No evidence of Bartonella infections in host-seeking Ixodes scapularis and I. pacificus ticks in the United States

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McClung, Lynn M. Osikowicz, Sarah Maes, Rebecca J. Eisen This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4492797/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 19 Aug, 2024 Read the published version in Parasites & Vectors → Version 1 posted 9 You are reading this latest preprint version Abstract Background Bartonella spp. infect a variety of vertebrates throughout the world with generally high prevalence. Several Bartonella spp. are known to cause diverse clinical manifestations in humans and have been recognized as emerging pathogens. These bacteria are mainly transmitted by blood sucking arthropods, such as fleas and lice. The role of ticks in the transmission of Bartonella sp. is unclear. Methods A recently developed quadruplex PCR amplicon next generation sequencing approach that targets Bartonella -specific fragments on gltA , ssrA , rpoB , and groEL was applied to test host-seeking Ixodes scapularis ticks (n = 1641; consisting of 886 nymphs and 755 adults) collected in 23 states of the eastern United States and Ixodes pacificus ticks (n = 966; all nymphs) collected in California in the western United States for the presence of Bartonella DNA. These species were selected because they are common human biters and serve as vectors of pathogens causing the greatest number of vector-borne diseases in the United States. Results No Bartonella DNA was detected in any of the ticks tested by any target. Conclusions Owing to the lack of Bartonella detection in a large number of host-seeking Ixodes sp. ticks tested across a broad geographic region, our results strongly suggest that I. scapularis and I. pacificus are unlikely to contribute more than minimally, if at all, to transmission of Bartonella spp.. Bartonella spp. Ixodes scapularis I. pacificus host-seeking next generation sequencing United States Background Bartonella is a diverse genus of gram-negative bacteria that are highly adapted to intracellular persistence in a wide range of vertebrates. Many mammalian species are natural reservoirs for Bartonella spp. and experience chronic, asymptomatic, intra-erythrocytic bacteremia when infected [ 1 ]. During the last two decades, over 40 species belonging to the genus Bartonella have been described from different mammalian hosts, with more than a dozen having been associated with diverse clinical manifestations of diseases in humans and recognized as emerging pathogens [ 2 – 9 ]. Many Bartonella spp. are host-specific, suggesting maintenance of individual species in independent enzootic cycles [ 10 ]. Bartonella spp. are typically vector-borne, transmitted between mammalian hosts by hematophagous arthropods. To date, several arthropods have been proven to be vectors for Bartonella spp. transmission. Sandflies ( Lutzomyia verrucarum ), human body lice ( Pediculus humanis corporis ), and cat fleas ( Ctenocephalides felis ) are well known vectors that transmit B. bacilliformis (Carrión’s disease) [ 11 ], B. quintana (trench fever) [ 12 , 13 ], and B. henselae (cat scratch disease) [ 14 ], respectively. Additionally, the Oriental rat flea ( Xenopsylla cheopis ) and other rodent fleas have been implicated as potential vectors transmitting rodent associated Bartonella spp., such as B. elizabethae , B. grahamii , and B. taylorii [ 15 , 16 ]. In the United States (US), the majority of reported vector-borne disease cases are caused by pathogens spread by ticks [ 17 ]. There has been considerable interest in ticks as potential vectors for Bartonella species. Numerous molecular surveys to detect Bartonella DNA in various tick species have been conducted from around the world in recent years [ 18 – 39 ]. Studies focused on host-seeking ticks are particularly intriguing because detection of Bartonella in unfed ticks would suggest the bacterium can survive the transstadial molt from larvae to nymphs or from nymphs to adults; demonstration of transstadial transmission is one component in demonstrating vector competency [ 40 ]. Results from these studies were discordant. Some studies reported detection of Bartonella DNA with high prevalence in host-seeking ticks. For example, Chang et al. [ 19 ] detected Bartonella quintana and several other Bartonella species by gltA in 19.2% of host-seeking Ixodes pacificus ticks in California, US; Adelson et al. [ 21 ] reported 34.5% of host-seeking Ixodes scapularis ticks collected in New Jersey, US were infected with Bartonella spp. using 16S primers; Morozova et al. [ 22 ] identified Bartonella henselae by groEL in as high as 44% of host-seeking Ixodes persulcatus ticks from Russia. By contrast, other studies reported very low prevalence of Bartonella in ticks. For example, only 0.9% of host-seeking I. scapularis ticks from Maryland, United States (US) [ 27 ] and 0.2% of host-seeking I. ricinus ticks from France [ 29 ], harbored Bartonella DNA; both studies were based on gltA results. Moreover, absence of Bartonella in host-seeking ticks was reported in studies from Hungary, Korea, and Finland based on results of gltA , groEL , or ssrA [ 25 , 28 , 41 ]. With these varied results, it has been debated whether ticks can serve as vectors of Bartonella spp.. In the US, the blacklegged tick ( Ixodes scapularis ) and the western blacklegged tick ( Ixodes pacificus ) are frequent human-biters in the eastern and far western US, respectively [ 42 ]. Both species serve as vectors of the Lyme disease spirochete, as well as several other human pathogens [ 43 – 45 ]. In this study, we tested host-seeking I. scapularis and I. pacificus nymphs and adults collected across 24 states for the presence of Bartonella DNA. Previous surveys mainly used traditional PCR with a single target [ 18 – 38 ]. Due to the genetic diversity of Bartonella spp., a single target may not be able to detect all species, and in some cases, single targets might not be adequately specific to accurately detect Bartonella , yielding false positive results. In our study, we applied a recently described quadruplex PCR amplicon next generation sequencing approach which amplifies and sequences four Bartonella -specific gene targets ( gltA , ssrA , rpoB , and groEL ) [ 46 ]. Using multiple target sequences increases detection success and also improves the accuracy of species identification compared to single target methods. Our objective was to elucidate whether Bartonella DNA is present in the host-seeking Ixodes ticks using this powerful detection method and to infer whether the ticks can serve as vectors for transmission of Bartonella spp. based on prevalence of infection in ticks collected over a broad geographic coverage in the US. Methods Tick collections and DNA templates The ticks tested in this study were from our archived DNA samples from ticks collected as part of national tick surveillance [47] or research efforts. Host-seeking I. scapularis were collected in 23 eastern states of the US by dragging or flagging during 2013-2023. Ticks included in this study were chosen based on life stage, sex, and collection sites. We were aiming to include ~100 ticks from each state, but the actual number of ticks per state varied between 13-153 due to low availability in some states or inclusion of more collection sites in some other states. As a result, a total of 1641 I. scapularis ticks consisting of 886 nymphs and 755 adults (330 males and 425 females) were tested for the presence of Bartonella spp. (Table 1). DNA was extracted previously using the KingFisher DNA extraction system (Thermo Fisher Scientific, Waltham, MA, USA) and the MagMAX™ Core Kit (Thermo Fisher Scientific) [48] for tick surveillance testing and stored at -80℃ afterwards. Residual DNA was used for the present study. Host-seeking I. pacificus ticks were collected from different woodland habitat types in highly climatically and ecologically diverse Mendocino County, California by dragging in 2004 as previously described [49]. A total of 966 Ixodes pacificus nymphs with representatives of all woodland habitat types described in the county were tested for the presence of Bartonella spp. (Table 2). Total DNA was extracted from individual nymphs previously using the DNeasy Blood and Tissue Kit (Qiagen, Valencia, California, USA) [50] and archived DNA was used for the present study. PCR amplification, library preparation, and next generation sequencing Ticks were tested for the presence of Bartonella DNA using a quadruplex PCR amplicon next generation sequencing assay that targets gltA , ssrA , rpoB , and groEL using Bartonella -specific primers [46]. Additionally, an internal control using 16S rRNA primer (forward 5’-CTGCTCAATGATTTTTTAAATTGCTGTGG-3’ and reverse 5’-CCGGTCTGAACTCAGATCAAGT-3’) [51] was included to ensure the presence of tick DNA and to confirm the original morphological identification of tick species. Detailed procedures follow those described in Bai et al. [46]. Briefly, a primary PCR reaction containing 12.5 μLof TEMPase 2x master mix (AMPLICON, Denmark), the four pairs of Bartonella- specific primers and the 16S rRNA tick-specific primer (final concentration of 300nM each), and 5 μL of tick DNA was first amplified. Positive and negative controls were always included in each PCR run to evaluate performance and detect contamination. Upon completion of amplification, the PCR products were purified with AMpure XP magnetic beads (Beckman Coulter, Brea, CA, USA), followed by index PCR using dual unique barcoded indices (Nextera XT index kit V2, Illumina, San Diego, CA), then purified with MagSi-DNA allround magnetic beads (BOCA Scientific, Westwood, MA). The purified products were then pooled, quantified, normalized, and denatured to generate the final library to be loaded into an MiSeq Nano v2 (500 cycles) reagent cassette (Illumina, San Diego, CA) to start sequencing on an Illumina Miseq instrument (Illumina, San Diego, CA). Tick DNA interference testing Inhibitory effect is an often-observed issue during PCR amplification, which sometimes causes false negative results for pathogen detection [52]. Before applying the assay for field tick testing, we evaluated the assay performance to check if tick DNA would cause any inhibition or interference during PCR amplification. Clean tick DNA was extracted from I. scapularis ticks raised at the CDC colony using the KingFisher DNA extraction system; Bartonella DNA from pure culture of B. henselae , B. koehlerae , and B. grahamii (Collections at CDC Bacterial Diseases Branch in Fort Collins, CO) was prepared by heating a heavy suspension of microorganisms for 15 minutes at 95°C followed by centrifugation of the lysed cells for 1 minute at 8,000 rpm. The supernatant was transferred to a clean centrifuge tube to be used as the DNA template. The DNA concentration was measured using Invitrogen™ Qubit™ 4 Fluorometer dsDNA HS Assay (Fisher Scientific, Pittsburgh, PA). Then a high concentration of the clean tick DNA (5 ng/ μL) was mixed with DNA of B. henselae (10 pg/μL), B. koehlerae (10 pg/μL), and B. grahamii (10 pg/μL), respectively. Five μL of each mixture was then used for the interference testing following the procedures described in the section above. Triplicates of each mixture were tested. Bioinformatics analysis After sequencing was completed, the raw sequences were analyzed with a custom Nextflow bioinformatics pipeline described by Osikowicz et al. [53]. Briefly, quality control analysis and primer trimming were first performed followed by error correction, paired reads merging, and amplicon sequence variants (ASVs) grouping. The observed ASVs are then aligned to reference sequences with BLASTn [54, 55]. The minimum read cut-off for a sample to be considered positive was set to be 50 reads. A 95% sequence similarity and 90% minimum sequence alignment length was used to align the observed ASVs to the reference sequences. Sequences that represent different Bartonella species for each target were obtained from GenBank and used as reference sequences. Results Tick DNA interference testing Spiked DNA from B. henselae , B. koehlerae , and B. grahamii were successfully detected and identified by each of the four targets used in the quadruplex sequencing in all triplicates of each mixture of tick DNA and Bartonella DNA, showing no interference from tick DNA. Host-seeking tick testing The internal control with the 16S rRNA tick primer showed that tick DNA was present in all samples. All ticks from the eastern US were confirmed to be I. scapularis ticks; and all ticks from Mendocino County, California were confirmed to be Ixodes pacificus . No Bartonella DNA was detected in any ticks, nymph or adult, by any target (Table 1 and Table 2 ). Discussion Ixodes scapularis and I. pacificus ticks are important vectors in the US that are responsible for transmission of Borrelia burgdorferi (the Lyme disease agent) and several other human pathogens [ 43 – 45 ]. However, the role of these ticks in Bartonella transmission was not clear. In the present study, we tested more than 2,600 host-seeking I. scapularis and I. pacificus ticks for the presence of Bartonella DNA using a sensitive and specific quadruplex PCR amplicon sequencing approach. No Bartonella DNA was detected in any ticks, nymphs or adults, by any target. Similar results have been reported by other investigators who tested host-seeking Ixodes ticks across more limited spatial scales but found no detectable Bartonella DNA [ 25 , 28 , 41 ]. These results demonstrated that host-seeking Ixodes ticks are unlikely to contribute to transmission of Bartonella spp.. Previous studies that tested blood-fed Ixodes ticks collected from different animals were able to detect Bartonella infections [ 56 , 57 ], demonstrating that Ixodes ticks are exposed to Bartonella through blood-feeding on infected hosts. Bartonella infections are prevalent in rodent species that commonly serve as bloodmeal sources for I. scapularis and I. pacificus [ 58 , 59 ]. However, the lack of detection of Bartonella in unfed Ixodes ticks, which take only a single bloodmeal per life stage, suggests that the bacteria seldom, if ever, survive the transstadial molt from larva to nymph or nymph to adult. The lack of Bartonella DNA in adult ticks is particularly compelling to support the conclusion that Bartonella does not survive the transstadial molt because adult ticks could have been exposed to any potential infections twice by taking two blood meals. Bartonella spp. typically cause long-lasting hemotrophic bacteremia in their mammalian reservoir hosts. The reservoir hosts, however, may clear the infection after being bacteremic for several months, but later may acquire the infection again, with the same or different strain [ 58 , 60 ]. The temporal dynamics observed in mammals may apply to the ticks as well. Both I. scapularis and I. pacificus ticks go through four life stages (egg, larva, nymph, and adult) to complete their lifecycles. After a blood meal, the ticks take months to a year to digest and molt into the next life stage. During the long molting process, Bartonella infection might have been cleared, assuming the ticks were infected during the previous life stage. This may explain the absence of Bartonella DNA in host-seeking ticks. Alternatively, Bartonella could have deleterious effects on tick survival, resulting in very low pathogen prevalence in surviving host-seeking ticks. Laboratory transmission studies are needed to elucidate acquisition, survival, and transmission rates. Notably, molecular detection of Bartonella DNA has been reported in ticks (primarily Ixodes spp.) collected at various locations in the US, Europe, and other parts of the world, with high or low infection rates. It is worth noting that high Bartonella prevalence in ticks (> 30%) were mostly from studies using 16S rRNA [ 21 , 30 ]. Such results are questionable due to the highly conserved 16S rRNA target used, which had little genetic diversity and shared homology with non-target microbes [ 61 , 62 ]. Studies utilizing non-specific targets could yield falsely high prevalence of Bartonella infections in ticks. On the other hand, lower Bartonella prevalence in host-seeking Ixodes ticks (< 1%) has been reported, mostly in studies using much more specific genes, for example, gltA [ 27 , 29 , 37 ]. Such low prevalence is consistent with our findings. Although we tested over 2,600 ticks in our study, we cannot rule out the possibility that Bartonella infections could occur at very low prevalence in host-seeking Ixodes sp. ticks. However, it is important to emphasize that the mere presence of Bartonella DNA within a tick does not imply that the bacteria are viable or that the tick could transmit it during the course of blood feeding [ 61 ]. Conclusions Although sample sizes were low for many states, our testing spanned a broad geographic region and cumulatively a large number of ticks were tested. Using a highly sensitive and specific NGS approach, we tested more than 2,600 host-seeking I. scapularis and I. pacificus ticks from 24 states. No Bartonella DNA was detected in the ticks. Our data, together with previous studies from more limited geographic regions [ 27 , 38 , 39 ] strongly suggest that I. scapularis and I. pacificus are unlikely to contribute more than minimally, if at all, to transmission of Bartonella spp. in the US. Declarations Acknowledgements This project was supported in part by an appointment to the Research Participation Program at the Centers for Disease Control and Prevention administered by the Oak Ridge Institute for Science and Education through an interagency agreement between the U.S. Department of Energy and the Centers for Disease Control and Prevention. Author contributions YB and RJB conceived and designed the study. YB and KM performed the lab testing. LMO, YB, and KM conducted data analysis. SM prepared and organized samples. YB prepared the manuscript draft. All authors reviewed and edited the manuscript. Funding This research was supported by intramural funding within the Centers of Disease Control and Prevention. Availability of data and materials All data of this study are presented in the manuscript. Ethics approval and consent to participate Not applicable. Consent for publication Not applicable. 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Microbiome analysis of Ixodes scapularis ticks from New York and Connecticut. Ticks Tick Borne Dis. 2019;10:894–900. Tables Table 1. Host-seeking I. scapularis ticks collected in 23 eastern states of the US during 2013-2023 for Bartonella DNA presence testing Region*/State No. of ticks tested No. of nymphs tested No. of adults tested Year collected Bartonella DNA presence Female Male gltA ssrA rpoB groEL Northeast Connecticut 45 45 2023 neg neg neg neg Maine 57 57 2015 neg neg neg neg Maryland 36 36 2014 neg neg neg neg New York 96 96 2014-2015 neg neg neg neg Pennsylvania 59 59 2014-2015 neg neg neg neg Vermont 104 9 95 2018 neg neg neg neg New Hampshire 45 38 7 2022 neg neg neg neg Northern Rockies and Plains Nebraska 78 1 33 44 2021-2022 neg neg neg neg Ohio Valley Indiana 88 88 2017-2019 neg neg neg neg Tennessee 35 23 12 2018-2019 neg neg neg neg West Virginia 98 98 2020-2022 neg neg neg neg Kentucky 13 13 2019 neg neg neg neg Ohio 104 75 29 2019, 2021-2022 neg neg neg neg Southeast North Carolina 121 50 28 43 2016-2019, 2022-2023 neg neg neg neg Virginia 97 75 7 15 2013-2015, 2018-2019 neg neg neg neg Alabama 33 5 20 8 2017 neg neg neg neg South Carolina 53 3 26 24 2020-2021 neg neg neg neg South Mississippi 49 49 2013, 2015-2016 neg neg neg neg Oklahoma 60 32 28 2021 neg neg neg neg Upper Midwest Iowa 153 56 36 61 2019-2021 neg neg neg neg Michigan 74 17 22 35 2016-2018 neg neg neg neg Minnesota 43 43 2015-2016, 2022 neg neg neg neg Wisconsin 100 48 21 31 2018-2019 neg neg neg neg Total 1641 886 425 330 2013-2023 neg neg neg neg * Climate regions of the eastern United States, as defined by the National Oceanic and Atmospheric Administration (NOAA, 2023). Table 2. Host-seeking I. pacificus nymphal ticks collected from different woodland habitat types in Mendocino County, California, 2004 for Bartonella DNA presence testing Habitat type No. of nymphs tested Bartonella DNA presence gltA ssrA rpoB groEL Redwood 74 neg neg neg neg Coastal Pine 13 neg neg neg neg Inland Pine 149 neg neg neg neg Oak-madrone (Hardwood) 278 neg neg neg neg Hardwood-conifer 305 neg neg neg neg Tanoak 107 neg neg neg neg Tanoak-madrone-conifer (Mixed Class) 40 neg neg neg neg Total 966 neg neg neg neg Additional Declarations No competing interests reported. Cite Share Download PDF Status: Published Journal Publication published 19 Aug, 2024 Read the published version in Parasites & Vectors → Version 1 posted Editorial decision: Revision requested 22 Jun, 2024 Reviews received at journal 17 Jun, 2024 Reviewers agreed at journal 06 Jun, 2024 Reviews received at journal 03 Jun, 2024 Reviewers agreed at journal 03 Jun, 2024 Reviewers invited by journal 01 Jun, 2024 Editor assigned by journal 29 May, 2024 Submission checks completed at journal 29 May, 2024 First submitted to journal 28 May, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4492797","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":311290893,"identity":"9644592f-4005-42d3-9885-f9c704210c82","order_by":0,"name":"Ying Bai","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAn0lEQVRIiWNgGAWjYBACAwkG9g8fGJhJ08LGOINkLcw8JGkxl+4xe2xTYy3PwL/4mARRWiznnDE3zjmWbtgg8SyNOC0GN3IMpHMbDicwSJwxNiBeiyWpWsykGUFa+HsMHxClxXLOsWLDHqBf2iTYEonTYi7dvPHBD2CI8fMfPnCAKC0MDBwQH7BJJBCpgYGBHeocfmLtGAWjYBSMghEHAMCZLAMRAXD0AAAAAElFTkSuQmCC","orcid":"","institution":"Centers for Disease Control and Prevention","correspondingAuthor":true,"prefix":"","firstName":"Ying","middleName":"","lastName":"Bai","suffix":""},{"id":311290894,"identity":"09997761-4c99-440d-9521-9686d2a8cacf","order_by":1,"name":"Kristin L. McClung","email":"","orcid":"","institution":"United States Department of Agriculture","correspondingAuthor":false,"prefix":"","firstName":"Kristin","middleName":"L.","lastName":"McClung","suffix":""},{"id":311290895,"identity":"3941a748-c5a2-436a-a69d-455463132efd","order_by":2,"name":"Lynn M. Osikowicz","email":"","orcid":"","institution":"Centers for Disease Control and Prevention","correspondingAuthor":false,"prefix":"","firstName":"Lynn","middleName":"M.","lastName":"Osikowicz","suffix":""},{"id":311290897,"identity":"9bbcffea-ccc0-420d-b9b1-5b823f0760b2","order_by":3,"name":"Sarah Maes","email":"","orcid":"","institution":"Centers for Disease Control and Prevention","correspondingAuthor":false,"prefix":"","firstName":"Sarah","middleName":"","lastName":"Maes","suffix":""},{"id":311290900,"identity":"b573e0e9-6fec-46cf-8d82-3606313ca2ea","order_by":4,"name":"Rebecca J. Eisen","email":"","orcid":"","institution":"Centers for Disease Control and Prevention","correspondingAuthor":false,"prefix":"","firstName":"Rebecca","middleName":"J.","lastName":"Eisen","suffix":""}],"badges":[],"createdAt":"2024-05-28 19:06:18","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4492797/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4492797/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s13071-024-06386-3","type":"published","date":"2024-08-19T15:57:14+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":63300097,"identity":"51c21528-b07d-4b5c-9f2c-e31936c756db","added_by":"auto","created_at":"2024-08-26 16:11:03","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":667227,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4492797/v1/0a2c9a27-6730-4180-bd0d-76f1e782b384.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"No evidence of Bartonella infections in host-seeking Ixodes scapularis and I. pacificus ticks in the United States","fulltext":[{"header":"Background","content":"\u003cp\u003e \u003cem\u003eBartonella\u003c/em\u003e is a diverse genus of gram-negative bacteria that are highly adapted to intracellular persistence in a wide range of vertebrates. Many mammalian species are natural reservoirs for \u003cem\u003eBartonella\u003c/em\u003e spp. and experience chronic, asymptomatic, intra-erythrocytic bacteremia when infected [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. During the last two decades, over 40 species belonging to the genus \u003cem\u003eBartonella\u003c/em\u003e have been described from different mammalian hosts, with more than a dozen having been associated with diverse clinical manifestations of diseases in humans and recognized as emerging pathogens [\u003cspan additionalcitationids=\"CR3 CR4 CR5 CR6 CR7 CR8\" citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Many \u003cem\u003eBartonella\u003c/em\u003e spp. are host-specific, suggesting maintenance of individual species in independent enzootic cycles [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003cem\u003eBartonella\u003c/em\u003e spp. are typically vector-borne, transmitted between mammalian hosts by hematophagous arthropods. To date, several arthropods have been proven to be vectors for \u003cem\u003eBartonella\u003c/em\u003e spp. transmission. Sandflies (\u003cem\u003eLutzomyia verrucarum\u003c/em\u003e), human body lice (\u003cem\u003ePediculus humanis corporis\u003c/em\u003e), and cat fleas (\u003cem\u003eCtenocephalides felis\u003c/em\u003e) are well known vectors that transmit \u003cem\u003eB. bacilliformis\u003c/em\u003e (Carri\u0026oacute;n\u0026rsquo;s disease) [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e], \u003cem\u003eB. quintana\u003c/em\u003e (trench fever) [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e], and \u003cem\u003eB. henselae\u003c/em\u003e (cat scratch disease) [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e], respectively. Additionally, the Oriental rat flea (\u003cem\u003eXenopsylla cheopis\u003c/em\u003e) and other rodent fleas have been implicated as potential vectors transmitting rodent associated \u003cem\u003eBartonella\u003c/em\u003e spp., such as \u003cem\u003eB. elizabethae\u003c/em\u003e, \u003cem\u003eB. grahamii\u003c/em\u003e, and \u003cem\u003eB. taylorii\u003c/em\u003e [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn the United States (US), the majority of reported vector-borne disease cases are caused by pathogens spread by ticks [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. There has been considerable interest in ticks as potential vectors for \u003cem\u003eBartonella\u003c/em\u003e species. Numerous molecular surveys to detect \u003cem\u003eBartonella\u003c/em\u003e DNA in various tick species have been conducted from around the world in recent years [\u003cspan additionalcitationids=\"CR19 CR20 CR21 CR22 CR23 CR24 CR25 CR26 CR27 CR28 CR29 CR30 CR31 CR32 CR33 CR34 CR35 CR36 CR37 CR38\" citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. Studies focused on host-seeking ticks are particularly intriguing because detection of \u003cem\u003eBartonella\u003c/em\u003e in unfed ticks would suggest the bacterium can survive the transstadial molt from larvae to nymphs or from nymphs to adults; demonstration of transstadial transmission is one component in demonstrating vector competency [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. Results from these studies were discordant. Some studies reported detection of \u003cem\u003eBartonella\u003c/em\u003e DNA with high prevalence in host-seeking ticks. For example, Chang et al. [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e] detected \u003cem\u003eBartonella quintana\u003c/em\u003e and several other \u003cem\u003eBartonella\u003c/em\u003e species by \u003cem\u003egltA\u003c/em\u003e in 19.2% of host-seeking \u003cem\u003eIxodes pacificus\u003c/em\u003e ticks in California, US; Adelson et al. [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e] reported 34.5% of host-seeking \u003cem\u003eIxodes scapularis\u003c/em\u003e ticks collected in New Jersey, US were infected with \u003cem\u003eBartonella\u003c/em\u003e spp. using 16S primers; Morozova et al. [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e] identified \u003cem\u003eBartonella henselae\u003c/em\u003e by \u003cem\u003egroEL\u003c/em\u003e in as high as 44% of host-seeking \u003cem\u003eIxodes persulcatus\u003c/em\u003e ticks from Russia. By contrast, other studies reported very low prevalence of \u003cem\u003eBartonella\u003c/em\u003e in ticks. For example, only 0.9% of host-seeking \u003cem\u003eI. scapularis\u003c/em\u003e ticks from Maryland, United States (US) [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e] and 0.2% of host-seeking \u003cem\u003eI. ricinus\u003c/em\u003e ticks from France [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e], harbored \u003cem\u003eBartonella\u003c/em\u003e DNA; both studies were based on \u003cem\u003egltA\u003c/em\u003e results. Moreover, absence of \u003cem\u003eBartonella\u003c/em\u003e in host-seeking ticks was reported in studies from Hungary, Korea, and Finland based on results of \u003cem\u003egltA\u003c/em\u003e, \u003cem\u003egroEL\u003c/em\u003e, or \u003cem\u003essrA\u003c/em\u003e [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eWith these varied results, it has been debated whether ticks can serve as vectors of \u003cem\u003eBartonella\u003c/em\u003e spp.. In the US, the blacklegged tick (\u003cem\u003eIxodes scapularis\u003c/em\u003e) and the western blacklegged tick (\u003cem\u003eIxodes pacificus\u003c/em\u003e) are frequent human-biters in the eastern and far western US, respectively [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. Both species serve as vectors of the Lyme disease spirochete, as well as several other human pathogens [\u003cspan additionalcitationids=\"CR44\" citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. In this study, we tested host-seeking \u003cem\u003eI. scapularis\u003c/em\u003e and \u003cem\u003eI. pacificus\u003c/em\u003e nymphs and adults collected across 24 states for the presence of \u003cem\u003eBartonella\u003c/em\u003e DNA. Previous surveys mainly used traditional PCR with a single target [\u003cspan additionalcitationids=\"CR19 CR20 CR21 CR22 CR23 CR24 CR25 CR26 CR27 CR28 CR29 CR30 CR31 CR32 CR33 CR34 CR35 CR36 CR37\" citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. Due to the genetic diversity of \u003cem\u003eBartonella\u003c/em\u003e spp., a single target may not be able to detect all species, and in some cases, single targets might not be adequately specific to accurately detect \u003cem\u003eBartonella\u003c/em\u003e, yielding false positive results. In our study, we applied a recently described quadruplex PCR amplicon next generation sequencing approach which amplifies and sequences four \u003cem\u003eBartonella\u003c/em\u003e-specific gene targets (\u003cem\u003egltA\u003c/em\u003e, \u003cem\u003essrA\u003c/em\u003e, \u003cem\u003erpoB\u003c/em\u003e, and \u003cem\u003egroEL\u003c/em\u003e) [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. Using multiple target sequences increases detection success and also improves the accuracy of species identification compared to single target methods. Our objective was to elucidate whether \u003cem\u003eBartonella\u003c/em\u003e DNA is present in the host-seeking \u003cem\u003eIxodes\u003c/em\u003e ticks using this powerful detection method and to infer whether the ticks can serve as vectors for transmission of \u003cem\u003eBartonella\u003c/em\u003e spp. based on prevalence of infection in ticks collected over a broad geographic coverage in the US.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e\u003cstrong\u003eTick collections and DNA templates\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe ticks tested in this study were from our archived DNA samples from ticks collected as part of national tick surveillance [47] or research efforts. \u0026nbsp;Host-seeking \u003cem\u003eI. scapularis\u003c/em\u003e were collected in 23 eastern states of the US\u0026nbsp;by dragging or flagging\u0026nbsp;during 2013-2023. Ticks included in this study were chosen based on life stage, sex, and collection sites. We were aiming to include ~100 ticks from each state, but the actual number of ticks per state varied between 13-153 due to low availability in some states or inclusion of more collection sites in some other states. As a result, a total of 1641 \u003cem\u003eI. scapularis\u003c/em\u003e ticks consisting of 886 nymphs and 755 adults (330 males and 425 females) were tested for the presence of \u003cem\u003eBartonella\u0026nbsp;\u003c/em\u003espp. (Table 1).\u0026nbsp;DNA was extracted previously using\u0026nbsp;the KingFisher DNA extraction system (Thermo Fisher Scientific, Waltham, MA, USA) and the MagMAX\u0026trade; Core Kit (Thermo Fisher Scientific)\u0026nbsp;[48]\u0026nbsp;for tick\u0026nbsp;surveillance testing\u0026nbsp;and stored at\u0026nbsp;-80℃ afterwards.\u0026nbsp;Residual DNA was used for the present study.\u003c/p\u003e\n\u003cp\u003eHost-seeking\u0026nbsp;\u003cem\u003eI. pacificus\u003c/em\u003e ticks were collected from different woodland habitat types in highly climatically and ecologically diverse Mendocino County, California by dragging in 2004 as previously described [49]. A total of 966 \u003cem\u003eIxodes pacificus\u003c/em\u003e nymphs with representatives of all woodland habitat types described in the county were\u0026nbsp;tested for the presence of \u003cem\u003eBartonella\u0026nbsp;\u003c/em\u003espp.\u0026nbsp;(Table 2).\u0026nbsp;Total DNA was extracted from individual nymphs previously using the DNeasy Blood and Tissue Kit (Qiagen, Valencia, California, USA) [50] and archived\u0026nbsp;DNA was used for the present study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePCR amplification, library preparation, and next generation sequencing\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTicks were tested for the presence of \u003cem\u003eBartonella\u003c/em\u003e DNA using a quadruplex PCR amplicon next generation sequencing assay that targets \u003cem\u003egltA\u003c/em\u003e, \u003cem\u003essrA\u003c/em\u003e, \u003cem\u003erpoB\u003c/em\u003e, and \u003cem\u003egroEL\u0026nbsp;\u003c/em\u003eusing \u003cem\u003eBartonella\u003c/em\u003e-specific primers [46]. Additionally, an internal control using 16S rRNA primer (forward\u0026nbsp;5\u0026rsquo;-CTGCTCAATGATTTTTTAAATTGCTGTGG-3\u0026rsquo; and reverse 5\u0026rsquo;-CCGGTCTGAACTCAGATCAAGT-3\u0026rsquo;)\u0026nbsp;[51] was included to ensure the presence of tick DNA and to confirm the original morphological identification of tick species.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eDetailed procedures follow those described in Bai et al. [46]. Briefly, a primary PCR reaction containing 12.5\u0026nbsp;\u0026mu;Lof\u0026nbsp;TEMPase 2x master mix (AMPLICON, Denmark),\u0026nbsp;the four pairs of \u003cem\u003eBartonella-\u003c/em\u003especific primers and the\u0026nbsp;16S rRNA tick-specific primer (final concentration of 300nM each), and\u0026nbsp;5\u0026nbsp;\u0026mu;L of\u0026nbsp;tick DNA\u0026nbsp;was first amplified. Positive and negative controls were\u0026nbsp;always\u0026nbsp;included in each PCR run to evaluate\u0026nbsp;performance\u0026nbsp;and\u0026nbsp;detect\u0026nbsp;contamination.\u0026nbsp;Upon completion of amplification, the PCR products were purified with AMpure XP magnetic beads (Beckman Coulter, Brea, CA, USA),\u0026nbsp;followed by index PCR using\u0026nbsp;dual unique barcoded indices (Nextera XT index kit V2, Illumina, San Diego, CA), then purified\u0026nbsp;with MagSi-DNA allround magnetic beads (BOCA Scientific, Westwood, MA). The purified products\u0026nbsp;were then pooled, quantified, normalized, and denatured to generate the final library to be loaded\u0026nbsp;into an MiSeq Nano v2 (500 cycles) reagent cassette\u0026nbsp;(Illumina, San Diego, CA)\u0026nbsp;to start sequencing on an Illumina Miseq instrument\u0026nbsp;(Illumina, San Diego, CA).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTick DNA interference testing\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;Inhibitory effect is an often-observed issue during PCR amplification, which sometimes causes false negative results for pathogen detection [52].\u0026nbsp;Before applying the assay for field tick testing, we evaluated the assay performance to check\u0026nbsp;if tick DNA would cause any inhibition or interference during PCR amplification. Clean tick\u0026nbsp;DNA was\u0026nbsp;extracted\u0026nbsp;from \u003cem\u003eI. scapularis\u003c/em\u003e ticks raised at the CDC colony\u0026nbsp;using\u0026nbsp;the KingFisher DNA extraction system;\u0026nbsp;\u003cem\u003eBartonella\u003c/em\u003e DNA from pure culture of \u003cem\u003eB. henselae\u003c/em\u003e, \u003cem\u003eB. koehlerae\u003c/em\u003e, and \u003cem\u003eB. grahamii\u003c/em\u003e (Collections at CDC Bacterial Diseases Branch in Fort Collins, CO) was prepared by heating a heavy suspension of microorganisms for 15 minutes at 95\u0026deg;C followed by centrifugation of the lysed cells for 1 minute at 8,000 rpm. The supernatant was transferred to a clean centrifuge tube to be used as the DNA template.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe DNA concentration was measured using\u0026nbsp;Invitrogen\u0026trade;\u0026nbsp;Qubit\u0026trade; 4 Fluorometer\u0026nbsp;dsDNA HS Assay\u0026nbsp;(Fisher Scientific, Pittsburgh, PA). Then a high concentration of the clean tick DNA (5 ng/\u0026nbsp;\u0026mu;L) was mixed with DNA of \u003cem\u003eB. henselae\u003c/em\u003e (10 pg/\u0026mu;L), \u003cem\u003eB. koehlerae\u003c/em\u003e (10 pg/\u0026mu;L), and \u003cem\u003eB. grahamii\u003c/em\u003e (10 pg/\u0026mu;L), respectively. Five\u0026nbsp;\u0026mu;L of each\u0026nbsp;mixture was then used for the interference testing following the procedures described in the section above. Triplicates of each mixture were tested.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eBioinformatics analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAfter sequencing was completed, the raw sequences\u0026nbsp;were analyzed with a custom Nextflow bioinformatics pipeline described by Osikowicz et al. [53]. Briefly, quality control analysis and primer trimming were first performed followed by error correction, paired reads merging, and amplicon sequence variants (ASVs) grouping.\u0026nbsp;The observed ASVs are then aligned to reference sequences with\u0026nbsp;BLASTn [54, 55].\u0026nbsp;The minimum read cut-off for a sample to be considered positive was set to be 50 reads. A 95% sequence similarity and 90% minimum sequence alignment length was used to align the observed ASVs to the reference sequences. Sequences that represent different \u003cem\u003eBartonella\u003c/em\u003e species for each target were obtained from GenBank and used as reference\u0026nbsp;sequences.\u0026nbsp;\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eTick DNA interference testing\u003c/h2\u003e \u003cp\u003eSpiked DNA from \u003cem\u003eB. henselae\u003c/em\u003e, \u003cem\u003eB. koehlerae\u003c/em\u003e, and \u003cem\u003eB. grahamii\u003c/em\u003e were successfully detected and identified by each of the four targets used in the quadruplex sequencing in all triplicates of each mixture of tick DNA and \u003cem\u003eBartonella\u003c/em\u003e DNA, showing no interference from tick DNA.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eHost-seeking tick testing\u003c/h3\u003e\n\u003cp\u003eThe internal control with the 16S rRNA tick primer showed that tick DNA was present in all samples. All ticks from the eastern US were confirmed to be \u003cem\u003eI. scapularis\u003c/em\u003e ticks; and all ticks from Mendocino County, California were confirmed to be \u003cem\u003eIxodes pacificus\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eNo \u003cem\u003eBartonella\u003c/em\u003e DNA was detected in any ticks, nymph or adult, by any target (Table\u0026nbsp;1 and Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003e \u003cem\u003eIxodes scapularis\u003c/em\u003e and \u003cem\u003eI. pacificus\u003c/em\u003e ticks are important vectors in the US that are responsible for transmission of \u003cem\u003eBorrelia burgdorferi\u003c/em\u003e (the Lyme disease agent) and several other human pathogens [\u003cspan additionalcitationids=\"CR44\" citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. However, the role of these ticks in \u003cem\u003eBartonella\u003c/em\u003e transmission was not clear. In the present study, we tested more than 2,600 host-seeking \u003cem\u003eI. scapularis\u003c/em\u003e and \u003cem\u003eI. pacificus\u003c/em\u003e ticks for the presence of \u003cem\u003eBartonella\u003c/em\u003e DNA using a sensitive and specific quadruplex PCR amplicon sequencing approach. No \u003cem\u003eBartonella\u003c/em\u003e DNA was detected in any ticks, nymphs or adults, by any target. Similar results have been reported by other investigators who tested host-seeking \u003cem\u003eIxodes\u003c/em\u003e ticks across more limited spatial scales but found no detectable \u003cem\u003eBartonella\u003c/em\u003e DNA [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. These results demonstrated that host-seeking \u003cem\u003eIxodes\u003c/em\u003e ticks are unlikely to contribute to transmission of \u003cem\u003eBartonella\u003c/em\u003e spp..\u003c/p\u003e \u003cp\u003ePrevious studies that tested blood-fed \u003cem\u003eIxodes\u003c/em\u003e ticks collected from different animals were able to detect \u003cem\u003eBartonella\u003c/em\u003e infections [\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e, \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e], demonstrating that \u003cem\u003eIxodes\u003c/em\u003e ticks are exposed to \u003cem\u003eBartonella\u003c/em\u003e through blood-feeding on infected hosts. \u003cem\u003eBartonella\u003c/em\u003e infections are prevalent in rodent species that commonly serve as bloodmeal sources for \u003cem\u003eI. scapularis\u003c/em\u003e and \u003cem\u003eI. pacificus\u003c/em\u003e [\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e, \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e]. However, the lack of detection of \u003cem\u003eBartonella\u003c/em\u003e in unfed \u003cem\u003eIxodes\u003c/em\u003e ticks, which take only a single bloodmeal per life stage, suggests that the bacteria seldom, if ever, survive the transstadial molt from larva to nymph or nymph to adult. The lack of \u003cem\u003eBartonella\u003c/em\u003e DNA in adult ticks is particularly compelling to support the conclusion that \u003cem\u003eBartonella\u003c/em\u003e does not survive the transstadial molt because adult ticks could have been exposed to any potential infections twice by taking two blood meals.\u003c/p\u003e \u003cp\u003e \u003cem\u003eBartonella\u003c/em\u003e spp. typically cause long-lasting hemotrophic bacteremia in their mammalian reservoir hosts. The reservoir hosts, however, may clear the infection after being bacteremic for several months, but later may acquire the infection again, with the same or different strain [\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e, \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e]. The temporal dynamics observed in mammals may apply to the ticks as well. Both \u003cem\u003eI. scapularis\u003c/em\u003e and \u003cem\u003eI. pacificus\u003c/em\u003e ticks go through four life stages (egg, larva, nymph, and adult) to complete their lifecycles. After a blood meal, the ticks take months to a year to digest and molt into the next life stage. During the long molting process, \u003cem\u003eBartonella\u003c/em\u003e infection might have been cleared, assuming the ticks were infected during the previous life stage. This may explain the absence of \u003cem\u003eBartonella\u003c/em\u003e DNA in host-seeking ticks. Alternatively, \u003cem\u003eBartonella\u003c/em\u003e could have deleterious effects on tick survival, resulting in very low pathogen prevalence in surviving host-seeking ticks. Laboratory transmission studies are needed to elucidate acquisition, survival, and transmission rates.\u003c/p\u003e \u003cp\u003eNotably, molecular detection of \u003cem\u003eBartonella\u003c/em\u003e DNA has been reported in ticks (primarily \u003cem\u003eIxodes\u003c/em\u003e spp.) collected at various locations in the US, Europe, and other parts of the world, with high or low infection rates. It is worth noting that high \u003cem\u003eBartonella\u003c/em\u003e prevalence in ticks (\u0026gt;\u0026thinsp;30%) were mostly from studies using 16S rRNA [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Such results are questionable due to the highly conserved 16S rRNA target used, which had little genetic diversity and shared homology with non-target microbes [\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e, \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e]. Studies utilizing non-specific targets could yield falsely high prevalence of \u003cem\u003eBartonella\u003c/em\u003e infections in ticks. On the other hand, lower \u003cem\u003eBartonella\u003c/em\u003e prevalence in host-seeking \u003cem\u003eIxodes\u003c/em\u003e ticks (\u0026lt;\u0026thinsp;1%) has been reported, mostly in studies using much more specific genes, for example, \u003cem\u003egltA\u003c/em\u003e [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. Such low prevalence is consistent with our findings. Although we tested over 2,600 ticks in our study, we cannot rule out the possibility that \u003cem\u003eBartonella\u003c/em\u003e infections could occur at very low prevalence in host-seeking \u003cem\u003eIxodes\u003c/em\u003e sp. ticks. However, it is important to emphasize that the mere presence of \u003cem\u003eBartonella\u003c/em\u003e DNA within a tick does not imply that the bacteria are viable or that the tick could transmit it during the course of blood feeding [\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e].\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eAlthough sample sizes were low for many states, our testing spanned a broad geographic region and cumulatively a large number of ticks were tested. Using a highly sensitive and specific NGS approach, we tested more than 2,600 host-seeking \u003cem\u003eI. scapularis\u003c/em\u003e and \u003cem\u003eI. pacificus\u003c/em\u003e ticks from 24 states. No \u003cem\u003eBartonella\u003c/em\u003e DNA was detected in the ticks. Our data, together with previous studies from more limited geographic regions [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e] strongly suggest that \u003cem\u003eI. scapularis\u003c/em\u003e and \u003cem\u003eI. pacificus\u003c/em\u003e are unlikely to contribute more than minimally, if at all, to transmission of \u003cem\u003eBartonella\u003c/em\u003e spp. in the US.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis project was supported in part by an appointment to the Research Participation Program at the Centers for Disease Control and Prevention administered by the Oak Ridge Institute for Science and Education through an interagency agreement between the U.S. Department of Energy and the Centers for Disease Control and Prevention.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eYB and RJB conceived and designed the study. YB and KM performed the lab testing. LMO, YB, and KM conducted data analysis. SM prepared and organized samples. YB prepared the manuscript draft. All authors reviewed and edited the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research was supported by intramural funding within the Centers of Disease Control and Prevention.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data of this study are presented in the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eDeng H, Le Rhun D, Buffet JP, Cott\u0026eacute; V, Read A, Birtles RJ, et al. 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J Vet Sci. 2008;9:285\u0026ndash;93.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCott\u0026eacute; V, Bonnet S, Cote M, Vayssier-Taussat M. Prevalence of five pathogenic agents in questing \u003cem\u003eIxodes ricinus\u003c/em\u003e ticks from western France. Vector Borne Zoonotic Dis. 2010;10:723\u0026ndash;30.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDietrich F, Schmidgen T, Maggi RG, Richter D, Matuschka FR, Vonthein R, et al. Prevalence of \u003cem\u003eBartonella henselae\u003c/em\u003e and \u003cem\u003eBorrelia burgdorferi\u003c/em\u003e sensu lato DNA in \u003cem\u003eIxodes ricinus\u003c/em\u003e ticks in Europe. Appl Environ Microbiol. 2010;76:1395\u0026ndash;8.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTijsse-Klasen E, Fonville M, Gassner F, Nijhof AM, Hovius EK, Jongejan F, et al. Absence of zoonotic \u003cem\u003eBartonella\u003c/em\u003e species in questing ticks: first detection of \u003cem\u003eBartonella clarridgeiae\u003c/em\u003e and \u003cem\u003eRickettsia felis\u003c/em\u003e in cat fleas in the Netherlands. Parasit Vectors. 2011;4:61.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCorrain R, Drigo M, Fenati M, Menandro ML, Mondin A, Pasotto D, et al. Study on ticks and tick-borne zoonoses in public parks in Italy. Zoonoses Public Health 2012;59:468\u0026ndash;76.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJanecek E, Mietze A, Goethe R, Schnieder T, Strube C. \u003cem\u003eBartonella\u003c/em\u003e spp. infection rate and \u003cem\u003eB. grahamii\u003c/em\u003e in ticks. Emerg Infect Dis. 2012;18:1689\u0026ndash;90.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSytykiewicz H, Karbowiak G, Werszko J, Czerniewicz P, Sprawka I, Mitrus J. Molecular screening for \u003cem\u003eBartonella henselae\u003c/em\u003e and \u003cem\u003eBorrelia burgdorferi\u003c/em\u003e sensu lato co-existence within \u003cem\u003eIxodes ricinus\u003c/em\u003e populations in central and eastern parts of Poland. Ann Agric Environ Med. 2012;19:451\u0026ndash;6.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVayssier-Taussat M, Moutailler S, F\u0026eacute;m\u0026eacute;nia F, Raymond P, Croce O, La Scola B, et al. Identification of Novel Zoonotic Activity of \u003cem\u003eBartonella\u003c/em\u003e spp., France. Emerg Infect Dis. 2016;22:457\u0026ndash;62.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eM\u0026uuml;ller A, Reiter M, Sch\u0026ouml;tta AM, Stockinger H, Stanek G. Detection of \u003cem\u003eBartonella\u003c/em\u003e spp. in \u003cem\u003eIxodes ricinus\u003c/em\u003e ticks and \u003cem\u003eBartonella\u003c/em\u003e seroprevalence in human populations. Ticks Tick Borne Dis. 2016;7:763\u0026ndash;7.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBonnet SI, Paul RE, Bischoff E, Cote M, Le Naour E. First identification of \u003cem\u003eRickettsia helvetica\u003c/em\u003e in questing ticks from a French Northern Brittany Forest. PLoS Negl Trop Dis. 2017;11:e0005416\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMaggi RG, Toliver M, Richardson T, Mather T, Breitschwerdt EB. Regional prevalences of \u003cem\u003eBorrelia burgdorferi\u003c/em\u003e, \u003cem\u003eBorrelia bissettiae\u003c/em\u003e, and \u003cem\u003eBartonella henselae\u003c/em\u003e in \u003cem\u003eIxodes affinis\u003c/em\u003e, \u003cem\u003eIxodes pacificus\u003c/em\u003e and \u003cem\u003eIxodes scapularis\u003c/em\u003e in the USA. Ticks Tick Borne Dis. 2019;10:360\u0026ndash;364.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLivengood J, Hutchinson ML, Thirumalapura N, Tewari D. Detection of Babesia, Borrelia, Anaplasma, and Rickettsia spp. in Adult Black-Legged Ticks (Ixodes scapularis) from Pennsylvania, United States, with a Luminex Multiplex Bead Assay. Vector Borne Zoonotic Dis. 2020;20:406\u0026ndash;11.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eEisen L. Vector competence studies with hard ticks and Borrelia burgdorferi sensu lato spirochetes: A review. Ticks Tick Borne Dis. 2020;11:101359.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSormunen JJ, Penttinen R, Klemola T, Hanninen J, Vuorinen I, Laaksonen M, et al. Tick-borne bacterial pathogens in southwestern Finland. Parasit Vectors. 2016;9:168.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eEisen L. Tick species infesting humans in the United States. Ticks Tick Borne Dis. 2022;13:102025.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePiesman, J., Eisen, L. Prevention of tick-borne diseases. Annu Rev Entomol. 2008;53:323\u0026ndash;343.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKrause PJ, Fish D, Narasimhan S, Barbour AG. \u003cem\u003eBorrelia miyamotoi\u003c/em\u003e infection in nature and in humans. Clin Microbiol Infect. 2015;21:631\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eEisen RJ, Eisen L. The blacklegged tick, Ixodes scapularis: an increasing public health concern. Trends Parasitol. 2018;34:295\u0026ndash;309.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBai Y, Osikowicz LM, Hojgaard A, Eisen RJ. Development of a quadruplex PCR amplicon next generation sequencing assay for detection and differentiation of \u003cem\u003eBartonella\u003c/em\u003e spp. Front Microbiol. 2023;14:1243471.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eEisen RJ, Paddock CD. Tick and Tickborne Pathogen Surveillance as a Public Health Tool in the United States. J Med Entomol. 2021;58:1490\u0026ndash;502.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLehane A, Maes SE, Graham CB, Jones E, Delorey M, Eisen RJ. Prevalence of single and coinfections of human pathogens in Ixodes ticks from five geographical regions in the United States, 2013\u0026ndash;2019. Ticks Tick Borne Dis. 2021;12:101637\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eEisen L, Eisen RJ, Lane RS. Geographical distribution patterns and habitat suitability models for presence of host-seeking ixodid ticks in dense woodlands of Mendocino County, California. J Med Entomol. 2006;43:415\u0026ndash;27.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eEisen RJ, Eisen L, Girard YA, Fedorova N, Mun J, Slikas B, et al. A spatially-explicit model of acarological risk of exposure to Borrelia burgdorferi-infected Ixodes pacificus nymphs in northwestern California based on woodland type, temperature, and water vapor. Ticks Tick Borne Dis. 2010;1:35\u0026ndash;43.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNorris DE, Klompen JS, Keirans JE, Black WC 4th. Population genetics of \u003cem\u003eIxodes scapularis\u003c/em\u003e (Acari: Ixodidae) based on mitochondrial 16S and 12S genes. J Med Entomol. 1996;33:78\u0026ndash;89.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSchrader C, Schielke A, Ellerbroek L, Johne R. PCR inhibitors - occurrence, properties and removal. J Appl Microbiol. 2012;113:1014\u0026ndash;26.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eOsikowicz LM, Hojgaard A, Maes S, Eisen RJ, Stenglein MD. A bioinformatics pipeline for a tick pathogen surveillance multiplex amplicon sequencing assay. Ticks Tick Borne Dis. 2023;14:102207.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAltschul SF, Gish W, Miller W, Myers EW, Lipman DJ. Basic local alignment search tool. J Mol Biol. 1993;doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/S0022-2836(05)80360-2\u003c/span\u003e\u003cspan address=\"10.1016/S0022-2836(05)80360-2\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCamacho C, Coulouris G, Avagyan V, Ma N, Papadopoulos J, Bealer K, et al. BLAST+: Architecture and applications. BMC Bioinformatics 10. 2009;doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1186/1471-2105-10-421\u003c/span\u003e\u003cspan address=\"10.1186/1471-2105-10-421\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBogumiła S, Adamska M. Capreolus capreolus and Ixodes ricinus as a reservoir of Bartonella in north-western Poland. Wiad Parazytol. 2005;51:139\u0026ndash;43.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDuplan F, Davies S, Filler S, Abdullah S, Keyte S, Newbury H, et al. Anaplasma phagocytophilum, Bartonella spp., haemoplasma species and Hepatozoon spp. in ticks infesting cats: a large-scale survey. Parasit Vectors. 2018;11:201.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBai Y, Calisher CH, KosoyMY, Root JJ, Doty JB. Persistent infection or successive reinfection of deer mice with \u003cem\u003eBartonella vinsonii\u003c/em\u003e subsp. \u003cem\u003earupensis\u003c/em\u003e. Appl Environ Microbio. 2011;77:1728\u0026ndash;31.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZiedins AC, Chomel BB, Kasten RW, Kjemtrup AM, Chang CC. Molecular epidemiology of Bartonella species isolated from ground squirrels and other rodents in northern California. Epidemiol Infect. 2016;144(9):1837\u0026ndash;44.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKosoy M, Mandel E, Green D, Marston E, Jones D, Childs J. Prospective studies of \u003cem\u003eBartonella\u003c/em\u003e of rodents. Part II. Diverse infections in a single rodent community. Vector Borne Zoonotic Dis. 2004;4:296\u0026ndash;305.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTelford SR 3rd, Wormser GP. \u003cem\u003eBartonella\u003c/em\u003e spp. transmission by ticks not established. Emerg Infect Dis. 2010;16:379\u0026ndash;84.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTokarz R, Tagliafierro T, Sameroff S, Cucura DM, Oleynik A, Che X, et al. Microbiome analysis of Ixodes scapularis ticks from New York and Connecticut. Ticks Tick Borne Dis. 2019;10:894\u0026ndash;900.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Tables","content":"\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"758\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd width=\"100%\" colspan=\"12\"\u003e\n \u003cp\u003eTable 1. Host-seeking \u003cem\u003eI. scapularis\u003c/em\u003e ticks collected in 23 eastern states of the US during 2013-2023 for \u003cem\u003eBartonella\u003c/em\u003e DNA presence testing\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"0%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"18.601583113456464%\" colspan=\"2\" rowspan=\"2\"\u003e\n \u003cp\u003eRegion*/State\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.686015831134565%\" rowspan=\"2\"\u003e\n \u003cp\u003eNo. of ticks tested\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.192612137203167%\" rowspan=\"2\"\u003e\n \u003cp\u003eNo. of nymphs tested\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.094986807387862%\" colspan=\"2\"\u003e\n \u003cp\u003eNo. of adults tested\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.20580474934037%\" rowspan=\"2\"\u003e\n \u003cp\u003eYear collected\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"23.218997361477573%\" colspan=\"6\"\u003e\n \u003cp\u003e\u003cem\u003eBartonella\u003c/em\u003e DNA presence\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"20.875420875420875%\"\u003e\n \u003cp\u003eFemale\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.2020202020202%\"\u003e\n \u003cp\u003eMale\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.151515151515152%\"\u003e\n \u003cp\u003e\u003cem\u003egltA\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.794612794612794%\"\u003e\n \u003cp\u003e\u003cem\u003essrA\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.804713804713804%\" colspan=\"2\"\u003e\n \u003cp\u003e\u003cem\u003erpoB\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.171717171717173%\" colspan=\"2\"\u003e\n \u003cp\u003e\u003cem\u003egroEL\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"18.626155878467635%\" colspan=\"2\"\u003e\n \u003cp\u003eNortheast\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.700132100396301%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"13.21003963011889%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"8.190224570673712%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"7.926023778071334%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"18.229854689564068%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"5.9445178335535%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"5.019815059445178%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"5.416116248348745%\" colspan=\"2\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"6.737120211360634%\" colspan=\"2\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"1.9815059445178336%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"16.6446499339498%\"\u003e\n \u003cp\u003eConnecticut\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.700132100396301%\"\u003e\n \u003cp\u003e45\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.21003963011889%\"\u003e\n \u003cp\u003e45\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.190224570673712%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"7.926023778071334%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"18.229854689564068%\"\u003e\n \u003cp\u003e2023\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.9445178335535%\"\u003e\n \u003cp\u003eneg\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.019815059445178%\"\u003e\n \u003cp\u003eneg\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.416116248348745%\" colspan=\"2\"\u003e\n \u003cp\u003eneg\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.737120211360634%\" colspan=\"2\"\u003e\n \u003cp\u003eneg\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"1.9815059445178336%\" valign=\"bottom\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"16.6446499339498%\" valign=\"bottom\"\u003e\n \u003cp\u003eMaine\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.700132100396301%\"\u003e\n \u003cp\u003e57\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.21003963011889%\"\u003e\n \u003cp\u003e57\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.190224570673712%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"7.926023778071334%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"18.229854689564068%\"\u003e\n \u003cp\u003e2015\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.9445178335535%\"\u003e\n \u003cp\u003eneg\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.019815059445178%\"\u003e\n \u003cp\u003eneg\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.416116248348745%\" colspan=\"2\"\u003e\n \u003cp\u003eneg\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.737120211360634%\" colspan=\"2\"\u003e\n \u003cp\u003eneg\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"1.9815059445178336%\" valign=\"bottom\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"16.6446499339498%\" valign=\"bottom\"\u003e\n \u003cp\u003eMaryland\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.700132100396301%\"\u003e\n \u003cp\u003e36\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.21003963011889%\"\u003e\n \u003cp\u003e36\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.190224570673712%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"7.926023778071334%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"18.229854689564068%\"\u003e\n \u003cp\u003e2014\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.9445178335535%\"\u003e\n \u003cp\u003eneg\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.019815059445178%\"\u003e\n \u003cp\u003eneg\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.416116248348745%\" colspan=\"2\"\u003e\n \u003cp\u003eneg\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.737120211360634%\" colspan=\"2\"\u003e\n \u003cp\u003eneg\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"1.9815059445178336%\" valign=\"bottom\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"16.6446499339498%\" valign=\"bottom\"\u003e\n \u003cp\u003eNew York\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.700132100396301%\"\u003e\n \u003cp\u003e96\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.21003963011889%\"\u003e\n \u003cp\u003e96\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.190224570673712%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"7.926023778071334%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"18.229854689564068%\"\u003e\n \u003cp\u003e2014-2015\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.9445178335535%\"\u003e\n \u003cp\u003eneg\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.019815059445178%\"\u003e\n \u003cp\u003eneg\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.416116248348745%\" colspan=\"2\"\u003e\n \u003cp\u003eneg\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.737120211360634%\" colspan=\"2\"\u003e\n \u003cp\u003eneg\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"1.9815059445178336%\" valign=\"bottom\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"16.6446499339498%\" valign=\"bottom\"\u003e\n \u003cp\u003ePennsylvania\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.700132100396301%\"\u003e\n \u003cp\u003e59\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.21003963011889%\"\u003e\n \u003cp\u003e59\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.190224570673712%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"7.926023778071334%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"18.229854689564068%\"\u003e\n \u003cp\u003e2014-2015\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.9445178335535%\"\u003e\n \u003cp\u003eneg\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.019815059445178%\"\u003e\n \u003cp\u003eneg\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.416116248348745%\" colspan=\"2\"\u003e\n \u003cp\u003eneg\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.737120211360634%\" colspan=\"2\"\u003e\n \u003cp\u003eneg\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"1.9815059445178336%\" valign=\"bottom\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"16.6446499339498%\" valign=\"bottom\"\u003e\n \u003cp\u003eVermont\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.700132100396301%\"\u003e\n \u003cp\u003e104\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.21003963011889%\"\u003e\n \u003cp\u003e9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.190224570673712%\"\u003e\n \u003cp\u003e95\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.926023778071334%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"18.229854689564068%\"\u003e\n \u003cp\u003e2018\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.9445178335535%\"\u003e\n \u003cp\u003eneg\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.019815059445178%\"\u003e\n \u003cp\u003eneg\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.416116248348745%\" colspan=\"2\"\u003e\n \u003cp\u003eneg\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.737120211360634%\" colspan=\"2\"\u003e\n \u003cp\u003eneg\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"1.9815059445178336%\" valign=\"bottom\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"16.6446499339498%\" valign=\"bottom\"\u003e\n \u003cp\u003eNew Hampshire\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.700132100396301%\"\u003e\n \u003cp\u003e45\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.21003963011889%\"\u003e\n \u003cp\u003e38\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.190224570673712%\"\u003e\n \u003cp\u003e7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.926023778071334%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"18.229854689564068%\"\u003e\n \u003cp\u003e2022\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.9445178335535%\"\u003e\n \u003cp\u003eneg\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.019815059445178%\"\u003e\n \u003cp\u003eneg\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.416116248348745%\" colspan=\"2\"\u003e\n \u003cp\u003eneg\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.737120211360634%\" colspan=\"2\"\u003e\n \u003cp\u003eneg\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"29.326287978863938%\" colspan=\"3\" valign=\"bottom\"\u003e\n \u003cp\u003eNorthern Rockies and Plains\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.21003963011889%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"8.190224570673712%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"7.926023778071334%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"18.229854689564068%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"5.9445178335535%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"5.019815059445178%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"5.416116248348745%\" colspan=\"2\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"6.737120211360634%\" colspan=\"2\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"1.9815059445178336%\" valign=\"bottom\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"16.6446499339498%\" valign=\"bottom\"\u003e\n \u003cp\u003eNebraska\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.700132100396301%\"\u003e\n \u003cp\u003e78\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.21003963011889%\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.190224570673712%\"\u003e\n \u003cp\u003e33\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.926023778071334%\"\u003e\n \u003cp\u003e44\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.229854689564068%\"\u003e\n \u003cp\u003e2021-2022\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.9445178335535%\"\u003e\n \u003cp\u003eneg\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.019815059445178%\"\u003e\n \u003cp\u003eneg\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.416116248348745%\" colspan=\"2\"\u003e\n \u003cp\u003eneg\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.737120211360634%\" colspan=\"2\"\u003e\n \u003cp\u003eneg\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"18.626155878467635%\" colspan=\"2\" valign=\"bottom\"\u003e\n \u003cp\u003eOhio Valley\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.700132100396301%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"13.21003963011889%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"8.190224570673712%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"7.926023778071334%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"18.229854689564068%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"5.9445178335535%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"5.019815059445178%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"5.416116248348745%\" colspan=\"2\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"6.737120211360634%\" colspan=\"2\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"1.9815059445178336%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"16.6446499339498%\"\u003e\n \u003cp\u003eIndiana\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.700132100396301%\"\u003e\n \u003cp\u003e88\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.21003963011889%\"\u003e\n \u003cp\u003e88\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.190224570673712%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"7.926023778071334%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"18.229854689564068%\"\u003e\n \u003cp\u003e2017-2019\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.9445178335535%\"\u003e\n \u003cp\u003eneg\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.019815059445178%\"\u003e\n \u003cp\u003eneg\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.416116248348745%\" colspan=\"2\"\u003e\n \u003cp\u003eneg\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.737120211360634%\" colspan=\"2\"\u003e\n \u003cp\u003eneg\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"1.9815059445178336%\" valign=\"bottom\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"16.6446499339498%\" valign=\"bottom\"\u003e\n \u003cp\u003eTennessee\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.700132100396301%\"\u003e\n \u003cp\u003e35\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.21003963011889%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"8.190224570673712%\"\u003e\n \u003cp\u003e23\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.926023778071334%\"\u003e\n \u003cp\u003e12\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.229854689564068%\"\u003e\n \u003cp\u003e2018-2019\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.9445178335535%\"\u003e\n \u003cp\u003eneg\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.019815059445178%\"\u003e\n \u003cp\u003eneg\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.416116248348745%\" colspan=\"2\"\u003e\n \u003cp\u003eneg\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.737120211360634%\" colspan=\"2\"\u003e\n \u003cp\u003eneg\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"1.9815059445178336%\" valign=\"bottom\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"16.6446499339498%\" valign=\"bottom\"\u003e\n \u003cp\u003eWest Virginia\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.700132100396301%\"\u003e\n \u003cp\u003e98\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.21003963011889%\"\u003e\n \u003cp\u003e98\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.190224570673712%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"7.926023778071334%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"18.229854689564068%\"\u003e\n \u003cp\u003e2020-2022\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.9445178335535%\"\u003e\n \u003cp\u003eneg\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.019815059445178%\"\u003e\n \u003cp\u003eneg\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.416116248348745%\" colspan=\"2\"\u003e\n \u003cp\u003eneg\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.737120211360634%\" colspan=\"2\"\u003e\n \u003cp\u003eneg\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"1.9815059445178336%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"16.6446499339498%\"\u003e\n \u003cp\u003eKentucky\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.700132100396301%\"\u003e\n \u003cp\u003e13\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.21003963011889%\"\u003e\n \u003cp\u003e13\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.190224570673712%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"7.926023778071334%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"18.229854689564068%\"\u003e\n \u003cp\u003e2019\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.9445178335535%\"\u003e\n \u003cp\u003eneg\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.019815059445178%\"\u003e\n \u003cp\u003eneg\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.416116248348745%\" colspan=\"2\"\u003e\n \u003cp\u003eneg\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.737120211360634%\" colspan=\"2\"\u003e\n \u003cp\u003eneg\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"1.9815059445178336%\" valign=\"bottom\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"16.6446499339498%\" valign=\"bottom\"\u003e\n \u003cp\u003eOhio\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.700132100396301%\"\u003e\n \u003cp\u003e104\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.21003963011889%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"8.190224570673712%\"\u003e\n \u003cp\u003e75\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.926023778071334%\"\u003e\n \u003cp\u003e29\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.229854689564068%\"\u003e\n \u003cp\u003e2019, 2021-2022\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.9445178335535%\"\u003e\n \u003cp\u003eneg\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.019815059445178%\"\u003e\n \u003cp\u003eneg\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.416116248348745%\" colspan=\"2\"\u003e\n \u003cp\u003eneg\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.737120211360634%\" colspan=\"2\"\u003e\n \u003cp\u003eneg\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"18.626155878467635%\" colspan=\"2\" valign=\"bottom\"\u003e\n \u003cp\u003eSoutheast\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.700132100396301%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"13.21003963011889%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"8.190224570673712%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"7.926023778071334%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"18.229854689564068%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"5.9445178335535%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"5.019815059445178%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"5.416116248348745%\" colspan=\"2\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"6.737120211360634%\" colspan=\"2\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"1.9815059445178336%\" valign=\"bottom\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"16.6446499339498%\" valign=\"bottom\"\u003e\n \u003cp\u003eNorth Carolina\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.700132100396301%\"\u003e\n \u003cp\u003e121\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.21003963011889%\"\u003e\n \u003cp\u003e50\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.190224570673712%\"\u003e\n \u003cp\u003e28\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.926023778071334%\"\u003e\n \u003cp\u003e43\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.229854689564068%\"\u003e\n \u003cp\u003e2016-2019, 2022-2023\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.9445178335535%\"\u003e\n \u003cp\u003eneg\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.019815059445178%\"\u003e\n \u003cp\u003eneg\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.416116248348745%\" colspan=\"2\"\u003e\n \u003cp\u003eneg\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.737120211360634%\" colspan=\"2\"\u003e\n \u003cp\u003eneg\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"1.9815059445178336%\" valign=\"bottom\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"16.6446499339498%\" valign=\"bottom\"\u003e\n \u003cp\u003eVirginia\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.700132100396301%\"\u003e\n \u003cp\u003e97\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.21003963011889%\"\u003e\n \u003cp\u003e75\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.190224570673712%\"\u003e\n \u003cp\u003e7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.926023778071334%\"\u003e\n \u003cp\u003e15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.229854689564068%\"\u003e\n \u003cp\u003e2013-2015, 2018-2019\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.9445178335535%\"\u003e\n \u003cp\u003eneg\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.019815059445178%\"\u003e\n \u003cp\u003eneg\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.416116248348745%\" colspan=\"2\"\u003e\n \u003cp\u003eneg\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.737120211360634%\" colspan=\"2\"\u003e\n \u003cp\u003eneg\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"1.9815059445178336%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"16.6446499339498%\"\u003e\n \u003cp\u003eAlabama\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.700132100396301%\"\u003e\n \u003cp\u003e33\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.21003963011889%\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.190224570673712%\"\u003e\n \u003cp\u003e20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.926023778071334%\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.229854689564068%\"\u003e\n \u003cp\u003e2017\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.9445178335535%\"\u003e\n \u003cp\u003eneg\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.019815059445178%\"\u003e\n \u003cp\u003eneg\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.416116248348745%\" colspan=\"2\"\u003e\n \u003cp\u003eneg\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.737120211360634%\" colspan=\"2\"\u003e\n \u003cp\u003eneg\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"1.9815059445178336%\" valign=\"bottom\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"16.6446499339498%\" valign=\"bottom\"\u003e\n \u003cp\u003eSouth Carolina\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.700132100396301%\"\u003e\n \u003cp\u003e53\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.21003963011889%\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.190224570673712%\"\u003e\n \u003cp\u003e26\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.926023778071334%\"\u003e\n \u003cp\u003e24\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.229854689564068%\"\u003e\n \u003cp\u003e2020-2021\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.9445178335535%\"\u003e\n \u003cp\u003eneg\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.019815059445178%\"\u003e\n \u003cp\u003eneg\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.416116248348745%\" colspan=\"2\"\u003e\n \u003cp\u003eneg\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.737120211360634%\" colspan=\"2\"\u003e\n \u003cp\u003eneg\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"18.626155878467635%\" colspan=\"2\" valign=\"bottom\"\u003e\n \u003cp\u003eSouth\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.700132100396301%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"13.21003963011889%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"8.190224570673712%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"7.926023778071334%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"18.229854689564068%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"5.9445178335535%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"5.019815059445178%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"5.416116248348745%\" colspan=\"2\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"6.737120211360634%\" colspan=\"2\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"1.9815059445178336%\" valign=\"bottom\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"16.6446499339498%\" valign=\"bottom\"\u003e\n \u003cp\u003eMississippi\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.700132100396301%\"\u003e\n \u003cp\u003e49\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.21003963011889%\"\u003e\n \u003cp\u003e49\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.190224570673712%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"7.926023778071334%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"18.229854689564068%\"\u003e\n \u003cp\u003e2013, 2015-2016\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.9445178335535%\"\u003e\n \u003cp\u003eneg\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.019815059445178%\"\u003e\n \u003cp\u003eneg\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.416116248348745%\" colspan=\"2\"\u003e\n \u003cp\u003eneg\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.737120211360634%\" colspan=\"2\"\u003e\n \u003cp\u003eneg\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"1.9815059445178336%\" valign=\"bottom\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"16.6446499339498%\" valign=\"bottom\"\u003e\n \u003cp\u003eOklahoma\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.700132100396301%\"\u003e\n \u003cp\u003e60\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.21003963011889%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"8.190224570673712%\"\u003e\n \u003cp\u003e32\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.926023778071334%\"\u003e\n \u003cp\u003e28\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.229854689564068%\"\u003e\n \u003cp\u003e2021\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.9445178335535%\"\u003e\n \u003cp\u003eneg\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.019815059445178%\"\u003e\n \u003cp\u003eneg\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.416116248348745%\" colspan=\"2\"\u003e\n \u003cp\u003eneg\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.737120211360634%\" colspan=\"2\"\u003e\n \u003cp\u003eneg\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"18.626155878467635%\" colspan=\"2\" valign=\"bottom\"\u003e\n \u003cp\u003eUpper Midwest\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.700132100396301%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"13.21003963011889%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"8.190224570673712%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"7.926023778071334%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"18.229854689564068%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"5.9445178335535%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"5.019815059445178%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"5.416116248348745%\" colspan=\"2\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"6.737120211360634%\" colspan=\"2\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"1.9815059445178336%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"16.6446499339498%\"\u003e\n \u003cp\u003eIowa\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.700132100396301%\"\u003e\n \u003cp\u003e153\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.21003963011889%\"\u003e\n \u003cp\u003e56\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.190224570673712%\"\u003e\n \u003cp\u003e36\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.926023778071334%\"\u003e\n \u003cp\u003e61\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.229854689564068%\"\u003e\n \u003cp\u003e2019-2021\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.9445178335535%\"\u003e\n \u003cp\u003eneg\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.019815059445178%\"\u003e\n \u003cp\u003eneg\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.416116248348745%\" colspan=\"2\"\u003e\n \u003cp\u003eneg\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.737120211360634%\" colspan=\"2\"\u003e\n \u003cp\u003eneg\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"1.9815059445178336%\" valign=\"bottom\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"16.6446499339498%\" valign=\"bottom\"\u003e\n \u003cp\u003eMichigan\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.700132100396301%\"\u003e\n \u003cp\u003e74\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.21003963011889%\"\u003e\n \u003cp\u003e17\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.190224570673712%\"\u003e\n \u003cp\u003e22\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.926023778071334%\"\u003e\n \u003cp\u003e35\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.229854689564068%\"\u003e\n \u003cp\u003e2016-2018\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.9445178335535%\"\u003e\n \u003cp\u003eneg\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.019815059445178%\"\u003e\n \u003cp\u003eneg\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.416116248348745%\" colspan=\"2\"\u003e\n \u003cp\u003eneg\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.737120211360634%\" colspan=\"2\"\u003e\n \u003cp\u003eneg\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"1.9815059445178336%\" valign=\"bottom\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"16.6446499339498%\" valign=\"bottom\"\u003e\n \u003cp\u003eMinnesota\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.700132100396301%\"\u003e\n \u003cp\u003e43\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.21003963011889%\"\u003e\n \u003cp\u003e43\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.190224570673712%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"7.926023778071334%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"18.229854689564068%\"\u003e\n \u003cp\u003e2015-2016, 2022\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.9445178335535%\"\u003e\n \u003cp\u003eneg\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.019815059445178%\"\u003e\n \u003cp\u003eneg\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.416116248348745%\" colspan=\"2\"\u003e\n \u003cp\u003eneg\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.737120211360634%\" colspan=\"2\"\u003e\n \u003cp\u003eneg\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"1.9815059445178336%\" valign=\"bottom\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"16.6446499339498%\" valign=\"bottom\"\u003e\n \u003cp\u003eWisconsin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.700132100396301%\"\u003e\n \u003cp\u003e100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.21003963011889%\"\u003e\n \u003cp\u003e48\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.190224570673712%\"\u003e\n \u003cp\u003e21\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.926023778071334%\"\u003e\n \u003cp\u003e31\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.229854689564068%\"\u003e\n \u003cp\u003e2018-2019\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.9445178335535%\"\u003e\n \u003cp\u003eneg\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.019815059445178%\"\u003e\n \u003cp\u003eneg\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.416116248348745%\" colspan=\"2\"\u003e\n \u003cp\u003eneg\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.737120211360634%\" colspan=\"2\"\u003e\n \u003cp\u003eneg\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"1.9815059445178336%\" valign=\"bottom\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"16.6446499339498%\" valign=\"bottom\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"10.700132100396301%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"13.21003963011889%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"8.190224570673712%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"7.926023778071334%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"18.229854689564068%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"5.9445178335535%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"5.019815059445178%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"5.416116248348745%\" colspan=\"2\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"6.737120211360634%\" colspan=\"2\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"18.626155878467635%\" colspan=\"2\" valign=\"bottom\"\u003e\n \u003cp\u003eTotal\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.700132100396301%\"\u003e\n \u003cp\u003e1641\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.21003963011889%\"\u003e\n \u003cp\u003e886\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.190224570673712%\"\u003e\n \u003cp\u003e425\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.926023778071334%\"\u003e\n \u003cp\u003e330\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.229854689564068%\"\u003e\n \u003cp\u003e2013-2023\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.9445178335535%\"\u003e\n \u003cp\u003eneg\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.019815059445178%\"\u003e\n \u003cp\u003eneg\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.416116248348745%\" colspan=\"2\"\u003e\n \u003cp\u003eneg\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.737120211360634%\" colspan=\"2\"\u003e\n \u003cp\u003eneg\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"93.26287978863937%\" colspan=\"10\"\u003e\n \u003cp\u003e* Climate regions of the eastern United States, as defined by the National Oceanic and Atmospheric Administration (NOAA, 2023).\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.737120211360634%\" colspan=\"2\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"0%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"471\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd width=\"100%\" colspan=\"6\"\u003e\n \u003cp\u003eTable 2. Host-seeking \u003cem\u003eI. pacificus\u0026nbsp;\u003c/em\u003enymphal ticks collected from different woodland habitat types in Mendocino County, California, 2004 for \u003cem\u003eBartonella\u003c/em\u003e DNA presence testing\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"33.12101910828026%\" rowspan=\"2\"\u003e\n \u003cp\u003eHabitat type\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25.05307855626327%\" rowspan=\"2\"\u003e\n \u003cp\u003eNo. of nymphs tested\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"41.825902335456476%\" colspan=\"4\"\u003e\n \u003cp\u003e\u003cem\u003eBartonella\u003c/em\u003e DNA presence\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"25.380710659898476%\"\u003e\n \u003cp\u003e\u003cem\u003egltA\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"21.82741116751269%\"\u003e\n \u003cp\u003e\u003cem\u003essrA\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"23.3502538071066%\"\u003e\n \u003cp\u003e\u003cem\u003erpoB\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"29.441624365482234%\"\u003e\n \u003cp\u003e\u003cem\u003egroEL\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"33.12101910828026%\"\u003e\n \u003cp\u003eRedwood\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25.05307855626327%\"\u003e\n \u003cp\u003e74\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.615711252653927%\"\u003e\n \u003cp\u003eneg\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.129511677282379%\"\u003e\n \u003cp\u003eneg\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.766454352441613%\"\u003e\n \u003cp\u003eneg\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.314225053078557%\"\u003e\n \u003cp\u003eneg\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"33.12101910828026%\"\u003e\n \u003cp\u003eCoastal Pine\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25.05307855626327%\"\u003e\n \u003cp\u003e13\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.615711252653927%\"\u003e\n \u003cp\u003eneg\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.129511677282379%\"\u003e\n \u003cp\u003eneg\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.766454352441613%\"\u003e\n \u003cp\u003eneg\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.314225053078557%\"\u003e\n \u003cp\u003eneg\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"33.12101910828026%\"\u003e\n \u003cp\u003eInland Pine\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25.05307855626327%\"\u003e\n \u003cp\u003e149\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.615711252653927%\"\u003e\n \u003cp\u003eneg\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.129511677282379%\"\u003e\n \u003cp\u003eneg\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.766454352441613%\"\u003e\n \u003cp\u003eneg\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.314225053078557%\"\u003e\n \u003cp\u003eneg\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"33.12101910828026%\"\u003e\n \u003cp\u003eOak-madrone (Hardwood)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25.05307855626327%\"\u003e\n \u003cp\u003e278\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.615711252653927%\"\u003e\n \u003cp\u003eneg\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.129511677282379%\"\u003e\n \u003cp\u003eneg\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.766454352441613%\"\u003e\n \u003cp\u003eneg\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.314225053078557%\"\u003e\n \u003cp\u003eneg\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"33.12101910828026%\"\u003e\n \u003cp\u003eHardwood-conifer\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25.05307855626327%\"\u003e\n \u003cp\u003e305\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.615711252653927%\"\u003e\n \u003cp\u003eneg\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.129511677282379%\"\u003e\n \u003cp\u003eneg\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.766454352441613%\"\u003e\n \u003cp\u003eneg\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.314225053078557%\"\u003e\n \u003cp\u003eneg\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"33.12101910828026%\"\u003e\n \u003cp\u003eTanoak\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25.05307855626327%\"\u003e\n \u003cp\u003e107\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.615711252653927%\"\u003e\n \u003cp\u003eneg\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.129511677282379%\"\u003e\n \u003cp\u003eneg\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.766454352441613%\"\u003e\n \u003cp\u003eneg\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.314225053078557%\"\u003e\n \u003cp\u003eneg\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"33.12101910828026%\"\u003e\n \u003cp\u003eTanoak-madrone-conifer (Mixed Class)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25.05307855626327%\"\u003e\n \u003cp\u003e40\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.615711252653927%\"\u003e\n \u003cp\u003eneg\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.129511677282379%\"\u003e\n \u003cp\u003eneg\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.766454352441613%\"\u003e\n \u003cp\u003eneg\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.314225053078557%\"\u003e\n \u003cp\u003eneg\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"33.12101910828026%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"25.05307855626327%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"10.615711252653927%\" valign=\"bottom\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"9.129511677282379%\" valign=\"bottom\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"9.766454352441613%\" valign=\"bottom\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"12.314225053078557%\" valign=\"bottom\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"33.12101910828026%\"\u003e\n \u003cp\u003eTotal\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25.05307855626327%\"\u003e\n \u003cp\u003e966\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.615711252653927%\"\u003e\n \u003cp\u003eneg\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.129511677282379%\"\u003e\n \u003cp\u003eneg\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.766454352441613%\"\u003e\n \u003cp\u003eneg\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.314225053078557%\"\u003e\n \u003cp\u003eneg\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"parasites-and-vectors","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"parv","sideBox":"Learn more about [Parasites \u0026 Vectors](http://parasitesandvectors.biomedcentral.com/)","snPcode":"13071","submissionUrl":"https://submission.nature.com/new-submission/13071/3","title":"Parasites \u0026 Vectors","twitterHandle":"@bugbittentweets","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Bartonella spp., Ixodes scapularis, I. pacificus, host-seeking, next generation sequencing, United States","lastPublishedDoi":"10.21203/rs.3.rs-4492797/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4492797/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003e \u003cem\u003eBartonella\u003c/em\u003e spp. infect a variety of vertebrates throughout the world with generally high prevalence. Several \u003cem\u003eBartonella\u003c/em\u003e spp. are known to cause diverse clinical manifestations in humans and have been recognized as emerging pathogens. These bacteria are mainly transmitted by blood sucking arthropods, such as fleas and lice. The role of ticks in the transmission of \u003cem\u003eBartonella\u003c/em\u003e sp. is unclear.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eA recently developed quadruplex PCR amplicon next generation sequencing approach that targets \u003cem\u003eBartonella\u003c/em\u003e-specific fragments on \u003cem\u003egltA\u003c/em\u003e, \u003cem\u003essrA\u003c/em\u003e, \u003cem\u003erpoB\u003c/em\u003e, and \u003cem\u003egroEL\u003c/em\u003e was applied to test host-seeking \u003cem\u003eIxodes scapularis\u003c/em\u003e ticks (n\u0026thinsp;=\u0026thinsp;1641; consisting of 886 nymphs and 755 adults) collected in 23 states of the eastern United States and \u003cem\u003eIxodes pacificus\u003c/em\u003e ticks (n\u0026thinsp;=\u0026thinsp;966; all nymphs) collected in California in the western United States for the presence of \u003cem\u003eBartonella\u003c/em\u003e DNA. These species were selected because they are common human biters and serve as vectors of pathogens causing the greatest number of vector-borne diseases in the United States.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eNo \u003cem\u003eBartonella\u003c/em\u003e DNA was detected in any of the ticks tested by any target.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eOwing to the lack of \u003cem\u003eBartonella\u003c/em\u003e detection in a large number of host-seeking \u003cem\u003eIxodes\u003c/em\u003e sp. ticks tested across a broad geographic region, our results strongly suggest that \u003cem\u003eI. scapularis\u003c/em\u003e and \u003cem\u003eI. pacificus\u003c/em\u003e are unlikely to contribute more than minimally, if at all, to transmission of \u003cem\u003eBartonella\u003c/em\u003e spp..\u003c/p\u003e","manuscriptTitle":"No evidence of Bartonella infections in host-seeking Ixodes scapularis and I. pacificus ticks in the United States","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-06-11 07:21:00","doi":"10.21203/rs.3.rs-4492797/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-06-22T20:46:44+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-06-17T11:32:53+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"46519140746043697131388003051504172374","date":"2024-06-06T10:19:51+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-06-03T19:50:32+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"314093282544439078801507578160182476665","date":"2024-06-03T14:07:10+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-06-01T18:04:49+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-05-29T07:22:40+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-05-29T07:17:14+00:00","index":"","fulltext":""},{"type":"submitted","content":"Parasites \u0026 Vectors","date":"2024-05-28T19:02:55+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"parasites-and-vectors","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"parv","sideBox":"Learn more about [Parasites \u0026 Vectors](http://parasitesandvectors.biomedcentral.com/)","snPcode":"13071","submissionUrl":"https://submission.nature.com/new-submission/13071/3","title":"Parasites \u0026 Vectors","twitterHandle":"@bugbittentweets","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"ffc0703d-2d18-449b-85c1-f45b7d296932","owner":[],"postedDate":"June 11th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-08-26T16:00:40+00:00","versionOfRecord":{"articleIdentity":"rs-4492797","link":"https://doi.org/10.1186/s13071-024-06386-3","journal":{"identity":"parasites-and-vectors","isVorOnly":false,"title":"Parasites \u0026 Vectors"},"publishedOn":"2024-08-19 15:57:14","publishedOnDateReadable":"August 19th, 2024"},"versionCreatedAt":"2024-06-11 07:21:00","video":"","vorDoi":"10.1186/s13071-024-06386-3","vorDoiUrl":"https://doi.org/10.1186/s13071-024-06386-3","workflowStages":[]},"version":"v1","identity":"rs-4492797","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4492797","identity":"rs-4492797","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}