Host-utilization differences between larval and nymphal deer ticks in northeastern U.S. sites enzootic for Borrelia burgdorferi sensu stricto.

In the northeastern U.S., Borrelia burgdorferi sensu stricto, the agent of Lyme disease, is maintained between vertebrate hosts and subadult deer ticks (the northern clade of Ixodes scapularis, formerly known as Ixodes dammini). Theoretical arguments suggest that the force of transmission would be greatest when infected nymphal ticks focus their bites on the same host as the uninfected larvae. Stage-specific differences in host utilization would reduce the force of transmission, but to date such differences remain understudied. We determined the host utilization differences of larval and nymphal deer ticks using bloodmeal analysis of host-seeking nymphs and adults collected from 5 field sites in New England. Matched cohorts of ticks (nymphs=506, adults=451), i.e. ticks that had fed during the same summer season, were used to control for yearly host population variations. Infection status of all ticks was determined by real time PCR. Nymphal deer ticks were more likely to have fed on birds and sciurids (13% vs 3%, and 41% vs 9%, respectively p<0.001) and larvae were more likely to have fed on shrews (26% vs 3%, p<0.001). Similarly, ticks that had fed on a mouse or a shrew as larvae were likely to become infected (OR= 3.195, 95% CI [1.9, 5.1] and OR=2.5[1.6,3.8] respectively), and they were positively associated with infection prevalence at our sites. However, very few nymphs fed on shrews, and they were not associated with infection, raising the question of how uninfected shrews acquire infection each year. Sciurids did not appear to contribute to the enzootic cycle at our sites, which may be due to the low numbers of larvae that fed on them. Sciurid-fed ticks of either stage were not associated with infection. Both stages of ticks were less likely to be infected if they had fed on deer (OR=0.08 [0.02.0.3] and OR=0.4 [0.2,0.7] tested as nymphs and adults, respectively) and thus deer likely served to reduce the force of transmission at our sites. Site-specific analysis of differential host utilization by subadult deer ticks may contribute to appropriate targeting of interventions and thereby promote reducing risk of acquiring Lyme disease and the other deer tick-transmitted infections.

Phylodynamics of deer tick virus in North America.

The burden of ticks and the pathogens they carry is increasing worldwide. Powassan virus (POWV; Flaviviridae: Flavivirus), the only known North American tick-borne flavivirus, is of particular concern due to rising cases and the severe morbidity of POWV encephalitis. Here, we use a multifaceted approach to evaluate the emergence of the II POWV lineage, known as deer tick virus (DTV), in parts of North America where human cases occur. We detected DTV-positive ticks from eight of twenty locations in the Northeast USA with an average infection rate of 1.4 per cent. High-depth, whole-genome sequencing of eighty-four POWV and DTV samples allowed us to assess geographic and temporal phylodynamics. We observed both stable infection in the Northeast USA and patterns of geographic dispersal within and between regions. A Bayesian skyline analysis demonstrated DTV population expansion over the last 50 years. This is concordant with the documented expansion of Ixodes scapularis tick populations and suggests an increasing risk of human exposure as the vector spreads. Finally, we isolated sixteen novel viruses in cell culture and demonstrated limited genetic change after passage, a valuable resource for future studies investigating this emerging virus.

Limited Capacity of Deer To Serve as Zooprophylactic Hosts for Borrelia burgdorferi in the Northeastern United States.

Because deer are considered to be incompetent reservoirs of the agent of Lyme disease (Borrelia burgdorferi sensu stricto) in the northeastern United States, they may serve as zooprophylactic or "dilution" hosts if larvae of the deer tick vector (Ixodes dammini, "northern" clade of Ixodes scapularis) frequently feed on them. To determine whether host-seeking nymphal deer ticks commonly feed on deer as larvae, we used a real-time PCR host bloodmeal remnant identification assay to identify the host on which these ticks had fed. Nymphal lone star ticks (Amblyomma americanum) were collected simultaneously in our sites and provided an index of the availability of deer in these sites. At 3 of the 4 sites, Ixodes nymphs had fed as larvae on a variety of hosts, including mice, birds, and shrews, but rarely on deer (<6% for all sites); in contrast, lone star tick nymphs had commonly fed on deer (31 to 78%). Deer were common larval hosts for Ixodes ticks (39% of bloodmeals) in only one site. The prevalence of B. burgdorferi in host-seeking nymphal deer ticks was associated with mouse-fed ticks (P = 0.007), but there was no association with deer-fed ticks (P = 0.5). The diversity and prevalence of hosts that were identified differed between deer ticks and lone star ticks that were collected simultaneously, demonstrating that there is no confounding of host bloodmeal identification by contaminating environmental DNA (eDNA). We conclude that deer were not common hosts for larval deer ticks, thus limiting their zooprophylactic role in our sites. IMPORTANCE Because deer are incompetent reservoirs for B. burgdorferi, their presence may modulate the force of enzootic transmission by serving as zooprophylactic or "dilution" hosts. Such an effect would depend on the extent to which subadult deer ticks feed on other hosts. We used bloodmeal analysis on nymphal deer ticks to identify the host upon which larvae had fed. We found that lone star ticks collected at the same time as deer ticks commonly fed on deer, but deer ticks did not. We conclude that deer are not a preferred host for larval deer ticks and, thus, are not necessarily zooprophylactic.

Host Contributions to the Force of Borrelia burgdorferi and Babesia microti Transmission Differ at Edges of and within a Small Habitat Patch.

In the northeastern United States, the emergence of Lyme disease has been associated, in part, with the increase of small forest patches. Such disturbed habitat is exploited by generalist species, such as white-footed mice, which are considered the host with the greatest reservoir capacity for the agents of Lyme disease (Borrelia burgdorferi sensu stricto) and human babesiosis (Babesia microti). Spatial risk analyses have identified edge habitat as particularly risky. Using a retrotransposon-based quantitative PCR assay for host bloodmeal remnant identification, we directly measured whether the hosts upon which vector ticks fed differed at the edge or within the contiguous small habitat patch. Questing nymphal deer ticks, Ixodes dammini, the northern clade of Ixodes scapularis, were collected from either the edge or within a thicket on Nantucket Island over 3 transmission seasons and tested for evidence of infection as well as bloodmeal hosts. Tick bloodmeal hosts significantly differed by site as well as by year. Mice and deer were identified most often (49.9%), but shrews, rabbits, and birds were also common. Ticks from the edge fed on a greater diversity of hosts than those from the thicket. Surprisingly, mice were not strongly associated with either infection at either sampling site (odds ratio [OR] < 2 for all). Although shrews were not the most common host utilized by ticks, they were highly associated with both infections at both sites (OR = 4.5 and 11.0 for B. burgdorferi and 7.9 and 19.0 for B. microti at the edge and thicket, respectively). We conclude that reservoir hosts may differ in their contributions to infecting ticks between edge and contiguous vegetated patches. IMPORTANCE Habitat fragmentation is thought to be a main factor in the emergence of Lyme disease and other deer tick-transmitted infections. The patchwork of forest and edges promotes altered biodiversity, favoring the abundance of generalist rodents, such as white footed mice, heretofore considered a key tick and reservoir host in the northeastern United States. We used tick bloodmeal analyses to directly identify the hosts from which nymphal deer ticks became infected. We demonstrate that there is considerable microfocality in host contributions to the cohort of infected ticks and that shrews, although they fed fewer ticks than mice, disproportionately influenced the force of pathogen transmission in our site. The venue of transmission of certain deer tick-transmitted agents may comprise a habitat scale of 10 m or fewer and depend on alternative small mammal hosts such as shrews.

Incrimination of shrews as a reservoir for Powassan virus.

Powassan virus lineage 2 (deer tick virus) is an emergent threat to American public health, causing severe neurologic disease. Its life cycle in nature remains poorly understood. We use a host-specific retrotransposon-targeted real time PCR assay to test the hypothesis that white-footed mice, considered the main eastern U.S. reservoir of the coinfecting agent of Lyme disease, is the reservoir for deer tick virus. Of 20 virus-infected host-seeking nymphal black-legged ticks 65% fed on shrews and none on mice. The proportion of ticks feeding on shrews at a site is positively associated with prevalence of viral infection, but not the Lyme disease agent. Viral RNA is detected in the brain of one shrew. We conclude that shrews are a likely reservoir host for deer tick virus and that host bloodmeal analysis can provide direct evidence to incriminate reservoir hosts, thereby promoting our understanding of the ecology of tick-borne infections.

Semicentennial of Human Babesiosis, Nantucket Island.

Fifty years ago, the index case of human babesiosis due to Babesia microti was diagnosed in a summer resident of Nantucket Island. Human babesiosis, once called "Nantucket fever" due to its seeming restriction to Nantucket and the terminal moraine islands of southern New England, has emerged across the northeastern United States to commonly infect people wherever Lyme disease is endemic. We review the history of babesiosis on Nantucket, analyze its epidemiology and ecology there, provide summaries of the first case histories, and comment on its future public health burden.

What Babesia microti Is Now.

Parasites from diverse hosts morphologically identified as Babesia microti have previously been shown to belong to a paraphyletic species complex. With a growing number of reports of B. microti-like parasites from across the world, this paper seeks to report on the current knowledge of the diversity of this species complex. Phylogenetic analysis of 18S rDNA sequences obtained from GenBank shows that the diversity of the B. microti species complex has markedly increased and now encompasses at least five distinct clades. This cryptic diversity calls into question much of our current knowledge of the life cycle of these parasites, as many biological studies were conducted before DNA sequencing technology was available. In many cases, it is uncertain which B. microti-like parasite was studied because parasites from different clades may occur sympatrically and even share the same host. Progress can only be made if future studies are conducted with careful attention to parasite identification and PCR primer specificity.

Perpetuation of Borreliae.

With one exception (epidemic relapsing fever), borreliae are obligately maintained in nature by ticks. Although some Borrelia spp. may be vertically transmitted to subsequent generations of ticks, most require amplification by a vertebrate host because inheritance is not stable. Enzootic cycles of borreliae have been found globally; those receiving the most attention from researchers are those whose vectors have some degree of anthropophily and, thus, cause zoonoses such as Lyme disease or relapsing fever. To some extent, our views on the synecology of the borreliae has been dominated by an applied focus, viz., analyses that seek to understand the elements of human risk for borreliosis. But, the elements of borrelial perpetuation do not necessarily bear upon risk, nor do our concepts of risk provide the best structure for analyzing perpetuation. We identify the major global themes for the perpetuation of borreliae, and summarize local variations on those themes, focusing on key literature to outline the factors that serve as the basis for the distribution and abundance of borreliae.

Powassan Virus Encephalitis Following Brief Attachment of Connecticut Deer Ticks.

BACKGROUND: Powassan virus (POWV) is a tick-transmitted pathogen that may cause severe encephalitis; experimentally, it can be transmitted within just 15 minutes following a tick bite. The deer tick virus subtype of POWV (DTV) is transmitted by the deer tick and is the likely cause of the increase in the number of POWV cases reported in the United States. However, DTV has only been definitively documented in 6 patients by molecular analysis of the virus. METHODS: Two patients from Connecticut with encephalitis, who had a recent deer tick bite, were evaluated by the relevant serologic tests to determine if they had been infected with POWV. Evaluation also included molecular testing of an adult deer tick that had been removed from one of the patients. RESULTS: We documented neuroinvasive POWV infection in 2 children from Connecticut. Based on the results of testing the tick removed from case 2, this patient was infected by DTV, representing the 7th reported case and the first documented case of DTV infection in a child. Of note, the duration of the tick bites in both cases was very short. CONCLUSIONS: We provide the first clinical and epidemiologic evidence that POWV/DTV can be rapidly transmitted to a human host, that is, within hours of tick attachment, which is distinctive when compared to other deer tick-transmitted infections such as Lyme disease.

Retrotransposon-Based Blood Meal Analysis of Nymphal Deer Ticks Demonstrates Spatiotemporal Diversity of Borrelia burgdorferi and Babesia microti Reservoirs.

Deer tick-transmitted Borrelia burgdorferisensu stricto (Lyme disease) and Babesia microti (babesiosis) increasingly burden public health across eastern North America. The white-footed mouse is considered the primary host for subadult deer ticks and the most important reservoir host for these and other disease agents. Local transmission is thought to be modulated by less reservoir-competent hosts, such as deer, diverting ticks from feeding on mice. We measured the proportion of mouse-fed or deer-fed host-seeking nymphs from 4 sites during 2 transmission seasons by blood meal remnant analysis using a new retrotransposon-based quantitative PCR (qPCR) assay. We then determined the host that was associated with the infection status of the tick. During the first year, the proportion of mouse-fed ticks ranged from 17% on mainland sites to 100% on an island, while deer-fed ticks ranged from 4% to 24%. The proportion of ticks feeding on mice and deer was greater from island sites than mainland sites (on average, 92% versus 43%). Mouse-fed ticks decreased significantly during year 2 in 3 of 4 sites (most were <20%), while deer-fed ticks increased for all sites (75% at one site). Overall, ticks were more likely to be infected when they had fed on mice (odds ratio [OR] of 2.4 and 1.6 for Borrelia and Babesia, respectively) and were less likely to be infected if they had fed on deer (OR, 0.8 and 0.4). We conclude that host utilization by deer ticks is characterized by significant spatiotemporal diversity, which may confound efficacy tests of interventions targeting reservoir hosts.IMPORTANCE White-footed mice are thought to be the most important reservoir host for the deer tick-transmitted pathogens that cause Lyme disease and human babesiosis because they are the primary host for immature ticks. Transmission would be reduced, however, if ticks feed on deer, which are not capable of infecting ticks with either pathogen. By directly measuring whether ticks had fed on either mice or deer using a new quantitative PCR (qPCR) assay to detect remnants of host DNA leftover from the larval blood meal, we demonstrate that host utilization by ticks varies significantly over time and space and that mice often feed fewer ticks than expected. This finding has implications for our understanding of the ecology of these diseases and for the efficacy of control measures.

Ecology of Francisella tularensis.

Tularemia is a Holarctic zoonosis caused by the gamma proteobacterium Francisella tularensis and is considered to be a vector-borne disease. In many regions, human risk is associated with the bites of flies, mosquitoes, or ticks. But the biology of the agent is such that risk may be fomite related, and large outbreaks can occur due to inhalation or ingestion of contaminated materials. Such well-documented human risk factors suggest a role for these risk factors in the enzootic cycle as well. Many arthropods support the growth or survival of the agent, but whether arthropods (ticks in particular) are obligately required for the perpetuation of F. tularensis remains to be demonstrated. As with most zoonoses, our knowledge of the ecology of F. tularensis has been driven with the objective of understanding human risk. In this review, we focus on the role of the arthropod in maintaining F. tularensis, particularly with respect to long-term enzootic persistence.

Babesial infection in the Madagascan flying fox, Pteropus rufus É. Geoffroy, 1803.

BACKGROUND: Babesiae are erythrocytic protozoans, which infect the red blood cells of vertebrate hosts to cause disease. Previous studies have described potentially pathogenic infections of Babesia vesperuginis in insectivorous bats in Europe, the Americas and Asia. To date, no babesial infections have been documented in the bats of Madagascar, or in any frugivorous bat species worldwide. RESULTS: We used standard microscopy and conventional PCR to identify babesiae in blood from the endemic Madagascan flying fox (Pteropus rufus). Out of 203 P. rufus individuals captured between November 2013 and January 2016 and screened for erythrocytic parasites, nine adult males (4.43%) were infected with babesiae. Phylogenetic analysis of sequences obtained from positive samples indicates that they cluster in the Babesia microti clade, which typically infect felids, rodents, primates, and canids, but are distinct from B. vesperuginis previously described in bats. Statistical analysis of ecological trends in the data suggests that infections were most commonly observed in the rainy season and in older-age individuals. No pathological effects of infection on the host were documented; age-prevalence patterns indicated susceptible-infectious (SI) transmission dynamics characteristic of a non-immunizing persistent infection. CONCLUSIONS: To our knowledge, this study is the first report of any erythrocytic protozoan infecting Madagascan fruit bats and the first record of a babesial infection in a pteropodid fruit bat globally. Given the extent to which fruit bats have been implicated as reservoirs for emerging human pathogens, any new record of their parasite repertoire and transmission dynamics offers notable insights into our understanding of the ecology of emerging pathogens.

Blood Smears Have Poor Sensitivity for Confirming Borrelia miyamotoi Disease.

Borrelia miyamotoi disease (BMD) is a newly recognized borreliosis that is cotransmitted by ticks wherever Lyme disease is zoonotic. Unlike Borrelia burgdorferisensu lato, the agent of Lyme disease, B. miyamotoi is closely related to relapsing fever spirochetes, such as Borrelia hermsii Some authors have suggested that the disease caused by B. miyamotoi should be considered a hard-tick-transmitted relapsing fever, and thus, the main mode of confirming a diagnosis for that infection, microscopy to analyze a blood smear, may have clinical utility. To determine whether blood smears may detect B. miyamotoi in the blood of acute BMD patients, we made standard malariological thick smears from anticoagulated blood samples that were previously determined to contain this agent (by PCR) and analyzed them for morphological evidence of spirochetes. Spirochetes were not detected in the blood smears from 20 PCR positive patient blood samples after examination of 100 thick smear fields and only 2 of 20 demonstrated spirochetes when the examination was extended to 300 thick smear fields. Inoculation of severe combined immunodeficient (SCID) mice yielded isolates from 5 of 5 samples, but 0 of 3 BALB/c mice became infected. We conclude that in strong contrast to the diagnosis of typical relapsing fever, microscopy of blood smears is not sensitive enough for confirming a diagnosis of BMD but that SCID mouse inoculation could be a useful complement to PCR.

Zoonotic Babesia microti in the northeastern U.S.: Evidence for the expansion of a specific parasite lineage.

The recent range expansion of human babesiosis in the northeastern United States, once found only in restricted coastal sites, is not well understood. This study sought to utilize a large number of samples to examine the population structure of the parasites on a fine scale to provide insights into the mode of emergence across the region. 228 B. microti samples collected in endemic northeastern U.S. sites were genotyped using published Variable number tandem repeat (VNTR) markers. The genetic diversity and population structure were analysed on a geographic scale using Phyloviz and TESS, programs that utilize two different methods to identify population membership without predefined population data. Three distinct populations were detected in northeastern US, each dominated by a single ancestral type. In contrast to the limited range of the Nantucket and Cape Cod populations, the mainland population dominated from New Jersey eastward to Boston. Ancestral populations of B. microti were sufficiently isolated to differentiate into distinct populations. Despite this, a single population was detected across a large geographic area of the northeast that historically had at least 3 distinct foci of transmission, central New Jersey, Long Island and southeastern Connecticut. We conclude that a single B. microti genotype has expanded across the northeastern U.S. The biological attributes associated with this parasite genotype that have contributed to such a selective sweep remain to be identified.

Fatal Deer Tick Virus Infection in Maine.

Deer tick virus (DTV), a genetic variant (lineage II) of Powassan virus, is a rare cause of encephalitis in North America. We report a fatal case of DTV encephalitis following a documented bite from an Ixodes scapularis tick and the erythema migrans rash associated with Lyme disease.

A global map of genetic diversity in Babesia microti reveals strong population structure and identifies variants associated with clinical relapse.

Human babesiosis caused by Babesia microti is an emerging tick-borne zoonosis of increasing importance due to its rising incidence and expanding geographic range(1). Infection with this organism, an intraerythrocytic parasite of the phylum Apicomplexa, causes a febrile syndrome similar to malaria(2). Relapsing disease is common among immunocompromised and asplenic individuals(3,4) and drug resistance has recently been reported(5). To investigate the origin and genetic diversity of this parasite, we sequenced the complete genomes of 42 B. microti samples from around the world, including deep coverage of clinical infections at endemic sites in the continental USA. Samples from the continental USA segregate into a Northeast lineage and a Midwest lineage, with subsequent divergence of subpopulations along geographic lines. We identify parasite variants that associate with relapsing disease, including amino acid substitutions in the atovaquone-binding regions of cytochrome b (cytb) and the azithromycin-binding region of ribosomal protein subunit L4 (rpl4). Our results shed light on the origin, diversity and evolution of B. microti, suggest possible mechanisms for clinical relapse, and create the foundation for further research on this emerging pathogen.

Borrelia miyamotoi Disease: Neither Lyme Disease Nor Relapsing Fever.

Borrelia miyamotoi disease (BMD) is a newly recognized borreliosis globally transmitted by ticks of the Ixodes persulcatus species complex. Once considered to be a tick symbiont with no public health implications, B miyamotoi is increasingly recognized as the agent of a nonspecific febrile illness often misdiagnosed as acute Lyme disease without rash, or as ehrlichiosis. The frequency of its diagnosis in the northeastern United States is similar to that of human granulocytic ehrlichiosis. A diagnosis of BMD is confirmed by polymerase chain reaction analysis of acute blood samples, or by seroconversion using a recombinant glycerophosphodiester phosphodiesterase enzyme immunoassay. BMD is successfully treated with oral doxycycline or amoxicillin.

Borrelia miyamotoi Disease in the Northeastern United States: A Case Series.

BACKGROUND: The first recognized cases of Borrelia miyamotoi disease (BMD) in North America were reported in the northeastern United States in 2013. OBJECTIVE: To further describe the clinical spectrum and laboratory findings for BMD. DESIGN: Case series. SETTING: Patients presenting to primary care offices, emergency departments, or urgent care clinics in 2013 and 2014. PARTICIPANTS: Acutely febrile patients from the northeastern United States in whom the treating health care providers suspected and ordered testing for tick-transmitted infections. MEASUREMENTS: Whole-blood polymerase chain reaction (PCR) testing was performed for the presence of specific DNA sequences of common tickborne infections (including BMD). Serologic testing for B. miyamotoi was performed using a recombinant glycerophosphodiester phosphodiesterase (rGlpQ) protein. Clinical records were analyzed to identify the major features of acute disease. RESULTS: Among 11,515 patients tested, 97 BMD cases were identified by PCR. Most of the 51 case patients on whom clinical histories were reviewed presented with high fever, chills, marked headache, and myalgia or arthralgia. Twenty-four percent were hospitalized. Elevated liver enzyme levels, neutropenia, and thrombocytopenia were common. At presentation, 16% of patients with BMD were seropositive for IgG and/or IgM antibody to B. miyamotoi rGlpQ. Most (78%) had seropositive convalescent specimens. Symptoms resolved after treatment with doxycycline, and no chronic sequelae or symptoms were observed. LIMITATION: Findings were based on specimens submitted for testing to a reference laboratory, and medical records of only 51 of the 97 case patients with BMD were reviewed. CONCLUSION: Patients with BMD presented with nonspecific symptoms, including fever, headache, chills, myalgia, and arthralgia. Laboratory confirmation of BMD was possible by PCR on blood from acutely symptomatic patients who were seronegative at presentation. Borrelia miyamotoi disease may be an emerging tickborne infection in the northeastern United States. PRIMARY FUNDING SOURCE: IMUGEN.

Not "out of Nantucket": Babesia microti in southern New England comprises at least two major populations.

BACKGROUND: Deer tick-transmitted human babesiosis due to Babesia microti appears to be expanding its distribution and prevalence in the northeastern United States. One hypothesis for this emergence is the introduction of parasites into new sites from areas of long-standing transmission, such as Nantucket Island, Massachusetts. METHODS: We developed a typing system based on variable number tandem repeat loci that distinguished individual B. microti genotypes. We thereby analyzed the population structure of parasites from 11 sites, representing long-standing and newly emerging transmission in southern New England (northeastern United States), and compared their haplotypes and allele frequencies to determine the most probable number of B. microti populations represented by our enzootic collections. We expected to find evidence for a point source introduction across southern New England, with all parasites clearly derived from Nantucket, the site with the most intense longstanding transmission. RESULTS: B. microti in southern New England comprises at least two major populations, arguing against a single source. The Nantucket group comprises Martha's Vineyard, Nantucket and nearby Cape Cod. The Connecticut/Rhode Island (CT/RI) group consists of all the samples from those states along with samples from emerging sites in Massachusetts. CONCLUSIONS: The expansion of B. microti in the southern New England mainland is not due to parasites from the nearby terminal moraine islands (Nantucket group), but rather from the CT/RI group. The development of new B. microti foci is likely due to a mix of local intensification of transmission within relict foci across southern New England as well as long distance introduction events.

Borrelia miyamotoi infection presenting as human granulocytic anaplasmosis: a case report.

BACKGROUND: The diverse tickborne infections of the northeastern United States can present as undifferentiated flu-like illnesses. In areas endemic for Lyme and other tickborne diseases, patients presenting with acute febrile illness with myalgia, headache, neutropenia, thrombocytopenia, and elevated hepatic aminotransferase levels are presumptively diagnosed as having human granulocytic anaplasmosis (HGA). OBJECTIVE: To assign a cause for illness experienced by 2 case patients who were initially diagnosed with HGA but did not rapidly defervesce with doxycycline treatment and had no laboratory evidence of Anaplasma phagocytophilum infection. DESIGN: Case report. SETTING: 2 primary care medical centers in Massachusetts and New Jersey. PATIENTS: 2 case patients acutely presenting with fever. MEASUREMENTS: Identification of the causative agent by polymerase chain reaction and DNA sequencing. RESULTS: Molecular diagnostic assays detected Borrelia miyamotoi in the peripheral blood of both patients. There was no evidence of infection with other tickborne pathogens commonly diagnosed in the referral areas. LIMITATION: One of the case patients may have had concurrent Lyme disease. CONCLUSION: The presence of B. miyamotoi DNA in the peripheral blood and the patients' eventual therapeutic response to doxycycline are consistent with the hypothesis that their illness was due to this newly recognized spirochete. Samples from tick-exposed patients acutely presenting with signs of HGA but who have a delayed response to doxycycline therapy or negative confirmatory test results for HGA should be analyzed carefully for evidence of B. miyamotoi infection.