| ObjectivesTo understand the infection status of Anaplasma phagocytophilum in wild small mammals and the relationship between the Anaplasma phagocytophilum infection status and the environmental factors,and to analyze the Anaplasma phagocytophilum gene polymorphism for providing the theoretical foundation to control the prevalence of infectious disease in the plague foci of Yunnan Province.Methods1.Study setting:A total of 2611 wild small mammals were captured in the plague foci of Yunnan Province.One thousand six hundred and five tissue(liver and spleen)of samples from 2549 wild small mammals were selected by stratified sampling method as study objects.2.DNA Extraction:DNA extracted from tissue(liver and spleen)of wild small mammals according to the instructions of MagBeads Genomic DNA Kit,DNA Concentration of samples were detected using NanoDrop-1000 Nucleic Concentration Meter.The non-conformed DNA was extracted again,and the conformed DNA was amplified using PCR.3.PCR amplification:The 16SrRNA gene fragment of Anaplasma phagocytophilum was amplified using nested PCR,and the gltA gene fragment of Anaplasma phagocytophilum was amplified using real-time quantitative PCR.4.Gene sequencing and phylogenetic tree analysis:Gene sequences were compared using the BLAST“Sequence Similarity Searching”program(http://www.ncbi.nlm.nih.gov)and phylogenetic tree was constructed using software MEGA7.0.5.Statistical analysis:Data was coded and computerized with Excel 2007 software and Analyzed using R Software.Anaplasma phagocytophilum infection of wild small mammals in different areas,species,altitude,gradient,sex,habitats and seasons were compared using Chi-square Test and Fisher’s Exact Test,α=0.05.Results1.One thousand six hundred and five samples including 30 species,19 genera,6families,3 orders.Among them,The proportion of Apodemus chevrieri were 29.35%(471/1605),and the proportion of Eothenomys mileyus were19.00%(305/1605),the proportion of other else species of wild small mammals were less than 10%.2.The infection rate of Anaplasma phagocytophilum in wild small mammals in the plague foci of Yunnan Province were 0.93%(15/1605).The infection rate of Lianghe plague foci were 3.19%(13/407)significantly higher than Yulong plague focus 0.36%(2/550)and Jianchuan plague foci 0%(0/648)(c~2=30.493,(49)<0.001),and the infection rate of ommensal plague foci 3.19%(13/407)was significantly higher than wild plague foci 0.17%(2/1198)(c~2=26.888,(49)<0.001).3.There are five species of wild small mammals infected with Anaplasma phagocytophilum in the plague foci of Yunnan Province.The infection rates were 0.21%(1/471),0.33%(1/305),5.77%(6/104),4.88%(4/82)and 20%(3/15)in Apodemus chevrieri,Eothenomys mileyus,Rattus tanezumi,Rattus steini and Rattus nitidus,respectively.The infection rate of mammals species 1.57%(13/829)which proportion less than 10%is significantly higher than the dominant species 0.26%(2/776)(c~2=7.434,P=0.006).4.The infection rates in four altitudinal gradients of Yulong foci(2400~2600m、2600~2800m、2800~3000m and≥3000m)were 0%(0/61)、1.32%(1/76)、0%(0/104)and 0.32%(1/309)respectively,there were no relationship in prevalence between different altitudinal gradients(P=0.472),and in three altitudinal gradients of Jianchuan foci(2250-2450m,2450-2650m and 2650-2850m)were 0%,and in four altitudinal gradients of Lianghe foci(1000~1200m、1200~1400m、1400~1600m and≥1600m)were 5.13%(6/117)、4.20%(5/119)、1.82%(2/110)and 0%(0/61)respectively,there were no relationship in prevalence between different altitudinal gradients(P=0.219).5.The infection rates of Anaplasma phagocytophilum in wild small mammals were1.95%(7/359),0.27%(1/368),0.64%(3/469)and 0.98%(4/409)in spring,summer,autumn and winter,respectively.There was no relationship in prevalence between different seasons(P=0.130).6.The infection rates of Anaplasma phagocytophilum in wild small mammals were0.67%(5/747)for females and 1.17%(10/852)for males,respectively.There was no relationship in prevalence between different genders(c~2=1.090,P=0.297).7.The infection rates of samples collected from forest land was 1.45%(15/1036).However,no small mammals were infected at the farmland,shrub,farmland forest boundary and farmland shrub boundary.There was no relationship in prevalence between different habitats(P=0.217).8.Evolution analysis showed that the Anaplasma phagocytophilum 16SrRNA gene sequences of positive samples and host animals(such as rats,ticks,etc.)were clustered in the same branch except Yulong foci YL-3-279 samples,including Northeast China(GQ412339.1),Russia(HM366583.1),Zhejiang(HM439430.1),Jilin(DQ449948.1),Canada(HG916766.1),Yunnan(MF134887.1)and Anhui(EF211110.2).The positive samples had high homology with Japanese(AB196721.1)and Korean(AF70699.1),there are in a large branch,but far away from each other.YL-3-279 clustered in a single branch,Hubei A.marginale(HM538192.1)and Italy A.centrale(EF520690.1)were clustered in the same branch.U.S.A Rickettsia rickettsii(DQ150682.1)is on the periphery.It is indicated that the detected Anaplasma phagocytophilum strains had genetic diversity and regional differences.9.Real-time quantitative PCR was used to amplificate the gene sequence of gltA in Anaplasma phagocytophilum and the sequencing results showed that the sequence of all samples was not the gltA gene fragment of Anaplasma phagocytophilum.Conclusions1.The infection rate of Anaplasma phagocytophilum of wild small mammals in the plague foci of Yunnan province were 0.93%.The infection rate of Anaplasma phagocytophilum of wild small mammals in commensal plague foci was significantly higher than wild plague foci.2.There are Apodemus chevrieri,Eothenomys mileyus,Rattus tanezumi,Rattus steini and Rattus nitidus,infected with Anaplasma phagocytophilum in the plague foci of Yunnan Province.3.Anaplasma phagocytophilum strains infected in wild small mammals in the plague foci of Yunnan Province had genetic diversity and regional differences. |
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