Analysis Of Drug Resistance Of Duck Main Pathogens And Isolation And Identification Of Phage

Riemerella anatipestifer,Escherichia coli,Pasteurella multocida and Salmonella enteriditis are bacterial diseases pathogen that seriously endanger the development of the duck industry,which can lead to a large number of duck deaths and cause huge economic losses.This study was conducted to study the multiplex PCR detection technology for the production of RA,E.coli,Pm and SE in the ducks,and provide technical support for the mixed infection and differential diagnosis of the four pathogens of ducks.Drug treatment is the main measure to deal with bacterial diseases.Due to the abuse of antibacterial drugs,the bacterial resistance is increased,so isolation and identification of pathogenic bacteria to diseased duck in duck farm,and then mastering the resistance status of isolates by the detection of resistant phenotypes and drug resistance genes in isolates,which can provides scientific guidance for clinical use.There is an urgent need to find alternatives to antibiotics for No anti-breeding in food animals,and phage are widely concerned,so in this study RA,E.coli,Pm and SE in duck were used as host bacteria to isolate and identify phage and study the biological characteristics,which laid a foundation for the development and application of phage.1.Identification of multiplex PCR for identification of four duck-intestinal susceptible pathogens:Four pairs of specific primers were designed respectively referring to SE inv A gene,RA Omp A gene,Pm kmt1 gene and E.coli Pho A gene sequence in GenBank.Multiplex PCR methods was established by optimizing primer concentration and annealing temperature with the extracted four kinds of bacterial DNA.And then test for specificity,sensitivity,and reproducibility were performed,and applied to clinical sample testing.Results:The four pairs of primers can respectively amplify the target bands corresponding to the expected size with the sequence of the SE inv A gene,RA Omp A gene,Pm kmt1 gene and E.coli Pho A gene fragment were 830 bp,664 bp,456 bp,and 261 bp,which the homology with the reference gene were 98.98%,99.68%,98.76%,and 98.33%,respectively.Optimizing primer concentration was SE inv A 0.25?mol/L,RA ompA 0.2?mol/L,Pm kmt1 0.15?mol/L and E.coli phoA 0.2?mol/L;annealing temperature was 51.4°C.Only specific bands of Salmonella,R.anatipestifer,Pasteurella and Escherichia coli could be amplified by multiplex PCR method,and the nucleic acid amplification results for Staphylococcus,Duck Plague Virus,Muscovy Duck Parvovirus and Goose Parvovirus were negative.The minimum detectable nucleic acid content is SE 8.6 pg,RA 4.4 pg,Pm 6.0 pg and E.coli 10 pg.The positive rate in 58 clinical nucleic acid samples were RA 12.07%(7/58),E.coli 12.07%(7/58),SE+RA 3.45%(2/58),RA+E.coli 6.90%(4/58),Pm+E.coli 5.18%(3/58).It is showed that the established SE-RA-Pm-E.coli multiplex PCR method has the characteristics of high specificity,high sensitivity,rapid and simple,and could be applied to the combined detection and differential diagnosis of RA,E.coli,Pm and SE clinical infection cases.2.Isolation,Identification and Drug Resistance Analysis of Duck Pathogenic Bacteria:The clinical disease materials were collected for isolation and culture of pathogenic bacteria,and the isolates were identified by morphological observation,biochemical identification,PCR detection and animal infection test.The sensitivity of20 antibacterial drugs was tested by KB paper method,and 16 kinds of drug resistance genes for aminoglycosides,?-lactams,quinolones and macrolide antibiotics was detected by PCR method.Results:Six strains of both ends Gram-negative bacillus whose colony is smooth and moist,slightly bulged,the edges are neat,translucent and were isolated.The 6 isolates were positive for oxidase and catalase tests both,and could decompose urea;no lactose and maltose were fermented,and some strains could ferment glucose(1/3)and sucrose(1/6);mannitol,methyl red Test,VP test,hydrogen sulfide production,nitrate reduction and citrate test were negative;no hydrolysis of lysine.Only RA-specific band was detected by SE-RA-Pm-E.coli multiplex PCR,and the bacterial 16S rRNA gene was amplified by PCR,and the sequencing results of the amplified products were more than 98%homologous to the R.anatipestifers published on NCBI.The lethality rate of the isolates infected with ducklings was 100%(18/18),so it was confirmed that the six isolates were pathogenic R.anatipestifers,named RA-SS1,RA-SS2,RA-SS3,RA-SS4,RA-SS5,RA-SS6.RA-SS1 is sensitive to ofloxacin and sulfamethoxazole,RA-SS2 is sensitive to cephalexin and sulfisoxazole,RA-SS3 is sensitive to cephalexin,and RA-SS4 is sensitive to cephalosporin,ofloxacin,ceftazidime,ceftriaxone,cephalexin,sulfisoxazole and rifampicin sensitive,RA-SS5 sensitive to cephalexin and rifampicin,RA-SS6 sensitive to ceftazidime,cephalexin and rifampicin among the 20antimicrobial agents selected.As for the 20 resistance genes selected,aac(6')-Ib,aph(3')-IIa,rmt C,qnr B,oqx A,oqx B and erm F genes were detected in RA-SS1,aac(6')-Ib,aph(3')-IIa,rmt C,qnr A,oqx A,oqx B and erm F genes were detected in RA-SS2,rmt C,?-DHA,oqx A,oqx B And erm F gene were detected in RA-SS3,aph(3')-IIa,rmt C,?-OXA,qnr B,oqx A,oqx B and erm F genes were detected in RA-SS4,aph(3')-IIa,qnr B,oqx A,oqx B and erm F genes were detected in RA-SS5,and aac(6')-Ib,aph(3')-IIa,rmt C,oqx A,oqx B and erm F genes were detected in RA-SS6.3.Isolation,Identification and Biological Characteristics of the Main Pathogens of Ducks:Collecting samples of farm sewage,feces and litter,and then separating phage using double-layer agar plate method with the host bacteria of Salmonella,Escherichia coli,Pasteurella and R.anatipestifer,Separately.The phage morphology was observed by electron microscopy,and the biological characteristics of phage host spectrum,titer,temperature and pH stability,optimal multiplicity of infection,one-step growth curve and antibacterial effect in vitro were studied.Results:Clear plaques with neat edges and a diameter of about 1~2 mm were observed on the double-layer agar plates with Salmonella as the host bacteria,while plaques were not observed on the double-layer agar plates with E.coli,Pm and RA as host bacteria.The phage,named Bp S,showed a sputum shape that the head plane was hexagonal,with a diameter of about 100 nm,a tail of about 125 nm,and a diameter of about 20nm by electron microscopy.The Bp S titer was 1.10×10~100 Pfu/mL;and it could maintain high activity at temperature from 30°C to 50°C(highest at 30°C),and pH among 6~9(highest at 7);The optimal muhiplieity of infection(MOI)was 0.01;The incubation period of Bp S was 20 min,the lysis period was 70 min,and the average lysis amount was 42 pfu/cell.Bp S had strong antibacterial activity against SE within3 h.Conclusion:A multiplex PCR method for rapid identification of SE,E.coli,Pm and RA was successfully established in this study.Six strains of R.anatipestifer were isolated from dead ducks,which were high sensitivity to ofloxacin,ceftriaxone,cephalexin,sulfisoxazole and rifampicin antibiotics,and were resistant for amoxicillin,enrofloxacin,streptomycin,kanamycin,Qing Adriamycin,amikacin,azithromycin,erythromycin and neomycin antibiotics.A strain of Salmonella phage was successfully isolated and identified,and biological characteristics were studied.