during april  october 2009 , bird bandings were conducted in the protected area of finca ribavellosa in la rioja , spain ( 4214n , 254w ) .
ticks were collected from birds and classified through taxonomic keys ( 7 ) and molecular methods ( 8) .
dna was individually extracted by using 2 incubations of 20 minutes each with ammonium hydroxide ( 1 ml of 25% ammonia and 19 ml of milli - q water that had been autoclaved ) at 100c and 90c .
dna extracts were used as templates for pcrs targeting fragment genes for tick classification and for bacteria detection ( table 1 ) .
two negative controls , 1 containing water instead of template dna and the other with template dna but without primers , and a positive control ( a tick extract , a. phagocytophilum , b. burgdorferi sensu stricto , or r. slovaca ) were included in all pcrs .
amplification products were sequenced , and nucleotide sequences were compared with those available in genbank by using a blast search ( www.ncbi.nlm.nih.gov/blast/blast.cgi ) .
phylogenetic and molecular evolutionary analyses were conducted by using mega4 ( 16 in technical appendix ) .
, reference ; msp , p44 major surface protein gene ; flab , flagellin gene ; ompb , 120-kda genus common antigen gene ; ompa , 190-kda protein antigen gene ; glta , citrate synthase gene . 
r  =  a / g ; w  =  a / t .
a total of 222 ticks belonging to the species haemaphysalis punctata ( n = 1 ) , ixodes frontalis ( n = 7 ) , i. arboricola ( n = 26 ) , i. ricinus ( n = 181 ) , and other ixodes spp .
two nucleotide sequences for the 16s rrna fragment gene of i. arboricola ticks were recorded ( genbank accession nos . jf791812 and jf791813 ) ( table 2 ) . *
l , larva ; msp , p44 major surface protein gene ; n , nymph ; flab , flagellin gene ; 5s-23s is , 5s-23s rrna intergenic spacer ; ompb , 120-kda genus common antigen gene ; ompa , 190-kda protein antigen gene ; glta , citrate synthase gene . 
a. phagocytophilum was detected only in 1 larva of an i. ricinus tick ( 0.5% ) .
the most prevalent genospecies was b. garinii ( n = 19 ) , which was detected in i. ricinus ( n = 16 ) , h. punctata ( n = 1 ) , i. frontalis ( n = 1 ) , and ixodes sp .
b. valaisiana was amplified in 9 samples ( 8 i. ricinus and 1 ixodes sp .
r. monacensis ( n = 1 ) , r. helvetica ( n = 1 ) , r. sibirica sibirica ( n = 1 ) , and rickettsia spp .
furthermore , according to glta , ompa , and ompb sequence analysis , a possible new rickettsia sp . was found in 25 i. arboricola ticks and 2 i. ricinus ticks .
for these 27 samples , highest identities with r. heilongjiangensis ( 97.1% ) and r. japonica ( 99.1% ) were found for ompa ( genbank accession no .
jf758826 ) nucleotide sequences , respectively , whereas glta nucleotide sequences were identical to those from both rickettsia spp . according to multilocus sequence typing ( data not shown ) and genetic criteria agreed on by experts , a candidatus status could be assigned .
we named it candidatus rickettsia vini ( 17 in technical appendix ) ( table 2 ) .
( figure ) . the phylogenetic position of candidatus rickettsia vini based on the ompa nucleotide sequences in a study of the role of birds in dispersal of etiologic agents of tick - borne zoonoses , spain , 2009 .
the percentage of replicate trees in which the associated taxa clustered in the bootstrap test ( 1,000 replicates ) is shown next to the branches .
the tree is drawn to scale , with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree .
the evolutionary distances were computed by using the kimura 2-parameter method and are in the units of the number of base substitutions per site .
the presence of anaplasma , borrelia , and rickettsia species in ticks removed from passerine birds corroborates the role of these vertebrates in the epidemiology and dispersion of tick - borne pathogens in spain and in other zones of the planet .
some of the parasitized birds in our study , such as the european robin ( erithacus rubecula ) or eurasian blackcap ( sylvia atricapilla ) , are considered migratory or partial migratory birds .
in addition , these species share an ecologic niche and ectoparasites ( horizontal transmission ) with other migratory birds that cover long distances from africa to the eurasian region . except for i. arboricola , the tick species captured in this study previously had been found on birds in spain ( 18 in technical appendix ) .
the high prevalence of i. ricinus ticks was expected because it is the most frequent tick in this area , and the immature stages of this tick frequently parasitize birds .
i. ricinus ticks are the main vectors of a. phagocytophilum in europe , and this microorganism has been detected on vegetation in the studied area ( 1 ) .
however , the low prevalence ( 0.5% ) of a. phagocytophilum in the ticks in our study corroborates data from other studies ( 19,20 in technical appendix ) .
the presence of a. phagocytophilum in a larva in our study supports the role of birds as reservoirs of a. phagocytophilum .
the prevalence ( 13.1% ) of b. burgdorferi in our samples is similar to prevalences reported in other studies in europe in which i. ricinus is the main species of tick captured from birds ( 19 in technical appendix ) . in spain , b. garinii , b. valaisiana , and b. afzelii
have been detected in ticks from birds ( 18 in technical appendix ) . according to our data ,
the human pathogen b. garinii was the most prevalent species , as reported in birds from europe ( 21 in technical appendix ) .
although it has been recently detected in ticks from birds in norway ( 22 in technical appendix ) , its finding in spain was unexpected .
regarding rickettsia species , r. monacensis and r. helvetica are among the human pathogens detected in our study .
both species have been identified in ticks from birds in europe ( 19,20,23 in technical appendix ) . on the contrary ,
candidatus rickettsia vini , a potential new rickettsia species , also detected in our study , has not been related to human disease ( 17 in technical appendix ) .
several genospecies closely related to r. heilongjiangensis and r. japonica have been identified in ixodes spp .
r. sibirica sibirica , responsible for siberian tick typhus in western people s republic of china and in siberia , was also amplified in an i. ricinus larva in this study .
our data confirm the involvement of birds in the cycle of human tick - borne diseases .
 we amplified gene sequences from anaplasma phagocytophilum , borrelia garinii , b. valaisiana , b. turdi , rickettsia monacensis , r. helvetica , r. sibirica sibirica , and rickettsia spp . 
 ( including candidatus rickettsia vini ) in ticks removed from birds in spain . 
 the findings support the role of passerine birds as possible dispersers of these tick - borne pathogens . 
The Study
Conclusions
Supplementary Material