blood samples : heparinized blood samples were collected from cattle with 
 swallowing difficulty and from asymptomatic cattle from the same cowsheds where the affected 
 cattle were raised . the sampling was conducted on the two farms ( farms a and b ) where the 
 clinical cases were observed , on october 25 and 31 , respectively . the blood samples were 
 separated into plasma and blood cells by centrifugation at 2,150 g for 10 
 min .
the blood cells were washed three times with phosphate - buffered saline ( pbs ) and 
 resuspended in pbs .
serum samples were obtained from the above - mentioned cattle at the same 
 time and 2 weeks later . in june 2013
, 120 calves that had not experienced summer were selected from all over 
 kagoshima prefecture for sentinel arbovirus surveillance . heparinized blood and serum 
 samplings were obtained from the calves in june , august , september and november .
the 
 processed plasmas and blood cells were kept at 80c until the virus isolation and viral 
 genome detection .
the serum samples were preserved at 20c until they were used for the 
 virus neutralization test ( vnt ) .
virus isolation : hamster lung ( hmlu-1 ) and baby hamster kidney ( bhk-21 ) 
 cells were grown in eagle s minimum essential medium ( mem ; nissui , tokyo , japan ) 
 supplemented with 0.295% tryptose phosphate broth , 0.015% sodium bicarbonate and 5% bovine 
 serum at 37c .
virus isolation was conducted with the processed blood samples as described 
 . in brief ,
tube - cultured cells were washed 
 three times with earl s solution , inoculated with the processed samples and incubated for 1 
 hr at 37c .
after the inocula were replaced with maintenance medium ( mem containing 0.295% 
 tryptose phosphate broth and 0.015% sodium bicarbonate ) , the inoculated cultures were 
 maintained rolling and observed for cytopathic effects ( cpes ) over 7 days . if no cpes were 
 observed , two further passages were allowed for the supernatant of the primary inoculated 
 culture .
virus neutralization test : the serum samples were heat - inactivated at 56c 
 for 30 min .
fifty l of serial two - fold dilutions of serum , in duplicate 
 rows , were mixed with an equal volume of cultured medium containing 100 tcid50 of 
 the virus in flat - bottomed 96-well plates .
the mixtures were incubated at 37c for 1 hr , and 
 then , 100 l of the suspension of hmlu-1 cells in serum - free medium ( git ; 
 wako pure chemical industries , osaka , japan ) was added to each well .
after incubation at 
 37c for 7 days under a 5% co2 atmosphere in a humid chamber , the plates were 
 microscopically observed for the presence of cpes .
the antibody titer is expressed as the 
 reciprocal of the highest serum dilution at which cpes were inhibited .
viral rna extraction and rt - pcr : viruses were propagated in hmlu-1 and 
 bhk-21 cells .
viral rna was extracted from cultured fluid and blood cell suspensions with 
 the high pure viral rna kit ( roche , mannheim , germany ) per the manufacturer s instructions . 

reverse transcription - polymerase chain reaction ( rt - pcr ) was conducted with the titan one 
 tube rt - pcr kit ( roche ) using the following conditions .
cdna synthesis was carried out for 
 30 min at 50c , followed by heat inactivation for 2 min at 94c .
the pcr conditions applied 
 were 10 cycles of 30 sec at 94c , 30 sec at 55c and 45 sec at 68c , followed by 25 cycles 
 of 30 sec at 94c , 30 sec at 55c and 45 sec at 68c , with the latter time increased by 5 
 sec per cycle . for rna extracted from the blood samples , 10 additional cycles were used in 
 the latter condition .
the partial sequence of segment 3 of ehdv was detected with a group - specific primer set , 
 ep3u1416f and e31931r .
ehdibal2f + ehdibal2r-2 and ehd97l2f + 
 ehd97l2r were designed for ibav and the ibav 1997 variant , respectively ( table 1table 1.oligonucleotide sequences for rt - pcr assays and sequencing for segment 2 of 
 ehdvsoligonucleotidesequence ( 53)positionpurposereferencesfor ibavibavl2 - 1 - 17fgttaaattgtccccaga 117rt - pcr , sequencingibavl2 - 552 - 571fctatgcatcgggtcaggaac552571sequencingibavl2 - 1107 - 1126fgccatatggggagataatag11071126sequencingehdibal2ftacgcaaggtaagagaccaga16671687rt - pcr , sequencingibavl2 - 2284 - 2305fatgaggacctattaccaatgta22842305sequencingibavl2 - 661 - 642rgttcttcctgaaacatcaac661642sequencingibavl2 - 1212 - 1193rgccacatcatatctgttcgc12121193sequencingibavl2 - 1785 - 1766rcagtcatacagaacgctcgg17851766sequencingehdibal2r-2tcttctccacctcttgcatt22832264rt - pcr , sequencingibal2rgtaagtttgttgttcccagtaaacc30022978rt - pcr , sequencingfor ehdv serotype 1ehdv-1/s2/103 - 124fatatcctggcggaaccatatgg103124rt - pcr , sequencingehdv-1/s2/462 - 481fgctcgcatacacctatgaat462481sequencingehdv-1/s2/609 - 590racatctggtcgactatgcct609590sequencingehdv-1/s2/1021 - 1001ratctgcctgatgtggtgtttg10211001rt - pcr , sequencingfor ibav 1997 variantehd97l2fgatgcgatcctatacaaccatc499520rt - pcrehd97l2raatcgtcagcactctggttatc868847rt - pcra ) oligonucleotide primers were designed from ibaraki ( genbank accession number : 
 ibaraki-2 ( am745078 ) , dpp2209 ( hm156728 ) and ksb-14/e/97 ( ab078628 ) strain / isolates 
 for ibav , ehdv serotype 1 and ibav1997 variant , respectively . ) .
a published primer set , ehdv-1/s2/103 - 124f and ehdv-1/s2/1021 - 1001r , was 
 used for specific detection directed to segment 2 of ehdv serotype 1 .
ibavl2 - 1 - 17f and ehdibal2r-2 , and ehdibav2f and ibal2r ( table 1 ) were applied for the amplification of the 
 entire sequence of segment 2 of ibav . the annealing temperature on the pcr
the rt - pcr products were electrophoresed in 1.5% agarose and 
 tae ( tris - acetate - edta ) gel , and ethidium bromide - stained gels were visualized with 
 ultraviolet light .
a ) oligonucleotide primers were designed from ibaraki ( genbank accession number : 
 ibaraki-2 ( am745078 ) , dpp2209 ( hm156728 ) and ksb-14/e/97 ( ab078628 ) strain / isolates 
 for ibav , ehdv serotype 1 and ibav1997 variant , respectively .
sequencing and phylogenetic analysis : the rt - pcr products were processed 
 with the qiaquick pcr purification kit ( qiagen , hilden , germany ) and then sequenced directly 
 using bigdye terminator cycle chemistry on an abi 3100-avanti genetic analyzer ( life 
 technologies , carlsbad , ca , u.s.a . ) .
the oligonucleotide primers used for the cdna synthesis 
 and the sequencing for segment 2 of ibav and ehdv serotype 1 are listed in table 1 .
10.1.3 software ( genetyx , tokyo , japan ) and compared with the 
 deposited sequences in genbank by using the genetyx homology search tools .
neighbor - joining 
 ( nj ) , maximum likelihood ( ml ) and maximum parsimony ( mp ) consensus trees were constructed 
 using mega 6 from clustal sequence alignments of ehdv segment 3 nt sequences and muscle 
 sequence alignments of ehdv vp2 aa sequences ( table 
 2table 2.list of ehdvs used for phylogenetic analysis in this studystrain / isolateserotypelocation ( year)isolation sourcegenbank accession no.segment 2segment 3ibaraki-22japan ( 1959)affected cattleab078620ab078621y870612japan ( 1987)cattleab078624ab078625ksb-14/e/97untypedjapan ( 1997)cattleab078628ab078629ks-7/e/13untypedjapan ( 2013)cattlelc018714lc042608kawanabe1japan ( 1984)cattle  lc042606ks-1/e/13untypedjapan ( 2013)cattle  lc042607new jersey1u.s.a .
( 1955)white - tailed deernc_013397am744979ib ar 226191nigeria ( 1967)culicoides spp.am745008am745009alberta2canada ( 1962)white - tailed deeram744998am744999csiro 4392australia ( 1979)cattleam744988am744989ib ar 338534nigeria ( 1968)culicoides spp.am745018am745019csiro 1575australia ( 1977)cattleam745028am745029csiro 7536australia ( 1981)cattleam745038am7450393186bahrain ( 1983)cattleam745068am745069csiro 7757australia ( 1981)cattleam745048am745049dpp598australia ( 1982)cattleam745058am745059 ) with default parameters .
the 
 reliability of the inferred consensus tree was tested by a boot - strap resampling of 1,000 
 pseudo - replicate data sets .
clinical cases : in october 2013 , suspected cases of ibaraki disease were 
 reported at two cattle breeding farms ( farms a and b ) located in the northwestern part of 
 the mainland region of kagoshima prefecture ( fig . 

1.geographical distribution of clinical cases and ibav seroprevalence in kagoshima 
 prefecture , japan . ) .
farms a and b were 10 km apart and maintained 450 and 15 japanese black beef 
 cattle , respectively .
the affected cow at farm a developed a fever over 40c , foamy 
 salivation , dysphagia , exophthalmos and swelling of the tongue .
a cow at farm b showed 
 fever , anorexia , foamy salivation , dysphagia and abdominal distension during the same 
 period .
the 
 diseased cattle recovered after 2224 days with supportive care and antibiotic treatment . 

rt - pcr for ehdv detection : we tested samples from the two diseased cattle 
 ( a-1 of farm a and b-1 of farm b ) and 13 asymptomatic cattle ( a-2 to -6 of farm a and b-2 to 
 -9 of farm b ) by rt - pcr using primer sets for the detection of ehdv segment 3 
 ( group - specific ) and ibav segment 2 ( strain - specific ) ( table 3table 3.rt-pcrs and serological tests to detect the prevalence of ehdv in cattle sampled 
 in the farms where clinical cases were observedfarmcattlert - pcrvirus isolationtiter of neutralizing antibodyibaraki-2ks-1/e/13ehdv segment 3ibav segment 2hmlu-1bhk-21prepostprepostaa-1++nt1,0241,02422a-2+nt282256a-3++nt2512<2<2a-4nt<216<2<2a-5nt<2<2<2<2a-6nt<2<2<2<2bb-1++1281,02442b-2<2512<22b-3+51225622b-4<2512<22b-5+2416128b-6++64128<28b-7+++ ( ks-7/e/13)8256<2<2b-8<22<2<2b-9++ ( ks-8/e/13)<2128<22a ) cattle showing swallowing difficulty ,
b ) parenthesis indicates virus isolate , c ) 
 ehdv isolates used for vnts , nt : not tested . ) . the group - specific primer set successfully amplified cdna of the expected 
 size from both of the diseased cattle and seven of the asymptomatic cattle .
a ) cattle showing swallowing difficulty , b ) parenthesis indicates virus isolate , c ) 
 ehdv isolates used for vnts , nt : not tested . of the ehdv - positive cattle ,
the two diseased cattle and three of the asymptomatic cattle 
 ( a-1 and -3 , and b-1 , -6 and -7 , respectively ) tested positive by the ibav - specific rt - pcr , 
 but the other four asymptomatic cattle tested negative .
sequences of the cdna by the 
 group - specific rt - pcr were divided into two groups with 9% nt diversity .
the sequence that 
 was detected in the two diseased cattle and five asymptomatic cattle ( a-1 and -3 , and b-1 , 
 -3 , -6 , -7 and -9 ) showed high identity with segment 3 of previous ibav isolates , ibaraki-2 
 and y87061 ( 97.297.8% nt identity ) .
the other sequence amplified from two asymptomatic 
 cattle ( a-2 and b-5 ) had relatively low nt identities (  92% ) with the ehdv segment 3 
 sequence deposited in genbank .
the cdna from segment 2 in the blood samples had 95.595.6% 
 nt identity with that of the previous ibav isolates .
virus isolation : two viruses were isolated in bhk-21 cells from blood 
 cells obtained from two of the asymptomatic cattle ( b-7 and b-9 ) on farm b ( table 3 ) and designated as ks-7/e/13 and ks-8/e/13 , 
 respectively . the group - specific rt - pcr successfully amplified cdna from these viruses .
six 
 additional viruses were isolated from sentinel cattle blood samples collected between 
 september and november 2013 . of these virus
isolates , three were identified as ehdv by the 
 group - specific rt - pcr and designated as ks-1/e13 , ks-2/e/13 and ks-5/p/13 .
the other three 
 isolates were identified as daguilar virus of the genus orbivirus using a 
 specific rt - pcr assay as described   ( data not 
 shown ) .
we performed a strain - specific rt - pcr directed to segment 2 of ibav , the ibav 1997 variant 
 and ehdv serotype 1 using the ehdv isolates ( fig . 

2.electrophoretic analysis of the rt - pcr product from segment 2 of ehdv isolates using 
 ibav - specific ( a ) , ehdv serotype 1-specific ( b ) and ibav 1997 variant - specific ( c ) 
 primer pairs .
each primer set expectedly generates a specific product with the lengths 
 617 , 919 and 370 bp , respectively .
lanes : 1 , ks-1/e/13 ; 2 , ks-2/e/13 ; 3 , ks-5/p/13 ; 4 , 
 ks-7/e/13 ; 5 , ks-8/e/13 ; 6 , ibaraki-2 ( positive control for ibav ) ; 7 , kawanabe 
 ( positive control for ehdv serotype 1 ) ; 8 , ksb-14/e/97 ( positive control for ibav 1997 
 variant ) ; 9 , negative water control ; m , 100 bp dna ladder . ) .
the rt - pcr product was amplified with the ibav - specific primer set from ks-7/e/13 
 and ks-8/e/13 .
in contrast , ks-1/e/13 , ks-2/e/13 and ks-5/p/13 tested positive by the ehdv 
 serotype 1-specific rt - pcr .
no amplification of the ehdv isolates was detected by the ibav 
 1997 variant - specific rt - pcr .
electrophoretic analysis of the rt - pcr product from segment 2 of ehdv isolates using 
 ibav - specific ( a ) , ehdv serotype 1-specific ( b ) and ibav 1997 variant - specific ( c ) 
 primer pairs .
each primer set expectedly generates a specific product with the lengths 
 617 , 919 and 370 bp , respectively .
lanes : 1 , ks-1/e/13 ; 2 , ks-2/e/13 ; 3 , ks-5/p/13 ; 4 , 
 ks-7/e/13 ; 5 , ks-8/e/13 ; 6 , ibaraki-2 ( positive control for ibav ) ; 7 , kawanabe 
 ( positive control for ehdv serotype 1 ) ; 8 , ksb-14/e/97 ( positive control for ibav 1997 
 variant ) ; 9 , negative water control ; m , 100 bp dna ladder .
sequences of segments 2 and 3 of ks-7/e/13 and ks-8/e/13 were identical to those detected 
 in the two diseased cattle and three of the asymptomatic cattle ( a-1 and -3 , and b-1 , -6 and 
 -7 ) .
the amplicons by the group - specific rt - pcr from the two asymptomatic cattle ( b-3 and 
 -9 ) that tested negative in the ibav - specific rt - pcr were also identical to those of segment 
 3 of ks-7/e/13 and ks-8/e/13 .
the rt - pcr product from segment 3 of ks-1/e/13 , ks-2/e/13 and 
 ks-5/p/13 matched the sequence from the two asymptomatic cattle ( a-2 and b-5 ) from which the 
 ibav - related sequences were negative .
the rt - pcr product from segment 2 of the isolates 
 shared 97.3% and 96.8% nt identity with the corresponding region of japanese ( kawanabe ) and 
 australian ( dpp2209 ) strains of ehdv serotype 1 , respectively . in our comparative analysis 
 of the aa sequence of vp2 , the isolates shared 99.3% and 98.3% identity with the japanese 
 and australian strains , respectively .
vnts for bovine sera : in a vnt using ibav , high neutralization antibody 
 ( na ) titers ranging from 1:128 to 1:1,024 were observed in post - sera of the two diseased 
 cattle and the seven asymptomatic cattle on farms a and b ( table 3 ) .
in contrast , the na titers 1:128 and 1:256 were detected 
 against ks-1/e/13 in post - sera of two of the asymptomatic cattle ( a-2 and b-5 ) . a 
 significant rise in the na titers against both ibav and ks-1/e/13
was observed between pre- 
 and post - serum collections from the studied farms , indicating that the viruses spread 
 simultaneously in those days .
the cross - reactions between the two viruses in the vnt were 
 not significant (  1:8 ) , and none of the serum samples contained high na titers against both 
 of the viruses at once .
the seroconversion of sentinel cattle to ibav was found in october 
 and november 2013 at three other farms located in the northwestern part of kagoshima 
 prefecture ( fig .
1 ) . phylogenetic analysis of ehdv : the phylogenetic tree based on the partial 
 sequence of segment 3 shows that all of the japanese and australian isolates / strains 
 composed a single cluster ( fig . 3fig .
3.phylogenetic comparison of the partial nucleotide sequence of segment 3 of japanese 
 field isolates and reference strains of ehdv .
however , kawanabe and ks-1/e/13 formed a single clade and were relatively less 
 related to other members in the cluster .
we identified 2,960 nt of segment 2 of ks-7/e/13 , 
 and they were shown to code for a deduced 982-aa length of vp2 protein , similar to the 
 previously reported ibav isolates .
the vp2 sequence from ks-7/e/13 shared 95.6% nt and 97.5% 
 aa identities with ibaraki-2 , and 96.1% nt and 98.2% aa identities with y87061 .
phylogenetic comparison of the partial nucleotide sequence of segment 3 of japanese 
 field isolates and reference strains of ehdv .
the sequence identity was also high between ks-7/e/13 and an australian strain ( csiro 439 ) 
 of ehdv serotype 2 ( 93.0% nt and 96.0% aa identities ) .
relatively low identities were 
 observed with a north american strain ( alberta ) of ehdv serotype 2 ( 71.3% nt and 73.6% aa 
 identities ) .
in contrast , ks-7/e/13 was highly diverged from the ibav 1997 variant 
 ( ksb-14/e/97 ) ( 69.7% nt and 67.5% aa identities ) . the nj consensus tree based on the aa 
 sequences of ehdv vp2 demonstrated that ks-7/e/13 clustered with previous ibav isolates 
 ( fig .
4.phylogenetic tree of full - length amino acid sequence of vp2 between reference strains 
 and japanese field isolates of ehdv listed in table 2 .
ksb-14/e/97 was less related to ehdv serotype 2 isolates , but it formed a distinct 
 clade with ehdv serotype 7 ( csiro 775 ) .
the trees constructed by ml and mp supported the nj 
 consensus tree ( data not shown ) .
phylogenetic tree of full - length amino acid sequence of vp2 between reference strains 
 and japanese field isolates of ehdv listed in table 2 .
our observation of typical ibaraki disease manifestations , such as fever and dysphagia , and 
 the detection of ibav - related sequences and antibodies in the diseased cattle clearly 
 demonstrated the occurrence of ibaraki disease in the northwestern part of kagoshima 
 prefecture in 2013 .
the ehdv isolates from one of the studied farms were genetically close 
 to previously reported ibav isolates and caused the typical clinical signs in the infected 
 cattle .
in contrast , the present phylogenetic 
 comparison showed that ksb-14/e/97 is less related to ehdv serotype 2 ( including ibavs ) and 
 should be classified as ehdv serotype 7 . a low level of cross - reaction in the vnt between 
 ibav and ehdv serotype 7 might have led to the misidentification at the time , when the vp2 
 sequence from all serotypes was not available [ 3 , 
 20 , 22 ] . 
 because the 199798 epidemic was caused by a different ehdv serotype , it should be 
 distinguished from the other outbreaks of ibaraki disease .
of interest is that ehdv serotype 1 also spread in the same area ( even on the same farms ) 
 during the instances of ibaraki disease in 2013 .
ehdv serotype 1 caused hemorrhagic disease 
 of wild deer in north america and may have been involved in the febrile disease of cattle on 
 reunion island [ 6 , 19 ] , but no association of ruminant morbidity was reported in other regions .
it is likely that ehdv serotype 1 was not involved 
 in the cattle morbidity in 2013 , as shown in its previous spread in an overlapping region 
 .
the phylogenetic tree based on segment 3 
 showed that ehdv serotype 1 isolates from japan were primarily sorted into the eastern 
 topotype , but diverged from other japanese and australian isolates .
this finding suggested 
 that these isolates had been geographically separated from the other isolates in their 
 evolution for a certain period of time .
like other segmented viruses , reassortants between heterogeneous serotypes of ehdv were 
 sometimes recovered in the field [ 1 , 2 ] .
co - circulation of the ibav and ehdv serotype 1 
 suggested the potential reassortment between them .
a full genome analysis of ehdv isolates 
 in 2013 is needed to determine whether reassortment has occurred .
surprisingly , the 
 co - infection was not found in the ehdv - positive cattle by the genetic and serological tests , 
 although the viruses spread in the studied farms during the same period . interference 
 between two viruses might restrict the replication of either virus , as shown by an 
 experimental co - infection of calves with bluetongue virus serotypes 1 and 8 .
although seroconversion in cattle to ibav has been detected sporadically , no occurrence of ibaraki disease has been seen in 
 japan for 26 years , until the present study .
reports of clinical cases and the sentinel 
 seroconversion to ibav in the northwestern part of kagoshima prefecture are limited .
no 
 occurrence of ibaraki disease was reported in the other 46 prefectures of japan in 2013 
 , suggesting the local spread of ibav .
the 
 present sentinel surveillance suggested that the incursion of ibav happened in october 2013 . 

the mean of the average 
 temperatures at four observation points close to the studied and seroconverted farms 
 generally reached 20c or above during the beginning and middle of october , but rapidly 
 decreased in november ( japan meteorological agency ) .
the inadequate temperatures for 
 culicoides flight behavior might have prevented the transmission cycle 
 and thus restricted the viral spread to within the small area . in the 1959 epizootic 
 outbreak , ibaraki disease was first recognized in southern japan in august and swept over 
 the southern half of the nation in the following four months .
the history of outbreaks implied that the earlier incursion of the 
 virus may have caused the larger outbreak of the disease in japan .
the current live attenuated and inactivated vaccines against ibaraki disease were developed 
 based on ibaraki-2 .
the small aa sequence diversity of vp2 between ibaraki-2 and ks-7/e/13 
 suggested that the current commercial vaccines are still effective for the prevention of 
 ibaraki disease .
the reemergence of ibaraki disease reminds us of the importance of 
 continuous vaccination in cattle populations to minimize economic losses . in the meantime
, 
 diagnostic and preventive measures should be prepared for other ehdv serotypes that are 
 associated with cattle morbidity in japan and other countries [ 22 , 26 , 31 ] .
sentinel surveillance will be useful to identify the incursion and 
 circulation of ehdvs in southern japan with a high risk of arbovirus infections .
 in japan in 2013 , two cattle in the northwestern part of kagoshima prefecture developed 
 fever and swallowing difficulty and were suspected of having ibaraki disease . 
 the 
 epizootic hemorrhagic virus ( ehdv ) genome was detected from diseased and asymptomatic 
 cattle by reverse transcription - polymerase chain reaction ( rt - pcr ) . 
 high neutralization 
 antibody titers to ibaraki virus ( ibav ) ranging from 1:128 to 1:1,024 were observed in the 
 rt - pcr - positive cattle , and the virus was isolated in one of the ibav - positive farms . 
 a 
 pairwise alignment and phylogenetic analysis based on the major outer coat protein vp2 
 encoded in segment 2 revealed a close relationship between the isolated viruses and 
 previous ibav isolates . 
 the phylogeny of vp2 also suggested that an ibav variant isolated 
 in 1997 was distinct from ibav and sorted into a heterogeneous serotype , ehdv serotype 7 . 
 
 the findings revealed the reemergence of ibaraki disease in japan after a 26-year absence . 
 
 interestingly , the co - circulation of ehdv serotype 1 with ibav was observed in the 
 affected region , suggesting the potential reassortment between two heterogeneous serotypes 
 in the field . 
 sentinel surveillance in kagoshima prefecture indicated that the incursion 
 of ibav occurred in october 2013 and that its spread was limited within the small area . 
 
 inadequate environmental temperatures for vector transmission in late autumn might have 
 limited the virus spread to a wider region . 
 the reemergence of ibaraki disease showed us 
 the importance of continuous vaccination to prevent economic losses . 
MATERIALS AND METHODS
RESULTS
DISCUSSION