Infection Of Neoehrlichia Mikurensis In Rodents Based On GroEL Gene From Three Counties Of Commensal Rodent Plague Foci,Yunnan Province

ObjectivesTo confirm the infection of Neoehrlichia mikurensis in rodents in commensal rodent plague foci of Yunnan Province.To analyze the genotype of N.mikurensis and to explore the influence factors on the N.mikurensis infection in rodents,provide the scientific basis for the effective prevention and control of neoehrlichiosis.Methods1.656 rodent spleen samples were collected from Lianghe,Mangshi,and Mile in commensal rodent plague foci of Yunnan Province,there were 399 in Lianghe,191 in Mangshi,and 66 in Mile,respectively.2.DNA extracted from the spleen tissue of rodents according to the instructions of Mag Beads Genomic DNA Kit,DNA concentration,and purity of samples were detected by Ultra-micro Spectrophotometer.3.The gro EL gene fragment of N.mikurensis,was amplified by nested PCR,and the second round of PCR amplified products were subjected to Sanger bidirectional sequencing.The successfully sequenced nucleotide sequences and highly similar standard reference sequences selected in the Gen Bank were used for BLAST comparison analysis,DNAStar 7.1 software was used to carry out sequence homology alignment and MEGA 11.0 software was used to construct a phylogenetic tree to determine the genotype of N.mikurensis.4.N.mikurensis infection in rodents in different areas,species,sex,and habitats were compared using Chi-square Test and Fisher’s Exact Test.α=0.05.Results1.656 rodent samples including 19 species,13 genera,6 families,and 3 orders,the dominant species were Rattus tanezumi and Suncus murinus accounted for 44.51%(292/656)and 24.54%(161/656),respectively.Other else species of rodents accounted less than 10%.There were 339 females and 317 males,with a male-to-female ratio of1.07:1.2.656 whole genome DNA samples from rodent spleens were obtained.There were 12 positive samples detected from nested PCR,and the N.mikurensis positive rate for Lianghe,Mangshi,and Mile was 2.76%(11/399),0.52%(1/191)and 0(0/66),respectively.The infected rodent species included R.tanezumi,Rattus sladeni,Niviventer confucianus,and Berylmys bowersi.3.There was no significant difference in regions,sexes,species,and habitats for their infection with N.mikurensis(P>0.05).4.Phylogenetic analysis based on the gro EL gene showed that the internal homology of the 12 positive nucleotide sequences was between 99.5% and 100%,The positive sequences had high homology with Japan(AB084583),Fujian(JQ359063),and Henan Province(JQ359064),etc.infected by different hosts(such as rodents,ticks,etc.),which were in the same branch,correspond with genotype Cluster Ⅳ.Conclusion1.Rodents from Lianghe and Mangshi were infected with N.mikurensis in commensal rodent plague foci in Yunnan Province,and the total positive rate was 2.03%(12/590).The positive rate in Lianghe was 2.76%(11/399)and in Mangshi was 0.52%(1/191).N.mikurensis infection in rodents has not been found in Mile(0/66).2.The infected rodent species included R.tanezumi(the dominant species),and other species R.sladeni,B.bowersi,and N.confucianus.3.The molecular characteristics of gro EL gene sequences of N.mikurensis showed that 12 positive nucleotide sequences were highly similar,corresponded with genotype Cluster Ⅳ,which were different from Cluster Ⅲ genetype obtained from Eothenomys custos in Shangri-La.Further studies should be conducted to investigate its epidemiologic in rodents,ticks,and population from different regions of Yunnan Province and pathogenic features.4.The positive rate of rodents infection in woodland and shrub was more higher than other habitats.Human contact with rodents require high attention.