Molecular Epidemiological Investigation Of Tick-Borne Anaplasma In Qinghai

Anaplasma is an intracellular parasite in the blood cells and granulocytes of a host such as human,cattle or sheep,and its clinical symptom includes fever,decreasing of leukocytes and granulocytes,multifunctional organ failure,and death in severe cases.Ticks are the main vectors of Anaplasma,and they are active in Qinghai Province,In this study,the distribution of tick species and Anaplasma carrying conditions in Qinghai Province were investigated.Classification and identification of ticks.A cloth-flag method was used to collect the ticks in 13 counties and cities of Qinghai Province from March to June in 2017.Ticks were identified by morphology and molecular biology based on 16S rRNA.There are 1959 ticks belonging to haemaphysalis and dermacentor being collected in this study,including 1141 H.qinghaiensis,786 D.niveus,32 H.leporispalustris,and H.qinghaiensis was the dominant specie(58.24%).Establishment of nested PCR and real-time fluorescence quantitative PCR(RT-PCR).The GroEL gene sequences of different Anaplasma species were analyzed,then,two pairs of primers of nested PCR and RT-PCR were designed with conservative sequence.A SYBR RT-PCR method for the detection of Anaplasma was established by using the standard plasmid constructed by nested positive template.The sensitivity,repeat ability and specificity of the RT-PCR method were evaluated.The results showed that the real-time PCR method of high sensitivity could detected the lowest level of the standard plasmid of 1x10 copies/?L,which was 10 times in sensitivity higher than nested PCR.The coefficient of variation was less than 2%and good in repeatability.the DNA templates were used by the positive template and the other four pathogens.The samples of DNA template except positive template were negative and good in specificity.The slope of the established standard curve was-3.21,R~2=0.994,indicating good concentration of the linear relationship between the threshold value and template.Molecular epidemiological investigation of tick-borne Anaplasma in Qinghai Province.The samples of 211-tube ticks were obtained by grouping 10 ticks into one tube in the same area.The established RT-PCR method was used to detect the amorphous pathogens in tick samples.45 positive samples were detected,and the positive rate was 2.50%,and the highest infection rate was 6.44%in Datong County.Statistical analysis showed that the difference rate between different regions was significant(P<0.05).22 positive fragments were amplified by nested PCR and the infection rate was 1.17%.Nucleotide phylogenetic analysis showed that 14 A.bovis were 99%homology with Australian strain K2425499 and 8 A.ovis were 95% homology with CP015994,KJ410289,MG869402 and MG38383808,which were sister branches of these four strains.These data providing basis for cattle and sheep Anaplasma epidemiology in Qinghai area.