Tick-borne Pathogens in Shanxi Province,China＊

YAN ia a,YAN ui Jun,ZHAN in,HO ue Xia,MIAOGuan ing,CAOHong Bing,HAORui E,an AOQin,#

Tick r ecognize ne o he most importan ector ha aus isease n animal nd humans, and have significan ubli ealth impacts worldwide[1].The av wide rang ost mong terrestrial vertebrates, includin ammals,birds,amphibians. Th umerou oonoti athogens transmitte ick re the causative agentso yme disease,rickettsiosis, ehrlichioses,relapsin ever, Q fever,an ularemia. Case ick-born iseases ar ncreasin round the worl nd som merging or re-emergin athogen r eing discovered,whic ose grea hallenge o reventio nd control.

Tick-borneBorreliaspp.ar urrently divided into two taxonomi roups, Lyme disease spirochete nd relapsin eve pirochetes.Lyme disease,caused byBorrelia burgdorferisensu lato(B.b.s.l.),is widesprea roun h lobe, having been reporte ore than 70 countries.Phylogenetically,Borrelia miyamotoii ategorized a relapsing fever spirochete,bu t doe ot cause typica elapsin ever and is transmitte ardbodie icks. Thi pirochet asa globa istribution and often cocirculates with B.b.s.l., as they have overlappin ertebrate animal an ic ectors[2].Mos ickettsiales describe h iteratur re well-know oonoti merging pathogens,som f whic a aus eriousdisease in humans,including rickettsioses,anaplasmoses, an hrlichioses.Spotte eve ickettsiose r ause y spotted fever grou ickettsi SFGR).Anaplasma phagocytophilum(AP)is th ausativ gen f human granulocytic anaplasmosi HGA), wit ases reporte h SA, Netherlands,Sweden, France,and other countries.Ehrlichia chaffeensis(EC)can lea o huma onocytic ehrlichiosis(HME),an o clinical case er eported i hin ntil 2010[3].

Shanxi Province,located i h iddl f China,features a rag f different landforms, including mountains,hills, an lains.Shrubs an rasslands ar idel istribute hanxi and animal husbandr ell developed, which provides a suitable environmen o ic urvival.I hi tudy,five pathogen as identifie eing carrie y har ick ro hanx rovince,an ttempt re being mad ontro ick-borne pathogen his area.

In May 2019,w se h lagging method to collec ore than 600 tick he villag f Liangping,Qi County, Shanx rovince.Al ick ere identified base heir morphologies,an hese identifications wer erified base heir mitochondrial 16 DNA sequences. After washing by 75% alcohol an istilled water,genomi NA was individually extracted from 112 tick sin he DNeas lood & Tissu it(QIAGEN, Germany).Polymerase chain reaction(PCR nd real-tim CR assay er erforme dentif h athogens.Neste C as used t dentify 1)B.burgdorferibase n 5S(rrfA)-23S(rrlB)rRNA intergenic spacer(IGS), and 2)B.miyamotoibased on glycerophosphodieste hosphodiesterase gene(glpQ). T etect SFGR, conventional PCR was performed b mplifyin fragment o he outer membrane protei gen ompA)o ickettsia spp..Amplification o h onserved 16 RNA gen s an effectiv ethod for identifying AP.Fo C, we designed primer nd aqma G rob ased o he 16S rRN oding gen o eal-time PCR.Table 1 list he targe enes an rimer f five pathogens.PCRamplification er erforme n a Labcycler(Sensoquest, Germany),and th mplicon product er nalyzed b lectrophoresi n 1.5%agarose gel. The real-time PC ssay ere performe n ightCycler 480 (Roche, Switzerland).After purification an equencing,th btained nucleotid equence er nalyzed b he bioninformatics algrorith LAST(https://blast.ncbi.nlm.nih.gov/Blast.cgi), an he results were compare it hose o he sequences deposited at th ational Center for Biotechnology(NCBI). We als ownloaded referenc equence ro enban o enric ur data fo hylogenetic analysis. Th ucleotide sequence ere aligne sing th lustalWmethod,phylogenetic tree ere generate sin EGA 7.0 and the neighbor-joining algorithm,and bootstrap analysis of 1,000 replicates wa erforme o determin h eliabilit h rees.

Base n their morphological characteristics, the ticks collected in this area were identified as eitherHaemaphysalislongicornisorHaemaphysalis japonica. Using a combination o orphologica nd conserve en nalyse mprove h ccurac f tick identificatio n revents subjectiv rror. As such, the 112 ticks wer ubjecte itochondrial 16 RN dentification,and 103H.longicornisand 9H. japonicawer dentified.

Based on th este C ssays, 11 tick 9.82%)wer dentifie s positiv orB.burgdorferi(Table 2).Al equences from th ic amples matche y 99%–100% the registere equences deposited a h CBI. Fou h equences were identica ach othe SX-Q58),with one sequence(SX-Q8 howin 1-bp difference and another sequenc SX-Q112)showing a 3-b ifference.These si mplicon ere closel elate oBorrelia gariniifrom Heilongjiang i hina(JX888456.1).Anothe hre equences wer dentica ach othe SX-Q7 nd wer losely related toBorrelia gariniifro unna hin KP677524.1). In addition,tw he 11 sequences were identica o eac the SX-Q28)and wer losely relate oBorrelia afzeliifrom Russi MK118766.1 Figure 1A).A eas wo pathogeni enotypes(B. gariniiandB.afzelii)occur in Qi County, Shanx rovince,an re also epidemi enotype hina.

W etectedB. miyamotoiin 28 tic amples(25% Table 2). O hi otal,25 sequences were found to b dentica ac the SX-Q1), showing a 1-b ifferenc ne sequence(SX-Q27)and 1-bp deletio nothe equence(SX-Q44).B.miyamotoica ivide nt hre eographicall eparate genotypes,i.e., Siberia o sian), European,and American. These 27 amplicon al nt h ame branch asB. miyamotoifrom Chin KU749384.1,KU749386.1), Japa AY368276.1, LC164098.1), and Russia(KX898133.1,MK955928.1,KJ950108.1),which belon h iberia ype. However, one sequence(SX-Q63)showe n 87%–89% similarity wit h equence fB.miyamotoideposite he NCBI,wit relatively independen ranc he phylogeneti ree(Figure 1B).

Table 1. Target genesan rimerso ic dentificatio n athoge etection

Figure 1. Phylogeneti nalyses o arget gen equence f thre athogens fro hanx rovince. The sequence btained i hi tudy are marked as“●”. (A)Sequence analysis for 5S-23SrRN GSof B.burgdorferi. (B equence analysisfo lp ene of B.miyamotoi.(C equence analysisfor ompA gene of SFGR.

Table 2.Detectio iv athogen n 112 tickso ounty,Shanxi Province

Based o CR amplification an equence alignment o h mp ene o FGR, 12 tick ere positivel dentified(10.71% Table 2), nine o hich wer dentica ach othe SX-Q108 nd the other thre ad th am equenc SX-Q91).SX-Q108 and SX-Q91 wer istributed i ifferent clusters i he phylogenetic tre Figure 1C). These result uggest that ther ay be nove enotypes o FGRin this area. Isolation efforts combined with the characterization of othe enes(rrs, gltA, ompB,and geneD re warranted.We found no presence of AP o hes ic amples.

Coinfectio as observe eve ic amples(6.25%),includin n ick having tripl oinfection(B.burgdorferi,B. miyamotoi, SFGR nd si ith dual coinfection(tw ithB. BurgdorferiandB.miyamotoi, tw ithB. burgdorferiand SFGR,two withB.miyamotoian FGR). Tick a ecome infecte ith multipl athogen fte singl lood mea rom a coinfecte os eeding on single infecte osts during sequentia ife stages.Researchers hav oun ha ick r host animals ca imultaneously infecte ith multiple pathogens and case uman infectio ith multipathoge ick-borne diseas lso occu n some areas i hina. Coinfectio uman nduces nonspecifi ymptoms an ake iagnosis exceedingl ifficult. Moreover,coinfection enhance he severit linical diseas n oses treatmen hallenges. W ecommen hat practitioner pply iagnosti anel rather tha single tes hen a patien resent it ymptoms o ick-born isease.

In Shanx rovince,clinicall onfirmed case f Lym iseas n potte ever have been reported,an erologica xamination av ls hown evidenc nfectio n oinfectio ith Lyme spirochetes. However,n ther tic athogens have bee eporte hanxi. I his study,we screened and successfully identifie he presence o hree pathogens. It i emarkable tha u eam identifiedB.miyatotoian wo pathogeni enospecie f B.b.s.l.(B. garinii,B. afzelii)i uestin icks, which ca ause huma ick-borne disease n re associate it ignificant har uman health in China. Coinfection o hese pathogens wa bserved in seven samples. In China,B. miyamotoiwa irst reporte atient n icks in 2018 in th it f Mudanjian Heilongjiang Province)an here have bee the elate eports[10]. Thus, this represent he firs etectio fB.miyamotoiin ticks i entral China and the discover f coinfectio fB.miyamotoiwith other pathogens i hina.I as als h irst time thatB.miyamotoiwas detected fromH.longicornisandH. japonicai hina. The ticks collecte urin ur study also exhibite n unexpectedly high infectio at 25%, 28/112 fB.miyamotoi.This finding implies tha hi re ay be atural epidemic focu orB. miyamotoi.Further investigation hould be made regardin nfections amon umansand othe osts.

In China, the mai ecto ym iseas sIxodes persulcatus;D. nuttalliis the mai ecto or spotte ever;A.testudinarium, H. yeni,andD.silvarumar ectors fo ME; andIxodes persulcatus, H. longicornis,andH. concinnaare suspected t he main vector GA[3].However,all o he questin ick e collected belonge oHaemaphysalisspp., and ther ittle evidence thatH. longicornisandH. japonicaare vector or the transmissio f tick-borne spirochaetale n ickettsiale n humans.Many factor ffect whethe tick ca c vecto or diseases, includin h ick species,climate, the pathogen arrie h icks,th athogen arried b ivestoc nd othe osts,th ossibilit f experiencing tick bites,and huma mmunity,to nam few. The DN otentia athogen n questing tick o ndicato hei revalence,bu nl marke f the community o icktransmitted pathogen irculating i h arget territory.However,from th resence of important pathogen n ticks, we can at least infe ha ome host animal re reservoirs i hi rea,which may hav ignifican mplication or the detectio nd transmissio ick-borne pathogens.

To clarif he rol hes icks, the 112 tick sample his stud ay be insufficien or determinin he overall prevalenc athogen n tick hi egion. A uch,the sample siz us e increased t btai or eliable results. Further studies are als eede o characterize tick infections i umans and animals.A he numbe f tick-born isease a ontinue ncrease globally, th nfectious microbe ssociated with thes isease ose a major healthcar hreat.Details of the infectiou icroorganisms transmitted to human ick r o e ull lucidated, so further research i ecessary t ai better understanding o h omplexity o hes icktransmitte nzootic agent n huma ealth.Continuous monitorin f tick-born athogen nd emergin ick-borne disease arranted.

Author ContributionYAN ia erforme he experiments an rot h anuscript. YAN ui Jun,CA ong Bing, and HA u participate n th ollectio icks. ZHAN in,HOUXue Xia, and MIAO Guan in articipated i onductin he experiments.HAO Qi upervise he stud nd reviewed the manuscript.

&These authorscontributed equall hi ork.

#Correspondence should be addresse AO Qin,E-mail: haoqin@icdc.cn, Tel:86-10-58900772.

Biographica otes o h irs uthors: YANGXia a,female,born in 1994,Postgraduat tudent, majorin n the molecula etectio ick-born athogens an yme disease;YAN u un, male,bor n 1970,Associat hief Physician,mainl ngaged i h reventio n ontro f natural infectiou iseases.

Received: August 11,2020;

Accepted:December 4, 2020