samples : edta - anticoagulated blood samples were collected from 51 sika 
 deer ( cervus nippon yesoensis ) that were maintained in captivity for the 
 purpose of meat processing in the kushiro district ( hokkaido , japan ) in june and july 2011 . 

dna was extracted from 200 l of whole blood using the qiaamp dna blood 
 mini kit ( qiagen , hilden , germany ) , eluted with 200 l of buffer ae 
 according to the manufacturer s instructions , and stored at 30c until use .
screening and species - specific pcr : we screened all dna samples for the 
 presence of hemoplasma using a previously described protocol .
the f2/r2 primer set amplifies the 16s rrna gene of most hemoplasma 
 species , including m. haemofelis , m. haemocanis , 
 
a 20-l reaction 
 mixture for the screening pcr contained 2.5 l of 10x buffer , 2.5 
 l of 2 mm dntp , 1 l of 50 mm mgcl2 , 0.75 u 
 of taq polymerase ( invitrogen , foster city , ca , u.s.a . ) , 1.0 l of each 
 primer , 11.35 l of distilled water and 0.5 l of dna 
 template .
cycling conditions were as follows : initial denaturation at 95c for 5 min ; 35 
 cycles of denaturation at 95c for 30 sec , annealing at 60c for 30 sec , extension at 72c 
 for 90 sec ; and a final extension at 72c for 5 min and cooling to 4c .
all amplicons were 
 electrophoresed on a 2.0% agarose gel in tbe buffer and visualized under uv light . to distinguish between  candidatus m. haemocervae and 
  candidatus m. erythrocervae ,
primers for the species - specific pcr were designed based on 
 the 16s rrna gene sequences of  candidatus m. haemocervae ( ab558899 ) and 
  candidatus m. erythrocervae ( ab558897 and ab558898 ) .
the primer sets 
 were cmhc f ( 5-ccgcgagtaggatagcagcc-3 ) and r2 for  candidatus m. 
 haemocervae , and cmec f ( 5-gcaaggggttccgcgtaaaa-3 ) and r2 for 
  candidatus m. erythrocervae , respectively .
pcr conditions were as 
 previously described , except that the extension times were 60 sec . to evaluate their 
 specificity ,
the method was initially tested using dna samples from deer confirmed to be 
 positive for  candidatus m. haemocervae and  candidatus 
 m. erythrocervae in this study .
the specific pcr was performed for all deer samples which 
 were positive in the screening pcr that targeted the 16s rrna gene as mentioned above .
pcr assays for 16s rrna , 23s rrna and rnpb genes : sequences of the full 
 16s rrna gene were then assessed in several randomly selected samples that tested positive 
 in the screening pcr . to determine the longer 16s rrna gene sequence of hemoplasma
, 2 pcrs 
 were performed using the following 2 primer sets : fd1 and r2 , and f2 and rp2 .
pcr conditions were as previously described , except 
 that the annealing temperatures were 58c for fd1/r2 and 52c for f2/rp2 .
based on the 23s rrna genes of deer hemoplasmas , including m. ovis - like 
 sp . ( hq197750 , hq197751 and hq197752 ) , deerhemo f ( 5-aaagagtgcgtaacagctcac-3 ) and deerhemo 
 r ( 5-tcacgccggaattcttactt-3 ) were newly designed . to determine the 23s rrna gene sequence 
 of hemoplasma
, 2 pcrs were performed using the following 2 primer sets : 23s fw and deerhemo 
 r , and deerhemo f and 23s rv .
pcr conditions were 
 as previously described , except that the annealing temperatures were 55c .
the rnpb gene was amplified using a primer set described previously : 80f1 
 and 290r1 .
pcr conditions were as previously 
 described , except that the annealing temperature was 50c .
sequence and phylogenetic analyses : to determine nucleotide sequences of 
 the positive samples , pcr products were purified using a qiaquick pcr purification kit 
 ( qiagen ) or a qiaquick gel extraction kit ( qiagen ) .
direct sequence analysis was performed 
 using bigdye terminator v3.1 cycle sequencing kit ( applied biosystems , foster city , ca , 
 u.s.a . ) .
nucleotide sequence results were checked using the blast search program 
 ( http://www.ncbi.nlm.nih.gov/blast/blast.cgi ) for comparison with other known sequences and 
 aligned using clustal w software ( http://www.genome.jp/tools/clustalw/ ) .
phylogenetic trees 
 were created in mega software version 5.05   by 
 the neighbor - joining method .
nucleotide accession numbers : hemoplasma sequences from deer in this study 
 were registered in genbank with the following accession numbers : for 16s rrna gene : kf306246 
 ( no .
16 ) , kf306249 ( no . 33 ) , kf306250 ( no . 34 ) and 
 kf306251 ( no . 49 ) and for 23s rrna gene : kf306252 ( no .
49 ) and rnpb gene : ab836744 ( no . 13 ) , ab836745 ( no .
16 ) , ab836746 ( no . 
 33 ) , ab836747 ( no . 34 ) and ab836748 ( no . 49 ) .
prevalence of hemoplasma infection : a total of 23/51 ( 45% ) deer dna 
 samples were positive for hemoplasmas in the screening pcr .
the species - specific pcr was 
 performed using dna samples from deer which were confirmed as 
candidatus 
 m. haemocervae and  candidatus m. erythrocervae by sequence analysis in 
 this study .
two pcrs were used to amplify each gene , and 439 and 363 bp amplicons were 
 observed in  candidatus m. haemocervae and  candidatus m. 
 erythrocervae positive samples , respectively ( fig . 

candidatus 
 mycoplasma haemocervae and ( b )  candidatus m. erythrocervae. lanes 
 1 and 2 :  candidatus m. haemocervae ; lanes 3 and 4 : 
  candidatus m. erythrocervae ; lane 5 : dual infection ; lane 6 : 
 negative control ( dw ) ; lane m : molecular size marker ( 100 bp ) . ) .
when the species - specific pcr was performed to screen pcr - positive samples , 12 and 
 17 samples were found to be positive for  candidatus m. haemocervae and 
  candidatus m. erythrocervae , respectively .
six samples were found to be 
 dual infections ( table 1table 1.prevalence of hemoplasma infected deerpcr result ( n=51)no .
of deer ( % ) screening pcr positive23 ( 45.1 ) negative28 ( 54.9)species - specific pcrc .
candidatus 
 mycoplasma haemocervae and ( b )  candidatus m. erythrocervae. lanes 
 1 and 2 :  candidatus m. haemocervae ; lanes 3 and 4 : 
  candidatus m. erythrocervae ; lane 5 : dual infection ; lane 6 : 
 negative control ( dw ) ; lane m : molecular size marker ( 100 bp )
. molecular characterization of hemoplasmas : the complete 16s rrna gene 
 sequences were successfully determined for the 6 randomly chosen hemoplasma - positive 
 samples . among cervine hemoplasma isolates , the 16s rrna gene sequences obtained from deer 
 nos . 12 , 16 , 33 and 34 were most closely related to 
candidatus m. 
 haemocervae ( ab558899 ) with a percent identity of 99.86 to 99.32% .
16 which was 
 representative sequence of  candidatus m. haemocervae matched with a 
 m. ovis - like sp . from white - tailed deer in america ( fj824847 ) with a 
 percent identity of 98.32% .
candidatus m. erythrocervae ( ab558898 ) with a percent 
 identity of 99.79 to 99.71% .
49 which was representative sequence of 
  candidatus m. erythrocervae matched 97.75% with a 
 mycoplasma sp .
( hq634379 ) detected from marsh deer in brazil , 97.79% with 
 a mycoplasma sp .
( table 2table 2.sequence identities ( % ) among ( a ) 16s rrna , ( b ) 23s rrna and ( c ) 
 rnpb genes of  candidatus mycoplasma haemocervae 
 ( isolate 16 ) and  candidatus m. erythrocervae ( isolate 49 ) detected 
 in hokkaido sika deer . isolates 16 and 49 are one representative sequence of 
  candidatus m. haemocervae and  candidatus m. 
 erythrocervae. genbank accession numbers of each hemoplasma detected in the other 
 family cervidae are in parenthesissample no.m .
ovis - like sp.mycoplasma sp.mycoplasma sp.mycoplasma sp.mycoplasma sp.deer-fawnb62-2b88-3group bgroup c(a ) 16s rrna(fj824847)(hq634379)(hq634380)(kc512403)(kc512402)isolate 1698.3295.9195.196.395.77isolate 4995.9297.7593.5897.7997.8(b ) 23s rrna(hq197752)(hq634381)(hq634382)nanaisolate
; mycoplasma sp . isolate from white - tailed 
 deer in america ( group a ) ( 5 ) . ) .
; mycoplasma sp . isolate from white - tailed 
 deer in america ( group a ) ( 5 ) . the 23s rrna genes from sample nos .
16 showed the closest relationship to m. ovis - like sp . from white - tailed 
 deer in brazil ( hq197752 ) with a percent identity of 97.61% .
hemoplasma species in deer no . 
 49 were most closely related to mycoplasma sp . ( hq634381 ) of marsh deer in 
 brazil with a percent identity of 95.70% ( table 
 2 ) .
( jq610624 and jq610628 ) 
 recently described in a white - tailed deer from america   ( table 2 ) .
12 , 16 , 33 and 34 was on a cluster separate from 
 the m. ovis - like spp . in deer from brazil and america , and 
  candidatus m. erythrocervae from deer nos .
13 and 49 was also on a 
 cluster separate from the unclassified hemoplasma ( hq634379 ) from marsh deer ( fig .
12 , 13 , 16 , 33 , 34 and 
 49 ) and the other hemoplasma species based on 16s rrna gene using a neighbor - joining 
 method .
isolates b86 , b175 , deer - fawn , 0221 , 
 2385 ( hq634377 ; hq634378 ; fj824847 ; hq197746 ; hq197748 ) , m. ovis 
 ( jf931135 ) , 
 candidatus m. haemocervae ( ab558899 ) , m. 
 wenyonii(ay946266 ) , mycoplasma sp .
isolates b62 - 2 , 1585 , 
 b88 - 3 , group a , group b , group c ( hq634379 , hq197747 , hq634380 , kc512404 , kc512403 and 
 kc512402 ) ,  candidatus m. erythrocervae ( ab558898 ) , 
  candidatus m. haemominutum ( ay150980 ) , 
  candidatus m. haematoparvum ( ay383241 ) , m. 
 haemomuris ( u82963 ) ,  candidatus m. turicensis 
 ( dq825454 ) ,  candidatus m. haemobos ( eu367965 ) , m. 
 haemofelis ( u88563 ) , m. haemocanis ( ay150973 ) , m. 
 suis ( eu603330 ) ,  candidatus m. haemolamae ( nr074478 ) and 
  candidatus m. kahanei ( af338269 ) are shown . m. 
 pneumonia ( m29061 ) was used as an outgroup . ) .
the 23s rrna gene - based phylogenetic tree revealed a similar result , wherein 
 hemoplasmas from nos . 16 and 33 and from no .
49 were separated from the m. 
 ovis - like spp . and the unclassified mycoplasma sp .
16 , 33 and 49 ) and the 
 other hemoplasma species based on 23s rrna gene using a neighbor - joining method . 

mycoplasma ovis - like sp . isolates 0221 , b86 - 1 , deer - fawn ( hq197751 ; 
 hq634383 ; hq197752 ) , mycoplasma sp .
isolates b62 - 2 , 1585 , b88 - 3 
 ( hq634381 ; hq197750 ; hq634382 ) , m. wenyonii ( nr076982 ) , m. 
 suis ( nc015153 ) , m. haemocanis ( nr076944 ) , m. 
 haemofelis isolates ohio2 , langford1 ( nc017520 ; nc014970 ) and 
  candidatus m. haemolamae ( nr076983 ) are shown .
( jq610624 ) which are 
 closely related to a m. ovis - like spp .
13 , 16 , 33 , 34 and 49 ) 
 and the other hemoplasma species based on rnpb gene using a 
 neighbor - joining method .
isolates group a , group b , group c 
 ( jq610624 , jq610626 and jq610618 ) , m. ovis ( eu078612 ) , m. 
 wenyonii ( eu078610 ) , m. haemolamae ( eu078613 ) , 
  candidatus m. haemominutum ( eu078614 ) , 
  candidatus m. haematoparvum ( eu078616 ) , m. suis 
 ( eu078611 ) ,  candidatus m. turicensis ( ef212002 ) , m. 
 haemofelis ( eu078617 ) , m. haemocanis ( eu078618 ) and 
  candidatus m. kahanei ( eu078615 ) are shown . m. 
 fastidiosum ( eu078609 ) was used as an outgroup . ) .
12 , 13 , 16 , 33 , 34 and 
 49 ) and the other hemoplasma species based on 16s rrna gene using a neighbor - joining 
 method .
isolates b86 , b175 , deer - fawn , 0221 , 
 2385 ( hq634377 ; hq634378 ; fj824847 ; hq197746 ; hq197748 ) , m. ovis 
 ( jf931135 ) ,  candidatus m. haemovis ( jf931131 ) , 
  candidatus m. haemocervae ( ab558899 ) , m. 
 wenyonii(ay946266 ) , mycoplasma sp .
isolates b62 - 2 , 1585 , 
 b88 - 3 , group a , group b , group c ( hq634379 , hq197747 , hq634380 , kc512404 , kc512403 and 
 kc512402 ) ,  candidatus m. erythrocervae ( ab558898 ) , 
 
 candidatus m. haematoparvum ( ay383241 ) , m. 
 haemomuris ( u82963 ) ,  candidatus m. turicensis 
 ( dq825454 ) ,  candidatus m. haemobos ( eu367965 ) , m. 
 haemofelis ( u88563 ) , m. haemocanis ( ay150973 ) , m. 
 suis ( eu603330 ) ,  candidatus m. haemolamae ( nr074478 ) and 
  candidatus m. kahanei ( af338269 ) are shown .
16 , 33 and 49 ) and the 
 other hemoplasma species based on 23s rrna gene using a neighbor - joining method . 

mycoplasma ovis - like sp . isolates 0221 , b86 - 1 , deer - fawn ( hq197751 ; 
 hq634383 ; hq197752 ) , mycoplasma sp .
isolates b62 - 2 , 1585 , b88 - 3 
 ( hq634381 ; hq197750 ; hq634382 ) , m. wenyonii ( nr076982 ) , m. 
 suis ( nc015153 ) , m. haemocanis ( nr076944 ) , m. 
 haemofelis isolates ohio2 , langford1 ( nc017520 ; nc014970 ) and 
  candidatus m. haemolamae ( nr076983 ) are shown .
13 , 16 , 33 , 34 and 49 ) 
 and the other hemoplasma species based on rnpb gene using a 
 neighbor - joining method .
isolates group a , group b , group c 
 ( jq610624 , jq610626 and jq610618 ) , m. ovis ( eu078612 ) , m. 
 wenyonii ( eu078610 ) , m. haemolamae ( eu078613 ) , 

 candidatus m. haemominutum ( eu078614 ) , 
  candidatus m. haematoparvum ( eu078616 ) , m. suis 
 ( eu078611 ) ,
 candidatus m. turicensis ( ef212002 ) , m. 
 haemofelis ( eu078617 ) , m. haemocanis ( eu078618 ) and 
  candidatus m. kahanei ( eu078615 ) are shown .
in the present study , hemoplasma species were detected from 45% ( 23/51 ) of the blood 
 samples from sika deer in hokkaido , japan , by screening pcr .
a past study revealed that 
 hemoplasma infections were found in only 9% ( 13/147 ) of free - ranging sika deer in iwate 
 prefecture in the tohoku region of honshu island , japan .
however , hemoplasma infections were found in 89.0% ( 65/73 ) of white - tailed 
 deer in america   and 87.1% ( 27/31 ) of brazilian 
 deer , both groups of which were maintained in 
 captivity or confined area .
given that the sika deer in the present study had also been 
 maintained in captivity , it seems that the high prevalence of hemoplasma infections is 
 attributable to situations in which animals are in close contact with one another . as a result of the comparison of phylogenetic position of various hemoplasma strains which 
 were found in the family cervidae using 16s , 23s rrna and 
 rnpb genes , 
candidatus m. haemocervae obtained from 
 hokkaido sika deer was closely related to a previously reported species which are so - called 
 m. ovis - like spp .
on 16s rrna ( fj824847 ) and 23s rrna ( hq634381 ) gene 
 sequences , while low identity was shown on rnpb gene between those species . 

in addition ,  candidatus m. erythrocervae was closely related to a 
 mycoplasma sp .
( hq634379 ) detected from marsh deer in brazil on 16s rrna 
 gene , while homology of 23s rrna gene sequence between those two species seemed to be low 
 because the threshold of the 23s rrna gene sequence for identification between different 
 bacterial species was 1.15% .
moreover , 
 phylogenetic trees based on the three genes also indicate that  candidatus 
 m. haemocervae and  candidatus m. erythrocervae are independent from 
 other cervine hemoplasma found in america and brazil .
the phylogeny of hemoplasma species has been investigated based on the 
 rnpb gene sequences , a more suitable target for phylogenetic 
 discrimination of closely related taxa when compared with 16s rrna sequences .
the rna polymerase beta subunit 
 ( rpob ) and the 16s-23s rrna intergenic transcribed spacer ( its ) region were 
 found to be reliable and useful taxonomic tools for species differentiation within the 
 family mycoplasmataceae . 

further research is required to compare those genes among unclassified hemoplasma in the 
 family cervidae , including 
candidatus m. haemocervae , 
  candidatus m. erythrocervae , m. ovis - like sp . and 
 other hemoplasma of free - ranging deer in america and brazil .
however , a further 
 study using free - ranging hokkaido sika deer is necessary , because animals using in this 
 study were maintained in captivity .
lower percent identities and divergent phylogenetic 
 position support the notion that  candidatus m. haemocervae and 
  candidatus m. erythrocervae are potentially different species from the 
 other cervine hemoplasma found in america and brazil .
further investigation is needed to 
 clarify phylogenetic position , molecular characterization and pathogenicity of deer 
 hemoplasma in japan .
 abstracthemotropic mycoplasmas ( hemoplasmas ) are cell - wall deficient , epierythrocytic 
 bacteria that cause infectious anemia in several mammalian species . 
 the prevalence of 
 hemoplasma species was examined by screening and species - specific pcr using blood samples 
 collected from 51 sika deer in hokkaido , japan . 
 molecular analyses were performed for the 
 16s rrna , 23s rrna and rnase p rna ( rnpb ) gene sequences . 
 a total of 
 23/51 ( 45% ) deer dna samples were positive for hemoplasmas in the screening pcr . 
 using 
 species - specific pcr , 12 and 17 samples were positive for  candidatus 
 mycoplasma haemocervae and  candidatus m. erythrocervae , respectively . 
 
 sequencing and phylogenetic trees of those three genes indicate that the 
  candidatus m. haemocervae and  candidatus m. 
 erythrocervae detected in japanese deer are potentially different species from the 
 cervine hemoplasma found in deer from america and brazil . 
MATERIALS AND METHODS
RESULTS
DISCUSSION