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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 Ixodesticks 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.

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. phagocytophilumhave 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. phagocytophilumwithout segregating variants. Lack of discrimination among A. phagocytophilumvariants 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. phagocytophilumvariants (Ap-ha vs. non ha) in host-seeking I. scapularisnymphs 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. phagocytophilumbut could not differentiate variants. We retested A. phagocytophiluminfected 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.

Borrelia miyamotoiis a hard tick-associated relapsing fever spirochete that is geographically widespread in Ixodesspp. (Acari: Ixodidae) ticks, but typically occurs at low prevalence. Genetic variability has been described among strains derived from Asia, Europe, and North America, and among tick species that carry the infection, but little variability has been described within foci or tick species. Capitalizing on access to B. miyamotoinucleic acid extracted from host-seeking Ixodes scapularisSay or Ixodes pacificusCooley & Kohls from 16 states, we explored genetic variability based on sequence analysis of four amplicons described herein. Consistent with previous studies, we detected significant genetic differences between strains derived from I. scapularis(eastern United States) and I. pacificus(western United States) and identified two distinct sequences in the western United States (Am-West-1 and Am-West-2). Unique to this study, we identified two distinct sequences in the eastern United States (Am-East-1 and Am-East-2). Based on the 161 samples we analyzed, Am-East-1 was the only type represented in 50 B. miyamotoi-infected ticks collected from the Northeast (Vermont, Maine, New York, Connecticut, and Rhode Island), whereas ticks collected from the North-Central and Mid-Atlantic states harbored B. miyamotoicomprised of both Am-East-1 and Am-East-2. Further studies are needed to better characterize the phylogeography of B. miyamotoiand to discern if there are biologically meaningful differences among sequence types. To facilitate further exploration, we developed a polymerase chain reaction (PCR) assay designed to differentiate Am-East-1, Am-East-2, and Am-West sequence types without having to sequence the amplicon.

The white-footed mouse, Peromyscus leucopus(Rafinesque), is a reservoir for the Lyme disease spirochete Borrelia burgdorferisensu stricto in the eastern half of the United States, where the blacklegged tick, Ixodes scapularisSay (Acari: Ixodidae), is the primary vector. In the Midwest, an additional Lyme disease spirochete, Borrelia mayonii, was recorded from naturally infected I. scapularisand P. leucopus. However, an experimental demonstration of reservoir competence was lacking for a natural tick host. We therefore experimentally infected P. leucopuswith B. mayoniivia I. scapularisnymphal bites and then fed uninfected larvae on the mice to demonstrate spirochete acquisition and passage to resulting nymphs. Of 23 mice fed on by B. mayonii-infected nymphs, 21 (91%) developed active infections. The infection prevalence for nymphs fed as larvae on these infected mice 4 wk post-infection ranged from 56 to 98%, and the overall infection prevalence for 842 nymphs across all 21 P. leucopuswas 75% (95% confidence interval, 72–77%). To assess duration of infectivity, 10 of the P. leucopuswere reinfested with uninfected larval ticks 12 wk after the mice were infected. The overall infection prevalence for 480 nymphs across all 10 P. leucopusat the 12-wk time point was 26% (95% confidence interval, 23–31%), when compared with 76% (95% confidence interval, 71–79%) for 474 nymphs from the same subset of 10 mice at the 4-wk time point. We conclude that P. leucopusis susceptible to infection with B. mayoniivia bite by I. scapularisnymphs and an efficient reservoir for this Lyme disease spirochete.

Lyme disease is the most commonly reported vector-borne disease in the United States and is transmitted by Ixodes scapularisin the eastern US and I. pacificusin the west. The causative agents, Borrelia burgdorferisensu stricto (Bbss) and B. mayoniibelong to the B. burgdorferisensu lato (Bbsl) species complex. An additional eight species of Bbsl have been identified in Ixodesspecies 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 Ixodesspecies ticks in the US, we previously screened 29,517 host-seeking I. scapularisor I. pacificusticks and 692 ticks belonging to eight other Ixodesspecies for Borreliaspirochetes using a previously described tick testing algorithm that utilizes a combination of real-time PCR and Sanger sequencing for Borreliaspecies identification. The assay was designed to detect known human pathogens spread by Ixodesspecies 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 Borreliainfected ticks using a next generation sequencing multiplex PCR amplicon sequencing (MPAS) assay that can identify Borreliaspecies and Bbsl co-infections. The assay also includes targets that can molecularly confirm identifications of Ixodesspecies ticks to better inform pathogen-vector associations. We show that Bbss is the most prevalent species in I. scapularisand I. pacificus; other Bbsl species were rarely detected in I. scapularisand the only Bbsl co-infections identified in I. scapulariswere with Bbss and B. mayonii. We detected B. andersoniiin I. dentatusin the Mid-Atlantic and Upper Midwest regions, B. kurtenbachiiin I. scapularisin the Upper Midwest, B. bissettiaein I. pacificusand I. spinipalpisin the Northwest, and B. carolinensisin I. affinisin the Mid-Atlantic and Southeast, and B. laneiin I. spinipalpisin the Northwest. Twelve of 62 (19.4%) Borrelia-infected I. affinisfrom 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 Ixodesspecies ticks.

Anaplasmosis is increasingly common in the United States, with cases being reported over an expanding geographic area. To monitor for changes in risk of human infection, the U.S. Centers for Disease Control and Prevention monitors the distribution and abundance of host-seeking vector ticks (Ixodes scapularisand Ixodes pacificus) and their infection with Anaplasma phagocytophilum. While several variants of A. phagocytophilumcirculate in I. scapularis, only the human-active variant (Ap-ha) appears to be pathogenic in humans. Failure to differentiate between human and non-human variants may artificially inflate estimates of the risk of human infection. Efforts to differentiate the Ap-ha variant from the deer variant (Ap-V1) in ticks typically rely on traditional PCR assays coupled with sequencing of PCR products. However, laboratories are increasingly turning to Next Generation Sequencing (NGS) to increase testing efficiency, retain high sensitivity, and increase specificity compared with traditional PCR assays. We describe a new NGS assay with novel targets that accurately segregate the Ap-ha variant from other non-human variants and further identify unique clades within the human and non-human variants. Recognizing that not all investigators have access to NGS technology, we also developed a PCR assay based on one of the novel targets so that variants can be visualized using agarose gel electrophoresis without the need for subsequent sequencing. Such an assay may be used to improve estimates of human risk of developing anaplasmosis in North America.

‘CandidatusEhrlichia khabarensis’ was first described from rodents and insectivores in the Far East territory of Khabarovsk on the Russian Pacific Coast. Here we report the detection of DNA from this microorganism in rodents and fed ticks collected from rodents in British Columbia, Canada in 2013–2014. ‘CandidatusEhrlichia khabarensis’ was detected in (i) a female Ixodes angustustick collected from a Peromyscus maniculatus; (ii) a female Dermacentor andersonitick collected from a Perognathus parvus; (iii) a pool of 2 larval Ixodes pacificusticks collected from a single P. maniculatus; and (iv) a pool of 3 nymphal I. pacificusticks collected from a single P. maniculatus. Three of these four rodents (2 P. maniculatusand 1 P. parvus) with infected ticks also had evidence of ‘CandidatusEhrlichia khabarensis’ in at least one tissue type. The infected P. maniculatusand Ixodesticks came from the Vancouver area in western British Columbia and the P. parvusand Dermacentortick from an inland site in central British Columbia. Although it remains to be determined whether ‘CandidatusEhrlichia khabarensis’ has any negative impacts on wildlife, domestic animals or humans, we note that all three tick species found to contain the DNA of this microorganism are known to bite humans. Future detection of this microorganism either in ticks collected from rodents and allowed to molt to the next life stage prior to being tested, or from host-seeking ticks, is required to determine if it can survive the tick’s molt after being ingested via an infectious blood meal.

Rodent fleas from northwestern Chihuahua, Mexico, were analyzed for the presence of Bartonella and Yersinia pestis. In total, 760 fleas belonging to 10 species were tested with multiplex polymerase chain reaction analysis targeting the gltA (338-bp) and pla genes (478-bp) of Bartonella and Y. pestis, respectively. Although none was positive for Y. pestis, 307 fleas were infected with Bartonella spp., resulting in an overall prevalence of 40.4%. A logistic regression analysis indicated that the presence of Bartonella is more likely to occur in some flea species. From a subset of Bartonella-positive fleas, phylogenetic analyses of gltA gene sequences revealed 13 genetic variants clustering in five phylogroups (I–V), two of which were matched with known pathogenic Bartonella species (Bartonella vinsonii subsp. arupensis and Bartonella washoensis) and two that were not related with any previously described species or subspecies of Bartonella. Variants in phylogroup V, which were mainly obtained from Meringis spp. fleas, were identical to those reported recently in their specific rodent hosts (Dipodomys spp.) in the same region, suggesting that kangaroo rats and their fleas harbor other Bartonella species not reported previously. Considering the Bartonella prevalence and the flea genotypes associated with known pathogenic Bartonella species, we suggest that analysis of rodent and flea communities in the region should continue for their potential implications for human health. Given that nearby locations in the United States have reported Y. pestis in wild animals and their fleas, we suggest conducting larger-scale studies to increase our knowledge of this bacterium.