Proficiency at Tick Identification by Pathologists and Clinicians Is Poor.

OBJECTIVES: Prompt accurate identification of tick species is required for appropriate administration of single dose antimicrobial prophylaxis for Lyme disease in selected patients. To determine the proficiency of clinicians at tick identification in the northeastern United States where Lyme disease has its highest incidence, we undertook a survey. METHODS: We analyzed the results of a voluntary survey testing proficiency in identifying tick species using high-resolution photographs of ticks. RESULTS: Only 35% of ticks were correctly identified. Although 60% of respondents could identify a nonengorged adult blacklegged tick, only 34% could correctly identify a partially engorged blacklegged tick. Participants performed even worse at classifying brown dog, American dog, and Lone Star ticks. CONCLUSIONS: Proficiency of tick identification by pathologists and clinicians is poor.

Identification of Hard Ticks in the United States: A Practical Guide for Clinicians and Pathologists.

ABSTRACT: According to guidelines published by the Infectious Disease Society of America, Lyme disease prophylaxis is possible if a tick can be identified as Ixodes scapularis (nymphal or adult) within 72 hours of tick removal. However, a recent survey of medical practitioners indicates generally poor proficiency in tick identification. In this study, we provide a simple, practical guide to aid medical practitioners in identifying the most commonly encountered human biting ticks of North America.

Tick-Borne Disease Prevention Behaviors Among Participants in a Tick Surveillance System Compared with a Sample Of Master Gardeners.

Theory-based approaches to health communication and behavior are increasingly applied to interventions that address poor public tick-borne disease prevention knowledge and practices. We sought to understand the tick-borne disease prevention behaviors among participants in a crowdsourced passive tick surveillance system that employs theory-based messages about tick bite risk and prevention strategies. We administered an electronic survey to a randomly selected sample of passive surveillance system users and compared their responses to those from a nationwide sample of Master Gardeners (MG), a group with heighten tick exposure due to outdoor activity. Over 80% of TickSpotters respondents, and over 75% of MG respondents encountered a tick in the past year. Among both groups, tick checks were the most frequently practiced prevention behavior, with over 70% of people performing them most or all the time after outdoor activity. A greater proportion of MGs used skin repellents such as DEET or picaridin than TickSpotters users, but more than 70% of respondents from both groups reported that they never or only sometimes use permethrin-treatment on clothing, and nearly half of both groups reportedly used no peridomestic tick treatments. TickSpotters respondents overwhelmingly reported recording tick encounter information and saving specimens for identification and testing, while only a small percentage of MGs monitored their tick encounters. These findings suggest that while both TickSpotters and MG groups appear to be practicing some important tick bite prevention behaviors, there remain areas that could benefit from targeted theory-based interventional approaches.

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.

Application of M13 phage display for identifying immunogenic proteins from tick (Ixodes scapularis) saliva.

BACKGROUND: Ticks act as vectors for a large number of different pathogens, perhaps most notably Borrelia burgdorferi, the causative agent of Lyme disease. The most prominent tick vector in the United States is the blacklegged tick, Ixodes scapularis. Tick bites are of special public health concern since there are no vaccines available against most tick-transmitted pathogens. Based on the observation that certain non-natural host animals such as guinea pigs or humans can develop adaptive immune responses to tick bites, anti-tick vaccination is a potential approach to tackle health risks associated with tick bites. RESULTS: The aim of this study was to use an oligopeptide phage display strategy to identify immunogenic salivary gland proteins from I. scapularis that are recognized by human immune sera. Oligopeptide libraries were generated from salivary gland mRNA of 18 h fed nymphal I. scapularis. Eight immunogenic oligopeptides were selected using human immune sera. Three selected immunogenic oligopeptides were cloned and produced as recombinant proteins. The immunogenic character of an identified metalloprotease (MP1) was validated with human sera. This enzyme has been described previously and was hypothesized as immunogenic which was confirmed in this study. Interestingly, it also has close homologs in other Ixodes species. CONCLUSION: An immunogenic protein of I. scapularis was identified by oligopeptide phage display. MP1 is a potential candidate for vaccine development.

Identifying Trusted Sources of Lyme Disease Prevention Information Among Internet Users Connected to Academic Public Health Resources: Internet-Based Survey Study.

BACKGROUND: Misinformation about Lyme disease and other tick-transmitted pathogens circulates frequently on the internet and can compete with, or even overshadow, science-based guidance on tick-borne disease (TBD) prevention. OBJECTIVE: We surveyed internet users connected to academic tick-related resources to identify trusted sources of Lyme disease prevention information, explore confidence in tick bite prevention information, and examine associations of these responses with answers to commonly disputed issues. METHODS: The survey was conducted through social media and website pages for Western Connecticut State University Tickborne Disease Prevention Laboratory and the University of Rhode Island TickEncounter Resource Center. RESULTS: Respondents (N=1190) were predominantly female (903/1190, 76.3%), middle-aged (574/1182, 48.6%), and resided in New England states (663/1190, 55.7%). In total 984 of 1186 (83%) respondents identified conventional experts (eg, the Centers for Disease Control [CDC] or other government health agencies, physicians who follow Infectious Diseases Society of America guidelines for Lyme disease treatment guidelines, and academics) as trustworthy TBD prevention resources. However, nearly one-fourth of respondents would first consult personal contacts and web-based communities regarding prevention information before consulting conventional expert sources. The opinions of public health experts and physicians were rated among the top motivators underlying personal prevention decisions; yet, more than 50% of participants revealed distrustful attitudes toward, or were uncertain about, CDC-supported statements related to time to transmission of Lyme disease (708/1190, 59.5%), the safety of diethyltoluamide-based repellents for children (604/1183, 51.1%), and recommended use of antibiotic prophylaxis (773/1181, 65.4%). Multimodal regression models revealed that participants from high-Lyme-disease-incidence states were more likely to first seek TBD prevention information from personal networks and nontraditional sources before approaching conventional sources of TBD prevention information. We found that those reporting high rates of social media usage were more than twice as likely to first seek traditional expert sources of prevention information but were overall more likely to reject CDC-promoted Lyme disease information, in particular the established time to transmission of Lyme disease bacteria. Models also predicted that those participants who disagreed with the conventional scientific view on the antibiotic prophylaxis prevention statement were less likely to be confident in their ability to protect themselves from a tick bite. Overall, uncertainty in one's ability to protect oneself against tick bites was strongly associated with uncertainty about beliefs in CDC-promoted TBD prevention information. Self-reported trust in experts and frequency of social media use suggest that these platforms may provide opportunities to engage directly with the public about TBD prevention practices. CONCLUSIONS: Using strategies to improve public trust and provide information where the public engages on social media may improve prevention communication and adoption of best practices.

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.

Ixodes scapularis Is the Most Susceptible of the Three Canonical Human-Biting Tick Species of North America to Repellent and Acaricidal Effects of the Natural Sesquiterpene, (+)-Nootkatone.

Ticks are vectors of many human and animal zoonotic disease-causing agents causing significant global health and economic strain. Repellents and acaricides are integral to the human capacity for personal protection from tick bites. Nootkatone, a naturally occurring sesquiterpene found in the Alaskan cedar tree, grapefruit, and other sources, has been documented to be a potent acaricide. Research has also noted repellent effects against some tick species. In this study, our aim was to investigate the effect of synthetic, high-purity (+)-nootkatone on adult Ixodes scapularis, Dermacentor variabilis, and Amblyomma americanum ticks in an in vitro, vertical filter paper bioassay. (+)-nootkatone showed compelling tick repellency, but median effective concentrations (EC50) significantly differed among species. Ixodes scapularis were repelled at very low concentrations (EC50 = 0.87 ± 0.05 µg/cm2). Higher concentrations were required to repel D. variabilis (EC50 = 252 ± 12 µg/cm2) and A. americanum (EC50 = 2313 ± 179 µg/cm2). Significant post-exposure mortality, assessed 24 h after repellency trials, was also observed in I. scapularis but was absent entirely in D. variabilis and A. americanum. These tests demonstrate that nootkatone has a promising dual-action personal protection capacity against adult I. scapularis ticks, warranting further investigation in more natural environments and in the presence of host cues.

Assessing Durability and Safety of Permethrin Impregnated Uniforms Used by Outdoor Workers to Prevent Tick Bites after One Year of Use.

Long lasting permethrin-impregnated (LLPI) clothing can retain permethrin and repel ticks for up to three months and without exceeding EPA-approved safe levels; however, little is known about longer term effects of wearing LLPI clothing. Here, permethrin content was measured in new forester pants soon after initial impregnation (Insect Shield) and again one year later after being repeatedly worn by foresters in the field. Urine samples were collected from foresters for biomonitoring of permethrin metabolites at multiple time intervals (pre-use, one-month, three-to-four-months, and one-year post-use). Lethality against nymphal Ixodes scapularis Say was measured in clothing after one year of wear by foresters. Furthermore, to test potential variability in permethrin impregnation of different batches of clothing, separate sets of clothing were anonymously sent to Insect Shield for permethrin treatment over a period of three months and permethrin was quantified. Results demonstrated 33% of participants' pants had no measurable permethrin after one year of wear and permethrin content and tick mortality varied significantly between clothing. Only two of the participants' clothing resulted in ≥ 30% tick mortality after one year of wear. Significant differences were observed in 3-PBA and trans-DCCA, but not cis-DCCA metabolites in participants over the four measured time points and were higher than general United States population levels. This study provides practical information on the safety (measured by urinary metabolites) over time of LLPI clothing. It also provides snapshots (pre-washing and after one year of wear) of effectiveness of LLPI clothing as personal protective equipment against ticks for outdoor workers.

Molecular characterization of novel sulfotransferases from the tick, Ixodes scapularis.

BACKGROUND: Ixodes scapularis, commonly known as the blacklegged or deer tick, is the main vector of Lyme disease in the United States. Recent progress in transcriptome research has uncovered hundreds of different proteins expressed in the salivary glands of hard ticks, the majority of which have no known function, and include many novel protein families. We recently identified transcripts coding for two putative cytosolic sulfotransferases in these ticks which recognized phenolic monoamines as their substrates. In this current study, we characterize the genetic expression of these two cytosolic sulfotransferases throughout the tick life cycle as well as the enzymatic properties of the corresponding recombinant proteins. Interestingly, the resultant recombinant proteins showed sulfotransferase activity against both neurotransmitters dopamine and octopamine. RESULTS: The two sulfotransferase genes were coded as Ixosc SULT 1 & 2 and corresponding proteins were referred as Ixosc Sult 1 and 2. Using gene-specific primers, the sulfotransferase transcripts were detected throughout the blacklegged tick life cycle, including eggs, larvae, nymphs, adult salivary glands and adult midgut. Notably, the mRNA and protein levels were altered upon feeding during both the larval and nymphal life stages. Quantitative PCR results confirm that Ixosc SULT1 was statistically increased upon blood feeding while Ixosc SULT 2 was decreased. This altered expression led us to further characterize the function of these proteins in the Ixodid tick. The sulfotransferase genes were cloned and expressed in a bacterial expression system, and purified recombinant proteins Ixosc Sult 1(R) and 2(R) showed sulfotransferase activity against neurotransmitters dopamine and octopamine as well as the common sulfotransferase substrate p-nitrophenol. Thus, dopamine- or octopamine-sulfonation may be involved in altering the biological signal for salivary secretion in I. scapularis. CONCLUSIONS: Collectively, these results suggest that a function of Ixosc Sult 1 and Sult 2 in Ixodid tick salivary glands may include inactivation of the salivation signal via sulfonation of dopamine or octopamine.

Tick bite protection with permethrin-treated summer-weight clothing.

The number of tick bites received by individuals wearing either permethrin-treated or untreated summer clothing (T-shirt, shorts, socks, and sneakers) was compared during a controlled indoor study. Pathogen-free nymphal Ixodes scapularis Say were placed on the left shoe, right leg, and left arm of 15 (5/treatment group/d) human volunteers wearing untreated outfits or outfits treated with permethrin either commercially or using a do-at-home treatment kit. The number and location of ticks attached to subjects' skin were recorded 2.5 h postinfestation. Subjects wearing outfits treated with permethrin received 3.36 times fewer tick bites than subjects wearing untreated outfits. No statistically significant differences in number of tick bites were detected between commercial permethrin treatment (19.33%) and the do-at-home permethrin application method (24.67%). The success of permethrin-treated clothing in reducing tick bites varied depending on the specific article of clothing. Subjects wearing permethrin-treated sneakers and socks were 73.6 times less likely to have a tick bite than subjects wearing untreated footware. Subjects wearing permethrin-treated shorts and T-shirts were 4.74 and 2.17 times, respectively, less likely to receive a tick bite in areas related to those specific garments than subjects wearing untreated shorts and T-shirts. Ticks attached to subjects were classified as alive or dead before removal. On subjects wearing untreated outfits, 97.6% of attached nymphs were alive, whereas significantly fewer (22.6%) attached nymphs were alive on subjects wearing repellent-treated outfits. Results of this study demonstrate the potential of permethrin-treated summer clothing for significantly reducing tick bites and tick-borne pathogen transmission.

An analysis of companion animal tick encounters as revealed by photograph-based crowdsourced data.

BACKGROUND: Community science is increasingly utilized to track important vectors of companion animal disease, providing a scalable, cost-effective strategy for identifying new foci, changing phenology, and disease prevalence across wide geographies. OBJECTIVES: We examined photographs of ticks found attached to predominately dogs and cats reported to a photograph-based tick surveillance program to identify potential areas for improvements in tick prevention education and risk intervention. METHODS: We compared estimated days of tick attachment using a Kruskal-Wallis one-way analysis of variance, and a Pearson's chi-square analysis of variance on the number of submissions by host type submitted for each season. RESULTS: The blacklegged tick (Ixodes scapularis) was the most common species reported (39.8%). Tick photographs submitted were almost entirely adults (89.5%), and ticks found on companion animals exhibited an estimated median engorgement time of 2.5 days. Ixodes scapularis displayed the highest median engorgement of the top tick species found feeding on companion animals (χ2  = 98.96, p < 0.001). Ticks were spotted year-round; during spring and summer, ticks collected from pets represented 15.4 and 12.8% of all submissions, but increased to 28.5 and 35.2% during autumn and winter, respectively. CONCLUSIONS: Crowdsourced data reveal that mostly adult ticks are detected on pets, and they are found at a point in the blood-feeding process that puts pets at heightened risk for disease transmission. The increase in proportion of ticks found on pets during colder months may reveal a critical knowledge gap amongst pet owners regarding seasonal activity of I. scapularis, a vector of Lyme disease, providing an opportunity for prevention-education.

Crowdsourced Tick Image-Informed Updates to U.S. County Records of Three Medically Important Tick Species.

Burgeoning cases of tick-borne disease present a significant public health problem in the United States. Passive tick surveillance gained traction as an effective way to collect epidemiologic data, and in particular, photograph-based tick surveillance can complement in-hand tick specimen identification to amass distribution data and related encounter demographics. We compared the Federal Information Processing Standards (FIPS) code of tick photos submitted to a free public identification service (TickSpotters) from 2014 to 2019 to published nationwide county reports for three tick species of medical concern: Ixodes scapularis Say (Ixodida: Ixodidae), Ixodes pacificus Cooley and Kohls (Ixodida: Ixodidae), and Amblyomma americanum Linneaus (Ixodida: Ixodidae). We tallied the number of TickSpotters submissions for each tick species according to "Reported" or "Established" criteria per county, and found that TickSpotters submissions represented more than half of the reported counties of documented occurrence, and potentially identified hundreds of new counties with the occurrence of these species. We detected the largest number of new county reports of I. scapularis presence in Michigan, North Carolina, and Texas. Tick image submissions revealed potentially nine new counties of occurrence for I. pacificus, and we documented the largest increase in new county reports of A. americanum in Kentucky, Illinois, Indiana, and Ohio. These findings demonstrate the utility of crowdsourced photograph-based tick surveillance as a complement to other tick surveillance strategies in documenting tick distributions on a nationwide scale, its potential for identifying new foci, and its ability to highlight at-risk localities that might benefit from tick-bite prevention education.

Assessing Public Tick Identification Ability and Tick Bite Riskiness Using Passive Photograph-Based Crowdsourced Tick Surveillance.

Tick identification is critical for assessing disease risk from a tick bite and for determining requisite treatment. Data from the University of Rhode Island's TickEncounter Resource Center's photo-based surveillance system, TickSpotters, indicate that users incorrectly identified their submitted specimen 83% of the time. Of the top four most commonly submitted tick species, western blacklegged ticks (Ixodes pacificus Cooley & Kohls [Ixodida: Ixodidae]) had the largest proportion of unidentified or misidentified submissions (87.7% incorrectly identified to species), followed by lone star ticks (Amblyomma americanum Linneaus [Ixodida: Ixodidae]; 86.8% incorrect), American dog ticks (Dermacentor variabilis Say [Ixodida: Ixodidae]; 80.7% incorrect), and blacklegged ticks (Ixodes scapularis Say [Ixodida: Ixodidae]; 77.1% incorrect). More than one quarter of participants (26.3%) submitted photographs of ticks that had been feeding for at least 2.5 d, suggesting heightened risk. Logistic regression generalized linear models suggested that participants were significantly more likely to misidentify nymph-stage ticks than adult ticks (odds ratio [OR] = 0.40, 95% confidence interval [CI]: 0.23, 0.68, P < 0.001). Ticks reported on pets were more likely to be identified correctly than those found on humans (OR = 1.07, 95% CI: 1.01-2.04, P < 0.001), and ticks feeding for 2.5 d or longer were more likely to be misidentified than those having fed for one day or less (OR = 0.43, 95% CI: 0.29-0.65, P < 0.001). State and region of residence and season of submission did not contribute significantly to the optimal model. These findings provide targets for future educational efforts and underscore the value of photograph-based tick surveillance to elucidate these knowledge gaps.

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.

Potential Effects of Climate Change on Tick-borne Diseases in Rhode Island.

Human cases of tick-borne diseases have been increasing in the United States. In particular, the incidence of Lyme disease, the major vector-borne disease in Rhode Island, has risen, along with cases of babesiosis and anaplasmosis, all vectored by the blacklegged tick. These increases might relate, in part, to climate change, although other environmental changes in the northeastern U.S. (land use as it relates to habitat; vertebrate host populations for tick reproduction and enzootic cycling) also contribute. Lone star ticks, formerly southern in distribution, have been spreading northward, including expanded distributions in Rhode Island. Illnesses associated with this species include ehrlichiosis and alpha-gal syndrome, which are expected to increase. Ranges of other tick species have also been expanding in southern New England, including the Gulf Coast tick and the introduced Asian longhorned tick. These ticks can carry human pathogens, but the implications for human disease in Rhode Island are unclear.

Evaluating the Effects of Minimal Risk Natural Products for Control of the Tick, Ixodes scapularis (Acari: Ixodidae).

Knockdown and residual activity of 10 minimal risk natural products (MRNPs), one experimental formulation of nootkatone, and two bifenthrin labels were evaluated against host-seeking nymphal Ixodes scapularis Say using a novel micro-plot product screening system placed in a landscape setting similar to a wooded residential property. The MRNPs evaluated included Tick Stop, EcoPCO EC-X, Met52 EC, CedarCide PCO Choice, EcoEXEMPT IC2, EcoSMART Organic Insecticide, Essentria IC3, privately labeled products 1 and 2 (based on EcoEXEMPT IC2 and sold as a professional pest control application), and Tick Killz. Just the nootkatone and 4 of these 10 products tested (EcoPCO EC-X, Met52 EC, EcoEXEMPT IC2, and Essentria IC3) had statistically significant (P < 0.05) knockdown effects (killed ticks while active in the arenas) when compared to water-only controls, but only 2 of these, EcoPCO EC-X and nootkatone, displayed significant residual tick-killing activity after weathering naturally in the landscape for 2 wk prior to tick application/testing. Moreover, botanical oil-based products with the same active ingredients provided inconsistent results when tested multiple times across study years.

Vectra 3D (dinotefuran, pyriproxyfen and permethrin) prevents acquisition of Borrelia burgdorferi sensu stricto by Ixodes ricinus and Ixodes scapularis ticks in an ex vivo feeding model.

BACKGROUND: We evaluated the efficiency of an ex vivo feeding technique using a silicone membrane-based feeding chamber to (i) assess the anti-feeding and acaricidal efficacy of a spot-on combination of dinotefuran, pyriproxyfen and permethrin (DPP, Vectra® 3D) against adult Ixodes scapularis and Ixodes ricinus ticks, and to (ii) explore its effect on blocking the acquisition of Borrelia burgdorferi sensu stricto. METHODS: Eight purpose-bred dogs were randomly allocated to two equal-size groups based on body weight assessed on day 2. DPP was administered topically, as spot-on, to four dogs on day 0. Hair from the eight dogs was collected individually by brushing the whole body on days 2, 7, 14, 21, 28 and 35. On each day of hair collection, 0.05 g of sampled hair was applied on the membrane corresponding to each feeding unit (FU). Seventy-two FU were each seeded with 30 adults of I. scapularis (n = 24 FU) or I. ricinus ticks (n = 48 FU). Bovine blood spiked with B. burgdorferi sensu stricto (strain B31) was added into each unit and changed every 12 h for 4 days. Tick mortality was assessed 1 h after seeding. One additional hour of incubation was added for live/moribund specimens and reassessed for viability. All remaining live/moribund ticks were left in the feeders and tick engorgement status was recorded at 96 h after seeding, and the uptake of B. burgdorferi s.s. was examined in the collected ticks by applying quantitative real-time PCR. RESULTS: Exposure to DPP-treated hair was 100% effective in blocking B. burgdorferi s.s. acquisition. The anti-feeding efficacy remained stable (100%) against both Ixodes species throughout the study. The acaricidal efficacy of DPP evaluated at 1 and 2 h after exposure was 100% throughout the study for I. ricinus, except the 1-h assessment on day 28 (95.9%) and day 35 (95.3%). The 1-h assessment of acaricidal efficacy was 100% at all time points for I. scapularis. CONCLUSIONS: The ex vivo feeding system developed here demonstrated a protective effect of DPP against the acquisition of B. burgdorferi without exposing the animals to the vectors or to the pathogen.

Functional genomics tool: gene silencing in Ixodes scapularis eggs and nymphs by electroporated dsRNA.

BACKGROUND: Ticks are blood-sucking arthropods responsible for transmitting a wide variety of disease-causing agents, and constitute important public health threats globally. Ixodes scapularis is the primary vector of the Lyme disease agent in the eastern and central U.S. RNAi is a mechanism by which gene-specific double-stranded RNA (dsRNA) triggers degradation of homologous mRNA transcripts. Here, we describe an optimized protocol for effectively suppressing gene expression in the egg and nymphal stages of I. scapularis by electroporation. RESULTS: The genes encoding the putative Phospholipase A2 (PLA2), cytoplasmic Cystatin, Syntaxin-5, beta-Actin and Calreticulin were targeted by delivering the dsRNA encoding the specific gene coding regions in the unfed nymphs. Silencing was measured using real time qRT-PCR. Electroporation as a mode of dsRNA delivery appears to be substantially efficient and less traumatic to the tick than dsRNA microinjection in the unfed nymphs. Using Cy3-labeled dsRNA to monitor the movement, electroporated dsRNA entered the nymphs and spread to salivary glands and other tissues. The significant disruption of beta-actin and cytoplasmic Cystatin transcripts in tick eggs demonstrate the applicability of this technique. The PLA2, cytoplasmic Cystatin, Syntaxin-5, beta-Actin and Calreticulin genes were also significantly silenced, suggesting that this method has the potential to introduce dsRNA in eggs and unfed nymphs. CONCLUSIONS: Our study demonstrates that electroporation can be used as a simple dsRNA delivery tool in assessing the functional role of tick genes in the vector-host interactions. This technique represents a novel approach for specific gene suppression in immature stages of ticks.

Cutting edge: Immunity against a "silent" salivary antigen of the Lyme vector Ixodes scapularis impairs its ability to feed.

Ixodes scapularis ticks transmit the Lyme disease agent in the United States. Although strong antitick immunity mediates tick rejection by certain vertebrates, only a few Ags have been molecularly characterized. We show that guinea pig vaccination against a secreted tick salivary immunomodulator, sialostatin L2, can lead to decreased feeding ability of I. scapularis nymphs. Increased rejection rate, prolonged feeding time, and apparent signs of inflammation were observed for nymphs attached to vaccinated animals, indicating a protective host immune response. Interestingly, sialostatin L2 humoral recognition does not take place upon repeated tick exposure in control animals, but only in the vaccinated animals that neutralize sialostatin L2 action. Therefore, we demonstrate an essential sialostatin L2 role upon nymphal infestation that can be blocked by vertebrate immunity and propose the discovery of similarly "silent" Ags toward the development of a multicomponent vaccine that will protect against tick bites and the pathogens they transmit.