| Avian colibacillosis refers to a series of local or systemic multi-system injury diseases caused by Avian pathogenic Escherichia coli（APEC）infection of poultry,including septicemia,peritonitis,arthritis,omphalitis or vitellocystitis.And APEC serotype of numerous and complicated,the lack of cross protection between different serotype,increased the vaccine and the difficulty of the research and development of antibacterial agents,Combined with the irrational use of antibiotics and other factors lead to drug-resistant E.coli and multiple drug resistance situation is becoming more and more serious,makes the disease prevention and control is very difficult.At the same time,APEC is regarded as a potential zoonotic bacteria,threat to human and animal life and health.Strengthen the pathogenic avian pathogenic e.coli,therefore,research and development of traditional Chinese medicine,probiotics,phage efficient green"for resistance to products"for the development of aquaculture,and public health security is of great significance.Phages are highly host specificity,and coevolution to overcome bacteria and bacteria to resist the ability of the phage,so the phage has potential as an alternative to clinical antibiotic effectively.In this study,a strain of Escherichia coli 21EC78 was screened from avian pathogenic Escherichia coli preserved in the laboratory.By analyzing its biological characteristics,genomic bioinformatics and pathogenicity,as well as exploring the isolation and cleavage of related phages,data support was provided for the prevention and control of colibacillosis.The details are as follows:1.21EC78 screening,the biological characteristics of E.coli,bioinformatics analysisThis test in Shandong province in the early stage of the laboratory from a white feather chicken farm samples randomly selected from eight separate avian pathogenic E.coli strains tested motility and biological membrane,1 strain screening motility and the strong ability of biofilm formation of 21EC78 strains.Through four heavy polymerase chain reaction（PCR）to measure its system development group,the results show that it belongs to B2 serogroup;By drug sensitive piece of paper and trace the broth dilution method to determine the beta lactam,penicillium carbon alkene,tetracycline,quinolone,large ring lactone class,aminoglycoside,polypeptide,15 of the sulfa eight classes of antibiotics clinical commonly used antibiotics resistant phenotypes,Results showed that E.coli 21EC78 for beta lactam,tetracycline,quinolone,large ring lactone class and sulfa antibiotics fall into five categories of 12 kinds of drug resistance,for multiple drug-resistant E.coli;Through genome sequence analysis its serotype O83:H28,at the same time carry 16 kinds of virulence genes and 14 kinds of drug resistance,in addition the Center for Genomic Epidemiology（CGE）website tool predicted that it had an 84.9%probability of being a human pathogen.These results may indicate 21EC78drug-resistant E.coli and severe cases may have stronger pathogenicity.2.Pathogenicity test of E.coli 21EC78 in chicks and G.mellonella modelsThis study has carried on the 21EC78 Escherichia coli in chickens and big wax moth animal pathogenicity test on two different models.Results One-day-old SPF White Leghorn chickens were infected with 2×105～2×109 CFU Escherichia coli 21EC78 by intrapitoneal injection.All chickens died within 3 days.And in the form of oral infection 1 day-old SPF white leghorn 2×105～2×107 cfu Escherichia coli after 21EC78,each group spirit within 7 d in good condition,no abnormal clinical symptoms and death;For 3 days of age SPF white leghorn infection in the form of intraperitoneal injection of 2×102～2×106 CFU Escherichia coli 21EC78,according to survival data to calculate LD50 of 1.78×104,autopsy results found bleeder liver;1day old Luhua chickens were infected with 2×105～2×109 CFU Escherichia coli 21EC78 by intrapitoneal injection,and the LD50 was 6.76×107 CFU Necroautopsy showed liver diffuse bleeding and pericardial effusion.The LD50 of Escherichia coli 21EC78 was 7.9×104 CFU.All known avian Escherichia coli 21EC78 of SPF animal models with white leghorn chicken,photo and big wax moth larvae has higher ability of disease;The oral way by intraperitoneal injection is easier to make 1 day-old SPF white leghorn chickens infected with E.coli,and the infection is more serious;SPF after infection are white leghorn chicken photo of mortality is higher,lead to different lesions.3.21EC78 E.coli phage LF2 isolation,identification,security,and in vitro biological characteristics analysis of cracked effectThis test 21EC78 avian pathogenic Escherichia coli as the host,by double plate method from our laboratory strain of Escherichia coli isolated screening the phage library cracking wide Escherichia coli phage,named LF2.The phage can form a circular on 0.3%double plate,edge tidy,transparent,plaque is about 1 mm in diameter;The temperature and p H stability,adsorption rate,optimum Multiplicity of infection（MOI）and one-step growth curve of LF2were measured.The results showed that LF2 had high stability under 4～55°C for 2 h.Under the condition of 3～12 p H,high activity can be maintained within 2 h.When MOI was 0.01,phage multiplication was the highest.According to the one-step growth curve,the incubation period of phage LF2 was short,the burst period was 80 min,and the calculated cleavage amount was 46.5 PFU/cell.That is,phage Escherichia coli LF2 has good temperature stability,acid and alkaline resistance and strong cleavage ability,which lays a foundation for the subsequent clinical application of phage Escherichia coli.Through large wax moth and chickens model to determine the safety of the phage,results show that the big wax moth infection within 48 h of phage LF2 without black and death;Chickens within 7 d after infection phage LF2 no death,chicken no abnormal clinical manifestations and pathological changes of eyes and explain the Escherichia coli phage in the host is security and stability.Through the determination of 21EC78 host and the in vitro cleavage of phage LF2 curve,in MOI=10,1,0.1 in vitro cracking within 8 h,basic not host growth,showing that the phage host bacteria showed a good sterilizing effect;At MOI=0.001,compared with MOI=10,1and 0.1,the effect was relatively poor,but still showed better bactericidal effect compared with the bacterial solution control group.The research results show that the E.coli 21EC78 resistance in severe cases,has the strong pathogenic,the popularity of the bacterium could cause serious symptoms,death and cause serious public health impact to people and environment,in clinical should be given a high degree of attention.The isolated related phage LF2 showed good environmental stability and safety stability,and showed good bactericidal and bacteriostatic effect against Escherichia coli21EC78 in vitro,which provided theoretical guidance for clinical phage prevention and control of colibacillosis. |
PDF Full Text Request