Combined bacteriophage preparation for treating piglet diarrhea

A phage and combined technology, applied in the application field of treating piglet diarrhea, can solve problems such as narrow lysis spectrum, weak lysis activity, and unstable effect, so as to reduce the occurrence probability, prevent and control piglet diarrhea disease, and reduce the abundance Effect

Active Publication Date: 2020-08-04
JILIN ACAD OF AGRI SCI +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, phage therapy also has problems such as too narro

Method used

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  • Combined bacteriophage preparation for treating piglet diarrhea
  • Combined bacteriophage preparation for treating piglet diarrhea
  • Combined bacteriophage preparation for treating piglet diarrhea

Examples

Experimental program
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Effect test

Embodiment 1

[0022] Phage Isolation and Screening

[0023] (1) Preparation of host bacteria

[0024] The present invention relates to three host bacteria, wherein enterotoxigenic Escherichiacoli k88 (CVCC 83902) was purchased from China Veterinary Microbial Culture Collection Center (China Veterinary Culture Collection Center, CVCC), Shigella dysenteriae (CGMCC 21535 ) was purchased from China General Microbiological Culture Collection Center (CGMCC); multidrug-resistant Escherichia coli F2 (JIsnky-E.coli F2) was isolated from Jilin Academy of Agricultural Sciences (JAAS) Piglet diarrhea feces from pig farms were independently preserved by the Antibiotic Substitute Laboratory of the Animal Husbandry Branch of the Jilin Academy of Agricultural Sciences, and were identified by drug sensitivity tests against ampicillin, sulbactam, piperacillin, piperacillin / tazobactam, Nine antibiotics including cefuroxime, cefuroxime axetil, cefotetan, imipenem and co-trimoxazole were resistant.

[0025] T...

Embodiment 2

[0042] Phage temperature, pH stability and lysis profile determination.

[0043] Take 1mL of phage lysate and add them into 1.5mL sterile centrifuge tubes, and then place the centrifuge tubes in water baths at 40°C, 50°C, 60°C, 70°C, and 80°C, and select 20, 40, 60, 80, At different time points of 100 and 120 min, 100 μL of phage lysate was taken from the centrifuge tube, cooled to room temperature first, and then the titer was measured by the double-layer plate method, and each group was repeated three times. The results showed that the phages vB_EcoP_E21, vB_EcoM_F2 and vB_SsoM_Z31 had a strong tolerance to temperature, and their titers remained basically stable at 40°C, 50°C, and 60°C for 120 minutes, except for phage vB_EcoM_F2, whose titer began to increase after 40 minutes at 70°C. The titer decreased sharply, and the titer dropped to 0 after 80 minutes of action, and the phages vB_EcoP_E21 and vB_SsoM_Z31 could still maintain a certain activity in the environment of 80°...

Embodiment 3

[0051] Determination of optimal multiplicity of infection of phages.

[0052] Phage multiplicity of infection (MOI) refers to the ratio of the number of phages to host bacteria at the time of infection, that is, the number of phages per bacterial infection. Determination method: (1) Use an inoculation loop to pick a single colony of the host bacteria and inoculate it in LB liquid medium, and culture it with shaking at 37°C and 160r / min until the logarithmic growth phase of the host bacteria; Six different ratios of 0.0001, 0.001, 0.01, 0.1, 1 and 10 were mixed with the above bacterial liquid; (3) 37°C, 160r / min shaking culture for 5h, 12000r / min, centrifugation for 2min, the supernatant was collected with 0.22μm Sterilize by membrane filtration; (4) Get three groups of parallel filtrate gradient dilutions, use the double-layer plate method to measure the phage titer, and the MOI group with the highest phage titer measured is the best MOI. The multiplicity of infection of the ...

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Abstract

The invention belongs to the technical field of biology, and particularly relates to a combined bacteriophage preparation for treating piglet diarrhea. The bacteriophage comprises an escherichia colibacteriophage vB _ EcoP _ E21, an escherichia coli bacteriophage vB _ EcoM _ F2 and a shigella bacteriophage vB _ SsoM _ Z31, the preservation number of the escherichia coli bacteriophage vB_EcoP_E21is shown as follows: vB_EcoP_E21; the preservation number of the escherichia coli bacteriophage vB _ EcoM _ F2 is CGMCC 18871, and the preservation number of the shigella bacteriophage vB _ SsoM _ Z31is CGMCC 18870. The combined bacteriophage preparation is applied in a direct drenching mode, the abundance of escherichia coli and shigella can be reduced, diarrhea diseases of piglets in the breeding process are effectively prevented and treated, the survival rate of the piglets is increased, and economic losses are reduced. The combined bacteriophage preparation provided by the invention can replace traditional feed antibiotics, has the characteristics of safety, high efficiency, no residue and the like, and is an ideal antibiotic substitute.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to three phage isolates capable of specifically lysing piglet enterotoxigenic Escherichia coli, multidrug-resistant Escherichia coli and Shigella dysenteriae strains and their application in treating piglet diarrhea. Background technique [0002] Newborn piglets have low self-resistance, are sensitive to external stimuli, and are susceptible to infection by pathogenic microorganisms and various stress factors due to the lack of a stable micro-ecological system in the intestinal tract. There are two types of infectious factors that cause diarrhea in piglets: viral and bacterial. Bacterial diarrhea is mainly caused by Escherichia coli and Shigella. Escherichia coli infection in piglets has the characteristics of wide prevalence, high morbidity and mortality, and often leads to decreased survival rate, stunted growth and even death of piglets. Clinically, the basic method of treating Escher...

Claims

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Application Information

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IPC IPC(8): A61K35/76A61P31/04
CPCA61K35/76A61P31/04Y02A50/30
Inventor 魏炳栋徐永平于维郑琳陈龙李淑英李纪彬杨欢
Owner JILIN ACAD OF AGRI SCI
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