Lyme disease has expanded into the Great Plains of the USA. To investigate local enzootic transmission, small mammals were trapped in two forested tracts in northeastern North Dakota during 2012 and 2013. Peromyscus mice and southern red-backed voles, Myodes gapperi, comprised over 90% of all mammals captured. One site was dominated by Peromyscus (79% of 100 mammals captured). At the other site, M. gapperi (59% of 107 mammals captured) was more abundant than Peromyscus (36%). Immature stages of two tick species parasitized small mammals: Dermacentor variabilis and Ixodes scapularis. Larval I. scapularis ectoparasitism was significantly higher on Peromyscus (81% infested; 3.7 larvae per infested mouse) than M. gapperi (47% infested; 2.6 larvae per infested vole) whereas larval and nymphal D. variabilis ectoparasitism were highest on M. gapperi. Over 45% of infested rodents were concurrently infested with both tick species. Testing engorged I. scapularis larvae from Peromyscus (n = 66) and M. gapperi (n = 20) yielded xenopositivity prevalence for Borrelia burgdorferi sensu lato (s.l.) in these rodents of 6% and 5%, respectively. Progeny of field collected M. gapperi were used to determine host infectivity for a local isolate of B. burgdorferi sensu stricto (s.s.). Five M. gapperi were injected with spirochetes, infested with pathogen-free I. scapularis larvae on days 10, 20, and 40 after infection, and engorged larvae molted to nymphs. Subsamples of nymphs were tested by PCR for B. burgdorferi s. s. DNA and yielded infection rates of 56% (n = 100 nymphs tested), 75% (n = 8) and 64% (n = 31), respectively. The remaining infected nymphs were fed on BALB/c Mus musculus mice and 7 d later, mice were euthanized, and tissues were cultured for B. burgdorferi s.s. Nymphs successfully transmitted spirochetes to 13 of 18 (72%) mice that were exposed to 1-5 infected ticks. Theoretical reservoir potentials - i.e., ability to generate B. burgdorferi infected nymphs - were compared between Peromyscus and M. gapperi. At one site, Peromyscus accounted for nearly all Borrelia-infected nymphs produced (reservoir potential value of 0.935). At the other site, the reservoir potentials for Peromyscus (0.566) and M. gapperi (0.434) were comparable. The difference was attributed to differences in the relative abundance of voles versus mice between sites and the higher level of ectoparasitism by larval I. scapularis on Peromyscus versus M. gapperi at both sites. The southern red-backed vole, M. gapperi, contributes to the enzootic maintenance of Lyme disease spirochetes in North Dakota and possibly other areas where this rodent species is abundant.

Ixodes scapularis is a blood-feeding obligate ectoparasite responsible for transmitting the Lyme disease (LD) agent, Borrelia burgdorferi. During the feeding process, I. scapularis injects B. burgdorferi into the host along with its saliva, facilitating the transmission and colonization of the LD agent. Tick calreticulin (CRT) is one of the earliest tick saliva proteins identified and is currently utilized as a biomarker for tick bites. Our recent findings revealed elevated levels of CRT in the saliva proteome of B. burgdorferi-infected I. scapularis nymphs compared to uninfected ticks. Differential precipitation of proteins (DiffPOP) and LC-MS/MS analyses were used to identify the interactions between Ixs (I. scapularis) CRT and human plasma proteins and further explore its potential role in shielding B. burgdorferi from complement killing. We observed that although yeast-expressed recombinant (r) IxsCRT binds to the C1 complex (C1q, C1r, and C1s), the activator of complement via the classical cascade, it did not inhibit the deposition of the membrane attack complex (MAC) via the classical pathway. Intriguingly, rIxsCRT binds intermediate complement proteins (C3, C5, and C9) and reduces MAC deposition through the lectin pathway. Despite the inhibition of MAC deposition in the lectin pathway, rIxsCRT did not protect a serum-sensitive B. burgdorferi strain (B314/pBBE22Luc) from complement-induced killing. As B. burgdorferi establishes a local dermal infection before disseminating to secondary organs, it is noteworthy that rIxsCRT promotes the replication of B. burgdorferi in culture. We hypothesize that rIxsCRT may contribute to the transmission and/or host colonization of B. burgdorferi by acting as a decoy activator of complement and by fostering B. burgdorferi replication at the transmission site.

Ixodes scapularis, the black-legged tick, is a major arthropod vector that transmits the causative agents of Lyme disease and several other pathogens of human significance. The tick midgut is the main tissue involved in blood acquisition and digestion and the first organ to have contact with pathogens ingested through the blood meal. Gene expression in the midgut before, during, and after a blood meal may vary in response to the physiological changes due to blood feeding. A systems biology approach based on RNA and protein sequencing was used to gain insight into the changes in tick midgut transcripts and proteins during blood ingestion (unfed and partially fed) and digestion (1-, 2-, 7-, and 14 days post detachment from the host) by the Ixodes scapularis female ticks. A total of 2,726 differentially expressed transcripts, and 449 proteins were identified across the time points. Genes involved in detoxification of xenobiotics, proteases, protease inhibitors, metabolism, and immunity were differentially expressed in response to blood feeding. Similarly, proteins corresponding to the same groups were also differentially expressed. Nine genes from major gene categories were chosen as potential vaccine candidates, and, using RNA interference, the effect of these gene knockdowns on tick biology was investigated. Knockdown of these genes had variable negative impacts on tick physiology, such as the inability to engorge fully and to produce eggs and increased mortality. These and additional gene targets provide opportunities to explore novel tick control strategies.

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.

A promising alternative approach to conventional vector and rodent control practices is the use of a bait containing a rodenticide and acaricide in controlling vectors and pathogen reservoirs concurrently. In the United States, Lyme disease continues to be the most prevalent vector-borne disease with approximately 500,000 Lyme disease cases estimated each year. Previous research has demonstrated the usefulness of a low dose fipronil bait in controlling Ixodes scapularis larvae feeding on white-footed mice. However, considering white-footed mice can be an unwanted species because of their association with tick-borne disease and hantaviruses, a combination rodent and tick bait (RTB) might provide a useful alternative to encourage additional community participation in integrated tick management (ITM) efforts. The purpose of this research was to evaluate the use of RTB (0.025 % warfarin, 0.005 % fipronil) in controlling white-footed mice and I. scapularis larvae. Studies were designed in part based on Environmental Protection Agency (EPA) guidelines. A laboratory choice test was conducted to evaluate the use of RTB in controlling white-footed mice over 15-day exposure when they were exposed to an alternative diet. Mice were observed every day for mortality and signs of warfarin toxicity. A simulated field test was conducted to evaluate the use of RTB, presented in the presence of an alternative diet, in controlling I. scapularis parasitizing white-footed mice over 4-day exposure. Mice were fitted with capsules and manually infested with I. scapularis larvae. The inside of each capsule was observed to evaluate tick attachment. Replete larvae detaching from each mouse were collected. Blood was collected from all treatment group mice via cardiac puncture to determine the fipronil sulfone concentration in plasma for each animal. Results indicated that RTB would be adequately consumed in the presence of an alternative diet under laboratory and simulated field conditions. Treatment with RTB resulted in 100 % mortality of white-footed mice during 15-day exposure and prevented 100 % larvae from feeding to repletion during 4-day exposure. All mice succumbing to RTB showed signs of warfarin toxicity. All mice parasitized with ticks that were exposed to RTB had fipronil sulfone detectable in plasma, with even the lowest concentration detected (8.1 parts per billion) controlling 100 % parasitizing I. scapularis larvae. The results suggest that RTB could be a useful means of rodent and tick control for use in ITM programs.

Emerging tick-borne viruses such as Powassan virus (POWV), Bourbon virus (BRBV), and Heartland virus (HRTV), whilst rare, can cause severe health problems in humans. While limited clinical cases have been reported thus far in Virginia, the presence of tick-borne viruses poses a serious health threat, and the extent of their prevalence in Virginia is unknown. Here, we sought evidence of POWV, BRBV, and HRTV exposure in Virginia via a serological assessment of wildlife and livestock. Wildlife in Virginia were found to be seropositive against POWV (18%), BRBV (8%), and HRTV (5%), with western and northern regions of the state having a higher prevalence. Multiple wildlife species were shown to have been exposed to each virus examined. To a lesser extent, cattle also showed exposure to tick-borne viruses, with seroprevalences of 1%, 1.2%, and 8% detected in cattle against POWV, BRBV, and HRTV, respectively. Cross-reactivity against other known circulating mosquito-borne flaviviruses was ruled out. In conclusion, there is widespread exposure to tick-borne viruses in western and northern Virginia, with exposure to a diverse range of animal populations. Our study provides the first confirmation that HRTV is circulating in the Commonwealth. These findings strengthen the existing evidence of emerging tick-borne viruses in Virginia and highlight the need for public health vigilance to avoid tick bites.

Background: Tick-borne diseases are an emerging threat to public health throughout the temperate world, leading to a growing field of research aimed at developing and testing intervention strategies for reducing human-tick encounters or prevalence of infection in ticks. Various wide-spectrum chemical acaricides have proven effective for controlling tick populations, but many of these have potential deleterious side-effects on health and the environment. In addition to chemical acaricides, certain compounds such as diatomaceous earth have been shown to have physical acaricidal properties. We hypothesized that dolomitic lime (CaMg(CO3)2, a corrosive, desiccant mineral that is already used extensively in agricultural and forestry contexts to balance the pH of soils, may affect ticks\' locomotory activity, habitat position, or survival and that this should manifest as a reduction in the number of questing ticks collected by dragging. Objective: This study aimed to formally assess this hypothesis in a controlled laboratory setting. Methods: We carried out a microcosm experiment, with one control and three treated microcosm trays, each replicating the natural substrate characterizing I. scapularis habitat in northeastern North America. Each tray was infested with 200 living larvae and 50 nymphs, and then treated with 0 (control), 50, 100, or 500 g/m2 of lime powder. Ticks were collected by microdragging 24 and 72 h postliming. Results: Efficacy of liming at reducing the number of collected questing ticks ranged from 87% to 100% for larvae and 0% to 69% for nymphs 24 h postliming and from 91% to 93% for larvae and -47% to 65% for nymphs 72 postliming. Conclusion: This study provides the first experimental evidence of the potential efficacy of liming for impairing activity of questing immature ticks. Given that lime is a low-cost material, that methods for widespread application in deciduous woodlands already exist, and that it has been documented as having a limited negative impact on the environment, further assessment of lime application as a public health risk reduction intervention for tick-borne diseases is warranted.

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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 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.

Powassan virus (POWV) is a tick-borne flavivirus endemic in North America and Russia. Experimental infections with POWV have confirmed horizontal, transstadial, vertical, and cofeeding transmission routes for potential virus maintenance. In the field, vertical transmission has never been observed. During New York State tick-borne pathogen surveillance, POWV RNA and/or infectious POWV was detected in five pools of questing Ixodes scapularis larvae. Additionally, engorged female I. scapularis adults were collected from hunter-harvested white-tailed deer (Odocoileus virginianus) in a region with relatively high tick infection rates of POWV and allowed to oviposit under laboratory conditions. POWV RNA was detected in three female adult husks and one pool of larvae from a positive female. Infectious virus was isolated from all three RNA-positive females and the single positive larval pool. The detection of RNA and infectious virus in unfed questing larvae from the field and larvae from replete females collected from the primary tick host implicates vertical transmission as a potential mechanism for the maintenance of POWV in I. scapularis in nature, and elucidates the potential epidemiological significance of larval ticks in the transmission of POWV to humans.