Sika Deer Carrying Babesia Parasites Closely Related to B. divergens, Japan

To the Editor: Human babesiosis caused by Babesia divergens, a zoonotic pathogen of bovines in Europe, is an emerging tickborne disease (1). In the United States, a closely related Babesia sp. was identified in persons in Missouri and Kentucky and in eastern cottontail rabbits (Sylvilagus floridanus) on Nantucket Island, Massachusetts (2–5). We report that sika deer (Cervus nippon) in Japan also carry parasites genetically closely related to B. divergens. During November 2007–February 2008 and November 2008–February 2009, we collected blood samples from 96 wild sika deer throughout Japan. We purified DNA from the blood, which had been stored in a freezer, by phenol/chloroform extraction and performed nested PCR for B. divergens 18S rRNA (rDNA), chaperonin-containing T-complex protein 1 eta subunit (CCT7, formerly described as CCTη) (6), and β-tubulin (7) genes. Primers for rDNA were designed from the sequences of related Babesia spp. (GenBank accession nos. U16370, U16369, and AY046575): dv101F (5′-ACAAC A G T T A T A G T T T C T T T G GTATTCG-3′) and dv1353R (5′-GCCTTAAACTTCCTTGCGGCTTAGAGC-3′), and dv159F (5 ′ -GCTAATGCAAGTTCGAGGCCTTTTGGCG-3′) and dv1296R (5′-CGGACGAACCTTTTTACGGACACTAG-3′) for the first and second rounds, respectively. CCT7 primers were similarly designed (GenBank accession nos. AB367924 and AB367925): Bdiv/odoCCTF1 (5′-CAAAATGAGYCACCTMCTCAACCTACC-3′) and BdivCCTR1 (5 ′ -ATCTCAGCAGCTCACTA CAGTGACCACCTC-3′), and Bdiv/ odoCCTF2 (5′CAACCTACCRATTCTCCTYYTGAAGGAGGG-3′) and BdivCCTR2 (5′-GGCTAATAA G T C G ATAT T G C G G G G C TCACG-3′) for the first and second rounds, respectively. The β-tubulin PCR protocol has been described (7). Of the 96 blood samples, 12 from 5 prefectures (Hokkaido, Iwate, Tochigi, Nagano, and Miyazaki) were positive for Babesia rDNA (online Technical Appendix, http://wwwnc. cdc.gov/EID/article/20/8/13-0061Techapp1.pdf). The sequence for sample 08–22 from Hokkaido (GenBank accession no. KC465978) was distinct from sequences of the other 11 samples (97.5%–97.6% identity in 1,041 bp), which consisted of 7 variant sequences (GenBank accession nos. KC465973–7 and AB857845–6) and 5 identical sequences (GenBank accession nos. KC465977 and AB861504–7) (99.7%–100% identity). The 5 identical sequences varied in only 1 of 909 bp from B. divergens rDNA from an Ixodes persulcatus tick in Russia [GenBank accession no. GU057385] (8). β-tubulin (900 bp) was also amplified from the 12 Babesia rDNA– positive samples. Ten of the sequences consisted of 3 sequence variants (99.9% identity; GenBank accession nos. KC465969, KC465970, and KC465968/AB861508–14). The 2 divergent sequences (GenBank accession nos. KC465971 [08–22] and KC465989 [08–25]) were most similar to B. odocoilei (GenBank accession no. KC465972; 91% identity) and Theileria orientalis (GenBank accession no. AP011947; 79.9% identity), respectively. Thus, at least 1 deer likely had Babesia and Theileria spp. infections. CCT7 was amplified from 10 of the 12 Babesia-positive blood samples. The sequences (GenBank accession nos. KC465979–88) were more heterogeneous (98.7%–99.9% identity) than those for rDNA and β-tubulin; this finding was expected because CCT7 evolves more quickly (6). We generated a neighbor-joining phylogenetic tree (ClustalW, http://clustalw.ddbj.nig.ac.jp/index. php?lang=ja) from the Babesia rDNA sequences from our study and from GenBank (Figure, panel A). The tree shows a distinct lineage (Asian) cluster for the deer parasites, except for 08–22 (GenBank accession no. KC465978), within a clade also holding the B. divergens strains (human and bovine) from Europe (European Union lineage). The tree also shows a cluster encompassing Babesia spp. (human and rabbit) from the United States; B. divergens (deer), B. capreoli (deer), and Babesia sp. (chamois) from Europe; and B. divergens (human) from Portugal. Sequence 08–22 branches with Babesia spp. in Ix. ovatus ticks from Japan (GenBank accession nos. AY190123 and AY190124) (9). The branch lengths indicate clear separation between the isolates from sika deer and ticks, suggesting that the clustering may be attributable to the limited number of available related sequences. We also generated a phylogenetic tree of β-tubulin sequences (900 bp), which produced similar topology and high bootstrap support (Figure, panel B). However, the limited number of relevant β-tubulin sequences precludes conclusions regarding the phylogeny of Babesia parasites. A CCT7 phylogenetic tree was not generated because of the paucity of sequences. Overall, the phylogenies suggest that B. divergens– and B. capreoli–related parasites are found worldwide in temperate zones of the Northern Hemisphere, including Europe (1), the United States (2–5), Russia (8), and Japan. We showed the presence of B. divergens–like rDNA, β-tubulin, and CCT7 genes in sika deer from different Japanese prefectures, confirming the presence of this parasite in Japan. B. capreoli, which is serologically indistinguishable from B. divergens, was previously reported in sika deer (10). However, no molecular data for the B. capreoli isolate exist, so no