birds were caught by mist - nets , banded ( ringed ) , and examined for ticks at the cernek bird ringing station ( 4136n , 3605e ) in the kizilirmak delta in turkey , an internationally important wetland area for birds ( 4 ) .
we conducted the study during the spring and autumn migration seasons in 2010 and 2011 and in spring 2012 .
the identified ticks were placed in tubes with steel beads and homogenized at the maximum speed ( 50 hz ) for 10 min in tissuelyser lt device ( qiagen , hilden , germany ) .
rna was isolated according to the manufacturer s recommendations by using high pure viral nucleic acid kit ( roche applied science , mannheim , germany ) , but as a small modification , the homogenized tick mixture was kept at 37c for 1 h. in accordance with the manufacturer s recommendations , we obtained viral cdna using the revertaid first strand cdna synthesis kit ( thermo scientific , vilnius , lithuania ) .
real - time pcr was performed by using the combination of primer pairs and the faststart taqman probe master kit ( roche applied science ) , as described by yapar et al . ( 6 ) , for each tick sample .
cdna from patient samples , which previously had been determined as positive , were used as the positive control sample .
cchfv small ( s ) segment ( encoding for the nucleocapsid protein ) specific primer pairs ( f3 : 3-gaatgtgcatgggttagctc-5 and r2 : 3-gacatcacaatttcaccagg-5 ) and same pcr conditions defined by schwarz et al . ( 7 ) were used in the pcr .
sequence analysis was performed on the 260-bp pcr product , when positivity was detected , by using the primers of f3 and r2 in the abi 310 genetic analyzer ( applied biosystems , foster city , ca , usa ) .
our sequences and genbank sequences were aligned in mega 5.1 ( http://www.megasoftware.net ) , and the phylogenetic tree was drawn on the basis of the 260 bp of the s segment of the cchfv genome . to compare the sequences and phylogenetic analysis , we used the maximum - likelihood method .
we found attached ticks on 65 ( 0.5% ) of the 13,377 captured and banded birds , which represented 17 species .
a total of 188 ticks collected on these birds belonged to ixodes , hyalomma , haemaphysalis , and rhipicephalus genera ( table ) .
( nymphs ) ( 127 [ 0.8% ] ) on great reed warblers ( acrocephalus arundinaceus ) ( 9.7% of banded birds ) and on european robins ( erithacus rubecula ) ( 0.5% of banded birds ) , respectively .
the samsun cchfv partial sequences of the s segments obtained in this study have been deposited in genbank under the accession nos . kf727976 and kf727977 .
the cchfv sequences obtained in the present study belong to genotype 4 ( figure 1 ) .
phylogenetic tree based on the 260 bp of the small segment of the cchfv genome .
the multiple sequence alignment was obtained by using mega 5.1 ( http://www.megasoftware.net ) , and the phylogenetic tree was constructed by the maximum - likelihood method using 1,000 bootstrap replicates of the sequence data .
the tree is drawn to scale with branch length in the same unit as those of the evolutionary distance used to infer the phylogenetic tree .
cchfv , crimean - congo hemorrhagic fever ; s. , south ; c. , central .
( nymphs ) on european robins , which migrate across turkey twice a year en route from their breeding sites to their wintering sites ( figure 2 ) and back .
the probability of cchfv transportation by ticks among different regions and countries is high during migration of both bird species . because these birds stop several times during migration ( 9 ) , cchf in europe possibly could increase , especially at the stopover sites in southern europe , which provide suitable ecologic environments .
a ) great reed warbler ( acrocephalus arundinaceus ) eastern migration routes ( red lines ) , breeding grounds ( yellow ) and wintering areas ( purple ) .
bodies of water are blue , and nonbreeding / nonwintering areas are light green .. b ) european robin ( erithacus rubecula ) eastern migration routes ( red lines ) , resident grounds ( green ) , breeding grounds ( yellow ) , and wintering areas ( purple ) . bodies of water are blue , and nonbreeding / nonwintering areas are light green . although hyalomma ticks are the most commonly encountered ticks that carry cchfv in turkey , the virus also was detected in ixodid ticks , such as rhipicephalus spp . and haemaphysalis spp . picked up from humans and animals ( 10 ) .
also , albayrak et al . ( 11 ) detected cchfv in i. ricinus ticks . because the 2 sequences detected showed similarity with cchfv genotype 4 , which was widespread in turkey ( 1 ) , whether the ticks were infected in turkey or infected earlier during bird migration is impossible to say .
other studies have shown that cchfv could be transported by ticks on birds ( 1214 ) . by itself , transportation of infected ticks by birds might not be sufficient to cause the epidemics in turkey , but along with this , climate changes , environmental changes , increased number of sensitive animals , and tick and animal movements might play a role in spreading cchf ( 1 ) . in ecologically important regions , such as the kizilirmak delta ,
where resident and migratory birds are mixed , different microorganisms or ticks can be transferred among birds , and then carried by birds for long distances .
therefore , knowing migration routes and what pathogens birds are infected with may help predict future epidemics in various countries and provide highlight the geographic regions where diseases could be detected ( 15 ) .
 we investigated migratory birds role in spreading crimean - congo hemorrhagic fever virus ( cchfv ) through attached ticks . 
 we detected cchfv rna in ticks on migratory birds in turkey . 
 two isolates showed similarity with cchfv genotype 4 , suggesting a role for ticks in cchfv epidemics in turkey and spread of cchfv by birds . 
The Study
Conclusions