Diversity of Ticks and Rickettsiae in the Southwestern United States: Implications for Public Health

Introduction

With increasing numbers of cases of tickborne diseases (Bishop et al., 2022; Eisen and Paddock, 2021), there is a pressing need for surveillance of ticks and the pathogens they transmit. The southwestern U.S. presents an ideal yet challenging landscape for studying tickborne diseases due to abundant tick species and diversity in ecologies. Important ticks in the region include Ixodes pacificus (I. pacificus), Dermacentor similis (D. similis), Rhipicephalus sanguineus (R. sanguineus), Amblyomma maculatum (A. maculatum), and Dermacentor occidentalis (D. occidentalis) (Hecht et al., 2020; Furman and Loomis, 1984).

Each of these ticks can vector gram-negative bacteria in the family Rickettsiaceae. Rickettsiae were some of the first vector-borne diseases discovered (Parola et al., 2005). The genus Rickettsia (R) consists of the spotted fever group (SFG), typhus group (TG), ancestral, and transitional groups of species (Merhej and Raoult, 2011). At least six zoonotic Rickettsia species have been identified along the U.S.–Mexico border, including SFG Rickettsia 364D (“R. phillipii”) transmitted by D. occidentalis, Rickettsia parkeri in A. maculatum, Rickettsia massiliae (R. massiliae) and Rickettsia rickettsia (R. rickettsia) in R. sanguineus (López-Pérez et al., 2021), and transitional group Rickettsia felis (R. felis) disseminated by fleas (Reif and Macaluso, 2009). Rocky Mountain spotted Fever (RMSF) caused by Rickettsia rickettsii (R. rickettsii) is a particularly dangerous threat to human health and is found across North, Central, and South America (Foley et al., 2024a). Before antibiotics were available, the mortality rate for RMSF was 23%, but even now, heavily impacted areas with marginalized populations experience case fatalities as high as 50% (Foley et al., 2024a).

There is a lack of knowledge about Rickettsia-caused disease incidence, geographic distribution, and contributing factors in the southwestern U.S., prompting targeted research and surveillance. In response, the Border Tick Rickettsia Surveillance Program (BiTeRS) was established as a collaboration among the Universities of California and Arizona and stakeholders in California and Arizona. This partnership offered tick collection and identification, pathogen testing, and training and provision of educational resources. The primary focus of the study was to enhance the detection of R. rickettsii to improve RMSF surveillance, with a secondary goal to identify other pathogenic rickettsial species in the region. This study documents tick species obtained during BiTeRS surveillance during 2021–2022, the prevalence of rickettsiae, temporal, spatial, and host characteristics associated with the submissions.

Materials and Methods

Results

From 2021 to 2022, we assessed 913 ticks from Arizona and California from kennels, parks, people, and other animals provided by 17 POs. Figure 1 employs graduated symbols to represent tick samples’ geographic distribution and count. We observed notable differences between the two states’ annual tick collection numbers. In 2021, Arizona contributed 309 (53.00% of the total ticks collected in Arizona) ticks, while California accounted for 182 (55.15% of California’s total). The following year saw a minor shift in these numbers, with Arizona’s contribution decreasing to 274 ticks (47.00% of Arizona’s total) and California’s decreasing to 148 ticks (44.85% of California’s total).

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FIG. 1.

Map of counties in the southwestern U.S. participating in the 2021–2022 BiTeRS program, highlighting sampling sites and frequency of tick collections.

Ticks from Arizona included R. sanguineus (90.05%, 525 of 583) and Argas sp. (9.95%, 58 of 583), compared with California with D. similis (47.40%, 155 of 330), brown dog ticks (43.70%, 143 of 330), I. pacificus (7.88%, 26 of 330), O. megnini (0.61%, 2 of 330), and H. leporispalustris (0.30%, 1 of 330) (Table 1). Due to missing or damaged body parts, three ticks were not identified as species. Figure 2 is a barplot of the tick species found during each season.

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FIG. 2.

Counts of R. sanguineus, D. similis, Argas sp., H. leporispalustris, I. pacificus, and O. megnini ticks collected for the BiTeRS project in Arizona and California, 2021–2022. BiTeRS, border tick rickettsia surveillance; D. similis, Dermacentor similis; H. leporispalustris, Haemaphysalis leporispalustris; I. pacificus, Ixodes pacificus; O. megnini, Otobius megnini; R. sanguineus, Rickettsia sanguineus.

Forty (5.99%) of the R. sanguineus were PCR-positive for Rickettsia spp. (Table 1). The prevalence in 2021 was 8.66% compared with 4.09% in 2022. In Arizona, the prevalence was 5.71% similar to the 6.99% from California. More R. sanguineus (48.05%, 321 of 668) were collected from Navajo County than any other county, and these ticks had a 9.03% Rickettsia PCR-prevalence. R. sanguineus collections were seasonal, with none collected in winter and the highest number of ticks (65.12%, 435 of 668) with a PCR-prevalence of 6.67% in summer. Dogs were the host species for 98.35% (657 of 668) of R. sanguineus, with 5.94% PCR-positive. Ticks found feeding and attached constituted 98.80% (660 of 668) of the samples, with a Rickettsia spp. prevalence of 5.91%. Adult male ticks (48.65%, 325 of 668) and adult females (38.02%, 254 of 668) had a 4.92% and 8.66% prevalence, respectively, though the slightly elevated prevalence in females was not statistically significant ([odds ratio] OR: 1.84, 95% [confidence interval] CI: 0.95–3.64, p-value = 0.073).

Of the 155 adult D. similis collected, 18 (11.61%) were PCR-positive (Table 1). The majority (83.23%, 129 of 155) of these ticks were collected in 2021, with a Rickettsia spp. prevalence of 7.75%, compared with 16.77% (26 of 155) collected in 2022, with a higher prevalence of 30.77%. Almost the entire sample (99.35%, 154 of 155) was from San Diego County, with a PCR-prevalence of 11.69%. Most D. similis (77.42%, 120 of 155) were collected in summer. Seventy-one percent (70.97%, 110 of 155) were sourced from natural areas and had a pan-Rickettsia prevalence of 9.09% which was significantly lower compared to those ticks that were collected from dogs (OR: 0.31, 95% CI: 0.11–0.95, p-value = 0.034). Ticks collected from dogs accounted for 18.71% (29 of 155) of the samples with a 24.14% Rickettsia prevalence. About a quarter of the ticks, 24.52% (38 of 155), were attached and feeding, with a prevalence of 18.42% compared with 72.26% (112 of 155) not feeding, with a prevalence of 9.82%. These were not significantly different (OR: 0.48, 95% CI: 0.17–1.41, p-value = 0.165). Adult female ticks made up 87.10% (135 of 155) of the samples, with a prevalence of 10.37%, and had a lower pan-Rickettsia prevalence than adult males (prevalence of 20.00%), though this difference was not statistically significant (OR: 2.62, 95% CI: 0.67–8.63, p-value = 0.132). All D. similis that were submitted were adults.

A total of 26 western black-legged ticks were evaluated, 20 of which (76.92%) were positive for Rickettsia spp. Most (24/26, 92.31%) were collected in 2021, and only two (7.69%) in 2022. All were from San Diego County, California. Most (88.77%, 21 of 26) were collected during fall from a single individual dog host. Most I. pacificus (84.62%, 22 of 26) were adult females and the remaining four were adult males.

Among 58 Argas sp. ticks, all of which were collected from birds in Pima County, Arizona in spring and summer 2021, 24 (41.38%) were PCR-positive. All collected ticks were morphologically consistent larvae and were attached and fed on six avian host species. Comparison of a portion of the 16S rDNA gene to existing Genbank sequences showed the highest similarity to Argas giganteus (A. giganteus) also collected from raptors in Arizona (Genbank accession MK078102), but with only 86.6% identity and 100% coverage, the species could not be determined definitively. The majority of the Argas larvae were collected from western screech owls (27.59% of ticks collected, 16 of 58), Harris hawks (27.59%, 16 of 58), and mourning doves (17.24%, 10 of 48). PCR-prevalence was highest in the ticks collected from Cooper’s hawks (83.33%) and was 12.50% in ticks from the western screech owl and 31.25% in ticks from the Harris hawk.

We found only two spinose ears and one rabbit tick larvae, all from San Diego, California in 2021. Spinose ear ticks were found in winter from dogs, and rabbit ticks in summer from a natural area. All were PCR-negative for Rickettsia sp.

None of the 102 pan-Rickettsia PCR-positive samples yielded a positive result on R. rickettsii-specific PCR. Of the positive samples, DNA sequencing of the 17 kDa gene was attempted for 38 samples and successful for 30, while four were also sequenced successfully for the gltA gene. Phylogenetic trees can be seen for the 17 kDa gene in Figure 3 and gltA in Figure 4. The most commonly identified Rickettsia species in our collection was Rickettsia rhipicephali (R. rhipicephali): it was found in both R. sanguineus and D. similis from San Diego County (Table 2). Two additional D. similis from San Diego were found to be infected with a Rickettsial species that could not be differentiated from Rickettsia raoultii and Rickettsia montanensis via 17 kDa sequencing (Fig. 3). The Rickettsia species derived from I. pacificus matched closely with 17 kDa sequences from both R. monacensis (Rickettsia monacensis) and Rickettsia tamurae (R. tamurae) (99.02% similarity to both, multiple GenBank submissions). In Navajo County, R. massiliae was found in R. sanguineus. The gltA gene sequencing indicated that Argas sp. ticks from Pima County were infected with a rickettsial species most closely related to Candidatus Rickettsia tarasevichiae (R. tarasevichiae) (GenBank: MN450397, 97.79% identity) and had only 90% identity (70% coverage) to the Rickettsia hoogstraalii (R. hoogstraalii) previously identified in Argas spp. ticks collected from raptors in the same region (Genbank: MH717094).

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FIG. 3.

Phylogenetic tree based on comparison of 200 base pair portion of the 17 kDa gene of Rickettsia species showing relationship between samples collected as part of the BiTeRS program from 2021 to 2022 in Arizona and California (in bold) and reference samples collected from GenBank (included with accession numbers). Tree constructed using the maximum likelihood method and Tamura–Nei model in Mega 11. BiTeRS, border tick rickettsia surveillance.

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FIG. 4.

Phylogenetic tree based on comparison of an approximately 800 base pair portion of the gltA gene of Rickettsia species showing the relationship between samples collected as part of the BiTeRS program from 2021 to 2022 in Arizona and California (in bold) and reference samples collected from GenBank (included with accession numbers). Tree constructed using the maximum likelihood method and Tamura–Nei model in Mega 11. BiTeRS, border tick rickettsia surveillance program.

Discussion

Tickborne diseases pose an urgent public health concern, demanding rigorous tick surveillance to tackle this critical issue, particularly in the southwestern U.S. with its diverse tick population. In this study, through an opportunistic, partner-based approach, we identified six species of ticks carrying five species of Rickettsia, none of which were R. rickettsii, the species of greatest public health concern. Our study utilized a hybrid opportunistic and passive surveillance system, allowing us to tap into the benefits of working with partners and large-scale data collection via passive monitoring while incorporating active surveillance elements when partners had the motivation and resources to do so. Partners were offered resources to develop an active surveillance plan, the time, but differed in their ability to devote resources to this work. The approach, therefore, was to encourage tick collection as a corollary to activities in which the partner was already engaged, resulting in largely opportunistic sampling. Passive, opportunistic data gathering has shown significant success in large-scale tick and pathogen surveillance (Stromdahl et al., 2001; Barrett et al., 2015; Xu et al., 2016; Nieto et al., 2018) because it can help create large networks of stakeholders and provide more extensive geographic coverage with less requirement for data collection person-power. For example, New York state has a program of community-engaged tick surveillance which has fostered the development of an interactive mapping tool, tickMAP, to monitor tick emergence and tickborne pathogens (Hart et al., 2022). Programs like BiTeRS also serve to educate and remind stakeholders and the public about the risks of tick-borne diseases. However, passive, partner-engaged surveillance can be hampered by variables such as population density and the extent of community engagement and education (Cortinas and Spomer, 2014; Nelder et al., 2014) and relies heavily on the willingness and capacity of local communities to contribute. It also is difficult to manage the variability of the data. It would be very difficult with data such as ours to infer underlying tick density or other key attributes of the population’s risk. There were inherently self-selection biases for partners who decided to participate, and while data was not collected on partners’ decisions to participate, it is likely that entities with pre-existing concerns about ticks and tick-borne diseases were more likely to participate and submit ticks. While active surveillance arguably provides more precise estimates of pathogen prevalence (Nsubuga et al., 2011), its high cost and labor intensity often prevent large-scale application (Ogden et al., 2014; Centers for Disease Control and Prevention, 2008).

The reason R. rickettsii was not identified in any tick sample submissions is unlikely to represent total absence in the region and is very likely a product of where samples were submitted from and very low prevalence. Even in RMSF epidemic regions, the prevalence of R. sanguineus is typically less than 1% (Eremeeva et al., 2011)(Foley et al., 2019; Foley et al., 2024b) and while R. rickettsii may occur in Dermacentor ticks in California, it is found infrequently (Osborne et al., 2020). It may also represent an additional form of bias, where due to social or economic factors, detection and reporting of ticks is less likely to occur in the circumstances most likely to drive R. rickettsii transmission (Nicholson et al., 2006), and presents an additional limitation to the surveillance methods used here.

Even in the absence of R. rickettsii detection, however, several other important findings arose from this work. The study supports that there is no specific “tick season” in southern California, with ticks collected and submitted in every season. We received R. sanguineus and D. similis primarily during the fall and summer, and I. pacificus mainly in winter and fall. This pattern aligns with known seasonality for these ticks in the state. Dermacentor species are known to tolerate some aridity and elevated temperatures, remaining active into summer months in California, in contrast with I. pacificus which requires a tight, relatively cool, and high humidity environmental window (MacDonald, 2018). However, an important caveat to these inferences is that systematic prospective sampling was not done, limiting possible positive data points during seasons when partners were not undertaking surveillance. Moreover, there are too few data on juvenile ticks to allow for analysis of the seasonality of the different life stages. We also found the single H. leporispalustris, which rarely bites humans but is a competent vector for R. rickettsii (Freitas et al., 2009) and has been implicated in human RMSF in Costa Rica (Hun et al., 2008). O. megnini feeds primarily on livestock with many records of it feeding on other hosts as well (Keirans and Pound, 2003; Nava et al., 2009). The agents of Q-fever and RMSF have been associated with O. megnini in older literature (Bustamante et al., 1946; Jellison et al., 1948).

Of concern is the finding of R. sanguineus in the summer and fall, given suggestions that this species may demonstrate increased aggression towards humans at elevated temperatures (Parola et al., 2008; Backus et al., 2021). The data also show the widespread presence of R. sanguineus across multiple counties within the southwestern U.S., corroborating previous findings that have indicated a significant prevalence of this tick species along the U.S.–Mexico border, particularly in arid regions of the western border (Eremeeva and Dasch, 2015). However, none of the 945 R. sanguineus collected during this study tested positive for R. rickettsii, in contrast with other studies in northern Mexico (López-Pérez et al., 2021; Álvarez-Hernández et al., 2017; Zazueta et al., 2021). This may be due to the reduced presence of roaming dogs in the southwestern U.S. (Rowan and Kartal, 2018) compared to parts of northern Mexico (Tinoco-Gracia et al., 2009), which can amplify R. rickettsii and R. sanguineus (Demma et al., 2005; Nicholson et al., 2006; Drexler et al., 2014). It is possible that sampling closer to the U.S.–Mexico border could have resulted in a higher R. rickettsii infection prevalence in our samples (Eremeeva et al., 2011; Bustamente Moreno and Pon Méndez, 2010). This highlights the need for further research with larger sample sizes and uniform sampling methods across different geographical regions.

All of the Argas sp. ticks in this study came from a facility in Tucson that cares for wildlife, and elevated numbers of these ticks have been reported previously (Latas et al., 2020). However, both the tick species A. giganteus identified in that study differ markedly from what we identified here. Our research broadens our understanding of the relationship between bird species and the soft tick genus Argas sp. in Arizona, which has also been documented in California, New Mexico, Utah, and Sonora, Mexico (Cooley et al., 1944; Furman and Loomis, 1984). The ticks are often hosted by a number of neotropical bird species that migrate through or nest in the Sonoran Desert (eBird, 2024; Rappole et al., 2011). Some of the estimated 19 million neotropical ticks annually introduced to the U.S. via bird migration may contribute to expanding tick species’ geographical ranges, potentially exacerbated by climate change (Cohen et al., 2015; Sonenshine, 2018). Although our study identified ticks, particularly Cooper’s hawk, Harris’s hawk, and red-tailed hawk, the challenges of comprehensive tick sampling from migratory birds may mean that the actual diversity and distribution of Argas ticks in the area could be underrepresented. We also likely have only a biased sense of the tick’s geographical distribution given our limited sampling space.

The study found five species of rickettsiae in various tick species. Infections from SFG R. massiliae are milder compared to RMSF and typically not life-threatening (Biggs et al., 2016). R. massiliae is reported to be a pathogen of both humans and dogs (Beeler et al., 2011; Eremeeva et al., 2006). It is often reported from the Mediterranean (Beati and Raoult, 1993), and has been reported before in R. sanguineus from California (Beeler et al., 2011), Virginia (Fornadel et al., 2013), and Eastern Arizona (Eremeeva et al., 2006). In this dataset, only R. sanguineus from Arizona tested positive for R. massiliae. R. rhipicephali is not considered pathogenic but is closely related to R. massiliae, nesting within the same clade (Fig. 3) and was found in both Dermacentor and Rhipicephalus ticks and in both California and Arizona in this study.

A rickettsial species in I. pacificus in San Diego County most closely matched zoonotic R. tamurae and R. monacensis (Jado et al., 2007; Imaoka et al., 2011; Kim et al., 2017). R. monacensis has a broad range of vectors including Ixodes ricinus (I. ricinus) in Germany (Simser et al., 2002), Ixodes boliviensis in Costa Rica (Springer et al., 2018), and I. pacificus in northern California (Osborne et al., 2020). The rickettsiae in the Argus ticks are closely related to Candidatus R. tarasevichiae, which has been primarily detected in Ixodes persulcatus ticks from China and Russia (Shpynov et al., 2003; Li et al., 2016) and thought to be an emerging human pathogen. It falls within the Ancestral group in a clade with Rickettsia canadensis (Fig. 4). The species identified here differs markedly in the gltA sequence from the R. hoogstraalii found in A. giganteus on raptors in the region previously (Latas et al., 2020), and R. hoogstraalii has only 90% identity with Candidatus R. tarasevichiae, indicating a separate species found here. A closely related organism was found in I. pacificus ticks from a gray fox in Ensenada, Baja California (López-Pérez et al., 2021). Definitively identifying the Rickettsia species would require culture and further molecular assays.

It is clear that the full scope of tick-host associations in the American Southwest has not yet been revealed. Pressing concerns are tick and infection seasonality and that poorly studied Argas sp. ticks and their rickettsiae may be spread by neotropical birds. Regular monitoring of R. sanguineus is crucial. Further study of rickettsial and tick diversity could illuminate roles in disease dynamics and opportunities for management. Hybrid active, passive, sentinel, and community-based programs will enable us to detect early signs of disease spread, mitigate potential impacts, and protect public health more effectively.