---
title: "Isolation of Orientia tsutsugamushi from Bat-Associated Chiggers on Yonaguni Island, Japan"
id: "cdc-emerging-infectious-diseases-journal-0-isolation-of-orientia-tsutsugamushi-from-bat-associated-chiggers-japan"
canonical_url: "https://medichelpline.com/clinical-feed/cdc-emerging-infectious-diseases-journal-0-isolation-of-orientia-tsutsugamushi-from-bat-associated-chiggers-japan"
content_type: "clinical_feed_article"
specialty: "Infectious Disease"
source_name: "CDC Emerging Infectious Diseases Journal"
source_url: "https://wwwnc.cdc.gov/eid/article/32/12/26-1156_article"
published_at: "2026-10-22T04:00:00.000Z"
evidence_level: "Agency Feed"
license: "CC-BY-NC-4.0 / Informational Use"
---
# Isolation of Orientia tsutsugamushi from Bat-Associated Chiggers on Yonaguni Island, Japan
## Provenance & Clinical Metadata
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- **Specialty:** [Infectious Disease](https://medichelpline.com/clinical-feed/infectious-disease.md)
- **Primary Source:** CDC Emerging Infectious Diseases Journal
- **Source URL:** [Original Journal Publication](https://wwwnc.cdc.gov/eid/article/32/12/26-1156_article)
- **Published At:** 2026-10-22T04:00:00.000Z
- **Evidence Rating:** Agency Feed
## Executive GIST (TL;DR)
- Investigators collected larval chiggers from 80 Hipposideros turpis bats on Yonaguni Island during September 2024 and 2025; the chiggers were morphologically identified as **Whartonia prima**. - The study team tested 68 chiggers individually using real-time PCR for 16S rRNA and nested PCR for the TSA56 gene and found high detection rates by molecular methods. - Real-time PCR detected **Orientia tsutsugamushi** DNA in 64 of 68 chiggers (reported 94%); nested PCR detected O. tsutsugamushi in 55 of 68 chiggers (reported 81%). - Full-length TSA56 sequences were obtained from two chigger-derived samples and clustered within the **Karp-related lineage**; partial TSA56 sequences from isolates matched these full-length sequences. - For isolation attempts, investigators pooled 129 chiggers by individual bat (2–5 chiggers per pool) and inoculated homogenates onto L929 cells; four pools produced cultivable isolates (Donan 18-3R, 17-2L, 14-1L, 14-2R). - The authors maintained isolates through four serial passages and validated identity by sequencing and multilocus sequence analysis (MLSA) using 11 housekeeping genes; Donan 18-3R and Donan 17-2L showed identical concatenated MLSA sequences. - Microscopy of fourth-passage Donan 18-3R-infected L929 cells showed intracellular, punctate basophilic structures consistent with O. tsutsugamushi; quantitative PCR-based growth curves showed increasing bacterial load from day 1 to day 8. - The study documents viable O. tsutsugamushi in bat-associated chiggers, notes clustering within the Karp-related lineage, and states that questions about vector competence and vertebrate host involvement remain unresolved; the source did not report experimental vector competence tests or vertebrate infection outcomes.
## Clinical Analysis & Structured Key Points
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[EID Journal](https://wwwnc.cdc.gov/eid/) 2. [Volume 32](https://wwwnc.cdc.gov/eid/early-release) 3. [Early Release](https://wwwnc.cdc.gov/eid/early-release#issue-1348) 4. [Main Article](https://wwwnc.cdc.gov/eid/article/32/12/26-1156_article) * [Facebook](https://www.facebook.com/sharer/sharer.php?u=%2Feid%2Farticle%2F32%2F12%2F26-1156_article "Share to Facebook") * [Twitter](http://twitter.com/share?url=%2Feid%2Farticle%2F32%2F12%2F26-1156_article&text= "Share to Twitter") * [LinkedIn](https://www.linkedin.com/shareArticle?url=%2Feid%2Farticle%2F32%2F12%2F26-1156_article&title= "Share to LinkedIn") * [Syndicate](https://tools.cdc.gov/medialibrary/index.aspx#/sharecontent//eid/article/32/12/26-1156_article "Embed this Page") [ Emerging Infectious Disease journal ISSN: 1080-6059 ](https://wwwnc.cdc.gov/eid/) _Disclaimer: Early release articles are not considered as final versions. Any changes will be reflected in the online version in the month the article is officially released._ #### Volume 32, Number 12—December 2026 ##### _Dispatch_ ### Isolation of _Orientia tsutsugamushi_ from Bat-Associated Chiggers, Japan On This Page [The Study](https://wwwnc.cdc.gov/eid/article/32/12/26-1156_article) * * * [Conclusions](https://wwwnc.cdc.gov/eid/article/32/12/26-1156_article) * * * [Suggested Citation](https://wwwnc.cdc.gov/eid/article/32/12/26-1156_article) Figures [Figure 1](https://wwwnc.cdc.gov/eid/article/32/12/26-1156-f1) * * * [Figure 2](https://wwwnc.cdc.gov/eid/article/32/12/26-1156-f2) * * * [Figure 3](https://wwwnc.cdc.gov/eid/article/32/12/26-1156-f3) Downloads [Appendix ](https://wwwnc.cdc.gov/eid/article/32/12/26-1156-app1.pdf) * * * [RIS [TXT - 2 KB] ](https://wwwnc.cdc.gov/eid/article/32/12/26-1156.ris) Article Metrics [Metric Details](https://wwwnc.cdc.gov/eid/article/32/12/26-1156_article) Yongjin Qiu[![Comments to Author](https://wwwnc.cdc.gov/eid/content/images/icon/email.gif)](https://wwwnc.cdc.gov/eid/article/32/12/26-1156_article#comment) , Kuniko Kawai, Paula Andrea Jiménez, Hisao Tamura, Mamoru Takahashi, Aiko Ohnuma, Shumpei Kamiyama, Tsutomu Kakuda, Nariaki Nonaka, and Ryo Nakao Author affiliation: Hokkaido University, Sapporo, Japan (Y. Qiu, P.A. Jiménez, A. Ohnuma, N. Nonaka, R. Nakao); Kitasato University, Towada, Japan (Y. Qiu, T. Kakuda); Tokai University, Sapporo (K. Kawai); The Asian Bat Research Institute, Nara, Japan (H. Tamura); Saitama Medical University, Moroyama, Japan (M. Takahashi); Zoukei Co., Ltd., Obihiro, Japan (S. Kamiyama) [Suggested citation for this article](https://wwwnc.cdc.gov/eid/article/32/12/26-1156_article#suggestedcitation) ### Abstract We isolated _Orientia tsutsugamushi_ bacteria from bat-associated _Whartonia prima_ chiggers collected from _Hipposideros turpis_ bats on Yonaguni Island, Japan. Serial passage, microscopic examination, growth kinetics, and multilocus sequence analysis confirmed bacterial propagation and identity. Our findings highlight an overlooked bat–chigger interface with potential relevance to broader ecologic surveillance of scrub typhus. Scrub typhus, or tsutsugamushi disease, is a vectorborne zoonosis caused by _Orientia tsutsugamushi_ bacteria. _O. tsutsugamushi_ is transmitted by larval trombiculid mites, or chiggers, and is primarily maintained within chigger populations through transovarial and transstadial transmission ([_1_](https://wwwnc.cdc.gov/eid/article/32/12/26-1156_article#r1 "1")). Ecologic studies of scrub typhus have largely focused on rodent-associated _Leptotrombidium_ chiggers, which are the principal vectors in many endemic areas ([_1_](https://wwwnc.cdc.gov/eid/article/32/12/26-1156_article#r1 "1")). In Japan, where ≈300–500 scrub typhus cases are reported annually ([_2_](https://wwwnc.cdc.gov/eid/article/32/12/26-1156_article#r2 "2")), the ecologic diversity of _O. tsutsugamushi_ in wildlife-associated chiggers remains poorly understood. Recent molecular detection of _O. tsutsugamushi_ DNA in bats from the China–Myanmar border raised questions about the roles of bats and their ectoparasites in _Orientia_ ecology ([_3_](https://wwwnc.cdc.gov/eid/article/32/12/26-1156_article#r3 "3")). Bat-associated chiggers are diverse and widely distributed ([_4_](https://wwwnc.cdc.gov/eid/article/32/12/26-1156_article#r4 "4")), but their involvement in _Orientia_ ecology remains limited. We report the isolation and propagation of _O. tsutsugamushi_ from _Whartonia prima_ chiggers collected on Yonaguni Island, Japan, from _Hipposideros turpis_ , a cave-dwelling insectivorous bat endemic to the Yaeyama Islands ([_5_](https://wwwnc.cdc.gov/eid/article/32/12/26-1156_article#r5 "5")). ### The Study We collected larval chiggers from _H. turpis_ bats on Yonaguni Island in September 2024 and 2025. Bat capture and handling were approved by the Ministry of the Environment, Tokyo, Japan (permission nos. 2402141 and 2502282), and complied with relevant institutional and national guidelines. We examined a total of 80 _H. turpis_ bats, yielding 212 chiggers: 31 in 2024 and 181 in 2025. We identified all chigger mites morphologically as _W. prima_ by using taxonomic descriptions and keys ([_6_](https://wwwnc.cdc.gov/eid/article/32/12/26-1156_article#r6 "6"),[_7_](https://wwwnc.cdc.gov/eid/article/32/12/26-1156_article#r7 "7")). We used the chigger mites collected in 2024 for molecular screening. Of the 181 chigger mites we collected in 2025, we used 37 mites for molecular screening, 129 mites for isolation attempts, and 4 mites for scanning electron microscopy; we excluded 11 mites because of poor specimen condition. Figure 1 ![Phylogenetic analyses of Orientia tsutsugamushi detected and isolated from bat-associated Whartonia prima chiggers, Yonaguni Island, Japan. A\) Maximum-likelihood tree on the basis of full-length 56-kDa type-specific antigen gene sequences from 2 O. tsutsugamushi isolates, Donan 18-3R and Donan 17-2L, and selected PCR-positive chigger specimens. B\) Maximum-likelihood tree on the basis of concatenated sequences of 11 housekeeping genes from 2 O. tsutsugamushi isolates, Donan 18-3R and Donan 17-2L, and selected PCR-positive chigger specimens. Bootstrap values were calculated from 1,000 replicates. Blue indicates isolates obtained in this study and red indicates sequences obtained directly from selected PCR-positive W. prima chiggers. Scale bars indicate the number of nucleotide substitutions per site. JG, Japanese Gilliam; JP, Japanese Karp; Wp, prefix used for DNA sample identifiers from Whartonia prima chiggers.](https://wwwnc.cdc.gov/eid/images/26-1156-F1-tn.jpg) [Figure 1](https://wwwnc.cdc.gov/eid/article/32/12/26-1156-f1 "Figure 1"). Phylogenetic analyses of _Orientia tsutsugamushi_ detected and isolated from bat-associated _Whartonia prima_ chiggers, Yonaguni Island, Japan. A) Maximum-likelihood tree on the basis of full-length 56-kDa type-specific antigen... We tested 68 chigger mites individually for _O. tsutsugamushi_ by using real-time PCR targeting the 16S rRNA gene ([_8_](https://wwwnc.cdc.gov/eid/article/32/12/26-1156_article#r8 "8")) and nested PCR targeting the 56-kDa type-specific antigen (TSA56) gene ([_9_](https://wwwnc.cdc.gov/eid/article/32/12/26-1156_article#r9 "9")). We sequenced amplicons from all nested PCR-positive chigger mite samples, revealing 2 partial TSA56 genotypes. We amplified full-length _O. tsutsugamushi_ TSA56 from 10 mite samples representing both partial genotypes by using previously described primers ([Appendix](https://wwwnc.cdc.gov/eid/article/32/12/26-1156-app1.pdf)) ([_10_](https://wwwnc.cdc.gov/eid/article/32/12/26-1156_article#r10 "10")). Of the 68 chigger mites tested, 64 (94%, 95% CI 86%–98%) were positive for _O. tsutsugamushi_ by real-time PCR, and 55 (81%, 95% CI 70%–89%) were positive for _O. tsutsugamushi_ by nested PCR. We obtained full-length TSA56 sequences from 2 of the 10 selected chigger mite samples; both clustered within the Karp-related lineage ([Figure 1](https://wwwnc.cdc.gov/eid/article/32/12/26-1156-f1), panel A). For isolation, we pooled the 129 _W. prima_ chiggers collected in 2025 by individual bat host. Each pool consisted of 2–5 chiggers from the same bat and was homogenized in sucrose–phosphate–glutamate buffer. We inoculated the homogenates onto L929 cells maintained in Leibovitz’s L-15 medium supplemented with 2% fetal bovine serum without antimicrobials. We passaged the cultures every 2 weeks and monitored _O. tsutsugamushi_ DNA by using real-time PCR at each passage. We maintained uninfected L929 cell controls in parallel that remained negative for _O. tsutsugamushi_. We discarded contaminated cultures immediately. Of 28 chigger pools, 4 pools yielded _O. tsutsugamushi_ isolates: Donan 18-3R (3 chiggers), Donan 17-2L (2 chiggers), Donan 14-1L (5 chiggers), and Donan 14-2R (5 chiggers). Three cultures remained negative, and the remainder were excluded because of contamination. We maintained all 4 isolates through 4 passages and stored fourth-passage infected cell suspensions in sucrose–phosphate–glutamate buffer. We generated full-length TSA56 gene sequences for 2 _O. tsutsugamushi_ isolates, Donan 18-3R and Donan 17-2L, and compared them with sequences obtained directly from selected PCR-positive _W. prima_ chiggers (GenBank accession nos. LC896643, LC896644, LC939506, and LC939507) ([Figure 1](https://wwwnc.cdc.gov/eid/article/32/12/26-1156-f1), panel A). Partial TSA56 sequences from the remaining _O. tsutsugamushi_ isolates, Donan 14-1L and Donan 14-2R (accession nos. LC939530 and LC939531), were identical to the corresponding regions of the full-length sequences from Donan 18-3R and Donan 17-2L. We performed multilocus sequence analysis (MLSA) on the basis of 11 housekeeping genes for 10 chigger samples selected for full-length TSA56 analysis and 2 _O. tsutsugamushi_ isolates, Donan 18-3R and Donan 17-2L, by using primers described previously ([_11_](https://wwwnc.cdc.gov/eid/article/32/12/26-1156_article#r11 "11")) (accession nos. LC897744–55 and LC939508–29). The concatenated MLSA sequences of Donan 18-3R and Donan 17-2L were identical. The _O. tsutsugamushi_ isolate sequences and sequences obtained directly from PCR-positive chigger samples clustered within the _O. tsutsugamushi_ clade in MLSA, supporting their identification as _O. tsutsugamushi_ ([Figure 1](https://wwwnc.cdc.gov/eid/article/32/12/26-1156-f1), panel B). Figure 2 ![Microscopy of Orientia tsutsugamushi isolate Donan 18-3R detected and isolated from bat-associated Whartonia prima chiggers, Yonaguni Island, Japan. Giemsa-stained L929 cells 5 days after inoculation with fourth-passage Donan 18-3R, shows punctate, basophilic structures in the cytoplasm outside the nucleus, consistent with intracellular O. tsutsugamushi; the red arrow indicates representative intracellular bacteria. Oil immersion; scale bar represents 10 µM. ](https://wwwnc.cdc.gov/eid/images/26-1156-F2-tn.jpg) [Figure 2](https://wwwnc.cdc.gov/eid/article/32/12/26-1156-f2 "Figure 2"). Microscopy of _Orientia tsutsugamushi_ isolate Donan 18-3R detected and isolated from bat-associated _Whartonia prima_ chiggers, Yonaguni Island, Japan. Giemsa-stained L929 cells 5 days after inoculation with fourth-passage... Figure 3 ![Growth kinetics of Orientia tsutsugamushi isolate Donan 18-3R detected and isolated from bat-associated Whartonia prima chiggers, Yonaguni Island, Japan. Growth kinetics were assessed by quantitative PCR and expressed as log10 bacterial copies/well after inoculation of fourth-passage bacteria onto L929 cells in 24-well plates. Three independent growth experiments were performed.](https://wwwnc.cdc.gov/eid/images/26-1156-F3-tn.jpg) [Figure 3](https://wwwnc.cdc.gov/eid/article/32/12/26-1156-f3 "Figure 3"). Growth kinetics of _Orientia tsutsugamushi_ isolate Donan 18-3R detected and isolated from bat-associated _Whartonia prima_ chiggers, Yonaguni Island, Japan. Growth kinetics were assessed by quantitative PCR and... To characterize a representative isolate, we inoculated fourth-passage Donan 18-3R onto fresh L929 cells. We identified numerous punctate, basophilic structures in the cytoplasm outside the nucleus from Giemsa-stained cells 5 days postinoculation (dpi), a pattern consistent with intracellular _O. tsutsugamushi_ ([Figure 2](https://wwwnc.cdc.gov/eid/article/32/12/26-1156-f2)). For growth analysis, we inoculated fourth-passage Donan 18-3R onto L929 cells in 24-well plates. We extracted DNA daily from 1 well and calculated bacterial copy numbers by using quantitative PCR, which we independently performed 3 times. After an initial decrease at 1 dpi, mean +SD bacterial loads increased from 4.19 +0.21 log10 copies/well at 1 dpi to 6.93 +0.59 log10 copies/well at 8 dpi, demonstrating growth of Donan 18-3R in L929 cells ([Figure 3](https://wwwnc.cdc.gov/eid/article/32/12/26-1156-f3)). [Top](https://wwwnc.cdc.gov/eid/article/32/12/26-1156_article) ### Conclusions We provided evidence that bat-associated _W. prima_ chiggers can harbor viable _O. tsutsugamushi_ , demonstrated by isolation, serial propagation, microscopic examination, and growth kinetics. The _O. tsutsugamushi_ isolates clustered phylogenetically within the Karp-related lineage. Although vector competence, vertebrate host invol
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