Figure 1

Figure 1. Study area for investigation of Orientia spp. bacteria in field-collected free-living Eutrombiculachigger mites, North Carolina, USA. Free-living chiggers were collected from 10 sites in 8 counties....

In 2022, we collected free-living chiggers using the tile method (9) in different locations in North Carolina: we placed tiles on the ground and then visually inspected for the presence of chiggers after ≈1 minute. When chiggers were present, we collected them with forceps or a small paintbrush and transferred them into vials with 95% ethanol. We identified subsamples from each collection location based on morphological characteristics using published taxonomic keys (10). We identified chiggers as Eutrombicula on the basis of their morphology. In addition, we obtained chigger images of Eutrombicula using scanning electron microscopy at the Analytical Instrumentation Facility at North Carolina State University (Raleigh, North Carolina, USA) (Appendix Figure 1).

We surface-sterilized individual free-living chiggers and extracted total nucleic acids using the DNeasy Blood & Tissue Kit (QIAGEN, https://www.qiagen.com) (11). In total, 95 chiggers from 10 different locations (10 chiggers/location; 1 location had 5 chiggers) were subjected to microbiome analyses (Figure 1). We randomly selected 8 chiggers for molecular identification using previously described 18S ribosomal RNA gene primers and PCR (12). Amplicons were Sanger sequenced at Eton Bioscience, Inc. (https://www.etonbio.com). The sequences (GenBank accession nos. OQ789321–5) were submitted for BLASTn (https://blast.ncbi.nlm.nih.gov/Blast.cgi) analysis and showed 99–100% identity with homologous sequences of Eutrombicula spp. (accession no. KY922159).

Figure 2

Figure 2. Phylogenetic tree of Orientia tsutsugamushi16S rRNA gene sequences (≈399 bp) from free-living chiggers collected in North Carolina, USA, and their reference sequences in GenBank. The tree was constructed...

To determine the total bacteria present in the chiggers, we constructed a 16S rRNA sequence library of individual chiggers using the Illumina 16S rRNA (V3-V4 region) metagenomics sequencing library preparation protocol (Illumina, https://www.illumina.com) (13). Sequencing was performed at the University of North Carolina Microbiome Core Facility (Chapel Hill, North Carolina, USA). The Illumina FASTQ files were processed using Quantitative Insights into Microbial Ecology 2 (14) (Appendix). An analysis of amplicon sequence variants (ASVs) revealed that chigger mites contained reads of a bacterial sequence classified as O. tsutsugamushi. O. tsutsugamushi–positive chigger sequence reads were found in 5/10 sampling sites as follows: 3 sites in the Piedmont region (Falls Lake State Recreation Area [1 positive/10 chiggers], Kerr Lake State Recreation Area [8 positive/10 chiggers], and Morrow Mountain State Park [1 positive/10 chiggers]); and 2 sites in the Coastal Plains region (Lumber River State Park [9 positive/10 chiggers] and Merchant Millpond State Park [1 positive/5 chiggers]) (Figure 1). By using the Greengenes 16S rRNA database, we found 13 ASVs to O. tsutsugamushi. To further confirm those results, we extracted representative sequences from the sequencing data and conducted BLASTn searches against the National Center for Biotechnology Information (NCBI) databases. We found 12 ASVs showed a high nucleotide identity (99.5%–100%) to the O. tsutsugamushi strain Kato (D38624), isolated from a human scrub typhus case in Kurosawa village, Japan (https://u.osu.edu/scrubtyphus/the-kato-strain). One ASV exhibited 89.46% homology to an O. tsutsugamushi sequence that was excluded from the phylogenetic analysis. We then examined the approximate phylogenetic relationships for 12 of the O. tsutsugamushi sequence variants from our study sites with other O. tsutsugamushi sequences obtained from the NCBI database using BLASTn (accessed January 18, 2023). We performed multiple alignments with the ClustalW program and maximum-likelihood method tree with the Kimura 2-parameter method from the MEGA X (https://www.megasoftware.net) software package. The phylogenetic analysis revealed that all 12 of the O. tsutsugamushi ASVs clustered closely to O. tsutsugamushi strains from Asia (Figure 2).

To further verify the identity of O. tsutsugamushi detected in our free-living chigger samples, we amplified a 47-kDa htrA (high-temperature requirement A) gene in the 20 chigger samples that were positive for the O. tsutsugamushi 16S rRNA gene. The primers for the first round of PCR were Ot-145F and Ot-1780R, and for the second round, Ot-263F and Ot-1133R (15) (Appendix). The 47-kDa gene amplification products were Sanger sequenced at Eton Bioscience. Four samples (FC28, 36, 38, 109) were 92.6%–97.29% identical to the O. tsutsugamushi HN82 strain (GenBank accession no. LC431268) and 92%–97% identical to the O. tsutsugamushi Kato strain (accession no. LS398550) after trimming low-quality sequences. Sixteen samples yielded ambiguous sequences, suggesting the presence of multiple Orientia species or primer binding sites caused by high variation in the 47 kDa gene among Orientia species in our samples. Jiang et al. (15) studied the genetic variation of Orientia in this region of the genome and reported percent identity of 17 isolates of Orientia as 82.2%–83.3% (15). Among our 4 Orientia 47 kDa sequences from North Carolina chiggers, identity was 93.97%–98%. Phylogenetic analysis revealed that all 4 of those O. tsutsugamushi sequences clustered to O. tsutsugamushi strains from Asia (Appendix Figure 2).

This study identified Orientia species within the United States in free-living Eutrombicula chiggers that were collected in North Carolina. This result is epidemiologically significant because it indicates vertical circulation of Orientia species in chiggers collected within the continental United States. The presence of Orientia species in free-living larvae suggests that the bacteria are maintained through transovarial transmission. Further studies are needed to complete sequencing of the 47-kDa htrA gene (htrA) in our samples, determine how widely distributed Orientia spp.–infected free-living and host-attached chiggers are in the United States, and ascertain whether wild animals that serve as hosts for chiggers become infected and infectious and develop symptoms of illness. Clinicians in this region should be alert for possible human cases of illness resulting from Orientia spp. infection.