tick surveillance : the tick surveillance was undertaken at 14 fixed points 
 and in 5 wild boars in kyoto city ( fig . 1.geographical representation of the tick surveillance points and tick prevalence in 
 kyoto city , japan in 2011 . we captured the ticks by a flagging method on the mountain 
 slopes around the city , at locations indicated by letters a to n ( a ) . ticks were also collected from wild boars hunted in yamashina - ku ( point o ) . the 
 proportions of tick species collected at each surveillance point were plotted on a 
 city map ( b ) . the legend for tick species shown in each pie chart is given in the box 
 in panel d. we classified tick distribution into three general areas , i , ii and iii , 
 according to the proportion of tick species shown in panel b. the points at which the 
 detection of rickettisal dna was performed are indicated with an asterisk ( * ) . the 
 points where rickettisal dna was detected are indicated with a double asterisk ( * * ) 
 ( c ) . we then re - constructed the tick population graphs for each geographical area , i , 
 ii and iii ( d ) . a circle representing a population size of fifty ticks is shown in the box in panel 
 d. ) from may to september 2011 , to identify tick populations and tick species in human 
 habitats in kyoto city . we chose 14 surveillance points located on the mountainsides of the 
 northern ( a to e in fig . 1a and table 1table 1.ticks collected in the area around kyoto cityplants / wild boresarea of kyoto citycollection point ( altitude)collection datetick species collectednumbers of ticks collectedtotal numbermalefemalenymphlarvaplantsi ( north)a ( keihokuyuzuki)9 augh . testudinarium2530028total number666134627253198 ) , western ( f to k ) and eastern ( l to n ) parts of kyoto city . ticks were also 
 collected from 5 wild boars hunted in yamashina - ku ( o ) for the harmful wildlife control . adult and nymph ticks were placed in sample bottles containing wet filter paper and taken 
 back to our diagnostic laboratory . they were anesthetized with chloroform . geographical representation of the tick surveillance points and tick prevalence in 
 kyoto city , japan in 2011 . we captured the ticks by a flagging method on the mountain 
 slopes around the city , at locations indicated by letters a to n ( a ) . ticks were also collected from wild boars hunted in yamashina - ku ( point o ) . the 
 proportions of tick species collected at each surveillance point were plotted on a 
 city map ( b ) . the legend for tick species shown in each pie chart is given in the box 
 in panel d. we classified tick distribution into three general areas , i , ii and iii , 
 according to the proportion of tick species shown in panel b. the points at which the 
 detection of rickettisal dna was performed are indicated with an asterisk ( * ) . the 
 points where rickettisal dna was detected are indicated with a double asterisk ( * * ) 
 ( c ) . we then re - constructed the tick population graphs for each geographical area , i , 
 ii and iii ( d ) . a circle representing a population size of fifty ticks is shown in the box in panel 
 d. detection of rickettsial dna by pcr : a total of 201 ticks collected at 9 
 locations and from 5 wild boars in kyoto city from june to september 2011 were selected for 
 the detection of rickettsial dna by pcr ( table 
 2table 2.prevalence of ticks investigated for detection of rickettsiaplants/ wild boarsarea of kyoto citycollection point ( altitude)tick speciesno . of ticks of ticks positive for 
 rickettisano . of ticks positive for r. 
 japonicamalefemalenymphplantsi ( north)a ( keihokuyuzuki)h . longicornis10922155total number2431146201143125a ) number of ticks which were positive in the pcr using at least one primer pair for 
 rickettsial dna . b ) number of ticks which were positive in the pcr using primer pair , 
 rj5 and rj10 . ) . they belonged to 3 genera including 4 species : haemaphysalis 
 ( h. ) longicornis , h. flava , 
 amblyomma ( a. ) testudinarium and 
 dermacentor ( d. ) taiwanensis ( table 2 ) . one hundred and ninety four ticks ( 96.5% ) 
 were identified as h. longicornis . twenty four , 30 and 140 ticks were male , 
 female and nymph , respectively . two 
 h. flava nymphs , 4 a. testudinarium nymphs and 1 adult 
 d. taiwanensis female were also examined . to extract dna from the ticks , these ticks were 
 placed on slide glass and covered with 100 l of phosphate buffered saline 
 ( pbs ) . after placing cover glasses on top a pcr was 
 performed using the genus - specific primer pair of rpcs877 , 5-gggggcctgctcacggcgg and 
 rpcs1258 , 5-attgcaaaaagtacagtgaaca for the rickettsial citrate synthase 
 ( glta ) gene . the reaction was 
 carried out using takara ex taq ( takara bio , otsu , japan ) with a 20 l 
 reaction mixture containing 2 l of template dna , 200 m 
 each of dntp and 1 m each of primer . following initial denaturation at 
 95c for 10 min , 5 cycles of denaturation at 94c for 60 sec , annealing at 53c for 60 sec 
 and extension at 72c for 60 sec were performed . then , 30 cycles of denaturation at 94c for 
 60 sec , annealing at 60c for 60 sec and extension at 72c for 60 sec were followed . the rickettsia 17-kda 
 genus - common antigen ( 17k antigen ) gene was also amplified using the primer pair r1 ; 
 5-tcaattcacaacttgccatt-3 and r2 ; 5-tttacaaaattctaaaaacc-3 . nested pcr was performed with a primer pair , rj5 ; 
 5-cgccattctacgttactacc-3 and rj10 ; 5-attctaaaaaccatatactg-3 to detect the sequence of 
 r. japonica dna using the 100 times diluted pcr products which were 
 amplified with primer pair of r1 and r2 as a template . the reaction was carried out using takara ex taq with a 20 l 
 reaction mixture containing 2 l of template dna , 200 m 
 each of dntp and 1 m of each primer . after initial denaturation at 94c 
 for 5 min , 30 cycles of denaturation at 94c for 30 sec , annealing at 57c for 2 min and 
 extension at 70c for 2 min were carried out . the pcr products were purified using the pcr purification kit ( wizard sv 
 gel and pcr and clean - up system ; promega , madison , wi , u.s.a . ) , and the sequences of 
 amplified products were determined by cycle sequencing . a ) number of ticks which were positive in the pcr using at least one primer pair for 
 rickettsial dna . b ) number of ticks which were positive in the pcr using primer pair , 
 rj5 and rj10 . phylogenetic analysis : the sequences obtained were evaluated and aligned 
 with each other and those of previously characterized rickettsiae in genbank by using blastn 
 . multiple alignment analysis was carried out 
 using the program clustalw . phylogenetic analysis 
 was carried out for the approximately 450 bp of partial sequence of 17k antigen gene using 
 the genetyx 11.1 software . a total of 3,198 ticks belonging to 5 species of 3 genera were collected from 12 
 of the 14 sampling points in kyoto city ( fig . 1a ) . a breakdown of numbers by percentage shows that 99.2 , 6.6 , 0.3 , 
 0.2 and 0.03% of the collected ticks from the plants were h. longicornis 
 neumann , h. flava neumann 
 , a. testudinarium koch , h. kitaokai hoogstraal   and d. taiwanensis sugimoto , respectively ( table 1 ) . the dominant species among the ticks collected in this study were 
 h. longicornis and h. flava ( fig . 1 and table 
 1 ) . the proportions of each growth stage among these ticks were 1.9 , 
 11 and 87.1% for adults , nymphs and larvae , respectively ( table 1 ) . no significant difference in sex among the collected 
 population was observed in this survey ( table 
 1 ) . furthermore , the populations of the species collected were not 
 affected by the altitude of the surveillance points ( table 1 ) . sixty - eight ticks belonging to h. 
 longicornis , h. flava and a. testudinarium were 
 also collected from 5 wild boars ( table 
 1 ) . the proportions of tick species found at each surveillance point are shown on a map of 
 kyoto city ( fig . interestingly , the tick 
 population differed markedly according to surveillance point . in the northern mountains ( a , 
 b , c , d and e ) , almost all the ticks captured were h. longicornis ( fig . 1b ) . in the western mountains 
 ( f , g , h and i ) , h. longicornis and h. flava were the 
 dominant species ( fig . 1b ) , whereas in the eastern 
 mountains ( l and m ) , a. testudinarium was the second common species after 
 h. longicornis ( fig . a total of 201 ticks collected at 9 points in the mountainous area in kyoto city from july 
 to september 2011 were investigated for the presence of rickettsial genes using primer pairs 
 for glta and 17k antigen genes . of these , 143 ( 71.1% ) of the ticks appeared 
 to be positive for rickettsial dna amplified by at least one pcr primer pair ( table 2 ) . of the pcr- positive ticks , 125 ( 87.4% ) of 
 the samples were positive with the pcr using r. japonica - specific primers 
 rj5 and rj10 . rickettsial dna was detected in 141 ( 72.7% ) of the h. 
 longicornis consisting of 12 males , 12 females and 117 nymphs . of these , 1 male , 
 2 females and 2 nymphs were captured from wild boars . no rickettsial dna was 
 amplified in h. flava and d. taiwanensis with the primer 
 pairs ( rpcs877 and rpcs1258 , r1 and r2 , and/or rj5 and rj10 ) used in this study . rickettsial 
 dna was detected in the 8 surveillance points a , b , c , d , e , f , g and l ( table 2 and fig . the sequence analysis of selected pcr fragments using the r1 and r2 primer pair ( r1/r2 
 fragment ) obtained from h. longicornis showed similarity to each other , 
 with 0 to 3 nucleotide sequence differences in 475 bases ( 99.3100% homology ) . the 
 similarity of the r1/r2 fragment of a. testudinarium to that of h. 
 longicornis was 96.2% ( 457/475 ) . the r1/r2 fragments were subjected to 
 phylogenetic analysis to confirm the association with previously reported rickettsial 
 sequences ( fig . 2.phylogenetic tree of sfg rickettsiae isolated from ticks in kyoto city , japan derived 
 from sequences of pcr fragments of the 17k antigen gene . r17k - ti187f represents the pcr fragments of the 17k antigen 
 gene obtained in this study from h. longicornis . the r1/r2 fragment from h. longicornis ( r17k - ti187f ) clustered 
 into the spotted fever group of rickettsia , including r. japonica , 
 r. conorii and r. slovaca , whereas those from a. 
 testudinarium were divided into clusters other than sfg rickettsia . phylogenetic tree of sfg rickettsiae isolated from ticks in kyoto city , japan derived 
 from sequences of pcr fragments of the 17k antigen gene . r17k - ti187f represents the pcr fragments of the 17k antigen 
 gene obtained in this study from h. longicornis . the tick species collected in kyoto city are known to be able to transmit various zoonotic 
 pathogens to humans and animals . for instance , h. longicornis is well known 
 to harbor the pathogens of theileriosis , 
 babesiosis , japanese spotted fever [ 14 , 21 , 27 , 31 ] and many 
 zoonotic viral fevers , including sfts [ 20 , 35 ] . h. flava is a possible vector of 
 the etiological agents of anaplasmosis   and 
 japanese spotted fever . sfts viruses   and ehrlichial rickettsiae   have been detected in a. testudinarium , and r. 
 japonica , which causes japanese spotted fever , has been detected in d. 
 taiwanensis . according to the map shown in fig . 1b , the tick 
 distribution in kyoto city might be classified into at least three geographical areas as 
 shown in fig . 1c and 1d : i , northern mountain 
 slopes ( a , b , c , d and e ) , ii , western mountain slopes ( f , g , h and i ) and iii , eastern 
 mountain slopes ( l and m ) . further 
 studies are needed to characterize the distribution of this tick species in the city as the 
 sampling period was limited . the present study revealed that 71.1% of ticks present near the central city of kyoto were 
 found to be positive for rickettsial dna by pcr . rickettsial dna was detected at all the 8 
 surveillance locations where h. longicornis was collected ( table 2 and fig . h. longicornis , which was the most abundant tick in this study , and of 
 which 78% were positive for rickettsial dna , appeared to be the dominant carrier of 
 rickettsial dna in the area surveyed . h. longicornis is well known as a 
 human - biting tick and is considered to be a competent vector of r. japonica 
 in japan . previously , rickettsial dna was 
 detected in various amounts in h. longicornis obtained from different areas 
 in the north and south of japan [ 14 , 16 ] . the detection of r. japonica has 
 previously been reported in various areas in japan [ 14 , 27 ] . the study on the prevalence of 
 rickettsia in ticks in the ehime prefecture , where over 70 cases of japanese spotted fever have been reported during the past decade , 
 showed that rickettsial dna was detected in 12.6% of ticks using the same primer pairs as 
 used in this study . on the other hand , 62.2% of 
 the ticks appeared to be positive for pcr using rj5 and rj10 as the primer set for 
 r. japonica in the present study . the sequences of the detected dna 
 segments were analyzed and found to have 98.5% similarity with r. japonica 
 yh strain which was isolated from a patient with japanese spotted fever . the phylogenic tree 
 revealed their sequences clustered with the spotted fever group rickettsiae , including 
 r.rickettsii and r. conorii , the causative agent of 
 rocky mountain spotted fever and mediterranean spotted fever , respectively . as cases of 
 rickettsiosis have never been reported in the area where we studied , in spite of the high 
 prevalence of rickettsiae in ticks collected , their pathogenicity is still unclear . the 
 sequences of the 17k antigen gene fragment were identical to those of the rickettsia related 
 species , lon-13 , which was detected in ticks and considered to be of low pathogenicity 
 . a. testudinarium is also a 
 possible vector of rickettsia in this area , although further studies are needed because only 
 a limited number of ticks were tested in this study . unfortunately , only one 
 blood sample was available from the wild boars , and rickettsial dna was not detected in its 
 blood sample ( data not shown ) . the proportion of ticks obtained from wild boars that 
 harbored rickettsial dna ( 5/21 , 23.8% ) was less than that of ticks sampled in the plants 
 ( 138/180 , 76.7% ) . although there are no reports of japanese spotted fever in humans in kyoto city , many ticks 
 that can transmit japanese spotted fever pathogens were distributed across the whole the 
 city . therefore , we must stay alert and pay attention to tick - borne diseases to ensure the 
 safety of the citizens of the city as well as that of visitors .
abstractthe tick is a well - known 
 vector for arthropod - borne pathogens , such as tick - borne encephalitis , lyme disease , 
 japanese spotted fever and severe fever with thrombocytopenia syndrome . 
 it is therefore 
 important to know the tick population and distribution in our environment and wild animals 
 in order to prevent tick - borne diseases . here , we report the results of tick surveillance 
 from may to september 2011 at 14 geographical points and in 5 wild boars in kyoto city , 
 kyoto prefecture , japan . 
 we collected 3,198 ticks comprising 5 tick species , 
 haemaphysalis ( h. ) longicornis , 
 h. flava , h. kitaokai , amblyomma 
 testudinarium and dermacentor taiwanensis . 
 interestingly , the 
 proportion of tick species varied according to geographical region within the city . the 
 ticks collected in the city were reported as potential vectors of pathogens , such as 
 rickettsiosis . 
 we detected rickettsial dna by pcr in 71.1% of 201 ticks investigated . 
 the 
 ticks that carried rickettsiae were distributed across the whole the city . 
 the sequences 
 of pcr - amplified dna fragments were determined and showed similarities to spotted fever 
 group rickettsiae . although their pathogenicity for animals including humans is still 
 unclear , it is important to stay alert and pay attention to tick - borne diseases in order 
 to ensure the safety of the citizens of the city as well as that of visitors .