| ObjectivesTo understand the infection status of Rickettsia typhi and Spotted fever group rickettsiae of wild small mammals in the plague foci of Yunnan province,to clarify the differences regarding Rickettsia typhi and Spotted fever group rickettsiae infection of wild small mammals in various regions and types of plague foci,and to explore the association of eco-environmental factors such as geographical,climate,and vegetation and wild small mammals’ Rickettsia typhi and Spotted fever group rickettsiae infection.Methods1.Source of the sample: 2512 samples of liver and spleen tissue were collected from wild small mammals captured in spring,summer,autumn,and winter from 2015 to 2016 in the plague foci of Lianghe County,Jianchuan County,and Yulong County of Yunnan province.2.DNA extraction and concentration determination: DNA of 2512 samples were extracted by magnetic bead tissue genomic DNA extraction kit,and the DNA concentration was measured using determinator of nucleic acid concentration.3.PCR detection: The heat shock protein gro EL gene primers were used for nested PCR amplification,and the second round of PCR amplified products were subjected to gel electrophoresis,and the results were observed using a gel imaging system.4.Sequencing: Sequencing was performed for products of positive amplification in nested PCR by Shanghai Shenggong,Co..5.Sequence alignment and evolution analysis: DNAStar(Editseq,Seq Man,Megalign),NCBI(Blast),MEGA 6.0 software was used to perform homology alignment analysis on those successfully sequenced and the phylogenetic tree was then constructed.6.Data statistics and analysis: Excel 2010 was employed for dataset establishment and management,while SPSS 17.0 was used to perform statistical analysis like Fisher￠s Exact Test.Results1.A total of 2512 wild small mammals of 3 orders,6 families,19 genera and 30 species were captured in the plague foci of Yunnan province,with the largest number of Apodemus australis,Eothenomys miletus and Apodemus draca,with composition ratios of 31.01%(779/2512),19.31%(485/2512),and 10.91%(274/2512),respectively.2.No infection of Rickettsia typhi was detected in the wild small mammals of the plague foci of Yunnan province,and the infection rate of Spotted fever group rickettsiae was 0.16%(4/2512).3.The infection rate of Spotted fever group rickettsiae in the wild small mammals of Lianghe and Jianchuan plague foci in Yunnan province respectively were 0.49%(3/611)and 0.10%(1/1029),and no infection of Spotted fever group rickettsiae in Yulong plague foci.There was no significant difference in the infection rate(P = 0.061).4.The infection rate of Spotted fever group rickettsiae in the mice plague foci and the rats plague foci in Yunnan province respectively were 0.49%(3/611)and 0.05%(1/1901).The infection rate of wild small mammals in the mice plague foci was higher than that of the rats plague foci(P = 0.047).5.The wild small mammals infected with Spotted fever group rickettsiae in plague foci of Yunnan province were Mus pahari,Rattus steini,Crocidura attenuata,and Suncus murinus.And the infection rate respectively were 1.39%(1/72),0.86%(1/116),4.17%(1/24),2.04%(1/49),the infection rate was higher than that of the dominant rat species(P = 0.023).6.In the four altitude gradients(1000~1200m,1200~1400m,1400~1600m and ≥1600m)of the Lianghe plague foci,the wild small mammals infected with Spotted fever group rickettsiae were distributed 1400~1600m and ≥1600m altitude gradients,the infection rate respectively were 1.32% and 1.09%;in three altitude gradients(2250~2450m,2450~2650m and 2650~2850m)of the Jianchuan plague foci,the wild small mammals infected with Spotted fever group rickettsiae were distributed on an altitude gradient of 2650-2850 m,the infection rate was 0.43%;the four altitude gradients(2400~2600m,2600~2800m,2800~3000m,and ≥3000m)of the Yulong plague foci were not detected Spotted fever group rickettsiae;there was no significant difference in the infection rate at different altitude gradients(P > 0.05).7.The infection rate of Spotted fever group rickettsiae in female and male wild small mammals in Yunnan province plague foci respectively were 0.09%(1/1158)and 0.22%(3/1340).There was no significant difference in the infection rate(P =0.629).8.The wild small mammals captured from Yunnan province plague foci mainly consisted of six habitats: forest land,cultivated land,shrubland,forest land and cultivated land junction,forest land and shrub junction,cultivated land and shrub junction.Habitats of wild small mammals infected with Spotted fever group rickettsia were all forest land,and the infection rate was 0.25%(4/1599).9.Among wild small mammals captured in spring,summer,autumn,and winter seasons of the plague foci in Yunnan province,no infectious Spotted fever group rickettsiae was detected in spring.In summer,autumn,and winter the infection rate of Spotted fever group rickettsiae respectively were 0.37%(2/540),0.13%(1/793),and 0.16%(1/641),and there was no significant difference in infection rate(P = 0.619).10.The results of molecular genetic evolution analysis show that the four samples gro EL gene sequences detected in the experiment all belong to Spotted fever group rickettsiae,and there were a certain difference between the gene sequences of gro EL in the plague foci of Lianghe County and Jianchuan County of Yunnan province.Conclusion1.No infection of Rickettsia typhi was detected in the wild small mammals of the plague foci of Yunnan province.2.The infection rate of Spotted fever group rickettsiae in the wild small mammals of the plague foci of Yunnan province was low(0.16%),the infected wild small mammals were Mus pahari,Rattus steini,Crocidura attenuata,and Suncus murinus.3.The infection rate of Spotted fever group rickettsiae with the wild small mammals in the mice plague foci of Yunnan province was higher than that of the rats plague foci.4.There were differences in the gro EL gene fragments of Spotted fever group rickettsiae infected by wild small mammals in the plague foci of Yunnan province. |
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