Use of the Western blot technique to identify the immunogenic proteins of Borrelia burgdorferi for developing a Lyme disease vaccine.

BACKGROUND: Lyme disease is a serious infectious disease having a restricted worldwide distribution for which there is no vaccine available for human use. OBJECTIVE: This study was designed to determine common reactive antigens involved in Borrelia burgdorferi (Bb) infection that are recognized in mammalian sera that may be useful for vaccine development. METHODS: Blood samples were collected from patients with documented Lyme disease, and from rabbits and mice experimentally infected with either tick-transmitted or culture-grown Borrelia burgdorferi. All samples were then processed for sera. For performing the Western blots, sonicated Bb organisms (whole cell lysates) and protein ladders were separated by protein gel electrophoresis. Immune reactivities of the electrophoresed proteins with the serum samples were then probed with anti-HRP IgG reagent. RESULTS: Rabbit, mouse and human sera consistently reacted with the 41 kDa band of Bb which corresponded to the flagellin protein - the major protein component of this organism's periplasmic flagella, also known as axial filaments or fibrils. Various other Bb antigens of wide molecular weight ranges were also recognized by rabbit and human sera, and less frequently with mouse sera. CONCLUSION: The strong immune response to the 41 kDa flagellin protein by the different mammalian species suggests the utility of a possible vaccine targeting this protein, although other proteins may also be appropriate, for preventing Lyme disease following a bite from an infected tick.

First Detection of the Invasive Asian Longhorned Tick (Acari: Ixodidae) on Migratory Passerines in the Americas.

The Asian longhorned tick (Haemaphysalis longicornis Neumann), native to East Asia, was first reported in the United States in 2017 and is now established in at least 17 states. Haemaphysalis longicornis feeds on birds in its range outside of the United States, and migratory birds disperse this tick and tick-borne pathogens. However, early studies in the United States did not find H. longicornis on migrating passerine birds. The transport of the parthenogenetic H. longicornis on birds has the potential to greatly expand its range. We report the first discovery of H. longicornis on migratory passerine birds in the Americas.

A new genetic approach to distinguish strains of Anaplasma phagocytophilum that appear not to cause human disease.

Genetic diversity of Anaplasma phagocytophilum was assessed in specimens from 16 infected patients and 16 infected Ixodes scapularis ticks. A region immediately downstream of the 16S rRNA gene, which included the gene encoding SdhC, was sequenced. For the A. phagocytophilum strains from patients no sequence differences were detected in this region. In contrast, significantly fewer ticks had a sequence encoding SdhC that was identical to that of the human strains (11/16 vs. 16/16, p = 0.04). This variation is consistent with the premise that not all A. phagocytophilum strains present in nature are able to cause clinical illness in humans. A strain referred to as A. phagocytophilumVariant-1 that is regarded as non-pathogenic for humans was previously described using a different typing method. Data from the current study suggest that both typing methods are identifying the same non-pathogenic strains.

Seasonal Activity of Haemaphysalis longicornis (Acari: Ixodidae) in Southern New York State.

The Asian longhorned tick, Haemaphysalis longicornis Neumann, is a species native to eastern Asia that has recently been discovered in the United States. In its native range, H. longicornis transmits pathogens that cause disease in humans and livestock. It is currently unknown whether H. longicornis will act as a vector in the United States. Understanding its seasonal activity patterns will be important in identifying which times of the year represent greatest potential risk to humans and livestock should this species become a threat to animal or public health. A study site was established in Yonkers, NY near the residence associated with the first reported human bite from H. longicornis in the United States. Ticks were collected once each week from July 2018 to November 2019. Haemaphysalis longicornis larvae were most active from August to November, nymphs from April to July, and adult females from June to September. This pattern of activity suggests that H. longicornis is capable of completing a generation within a single year and matches the patterns observed in its other ranges in the northern hemisphere. The data presented here contribute to a growing database for H. longicornis phenology in the northeastern United States. Potential implications of the short life cycle for the tick's vectorial capacity are discussed.

Assessment of Duration of Tick Feeding by the Scutal Index Reduces Need for Antibiotic Prophylaxis After Ixodes scapularis Tick Bites.

Doxycycline is recommended for persons with Ixodes scapularis tick bites in certain geographic areas, if the tick had fed for at least 36 hours. Based on the scutal index, over 40% of I. scapularis tick bites from patients seen at the Lyme Disease Diagnostic Center did not warrant antibiotic prophylaxis.

Transient influence of blood meal and natural environment on blacklegged tick bacterial communities.

Blacklegged ticks (Ixodes scapularis) spend the majority of their life cycle off host, typically in woodland habitat, but require a blood meal at each of three life stages (larva, nymph, adult) to reach maturity and reproduce. Blood feeding usually lasts for several days each time and as blood is imbibed, a range of known pathogens from the host may also be acquired. Using next generation sequencing of 16S rRNA gene amplicons, we examined the influence of host blood meal on the internal bacterial community within nymphal blacklegged ticks across host-seeking, feeding, blood meal digestion, and after molting into the adult stage. Results demonstrate bacterial community structuring across host and ticks with 287 taxa found exclusively in ticks, suggesting the field environment plays a significant role in shaping the internal tick microbiome. A decrease in bacterial diversity was noted from unfed nymphs through feeding/digestion and after molting into adults, suggesting that bacterial species are lost during the corresponding physiological changes. The similarity in biochemical pathways across the different tick categories suggests that the loss of bacterial taxa does not mirror a large change in microbial function. Ticks likely lose bacterial taxa after feeding, but continual exposure to bacteria from the field environment counters this loss.

Energy Usage of Known-Age Blacklegged Ticks (Acari: Ixodidae): What Is the Best Method for Determining Physiological Age?

Ticks expend energy while host-seeking and must consume blood to advance to the next life stage. The energy required for activity is derived from the tick's lipid reserves, a valuable resource that sustains the tick until it finds the next host and can take another bloodmeal. The amount of lipid reserves in an unfed tick has been proposed as an index of tick biological age. Two different methods for aging nymphal blacklegged ticks, Ixodes scapularis Say, were analyzed in this study. To study lipid usage, colony-raised nymphs were held in lab-controlled chambers at 21.0 °C and ≥95% relative humidity, with a photoperiod of 12:12 (L:D) h. Samples of ticks were frozen at -80 °C every 2-3 wk, starting at 12-wk postmolt and continuing until 38-wk postmolt. Lipid reserves were determined indirectly through measurements of "physiological age" that estimate the energy a tick has based on the evaluation of morphometric size ratios of the tick scutum and alloscutum, and quantified directly through chloroform extractions of lipid from individual ticks. Morphometric age ratios and lipid amounts were compared to determine if morphometric measurements accurately estimated a tick's physiological state. Although the morphometric age ratio did correlate significantly with total tick lipid content, the predictive value of the ratio was not reliable; chloroform extraction results showed that lipid amounts declined steadily through the study and more accurately characterized the physiological condition of nymphal I. scapularis. The study of physiological aging of blacklegged ticks may lead to a better understanding of how changing environmental conditions affect tick longevity.

Microbiome changes through ontogeny of a tick pathogen vector.

Blacklegged ticks (Ixodes scapularis) are one of the most important pathogen vectors in the United States, responsible for transmitting Lyme disease and other tick-borne diseases. The structure of a host's microbial community has the potential to affect the ecology and evolution of the host. We employed high-throughput sequencing of the 16S rRNA gene V3-V4 hypervariable regions in the first study to investigate the tick microbiome across all developmental stages (larvae, nymphs, adults). In addition to field-collected life stages, newly hatched laboratory-reared larvae were studied to determine the baseline microbial community structure and to assess transovarial transmission. We also targeted midguts and salivary glands due to their importance in pathogen maintenance and transmission. Over 100 000 sequences were produced per life stage replicate. Rickettsia was the most abundant bacterial genus across all sample types matching mostly the Ixodes rickettsial endosymbionts, and its proportion decreased as developmental stage progressed, with the exception of adult females that harboured a mean relative abundance of 97.9%. Laboratory-reared larvae displayed the lowest bacterial diversity, containing almost exclusively Rickettsia. Many of the remaining bacteria included genera associated with soil, water and plants, suggesting environmental acquisition while off-host. Female organs exhibited significantly different ß-diversity than the whole tick from which they were derived. Our results demonstrate clear differences in both α- and ß-diversity among tick developmental stages and between tick organs and the tick as a whole. Furthermore, field-acquired bacteria appear to be very important to the overall internal bacterial community of this tick species, with influence from the host bloodmeal appearing limited.

No Observed Effect of Landscape Fragmentation on Pathogen Infection Prevalence in Blacklegged Ticks (Ixodes scapularis) in the Northeastern United States.

Pathogen prevalence within blacklegged ticks (Ixodes scapularis Say, 1821) tends to vary across sites and geographic regions, but the underlying causes of this variation are not well understood. Efforts to understand the ecology of Lyme disease have led to the proposition that sites with higher host diversity will result in lower disease risk due to an increase in the abundance of inefficient reservoir species relative to the abundance of species that are highly competent reservoirs. Although the Lyme disease transmission cycle is often cited as a model for this "dilution effect hypothesis", little empirical evidence exists to support that claim. Here we tested the dilution effect hypothesis for two pathogens transmitted by the blacklegged tick along an urban-to-rural gradient in the northeastern United States using landscape fragmentation as a proxy for host biodiversity. Percent impervious surface and habitat fragment size around each site were determined to assess the effect of landscape fragmentation on nymphal blacklegged tick infection with Borrelia burgdorferi and Anaplasma phagocytophilum. Our results do not support the dilution effect hypothesis for either pathogen and are in agreement with the few studies to date that have tested this idea using either a landscape proxy or direct measures of host biodiversity.

American Black Bears as Hosts of Blacklegged Ticks (Acari: Ixodidae) in the Northeastern United States.

Ticks and whole blood were collected from American black bears (Ursus americanus Pallas) between October 2011 and October 2012 across four counties in northwestern New Jersey, an area where blacklegged ticks (Ixodes scapularis Say) and their associated tick-borne pathogens are prevalent. Adult American dog ticks (Dermacentor variabilis Say) were the most frequently collected tick species in late spring, whereas adult and nymphal blacklegged ticks were found in both the late spring and fall months. Additionally, for blacklegged ticks, we determined the quality of bloodmeals that females acquired from black bears compared with bloodmeals from white-tailed deer (Odocoileus virginianus Zimmerman), the most important host for the adult stage of this tick species. Measures of fecundity after feeding on each host species were not significantly different, suggesting that the bloodmeal a female blacklegged tick acquires from a black bear is of similar quality to that obtained from a white-tailed deer. These results establish the American black bear as both a host and quality bloodmeal source to I. scapularis. Thus, black bears may help support blacklegged tick populations in areas where they are both present. In addition, samples of black bear blood were tested for DNA presence of three tick-borne pathogens. Anaplasma phagocytophilum Foggie and Babesia microti Franca were found in 9.2 and 32.3% of blood samples, respectively. All blood samples were quantitative polymerase chain reaction-negative for Borrelia burgdorferi Johnson, Schmid, Hyde, Steigerwalt, & Brenner. Although circulating pathogens were found in blood, the status of black bears as reservoirs for these pathogens remains unknown.

To beat or not to beat a tick: comparison of DNA extraction methods for ticks (Ixodes scapularis).

Background. Blacklegged ticks (Ixodes scapularis) are important disease vectors in the United States, known to transmit a variety of pathogens to humans, including bacteria, protozoa, and viruses. Their importance as a disease vector necessitates reliable and comparable methods for extracting microbial DNA from ticks. Furthermore, to explore the population genetics or genomics of this tick, appropriate DNA extraction techniques are needed for both the vector and its microbes. Although a few studies have investigated different methods of DNA isolation from ticks, they are limited in the number and types of DNA extraction and lack species-specific quantification of DNA yield. Methods. Here we determined the most efficient and consistent method of DNA extraction from two different developmental stages of I. scapularis-nymph and adult-that are the most important for disease transmission. We used various methods of physical disruption of the hard, chitinous exoskeleton, as well as commercial and non-commercial DNA isolation kits. To gauge the effectiveness of these methods, we quantified the DNA yield and confirmed the DNA quality via PCR of both tick and microbial genetic material. Results. DNA extraction using the Thermo GeneJET Genomic DNA Purification Kit resulted in the highest DNA yields and the most consistent PCR amplification when combined with either cutting or bead beating with select matrices across life stages. DNA isolation methods using ammonium hydroxide as well as the MoBio PowerSoil kit also produced strong and successful PCR amplification, but only for females. Discussion. We contrasted a variety of readily available methods of DNA extraction from single individual blacklegged ticks and presented the results through a quantitative and qualitative assessment.

Borrelia chilensis, a new member of the Borrelia burgdorferi sensu lato complex that extends the range of this genospecies in the Southern Hemisphere.

Borrelia burgdorferi sensu lato (s.l.), transmitted by Ixodes spp. ticks, is the causative agent of Lyme disease. Although Ixodes spp. ticks are distributed in both Northern and Southern Hemispheres, evidence for the presence of B. burgdorferi s.l. in South America apart from Uruguay is lacking. We now report the presence of culturable spirochetes with flat-wave morphology and borrelial DNA in endemic Ixodes stilesi ticks collected in Chile from environmental vegetation and long-tailed rice rats (Oligoryzomys longicaudatus). Cultured spirochetes and borrelial DNA in ticks were characterized by multilocus sequence typing and by sequencing five other loci (16S and 23S ribosomal genes, 5S-23S intergenic spacer, flaB, ospC). Phylogenetic analysis placed this spirochete as a new genospecies within the Lyme borreliosis group. Its plasmid profile determined by polymerase chain reaction and pulsed-field gel electrophoresis differed from that of B. burgdorferi B31A3. We propose naming this new South American member of the Lyme borreliosis group B. chilensis VA1 in honor of its country of origin.

Failure of topical antibiotics to prevent disseminated Borrelia burgdorferi infection following a tick bite in C3H/HeJ mice.

A prior study in mice has shown that the timely application of topical antibiotics to the skin at the tick bite site could eradicate Borrelia burgdorferi infection. That study, however, did not evaluate antibiotic preparations that are considered suitable for use in humans. In this murine study, topical application of 2% erythromycin and 3% tetracycline preparations that are acceptable for use in humans was found to be ineffective in eliminating B. burgdorferi from the tick bite site or in preventing dissemination to other tissues. Reasons for the discrepant findings are discussed.

Occurrence of soil- and tick-borne fungi and related virulence tests for pathogenicity to Ixodes scapularis (Acari: Ixodidae).

Ixodes scapularis Say, the blacklegged tick, vectors Borrelia burgdorferi Johnson et al. 1984, the bacterium that causes Lyme disease, the most important vector-borne disease in the United States. Efforts to reduce I. scapularis populations are shifting toward the development of biological control methods. Currently, only a few entomopathogenic fungal species are considered virulent to ticks. We hypothesized that these species may not represent the most abundant local taxa that would be pathogenic to ticks in situ. To identify potential entomopathogenic fungi at a study site in Westchester County, New York, we sampled soils and ticks, extracted and amplified the internal transcribed spacer region of nuclear ribosomal DNA (nrDNA), and compared sequences with those in GenBank. Over three sampling periods from June 2007 to May 2008, 70 fungal taxa were isolated and identified from soils (48 taxa) and ticks (27 taxa; 5 taxa were found both in soil and on ticks) collected in this study, encompassing species in 25 different genera. In laboratory bioassays, 15 fungal taxa were found to be significantly virulent, although none of these were previously considered common pathogens of I. scapularis. Two species, Hypocrea lixii Patouillard 1891 and Penicillium soppii K. M. Zalessky 1927, were tested in field trials by spraying suspensions on forested plots. Mean tick mortality was 71% after treatment with H. lixii, 58% after treatment with P. soppii, and 32% in the control plots. The complete diversity of entomopathogenic fungal species at this site is yet to be defined, but, in general, such fungi appear to be more common in forest habitats where I. scapularis resides than previously thought. Examination of intact fungal communities can provide information that serves as the foundation for site-specific biocontrol programs.

Acaricidal treatment of white-tailed deer to control Ixodes scapularis (Acari: Ixodidae) in a New York Lyme disease-endemic community.

The efficacy of topically treating white-tailed deer with an acaricide was evaluated in a Lyme disease-endemic community of southern New York State. Twenty-four 4-Poster feeders were placed in a 5.2 km(2) treatment area in Bedford, NY, while a site in Lewisboro, NY, 4.8 km distant, served as control. Treatment periods ran from 15 September to 15 December each fall from 1997 to 2001, and from 15 March to 15 May each spring from 1998 to 2002. Corn consumption averaged 15,779 kg in fall sessions and 9054 kg in spring sessions, and a mean of 89.6% of deer in the study area showed evidence of using the feeders. Deer densities, estimated by aerial snow counts, averaged 22 and 28 deer per km(2) in Bedford and Lewisboro, respectively, over a 3-year period. Significant reductions in tick numbers on deer captured in the treatment area were noted in fall 1999 compared to deer captured at the control site. Drag sampling for nymphal host-seeking ticks indicated 63.6% control in 2001, which dropped to 54.8% the following year, but reached 80% in 2003. Higher-than-normal acorn production in 2001 that likely caused a drop in deer visitation to the feeders may have reduced efficacy against larval ticks in 2002. The 4-Poster effectively reduced the density of Ixodes scapularis, though the level of control is dependent on environmental factors that affect feeding behavior of white-tailed deer.

Effects of tick control by acaricide self-treatment of white-tailed deer on host-seeking tick infection prevalence and entomologic risk for Ixodes scapularis-borne pathogens.

We evaluated the effects of tick control by acaricide self-treatment of white-tailed deer on the infection prevalence and entomologic risk for three Ixodes scapularis-borne bacteria in host-seeking ticks. Ticks were collected from vegetation in areas treated with the "4-Poster" device and from control areas over a 6-year period in five geographically diverse study locations in the Northeastern United States and tested for infection with two known agents of human disease, Borrelia burgdorferi and Anaplasma phagocytophilum, and for a novel relapsing fever-group spirochete related to Borrelia miyamotoi. Overall, 38.2% of adults and 12.5% of nymphs were infected with B. burgdorferi; 8.5% of adults and 4.2% of nymphs were infected with A. phagocytophilum; and 1.9% of adults and 0.8% of nymphs were infected with B. miyamotoi. In most cases, treatment with the 4-Poster device was not associated with changes in the prevalence of infection with any of these three microorganisms among nymphal or adult ticks. However, the density of nymphs infected with B. burgdorferi, and consequently the entomologic risk for Lyme disease, was reduced overall by 68% in treated areas compared to control areas among the five study sites at the end of the study. The frequency of bacterial coinfections in ticks was generally equal to the product of the proportion of ticks infected with a single bacterium, indicating that enzootic maintenance of these pathogens is independent. We conclude that controlling ticks on deer by self-application of acaricide results in an overall decrease in the human risk for exposure to these three bacterial agents, which is due solely to a reduction in tick density.

Evaluation of the United States Department Of Agriculture Northeast Area-wide Tick Control Project by meta-analysis.

As part of the Northeast Area-wide Tick Control Project (NEATCP), meta-analyses were performed using pooled data on the extent of tick-vector control achieved through seven concurrent studies, conducted within five states, using U.S. Department of Agriculture "4-Poster" devices to deliver targeted-acaricide to white-tailed deer. Although reductions in the abundance of all life-stages of Ixodes scapularis were the measured outcomes, this study focused on metrics associated with I. scapularis nymphal tick densities as this measure has consistently proven to directly correlate with human risk of acquiring Lyme disease. Since independent tick sampling schemes were undertaken at each of the five environmentally distinct study locations, a meta-analytic approach permitted estimation of a single true control-effect size for each treatment year of the NEATCP. The control-effect is expressed as the annual percent I. scapularis nymphal control most consistent with meta-analysis data for each treatment year. Our meta-analyses indicate that by the sixth treatment year, the NEATCP effectively reduced the relative density of I. scapularis nymphs by 71% on the 5.14 km(2) treatment sites, corresponding to a 71% lower relative entomologic risk index for acquiring Lyme disease.

The United States Department of Agriculture's Northeast Area-wide Tick Control Project: summary and conclusions.

From 1997 to 2002, the U.S. Department of Agriculture's Northeast Area-wide Tick Control Project used acaricide-treated 4-Poster Deer Treatment Bait Stations in five eastern states to control ticks feeding on white-tailed deer. The objectives of this host-targeted technology were to reduce free-living blacklegged (Ixodes scapularis Say) and lone star (Amblyomma americanum [L.]) tick populations and thereby to reduce the risk of tick-borne disease. During 2002 to 2004, treatments were suspended, and tick population recovery rates were assayed. Subsequently, the major factors that influenced variations in efficacy were extrapolated to better understand and improve this technology. Treatments resulted in significant reductions in free-living populations of nymphal blacklegged ticks at six of the seven sites, and lone star ticks were significantly reduced at all three sites where they were present. During the study, maximal significant (p < or = 0.05) efficacies against nymphal blacklegged and lone star ticks at individual sites ranged from 60.0 to 81.7 and 90.9 to 99.5%, respectively. The major environmental factor that reduced efficacy was the occurrence of heavy acorn masts, which provided an alternative food resource for deer. Although the 4-Poster technology requires 1 or more years to show efficacy, this host-targeted intervention was demonstrated to be an efficacious, economical, safe, and environment-friendly alternative to area-wide spraying of acaricide to control free-living populations of these tick species.

Isolation of entomopathogenic fungi from soils and Ixodes scapularis (Acari: Ixodidae) ticks: prevalence and methods.

Entomopathogenic fungi are commonly found in forested soils that provide tick habitat, and many species are pathogenic to Ixodes scapularis Say, the blacklegged tick. As a first step to developing effective biocontrol strategies, the objective of this study was to determine the best methods to isolate entomopathogenic fungal species from field-collected samples of soils and ticks from an Eastern deciduous forest where I. scapularis is common. Several methods were assessed: (1) soils, leaf litter, and ticks were plated on two types of media; (2) soils were assayed for entomopathogenic fungi using the Galleria bait method; (3) DNA from internal transcribed spacer (ITS) regions of the nuclear ribosomal repeat was extracted from pure cultures obtained from soils, Galleria, and ticks and was amplified and sequenced; and (4) DNA was extracted directly from ticks, amplified, and sequenced. We conclude that (1) ticks encounter potentially entomopathogenic fungi more often in soil than in leaf litter, (2) many species of potentially entomopathogenic fungi found in the soil can readily be cultured, (3) the Galleria bait method is a sufficiently efficient method for isolation of these fungi from soils, and (4) although DNA extraction from ticks was not possible in this study because of small sample size, DNA extraction from fungi isolated from soils and from ticks was successful and provided clean sequences in 100 and 73% of samples, respectively. A combination of the above methods is clearly necessary for optimal characterization of entomopathogenic fungi associated with ticks in the environment.

Molecular analysis of microbial communities identified in different developmental stages of Ixodes scapularis ticks from Westchester and Dutchess Counties, New York.

Ixodes scapularis ticks play an important role in the transmission of a wide variety of pathogens between various mammalian species, including humans. Pathogens transmitted by ticks include Borrelia, Anaplasma and Babesia. Although ticks may harbour both pathogenic and non-pathogenic microflora, little is known about how the diversity of the microflora within ticks may influence the transmission of pathogens. To begin addressing this question, we examined the composition of bacterial communities present in Ixodes scapularis collected from Westchester and Dutchess Counties, New York State, at different developmental and nutritional stages. Genetic fingerprints of bacterial populations were generated by temporal temperature gradient gel electrophoresis (TTGE) separation of individual polymerase chain reaction (PCR)-amplified 16S rRNA gene fragments, followed by DNA sequence analysis for bacterial identification. The fingerprints of the TTGE bands were grouped into five clusters. The most abundant DNA sequence found in all the samples was Rickettsia, followed by Pseudomonas and Borrelia. Ralstonia, Anaplasma, Enterobacterias, Moraxella, Rhodococcus and uncultured proteobacterium were present as well. We also determined the prevalence of Anaplasma phagocytophilum and Borrelia burgdorferi by PCR and DNA sequence analysis. Statistical analyses indicated significant variations in the bacterial communities depending on tick developmental stage and degree of engorgement. We suggest that these two elements affect microbial diversity within the tick and may in turn influence pathogen transmission to humans and animals after tick bite.