| In recent years,due to piglets diarrhea caused by a sharp increase in piglet mortality,to the pig industry in China caused huge economic losses.Piglet diarrhea is caused by a variety of factors,including infectious pathogens and non-infectious factors such as infection,and the high incidence of high mortality.Infectious factors causing piglets diarrhea,including bacteria,viruses and other pathogens,etc.,previous studies have shown that bacterial-induced piglet diarrhea in piglets infected diarrhea occupies a large proportion,and in recent years,the size of Guizhou Province,There was a higher degree of diarrhea in both piglets,and morbidity and mortality were elevated.Therefore,this study mainly carried out the epidemiological investigation of infectious diarrhea in piglets in Guizhou province,and established the molecular biology method to isolate and identify the main pathogens.The main pathogens serotype and virulence genes,drug resistance phenotype and drug resistance Genotype and other research and analysis,with a view to Guizhou Province,piglets bacterial diarrhea,scientific diagnosis and comprehensive prevention and control to provide an important basis.1.Epidemiological survey of infectious diarrhea in Guizhou piglets:From the aspects of pathogen type,type of infection,seasonal season and geographical distribution,the epidemiological survey of nine provinces(states,cities)41a total of 271 cases of infectious diarrhea in piglets were investigated and analyzed.The results showed that:? 48.3%of the bacterial infection caused by diarrhea in piglets in Guizhou province from 2011 to 2015(131/271),viral infection accounted for 31.0%(84/271),mixed infection accounted for 20.7%(56/271);?piglets infected diarrhea in the main pathogens,the main bacterial pathogens isolated Escherichia coli Bacteria,Salmonella,Streptococcus,etc.Accounted for 85.2%(231/271),the detection of the main viral pathogens of porcine epidemic diarrhea virus,swine fever virus,swine infectious gastroenteritis virus,a total of 57.9%(83/271).?In the main diseases of infectious diarrhea in piglets,bacterial diarrhea occurred in spring and summer,accounting for 30.6%(83/271).Viral diarrhea occurred in winter and spring,accounting for 34.7%(94/271),and the geographical distribution of the main epidemic of infectious diarrhea in our province was higher than that in other areas.2.Establishment of triplex PCR method for the main pathogens of piglets diarrhea:In this study,three pairs of specific primers were designed according to the universal identification of Escherichia coli,Salmonella and Shigella bacteria by Genbank,and the genomes of three bacterial positive strains DNA was used as template to establish the method of triple PCR for Escherichia coli,Salmonella and Shigella,and the reaction conditions were optimized.The results showed that the optimal annealing temperature was 58?,the optimum primer concentration was 0.16?M,0.16?M and 0.16?M,respectively.Salmonella,Shigella,Escherichia coli Fungi,the lowest DNA template detection were 0.3pg/?L,0.22pg/?L,1.24pg/?L,the established detection method specificity,good sensitivity,can be used for clinical testing.3.Isolation and identification of main pathogens of piglets diarrhea:A total of 128 diets of diarrhea were collected from 13 large scale pig farms in five provinces(states and cities)in Guizhou Province.The traditional pathogens were identified by molecular pathogens The detection and identification of the main pathogens were carried out by methods such as detection and animal pathogenicity test.The results showed that:?78 isolates of Escherichia coli were isolated and identified from the intestinal samples of diarrhea piglets,the separation rate was 60.9%(78/128),and the molecular biology compared with the traditional pathogen identification method;?and the isolation rate was 16.4%(21%).The results showed that the isolation rate of Escherichia coli was 85.2%(109/128)and the coincidence rate was higher.? 43 strains of Staphylococcus aureus were isolated and the isolation rate was 33.6%(43%),and the coincidence rate of Salmonella was 72.4%(21/29),and the coincidence rate was higher than that of traditional pathogen identification method.128),Streptococcus 21,the separation rate of 16.4%(21/128),and his pathogens such as Serratia,Klebsiella,Campylobacter,Citrobacter,Bacillus,Shigella and so on.4.Serotype and virulence genotype of the main pathogens of piglets diarrhea:serological and virulence genotypes of pathogenic Escherichia coli and Salmonella were identified by serological methods and molecular biology methods.The results showed that:?78 strains of pathogenic Escherichia coli isolated and identified were isolated and identified in addition to 27 strains.A total of 51pathogenic Escherichia coli serotypes were identified.A total of 62 strains of virulence genes were detected.The detection rate was 79.5%(62/78),and the toxicity was significantly higher than that of the control group The positive rate of eaeA,escV and elt was 38.4%(30/78),21.8%(17/78)and 28.2%(22/78)respectively,which were divided into 9 kinds of virulence genotypes,eaeA,eaeA + escV as the main virulence genotype,according to the type of virulence gene can be pathogenic Escherichia coli is divided into three types of EPEC,ETEC,EAEC;? isolation and identification of 21 disease Salmonella typhimurium,in addition to four unformed,were identified 17 strains of Salmonella serotype,including Salmonella typhimurium,Salmonella enteritidis majority,the separation rate of 42.9%(9/21).The detection rate of hirA,siiE and sopB were high,and the detection rate was 85.7%(18/21),76.2%(16/21)and76.2%(16/21),which were divided into seven kinds of virulence genotypes.SiiE/sopB/hilA and siiE/sopB were the main virulence genotypes.5.Drug resistance of the main pathogens of bacterial diarrhea in piglets:The antimicrobial resistance of 12 antimicrobial agents was determined by using the drug sensitive paper method for 78 isolates of Escherichia coli and 21 strains of Salmonella,And the resistance genes of each strain were detected,and the relationship between bacterial resistance phenotype and drug resistance genotype was analyzed.The results showed that:?78 strains of Escherichia coli were highly resistant to penicillin,ampicillin and gentamicin,and the resistance rate was 94.9%(74/78),92.3%(72/78),73.7%(70/78),and the isolates were multi-resistant strains.Among them,14 kinds of drug resistance genes were detected in 12 kinds,the resistance genes aphA,TEM,SHV detection rate,respectively 71.8%(56/78),44.9%(35/78),47.1%(37/78),pathogenic Escherichia coli on beta amides,aminoglycosides,sulfonamides 66.2%(49/74),82.9%(58/70),72.9%(35/48),75.9%(41/54)were significantly higher than those in the control group ?21 strains of Salmonella were isolated and resistant to amikacin,kanamycin and sulfamethoxazole.The resistant rates were 61.9%(12/21),57.1%(10/21),47.6%(9/21),and the isolates were multi-resistant strains.Among them,14 kinds of drug resistance genes were detected in 10 kinds,the resistance genes aacC2,aacC4,sull,SHV detection rate was the highest,respectively 66.7%(14/21),66.7%(14/21),52.4%(11/21),42.9%(9/21);Salmonella to beta amides,aminoglycosides,sulfonates 100.0%(9/9),62.8%(5/8),81.8%(9/11),73.3%(11/15)were significantly higher than those in the control group.In conclusion:1.The proportion of infectious diarrhea caused by bacterial infection in our province was higher than that of other infection types.The bacterial diarrhea occurred in piglets In spring and summer;and there were significant differences in the geographical distribution of the main epidemic diseases of infectious diarrhea in our province.2.The method of triple PCR for Escherichia coli,Salmonella and Shigella was successfully established in this study.The method has high sensitivity and good specificity and can be used for clinical diagnosis.3.The pathogenicity of Escherichia coli and Salmonella were identified as the main pathogens causing bacterial diarrhea in our province.Two major pathogens could be identified by molecular biology method and traditional method.Higher.4.The results showed that the major pathogens of bacterial diarrhea in Guizhou piglets had a variety of serotypes and virulence genotypes,among which EPEC and ETEC in pathogenic Escherichia coli were the most important colorectal Cytotoxicity and serotype to 0138,087 majority;caused in our province piglets diarrhea Salmonella virulence genes to siiE,sopB detection of the most sera to Salmonella typhimurium,Salmonella enteritidis as the main serotype.5.The main pathogens of Guizhou piglets diarrhea were highly resistant and the number of multi-drug resistant species was more than 8.There were many kinds of drug-resistant genes detected,and the coincidence rate of resistance phenotype and drug resistance genotype was highly unified. |
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