Phylogeographic reconstruction of the emergence and spread of Powassan virus in the northeastern United States.

Powassan virus is an emerging tick-borne virus of concern for public health, but very little is known about its transmission patterns and ecology. Here, we expanded the genomic dataset by sequencing 279 Powassan viruses isolated from Ixodes scapularis ticks from the northeastern United States. Our phylogeographic reconstructions revealed that Powassan virus lineage II was likely introduced or emerged from a relict population in the Northeast between 1940 and 1975. Sequences strongly clustered by sampling location, suggesting a highly focal geographical distribution. Our analyses further indicated that Powassan virus lineage II emerged in the northeastern United States mostly following a south-to-north pattern, with a weighted lineage dispersal velocity of ~3 km/y. Since the emergence in the Northeast, we found an overall increase in the effective population size of Powassan virus lineage II, but with growth stagnating during recent years. The cascading effect of population expansion of white-tailed deer and I. scapularis populations likely facilitated the emergence of Powassan virus in the northeastern United States.

Comparative reservoir competence of Peromyscus leucopus, C57BL/6J, and C3H/HeN for Borrelia burgdorferi B31.

Borrelia burgdorferi, a Lyme disease spirochete, causes a range of acute and chronic maladies in humans. However, a primary vertebrate reservoir in the United States, the white-footed deermouse Peromyscus leucopus, is reported not to have reduced fitness following infection. Although laboratory strains of Mus musculus mice have successfully been leveraged to model acute human Lyme disease, the ability of these rodents to model B. burgdorferi-P. leucopus interactions remains understudied. Here, we compared infection of P. leucopus with B. burgdorferi B31 with infection of the traditional B. burgdorferi murine models-C57BL/6J and C3H/HeN Mus musculus, which develop signs of inflammation akin to human disease. We find that B. burgdorferi was able to reach much higher burdens (10- to 30-times higher) in multiple M. musculus skin sites and that the overall dynamics of infection differed between the two rodent species. We also found that P. leucopus remained transmissive to larval Ixodes scapularis for a far shorter period than either M. musculus strain. In line with these observations, we found that P. leucopus does launch a modest but sustained inflammatory response against B. burgdorferi in the skin, which we hypothesize leads to reduced bacterial viability and rodent-to-tick transmission in these hosts. Similarly, we also observe evidence of inflammation in infected P. leucopus hearts. These observations provide new insight into reservoir species and the B. burgdorferi enzootic cycle.IMPORTANCEA Lyme disease-causing bacteria, Borrelia burgdorferi, must alternate between infecting a vertebrate host-usually rodents or birds-and ticks. In order to be successful in that endeavor, the bacteria must avoid being killed by the vertebrate host before it can infect a new larval tick. In this work, we examine how B. burgdorferi and one of its primary vertebrate reservoirs, Peromyscus leucopus, interact during an experimental infection. We find that B. burgdorferi appears to colonize its natural host less successfully than conventional laboratory mouse models, which aligns with a sustained seemingly anti-bacterial response by P. leucopus against the microbe. These data enhance our understanding of P. leucopus host-pathogen interactions and could potentially serve as a foundation to uncover ways to disrupt the spread of B. burgdorferi in nature.

Genome resequencing reveals population divergence and local adaptation of blacklegged ticks in the United States.

Tick vectors and tick-borne disease are increasingly impacting human populations globally. An important challenge is to understand tick movement patterns, as this information can be used to improve management and predictive modelling of tick population dynamics. Evolutionary analysis of genetic divergence, gene flow and local adaptation provides insight on movement patterns at large spatiotemporal scales. We develop low coverage, whole genome resequencing data for 92 blacklegged ticks, Ixodes scapularis, representing range-wide variation across the United States. Through analysis of population genomic data, we find that tick populations are structured geographically, with gradual isolation by distance separating three population clusters in the northern United States, southeastern United States and a unique cluster represented by a sample from Tennessee. Populations in the northern United States underwent population contractions during the last glacial period and diverged from southern populations at least 50 thousand years ago. Genome scans of selection provide strong evidence of local adaptation at genes responding to host defences, blood-feeding and environmental variation. In addition, we explore the potential of low coverage genome sequencing of whole-tick samples for documenting the diversity of microbial pathogens and recover important tick-borne pathogens such as Borrelia burgdorferi. The combination of isolation by distance and local adaptation in blacklegged ticks demonstrates that gene flow, including recent expansion, is limited to geographical scales of a few hundred kilometres.

Resource selection by New York City deer reveals the effective interface between wildlife, zoonotic hazards and humans.

Although the role of host movement in shaping infectious disease dynamics is widely acknowledged, methodological separation between animal movement and disease ecology has prevented researchers from leveraging empirical insights from movement data to advance landscape scale understanding of infectious disease risk. To address this knowledge gap, we examine how movement behaviour and resource utilization by white-tailed deer (Odocoileus virginianus) determines blacklegged tick (Ixodes scapularis) distribution, which depend on deer for dispersal in a highly fragmented New York City borough. Multi-scale hierarchical resource selection analysis and movement modelling provide insight into how deer's movements contribute to the risk landscape for human exposure to the Lyme disease vector-I. scapularis. We find deer select highly vegetated and accessible residential properties which support blacklegged tick survival. We conclude the distribution of tick-borne disease risk results from the individual resource selection by deer across spatial scales in response to habitat fragmentation and anthropogenic disturbances.

Characteristics of Hard Tick Relapsing Fever Caused by Borrelia miyamotoi, United States, 2013-2019.

Borrelia miyamotoi, transmitted by Ixodes spp. ticks, was recognized as an agent of hard tick relapsing fever in the United States in 2013. Nine state health departments in the Northeast and Midwest have conducted public health surveillance for this emerging condition by using a shared, working surveillance case definition. During 2013-2019, a total of 300 cases were identified through surveillance; 166 (55%) were classified as confirmed and 134 (45%) as possible. Median age of case-patients was 52 years (range 1-86 years); 52% were male. Most cases (70%) occurred during June-September, with a peak in August. Fever and headache were common symptoms; 28% of case-patients reported recurring fevers, 55% had arthralgia, and 16% had a rash. Thirteen percent of patients were hospitalized, and no deaths were reported. Ongoing surveillance will improve understanding of the incidence and clinical severity of this emerging disease.

Associations of Anaplasma phagocytophilum Bacteria Variants in Ixodes scapularis Ticks and Humans, New York, USA.

Anaplasmosis, caused by the tickborne bacterium Anaplasma phagocytophilum, is an emerging public health threat in the United States. In the northeastern United States, the blacklegged tick (Ixodes scapularis) transmits the human pathogenic genetic variant of A. phagocytophilum (Ap-ha) and a nonpathogenic variant (Ap-V1). New York has recently experienced a rapid and geographically focused increase in cases of anaplasmosis. We analyzed A. phagocytophilum-infected I. scapularis ticks collected across New York during 2008-2020 to differentiate between variants and calculate an entomological risk index (ERI) for each. Ap-ha ERI varied between regions and increased in all regions during the final years of the study. Space-time scan analyses detected expanding clusters of Ap-ha located within documented anaplasmosis hotspots. Ap-ha ERI was more positively correlated with anaplasmosis incidence than non-genotyped A. phagocytophilum ERI. Our findings help elucidate the relationship between the spatial ecology of A. phagocytophilum variants and anaplasmosis.

Riboflavin salvage by Borrelia burgdorferi supports carbon metabolism and is essential for survival in the tick vector.

The Lyme disease agent, Borrelia burgdorferi, harbors a significantly reduced genome and relies on the scavenging of critical nutrients from its tick and mammalian hosts for survival. Riboflavin salvage has been shown to be important for B. burgdorferi infection of mice, yet the contributions of riboflavin to B. burgdorferi metabolism and survival in the tick remain unknown. Using a targeted mass spectrometry approach, we confirmed the importance of bb0318, the putative ATPase component of an ABC-type riboflavin transporter, for riboflavin salvage and the production of FMN and FAD. This analysis further revealed that Δbb0318 B. burgdorferi displayed increased levels of glycerol 3-phosphate compared to the wild-type. The glycerol 3-phosphate dehydrogenase activity of GlpD was found to be FAD-dependent and the transcription and translation of glpD were significantly decreased in Δbb0318 B. burgdorferi. Finally, gene bb0318 was found to be important for maximal spirochete burden in unfed larvae and essential for survival in feeding ticks. Together, these data demonstrate the importance of riboflavin salvage for B. burgdorferi carbon metabolism and survival in ticks.

Limited detection of shared zoonotic pathogens in deer keds and blacklegged ticks co-parasitizing white-tailed deer in the eastern United States.

Deer keds, such as Lipoptena cervi Linnaeus (Diptera: Hippoboscidae), are blood-feeding flies from which several human and animal pathogens have been detected, including Borrelia burgdorferi sensu lato Johnson (Spirochaetales: Borreliaceae), the causative agent of Lyme disease. Cervids (Artiodactyla: Cervidae), which are the primary hosts of deer keds, are not natural reservoirs of B. burgdorferi sl, and it has been suggested that deer keds may acquire bacterial pathogens via co-feeding near infected ticks. We screened L. cervi (n = 306) and Ixodes scapularis Say (Ixodida: Ixodidae) (n = 315) collected from 38 white-tailed deer in Pennsylvania for the family Anaplasmataceae, Bartonella spp. (Hyphomicrobiales: Bartonellaceae), Borrelia spp., and Rickettsia spp. (Rickettsiales: Rickettsiaceae). Limited similarity in the bacterial DNA detected between these ectoparasites per host suggested that co-feeding may not be a mechanism by which deer keds acquire these bacteria. The feeding biology and life history of deer keds may impact the observed results, as could the season when specimens were collected. We separately screened L. cervi (n = 410), L. mazamae Róndani (n = 13), L. depressa Say (n = 10), and Neolipoptena ferrisi Bequaert (n = 14) collections from locations within the United States and Canada for the same pathogens. These results highlight the need to further study deer ked-host and deer ked-tick relationships.

Cytochrome b Drug Resistance Mutation Decreases Babesia Fitness in the Tick Stages But Not the Mammalian Erythrocytic Cycle.

Human babesiosis is an emerging tick-borne malaria-like illness caused by Babesia parasites following their development in erythrocytes. Here, we show that a mutation in the Babesia microti mitochondrial cytochrome b (Cytb) that confers resistance to the antibabesial drug ELQ-502 decreases parasite fitness in the arthropod vector. Interestingly, whereas the mutant allele does not affect B. microti fitness during the mammalian blood phase of the parasite life cycle and is genetically stable as parasite burden increases, ELQ-502-resistant mutant parasites developing in the tick vector are genetically unstable with a high rate of the wild-type allele emerging during the nymphal stage. Furthermore, we show that B. microti parasites with this mutation are transmitted from the tick to the host, raising the possibility that the frequency of Cytb resistance mutations may be decreased by passage through the tick vector, but could persist in the environment if present when ticks feed.

Effects of Tick-Control Interventions on Tick Abundance, Human Encounters with Ticks, and Incidence of Tickborne Diseases in Residential Neighborhoods, New York, USA.

Tickborne diseases (TBDs) such as Lyme disease result in ≈500,000 diagnoses annually in the United States. Various methods can reduce the abundance of ticks at small spatial scales, but whether these methods lower incidence of TBDs is poorly understood. We conducted a randomized, replicated, fully crossed, placebo-controlled, masked experiment to test whether 2 environmentally safe interventions, the Tick Control System (TCS) and Met52 fungal spray, used separately or together, affected risk for and incidence of TBDs in humans and pets in 24 residential neighborhoods. All participating properties in a neighborhood received the same treatment. TCS was associated with fewer questing ticks and fewer ticks feeding on rodents. The interventions did not result in a significant difference in incidence of human TBDs but did significantly reduce incidence in pets. Our study is consistent with previous evidence suggesting that reducing tick abundance in residential areas might not reduce incidence of TBDs in humans.

Lyme Disease, Anaplasmosis, and Babesiosis, Atlantic Canada.

In July 2021, a PCR-confirmed case of locally acquired Babesia microti infection was reported in Atlantic Canada. Clinical features were consistent with babesiosis and resolved after treatment. In a region where Lyme disease and anaplasmosis are endemic, the occurrence of babesiosis emphasizes the need to enhance surveillance of tickborne infections.

Economic Burden of Reported Lyme Disease in High-Incidence Areas, United States, 2014-2016.

Approximately 476,000 cases of Lyme disease are diagnosed in the United States annually, yet comprehensive economic evaluations are lacking. In a prospective study among reported cases in Lyme disease-endemic states, we estimated the total patient cost and total societal cost of the disease. In addition, we evaluated disease and demographic factors associated with total societal cost. Participants had a mean patient cost of ≈$1,200 (median $240) and a mean societal cost of ≈$2,000 (median $700). Patients with confirmed disseminated disease or probable disease had approximately double the societal cost of those with confirmed localized disease. The annual, aggregate cost of diagnosed Lyme disease could be $345-968 million (2016 US dollars) to US society. Our findings emphasize the importance of effective prevention and early diagnosis to reduce illness and associated costs. These results can be used in cost-effectiveness analyses of current and future prevention methods, such as a vaccine.

Thermostable Keystone Bacteria Maintain the Functional Diversity of the Ixodes scapularis Microbiome Under Heat Stress.

Variations in the composition and diversity of tick microbiome due to high temperatures may influence the hierarchy of community members as a response to environmental change. Modifications in the community structure are hypothesized to drive alterations in the presence and/or abundance of functional pathways in the bacterial metagenome. In this study, this hypothesis was tested by using published 16S rRNA datasets of Ixodes scapularis males incubated at different temperatures (i.e., 4, 20, 30, and 37 °C) in a laboratory setting. Changes in community structure and functional profiles in response to temperature shifts were measured using co-occurrence networks and metagenome inference. Results from laboratory-reared ticks were then compared with those of field-collected ticks. The results from laboratory-reared ticks showed that high temperature altered the structure of the microbial community and decreased the number of keystone taxa. Notably, four taxa were identified as keystone in all the temperatures, and the functional diversity of the tick microbiome was contained in the four thermostable keystone their associated bacterial taxa. Three of the thermostable keystone taxa were also found in free-living ticks collected in Massachusetts. Moreover, the comparison of functional profiles of laboratory-reared and field-collected ticks revealed the existence of an important set of metabolic pathways that were common among the different datasets. Similar to the laboratory-reared ticks, the keystone taxa identified in field-collected ticks alongside their consortia (co-occurring taxa) were sufficient to retain the majority of the metabolic pathways in the functional profile. These results suggest that keystone taxa are essential in the stability and the functional resiliency of the tick microbiome under heat stress.

Tick infestation effects on haemoglobin levels of deer mice (Peromyscus maniculatus).

Deer mice (Peromyscus maniculatus) are hosts to ixodid ticks as well as the associated tick-borne pathogens they can spread. As the ranges of black-legged ticks (Ixodes scapularis) and American dog ticks (Dermacentor variabilis) expand northwards, naïve host populations of deer mice are likely to become infested by ticks and experience the physiological effects that ticks can have on them via blood-feeding. The prevalence of these haematophagous ticks can affect the haemoglobin levels of the mice they infest. Haemoglobin levels were compared and analysed in deer mice populations at three different sites with varying tick exposure. These results suggested that without confounding effects, the abundance of black-legged and American dog ticks on individual mice had a significant negative effect on the hosts' haemoglobin levels, but only in an area with high tick infestation. This was seen across the average haemoglobin levels between populations, where there was a significant difference between the source population with the longest established tick populations and the source population where neither black-legged nor American dog ticks were prevalent. As the ticks' ranges expand and they become more abundant, it is important to understand how their prevalence and intensity can alter host physiology, potentially affecting their own range expansion and the spread of the diseases they may carry.

Integrating tick density and park visitor behaviors to assess the risk of tick exposure in urban parks on Staten Island, New York.

BACKGROUND: Public green spaces are important for human health, but they may expose visitors to ticks and tick-borne pathogens. We sought to understand, for the first time, visitors' exposure risk and drivers of tick-preventative behavior in three popular parks on Staten Island, New York City, NY, USA, by integrating tick hazard and park visitors' behaviors, risk perceptions and knowledge. METHODS: We conducted tick sampling in three parks, across three site types (open spaces, the edge of open spaces, and trails) and three within-park habitats (maintained grass, unmaintained herbaceous, and leaf litter) to estimate tick density during May-August 2019. Human behavior was assessed by observations of time spent and activity type in each site. We integrated the time spent in each location by park visitors and the tick density to estimate the probability of human-tick encounter. To assess visitors' tick prevention behaviors, a knowledge, attitude, and practices (KAP) survey was administered. RESULTS: Three tick species (Ixodes scapularis, Amblyomma americanum and Haemaphysalis longicornis) were collected. For all species, the density of nymphs was greatest in unmaintained herbaceous habitats and trails, however, the fewest people entered these hazardous locations. The KAP survey revealed that most respondents (N = 190) identified parks as the main location for tick exposure, but most believed they had minimal risk for tick encounter. Consequently, many visitors did not conduct tick checks. People were most likely to practice tick checks if they knew multiple prevention methods and perceived a high likelihood of tick encounter. CONCLUSIONS: By integrating acarological indices with park visitor behaviors, we found a mismatch between areas with higher tick densities and areas more frequently used by park visitors. However, this exposure risk varied among demographic groups, the type of activities and parks, with a higher probability of human-tick encounters in trails compared to open spaces. Furthermore, we showed that people's KAP did not change across parks even if parks represented different exposure risks. Our research is a first step towards identifying visitor risk, attitudes, and practices that could be targeted by optimized messaging strategies for tick bite prevention among park visitors.

Identification of microRNAs in the Lyme Disease Vector Ixodes scapularis.

MicroRNAs (miRNAs) are a class of small non-coding RNAs involved in many biological processes, including the immune pathways that control bacterial, parasitic, and viral infections. Pathogens probably modify host miRNAs to facilitate successful infection, so they might be useful targets for vaccination strategies. There are few data on differentially expressed miRNAs in the black-legged tick Ixodes scapularis after infection with Borrelia burgdorferi, the causative agent of Lyme disease in the United States. Small RNA sequencing and qRT-PCR analysis were used to identify and validate differentially expressed I. scapularis salivary miRNAs. Small RNA-seq yielded 133,465,828 (≥18 nucleotides) and 163,852,135 (≥18 nucleotides) small RNA reads from Borrelia-infected and uninfected salivary glands for downstream analysis using the miRDeep2 algorithm. As such, 254 miRNAs were identified across all datasets, 25 of which were high confidence and 51 low confidence known miRNAs. Further, 23 miRNAs were differentially expressed in uninfected and infected salivary glands: 11 were upregulated and 12 were downregulated upon pathogen infection. Gene ontology and network analysis of target genes of differentially expressed miRNAs predicted roles in metabolic, cellular, development, cellular component biogenesis, and biological regulation processes. Several Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways, including sphingolipid metabolism; valine, leucine and isoleucine degradation; lipid transport and metabolism; exosome biogenesis and secretion; and phosphate-containing compound metabolic processes, were predicted as targets of differentially expressed miRNAs. A qRT-PCR assay was utilized to validate the differential expression of miRNAs. This study provides new insights into the miRNAs expressed in I. scapularis salivary glands and paves the way for their functional manipulation to prevent or treat B. burgdorferi infection.

Rickettsial Pathogen Perturbs Tick Circadian Gene to Infect the Vertebrate Host.

Ixodes scapularis is a medically important tick that transmits several microbes to humans, including rickettsial pathogen Anaplasma phagocytophilum. In nature, these ticks encounter several abiotic factors including changes in temperature, humidity, and light. Many organisms use endogenously generated circadian pathways to encounter abiotic factors. In this study, we provide evidence for the first time to show that A. phagocytophilum modulates the arthropod circadian gene for its transmission to the vertebrate host. We noted a circadian oscillation in the expression of arthropod clock, bmal1, period and timeless genes when ticks or tick cells were exposed to alternate 12 h light: 12 h dark conditions. Moreover, A. phagocytophilum significantly modulates the oscillation pattern of expression of these genes. In addition, increased levels of clock and bmal1 and decreased expression of Toll and JAK/STAT pathway immune genes such as pelle and jak, respectively, were noted during A. phagocytophilum transmission from ticks to the vertebrate host. RNAi-mediated knockdown of clock gene expression in ticks resulted in the reduced expression of jak and pelle that increased bacterial transmission from ticks to the murine host. Furthermore, clock-deficient ticks fed late and had less engorgement weights. These results indicate an important role for circadian modulation of tick gene expression that is critical for arthropod blood feeding and transmission of pathogens from vector to the vertebrate host.

Repellency and toxicity of a CO2-derived cedarwood oil on hard tick species (Ixodidae).

The repellency and toxicity of a CO2-derived cedarwood oil (CWO) was evaluated against actively questing unfed nymphs of four species of hard ticks: Amblyomma americanum (L.), Dermacentor variabilis (Say), Ixodes scapularis Say, and Rhipicephalus sanguineus (Latreille). Using a vertical climb bioassay for repellency, nymphs of these species avoided a CWO-treated filter paper in proportional responses to treatment concentrations. At 60 min of exposure, I. scapularis nymphs were most sensitive with 50% repellency concentration (RC50) of 19.8 µg cm-2, compared with RC50 of 30.8, 83.8 and 89.6 µg cm-2 for R. sanguineus, D. variabilis and A. americanum, respectively. Bioassays determined the lethal concentration for 50% (LC50) and 90% (LC90) mortality of nymphs exposed to CWO in treated vials after 24- and 48-h exposure. After 24 h exposure, the LC50 values were 1.25, 3.45 and 1.42 µg cm-2 and LC90 values were 2.39, 7.59 and 4.14 µg cm-2 for D. variabilis, I. scapularis and R. sanguineus, respectively, but had minimal effect on A. americanum. After 48 h exposure, the LC50 values were 4.14, 0.78, 0.79 and 0.52 µg cm-2, and LC90 values were 8.06, 1.48, 1.54 and 1.22 µg cm-2 for A. americanum, D. variabilis, I. scapularis and R. sanguineus, respectively. The repellency of CWO on tick species decreased with time. The repellency and toxicity bioassays demonstrated concentration-dependent responses of tick nymphs to the oil, indicating the potential of the CO2-derived cedarwood oil be developed as an eco-friendly repellent and/or acaricide.

The Impacts of Climate Change on Ticks and Tick-Borne Disease Risk.

Ticks exist on all continents and carry more zoonotic pathogens than any other type of vector. Ticks spend most of their lives in the external environment away from the host and are thus expected to be affected by changes in climate. Most empirical and theoretical studies demonstrate or predict range shifts or increases in ticks and tick-borne diseases, but there can be a lot of heterogeneity in such predictions. Tick-borne disease systems are complex, and determining whether changes are due to climate change or other drivers can be difficult. Modeling studies can help tease apart and understand the roles of different drivers of change. Predictive models can also be invaluable in projecting changes according to different climate change scenarios. However, validating these models remains challenging, and estimating uncertainty in predictions is essential. Another focus for future research should be assessing the resilience of ticks and tick-borne pathogens to climate change.

Borrelia miyamotoi in Human-Biting Ticks, United States, 2013-2019.

During 2013-2019, Borrelia miyamotoi infection was detected in 19 US states. Infection rate was 0.5%-3.2%; of B. miyamotoi-positive ticks, 59.09% had concurrent infections. B. miyamotoi is homogeneous with 1 genotype from Ixodes scapularis ticks in northeastern and midwestern states and 1 from I. pacificus in western states.