Investigators captured 80 Hipposideros turpis bats on Yonaguni Island during September 2024 and 2025 and collected 212 larval chiggers. The study team identified all chiggers morphologically as Whartonia prima using recognized taxonomic keys. The authors reported that chiggers from the 2024 field season were used for molecular screening, while the 2025 collection supported molecular assays, isolation attempts, and electron microscopy. The source did not report any experimental infection of bats or direct testing of bat tissue for Orientia in this report.
The investigators tested 68 chiggers individually by real-time PCR targeting 16S rRNA and by nested PCR for the TSA56 gene. Real-time PCR detected Orientia tsutsugamushi DNA in 64 of 68 mites (reported 94%), and nested PCR returned positive results in 55 of 68 mites (reported 81%). The authors sequenced nested PCR amplicons and identified two partial TSA56 genotypes. They amplified full-length TSA56 from 10 selected mites; full-length sequences were recovered from two specimens and grouped within the Karp-related lineage. The publication states these molecular results but did not include data on any parallel testing of vertebrate hosts in the same publication.
For isolation work, the team pooled the 129 W. prima chiggers collected in 2025 by origin bat, with pools of 2–5 mites each. Pools were homogenized in sucrose–phosphate–glutamate buffer and inoculated onto L929 fibroblast cells maintained in Leibovitz’s L-15 medium with 2% fetal bovine serum, notably without antimicrobials. Cultures were passaged every two weeks; the investigators monitored O. tsutsugamushi DNA by real-time PCR at each passage and kept uninfected L929 controls in parallel. Contaminated cultures were discarded immediately, per the methods described. Of 28 chigger pools inoculated, four yielded cultivable isolates—designated Donan 18-3R, Donan 17-2L, Donan 14-1L, and Donan 14-2R—while three cultures remained negative and others were lost to contamination. All four isolates were maintained through four passages and fourth-passage infected cell suspensions were stored in sucrose–phosphate–glutamate buffer.
To characterize a representative isolate, investigators inoculated fourth-passage Donan 18-3R onto fresh L929 cells. Giemsa-stained preparations examined at five days postinoculation showed numerous punctate, basophilic cytoplasmic inclusions outside the nucleus—morphology the study team interpreted as consistent with intracellular Orientia tsutsugamushi. The authors performed growth-curve experiments by inoculating fourth-passage Donan 18-3R into 24-well plates and extracting DNA daily from replicate wells. Quantitative PCR, performed independently three times, yielded mean bacterial loads that decreased initially at day 1 but rose from a reported 4.19 ±0.21 log10 copies/well at day 1 to 6.93 ±0.59 log10 copies/well by day 8, indicating replication of the isolate in L929 cells under the conditions described.
The study team generated full-length TSA56 sequences for Donan 18-3R and Donan 17-2L and compared them with sequences obtained directly from PCR-positive W. prima chiggers. Partial TSA56 sequences from Donan 14-1L and Donan 14-2R matched corresponding regions of the two full-length sequences. For broader genomic context, investigators performed multilocus sequence analysis (MLSA) using concatenated sequences from 11 housekeeping genes for selected chigger samples and for Donan 18-3R and Donan 17-2L. The concatenated MLSA sequences of Donan 18-3R and Donan 17-2L were identical. In phylogenetic trees the isolate sequences and chigger-derived sequences clustered within the O. tsutsugamushi clade and specifically grouped with Karp-related lineages, according to the figures and sequence accession information provided.
The authors provided direct evidence that bat-associated W. prima chiggers can harbor and yield viable Orientia tsutsugamushi by culture, microscopy, serial passage, and growth kinetics. They noted, however, that vector competence of these chiggers and the role of bats as vertebrate hosts for transmission remain unresolved in this report. The source did not present experimental transmission studies, controlled vector competence assays, or vertebrate infection outcomes. The paper therefore leaves specific ecological and epidemiologic questions open: whether W. prima can naturally transmit O. tsutsugamushi to vertebrate hosts, and whether Hipposideros turpis bats act as reservoirs or incidental hosts. These items were identified in the article as unanswered and were not assessed within the methods or results presented.
Limitations documented by the authors also included losses of cultures to contamination and the small number of isolates fully sequenced; the source did not claim population-level prevalence in bats or vector competence. Next steps the authors implied but did not report would require targeted vector competence experiments, vertebrate infection studies, and expanded ecological surveillance across seasons and locations to determine the epidemiologic significance of bat-associated chiggers in scrub typhus ecology.