Molecular Detection of Tick-borne Pathogens in Ticks Collected from Hainan lsland,China＊

LU Miao , TANG Guang Peng , BAI Xiao Song , QIN Xin Cheng ,WANG Wen , GUO Wen Ping , and LI Kun,#

Pathogens like bacteria and protozoa,which affect human and animal health worldwide,can be transmitted by vectors like ticks.To investigate the epidemiology and genetic diversity of bacteria and protozoans carried by ticks in Chengmai county of Hainan province,China,285 adult hard ticks belonging to two species [Rhipicephalus sanguineus(sensu lato): 183,64.21% andRhipicephalus microplus:102,35.79%] from dogs,cattle,and goats were collected.Microbial families were identified in these ticks by amplifying the 18S rRNA,16S rRNA(rrs),citrate synthase (gltA),and heat shock protein(groEL) genes.Our data revealed the presence of four recognized species and two Candidatus spp.of Anaplasmataceae and Coxiellaceae.In sum,these data reveal an extensive diversity of Anaplasmataceae bacteria,Coxiellaceae bacteria,Babesiidae,and Hepatozoidae in ticks from Hainan Island,highlighting the need to understand the tickborne pathogen infection in local animals and humans.

Key words:Ticks; Rickettsiales bacteria;Protozoa;Coxiellaceae bacteria;Tick-borne disease;China

Ticks function as vectors for a variety of etiological agents of zoonotic diseases,including viruses,bacteria,and protozoa[1].Some of these tickborne diseases,such as rickettsiosis,anaplasmosis,and babesiosis,are of substantial concern for both humans and animals all over the world.In China,tick-borne pathogens pose a great threat to residents,especially those in rural areas and forests[1].However,most of these pathogens are still undetermined because of the limited epidemiological and clinical information about them.In addition,misdiagnosis is very common,as their clinical manifestation is often similar to other syndromes such as hemorrhagic fever with renal syndrome (HFRS)[1].Importantly,ticks also act as vectors for a variety of pathogens that infect companion animals and livestock[1].These include bovine anaplasmosis (Anaplasma marginaleandAnaplasma bovis),bovine babesiosis (Babesia bigeminaandBabesia bovis),canine babesiosis(Babesia canis),canine ehrlichiosis (Ehrlichia canis),caprine anaplasmosis (Anaplasma capra),and many more[2].These pathogens have significant economic impacts on livestock production and causing great losses each year.On the other hand,infection in these animals also increases the risk of developing tick-borne disease in their owners[1].

Hainan Island,the second largest island of China,is located in the South China Sea.It has a typical tropical climate and plentiful wildlife.It has an area of 33,900 km2and a population of 9.34 million (2018 estimations).In the recent decades,it has become a tourist attraction because of its beautiful scenery and geographical position. A previous study indicated that Hainan Island is the natural epidemic focus of North Asia tick-borne spotted fever (NASF),which is caused byRickettsia sibiricabelongs to spotted fever-causing group Rickettsiae (SFGR)[3].Investigation of NASF antibodies in locality,revealed an incidence of 38.3% and 53.0% in local human and mouse,separately[3].However,there are very few reports on other tick-borne pathogens in Hainan Island.In this study,to evaluate the potential risk of tick-borne pathogens to local residents,tourists,and domestic animals,we analyzed the occurrence and prevalence of bacteria and protozoans in ticks collected from Hainan Island,China.

A total of 285 adult tick samples were collected from 12 stray dogs,10 goats,and 15 cattle in Fushan town,located in the northwest of Hainan Island in September and October 2019.We chose domestic animals that had extensive contact with humans,and 5-10 ticks were obtained from each animal.Ticks were brought to China CDC (Chinese center for disease control and prevention) alive and stored individually at-80 °C before DNA extraction.All the animal experiments were approved by the ethics committee of the National Institute of Communicable Disease Control and Prevention of the China CDC,under the permit number ICDC:2020-018.

Before DNA extraction,the ticks were washed with 75% ethyl alcohol and 0.01 mol/L phosphate buffer solution (PBS) followed to be dried.They were divided into pools (3 ticks each) and homogenized in PBS.Genomic DNA was extracted using a QIAamp DNA Mini Kit (Qiagen,Hilden,Germany) according to the instructions of the manufacturer.All DNA extracts were stored at -20 °C.

These ticks were identified by morphological observation and sequence analysis of their cytochrome oxidase subunit 1 (COI) gene,and they were assigned to two tick species,Rhipicephalus sanguineussensu lato(183,64.21%) andRhipicephalus microplus(102,35.79%).The taxonomical keys used for tick identification mainly included the shape of basis capitulum,scutum,palp,coxae,eyes,anal groove,festoons and adanal plates,spiracle,and hypostomal teeth[4].All the 132 ticks from dogs and 51 ticks from goats wereR.sanguineussensu lato.They were divided into 44 pools and 17 pools,respectively,with 3 ticks for each pool.The 102 ticks from cattle were allR.microplusand they were divided into 34 pools (Supplementary Table S1,available in www.besjournal.com).

Supplementary Table S1. Detection of tick-borne pathogens from domestic animals in Hainan island,China,2019

All the tick samples were tested for the presence of Rickettsiales bacteria,Coxiellaceae bacteria,and parasites belonging to the families Babesiidae and Hepatozoidae.PCR targeting the 18S rRNA gene for the detection of Babesiidae and Hepatozoidae species was performed as described previously[5].Therrsgene of Rickettsiales bacteria and Coxiellaceae bacteria species were amplified by nested PCR using primers as described[6,7].For further exploration of the phylogenetic positioning of the bacterial strains,the 760 bp fragment of thegltAgene encoding citrate synthase and the 614 bp fragment of thegroELgene encoding the heat shock protein,were amplified for samples that were positive for these bacteria.Both negative and positive controls were used.

PCR amplicons were analyzed by electrophoresis in 1.0% agarose gels.The amplicons shorter than 600 bp were directly subjected to Sanger sequencing by Sangon Biotechnology Company (Shanghai,China).The PCR products longer than 600 bp were cloned into the pMD19-T cloning vector (TaKaRa,China),transformed intoE.coliand plated onto a culture dish.Then the obtained clones were picked and sent for bi-directional sequencing.

Nucleotide sequence similarities between the obtained 16S/18S rRNA,groEL,andgltAsequences and those from GenBank were calculated by DNAStar (ver.7.0).For phylogenetic analysis,the sequences were aligned with reference sequences using ClustalW (default parameters) within MEGA 5.2[6,8].Phylogenetic trees were then estimated using the Maximum Likelihood (ML) method implemented in PhyML,version 3[6].A total of 1,000 bootstrap replicates were used under the same procedure to estimate the support for each node.All trees were mid-point rooted.

The present study revealed that several species of ticks parasitize many wild animals and livestock in Hainan Island,China.We mainly foundR.sanguineussensu latoandR.microplusin Chengmai.Many kinds of tick species can be found around Hainan Island includingIxodes granulatus,R. sanguineus,R.microplus,Haemaphysalislongicornis,R.haemaphysaloides haemaphysaloides,Dermacentor auratus,Amblyomma javanense,A.testudinarium,H.lagrangei,H.hystricis,H.yeni,andH.doenitzi,and the main species areIxodes granulatus,R.sanguineus,R.microplus,andR.haemaphysaloides haemaphysaloidesin Chengmai County.In the current study we evaluated the presence of several pathogens like Rickettsiales bacteria,Coxiellaceae bacteria,Babesiidae,and Hepatozoidae in these two ticks.The results revealed the presence of four recognized species and twoCandidatusspp.of Anaplasmataceae and Coxiellaceae.Importantly,A.marginale,A.platys,H.canis,B.canis,andCoxiella burnetii,which were identified in this study,are known to be animal and/or human pathogens[1].Hence,our data clearly indicated that multiple bacteria and protozoa co-circulate in hard ticks in Hainan Island.As more tick species are present in Hainan Island,it is likely that more tickassociated pathogens are to be discovered.

Genetic analysis of the recovered 18S rRNA gene sequences using the BlastN with Nucleotide collection (nr/nt) revealed that they were most closely related to those ofBabesia canis vogeli(100.00%) andHepatozoon canisstrain SK-144(99.77%) (Table 1).Phylogenetic analysis of 18S RNA gene sequences revealed the co-circulation ofB.canisandH.canisin the 1 and 5R.sanguineussensu latopools from dogs,respectively (Table 2,Supplementary Figure S1,available in www.besjournal.com).The sequences ofB.canis vogeliHNRS/dog/B1 were closely related to those of the knownB.canis vogelifound inR.sanguineussensu latofrom domestic and foreign[2,6]in the 18S RNA tree (Supplementary Figure S1A).Additionally,the sequencesH. canisHNRS/dog/A2,H. canisHNRS/dog/B6,andH.canisHNRS/dog/C12 were closely related to each other,and they were also related toH.canisstrain SK-144 andH.canisstrain 9992-4[9]inCanis lupusspecimens from Israel,Saint Kitts,and Nevis,forming a distinct lineage in the 18S RNA tree (Supplementary Figure S1B). Canine hepatozoonosis is a tick-borne disease distributed worldwide triggered byH.canisin dogs[9].Canine babesiosis is a vector-borne disease caused byBabesiaspp.likeB.canis vogeli[9].Our data revealed that these two agents are co-circulating in arthropods,and the infection risk is high for dogs,as well as other animals in certain regions.

Supplementary Figure S1. Phylogenetic trees based on the partial 18S rRNA gene sequences of Babesia canis (A) and Hepatozoon canis (B).Both trees are mid-point rooted for clarity only.Bootstrap values (＞70%) are shown for appropriate nodes.The scale bar represents number of nucleotide substitutions per site.The sequences revealed in the present study are marked in red.

Genetic analysis of therrs,groEL,andgltAgene sequences from the bacterial pathogens revealedthat they were closely related to those of Coxiellaceae and Anaplasmataceae bacteria.Briefly,therrssequences recovered from the tick pools sampled from Hainan Island exhibited high sequence similarities to those from species of Coxiellaceae bacterium (100%),A.marginale(100%),A.platys(99.87%),andEhrlichia sp.(100%) (Table 1).The similarities between the sequences recovered from this study and known reference sequences from GenBank varied from 99.28% to 99.86% for thegltAgene sequences,and from 99.88% to 100.0% for thegroELgene sequences (Table 1).Hence,these data revealed the co-circulation ofEhrlichia,Anaplasma,and the proposedCandidatusCoxiellaceae bacterium in ticks collected from Hainan Island(Figure 1,Table 1,Supplementary Figure S2,available in www.besjournal.com).

Table 1.Bacterial and protozoal sequences obtained from ticks in Hainan island,China

Table 2.Prevalence of tick-borne pathogens in ticks in Hainan Island,China

Supplementary Figure S2. Phylogenetic trees based on partial Coxiellaceae bacteria rrs gene sequences.Trees are mid-point rooted for clarity only.Bootstrap values (＞ 70%) are shown for appropriate nodes.The scale bars represent number of nucleotide substitutions per site.The sequences revealed in the present study are marked in red.

Anaplasmataceae bacteria were identified in 5R.sanguineussensu latopools,and 6R.micropluspools (Table 2). Phylogenetic analysis of the sequences ofrrs,gltA,andgroELgenes revealed the circulation of three species of Anaplasmataceae bacteria in the ticks from Hainan Island.In therrs,gltA,andgroELgene trees (Figure 1),the sequences ofA.marginaleHNRM/cattle/D6,A.marginaleHNRM/cattle/D26,andA.marginaleHNRM/cattle/D29,were closely related to those of the knownA.marginalefound in ticks from cattle[6].Notably,A.platysHNRS/dog/C6 andA.platysHNRS/dog/C23 clustered with those ofA.platysdiscovered in ticks,dogs,and camels from China,Portugal,Italy,and Japan[6]in all three gene trees(Figure 1). Finally,the sequenceEhrlichiasp.HNRM/cattle/E8 andEhrlichiasp.HNRM/cattle/E24 recovered from twoR.microplustick pools exhibited a close relationship to those of the candidatusEhrlichiasp.identified in ticks from China (Fujian,Wuhan,Xinjiang,and Shenyang provinces),Niger,and Thailand[6](Figure 1).

Figure 1.Phylogenetic trees based on partial Anaplasmataceae rrs (A),gltA (B),and groEL (C) gene sequences.Trees were mid-point rooted for clarity only.Bootstrap values (＞ 70%) are shown for appropriate nodes.The scale bars represent number of nucleotide substitutions per site.The sequences revealed in the present study are marked in red.

Many members in the family Anaplasmataceae cause tick-borne diseases (i.e.Anaplasmosis and Ehrlichiosis) with a remarkable impact on human and animal health[2,10].For example,A.phagocytophilum,A.marginale,andA.platysare the important disease-producing pathogens in the genusAnaplasma[2].A.marginaleis a common pathogen among ruminants infecting buffalo and cattle distributed on six continents,especially in tropical and subtropical regions[10].In contrast,A.platyscauses cyclic thrombocytopenia in dogs and is the only classified Rickettsiales species known to infect platelets[2].Furthermore,A.platysinfection has been reported in cats,foxes (Vulpes vulpes),cattle,goats,camels,red deer,and humans[2].In our study,A.marginaleandA.platyswere identified inR.microplusandR.sanguineussensu latoticks,respectively.The cattle,which are the major hosts ofR.microplus,showed the high prevalence ofA.marginale.Therefore,these results suggest that more cattle surveillance in this area is necessary.R.sanguineus sensu latois considered the primary vector ofA.platys.Due to the close exposure between the dogs and others animals,the high infection rate ofA.platysin local dog ticks indicated high risk of cyclic thrombocytopenia affecting animals and humans.Therefore,more surveillance and research on dogs and its pathogens should be carried out in Hainan Province.

In the past decades,several novelEhrlichiaspecies have been discovered in ticks and vertebrate hosts[9].Herein,a tentative species was identified inR.microplusticks from cattle,which is mainly found inR.microplusticks from several provinces and countries.The genome sequence of this candidatus species is close to those ofE.chaffeensis,a widespread human pathogen. Therefore,more attention should be paid to its pathogenicity and potential public health risk.

Coxiella-like bacterial DNA was found in 15R.sanguineussensu latotick pools (Table 2).In therrsgene tree (Supplementary Figure S2),three positive samples (Coxiella-like bacteria HNRS/goat/Y5,Coxiella-like bacteria HNRS/goat/Y9,andCoxiella-like bacteria HNRS/goat/Y10) were analyzed,which were 100% similar to Coxiellaceae bacterium PH06(KM079622),found inPediculus humanusin Marseille,France in 2014.In this study,the positive sequences were clustered with those of Coxiellaceae bacterium discovered inR.sanguineussensu lato and R.bursaticks in Marseille,France,and Israel.Coxiella burnetiiis the causative agent of Q fever,which is a worldwide zoonotic disease.Normally,the infection is persistent in animals,whereas humans are often asymptomatic.It can manifest as a flu-like illness or pneumonia in its acute form,or in a chronic form like endocarditis[7].There are no typical symptoms when infection occurs in animals and humans except during pregnancy.The primary reservoirs ofC.burnetiiare goats,sheep,and cattle[7].In nature,ticks play an important part in the maintenance and transmission of the bacteria,whereas cattle and goats play a very important role in human infections.In this study,Coxiella-like bacteria were identified in 15R.sanguineus sensu latoticks sampled from Hainan Island. More attention should be paid on this pathogen due to its close relationship with the known human pathogenCoxiella burnetii.

In conclusion,two species (R.microplusandR.sanguineussensu lato) of hard ticks were sampled from dogs,cattle,and goats and four recognized species and twoCandidatusspp.of Anaplasmataceae and Coxiellaceae were discovered from these two hard ticks,indicating that this specific geographic region harbors a considerable diversity of Anaplasmataceae bacteria,Coxiellaceae bacteria,Babesiidae,and Hepatozoidae.The data from this study highlight the necessity for the surveillance of local arthropods,mammals,and humans,which may provide the evidence of the presence of several bacterial and protozoan pathogens.

Conflicts of InterestThe authors declare no competing interests.

Author ContributionLI Kun designed the research and supervised the experiments;TANG Guang Peng,BAI Xiao Song,LI Kun,and QIN Xin Cheng collected the samples and performed the experiments;LI Kun,LU Miao,and WANG Wen analyzed the data;LI Kun,LU Miao,and GUO Wen Ping wrote the manuscript.

#Correspondence should be addressed to LI Kun,Tel:86-10-58900783,Fax:86-10-58900700,E-mail:likun@icdc.cn

Biographical note of the first author:LU Miao,female,born in 1986,Doctoral Degree (PHD,MD),Associate Researcher,majoring in tick-borne diseases,especially in rickettsiales bacteria and rickettsioses.