Plague is a rare but potentially life-threatening fleaborne zoonotic disease caused by Yersinia pestis. Public health agencies in the United States use multiple concurrent epidemiologic and ecologic strategies to determine Y. pestis exposure sites. We reviewed 196 plague case files from 1991-2018 to describe effort and yield of implemented strategies. All files included an epidemiologic component, and 71% were followed up with environmental investigations. Environmental samples were collected for laboratory testing in 88% of investigations. The percentages of investigations yielding laboratory evidence of local transmission varied from 28% for testing live-trapped rodents to 50% for pet serology. We suggest that collection and laboratory testing of samples should be prioritized when epidemiologic investigations implicate potential exposure in an unusual setting, in areas where many people could be at risk of exposure to Y. pestis, or in situations where prevention activities extend beyond educational outreach and incur greater costs.

Borrelia miyamotoi Fukunaga (Spirochaetales: Spirochaetaceae), the etiological agent of hard tick relapsing fever, has been detected at low prevalence in Ixodes spp. (Acari: Ixodidae) ticks from the Northern Hemisphere. Genetically distinct populations of B. miyamotoi have been described across continents and among Ixodes tick species distributed within continents. Within the eastern United States, a previous study identified two B. miyamotoi genotypes infecting Ixodes scapularis Say (Acari: Ixodidae). Here, we expand upon that work by developing a new multiplex PCR amplicon sequencing assay to differentiate B. miyamotoi genotypes (Am-East-1 and Am-East-2), and we use the new assay to differentiate genotypes in an expanded sample of 186 B. miyamotoi ticks collected from 17 eastern states. All 80 infections derived from the Northeast (Delaware, Massachusetts, Maryland, Maine, New Hampshire, and Vermont) or northern states in the Southeast region (North Carolina and Virginia) were identified as Am-East-1. Among the 106 B. miyamotoi infections identified in I. scapularis from the Ohio Valley (Illinois, Indiana, Kentucky, Ohio, and West Virginia), Upper Midwest (Michigan, Minnesota, and Wisconsin), or Northern Rocky Mountains and Plains (Nebraska) regions, 10 (9.4%) were characterized as Am-East-1 and 96 (90.6%) were characterized as Am-East-2. We also identified a new B. miyamotoi genotype (Am-IxKi1) in a single I. keiransi Beati, Nava, Venzal, & Guglielmone (Acari: Ixodidae) collected from Virginia.

Efforts to monitor the expanding geographic distribution of pathogens associated with Ixodes scapularis Say (Acari: Ixodidae), and to evaluate the outcomes of environmental control interventions aiming to reduce the intensity of enzootic transmission of these pathogens, are dependent on sensitive and specific molecular assays for detection of pathogens in ticks and their rodent hosts. In this study, we modified a highly sensitive and specific multiplex PCR amplicon sequencing assay developed to identify pathogens in Ixodes ticks for use in screening blood or ear tissue from rodents (Peromyscus leucopus Rafinesque (Rodentia: Cricetidae)) to detect Borrelia burgdorferi sensu stricto Johnson, Schmid, Hyde, Steigerwalt and Brenner (Spirochaetales: Spirochaetaceae), Borrelia miyamotoi Fukunaga (Spirochaetales: Spirochaetaceae), Anaplasma phagocytophilum (Foggie, 1949) Dumler (Rickettsiales: Anaplasmataceae), and Babesia microti Franca, 1910 (Piroplasmida: Babesiidae). The modified assay retained similarly high sensitivity to the original assay for detection of Bo. burgdorferi sensu stricto, Bo. miyamotoi, A. phagocytophilum, and Ba. microti. However, the probability of detecting each of the pathogens was dependent on the sample type (ear tissue or blood) tested. Generally, pathogens were detected in either ear tissue or blood samples from an individual mouse, but not in both sample types highlighting the need to test both blood and ear tissue from all individual animals to accurately categorize their infection status.

Blacklegged tick (Ixodes scapularis)-associated diseases are increasing in incidence and geographic range in the United States. Pathogen range expansion may increase the risk of coinfections, which could complicate accurate diagnosis and treatment of tick-borne diseases. To identify regions of coinfection risk across the eastern United States, we used a national database of tick-borne pathogen occurrence to identify areas of co-occurrence between four I. scapularis-borne human pathogens: Anaplasma phagocytophilum human-active variant (Apha; anaplasmosis), Babesia microti (Bam; babesiosis), Borrelia burgdorferi sensu stricto (Bbss; Lyme disease), and Borrelia miyamotoi (Bmiya; hard tick relapsing fever). Using testing data from 13,437 nymphs submitted to the United States Centers for Disease Control and Prevention from 2013 to 2024, we summarized the mean and variation in observed coinfection prevalence from site to regional scales. Bbss-Bam, Bbss-Apha, and Bam-Apha co-occurred primarily in the Northeast and Upper Midwest, whereas Bbss-Bmiya co-occurrence extended from those regions into the Ohio Valley and southern Appalachia. Within zones of co-occurrence, Bbss-Bam was the most prevalent coinfection, followed by Bbss-Apha, Bam-Apha, and Bbss-Bmiya. Except for Bbss-Bmiya, resampling simulations for all coinfections revealed coinfections form more often than expected by chance. This study is a first step in delineating acarological risk for I. scapularis-borne coinfections in the United States.

Ticks and tick-borne diseases pose a significant public health threat in the United States, particularly in the Northeast and Upper Midwest. Tick control operations are predominately focused on private residential properties. Surveys of publicly funded vector control programs have indicated that high use public lands may be viable targets for future tick control activities if funding is available. However, little is known about the feasibility of implementing tick control activities on these properties. We administered a survey to understand current tick control and tick bite prevention educational practices and potential barriers to future tick control on public lands. The survey was distributed to public land managers in Minnesota, New York, Pennsylvania, and Wisconsin and we received 129 responses. The responses indicated that tick control was undertaken on only 10% of the public lands that respondents managed. Landscape management was the most common intervention. Some (40%) of the public land managers indicated interest in using tick control methods in the future, again with landscape management being the preferred intervention. Respondents indicated that there may be significant barriers for the use of acaricides and host-targeted interventions. Currently, tick bite prevention education appears to be the primary protective measure utilized on public land, with 63% of responding land managers offering education to staff or visitors. Our survey indicates that high use public lands may present potential targets for limited tick control operations, but the potential barriers must be addressed and additional evaluation of these interventions on high use public land is required.

Most vector-borne disease cases reported in the United States are caused by pathogens spread by blacklegged ticks, Ixodes scapularis. Of these, a majority are Lyme disease cases caused by Borrelia burgdorferi sensu stricto (s.s.). Because most human infections are associated with nymphal tick bites, the density of host-seeking B. burgdorferi s.s.-infected I. scapularis nymphs (DIN) is often used to estimate risk of Lyme disease cases. DIN combines estimates of nymphal infection prevalence with estimates of densities of host-seeking nymphs, making it a costly metric to obtain through tick surveillance. Thus, field-derived estimates of DIN are limited. To fill these gaps, we sought to estimate DIN across all counties in the eastern United States. We first estimated B. burgdorferi s.s. prevalence in host-seeking I. scapularis nymphs using generalized additive models and historical tick surveillance data reported to the Centers for Disease Control and Prevention’s ArboNET database (2004–2023). We then combined prevalence estimates with previously estimated densities of host-seeking nymphs to estimate DIN. We validated these model-based estimates against data reported to ArboNET: field-derived county estimates of prevalence and DIN as well as county records of B. burgdorferi s.s. presence and collection of host-seeking nymphs. We estimated higher average nymphal prevalence (20–30

The United States Centers for Disease Control and Prevention introduced the National Tick Surveillance Program in 2018 to better define areas of acarologic risk in response to the increasing burden of blacklegged tick (Ixodes scapularis, Acari: Ixodidae)-associated infections. The program coordinates surveillance efforts conducted by state and local public health programs and collates acarological data in the ArboNET Tick Module national database. Among the metrics collected, the density of infected host-seeking nymphs (DIN) is believed to be most closely correlated with the reported occurrence of tick-borne diseases. Here, we assess the completeness and geographic representativeness of pathogen-specific DIN data collected from 2004 to 2023 and reported to the ArboNET Tick Module. We summarize county, state, and regional variation in the density of host-seeking I. scapularis nymphs infected with 6 human pathogens: Borrelia burgdorfieri sensu stricto (Spirochaetales: Spirochaetaceae), Borrelia mayonii (Spirochaetales: Spirochaetaceae), Borrelia miyamotoi (Spirochaetales: Spirochaetaceae), Anaplasma phagocytophilum (Rickettsiales: Anaplasmataceae), Ehrlichia muris eauclairensis (Rickettsiales: Ehrlichiaceae), and Babesia microti (Piroplasmida:Babesiidae). Although DIN data submissions have increased from the first to the second decade of surveillance in some regions (Northeast, Ohio Valley, Northern Rockies and Plains), they have decreased in other regions (South, Southeast). For a majority of counties across all regions, county DIN estimates were largely based on only a single annual DIN estimate per pathogen over the nearly 20 yr of surveillance. Despite the sparseness of DIN records in ArboNET, we show that acarological risk for Lyme disease has expanded geographically over the past 2 decades, and we present acarological risk maps for other I. scapularis-borne infections across the eastern United States.

A previous laboratory study using Haemaphysalis longicornis Neumann (Acari: Ixodidae) ticks of North American origin showed that larvae could acquire the Lyme disease spirochete, Borrelia burgdorferi sensu stricto (s.s.) (Spirochaetales: Spirochaetaceae) while feeding to completion on infected mice. However, the infection was lost during the molt to the nymphal stage. Nonetheless, questing H. longicornis nymphs and adults collected by drag sampling in the northeastern United States have been reported infected with B. burgdorferi s.s. DNA; occasionally these ticks appeared to be partially engorged. This raises the question of whether H. longicornis ticks can (i) acquire B. burgdorferi s.s. during an interrupted, partial blood meal on an infected host and (ii) transmit spirochetes while completing the blood meal on a second host. In this laboratory study, we demonstrated that H. longicornis nymphs could acquire B. burgdorferi s.s. from infected Mus musculus mice during a partial blood meal. Borrelia burgdorferi s.s. was detected by a multiplex polymerase chain reaction amplicon sequencing assay in 2 of 32 (6.3%) nymphs allowed to remain attached to infected mice for 48 h but, paradoxically, not in any of 25 nymphs that remained attached to infected mice for 72 h. Unfortunately, due to the low percentage of infected nymphs, we were not able to examine if such partially fed, infected nymphs were able to transmit B. burgdorferi s.s. while completing their blood meal on a second, naïve host.

Plague is a rare, potentially fatal flea-borne zoonosis endemic in the western United States. A previous model described interannual variation in human cases based on temperature and lagged precipitation. We recreated this model in northeastern Arizona (1960-1997) to evaluate its capacity to predict recent cases (1998-2022). In recreating the original model, we found that future instead of concurrent temperature had inadvertently been used for the presented fit. Prediction from our revised models with lagged precipitation and temporally plausible temperature relationships aligned with low observed cases in 1998-2022. Elevated precipitation associated with high cases in historical data (>6 inches combined precipitation over two previous springs) was only observed once in the last quarter century, so we could not assess if these conditions were reliably associated with elevated (four or more) human plague cases. Observed weather conditions were similar to those previously associated with low (fewer than or equal to two) case counts, suggesting "baseline" conditions in the last quarter century.

In response to notable increases in tick-associated illnesses in the United States, recent public health policies encouraged multi-sector collaborative approaches to preventing vector-borne diseases. Primary prevention strategies focus on educating the public about risks for tick-borne diseases and encouraging adoption of personal protection strategies. Accurate descriptions of when and where people are at risk for tick-borne diseases aid in the optimization of prevention messaging. Tick and tick-borne pathogen data can be used to fill gaps in epidemiological surveillance. However, the utility of acarological data is limited by their completeness. National maps showing the distribution of medically important tick species and the pathogens they carry are often incomplete or non-existent. Recent policies encourage accelerated efforts to monitor changes in the distribution and abundance of medically important ticks and the presence and prevalence of human pathogens that they carry, and to provide actionable, evidence-based information to the public, health care providers and public health policy makers. In 2018, the Centers for Disease Control and Prevention initiated a national tick surveillance program focused on Ixodes ticks. The national program coordinated and expanded upon existing efforts led by public health departments and academic institutions. Here, we describe experiences of state public health departments engaged in Ixodes tick surveillance, including information on why they initiated Ixodes surveillance programs, programmatic objectives, and strategies for maintaining tick surveillance programs. We share experiences and challenges in interpreting or communicating tick surveillance data to stakeholders and explore how the acarological data are used to complement epidemiological data.

Tick-borne diseases pose a persistent and increasing threat to public health. In the United States, the majority of human infections are caused by pathogens spread by the blacklegged tick, Ixodes scapularis. Most infections are reported during the summer months, when nymphal ticks are active in states in the Northeast and Upper Midwest. The density of questing I. scapularis nymphs (DON) provides an estimate for the risk of human encounters with nymphs, but it is a resource intensive metric to obtain from field sampling. Thus, DON estimates are limited in the US national tick surveillance database, the ArboNET Tick Module. We estimated DON across all counties in the eastern US using a zero-inflated negative binomial model utilizing tick surveillance data reported to ArboNET (2004-2023) as well as climate and land cover data. The model estimated generally low DON across the southeastern US and Great Plains states with higher estimates in the Upper Midwest and Northeast regions. We assigned counties to relative acarological encounter risk categories based on estimated DON: zero or lower quartile DON estimates were scored as low risk, whereas inter- and upper-quartile DON estimates were scored as moderate-high risk. Counties with moderate-high DON reported from field sampling were accurately categorized by the model as moderate-high encounter risk (99 % sensitivity). However, 80 % of sampled counties reporting low DON were classified as moderate-high risk (20 % specificity). These misclassified counties were typically situated in recently colonized areas in the Northeast and Upper Midwest and likely indicated areas potentially suitable for tick population expansion. Our model yielded a very high negative predictive value (96 %) indicating the model did very well estimating low relative encounter risk in counties where no or few nymphs were collected, and a fair positive predictive value (60 %) indicated that densities may not have reached an expected peak in some locations, particularly in the Northeast, Upper Midwest, and northern states in the Southeast. Further tick surveillance is needed to evaluate and to refine these predictions. The resulting maps are useful for estimating relative risk of nymphal encounters across the eastern US where field data are sparse and may aid in efforts aimed at promoting the use of personal protective measures in communities that are at risk for nymphal tick encounters.

The burden of tick-borne diseases continues to increase in the United States. Tick surveillance has been implemented to monitor changes in the distribution and prevalence of human disease-causing pathogens in ticks that frequently bite humans. Such efforts require accurate identification of ticks to species and highly sensitive and specific assays that can detect and differentiate pathogens from genetically similar microbes in ticks that have not been demonstrated to be pathogenic in humans. We describe a modification to a next generation sequencing pathogen detection assay that includes a target that accurately identifies Ixodes ticks to species. We show that the replacement of internal control primers used to ensure assay performance with primers that also act as an internal control and can additionally differentiate tick species, retains high sensitivity and specificity, improves efficiency, and reduces costs by eliminating the need to run separate assays to screen for pathogens and for tick identification.

Ixodes scapularis (the blacklegged tick) is widely distributed in forested areas across the eastern United States. The public health impact of I. scapularis is greatest in the north, where nymphal stage ticks commonly bite humans and serve as primary vectors for multiple human pathogens. There were dramatic increases in the tick's distribution and abundance over the last half-century in the northern part of the eastern US, and climate warming is commonly mentioned as a primary driver for these changes. In this review, we summarize the evidence for the observed spread and proliferation of I. scapularis being driven by climate warming. Although laboratory and small-scale field studies have provided insights into how temperature and humidity impact survival and reproduction of I. scapularis, using these associations to predict broad-scale distribution and abundance patterns is more challenging. Numerous efforts have been undertaken to model the distribution and abundance of I. scapularis at state, regional, and global scales based on climate and landscape variables, but outcomes have been ambiguous. Across the models, the functional relationships between seasonal or annual measures of heat, cold, precipitation, or humidity and tick presence or abundance were inconsistent. The contribution of climate relative to landscape variables was poorly defined. Over the last half-century, climate warming occurred in parallel with spread and population increase of the white-tailed deer, the most important reproductive host for I. scapularis adults, in the northern part of the eastern US. There is strong evidence for white-tailed deer playing a key role to facilitate spread and proliferation of I. scapularis in the US over the last century. However, due to a lack of spatially and temporally congruent data, climate, landscape, and host variables are rarely included in the same models, thus limiting the ability to evaluate their relative contributions or interactions in defining the geographic range and abundance patterns of ticks. We conclude that the role of climate change as a key driver for geographic expansion and population increase of I. scapularis in the northern part of the eastern US over the last half-century remains uncertain.

BackgroundThe vast majority of vector-borne diseases in the USA are associated with mosquitoes or ticks. Mosquito control is often conducted as part of community programs run by publicly-funded entities. By contrast, tick control focuses primarily on individual residential properties and is implemented predominantly by homeowners and the private pest control firms they contract. We surveyed publicly-funded vector control programs (VCPs), presumed to focus mainly on mosquitoes, to determine what tick-related services they currently offer, and their interest in and capacity to expand existing services or provide new ones.MethodsWe distributed a survey to VCPs in the Northeast, Upper Midwest and Pacific Coast states of the USA, where humans are at risk for bites by tick vectors (Ixodes scapularis or Ixodes pacificus) of agents causing Lyme disease and other tick-borne diseases. The data we report are based on responses from 118 VCPs engaged in vector control and with at least some activities focused on ticks.ResultsDespite our survey targeting geographic regions where ticks and tick-borne diseases are persistent and increasing public health concerns, only 11% (12/114) of VCPs reported they took direct action to suppress ticks questing in the environment. The most common tick-related activities conducted by the VCPs were tick bite prevention education for the public (70%; 75/107 VCPs) and tick surveillance (48%; 56/116). When asked which services they would most likely include as part of a comprehensive tick management program, tick bite prevention education (90%; 96/107), tick surveillance (89%; 95/107) and tick suppression guidance for the public (74%; 79/107) were the most common services selected. Most VCPs were also willing to consider engaging in activities to suppress ticks on public lands (68%; 73/107), but few were willing to consider suppressing ticks on privately owned land such as residential properties (15%; 16/107). Across all potential tick-related services, funding was reported as the biggest obstacle to program expansion or development, followed by personnel.ConclusionsConsidering the hesitancy of VCPs to provide tick suppression services on private properties and the high risk for tick bites in peridomestic settings, suppression of ticks on residential properties by private pest control operators will likely play an important role in the tick suppression landscape in the USA for the foreseeable future. Nevertheless, VCPs can assist in this effort by providing locally relevant guidelines to homeowners and private pest control firms regarding best practices for residential tick suppression efforts and associated efficacy evaluations. Publicly-funded VCPs are also well positioned to educate the public on personal tick bite prevention measures and to collect tick surveillance data that provide information on the risk of human encounters with ticks within their jurisdictions.

Abstract Background Ixodes scapularis and Ixodes pacificus are important vectors of multiple pathogens in the United States. However, their role in transmission of Bartonella spp., which are commonly reported in rodents and fleas, has been debated. Our previous investigation on Bartonella spp. in host-seeking I. scapularis and I. pacificus showed Bartonella spp. were absent in the ticks, suggesting the two species are unlikely to contribute to Bartonella transmission. It is unclear whether the absence of Bartonella spp. in the host-seeking ticks was attributable to ticks not being exposed to Bartonella in nature or being exposed but unable to acquire or transstadially transmit the bacterium. To assess the likelihood of exposure and acquisition, we tested Ixodes spp. ticks collected from rodents for Bartonella infections. Methods Blood-fed I. scapularis ticks (n = 792; consisting of 645 larvae and 147 nymphs), I. pacificus ticks (n = 45, all larvae), and Ixodes angustus ticks (n = 16, consisting of 11 larvae and 5 nymphs) collected from rodents from Minnesota and Washington were tested for Bartonella spp. using a quadruplex polymerase chain reaction (PCR) amplicon next-generation sequencing approach that targets Bartonella-specific fragments on gltA, ssrA, rpoB, and groEL. In parallel, rodents and fleas collected from the same field studies were investigated to compare the differences of Bartonella distribution among the ticks, fleas, and rodents. Results Bartonella spp. were commonly detected in rodents and fleas, with prevalence of 25.6% in rodents and 36.8% in fleas from Minnesota; 27.9% in rodents and 45.2% in fleas from Washington. Of all tested ticks, Bartonella DNA was detected by gltA in only one larval I. scapularis tick from Minnesota. Conclusions The high prevalence of Bartonella spp. in rodents and fleas coupled with extremely low prevalence of Bartonella spp. in blood-fed ticks suggests that although Ixodes ticks commonly encounter Bartonella in rodents, they rarely acquire the infection through blood feeding. Notably, ticks were at various stages of feeding on rodents when they were collected. Laboratory transmission studies are needed to assess acquisition rates in fully blood-fed ticks and to assess transstadial transmission efficiency if ticks acquire Bartonella infections from feeding to repletion. Graphical Abstract

We report the genomic sequence of the hard tick relapsing fever spirochete Borrelia miyamotoi strain MN18-0001. B. miyamotoi causes human illness and is geographically widespread in Ixodes spp. (Acari: Ixodidae) ticks. This is a chromosome- and plasmid-resolved genome assembly of an Am-East-2 strain type isolate from the midwestern United States.

Lyme disease is the most commonly reported vector-borne disease in the United States and is transmitted by Ixodes scapularis in the eastern US and I. pacificus in the west. The causative agents, Borrelia burgdorferi sensu stricto (Bbss) and B. mayonii belong to the B. burgdorferi sensu lato (Bbsl) species complex. An additional eight species of Bbsl have been identified in Ixodes species ticks in the US, but their geographic distribution, vector associations, human encounter rates and pathogenicity in humans are poorly defined. To better understand the geographic distribution and vector associations of Bbsl spirochetes in frequent and infrequent human-biting Ixodes species ticks in the US, we previously screened 29,517 host-seeking I. scapularis or I. pacificus ticks and 692 ticks belonging to eight other Ixodes species for Borrelia spirochetes using a previously described tick testing algorithm that utilizes a combination of real-time PCR and Sanger sequencing for Borrelia species identification. The assay was designed to detect known human pathogens spread by Ixodes species ticks, but it was not optimized to detect Bbsl co-infections. To determine if such co-infections were overlooked particularly in ticks infected with Bbss, we retested and analyzed a subsample of 845 Borrelia infected ticks using a next generation sequencing multiplex PCR amplicon sequencing (MPAS) assay that can identify Borrelia species and Bbsl co-infections. The assay also includes targets that can molecularly confirm identifications of Ixodes species ticks to better inform pathogen-vector associations. We show that Bbss is the most prevalent species in I. scapularis and I. pacificus; other Bbsl species were rarely detected in I. scapularis and the only Bbsl co-infections identified in I. scapularis were with Bbss and B. mayonii. We detected B. andersonii in I. dentatus in the Mid-Atlantic and Upper Midwest regions, B. kurtenbachii in I. scapularis in the Upper Midwest, B. bissettiae in I. pacificus and I. spinipalpis in the Northwest, and B. carolinensis in I. affinis in the Mid-Atlantic and Southeast, and B. lanei in I. spinipalpis in the Northwest. Twelve of 62 (19.4%) Borrelia-infected I. affinis from the Mid-Atlantic region were co-infected with Bbss and B. carolinensis. Our data support the notion that Bbsl species are maintained in largely independent enzootic cycles, with occasional spill-over resulting in multiple Bbsl species detected in Ixodes species ticks.

Background The majority of vector-borne disease cases in the USA are caused by pathogens spread by ticks, most commonly the blacklegged tick, Ixodes scapularis . Personal protection against tick bites, including use of repellents, is the primary defense against tick-borne diseases. Tick repellents registered by the Environmental Protection Agency (EPA) are well documented to be safe as well as effective against ticks. Another group of tick repellent products, 25(b) exempt or minimum risk products, use alternative, mostly botanically derived, active ingredients. These are considered to pose minimal risk to human health and therefore are exempt from EPA registration; efficacy testing is not mandated for these products. Methods We used a finger bioassay to evaluate the repellency against I. scapularis nymphs for 11 formulated 25(b) exempt products together with two positive control DEET-based EPA registered products. Repellency was assessed hourly from 0.5 to 6.5 h after product application. Results The DEET-based products showed ≥ 97% repellency for all examined timepoints. By contrast, an average of 63% of ticks were repelled in the first 1.5 h after application across the 11 25(b) exempt products, and the average fell to 3% repelled between 2.5 and 6.5 h. Ten of the 11 25(b) exempt products showed statistically similar efficacy to DEET-based products at 30 min after application (repellency of 79–97%). However, only four 25(b) exempt products maintained a level of repellency similar to DEET-based products (> 72%) at the 1.5-h mark, and none of these products were effective in repelling ticks at the timepoints from 2.5 to 6.5 h after application. Conclusions Neither the claims on the labels nor specific active ingredients and their concentrations appeared to predict the duration of efficacy we observed for the 25(b) exempt products. These products are not registered with the EPA, so the methods used to determine the application guidelines on their labels are unclear. Consumers should be aware that both the level of efficacy and the duration of repellency may differ among unregulated 25(b) exempt repellent products labeled for use against ticks. We encourage more research on these products and the 25(b) exempt active ingredients they contain to help determine and improve their efficacy as repellents under different conditions. Graphical Abstract

Human anaplasmosis cases, caused by Anaplasma phagocytophilum, are increasing in the United States. This trend is explained, in part, by expansion in the geographic range of the primary vector, Ixodes scapularis. Multiple variants of A. phagocytophilum have been identified in field collected ticks, but only a single variant (human active, or “Ap-ha,” variant) has been shown to be pathogenic in humans. Until recently, laboratory methods used to differentiate variants were cumbersome and seldomly used in large scale assessments of the pathogen's geographic distribution. As a result, many surveys reported A. phagocytophilum without segregating variants. Lack of discrimination among A. phagocytophilum variants could lead to overestimation of anaplasmosis risk to humans. Next Generation Sequencing (NGS) assays were recently developed to efficiently detect multiple Ixodes scapularis-borne human pathogens including Ap-ha. In this study, we utilized NGS to detect and differentiate A. phagocytophilum variants (Ap-ha vs. non ha) in host-seeking I. scapularis nymphs and adults collected across 23 states in the eastern United States from 2012 to 2023 as part of national tick surveillance efforts and research studies. Many of the included ticks were tested previously using a TaqMan PCR assay that could detect A. phagocytophilum but could not differentiate variants. We retested A. phagocytophilum infected ticks with NGS to differentiate variants. Anaplasma phagocytophilum (any variant) was identified in 165 (35 %) of 471 counties from which ticks were tested, whereas Ap-ha was detected in 70 (15 %) of 469 counties where variants were differentiated. Both variants were identified in 32 % (n = 40) of 126 counties with either variant detected. Among states where A. phagocytophilum (any variant) was detected, prevalence ranged from 2 % to 19 % in unfed adults and from 0.2 % to 7.8 % in unfed nymphs; prevalence of Ap-ha variant ranged from 0.0 % to 16 % in adults, and 0.0 % to 4.6 % in nymphs.

The genus Bartonella includes a group of species that are associated with a wide range of mammalian species, including human. It is challenging to detect all Bartonella species using a single molecular target due to its high genetic diversity. To solve this issue, we developed a quadruplex PCR amplicon sequencing assay using next-generation sequencing (NGS) technology for the detection and differentiation of Bartonella species. Our objective was to obtain the specific sequences of a minimum of two of the four target genes as confirmation of the identity of a particular Bartonella species using the assay. Four pairs of primers targeting specific regions on gltA, groEL, rpoB, and ssrA were evaluated for their capability of differentiating Bartonella species individually and collectively by performing singular PCR amplicon sequencing and quadruplex PCR amplicon sequencing. Using the quadruplex PCR amplicon sequencing, 24 Bartonella reference species were tested, all of which were successfully differentiated by at least two targets. Bartonella species were accurately identified from the artificially mixed DNA templates developed to simulate coinfections. The limit of detection was determined to be 1 fg based on testing a series of 10-fold dilutions of DNA from the Bartonella species. Testing of high DNA concentrations of 19 non-Bartonella species showed high specificity with none of the non-Bartonella species misclassified as Bartonella. Finally, the assay was evaluated by testing DNA extracts from field-collected body lice (Pediculus humanus humanus) and Norway rats (Rattus norvegicus): Bartonella quintana was detected and confirmed by three targets in the lice and Bartonella tribocorum was detected and confirmed by two targets in the rats. These results demonstrated that Bartonella species could be accurately and rapidly detected and differentiated into different tissue types using the quadruplex sequencing assay.