Detection Of Pathogenic Yersinia From Some Plague Foci In Yunnan Province By PCR And Its Epidemiological Significance

ObjectiveTo understand the distribution and prevalence of Yersinia pestis among some plague foci in Yunnan Province;To improve the PCR genetic testing methods for three Yersinia;To explore the relationship among three Pathogenic Yersinia.Method1.Sample Collection: A total of 11 plague foci of Yunnan Province.The home plague foci include: Lianghe County,Yiliang County,Baoshan City,Mile County,and Midu County,Yuxi City,and Wenshan City.The wild plague foci include:Yulong County,Jianchuan County,Eryuan County,and Heqing County.The rodent trapping method was adopted,and some dog anal swabs were collected at the site of the home plague.2.Three Pathogenic Yersinia screening genes were identified: The target genes for the three pathogenic Yersinia species were screened by conducting experiments on collected part of samples.3.Preliminary Screening of PCR: A total of 4985 samples taken from the mouse cecum with its contents and the dog’s anal swab were placed in 10 m L of PBS,After cold-accumμLating bacteria at 4°C for 15-21 days,and then extract the DNA with its precipitate,and using fox A,inv,0392 gene for PCR screening.4.Bacterial Screening: The PCR positive samples were inocμLated on CIN agarplates and cultured at 28°C for 24 h.Suspicious colonies were picked for PCR screening of fox A,inv,0392 genes.5.Sterilization Treatment: For bacteria-positive samples,extract their DNA for PCR detection and preserve their pure bacteria samples,and store them in a refrigerator at-80°C.6.Analysis: Statistics and analysis of experimental data using chi-square test,statistical analysis using spss17.0,test level P <0.05 for statistical differences.Result1.Identification of three pathogenic Yersinia screening genes: From January to May 2016,1344 samples and other strains from four regions of Yiliang,Lijiang,Mile and Midu were collected for screening of fox A,inv,F1 and 0392 genes.The fox A,inv gene were screening specificity for Yersinia enterocolitica,Yersinia pseudotuberculosis,F1 gene screening Yersinia pestis will appear non-specific bands,0392 genes for screening Yersinia pestis will be specific and it can be used as a target gene for Yersinia pestis screening.2.Isolation of pathogenic Yersinia pestis in some plague foci in Yunnan Province:From January 2016 to October 2017,a total of 4985 samples of 11 districts in Yunnan Province were collected,A total of 248 strains of fox A positive strains were isolated with a separation rate of 4.97%;6 samples of positive strains were screened 0392,with a positive rate of 0.12%,and 0 positive samples were screened for inv positive samples.3.Statistical analysis of pathogenic Yersinia in some plague foci in Yunnan Province:The positive rates of three pathogenic Yersinia in 11 areas in Yunnan province were not statistically different(χ2=197.94,P<0.05).The statistical analysis of0392 gene(P>0.05)showed no statistical significance.Fox A gene positive samples(χ2=333.74,P<0.05)showed the specific statistical differences.Inv gene were negative no difference.4.Statistical analysis of some plague foci in Yunnan Province: A total of 4284 mouse samples and 701 dog samples were collected.Among them,there were 225 strains of fox A-positive strains in the mouse,and the isolation rate was 4.97%.There were 23 strains of fox A-positive strains in the dog anal swab,and the isolation rate was 3.28%.The statistical analysis(χ2=4.95,P=0.024<0.05)showed that the positive rate of fox A was higher than that of canine anal swabs Sub-fox A-positive rate.5.Quantitative analysis of the plague and mouse plague foci in Yunnan Province:The positive rate of three pathogenic Yersinia(χ2=164.51,P<0.05)are not exactly the same.The positive rate of Yersinia enterocolitica isolates from the plague foci of wild rats was higher than that from the plague foci of domestic rats.The isolation rate of fox A-positive strains in Yulong County was higher than that of the other three counties in wild plague foci,and the isolation rate of fox A-positive strains in Lianghe County and Mile County was higher in the plague foci of home rats.6.Distribution of fox A-positive strains in some plague foci in Yunnan Province:Different fox A-positive strains of different mouse types have different isolation rates(χ2 =86.20,P<0.05).The type of hamster was mainly Rattus norvegicus and Rattus flavipectus.The type of wild plague foci mainly consisted of Apodemus agrarius,Eupolyphaga nigrum and Apodemus agrarius.The fox A positive separation rate mainly distributed in the wild rat plague foci,mainly to Apodemus agkistrodon,mammoth and Apodemus aganum.7.Distribution of fox A-positive strains in Yulong County and Jianchuan: A total of 853 Yulong County rodents samples collected mainly from Luzi Village and Tai’an Township that with fox A positive rate highest.The results showed that the distribution of fox A positive rate in different mouse types in Yulong county was statistically significant(χ2= 31.80,P <0.05).There are differences between the two pairs of analysis,the results showed that the positive rate of fox A was higher than other types of rodents.The samples of Jianchuan County included 794 samples of rats,collected mainly in Jinhua Town and Shilong Village.The areas with high fox A positive rate were mainly located in Shilong Village and Changle Village.The statistically analyzed for the type of Jianchuan mouse without statistical difference(χ2=9.97,P>0.05).The positive rate of Yersinia enterocolitica in Jianchuan County was found to be more evenly distributed in the wild species.Conclusion1.Yunnan wild rat plague foci of Yersinia enterocolitica rodents overall isolation rate is higher than the Lianghe and other home rats plague foci.2.In the initial screening of on-site specimens of the epidemic source or processing of a plague epidemic,it is more convincing to use specific genes on the chromosomes of Y.pestis,such as YPO0392,for PCR than for F1 genes on plasmids.3.In the detection of three pathogenic Yersinia species in the epidemic area,three target genes,0392,fox A and inv,can be used to screen and screen the three pathogenic Yersinia species.