during july 2011april 2014 , a total of 45 blood samples were collected in edta - containing collection tubes from wild japanese macaques in aomori ( n = 25 ) , yamagata ( n = 5 ) , and wakayama ( n = 15 ) prefectures in japan .
the animals were captured by licensed trappers , in accordance with the wildlife protection and proper hunting act , by using large hand - made cage traps and commercial cage traps ( no .
the physical conditions of each animal were recorded before they were euthanized , according to the guidelines of the japanese veterinary medical association .
freeze - thawed blood samples were spread onto chocolate agar plates ( 8) for isolation of bartonella spp . and incubated at 35c under 5% co2 for up to 4 weeks .
bartonella - specific pcrs that targeted the glta ( 9 ) and rpob ( 10 ) genes and the 16s23s rdna intergenic transcribed spacer ( its ) regions ( 11 ) were used for identification of bartonella isolates ; genomic dna of b. alsatica strain ibs 382 and nuclease - free distilled water were used as positive and negative controls for the pcrs , respectively .
bartonella isolates were obtained from 6 ( 13.3% ) of 45 japanese macaques ; 1 ( 4.0% ) of 25 macaques in aomori , 1 ( 20.0% ) of 5 in yamagata , and 4 ( 26.7% ) of 15 in wakayama prefectures .
the bacteremia levels in the macaques ranged from 5.0  10 to 3.7  10 cfu / ml .
the dna sequences of all 30 isolates were identical in the glta ( 338 bp ) , rpob ( 825 bp ) , and its ( 1,297 bp ) regions ; the sequences were registered in genbank / european nucleotide archive in embl , and ddbj under accession nos .
lc031777 ( glta ) , lc031778 ( rpob ) , and lc031779 ( its ) .
blast searches ( http://blast.ncbi.nlm.nih.gov/blast.cgi ) indicated that the dna sequences of the isolates had the highest degree of similarity ( 100% for glta and rpob , 99.5% for its ) with those of b. quintana rm11 strain from rhesus macaques .
subsequently , mlst analysis with 9 loci ( 12 ) revealed that 6 representative strains ( mf11 , mf31 , mf101 , mf111 , mf191 , and mf341 strains ) from each culture - positive macaque were identical and belonged to a new sequence type ( st ) , st22 .
the allelic profiles of st22 and other sts are shown in the technical appendix table . a phylogenetic relationship between st22 and other known sts
was analyzed by using eburst version 3 ( http://eburst.mlst.net/default.asp ) in combination with the mlst data . a clonal complex
was defined as the group of sts that had identical alleles at 8 of 9 loci , and the lineage was defined as the group of sts that had identical alleles at 7 of the 9 loci . as
previously reported ( 6,12 ) , sts 14 , sts 6 and 7 , sts 810 and 14 , and sts 1521 formed clonal complexes 1 , 2 , 3 , and 4 , respectively , whereas st22 remained a singleton ( figure 1 ) . in terms of lineage classification ,
all sts , except st22 , were divided into 3 individual lineages by the host animal species : sts 17 for human strains , sts 814 for cynomolgus macaque strains , and sts 1521 for rhesus macaque strains . in contrast , st22 from japanese macaque strains belonged to a singleton lineage .
phylogenetic relationship among 1 to 22 sequence types ( sts ) of bartonella quintana strains based on eburst analysis ( http://eburst.mlst.net/default.asp ) .
clonal complexes 1 , 2 , 3 , and 4 consist of sts 14 , sts 67 , sts 810 and 14 , and sts 1521 , respectively . a lineage was defined as a group of sts that had 7 identical alleles .
we constructed a phylogenetic tree with the concatenated sequences ( 4,270 bp ) of the 9 loci in each st using the maximum - likelihood method in mega6 ( 13 ) .
the sts 17 from human strains , sts 814 from cynomolgus macaque strains , and sts 1521 from rhesus macaque strains were classified into groups 1 , 2 , and 3 , respectively , as with the lineage classification by eburst analysis .
all strains of st22 formed a monophyletic clade defined as group 4 ( figure 2 ) .
the tree was constructed from the concatenated sequences ( 4,270 bp ) of the 9 loci used for multilocus sequence typing by using the maximum - likelihood method based on the tamura 3-parameter model in mega6 ( 13 ) .
the 22 sequence types ( sts ) of b. quintana strains from humans ( sts 17 ) , cynomolgus macaques ( sts 84 ) , rhesus macaques ( sts 1521 ) , and japanese macaques ( st22 ) were included in the tree .
our study shows that natural infection with b. quintana can occur in free - ranging nonhuman primates in japan .
the japanese macaques harboring b. quintana showed no clinical abnormalities , although bacteremia levels were considerably high ( > 10 cfu / ml ) in 3 animals that tested positive .
these data suggest that japanese macaques are one of the natural reservoirs of b. quintana .  
all isolates from japanese macaques were more closely related to rhesus macaque strains than to human strains in the glta , rpob , and its regions . by mlst
analysis with 9 loci , all 6 representative strains from japanese macaques were identified as st22 ( 6 ) .
thus , st22 is likely a new genotype of b. quintana specific to japanese macaques .
because wild - caught japanese macaques from only 3 prefectures were examined for b. quintana bacteremia , a large - scale surveillance study would help elucidate the genetic diversity of japanese macaque strains . according to eburst analysis , human , cynomolgus macaque , and rhesus macaque lineages
as reported previously ( 6,12 ) , the primary founders in clonal complexes 1 , 3 , and 4 were reconfirmed as sts 2 , 9 , and 15 , respectively .
however , st22 from wild - caught japanese macaque strains was not found in any other clonal complex and formed an independent lineage . through phylogenetic analysis with concatenated mlst sequences ,
li et al . ( 6 ) showed that 3 groups were formed by each host species ; this finding was confirmed in our study .
are known to have an adaptive strategy of causing asymptomatic and prolonged bacteremia in their specific reservoirs ( 14 ) .
our data support the idea that b. quintana may have separately co - evolved with the macaque species and humans .
notably , bartonella dna was recently detected in gorillas from west africa , suggesting that nonhuman primates , including apes , could be naturally infected with bartonella spp .
further studies are necessary to clarify the prevalence of b. quintana and the vector of the organism in other nonhuman primates , and the potential of these primates to serve as a source of infection to humans .
 bartonella quintana bacteremia was detected in 6 ( 13.3% ) of 45 wild - caught japanese macaques ( macaca fuscata ) . 
 multilocus sequence typing of the isolates revealed that japanese macaques were infected with a new and specific b. quintana sequence type . 
 free - ranging japanese macaques thus represent another natural reservoir of b. quintana . 
The Study
Conclusions
None