Isolation And Identification Of Chicken Escherichia Coli And Phage

Avian pathogenic Escherichia coli(APEC)can infect chickens,turkeys and other birds,and cause local or systemic symptoms.Avian colibacillosis is currently a major cause of economic losses in the poultry industry.For a long time,the prevention and control of avian colibacillosis mainly depended on antibiotics.However,the long-term unscientific use of antimicrobial drugs leads to increased drug resistance in APEC and posed a threat to the safety of animal food products.Bacteriophage is the general name of a group of bacterial viruses that infect bacteria,fungi,mycoplasma,cyanobacteria,actinomycetes and Other microorganisms.Lytic bacteriophages are able to kill antibiotic-resistant bacteria at the end of the phage infection cycle.Thus,the development of phage therapy is potentially a way to improve the treatment of bacterial infections.In this study,livers from chickens which died from colibacillosis,feces of colibacillosis chickens and sewage from chicken farm,were collected from Shandong province.E.coli and bacteriophage were isolated and purified.The pathogenicity and virulence gene distribution of isolated Escherichia coli as well as the biological characteristics and host range of E.coli phage were studied.The study results can provide theoretical basis for clinical application of coliphage to control colibacillosis.Isolation and identification of chicken E.coli.Tissues from chickens which died from colibacillosis,feces of colibacillosis chickens and sewage from chicken farm,were collected from Shandong province.E.coli were isolated and purified.To identify the E.coli,the methods of Medium culture,Gram staining,biochemical identification and PCR were used.The serotype of isolated E.coli was detected to understand the distribution characteristics of the serotype of E.coli in this region.After Gram staining,PCR identification and biochemical tests,364 strains of chicken-derived E.coli were isolated.Among the 364 strains of chicken E.coli,251 isolates were able to detect the serotype,with a rate of 68.96%.The dominant serotypes were O₇₈(9.34%),O₁(7.42%),O₃₅(5.77%),O₂(4.67%),O₈(6.37%),O₁₅(4.40%),O₂₆(2.47%),O₇₆(2.20%),O₁₄(1.92%),O₁₂₇(1.92%).Virulence gene distribution and pathogenicity test.In order to master the distribution of virulence genes in chicken E.coli,the relationship between virulence genes and pathogenicity of E.coli.18 virulence genes of chicken E.coli were detected,and the distribution of virulence genes of chicken E.coli was analyzed.In order to study the relationship between virulence gene and pathogenicity of E.coli,some E.coli were selected for chicken embryo challenge test.The identification of 18 common virulence genes of chicken E.coli showed that the detection rate of the virulence genes yijp,fim C,mat and omp A was high,and no neu C gene was detected.E.coli with 5-9 virulence genes was the most common,accounting for 73%of the total.The results of the challenge test of chicken embryo showed that the E.coli isolated from dead chickens had high pathogenicity.Among the pathogenic strains,serotype O₇₈was the most pathogenic,followed by O₂serotype.Except for the O₁₅serotype strain,all the other serotypes had certain pathogenicity.The pathogenicity of strains with the same serotype and different virulence genes was significantly different.The number of virulence genes was positively correlated with the death rate of chicken embryo.Isolation and biological characteristics of bacteriophages.The bacteriophage of isolated chicken E.coli was isolated by double-layered plate method.The morphology was observed by electron microscopy,and the biological characteristics such as temperature stability,p H stability,optimal complex number of infections,one-step growth curve and optimal preservation temperature were investigated.155 strains of bacteriophage were isolated from 195 strains chicken E.coli.PLCF1 bacteriophage presents a polyhedral three-dimensional structure under the electron microscope.When viewed in a plane,the head is roughly a long hexagonal head with a long diameter of about 120 nm and a transverse diameter of about 120 nm.It has no obvious tail structure.The biological characteristics of two bacteriophages with large morphological differences were studied.The titer of the two bacteriophages can reach more than 10⁹,and the activity can be kept high within the temperature of 40?,and the optimal p H value is 7.The best multiplicity of PLCF1 infection was 0.001,and the best multiplicity of PLCF72 infection was 0.01.The one-step growth curve shows that the cracking performance of PLCF1 is better than that of PLCF72.The short-term preservation of phage can be 4?or-20?,and the long-term preservation of-80?is most suitable.Host spectrum and coverage of phage.The host range of phage was determined by double-layered plate drip.By analyzing the host range,phages were selected and made into phage mixture by scientific combination.Screening the coverage of chicken E.coli stored in the laboratory and collected in different areas by double-layered plate drip method.The host range of 83 bacteriophages against 282 E.coli showed that the lytic rate of bacteriophages could reach 78.1%,among which 49 were able to lyse more than 10 host bacteria,accounting for 60%of all bacteriophages.The mixture of 10 bacteriophages with the most extensive host range was screened,and the cracking rate of the preparation against randomly selected 100 strains of E.coli with the most virulence genes stored in the laboratory was 62%,and that against randomly collected E.coli strains from different regions was 41%.