Geographic variation in the distribution of Anaplasma phagocytophilum variants in host-seeking Ixodes scapularis nymphs and adults in the eastern United States elucidated using next generation sequencing.

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.

A next generation sequencing assay combining Ixodes species identification with pathogen detection to support tick surveillance efforts in the United States.

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.

Detection of Borrelia burgdorferi sensu lato species in host-seeking Ixodes species ticks in the 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.

Predicting distributions of blacklegged ticks (Ixodes scapularis), Lyme disease spirochetes (Borrelia burgdorferi sensu stricto) and human Lyme disease cases in the eastern United States.

Lyme disease is the most commonly reported vector-borne disease in the United States (US), with approximately 300,000 -to- 40,000 cases reported annually. The blacklegged tick, Ixodes scapularis, is the primary vector of the Lyme disease-causing spirochete, Borrelia burgdorferi sensu stricto, in high incidence regions in the upper midwestern and northeastern US. Using county-level records of the presence of I. scapularis or presence of B. burgdorferi s.s. infected host-seeking I. scapularis, we generated habitat suitability consensus maps based on an ensemble of statistical models for both acarological risk metrics. Overall accuracy of these suitability models was high (AUC = 0.76 for I. scapularis and 0.86 for B. burgdorferi s.s. infected-I. scapularis). We sought to compare which acarological risk metric best described the distribution of counties reporting high Lyme disease incidence (≥10 confirmed cases/100,000 population) by setting the models to a fixed omission rate (10%). We compared the percent of high incidence counties correctly classified by the two models. The I. scapularis consensus map correctly classified 53% of high and low incidence counties, while the B. burgdorferi s.s. infected-I. scapularis consensus map classified 83% correctly. Counties classified as suitable by the B. burgdorferi s.s. map showed a 91% overlap with high Lyme disease incidence counties with over a 38-fold difference in Lyme disease incidence between high- and low-suitability counties. A total of 288 counties were classified as highly suitable for B. burgdorferi s.s., but lacked records of infected-I. scapularis and were not classified as high incidence. These counties were considered to represent a leading edge for B. burgdorferi s.s. infection in ticks and humans. They clustered in Illinois, Indiana, Michigan, and Ohio. This information can aid in targeting tick surveillance and prevention education efforts in counties where Lyme disease risk may increase in the future.

Reported County-Level Distribution of Seven Human Pathogens Detected in Host-Seeking Ixodes scapularis and Ixodes pacificus (Acari: Ixodidae) in the Contiguous United States.

Tickborne disease cases account for over 75% of reported vector-borne disease cases in the United States each year. In addition to transmitting the agents of Lyme disease (Borrelia burgdorferi sensu strict [Spirochaetales: Spirochaetaceae] and Borrelia mayonii [Spirochaetales: Spirochaetaceae]), the blacklegged tick, Ixodes scapularis, and the western blacklegged tick, Ixodes pacificus collectively transmit five additional human pathogens. By mapping the distributions of tickborne pathogens in host-seeking ticks, we can understand where humans are at risk of contracting tickborne diseases and devise targeted strategies to prevent them. Using publicly available tickborne pathogen surveillance databases, internal CDC pathogen testing databases, and SCOPUS search records published since 2000, we mapped the county-level distribution of Borrelia miyamotoi (Spirochaetales: Spirochaetaceae), Anaplasma phagocytophilum (Rickettsiales: Anaplasmataceae), Ehrlichia muris eauclairensis (Rickettsiales: Ehrlichiaceae), Babesia microti (Piroplasmida: Babesiidae), and Powassan virus (Flaviviridae) reported in host-seeking I. scapularis or I. pacificus in the contiguous United States. We also updated recently published maps of the distributions of Borrelia burgdorferi sensu stricto and Borrelia mayonii. All seven pathogen distributions were more limited than the distributions of vector ticks, with at least one of the seven pathogens detected in 30 states out of 41 total states (73.2% of states) where vector ticks are considered to be established. Prevention and diagnosis of tickborne diseases rely on an accurate understanding by the public and health care providers of where people are at risk for exposure to infected ticks. Our county-level pathogen distribution maps expand on previous efforts showing the distribution of Lyme disease spirochetes and highlight counties where further investigation may be warranted.

First detection of human pathogenic variant of Anaplasma phagocytophilum in field-collected Haemaphysalis longicornis, Pennsylvania, USA.

The Asian longhorned tick, Haemaphysalis longicornis, an invasive species associated with human pathogens, has spread rapidly across the eastern USA. Questing H. longicornis ticks recovered from active surveillance conducted from 1 May to 6 September, 2019 throughout Pennsylvania were tested for rickettsial pathogens. Of 265 ticks tested by PCR for pathogens, 4 (1.5%) were positive for Anaplasma phagocytophilum. Sequence analysis of the 16S rRNA gene confirmed two positives as A. phagocytophilum-human agent variant. This is the first reported detection of A. phagocytophilum-human pathogenic strain DNA in exotic H. longicornis collected in the USA.

Reported County-Level Distribution of Lyme Disease Spirochetes, Borrelia burgdorferi sensu stricto and Borrelia mayonii (Spirochaetales: Spirochaetaceae), in Host-Seeking Ixodes scapularis and Ixodes pacificus Ticks (Acari: Ixodidae) in the Contiguous United States.

Lyme disease is the most common vector-borne disease in the United States. While Lyme disease vectors are widespread, high incidence states are concentrated in the Northeast, North Central and Mid-Atlantic regions. Mapping the distribution of Lyme disease spirochetes in ticks may aid in providing data-driven explanations of epidemiological trends and recommendations for targeting prevention strategies to communities at risk. We compiled data from the literature, publicly available tickborne pathogen surveillance databases, and internal CDC pathogen testing databases to map the county-level distribution of Lyme disease spirochetes reported in host-seeking Ixodes pacificus and Ixodes scapularis across the contiguous United States. We report B. burgdorferi s.s.-infected I. scapularis from 384 counties spanning 26 eastern states located primarily in the North Central, Northeastern, and Mid-Atlantic regions, and in I. pacificus from 20 counties spanning 2 western states, with most records reported from northern and north-coastal California. Borrelia mayonii was reported in I. scapularis in 10 counties in Minnesota and Wisconsin in the North Central United States, where records of B. burgdorferi s.s. were also reported. In comparison to a broad distribution of vector ticks, the resulting map shows a more limited distribution of Lyme disease spirochetes.

Borrelia burgdorferi Sensu Stricto DNA in Field-Collected Haemaphysalis longicornis Ticks, Pennsylvania, United States.

We collected questing Haemaphysalis longicornis ticks from southeastern counties of Pennsylvania, USA. Of 263 ticks tested by PCR for pathogens, 1 adult female was positive for Borrelia burgdorferi sensu stricto, yielding a 0.4% infection rate. Continued monitoring of this invasive tick is essential to determine its public health role.

Prevalence of single and coinfections of human pathogens in Ixodes ticks from five geographical regions in the United States, 2013-2019.

As the geographic distributions of medically important ticks and tick-borne pathogens continue to expand in the United States, the burden of tick-borne diseases continues to increase along with a growing risk of coinfections. Coinfection with multiple tick-borne pathogens may amplify severity of disease and complicate diagnosis and treatment. By testing 13,400 Ixodes ticks from 17 US states spanning five geographical regions for etiological agents of Lyme disease (Borrelia burgdorferi sensu stricto [s.s.] and Borrelia mayonii), Borrelia miyamotoi disease (Borrelia miyamotoi), anaplasmosis (Anaplasma phagocytophilum), and babesiosis (Babesia microti) we show that B. burgdorferi s.s. was the most prevalent and widespread pathogen. Borrelia miyamotoi, A. phagocytophilum, and B. microti were widespread but less prevalent than B. burgdorferi s.s. Coinfections with B. burgdorferi s.s. and A. phagocytophilum or B. microti were most common in the Northeast and occurred at rates higher than expected based on rates of single infections in that region.

Prevalence and distribution of seven human pathogens in host-seeking Ixodes scapularis (Acari: Ixodidae) nymphs in Minnesota, USA.

In the north-central United States, the blacklegged tick (Ixodes scapularis) is currently known to vector seven human pathogens. These include five bacteria (Borrelia burgdorferi sensu stricto, Borrelia mayonii, Borrelia miyamotoi, Anaplasma phagocytophilum, Ehrlichia muris eauclairensis), one protozoan (Babesia microti) and one virus (Powassan). We sought to assess the prevalence and distribution of these pathogens in host-seeking nymphs collected throughout Minnesota, a state on the northwestern edge of the tick's expanding range, where reported cases of I. scapularis-borne diseases have increased in incidence and geographic range over the past decade. Among the 1240 host-seeking I. scapularis nymphs that we screened from 64 sites, we detected all seven pathogens at varying frequencies. Borrelia burgdorferi s.s. was the most prevalent and geographically widespread, found in 25.24% of all nymphs tested. Anaplasma phagocytophilum and Babesia microti were also geographically widespread, but they were less prevalent than Bo. burgdorferi s.s. (detected in 6.29% and 4.68% of ticks, respectively). Spatial clusters of sites with high prevalence for these three pathogens were identified in the north-central region of the state. Prevalence was less than 1.29% for each of the remaining pathogens. Two or more pathogens were detected in 90 nymphs (7.26%); coinfections with Bo. burgdorferi s.s. and either A. phagocytophilum (51 nymphs, 4.11%) or Ba. microti (43 nymphs, 3.47%) were the most common combinations. The distribution and density of infected ticks mirrors the distribution of notifiable tick-borne diseases in Minnesota and provides information on the distribution and prevalence of recently described human pathogens.

Acquisition of Bartonella elizabethae by Experimentally Exposed Oriental Rat Fleas (Xenopsylla cheopis; Siphonaptera, Pulicidae) and Excretion of Bartonella DNA in Flea Feces.

Few studies have been able to provide experimental evidence of the ability of fleas to maintain rodent-associated Bartonella infections and excrete these bacteria. These data are important for understanding the transmission cycles and prevalence of these bacteria in hosts and vectors. We used an artificial feeding approach to expose groups of the oriental rat flea (Xenopsylla cheopis Rothschild; Siphonaptera, Pulicidae) to rat blood inoculated with varying concentrations of Bartonella elizabethae Daly (Bartonellaceae: Rhizobiales). Flea populations were maintained by membrane feeding on pathogen-free bloodmeals for up to 13 d post infection. Individual fleas and pools of flea feces were tested for the presence of Bartonella DNA using molecular methods (quantitative and conventional polymerase chain reaction [PCR]). The threshold number of Bartonellae required in the infectious bloodmeal for fleas to be detected as positive was 106 colony-forming units per milliliter (CFU/ml). Individual fleas were capable of harboring infections for at least 13 d post infection and continuously excreted Bartonella DNA in their feces over the same period. This experiment demonstrated that X. cheopis are capable of acquiring and excreting B. elizabethae over several days. These results will guide future work to model and understand the role of X. cheopis in the natural transmission cycle of rodent-borne Bartonella species. Future experiments using this artificial feeding approach will be useful for examining the horizontal transmission of B. elizabethae or other rodent-associated Bartonella species to naïve hosts and for determining the viability of excreted bacteria.

A molecular algorithm to detect and differentiate human pathogens infecting Ixodes scapularis and Ixodes pacificus (Acari: Ixodidae).

The incidence and geographic range of tick-borne illness associated with Ixodes scapularis and Ixodes pacificus have dramatically increased in recent decades. Anaplasmosis, babesiosis, and Borrelia spirochete infections, including Lyme borreliosis, account for tens of thousands of reported cases of tick-borne disease every year. Assays that reliably detect pathogens in ticks allow investigators and public health agencies to estimate the geographic distribution of human pathogens, assess geographic variation in their prevalence, and evaluate the effectiveness of prevention strategies. As investigators continue to describe new species within the Borrelia burgdorferi sensu lato complex and to recognize some Ixodes-borne Borrelia species as human pathogens, assays are needed to detect and differentiate these species. Here we describe an algorithm to detect and differentiate pathogens in unfed I. scapularis and I. pacificus nymphs including Anaplasma phagocytophilum, Babesia microti, Borrelia burgdorferi sensu stricto, Borrelia mayonii, and Borrelia miyamotoi. The algorithm comprises 5 TaqMan real-time polymerase chain reaction assays and 3 sequencing protocols. It employs multiple targets for each pathogen to optimize specificity, a gene target for I. scapularis and I. pacificus to verify tick-derived DNA quality, and a pan-Borrelia target to detect Borrelia species that may emerge as human disease agents in the future. We assess the algorithm's sensitivity, specificity, and performance on field-collected ticks.

Evaluating acarological risk for exposure to Ixodes scapularis and Ixodes scapularis-borne pathogens in recreational and residential settings in Washington County, Minnesota.

The distribution of I. scapularis, the tick vector of the bacteria that cause Lyme disease, has been expanding over the last two decades in the north-central United States in parallel with increasing incidence of human cases of Lyme disease in that region. However, assessments of residential risk for exposure to ticks are lacking from this region. Here, we measured the density of host-seeking I. scapularis nymphs in two suburban and two rural public recreational sites located in Washington County, Minnesota as well as in nearby residential properties. We sought to compare tick densities across land use types and to identify environmental factors that might impact nymphal density. We also assessed the prevalence of infection in the collected ticks with Lyme disease spirochetes (Borrelia burgdorferi sensu stricto, B. mayonii), and other I. scapularis-borne pathogens including B. miyamotoi, Babesia microti and Anaplasma phagocytophilum. Similar to studies from the eastern United States, on residential properties, I. scapularis nymphal densities were highest in the ecotonal areas between the forest edge and the lawn. Residences with the highest densities of nymphs were more likely to have a higher percentage of forest cover, log piles, and signs of deer on their property. In recreational areas, we found the highest nymphal densities both in the wooded areas next to trails as well as on mowed trails. Among the 303 host-seeking I. scapularis nymphs tested for pathogens, B. burgdorferi sensu stricto, A. phagocytophilum and B. miyamotoi were detected in 42 (13.8%), 14 (4.6%), and 2 (0.6%) nymphs, respectively.

Isolation of the Lyme Disease Spirochete Borrelia mayonii From Naturally Infected Rodents in Minnesota.

Borrelia mayonii is a newly described member of the Borrelia burgdorferi sensu lato complex that is vectored by the black-legged tick (Ixodes scapularis Say) and a cause of Lyme disease in Minnesota and Wisconsin. Vertebrate reservoir hosts involved in the enzootic maintenance of B. mayonii have not yet been identified. Here, we describe the first isolation of B. mayonii from naturally infected white-footed mice (Peromyscus leucopus Rafinesque) and an American red squirrel (Tamiasciurus hudsonicus Erxleben) from Minnesota, thus implicating these species as potential reservoir hosts for this newly described spirochete.

Prevalence and Diversity of Tick-Borne Pathogens in Nymphal Ixodes scapularis (Acari: Ixodidae) in Eastern National Parks.

Tick-borne pathogens transmitted by Ixodes scapularis Say (Acari: Ixodidae), also known as the deer tick or blacklegged tick, are increasing in incidence and geographic distribution in the United States. We examined the risk of tick-borne disease exposure in 9 national parks across six Northeastern and Mid-Atlantic States and the District of Columbia in 2014 and 2015. To assess the recreational risk to park visitors, we sampled for ticks along frequently used trails and calculated the density of I. scapularis nymphs (DON) and the density of infected nymphs (DIN). We determined the nymphal infection prevalence of I. scapularis with a suite of tick-borne pathogens including Borrelia burgdorferi, Borrelia miyamotoi, Anaplasma phagocytophilum, and Babesia microti. Ixodes scapularis nymphs were found in all national park units; DON ranged from 0.40 to 13.73 nymphs per 100 m2. Borrelia burgdorferi, the causative agent of Lyme disease, was found at all sites where I. scapularis was documented; DIN with B. burgdorferi ranged from 0.06 to 5.71 nymphs per 100 m2. Borrelia miyamotoi and A. phagocytophilum were documented at 60% and 70% of the parks, respectively, while Ba. microti occurred at just 20% of the parks. Ixodes scapularis is well established across much of the Northeastern and Mid-Atlantic States, and our results are generally consistent with previous studies conducted near the areas we sampled. Newly established I. scapularis populations were documented in two locations: Washington, D.C. (Rock Creek Park) and Greene County, Virginia (Shenandoah National Park). This research demonstrates the potential risk of tick-borne pathogen exposure in national parks and can be used to educate park visitors about the importance of preventative actions to minimize tick exposure.

Paired real-time PCR assays for detection of Borrelia miyamotoi in North American Ixodes scapularis and Ixodes pacificus (Acari: Ixodidae).

Borrelia miyamotoi is an emerging, tick-borne human pathogen. In North America, it is primarily associated with Ixodes scapularis and Ixodes pacificus, two species known to bite humans. Here we describe the development and evaluation of a pair of real-time TaqMan PCR assays designed to detect B. miyamotoi in North American ticks. We sought to achieve sensitivity to B. miyamotoi strains associated with ticks throughout North America, the full genetic diversity of which is unknown, by targeting sequences that are largely conserved between B. miyamotoi strains from the eastern United States and genetically distinct B. miyamotoi strains from Japan. The two assays target different loci on the B. miyamotoi chromosome and can be run side by side under identical cycling conditions. One of the assays also includes a tick DNA target that can be used to verify the integrity of tick-derived samples. Using both recombinant plasmid controls and genomic DNA from North American and Japanese strains, we determined that both assays reliably detect as few as 5 copies of the B. miyamotoi genome. We verified that neither detects B. burgdorferi, B. lonestari or B. turicatae. This sensitive and specific pair of assays successfully detected B. miyamotoi in naturally-infected, colony-reared nymphs and in field-collected I. scapularis and I. pacificus from the Northeast and the Pacific Northwest respectively. These assays will be useful in screening field-collected Ixodes spp. from varied regions of North America to assess the risk of human exposure to this emerging pathogen.

Evaluation of the effect of host immune status on short-term Yersinia pestis infection in fleas with implications for the enzootic host model for maintenance of Y. pestis during interepizootic periods.

Plague, a primarily flea-borne disease caused by Yersinia pestis, is characterized by rapidly spreading epizootics separated by periods of quiescence. Little is known about how and where Y. pestis persists between epizootics. It is commonly proposed, however, that Y pestis is maintained during interepizootic periods in enzootic cycles involving flea vectors and relatively resistant host populations. According to this model, while susceptible individuals serve as infectious sources for feeding fleas and subsequently die of infection, resistant hosts survive infection, develop antibodies to the plague bacterium, and continue to provide bloodmeals to infected fleas. For Y. pestis to persist under this scenario, fleas must remain infected after feeding on hosts carrying antibodies to Y. pestis. Studies of other vector-borne pathogens suggest that host immunity may negatively impact pathogen survival in the vector. Here, we report infection rates and bacterial loads for fleas (both Xenopsylla cheopis (Rothschild) and Oropsylla montana (Baker)) that consumed an infectious bloodmeal and subsequently fed on an immunized or age-matched naive mouse. We demonstrate that neither the proportion of infected fleas nor the bacterial loads in infected fleas were significantly lower within 3 d of feeding on immunized versus naive mice. Our findings thus provide support for one assumption underlying the enzootic host model of interepizootic maintenance of Y. pestis.