Molecular Epidemiology Investigation And Establishment Of Mass Spectrometry Bacteria Identification Method Among Waterfowl Major Bacteria Around Northern Jiangsu Province

Molecular epidemiology is to use relatively advanced technology to measure the distribution of biological markers,combined with epidemiological research methods,to clarify the epidemiological characteristics of diseases at the molecular or genetic level.The rapid molecular epidemiological investigation can help us to quickly analyze the type of epidemic strains,and then understand the epidemic characteristics of the strains,which is of great significance for the prevention and treatment of diseases.Multi-site sequence(MLST)typing,phylogenetic grouping,virulence gene detection,and molecular serotyping are relative analysis methods of molecular epidemiology.And MALDI-TOF MS is a new type of bacteria Identification methods have developed rapidly in recent years,and more and more studies have shown that MALDI-TOF MS may become a new and rapid tool for bacterial typing.At this stage,MALDI-TOF MS is relatively mature in the identification of E.coli,and the identification results are reliable,but its application in the veterinary field is limited,and in the identification of Riemerella anatipestifer,either.As a result,the self-built library of mass spectrometry is not perfect and the accuracy of identification results is low.In this thesis,two important bacteria(Avian Pathogenic Escherichia Coli and Riemerella anatipestifer)in the waterfowl breeding industry were studied.On the one hand,the traditional molecular epidemiological research methods were used to investigate the molecular epidemiology of avian pathogenic Escherichia coli isolates,which provided important reference value for the prevention and control of APEC.On the other hand,MALDITOF MS was applied to Riemerella anatipestifer and analyzed original off-machine data(m/z,Intensity)by R to establish a mass spectrometry identification method of Riemerella anatipestifer and try to explore the relationship between the serotypes.1.Molecular epidemiological investigation of avian pathogenic E.coliAvian Pathogenic Escherichia Coli(APEC)is the pathogen of Avian Escherichia coli disease,which can cause symptoms such as sepsis,peritonitis,air sac inflammation,salpingitis,swelling syndrome and even death of the diseased poultry,seriously harming poultry breeding Industry health.With the misuse and abuse of antibiotics,APEC resistance gradually increases,which makes it difficult to prevent and control.From February 2019 to September 2019,disease materials were collected from waterfowl farms in Jiangsu and surrounding areas,and 104 APEC strains were isolated.In order to study the epidemiological characteristics of APEC during this period,this study conducted a molecular epidemic,and could provide important reference value for APEC prevention and control.In order to clearly understand the epidemic situation,104 APEC strains were first classified by MLST,and seven housekeeping genes(adk,fumC,gyrB,icd,mdh,purA,recA)of APEC were sequenced and analyzed.The results showed that the most popular ST sequence was ST23(30.8%),and ST2505(17.3%),ST117(12.5%),ST224(8.7%),ST155(5.8%).ST156,ST602 and st2085 were 1.9%equally,ST648,ST949,ST227,ST3967,ST48,ST349,ST10,ST383 and ST140 were all 1 strain(0.96%).In addition,11 APEC strains were found to be new ST type,accounting for 10.6%.The results indicated that the dominant ST types of APEC epidemic in Jiangsu and its surrounding areas were ST23,ST2505,ST117,ST224 and ST155,of which ST23 was the most popular.Then,we used the molecular clustering method to carry out the phylogenetic clustering of APEC,and used PCR method to detect the presence of chuA gene,yiaA gene and DNA fragment tspe4.C2 of E.coli,and divided E.coli into A,B1,B2 and D groups.The results showed that there were 56 strains in group A,21 strains in group B1,3 strains in group B2,24 strains in group D,accounting for 53.8%,20.2%,2.9%and 23.1%,respectively.The results indicated that group A was the dominant evolution group of APEC in Jiangsu and its surrounding areas.Secondly,in order to understand the distribution of virulence genes in the isolates,9 representative APEC virulence genes were selected for PCR detection.These 9 virulence genes include astA,vat,hlyF,cvi/cva,iss,tsh,papC,irp2,and iroN.The results showed that the virulence genes of 104 APEC strains were as follows:astA gene was 15.4%,vat gene was 18.3%,hlyF gene was 97.1%,cvi/cva gene was 89.4%,tsh gene was 70.2%,iss gene was 97.1%,papC gene was 7.7%,irp2 gene was 68.3%,iroN was gene 94.2%.Finally,as a supplementary epidemiological data,According to the genome sequencing results of 21 APEC strains,wzx and WZY genes were compared with the database and serotype O antigen analysis showed that there were other serotypes except O1,O2 and O78,such as O9,O29,O33,O83 and O145,which indicated that there were many and complex serotypes in APEC.2.Establishment of mass spectrometry for identification of waterfowl bacterial diseasesRiemerella anatipestifer(RA)is the pathogen of infectious serositis,which mainly harms goose and duck,and is one of the most dangerous bacterial pathogens.The isolation and identification of RA is very important in clinical and epidemiological investigation,but the traditional detection method is costly in labor and time.In recent years,a new detection technology(MALDI-TOF MS)is gradually replacing the traditional bacterial detection method,compared with the traditional bacterial detection method,MALDI-TOF MS has many advantages,making bacterial detection more efficient and accurate.However,MALDI-TOF MS is rarely used in the field of veterinary medicine and in the identification of RA.Therefore,MALDI-TOF MS was used to identify the characteristic mass spectrum peak of RA,which is of great significance for the establishment of mass spectrometry bacteria identification method.In this study,16 strains of RA,8 strains of Salmonella,8 strains of Streptococcus suis and 8 strains of Escherichia coli were analyzed by using the Biomerieux VITEK mass spectrometer.The original peak data were analyzed by R(version 3.6.2).Finally,8 characteristic peaks belonging to RA were found.In addition,in order to explore the possibility of identifying serotypes by MALDI-TOF MS,we compared the characteristic peaks of the common dominant serotypes 1 and 2,and found that there are some regular characteristics in these characteristic peaks,which may be helpful for us to identify serotypes by mass spectrometry.