| Objective:To find out epidemic and pathogenic features of Yersinia enterocolitica in Yunnan plague foci, and to explore the relationship between Yersinia enterocolitica and Yersinia pestis in Yunnan plague foci.Methods:1. SpecimensWe selected three foci of wild rodent plague and two foci of rat plague in Yunnan province to capture mice(no less than 300 each region by mousetrap clip), meanwhile,we collected rectal swabs of some indicator animal.2. Isolation and identificationRectal swabs from dogs,root of tongue and intestines from captured mice and dead mice were cultivated in 10 ml peptone sorbitol bile broth(PBS) at 4℃ for 21 days. Then, DNA of specimens was extracted for screening of foxA gene by PCR.3. Preliminary screeningThe foxA positive samples were inoculated on CIN agar plate at 25℃. After 24 ~48h, we observed the culture and picked up the suspicious colonies(transparent edge,boundary clear, deep red in the center, "bull’s eye" shaped colonies) for further identification. Suspicious colonies were then screened of foxA, ail and inv gene.4. Biochemical identification and biotypingBiochemical identification of foxA positive strains were carried out using Api20 E biochemical article. The majority of the Biotype 1A are non pathogenic strains,so suspected colonies were inoculatied in bile-esculin plate at 25℃ for 24 h.5. Detection of virulent genesVirulent genes of ail, ystA, ystB, yadA, virF and rbfc were detected by PCR.6. SerotypeFour serotypes of O:3, O:5, O:8 and O: 9 were tested by slide agglutination to virulent strains with normal saline as control.7. Further identification to exceptional strains and pathogenic strains.8. Statistical analysis to experimental results.Results:1. Isolation status of Yersinia enterocolitica in Yunnan plague fociTotal 4853 samples were collected from five counties in Yunnan province. 464 strains were foxA positive(Positive rate 9.56%), and 266 strains of them were Yersinia enterocolitica by biochemical identification(Isolation rate 5.48%). In Yulong County, 1370 samples were collected, 274 strains were foxA positive(Positive rate19.56%), and 164 strains of them were Yersinia enterocolitica by biochemical identification(Isolation rate 11.97%). In Lijiang Gucheng area, 782 samples were collected, 57 strains were foxA positive(Positive rate 7.92%), and 17 strains of them were Yersinia enterocolitica by biochemical identification(Isolation rate 2.17%). In Jianchuan County, 1621 samples were collected, 116 strains were foxA positive(Positive rate 7.16%), and 80 strains of them were Yersinia enterocolitica by biochemical identification(Isolation rate 4.94%). In 2 Dehong Counties, 610 samples were collected, 12 strains were foxA positive(Positive rate 6.64%), and 1 strains of them were Yersinia enterocolitica by biochemical identification(Isolation rate0.16%). In dogs’ samples of Lijiang, 470 samples were collected, 5 strains were foxA positive(Positive rate 1.06%), and 4 strains of them were Yersinia enterocolitica by biochemical identification(Isolation rate 0.85%).2. Genotype of Virulent Yersinia enterocolitica strains in YunnanTotally there were 182 virulent strains in all 266 strains. There were five kinds of genotypes, they are 1 strain of “ail+, ystA-, ystB-, yadA-, virF-, rbfc-”, 10 strains of “ail+,ystA-, ystB+, yadA-, virF-, rbfc-”, 2 strains of “ail+, ystA+, yst B-, yadA+, virF+, rbfc+”,169 strains of “ail-, ystA-, ystB+, yadA-, virF-, rbfc-”, 84 strains of “ail-, ystA-, ystB-,yadA-, virF-, rbfc-”.3. Biotypes of Yersinia enterocolitica strains in YunnanA total of 7 bio/serotypes were identified, including Bio/Serotype 1A/O:3(2strains), 3/O:3(2 strains), 1A/O:8(4 strains), 1A/O:9(14 strains), 1A/O:5( 6 strains),non- Biotype 1A with undetermined serotype(4 strains), Biotype 1A with undetermined serotype(234 strains).4. PFGE molecular typing of strains containing virulence genesThe 12 pathogenic strains could be divided into three categories by PFGE molecular typing.The 3 strains isolated from Yulong mice(Serotype O:9)were the first category, the 2 strains isolated from dog’s anal swabs in Lijiang(Serotype O:3)were the second category, and the 3 strains isolated from Jianchuan rodents specimens and the one strain isolated from Yulong mouse(Serotype O:9)were the third category.5. Epidemiological analysis of Yersinia enterocolitica in Yunnan plague fociThe separation rates of the Yersinia enterocolitica strains isolated from different regions were not all same(c2=182.484, P<0.001) by SPSS17.0. The separation rate of Yulong county was higher than that of Lijiang Gucheng area(c2=62.027, P<0.001).The separation rate of Yulong county was higher than that of Jianchuan county(c2=49.054, P<0.001). The separation rate of Yulong county was higher than that of the 2 counties in Dehong(c2=78.286, P<0.001). The separation rate of Yulong county was higher than that of Lijiang collecting samples from dogs(c2=52.153, P<0.001).The separation rate of Jianchuan county was higher than that of Lijiang Gucheng area(c2=10.383, P=0.01). The separation rate of Lijiang Gucheng area was higher than that of the 2 counties in Dehong( c 2=11.053, P=0.001). The separation rates of Lijiang Gucheng area and Lijiang collecting samples from dogs were compared and there was no statistically significant difference between them(c2=3.115, P=0.11). The separation rates of Jianchuan county was higher than that of Dehong 2 counties(c2=29.338, P<0.001). The separation rates of Jianchuan county was higher than that of Lijiang collecting samples from dogs( c 2=15.762, P<0.001). There was no statistically significant difference between Dehong 2 counties separation rate and Lijiang separation rate from dogs(c2=2.786, P=0.095).Conclusion:Yersinia enterocolitica strains are widespread in Yunnan plague foci, and the isolating rate in wild rodent plague foci is 10%, much higher than the isolating rate that is less than 1% in rat plague foci. Yersinia enterocolitica strains isolated from Yunnan plague foci are mainly Biotype 1A and Serotype O:3、O:5、O:8、O:9. Theprimary Genotype is featured by “ail-, ystA-, ystB+, yadA-, virF-, rbfc-” and the second is featured by “ail-, ystA-, yst B+, yadA-, virF-, rbfc-”. |
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