Ticks transmit the greatest variety of arthropod-borne pathogens that cause the most cases of vector-borne disease in the United States and Northern Hemisphere, second only to mosquitoes worldwide (Socolovschi et al., 2009; Eisen et al., 2017; Sonenshine and Macaluso, 2017; MacDonald, 2018; Swei et al., 2020). California is home to several medically important ticks including the western black-legged tick, Ixodes pacificus (Eisen et al., 2017; Furman and Loomis, 1984), the primary vector of pathogens causing Lyme borreliosis, human anaplasmosis, and Borrelia miyamotoi disease in the western United States. While nymphal and adult I. pacificus ticks transmit pathogens, the nymphs are thought to account for more disease transmission to humans and other vertebrates due to their smaller size and their peak questing activities coinciding with increased outdoor activity by people (Clover and Lane, 1995; Padgett and Bonilla 2011). The nymphal stage of I. pacificus has also been shown to have a higher burden of certain pathogenic bacteria, such as Borrelia burgdorferi, than the adult stage (Clover and Lane, 1995; Lane and Quistad, 1998; Kuo et al., 2000).

I. pacificus is established in at least 55 of California’s 58 counties and is commonly found in dense woodlands with a moist microclimate (Eisen et al., 2006, 2016; MacDonald, 2018). Moreover, multiple biotic and abiotic factors have determined contemporary and likely future suitable habitats for this tick (Hahn et al., 2016, 2021; Eisen et al., 2018). Seasonal variability in temperature, relative humidity, and precipitation may lead to increases in suitability over time (Hahn et al., 2016, 2021; Eisen et al., 2018). Temperature and relative humidity are important factors influencing tick density and may also influence pathogen infection in I. pacificus (Eisen et al., 2018, 2006, 2016).

Globally, 20 species of Rickettsia are known human pathogens, 16 of which are transmitted by ticks (Centers for Disease Control and Prevention [CDC], 2019). There are more tick-associated rickettsiae with undetermined pathogenicity being discovered episodically (Parola et al., 2013). Although the identities of other bacterial pathogens I. pacificus harbors have been well researched, studies on Rickettsia sp. carried by this tick are limited (Lane et al., 1981a; Lane et al., 1981b; Philip et al., 1981; Parola et al., 2013; Xu et al., 2019; Salkeld et al., 2021). For example, Rickettsia tillamookensis was first described from I. pacificus in 1976 and although shown to be mildly virulent in guinea pigs and deadly to mice, it remained unstudied until recently (Hughes et al., 1976; Gauthier et al., 2021; Paddock et al., 2022). R. tillamookensis was originally a member of the spotted fever group, but current molecular and phylogenetic analyses placed this species in a transitional group related to R. asembonensis and R. felis strain Pedreira, two species carried by cat fleas (Hughes et al., 1976; Gauthier et al., 2021; Maina et al., 2016, 2019).

In a 2011 study, Rickettsia species phylotype G022 (hereinafter, G022) and Rickettsia monacensis str. Humboldt (also known as Rickettsia species phylotype G021, abbreviated as strain Humboldt to differentiate it from the IrR/Munich strain of R. monacensis found in Europe) were first identified from adult I. pacificus ticks collected in California (Phan et al., 2011; Simser et al., 2002). In a follow up study, 2 % of adult I. pacificus tested positive for G022 and 100 % were found infected with strain Humboldt (Cheng et al., 2013a). Further studies showed transovarial and transstadial transmission of strain Humboldt was 100 %, and it possessed the ability to synthesize folate de novo, supporting the hypothesis that strain Humboldt is an endosymbiont (Cheng et al., 2013a, 2013b; Hunter et al., 2015). Preliminary phylogenetics placed G022 as a novel spotted fever group Rickettsia, with its capability to impact human health currently unknown (Phan et al., 2011).

Here, we examined the prevalence of G022 in I. pacificus nymphs and adults from multiple locations in California. This study represents the first investigation of G022 in I. pacificus nymphs. Additionally, nymphs were tested for R. tillamookensis. The prevalence of infection between and among collection sites were compared to determine if there were significant differences based on habitat type and climatic factors.

Rickettsia species phylotype G022 and R. tillamookensis-infected I. pacificus nymphs were detected in multiple Northern California counties at an overall prevalence of 5.3 % and 1.6 %, respectively. G022-positive nymphs were detected in all but one county (Santa Cruz) while R. tillamookensis was detected in nymphs collected and tested from four counties. Comparison of G022 prevalence between adult and nymphal I. pacificus revealed that nymphs were less likely than adults of being infected with G022. The prevalence of G022 in adults vs nymphs varied significantly between Humboldt County (6.5 % in adults, 2.4 % in nymphs) and Contra Costa County (11.7 % in adults, 0.9 % in nymphs), compared to Napa County, where the prevalence was similar for both adults (12.9 %) and nymphs (12.3 %). This highlights distinct prevalence patterns between adult and nymphal stages in different collection areas.

G022 was originally identified in 2011 from I. pacificus adult ticks collected from the Napa Valley (Phan et al., 2011). Adult ticks in the current study were collected from counties that had G022 positive ticks in previous studies to increase the likelihood of finding additional positive ticks. Due to travel restrictions during the Covid-19 pandemic, most I. pacificus adults were collected from redwood habitat at the Green Diamond Resource Co. in Humboldt County. The calculated prevalence of G022 in adults in this study was 9.0 % (95 % CI = 7.2 %–11.3 %) overall, higher than the previous study’s 2 % (Cheng et al., 2013a) and similar to the prevalence of other well-known TBPs (Salkeld et al., 2021; Eshoo et al., 2015; MacDonald et al., 2017; Saunders and Kjemtrup, 2022; Holden et al., 2003; Lane et al., 2007, 2010; Sambado et al., 2020). The variation in G022 prevalence among adult ticks observed between this study and previous studies may be attributed to differences in collection locations and the larger number of ticks sampled from a single county. Humboldt County, with a prevalence of 3.2 % in adult I. pacificus, recorded the second-highest G022 prevalence among the seven counties surveyed, compared to the average prevalence of 2.0 % reported in 2013 (Cheng et al., 2013a).

Analyzing adult prevalence data by county, habitat, mean annual temperature, and mean annual relative humidity yielded no statistically significant correlations. Most specimens were collected either on walking trails or adjacent to them, so the chance of human encounters is high. Dense oak woodlands pose a higher risk for I. pacificus encounters in Southern and Northern California (MacDonald et al., 2017; Lane et al., 2007; Eisen et al., 2003, 2009, 2010). Apart from being present in oak woodland habitat, coastal chaparral was found to be another habitat for potentially acquiring tick-borne disease agents (Salkeld et al., 2021).

The prevalence of R. tillamookensis was 1.6 % (95 % CI = 0.8 %–3.3 %) in nymphal I. pacificus, which is similar to results of a recent study (Paddock et al., 2022). It is unsurprising that we detected G022 in I. pacificus nymphs since many rickettsiae are passed transstadially from one parasitic life stage to the next (Azad et al., 1998; Macaluso et al., 2002; Bonnet et al., 2017). Detecting G022 in questing nymphs indicates transstadial transmission from the larval stage. The overall prevalence was a modest 5.3 %, and all infected nymphs were derived from coastal oak woodlands. On the other hand, some significant differences were detected when the prevalence of G022 was compared among counties and mean annual temperatures. These findings could have future ramifications as climatic changes may reduce infection rates in certain areas while increasing them in others as habitat suitability shifts (Eisen et al., 2018, 2016; Padgett and Lane, 2001).

Analyses of mean annual temperature and mean annual relative humidity did not reveal significant associations between G022 prevalence and adult I. pacificus. There was a statistically significant association between the prevalence of G022 in I. pacificus nymphs and mean annual temperature, but R. tillamookensis infection was not correlated with either variable. The 11.7–13.3 °C (53–56°F) zone had an increased likelihood of having G022 positive nymphs than the 13.9–15 °C (57–59°F) zone. These ranges coincide with some inland areas north of San Francisco and the San Francisco Bay Area, respectively.

Since ticks spend more than 90 % of their life cycles off-host, environmental conditions impact their development and survival. In the San Francisco Bay Area, for example, I. pacificus females oviposited at temperatures ranging between 6.5 °C and 29 °C, whereas egg development proceeded within a narrower range spanning 9 to 25 °C and hatching success was lower in exposed sites versus sheltered plots (Peavey and Lane, 1996).

One reason why there was a significant association between G022 in I. pacificus nymphs and temperature that was not seen in adults or with R. tillamookensis could be due to the climate sections map overlay that was used instead of recording exact microclimatic temperature measurements during each collection trip. While the initial temperature was taken on all collection trips, it was not as precise a method as previous studies that measured temperature at the soil surface (Lane et al., 2007; Padgett and Lane, 2001; Peavey and Lane, 1996). Moreover, relative humidity was not logged on field trips. Adopting the use of temperature and relative humidity data loggers (Lane et al., 2007) to track diurnal changes during collections would increase accuracy, thereby elucidating how microclimatic conditions may influence pathogen prevalence in I. pacificus.

Our study found that nymphal I. pacificus can be infected with G022, albeit at a lower prevalence than adults (nymphs: 5.3 %). Moreover, the detection of G022 in questing nymphs and adult ticks suggests that it can be passed transstadially by I. pacificus larvae and nymphs. The prevalence of R. tillamookensis in nymphal I. pacificus is consistent with previous findings (1.6 %). The low prevalence of G022 and R. tillamookensis among adults and nymphs suggests horizontal transmission occurs between ticks and their vertebrate hosts for these Rickettsia species. Despite the high sensitivity of qPCR, false negatives were possible if the DNA concentration in a given tick extract was too low. Further studies are needed to determine the vertebrate host and their pathogenicity to humans. This study also preliminarily investigated whether two climatic variables, mean annual temperature and mean annual relative humidity, may influence the prevalence of the Rickettsia species in I. pacificus. The only significant outcome was G022 infected nymphs had a higher likelihood of being in the 11.7–13.3 °C zone than 13.9–15 °C, which merits further study. Of six counties surveilled, Napa County had the highest prevalence of G022 positive nymphs, whereas Humboldt County had the highest prevalence of R. tillamookensis-positive nymphs. A potential limitation of this study was that it may have been underpowered due to the relatively low numbers of ticks collected from the various locations.