain E. coli 107 / 86 were inoculated in liquid LB at 37°C for overnight shaking culture. The next day, the bacterial liquid was drawn and streaked on MacConkey plates for comparison of culture characteristics. At the same time, the three strains were subjected to sucrose, lactose, glucose, raffinose, maltose, mannitol, indigo matrix, mannose, citric acid, dulcitol, ornithine, lysine, potassium cyanide, hydrogen sulfide, Trace biochemical reactions such as urea, ONPG, MR test, V-P test, semi-solid agar, Calendula officinalis, nitrate reduction (shown in Table 4).

[0046] On the MacConkey plate, isolates F418, F4 virulent standard strain C83902 and F18ab standard strain E. coli 107 / 86 all grew pink round smooth colonies.

[0047] Table 4 Comparison of biochemical properties...

Embodiment 3

[0051] Example 3 Infection test of porcine-derived Escherichia coli avirulent isolate F418 to 30-day-old weaned piglets

[0052] Nine 5-day-old weaned piglets were randomly divided into 3 groups with 3 piglets in each group. 9 CFU / mL was orally administered orally, and a balanced salt buffer control group was set up, and the oral dose was 1 mL. Each group was reared in isolation, fed with antibiotic-free feed, and carefully observed for a week. Diarrhea symptoms and other clinical symptoms (such as mental state, feeding situation, etc.) of all experimental piglets were observed every day. At the same time, the mortality was recorded, and the organ lesions were observed by autopsy one week later. The duodenum, jejunum and ileum of piglets in each group were collected and sliced ​​to observe the pathological changes.

[0053] The results of the one-week test showed that the piglets in the isolated strain F418 infection group did not die, and the piglets were in good condition ...

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Abstract

The invention relates to a swine Escherichia coli non-toxic isolated strain capable of simultaneously expressing F4 and F18 pili and application of the swine Escherichia coli non-toxic isolated strain, the preservation number of the swine Escherichia coli non-toxic isolated strain F418 is CCTCC M 2022089, and the preservation date is January 18, 2022. The isolated strain is escherichia coli, can simultaneously express F4 and F18 fimbriae antigens and is a swine-derived natural isolated strain, PCR (Polymerase Chain Reaction) tests show that the strain lacks LT and STb enterotoxin genes, does not generate enterotoxin, can be well adhered and colonized to a host pig, has the potential of being developed into a bacterial non-toxic live vaccine candidate strain, and can be used for preparing a bacterial non-toxic live vaccine candidate strain. The strain can be well adhered and colonized to a host pig by oral administration, a piglet is induced to generate immune response for resisting F4 and F18 pilus antigens, a certain immune efficacy is exerted, the safety is guaranteed, and the strain is expected to become a potential live vaccine candidate strain for preventing and controlling F4 and F18 escherichia coli infection.

Description

technical field [0001] The invention belongs to the technical field of biomedicine, and in particular relates to a pig-derived Escherichia coli avirulent isolate F418 which simultaneously expresses F4 and F18 fimbriae and its application. Background technique [0002] Diarrhea in newborn or weaned piglets causes major losses in swine farming worldwide, with enterotoxogenic Escherichia coli (ETEC) being the main cause. Porcine-derived ETEC mainly includes two serotypes, F4 and F18, which can infect piglets of different ages, causing diseases such as yellow diarrhea, pullorum, edema, and diarrhea in weaned piglets. According to statistics, the morbidity and mortality of piglets caused by ETEC accounted for 56.2% and 24.7% of the total morbidity and mortality of piglets. [0003] The prevention and control strategy for ETEC in animal production and breeding is mainly the application of antibiotics. However, after the national policy of "limiting antibiotics" was introduced in ...

Claims

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Application Information

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IPC IPC(8): C12N1/20C07K16/12G01N33/569G01N33/68A61K39/108A61P31/04C12R1/19
CPCC12N1/20C07K16/1232G01N33/6854G01N33/56916A61K39/0258A61P31/04A61K2039/522A61K2039/542A61K2039/552G01N2469/20G01N2333/245Y02A50/30
Inventor 朱国强刘家奇张晓洁段强德武琥琮权国梅连思琪羊扬夏芃芃
Owner YANGZHOU UNIV
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