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Author affiliation: Author affiliations: California Department of Public Health, Richmond, California, USA (W.S. Probert, C. Kath, M.E.M Saunders, B. Bermudez, M.I. Cazas, A. Espinosa, H. Romo, J.K. Hacker); San Francisco State University, San Francisco, California, USA (N. Putirka)
Spotted fever group (SFG) rickettsioses are acute undifferentiated febrile illnesses caused by tickborne transmission of intracellular gram-negative bacteria belonging to SFG Rickettsia. Rocky Mountain spotted fever (RMSF) is caused by R. rickettsii subspecies rickettsii and Pacific Coast tick fever by R. rickettsii subsp. californica; those 2 SFG rickettsioses are the most frequently reported in California, USA (1,2). We recently described 2 cases, 1 with disease onset in 2023 and 1 identified retrospectively with onset in 2004, of severe RMSF-like illness in northern California caused by a newly recognized SFG rickettsial pathogen, Rickettsia sp. CA6269 (3). This novel rickettsial genotype was first identified in Haemaphysalis leporispalustris ticks in Sonoma County, California, and, more recently in H. leporispalustris ticks in Maine, USA (4,5). Subsequently, whole-genome sequencing of rickettsial strain HLP 7421, isolated in 1961 from a pool of H. leporispalustris ticks collected in Montana, USA, supported classification of the Rickettsia sp. CA6269 genotype as a new species, R. lanei (6). In this study, we report on the detection of R. lanei in Dermacentor occidentalis and H. leporispalustris ticks collected at or near locations of exposure for the 2004 and 2023 cases.
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Figure. Heat maps displaying the number of Dermacentor occidentalis (A) and Haemaphysalis leporispalustris (B) ticks tested for spotted fever group Rickettsiain 2024, by…
The California Department of Public Health monitors prevalence of SFG Rickettsia annually by collecting and testing Dermacentor spp. ticks (3,7). In 2024, SFG Rickettsia surveillance was enhanced to include collection of H. leporispalustris and additional Dermacentor spp. ticks from areas associated with exposures for the 2004 (Marin and San Mateo Counties) and 2023 (Alameda and Contra Costa Counties) cases. In addition, H. leporispalustris ticks were collected from an area in Sonoma County where R. lanei–positive ticks were originally described (4). In 2024, a total of 3,607 adult and nymphal ticks were tested for SFG Rickettsia: 2,872 D. occidentalis ticks collected from 34 of the 58 California counties and 69 D. similis and 666 H. leporispalustris ticks collected from 6 counties (Figure).
We extracted and purified tick nucleic acids and screened for Rickettsia spp. using PanR8 real-time PCR (rPCR) (3,8). We tested all positive samples for R. rickettsii subsp. californica using the nusG rPCR and R. rickettsii subsp. rickettsii/R. lanei using RRi6 rPCR (8,9). We used a fourth rPCR to distinguish R. lanei from R. rickettsii subsp. rickettsii (3). We detected Rickettsia spp. in 14% of D. occidentalis ticks, 20.3% of D. similis ticks, and 3.5% of H. leporispalustris ticks (Table 1). We detected R. rickettsii subsp. californica only in D. occidentalis ticks (1.3%) and did not detect R. rickettsii subsp. rickettsii in any ticks. Two H. leporispalustris nymphs (24-5179 and 24-6522) collected 34 days apart from the same site in San Mateo County and 1 D. occidentalis adult female tick (24-7980) from Contra Costa County were positive for R. lanei by rPCR with cycle threshold values of 27.5 (24-5179; DNA diluted 1:20), 27.5 (24-6522; DNA diluted 1:20), and 18.4 (24-7980; DNA neat) (Appendix). The overall prevalence of R. lanei was 0.3% in H. leporispalustris ticks and 0.03% in D. occidentalis ticks (Table 1).
We performed hybridization capture-based target enrichment sequencing to investigate genome relatedness of R. lanei strains from the 2023 case (strain CA23RL1) and ticks 24-5179, 24-6522, and 24-7980 (10). We amplified remnant nucleic acids from the plasma specimen collected on day 7 from the 2023 case using the REPLI-G whole genome amplification kit (QIAGEN, https://www.qiagen.com). Unfortunately, nucleic acids from the 2004 case had been depleted. We processed tick DNAs without whole-genome amplification. We performed DNA library preparation and hybridization capture-based target enrichment using a customized panel of ≈10,000 120-bp biotinylated oligonucleotides designed to span the genome of R. rickettsii subsp. californica (GenBank accession no. CP003308.1) end-to-end using the KAPA HyperCap workflow version 3 (Roche, https://www.roche.com). We sequenced enriched libraries on a MiSeq using reagent kit v2 (500 cycles) (Illumina, https://www.illumina.com).
We assembled a near whole-genome sequence of strain CA23RL1 from the 2023 case by mapping sequencing reads to the R. lanei type strain HLP 7421 genome (GenBank accession no. CP172233) using Geneious Prime v2022.0.2 (https://www.geneious.com). The percentage of CA23RL1 reads that mapped to HLP 7421 was 29.9%, providing 99.4% genome coverage with an average depth of 400 reads. We mapped unused reads to the genome of R. rickettsii subsp. californica and inserted the resultant mapped sequences into the CA23RL1 consensus sequence. We used Sanger sequencing to close genome sequence gaps and resolve repetitive regions, establishing a complete genome sequence of 1,270,942 bp for CA23RL1. Genome relatedness was 98.5% between CA23RL1 and R. rickettsii subsp. rickettsii, 98.7% between CA23RL1 and R. rickettsii subsp. californica, and 99.7% between CA23RL1 and R. lanei, as determined by the orthologous average nucleotide identity algorithm (11).
We assembled genome sequences for the tick samples by mapping reads to the CA23RL1 genome. Genome coverage for the tick samples was >99.4%; average depth was >1,100 sequencing reads. We resolved repetitive regions and genome sequence gaps using Sanger sequencing and aligned complete genome sequences for the tick samples to the CA23RL1 genome using Mauve version 1.1.3 to determine genetic relatedness (12). The D. occidentalis 24-7980 genome sequence was identical to the CA23RL1 genome sequence, whereas those of the H. leporispalustris samples (24-5179 and 24-6522) were identical to one another but differed from the CA23RL1 and 24–7980 genome sequences by 19 nt polymorphisms and 4 insertions/deletions (Table 2).
Sequencing data are available in the National Center for Biotechnology Information BioProject database (accession no. PRJNA1261853). Limitations to our study are the lack of long-read next generation sequencing data to confirm lengthy repetitive DNA regions and the potential bias in using closely related reference sequences for guiding capture probe design and genome assembly.
We investigated the role of Dermacentor spp. and H. leporispalustris ticks as reservoirs and potential vectors of R. lanei in California and found a very low prevalence of infection. Ticks infected with R. lanei were only detected in counties identified as locations of exposure for the 2 cases: 2 H. leporispalustris nymphs collected in San Mateo County near a location of exposure for the 2004 case and 1 D. occidentalis adult tick collected in Contra Costa County at a site of exposure for the 2023 case. The positive D. occidentalis tick was collected at 1 of 5 golf courses visited by the case-patient within 14 days of illness onset (3). This ecoepidemiologic association and identical R. lanei genomic sequence match between the 2023 case and the D. occidentalis tick strongly implicate this tick species as the source of disease transmission. This conclusion is further supported by the observation that D. occidentalis ticks infest humans much more frequently than do H. leporispalustris ticks (13).
The R. lanei genome sequence from the H. leporispalustris ticks shared a high degree of sequence identity (orthologous average nucleotide identity >99.9%) with the sequence from the 2023 case and the D. occidentalis tick. Although it is unlikely that H. leporispalustris plays a role in the human transmission of R. lanei given both its preference for lagomorphs and the rarity of human infestations, this tick species may serve as a key vector for maintenance of R. lanei in nature, much as it does for R. rickettsii (14,15). Future studies are warranted to confirm vector competency of D. occidentalis and H. leporispalustris for R. lanei transmission. Acknowledging the severity of the 2 R. lanei infections and the broad distribution of the tick species, our results highlight the role of ecoepidemiologic investigations in identifying risk factors and guiding mitigation strategies for preventing vectorborne diseases.
Dr. Probert is a research scientist with the Viral and Rickettsial Disease Laboratory of the California Department of Public Health, Center for Laboratory Sciences. He is interested in the development of molecular diagnostic assays for the detection and genotyping of microbial pathogens.