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Published in final edited form as: Ticks Tick Borne Dis. 2020 Apr 6;11(4):101422. doi: 10.1016/j.ttbdis.2020.101422

Rickettsia and Anaplasma species in Dermacentor andersoni ticks from Washington

Lily Francis a, Christopher D Paddock a, Elizabeth A Dykstra b, Sandor E Karpathy a,*
PMCID: PMC7430063  NIHMSID: NIHMS1617300  PMID: 32273163

Abstract

Dermacentor andersoni, the Rocky Mountain wood tick, occurs predominantly in the northwestern United States and southwestern Canada. There are relatively few contemporary data to evaluate the occurrence of Rickettsia and Anaplasma species in D. andersoni in western North America, and even less information about these associations in the state of Washington, where this tick species is widely distributed and often bites humans. We used PCR assays to detect DNA of Rickettsia and Anaplasmataceae bacteria in 203 adult D. andersoni ticks collected from 17 sites in 9 counties of Washington between May 2012 and May 2015. Of these, 56 (27.6 %) were infected with a Rickettsia species and 3 (5.4 %) with a member of the Anaplasmataceae family. Rickettsia peacockii, R. bellii and R. rhipicephali were found in 17.7 %, 4.9 %, and 4.4 % of the Rickettsia positive ticks, respectively. Coinfections of R. bellii with R. peacockii or R. rhipicephali were identified in 6 ticks. Of the Anaplasmataceae-positive ticks, one was identified as being infected with Anaplasma phagocytophilum AP-Variant 1. No ticks were infected with a recognized human or animal pathogen, including R. rickettsii, A. phagocytophilum-ha, A. bovis, orA. marginale.

Keywords: Dermacentor andersoni, Rickettsia, Anaplasma phagocytophilum

1. Introduction

Dermacentor andersoni, the Rocky Mountain wood tick, is found predominantly in the northwestern United States and southwestern Canada (Chapman et al., 2006). Nymphal and larval D. andersoni feed primarily on small mammals and adult ticks feed primarily on larger animals such as deer, cattle, horses, elk, and occasionally, humans (Eisen, 2007; James et al., 2006). Dermacentor andersoni is a historically recognized vector of Rickettsia rickettsii, the agent of Rocky Mountain spotted fever (RMSF). More recently, various other rickettsial agents have been identified in D. andersoni ticks in the western United States and Canada, including R. peacockii, R. bellii, and R. rhipicephali, none of which have caused known disease in humans (Parola et al., 2013). Additionally, Anaplasma bovis and A. marginale, which are of unknown pathogenicity to humans but cause disease in cattle and other wild ruminants, have been identified in D. andersoni (Rar and Golovljova, 2011).

There are relatively few contemporary data to evaluate rickettsial agents in D. andersoni in western North America and even less information about Rickettsia and Anaplasma species associated with D. andersoni in Washington state, where this tick species is distributed broadly and frequently bites humans (Dergousoff et al., 2009; Dworkin et al., 1999; Niebylski et al., 1997; Philip et al., 1983). Previous work by Gall et al. has highlighted the importance of examining ticks collected from different geographic sites as the bacterial microbiome composition was shown to be affected by the geographic origin of D. andersoni ticks (Gall et al., 2016, 2017). Thus, for this study a survey of Rickettsia and Anaplasma-infected D. andersoni from multiple sites in Washington was conducted to provide information about potential tick-borne pathogens in those areas which can better inform healthcare providers and local residents of some of the risks associated with bites of D. andersoni in this region.

2. Methods

2.1. Sample collection

Two hundred and three adult-stage D. andersoni ticks were collected from 17 sites in 9 counties (Benton, Chelan, Ferry, Klickitat, Kittitas, Lincoln, Okanogan, Spokane, and Yakima) of Washington between May 2012 and May 2015 and were identified using a standard taxonomic key (Brinton et al., 1965). The ticks were collected by several methods, including, dragging, flagging, and direct removal from the clothing of collectors while flagging or dragging; of these, 105 were female and 98 were male (Table 1). The ticks were stored in 70 % ethanol until DNA was extracted.

Table 1.

Collection location and source of Dermacentor andersoni ticks collected in Washington and evaluated by PCR for Rickettsia and Anaplasma species.

County Collection Site Source No. Tested
Benton 46.22374, − 119.28775 Host check 1
Chelan 47.56564, − 120.78279 Hand collection/host check 2
Ferry 48.60055, − 118.62843 Drag/flag 6
Klickitat 45.69707, − 121.29003 Drag/flag 2
Kittitas 46.9965, − 120.5478 Hand collection 1
Lincoln 47.7585, − 118.5197 Host check 52
47.29700, − 120.75960 Host check 1
Okanogan 48.9391, − 119.4356 Hand collection 1
48.7052, − 119.4395 Host check 7
Spokane 47.40705, − 117.58925 Drag 5
47.4874, − 117.5758 Host check 3
47.7443, − 117.0688 Hand collection 1
47.60825, − 117.05212 Host check 1
47.77628, − 117.5468 Hand collection 1
47.41386, − 117.59376 Drag/hand collection 115
Yakima 46.91500, − 121.05230 Host check 1
46.5351, − 120.8673 Host check 1
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2.2. DNA extraction and molecular evaluation

Ticks were minced individually using sterile scalpels and DNA was extracted using QIAGEN DNeasy Blood and Tissue Kit (QIAGEN, Valencia, California) following the manufacturer’s recommendations. Samples were eluted in a final volume of 100 μL and stored at 4 °C until polymerase chain reaction (PCR) analyses were performed. Samples were screened using a pan-Rickettsia real-time PCR assay (Kato et al., 2013). Samples were screened in duplicate and a negative control consisting of sterile water in place of template DNA and a positive control consisting of R. conorii DNA were included on every plate. Samples with a Cq < 40 were considered positive. Positive samples were tested for DNA of spotted fever group Rickettsia species using a semi-nested ompA assay (Eremeeva et al., 2006). Amplicons of the expected size (532 bp) were sequenced in both directions on an Applied Biosystems 3500 Genetic Analyzer and the reads were assembled using Geneious v10.2.2. All pan-Rickettsia positive samples were also screened using a R. bellii-specific real-time PCR assay in which both the primer pair and the probe were designed to be highly specific for R. bellii (Hecht et al., 2016; Szabo et al., 2013). Samples with a Cq < 40 were considered positive for R. bellii infection. DNA samples were also tested by using a real-time pan-Anaplasmataceae assay targeting the 16S rRNA gene (Eremeeva et al., 2007; Li et al., 2002). Amplicons (146–154 bp) were gel purified, sequenced, and analyzed as previously described to determine the bacterial species.

3. Results

Of the 203 ticks screened using the pan-Rickettsia and Anaplasmataceae assays, 56 (27.6 %) contained DNA of a Rickettsia sp. and three (5.4 %) contained DNA of a member of the Anaplasmataceae family (Table 2). Rickettsia peacockii infections were the most prevalent at 17.7 % (n = 36); sequences were 98.4 %–100 % identical to R.peacockii strain 5 (GenBank KJ675444). Rickettsia bellii was the next most prevalent rickettsial species identified, with 4.9 % (n = 10) of the ticks positive for DNA from this species. Rickettsia rhipicephali was identified in 4.4 % (n = 9) of the tick samples; all nine sequences were identical to each other and were 99.4 % identical to strain Do276 (GenBank EU109175). The AP-Variant 1 genotype of A. phagocytophilum (100 % identical to AP-Variant 1 isolate CRT38; GenBank APHI000002.1) was identified in 1 tick (0.5 %) while 2 ticks (0.9 %) were infected with undetermined Anaplasmataceae. PCR analysis revealed 10 ticks positive for R. bellii; six of these were co-infections with either R. peacockii (5) or R. rhipicephali (1). Seven of the Rickettsia-infected samples and two of the Anaplasmataceae-infected samples had high Cq values and the DNA concentrations were too low to sequence for species confirmation.

Table 2.

Results of PCR analysis for the detection of Rickettsia or Anaplasmataceae in 203 D. andersoni.

Agent No. (% of 203) No. Male (% of 98) No. Female (% of 105)
Rickettsia spp. 56 (27.6 %) 24 (24.5 %) 32 (30.5 %)
R. peacockii only 31 (15.3 %) 12 (12.2 %) 19 (18.1 %)
R. peacockii and R. bellii 5 (2.5 %) 3 (3.1 %) 2 (1.9 %)
R. rhipicephali only 8 (3.9 %) 2 (2.0 %) 6 (5.7 %)
R. rhipicephali and R. bellii 1 (0.5 %) 1 (1.0 %) 0 (0.00 %)
R. bellii only 4 (2.0 %) 3 (3.1 %) 1 (1.0 %)
Anaplasmataceae 3 (5.4%) 3 (3.1 %) 0 (0.00 %)
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Each of the Anaplasmataceae-infected ticks were collected at different sites in Chelan, Lincoln, and Spokane counties, while a majority of the Rickettsia-infected ticks (34 ticks, 60.7 %) were collected from two sites in Lincoln County.

4. Discussion

We identified three distinct Rickettsia species and an A. phagocytophilum variant among approximately 200 questing adult D. andersoni collected from 9 counties of Washington. Among these, R. peacockii was the most commonly identified agent. To our knowledge, we identified for the first time co-infections of D. andersoni comprising R. bellii and R. peacockii, and those comprising R. bellii and R. rhipicephali from Washington. Although uncommon, co-infections (especially those involving R. bellii) with multiple rickettsial species have been previously reported (Gall et al., 2017; Hecht et al., 2019; Paddock et al., 2018; Wikswo et al., 2008; Wright et al., 2015). We also identified a variant of A. phagocytophilum in D. andersoni, which has not been reported previously. Nonetheless, we did not detect any recognized human or animal pathogens described previously in D. andersoni, including R. rickettsii, A. marginale, or A. bovis

Rickettsia peacockii was originally identified in D. andersoni in Western Montana where 66.1 % of the collected ticks were infected (Niebylski et al., 1997). The high prevalence of R. peacockii in Western Montana and the provinces of Alberta and Saskatchewan (76 %) in Canada is similar to the relatively high percentage of D. andersoni infected with R. peacockii in Washington (Dergousoff et al., 2009). There is some evidence that indicates rickettsial infection within a vector inhibits transmission of a second Rickettsia (Macaluso et al., 2002). This has been described specifically in D. andersoni infected with R. peacockii inhibiting the transmission of R. rickettsii (Burgdorfer et al., 1981). It is possible that the relatively high prevalence of R. peacockii could be responsible for the absence of R. rickettsii from the sample set, but analysis of progeny of R. peacockii-infected ticks is necessary to confirm this hypothesis (Hayes and Burgdorfer, 1989).

The AP-Variant 1 genotype of A. phagocytophilum was identified; however, this variant is not believed to infect humans (Massung et al., 2006). Dermacentor andersoni is a known vector for A. bovis and A. marginale (Rar and Golovljova, 2011). Anaplasma marginale has been detected in infected calves from the same county as one of the sites where samples were collected in Washington (Palmer et al., 1989). Additionally, although no tick vector was identified, A. phagocytophilum has been identified in canines in Washington (Poitout et al., 2005).

Because only one tick was collected from many sites, data may be non-representative of these sites, county, or state as a whole. This precludes any estimation of frequency based on the findings of this study. However, these preliminary data suggest that rickettsial pathogens are infrequent among D. andersoni ticks encountered in Washington. While other researchers have found high frequency of rickettsial pathogens in D. andersoni in the western United States, ticks have specific ecological needs that determine their distribution (Niebylski et al., 1997). In a study from Bitterroot Valley, Montana, it was found that D. andersoni on one side of the valley were not infected with R. peacockii while a majority of D. andersoni collected from the other side of the valley were infected (Philip and Casper, 1981). These isolated populations seemed not to interact or transmit pathogens between each other. Additionally, animals may serve both as blood meals for the ticks and as reservoirs for the bacterial pathogens. Thus, the presence or absence of specific animal species may also play a role in determining the presence or absence of a particular rickettsial agent in the tick population (Burgdorfer et al., 1966; Eremeeva and Dasch, 2015; Parola et al., 2005). Because of trends like this, the small sample size and convenience sampling in this study are a limiting factor in defining Rickettsia and Anaplasma in Washington’s D. andersoni populations.

5. Conclusion

A total of 203 adult D. andersoni ticks from Washington were analyzed for the presence of rickettsial disease. The most prevalent Rickettsia species was R. peacockii, a non-pathogenic bacterium that could inhibit the transmission of the etiological agent of RMSF, R. rickettsii. Other Rickettsia species found in the samples included R. rhipicephali and R. bellii, although neither have been definitively linked with disease in humans. One tick was infected with a non-pathogenic variant of A. phagocytophilum. These results suggest that the frequency of medically important rickettsial pathogens in D. andersoni ticks in Washington state is low.

Acknowledgments

The findings and conclusions in this article are those of the authors and do not necessarily represent the official position of the Centers for Disease Control and Prevention.

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