Rickettsia are obligate intracellular bacteria responsible for various vector-borne diseases in humans and animals. A pangenomic meta-analysis showed that Rickettsia species diverged into five phylogenetically relevant categories including two spotted fever groups (SFGI and SFGII), the Typhus group (TG), the Canadensis group (CG), and the Bellii group (BG) (El Karkouri et al., 2022). Tick-borne rickettsioses are mainly caused by Rickettsia species within the SFG groups. The SFG rickettsioses incidence in the United States has generally increased over time with a distinct increase since the mid-1990s, rising from 1,985 cases in 2010 to 6,248 cases in 2017. However, reported cases slightly decreased to 5,207 in 2019 (Bishop et al., 2022). Cases from 2020 to 2022 were much lower, ranging from 1,100 to 1,300. This sharp decline was likely due in large part to a 2020 change in the case definition (Centers for Disease Control and Prevention, 2025).

In the United States, the most serious and commonly reported spotted fever is Rocky Mountain spotted fever (RMSF), caused by Rickettsia rickettsii. The surveillance case definition for RMSF was modified to encompass the broader category of spotted fever rickettsiosis in 2010. This change was made because some cases reported as RMSF might actually be caused by other SFG rickettsiae, rather than the RMSF pathogen specifically (Parola et al., 2013). Rocky Mountain spotted fever cases associated with Dermacentor variabilis are primarily found in the eastern United States, while Dermacentor andersoni is more commonly associated with RMSF in the Rocky Mountain region and farther west. Additionally, RMSF cases linked to Rhipicephalus sanguineus sensu lato are concentrated in the Southwest (Parola et al., 2013). Although D. variabilis has been historically treated as a major vector of the RMSF agent in the eastern United States, the infection rate of that tick species has been found to be extremely low (Stromdahl et al., 2011). Rhipicephalus sanguineus sensu lato may be a more important vector of the RMSF agent than previously recognized, especially in the southwestern United States (Demma et al., 2005). While RMSF remains the most significant spotted fever disease, several other rickettsial pathogens and their tick vectors have been identified as causing human disease in the United States. Rickettsia parkeri rickettsiosis, caused by R. parkeri transmitted by the Gulf Coast tick, Amblyomma maculatum, is distributed primarily in the southeastern United States (Paddock et al., 2004). Pacific Coast tick fever is caused by the bacterium Rickettsia rickettsii subspecies californica, which is transmitted by the Pacific Coast tick, Dermacentor occidentalis, in the western United States (Padgett et al., 2016). Genomic, phenotypic, ecological, and clinical data recently identified Rickettsia 364D (“R. philipii”) as a distinct subspecies of R. rickettsii (Paddock et al., 2024). Interestingly, a recently recognized SFG pathogen, determined to be the cause of a severe RMSF-like illness in California, was likely transmitted by Haemaphysalis leporispalustris (Eremeeva et al., 2018; Probert et al., 2024).

The rabbit tick (H. leporispalustris) is a three-host tick and the only tick species that is widely distributed throughout North and Central America to Argentina (Kohls, 1960). The adult and immature stages of H. leporispalustris primarily feed on wild rabbits (Sylvilagus spp.) and hares (Lepus spp.), while larvae and nymphs may also infest other small animals and several species of birds (Campbell and Glines, 1979; Kollars and Oliver, 2003). The tick can go through two life cycles in one year in the southern United States but has a 2-year life cycle in southwestern Nova Scotia in Canada (Campbell and Glines, 1979). Haemaphysalis leporispalustris does bite humans, but not frequently (Eisen, 2022; Harrison et al., 1997).

In North America, H. leporispalustris can transmit or carry several Rickettsia species, including human pathogenic R. rickettsii (Parker et al., 1951), R. parkeri (Henning et al., 2014), Rickettsia felis (Roth et al., 2017), non-pathogenic Rickettsia bellii (Backus et al., 2023) and Rickettsia canadensis (McKiel et al., 1967). This tick species is usually not considered the primary vector for human rickettsioses agents in the United States, but it plays an important role in the enzootic maintenance of R. rickettsii in nature (Parker et al., 1951). However, Fuentes et al. isolated two highly virulent isolates of R. rickettsii from H. leporispalustris in Costa Rica (Fuentes et al., 1985). These two isolates were physiologically and serologically indistinguishable from human strains. Moreover, Probert et al. found severe RMSF-like illness in two patients in northern California over a span of 20 years, and these two case patients might have encountered questing H. leporispalustris infected with a new Rickettsia species: Candidatus Rickettsia lanei (also known as CA6269/CA23RLA) or may have encountered other tick species infected with this pathogen (Eremeeva et al., 2018; Probert et al., 2024). The potential role of H. leporispalustris in the maintenance and transmission of SFG Rickettsia is underexplored.

In this study, we found that 18 of 296 questing H. leporispalustris ticks collected from Maine were Rickettsia spp. positive using RRi6 Taqman real-time PCR assays, which were originally designed to be R. rickettsii-specific (Kato et al., 2013). Further multilocus sequence typing (MLST) and phylogenetic analysis revealed that this is a new Rickettsia genotype closely related to Candidatus Rickettsia lanei.

Project ITCH collected 2,536 questing ticks from Maine, including six species: Ixodes scapularis, Ixodes cookei, Ixodes dentatus, Ixodes marxi, D. variabilis and H. leporispalustris. Among them, 296 (11.7%) H. leporispalustris ticks were collected from 38 towns across nine Maine counties (Table 1). All ticks were collected from June to August, 2020–2024. The species was identified as H. leporispalustris using both morphological characteristics and the molecular assay described in this paper. The collection comprised six adult females, nine adult males, 260 nymphs, and 21 larvae (Table 1).

Rickettsia spp. were detected by both RRi6 TaqMan real-time assays. The results from both assays were congruent. The overall prevalence of Rickettsia spp. in H. leporispalustris ticks was 6.1% (18 of 296 ticks, 95% confidence interval 3.6% to 9.4%). The Rickettsia spp. positive ticks were one adult male, two larvae, and 15 nymphs. The cycle threshold (Ct) values of these Rickettsia spp. positive samples averaged 23.90 (range 20.84 to 26.75).

Multilocus Sequence Typing (MLST) was further applied to characterize these Rickettsia spp. positive samples. Four gene fragments (gltA, ompA, ompB, and sca4) were successfully amplified and sequenced from 16 Rickettsia spp. positive ticks. The gltA, ompB, and sca4 sequences from 16 ticks were identical (GenBank accession no: PQ633344 - PQ633346). The ompA sequences from 15 of the 16 ticks were identical, whereas the last tick had a single nucleotide difference (C/T) (GenBank accession no: PQ633343 and PQ633347). These Rickettsia spp. positive samples appear to belong to the same Rickettsia genotype, which we describe herein as Rickettsia sp. ME2023.

Phylogenetic analysis showed that the 4-gene sequence concatenated fragment of Rickettsia sp. ME2023 not only belonged to the SFG group, but also clustered with three pathogenic Rickettsia species: R. rickettsii and two California species (R. rickettsii subspecies californica and Candidatus Rickettsia lanei) (Fig. 1). It formed a sister clade with Candidatus Rickettsia lanei. When comparing Rickettsia sp. ME2023 with Candidatus Rickettsia lanei, the nucleotide sequence identities were 99.7%, 98.8%, 99.2%, and 96.4% for the gltA, ompA, ompB, and sca4 genes, respectively (Table 2). The sequence identity of Rickettsia sp. ME2023 was more distant from R. rickettsii subspecies californica and R. rickettsii.

In this paper, we found a new spotted fever group Rickettsia genotype (Rickettsia sp. ME2023) in H. leporispalustris from Maine, USA. According to the sequence identity cutoff value guideline (Eremeeva et al., 2018; Fournier et al., 2003), Rickettsia sp. ME2023 may be classified as a new Rickettsia species or subspecies with support of additional genetic or phenotypic characteristics (Parola et al., 2005; Zhu et al., 2005). Recently, Paddock et al. combined phenotypic, ecological, and clinical data with whole-genome sequencing of 11 contemporary isolates and identified Rickettsia 364D as R. rickettsii subspecies californica (Paddock et al., 2024). The taxonomic designation of Rickettsia sp. ME2023 may also need an integrative taxonomic approach.

It has been demonstrated that larvae, nymphs, and adults of H. leporispalustris are able to acquire and maintain R. rickettsii infection by transstadial and transovarial transmissions within the tick population, with active transmission of the bacterium to rabbits by all parasitic stages (Freitas et al., 2009). Rickettsia sp. ME2023 may also be transmitted both transovarially and transstadially since it was found in questing larvae, nymphs and adults of H. leporispalustris. We did not find Rickettsia sp. ME2023 in I. scapularis, I. cookei, I. dentatus, I. marxi or D. variabilis ticks. However, it is worth looking for this Rickettsia in other tick species that feed on the same hosts as H. leporispalustris. Finding this Rickettsia genotype in five Maine counties indicated that this genotype is widely distributed in Maine and may also be distributed in the greater New England region where H. leporispalustris is endemic.

The newly discovered Rickettsia genotype is likely pathogenic. It is grouped together with three known pathogens: widely distributed R. rickettsii and two California species (R. rickettsii subspecies californica and Candidatus Rickettsia lanei). Further research is needed to clarify the possible human disease incidence, clinical manifestations, and epidemiology of this putative pathogen. Rickettsia rickettsii subspecies californica infection is associated with milder disease than RMSF and presents with a localized eschar (necrotic wound) but without a petechial rash (Padgett et al., 2016). Candidatus Rickettsia lanei is a rarely encountered pathogen in California and no typical rash was observed in the first human case in 2023 (Probert et al., 2024). Interestingly, a 65-year-old Maine resident tested positive for SFG-reactive IgG antibodies in 2020 but lacked the typical rash progression from wrists/ankles to trunk (Ray, 2020). Ray (2020) suspected that other less-severe SFG diseases with cross-reactive antibodies are possible causative agents. Whether this genotype is linked to probable or less-severe SFG diseases in Maine and New England warrants further investigation.

The identification of Rickettsia sp. ME2023 in the rabbit tick raises attention to the potential role for H. leporispalustris to serve as a vector for transmission or maintenance of rickettsial agents. The CG group rickettsiae: R. canadensis was first detected in H. leporispalustris from Canada (McKiel et al., 1967) and later was proven to be an established, ecologically stable Rickettsia species in North America (Philip et al., 1982). Burgdorfer noted the potential importance of this three-host species with broad distribution from Alaska to Argentina, remarking that “it is the only species of tick that occurs in all parts of North America where spotted fever has been recognized in man.” (Burgdorfer, 1969). The R. rickettsii isolates from H. leporispalustris in the United States are of low virulence for guinea pigs and serologically differ from isolates obtained from human patients (Parker et al., 1952; Parker et al., 1951). However, Fuentes et al. suggested that H. leporispalustris plays a role as an effective reservoir for maintenance of R. rickettsii in Costa Rica because they obtained two high virulent isolates from H. leporispalustris collected from the common tapeti Sylvilagus brasiliensis. These isolates were physiologically and serologically indistinguishable from human strains (Fuentes et al., 1985). Haemaphysalis leporispalustris has not been frequently reported to bite humans (Eisen, 2022). It tends to drop off hares and rabbits during the day when these hosts spend most of their time in their resting places (Burgdorfer, 1969). For these two reasons, this tick is usually not considered the primary vector for human rickettsioses agents in the United States. However, the recent finding of Candidatus Rickettsia lanei as the cause of severe RMSF-like illnesses in two patients suggests that these patients might have encountered Rickettsia infected H. leporispalustris (Eremeeva et al., 2018; Probert et al., 2024).

In New England, Cape Cod and the offshore islands were identified as a hotspot for RMSF as early as 1937. Hazard et al. studied 13 RMSF cases reported in this area, highlighting the region as an important endemic area (Hazard et al., 1969). Rocky Mountain spotted fever incidence now has significantly decreased in New England compared to historical trends (Chapman et al., 2006; Treadwell et al., 2000). During 2008–2012, 63% of reported SFG rickettsiosis cases originated from five states: Arkansas, Missouri, North Carolina, Oklahoma, and Tennessee (Biggs et al., 2016). Vector competition may play a role in the RMSF decrease in New England. For example, the increase in I. scapularis populations might have affected the distribution of D. variabilis, the primary vector for the RMSF agent. These two tick species show distinct host preferences: immature stages of I. scapularis primarily feed on white-footed mice (Peromyscus leucopus), with adults preferring white-tailed deer (Odocoileus virginianus), while D. variabilis immature stages feed almost exclusively on rodents, with adults targeting dogs and medium-sized mammals (Sonenshine, 2018). How H. leporispalustris will compete with these ticks is not clear, although it is a common tick species in Cape Cod (Snow et al., 2023). It is also worth noting that while D. variabilis is historically identified as the major vector for the RMSF agent in New England, none of the rickettsial strains isolated from Massachusetts populations of D. variabilis in past half century have been identified as R. rickettsii (Keirans, 1977; St John et al., 2016). The tick vectors and rickettsial species associated with SFG rickettsioses in New England and the effect of H. leporispalustris in the pathogen enzootic cycles and transmission also warrant further investigation.

The CDC RRi6 assay was designed specifically to detect R. rickettsii by targeting the RRi6 gene that is absent in other rickettsial genomes including R. rickettsii subspecies californica (Denison et al., 2014; Kato et al., 2013). The RRi6 gene was initially believed to be unique to R. rickettsii, leading to the development of an RRi6-based assay for R. rickettsii-specific detection (Denison et al., 2014). Homologues of this gene may, however, exist in other Rickettsia species. This has resulted in the potential misidentification of Candidatus Rickettsia lanei as R. rickettsii when using the RRi6 assay (Probert et al., 2024). Rickettsia sp. ME2023 and Candidatus Rickettsia lanei may have homologues of the RRi6 gene. The reverse primer and probe sequences of the newly developed Candidatus Rickettsia lanei-specific Taqman real-time PCR assay by Probert 2024 are identical to the sequence found in Rickettsia sp. ME2023. As a result, the assay may not be able to distinguish between Candidatus Rickettsia lanei and Rickettsia sp. ME2023, despite a two-nucleotide difference within the forward primer. DNA sequences of the ompA and ompB genes can provide unique distinguishing characteristics for Rickettsia sp. ME2023 and closely related Rickettsia species.

We report a new spotted fever group Rickettsia genotype (Rickettsia sp. ME2023) found in questing H. leporispalustris from Maine, USA. Multilocus phylogenetic evidence strongly supports the monophyly of this genotype with the pathogenic spotted fever group rickettsiae, including Candidatus Rickettsia lanei. This discovery expands our understanding of the diversity and distribution of rickettsial species in the northeastern United States. Future work is needed to understand the role of H. leporispalustris as a vector for enzootic maintenance and transmission of this putative pathogen. While its genetic relatedness to known pathogenic species points to its potential clinical relevance, further epidemiological investigation is warranted to evaluate its public health risk.