Search

Three tick species that can transmit pathogen causing disease are commonly found parasitizing people and animals in the mid-Atlantic United States: the blacklegged tick (Ixodes scapularis Say), the American dog tick (Dermacentor variabilis [Say]), and the lone star tick (Amblyomma americanum [L.]) (Acari: Ixodidae). The potential risk of pathogen transmission from tick bites acquired at schools in tick-endemic areas is a concern, as school-aged children are a high-risk group for tick-borne disease. Integrated pest management (IPM) is often required in school districts, and continued tick range expansion and population growth will likely necessitate IPM strategies to manage ticks on school grounds. However, an often-overlooked step of tick management is monitoring and assessment of local tick species assemblages to inform the selection of control methodologies. The purpose of this study was to evaluate tick species presence, abundance, and distribution and the prevalence of tick-borne pathogens in both questing ticks and those removed from rodent hosts on six school properties in Maryland. Overall, there was extensive heterogeneity in tick species dominance, abundance, and evenness across the field sites. A. americanum and I. scapularis were found on all sites in all years. Overall, A. americanum was the dominant tick species. D. variabilis was collected in limited numbers. Several pathogens were found in both questing ticks and those removed from rodent hosts, although prevalence of infection was not consistent between years. Borrelia burgdorferi, Ehrlichia chaffeensis, Ehrlichia ewingii, and Ehrlichia "Panola Mountain" were identified in questing ticks, and B. burgdorferi and Borrelia miyamotoi were detected in trapped Peromyscus spp. mice. B. burgdorferi was the dominant pathogen detected. The impact of tick diversity on IPM of ticks is discussed.

Borrelia miyamotoi is a relapsing fever spirochete carried by Ixodes spp. ticks throughout the northern hemisphere. The pathogen is acquired either transovarially (vertically) or horizontally through blood-feeding and passed transtadially across life stages. Despite these complementary modes of transmission, infection prevalence of ticks with B. miyamotoi is typically low (5%) in natural settings and the relative contributions of the two transmission modes have not been studied extensively. Horizontal transmission of B. miyamotoi (strain CT13-2396 or wild type strain) was initiated using infected Ixodes scapularis larvae or nymphs to expose rodents, which included both the immunocompetent CD-1 laboratory mouse (Mus musculus) and a natural reservoir host, the white-footed mouse (Peromyscus. leucopus), to simulate natural enzootic transmission. Transovarial transmission was evaluated using I. scapularis exposed to B. miyamotoi as either larvae or nymphs feeding on immunocompromised SCID mice (M. musculus) and subsequently fed as females on New Zealand white rabbits. Larvae from infected females were qPCR-tested individually to assess transovarial transmission rates. Tissue tropism of B. miyamotoi in infected ticks was demonstrated using in situ hybridization. Between 1 and 12% of ticks were positive (post-molt) for B. miyamotoi after feeding on groups of CD-1 mice or P. leucopus with evidence of infection, indicating that horizontal transmission was inefficient, regardless of whether infected larvae or nymphs were used to challenge the mice. Transovarial transmission occurred in 7 of 10 egg clutches from infected females. Filial infection prevalence in larvae ranged from 3 to 100% (median 71%). Both larval infection prevalence and spirochete load were highly correlated with maternal spirochete load. Spirochetes were disseminated throughout the tissues of all three stages of unfed ticks, including the salivary glands and female ovarian tissue. The results indicate that while multiple transmission routes contribute to enzootic maintenance of B. miyamotoi, transovarial transmission is likely to be the primary source of infected ticks and therefore risk assessment and tick control strategies should target adult female ticks.

The objective of the current study was to determine the requirement of standardized ileal digestible (SID) CP for maximal litter gain in high-yielding lactating sows due to insufficient supply of either His, Leu, Val, Ile, or Phe. The content of SID Lys was formulated at 95% of the recommended level, while that of Met, Met+Cys, Thr, and Trp was formulated at 100% of the recommended level or slightly greater using crystalline AA. A total of 540 parity 1 to 5 sows (L×Y, DanBred, Herlev, Denmark) were included in the study from day 3 after farrowing until weaning at day 26. Sows were allocated to six dietary treatments increasing in SID CP content (96, 110, 119, 128, 137, and 152 g/kg). Litters were standardized to 14 piglets at day 3 ± 2 after farrowing. At day 3 ± 2 after farrowing and at day 26 ± 3, sow BW and back fat, and litter weight were recorded. On a subsample of 72 sows (parity 2 to 4), litters were also weighed at days 10 and 17 ± 3, and milk and blood were sampled at day 3 ± 2 d, and 10, 17 and at 24 ± 3 d in lactation. Sow body pools of protein and fat were determined on the 72 sows at days 3 ± 2 and 26 ± 3 d using the D2O dilution technique. All data were subjected to ANOVA, and to linear and quadratic polynomial contrasts. Variables with quadratic effects or days in milk × treatment interactions were analyzed using linear regression or one-slope linear broken line using the NLMIXED procedure of SAS. Average daily litter gain reached a breakpoint at 125 g SID CP/kg (as-fed). Multiparous sows had a greater litter gain than primiparous sows (3.33 vs. 3.02 kg/d above the breakpoint; P < 0.001) but litter size (13.1 ± 0.1) at weaning were unaffected by dietary treatment (P = 0.62). Sow BW loss was minimized at 102 g SID CP/kg. Concentrations of protein and casein in milk increased linearly with increasing SID CP (P < 0.001). Milk urea reached a minimum at 111–118 g SID CP/kg (P < 0.05) and milk fat a maximum at 116 g SID CP/kg (P < 0.05). In conclusion, 125 g SID CP/kg feed was required to maximize litter gain.

Tickborne diseases are an increasing public health problem in the northeastern USA. Bait boxes that apply acaricide to rodents have been shown in small field studies to significantly reduce abundance of Ixodes scapularis ticks as well as their pathogen infection rates in treated areas. The effectiveness of this intervention for preventing human tickborne diseases (TBDs) has not been demonstrated. During 2012-2016, TickNET collaborators conducted a randomized, blinded, placebo-controlled trial among 622 Connecticut households. Each household received active (containing fipronil wick) or placebo (empty) bait boxes in their yards over two consecutive years. Information on tick encounters and TBDs among household members was collected through biannual surveys. Nymphal ticks were collected from a subset of 100 properties during spring at baseline, during treatment, and in the year post-intervention. Demographic and property characteristics did not differ between treatment groups. There were no significant differences post-intervention between treatment groups with respect to tick density or pathogen infection rates, nor for tick encounters or TBDs among household members. We found no evidence that rodent-targeted bait boxes disrupt pathogen transmission cycles or significantly reduce household risk of tick exposure or TBDs. The effectiveness of this intervention may depend on scale of use or local enzootic cycles.

Borrelia miyamotoi is a hard tick-associated relapsing fever spirochete that is geographically widespread in Ixodes spp. (Acari: Ixodidae) ticks, but typically occurs at low prevalence. Genetic variability has been described among strains derived from Asia, Europe, and North America, and among tick species that carry the infection, but little variability has been described within foci or tick species. Capitalizing on access to B. miyamotoi nucleic acid extracted from host-seeking Ixodes scapularis Say or Ixodes pacificus Cooley & Kohls from 16 states, we explored genetic variability based on sequence analysis of four amplicons described herein. Consistent with previous studies, we detected significant genetic differences between strains derived from I. scapularis (eastern United States) and I. pacificus (western United States) and identified two distinct sequences in the western United States (Am-West-1 and Am-West-2). Unique to this study, we identified two distinct sequences in the eastern United States (Am-East-1 and Am-East-2). Based on the 161 samples we analyzed, Am-East-1 was the only type represented in 50 B. miyamotoi-infected ticks collected from the Northeast (Vermont, Maine, New York, Connecticut, and Rhode Island), whereas ticks collected from the North-Central and Mid-Atlantic states harbored B. miyamotoi comprised of both Am-East-1 and Am-East-2. Further studies are needed to better characterize the phylogeography of B. miyamotoi and to discern if there are biologically meaningful differences among sequence types. To facilitate further exploration, we developed a polymerase chain reaction (PCR) assay designed to differentiate Am-East-1, Am-East-2, and Am-West sequence types without having to sequence the amplicon.

Lyme and other tick-borne diseases are increasing in the eastern United States and there is a lack of research on integrated strategies to control tick vectors. Here we present results of a study on tick-borne pathogens detected from tick vectors and rodent reservoirs from an ongoing 5-yr tick suppression study in the Lyme disease-endemic state of Maryland, where human-biting tick species, including Ixodes scapularis Say (Acari: Ixodidae) (the primary vector of Lyme disease spirochetes), are abundant. During the 2017 tick season, we collected 207 questing ticks and 602 ticks recovered from 327 mice (Peromyscus spp. (Rodentia: Cricetidae)), together with blood and ear tissue from the mice, at seven suburban parks in Howard County. Ticks were selectively tested for the presence of the causative agents of Lyme disease (Borrelia burgdorferi sensu lato [s.l.]), anaplasmosis (Anaplasma phagocytophilum), babesiosis (Babesia microti), ehrlichiosis (Ehrlichia ewingii, Ehrlichia chaffeensis, and ‘Panola Mountain’ Ehrlichia) and spotted fever group rickettsiosis (Rickettsia spp.). Peromyscus ear tissue and blood samples were tested for Bo. burgdorferi sensu stricto (s.s), A. phagocytophilum, Ba. microti, and Borrelia miyamotoi. We found 13.6% (15/110) of questing I. scapularis nymphs to be Bo. burgdorferi s.l. positive and 1.8% (2/110) were A. phagocytophilum positive among all sites. Borrelia burgdorferi s.s. was found in 71.1% (54/76) of I. scapularis nymphs removed from mice and 58.8% (194/330) of captured mice. Results from study on tick abundance and pathogen infection status in questing ticks, rodent reservoirs, and ticks feeding on Peromyscus spp. will aid efficacy evaluation of the integrated tick management measures being implemented.

Upon acquiring two unique plasmids (pMT1 and pPCP1) and genome rearrangement during the evolution from Yersinia pseudotuberculosis, the plague causative agent Y. pestis is closely related to Y. pseudotuberculosis genetically but became highly virulent. We developed a pentaplex real‐time PCR assay that not only detects both Yersinia species but also differentiates Y. pestis strains regarding their plasmid profiles. The five targets used were Y. pestis‐specific ypo2088, caf1, and pst located on the chromosome, plasmids pMT1 and pPCP1, respectively; Y. pseudotuberculosis‐specific chromosomal gene opgG; and 18S ribosomal RNA gene as an internal control for flea DNA. All targets showed 100% specificity and high sensitivity with limits of detection ranging from 1 fg to 100 fg, with Y. pestis‐specific pst as the most sensitive target. Using the assay, Y. pestis strains were differentiated 100% by their known plasmid profiles. Testing Y. pestis and Y. pseudotuberculosis‐spiked flea DNA showed there is no interference from flea DNA on the amplification of targeted genes. Finally, we applied the assay for testing 102 fleas collected from prairie dog burrows where prairie dog die‐off was reported months before flea collection. All flea DNA was amplified by 18S rRNA; no Y. pseudotuberculosis was detected; one flea was positive for all Y. pestis‐specific targets, confirming local Y. pestis transmission. Our results indicated the assay is sensitive and specific for the detection and differentiation of Y. pestis and Y. pseudotuberculosis. The assay can be used in field investigations for the rapid identification of the plague causative agent., We developed a pentaplex real‐time PCR assay including Yersinia pestis‐specific genes ypo2088, caf1, and pst located on the chromosome, as well as on plasmids pMT1 and pPCP1, respectively. Yersinia pseudotuberculosis‐specific chromosomal gene opgG and the 18S ribosomal RNA gene served as an internal control for flea DNA. All targets showed 100% specificity and high sensitivity with limits of detection ranging from 1 to 100 femtograms. Using the assay, Y. pestis was detected in field‐collected fleas and strains were differentiated 100% by their known plasmid profiles.

Tickborne diseases are an increasing public health concern in the United States, where the majority of notifiable cases are caused by pathogens vectored by Ixodes ticks. To better monitor changes in acarological risk of human encounters with these ticks and their associated pathogens, the Centers for Disease Control and Prevention (CDC) recently established a national tick and tickborne pathogen surveillance program. Here, we describe and evaluate a new Multiplex PCR Amplicon Sequencing (MPAS) assay for potential use in surveillance programs targeting two common human-biting vector ticks, Ixodes scapularis and Ixodes pacificus. The ability of the MPAS assay to detect five Ixodes-associated human pathogens (Borrelia burgdorferi sensu stricto, Borrelia mayonii, Borrelia miyamotoi, Anaplasma phagocytophilum and Babesia microti) was compared to that of a previously published and routinely used probe-based (TaqMan) PCR testing algorithm for pathogen detection in Ixodes ticks. Assay performance comparisons included a set of 175 host-seeking Ixodes nymphs collected in Connecticut as well as DNA from our pathogen reference collection. The MPAS assay and the CDC standard TaqMan PCR pathogen testing algorithm were found to have equivalent detection sensitivity for Ixodes-associated human pathogens. However, the MPAS assay was able to detect a broader range of tick-associated microorganisms, more effectively detected co-infections of multiple pathogens in a single tick (including different species within the Borrelia burgdorferi sensu lato complex), and required a smaller volume of test sample (thus preserving more sample for future testing).

Deer management (e.g., reduction) has been proposed as a tool to reduce the acarological risk of Lyme disease. There have been few opportunities to investigate Ixodes scapularis (blacklegged tick) and Borrelia burgdorferi sensu stricto dynamics in the absence of white-tailed deer (Odocoileus virginianus) in midwestern North America. A pair of islands in Lake Michigan presented a unique opportunity to study the role of alternative hosts for the adult stage of the blacklegged tick for maintaining a tick population as a deer herd exists on North Manitou Island but not on South Manitou Island, where coyotes (Canis latrans) and hares (Lepus americanus) are the dominant medium mammals. Additionally, we were able to investigate the maintenance of I. scapularis and B. burgdorferi in small mammal communities on both islands, which were dominated by eastern chipmunks (Tamias striatus). From 2011 to 2015, we surveyed both islands for blacklegged ticks by drag cloth sampling, bird mist netting, and small and medium-sized mammal trapping. We assayed questing ticks, on-host ticks, and mammal biopsies for the Lyme disease pathogen, B. burgdorferi. We detected all three life stages of the blacklegged tick on both islands. Of the medium mammals sampled, no snowshoe hares (Lepus americanus, 0/23) were parasitized by adult blacklegged ticks, but 2/2 coyotes (Canis latrans) sampled on South Manitou Island in 2014 were parasitized by adult blacklegged ticks, suggesting that coyotes played a role in maintaining the tick population in the absence of deer. We also detected I. scapularis ticks on passerine birds from both islands, providing support that birds contribute to maintaining as well as introducing blacklegged ticks and B. burgdorferi to the islands. We observed higher questing adult and nymphal tick densities, and higher B. burgdorferi infection prevalence in small mammals and in adult ticks on the island with deer as compared to the deer-free island. On the islands, we also found that 25% more chipmunks were tick-infested than mice, fed more larvae and nymphs relative to their proportional abundance compared to mice, and thus may play a larger role compared to mice in the maintenance of B. burgdorferi. Our investigation demonstrated that alternative hosts could maintain a local population of blacklegged ticks and an enzootic cycle of the Lyme disease bacterium in the absence of white-tailed deer. Thus, alternative adult blacklegged tick hosts should be considered when investigating deer-targeted management tools for reducing tick-borne disease risk, especially when the alternative host community may be abundant and diverse.

The recently recognized Lyme disease spirochete, Borrelia mayonii, has been detected in host-seeking Ixodes scapularis Say ticks and is associated with human disease in the Upper Midwest. Although experimentally shown to be vector competent, studies have been lacking to determine the duration of time from attachment of a single B. mayonii-infected I. scapularis nymph to transmission of spirochetes to a host. If B. mayonii spirochetes were found to be transmitted within the first 24 h after tick attachment, in contrast to Borrelia burgdorferi spirochetes (>24 h), then current recommendations for tick checks and prompt tick removal as a way to prevent transmission of Lyme disease spirochetes would need to be amended. We therefore conducted a study to determine the probability of transmission of B. mayonii spirochetes from single infected nymphal I. scapularis ticks to susceptible experimental mouse hosts at three time points postattachment (24, 48, and 72 h) and for a complete feed (>72–96 h). No evidence of infection with or exposure to B. mayonii occurred in mice that were fed upon by a single infected nymph for 24 or 48 h. The probability of transmission by a single infected nymphal tick was 31% after 72 h of attachment and 57% for a complete feed. In addition, due to unintended simultaneous feeding upon some mice by two B. mayonii-infected nymphs, we recorded a single occasion in which feeding for 48 h by two infected nymphs resulted in transmission and viable infection in the mouse. We conclude that the duration of attachment of a single infected nymphal I. scapularis tick required for transmission of B. mayonii appears to be similar to that for B. burgdorferi: transmission is minimal for the first 24 h of attachment, rare up to 48 h, but then increases distinctly by 72 h postattachment.

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

The recently described Lyme disease spirochete Borrelia mayonii is associated with human illness in the Upper Midwest of the United States. Experimental laboratory studies and field observations on natural infection indicate that B. mayonii is maintained by horizontal transmission between tick vectors and vertebrate reservoirs. While maintaining a colony of Ixodes scapularis Say (Acari: Ixodidae) ticks infected with the B. mayonii type strain (MN14-1420), we had an opportunity to examine whether infected females may pass this spirochete transovarially to their offspring. We found no evidence of B. mayonii infection in subsets of larvae originating from 18 infected I. scapularis females (grand total of 810 larvae tested), or in mice exposed to larval feeding.

The white-footed mouse, Peromyscus leucopus (Rafinesque), is a reservoir for the Lyme disease spirochete Borrelia burgdorferi sensu stricto in the eastern half of the United States, where the blacklegged tick, Ixodes scapularis Say (Acari: Ixodidae), is the primary vector. In the Midwest, an additional Lyme disease spirochete, Borrelia mayonii, was recorded from naturally infected I. scapularis and P. leucopus. However, an experimental demonstration of reservoir competence was lacking for a natural tick host. We therefore experimentally infected P. leucopus with B. mayonii via I. scapularis nymphal bites and then fed uninfected larvae on the mice to demonstrate spirochete acquisition and passage to resulting nymphs. Of 23 mice fed on by B. mayonii-infected nymphs, 21 (91%) developed active infections. The infection prevalence for nymphs fed as larvae on these infected mice 4 wk post-infection ranged from 56 to 98%, and the overall infection prevalence for 842 nymphs across all 21 P. leucopus was 75% (95% confidence interval, 72–77%). To assess duration of infectivity, 10 of the P. leucopus were reinfested with uninfected larval ticks 12 wk after the mice were infected. The overall infection prevalence for 480 nymphs across all 10 P. leucopus at the 12-wk time point was 26% (95% confidence interval, 23–31%), when compared with 76% (95% confidence interval, 71–79%) for 474 nymphs from the same subset of 10 mice at the 4-wk time point. We conclude that P. leucopus is susceptible to infection with B. mayonii via bite by I. scapularis nymphs and an efficient reservoir for this Lyme disease spirochete.

The invasive, human-biting Asian longhorned tick, Haemaphysalis longicornis, was detected in New Jersey in the eastern United States in August of 2017 and by November of 2018 this tick had been recorded from 45 counties across 9 states, primarily along the Eastern Seaboard. The establishment of H. longicornis in the United States has raised the questions of how commonly it will bite humans and which native pathogens may naturally infect this tick. There also is a need for experimental vector competence studies with native pathogens to determine if H. longicornis can acquire a given pathogen while feeding, pass it transstadially, and then transmit the pathogen in the next life stage. In this experimental study, we evaluated the vector competence of a population of H. longicornis originating from the United States (New York) for a native isolate (B31) of the Lyme disease spirochete, Borrelia burgdorferi sensu stricto (s.s.). In agreement with a previous experimental study on the vector competence of H. longicornis for Borrelia garinii, we found that uninfected H. longicornis larvae could acquire B. burgdorferi s.s. while feeding on infected Mus musculus mice (infection prevalence >50% in freshly fed larvae) but that the infection was lost during the molt to the nymphal stage. None of 520 tested molted nymphs were found to be infected, indicating that transstadial passage of B. burgdorferi s.s. is absent or rare in H. longicornis; and based on the potential error associated with the number of nymphs testing negative in this study, we estimate that the upper 95% limit for infection prevalence was 0.73%. An Ixodes scapularis process control showed both effective acquisition of B. burgdorferi s.s. from infected mice by uninfected larvae and transstadial passage to the nymphal stage (infection prevalence of 80-82% for both freshly fed larvae and molted nymphs). We also observed that although H. longicornis larvae could be compelled to feed on mice by placing the ticks within feeding capsules, attachment and feeding success was minimal (

The objective of the current study was to determine the optimal concentration of dietary standardized ileal digestible (SID) Lys required to maximize litter gain and minimize sow BW loss in modern high-yielding lactating sows when SID CP was kept constant across dietary treatments. A total of 396 parity 1 to 5 sows (L × Y, DanBred, Herlev, Denmark) were included in the study from day 3 after farrowing until weaning at day 26. Sows were allocated to 6 dietary treatments increasing in SID Lys concentration (6.19, 6.90, 7.63, 8.33, 9.04, and 9.76 g/kg). Diets were isoenergetic (14.04 MJ ME/kg as-fed). Litters were standardized to 14 piglets at day 3 ± 2 d postpartum. At day 3 ± 2 d and at day 26 ± 3 d in lactation, litter weight, and sow BW and back fat were registered. On a subsample of 72 parity 2 to 4 sows, litters were additionally weighed at days 10 and 17 ± 3 d, and milk and blood were sampled at day 3 ± 2 d, and 10, 17 and at 24 ± 3 d in lactation. For the 72 sows, body pools of fat and protein were also determined at days 3 ± 2 and 26 ± 3 d using the D2O dilution technique. All data were analyzed as a randomized complete block design using PROC MIXED in SAS. Furthermore, data were subjected to linear and quadratic polynomial contrasts. Variables with quadratic or linear effects or days in milk × treatment interactions were selected for analysis in PROC NLMIXED using linear broken-line models to evaluate optimal SID Lys concentrations. Only models that converged and the best fitting models were included. Average daily litter gain increased until a breakpoint at 8.11 g/kg of SID Lys (as-fed). At and above the breakpoint, multiparous and primiparous sows had litter gains of 3.36 and 2.93 kg/d, respectively. Weaning litter size (13.0 ± 0.1) was similar between the 6 dietary treatments (P = 0.28). Lactation sow BW loss was minimized to 0.17 kg/d at 9.05 g/kg of SID Lys and sow body protein loss was minimized to 0.23 kg at 9.22 g/kg of SID Lys. Linear broken-line analyses showed that for 3, 10, 17, and 24 DIM, plasma urea was minimized at 7.02, 8.10, 8.73, and 8.32 g/kg of SID Lys, respectively, and milk fat was maximized at 7.80 g/kg of SID Lys. In conclusion, in our conditions, high-yielding lactating sows required 8.11 g/kg of SID Lys to maximize litter gain and 9.05 g/kg of SID Lys to minimize sow BW loss. Based on plasma urea, the optimal dietary concentration of SID Lys was lowest in week 1, intermediate in week 2 and 4, and greatest in week 3 of lactation.

‘Candidatus Ehrlichia khabarensis’ was first described from rodents and insectivores in the Far East territory of Khabarovsk on the Russian Pacific Coast. Here we report the detection of DNA from this microorganism in rodents and fed ticks collected from rodents in British Columbia, Canada in 2013–2014. ‘Candidatus Ehrlichia khabarensis’ was detected in (i) a female Ixodes angustus tick collected from a Peromyscus maniculatus; (ii) a female Dermacentor andersoni tick collected from a Perognathus parvus; (iii) a pool of 2 larval Ixodes pacificus ticks collected from a single P. maniculatus; and (iv) a pool of 3 nymphal I. pacificus ticks collected from a single P. maniculatus. Three of these four rodents (2 P. maniculatus and 1 P. parvus) with infected ticks also had evidence of ‘Candidatus Ehrlichia khabarensis’ in at least one tissue type. The infected P. maniculatus and Ixodes ticks came from the Vancouver area in western British Columbia and the P. parvus and Dermacentor tick from an inland site in central British Columbia. Although it remains to be determined whether ‘Candidatus Ehrlichia khabarensis’ has any negative impacts on wildlife, domestic animals or humans, we note that all three tick species found to contain the DNA of this microorganism are known to bite humans. Future detection of this microorganism either in ticks collected from rodents and allowed to molt to the next life stage prior to being tested, or from host-seeking ticks, is required to determine if it can survive the tick’s molt after being ingested via an infectious blood meal.

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

A novel species within the Borrelia burgdorferi sensu lato complex, provisionally named Borrelia mayonii , was recently found to be associated with Lyme borreliosis in the Upper Midwest of the United States. Moreover, B. mayonii was detected from host-seeking Ixodes scapularis , the primary vector of B. burgdorferi sensu stricto in the eastern United States. We therefore conducted a study to confirm the experimental vector competence of I. scapularis for B. mayonii (strain MN14-1420), using colony ticks originating from adults collected in Connecticut and CD-1 white mice. Larvae fed on mice 10 weeks after needle-inoculation with B. mayonii acquired spirochetes and maintained infection through the nymphal stage at an average rate of 12.9%. In a transmission experiment, 40% of naive mice exposed to a single infected nymph developed viable infections, as compared with 87% of mice fed upon by 2–3 infected nymphs. Transmission of B. mayonii by one or more feeding infected nymphs was uncommon up to 48 h after attachment (one of six mice developed viable infection) but occurred frequently when nymphs were allowed to remain attached for 72–96 h or feed to completion (11 of 16 mice developed viable infection). Mice infected via tick bite maintained viable infection with B. mayonii , as determined by ear biopsy culture, for at least 28 weeks. Our results demonstrate that I. scapularis is capable of serving as a vector of B. mayonii . This finding, together with data showing that field-collected I. scapularis are infected with B. mayonii , indicate that I. scapularis likely is a primary vector to humans of this recently recognized Lyme borreliosis spirochete.

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

The relapsing fever spirochete, Borrelia miyamotoi, is increasingly recognized as a cause of human illness (hard tick-borne relapsing fever) in the United States. We previously demonstrated that single nymphs of the blacklegged tick, Ixodes scapularis, can transmit B. miyamotoi to experimental hosts. However, two recent epidemiological studies from the Northeastern United States indicate that human cases of hard tick-borne relapsing fever peak during late summer, after the spring peak for nymphal tick activity but coincident with the peak seasonal activity period of larval ticks in the Northeast. These epidemiological findings, together with evidence that B. miyamotoi can be passed from infected I. scapularis females to their offspring, suggest that bites by transovarially-infected larval ticks can be an important source of human infection. To demonstrate experimentally that transovarially-infected larval I. scapularis ticks can transmit B. miyamotoi, outbred Mus musculus CD1 mice were exposed to 1 or 2 potentially infected larvae. Individual fed larvae and mouse blood taken 10 d after larvae attached were tested for presence of B. miyamotoi DNA, and mice also were examined for seroreactivity to B. miyamotoi 8 wk after tick feeding. We documented B. miyamotoi DNA in blood from 13 (57%) of 23 mice exposed to a single transovarially-infected larva and in 5 (83%) of 6 mice exposed to two infected larvae feeding simultaneously. All 18 positive mice also demonstrated seroreactivity to B. miyamotoi. Of the 11 remaining mice without detectable B. miyamotoi DNA in their blood 10 d after infected larvae attached, 7 (64%) had evidence of spirochete exposure by serology 8 wk later. Because public health messaging for risk of exposure to Lyme disease spirochetes focuses on nymphal and female I. scapularis ticks, our finding that transovarially-infected larvae effectively transmit B. miyamotoi should lead to refined tick-bite prevention messages.

Ticks transmit infectious agents to humans and other animals. Genetic manipulation of vectors like ticks could enhance the development of alternative disease control strategies. Transgene expression using the phytopathogen Agrobacterium tumefaciens has been shown to promote the genetic modification of non-plant cells. In the present work we developed T-DNA constructs for A. tumefaciens to mediate transgene expression in HeLa cells as well as Rhipicephalus microplus tick cells. Translational fusions eGfp:eGfp or Salp15:eGfp, including the enhanced-green fluorescent protein and the Ixodes scapularis salivary factor SALP15 genes, were constructed using the CaMV 35S (cauliflower mosaic virus) promoter, "PBm" tick promoter (R. microplus pyrethroid metabolizing esterase gene) or the Simian Virus SV40 promoter. Confocal microscopy, RT-PCR and Western-blot assays demonstrated transgene(s) expression in both cell lines. Transgene expression was also achieved in vivo, in both R. microplus and I. scapularis larvae utilizing a soaking method including the A. tumefaciens donor cells and confirmed by nested-RT-PCR showing eGfp or Salp15 poly-A-mRNA(s). This strategy opens up a new avenue to express exogenous genes in ticks and represents a potential breakthrough for the study of tick-host pathophysiology.

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

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

Background Forests in urban landscapes differ from their rural counterparts in ways that may alter vector-borne disease dynamics. In urban forest fragments, tick-borne pathogen prevalence is not well characterized; mitigating disease risk in densely-populated urban landscapes requires understanding ecological factors that affect pathogen prevalence. We trapped blacklegged tick (Ixodes scapularis) nymphs in urban forest fragments on the East Coast of the United States and used multiplex real-time PCR assays to quantify the prevalence of four zoonotic, tick-borne pathogens. We used Bayesian logistic regression and WAIC model selection to understand how vegetation, habitat, and landscape features of urban forests relate to the prevalence of B. burgdorferi (the causative agent of Lyme disease) among blacklegged ticks. Results In the 258 nymphs tested, we detected Borrelia burgdorferi (11.2% of ticks), Borrelia miyamotoi (0.8%) and Anaplasma phagocytophilum (1.9%), but we did not find Babesia microti (0%). Ticks collected from forests invaded by non-native multiflora rose (Rosa multiflora) had greater B. burgdorferi infection rates (mean = 15.9%) than ticks collected from uninvaded forests (mean = 7.9%). Overall, B. burgdorferi prevalence among ticks was positively related to habitat features (e.g. coarse woody debris and total understory cover) favorable for competent reservoir host species. Conclusions Understory structure provided by non-native, invasive shrubs appears to aggregate ticks and reservoir hosts, increasing opportunities for pathogen transmission. However, when we consider pathogen prevalence among nymphs in context with relative abundance of questing nymphs, invasive plants do not necessarily increase disease risk. Although pathogen prevalence is greater among ticks in invaded forests, the probability of encountering an infected tick remains greater in uninvaded forests characterized by thick litter layers, sparse understories, and relatively greater questing tick abundance in urban landscapes. Electronic supplementary material The online version of this article (10.1186/s13071-018-2623-0) contains supplementary material, which is available to authorized users.

Borrelia miyamotoi sensu lato relapsing fever group spirochetes are emerging as causative agents of human illness ( Borrelia miyamotoi disease) in the United States. Host-seeking Ixodes scapularis ticks are naturally infected with these spirochetes in the eastern United States and experimentally capable of transmitting B. miyamotoi . However, the duration of time required from tick attachment to spirochete transmission has yet to be determined. We therefore conducted a study to assess spirochete transmission by single transovarially infected I. scapularis nymphs to outbred white mice at three time points post-attachment (24, 48, and 72 h) and for a complete feed ( > 72–96 h). Based on detection of B. miyamotoi DNA from the blood of mice fed on by an infected nymph, the probability of spirochete transmission increased from 10% by 24 h of attachment (evidence of infection in 3/30 mice) to 31% by 48 h (11/35 mice), 63% by 72 h (22/35 mice), and 73% for a complete feed (22/30 mice). We conclude that (i) single I. scapularis nymphs effectively transmit B. miyamotoi relapsing fever group spirochetes while feeding, (ii) transmission can occur within the first 24 h of nymphal attachment, and (iii) the probability of transmission increases with the duration of nymphal attachment.

Borrelia mayonii, a recently recognized species within the Borrelia burgdorferi sensu lato complex, has been detected in host-seeking Ixodes scapularis Say ticks and found to be associated with Lyme disease in the Upper Midwest. This spirochete has, to date, not been documented from the Northeast, but we previously demonstrated that I. scapularis ticks originating from Connecticut are capable of serving as a vector of B. mayonii. In this follow-up study, we compared the vector efficiency for B. mayonii (strain MN14-1420) of I. scapularis ticks originating from Minnesota in the Upper Midwest and Connecticut in the Northeast. CD-1 outbred white mice previously infected with B. mayonii via tick bite were exposed to simultaneous feeding by Minnesota and Connecticut larvae contained within separate feeding capsules. We found no difference in the ability of Minnesota and Connecticut larvae to acquire B. mayonii from infected mice and pass spirochetes to the nymphal stage (overall nymphal infection rates of 11.6 and 13.3%, respectively). Moreover, the efficiency of transmission of B. mayonii by single infected nymphs was similar for the Minnesota and Connecticut ticks (33 and 44%, respectively). We conclude that the examined I. scapularis ticks from the Upper Midwest and Northeast did not differ in their efficiency as vectors for B. mayonii.

A novel species within the Borrelia burgdorferi sensu lato complex, Borrelia mayonii, was recently described and found to be associated with Lyme borreliosis in the Upper Midwest of the United States. The blacklegged tick, Ixodes scapularis, is naturally infected with B. mayonii in the Upper Midwest and has been experimentally demonstrated to serve as a vector for this spirochete. Natural vertebrate reservoirs for B. mayonii remain unknown. In this study, we demonstrate that an experimental spirochete host, the CD-1 strain outbred white mouse, can maintain active infection with B. mayonii for up to 1year: infected mice consistently yielded ear biopsies containing motile spirochetes from 29 to 375days after they were first infected via tick bite. Infection rates in resultant nymphal ticks varied greatly both over time for larvae fed on the same individual mouse at different time points after infection (2-42%) and for larvae fed on different mice at a given time point up to 8 months after infection (0-48%). Infection rates were lower in nymphs fed as larvae on mice 10-12 months after infection (2-3% for 5 mice and 9.8% for 1 mouse). In addition to ear biopsies, B. mayonii was detected from bladder, heart, and spinal cord of infected mice when they were sacrificed 163-375days after initial infection via tick bite. Examination of blood from mice determined to be infected with B. mayonii by ear biopsy did not produce evidence of B. mayonii DNA in blood taken 8-375days after the mice were first infected via tick bite.

Members of the Coxiella genus are intracellular bacteria that can infect a variety of animals including humans. A symbiotic Coxiella was recently described in Amblyomma americanum ticks in the Northern Hemisphere with no further investigations of other Amblyomma species in other geographic regions. These ixodid ticks represent a group of important vectors for human infectious agents. In the present work, we have demonstrated that symbiotic Coxiella (SCox) are widespread, occurring in South America and infecting 100% of all life stages and eggs of the Cayenne ticks Amblyomma cajennense from Brazil and the USA. Using light microscopy, in situ hybridization, and PCR, we demonstrated SCox in salivary glands, ovaries, and the intestines of A. cajennense. These symbionts are vertically and transtadially transmitted in laboratory reared A. cajennense, and quantitative PCR analyses indicate that SCox are more abundant in adult female ticks, reaching values corresponding to an 11×, 38×, and 200× increase in SCox 16S rRNA gene copy number in unfed females, compared to unfed nymphs, larvae, and eggs, respectively. Phylogenetic analyses showed distinct SCox subpopulations in the USA and Brazil and demonstrated that SCox bacteria do not group with pathogenic Coxiella burnetii.

Lyme disease is the most common tick-borne disease in Europe and North America. In the hyperendemic Lyme disease regions of the eastern United States, nymphal Ixodes scapularis are the principal ticks transmitting the Lyme disease spirochete, Borrelia burgdorferi sensu stricto (s.s.). Approximately 25% of questing nymphs in endemic regions are infected with spirochetes. High throughput-sensitive and specific methods for testing nymphal I. scapularis for infection with B. burgdorferi are clearly needed. In the current study, we evaluated whether low-density microarrays could be adapted for the rapid and accurate detection and characterization of spirochetes in nymphal I. scapularis. Three different microarray platforms were developed and tested for the detection of spirochetes in ticks. They could both detect and differentiate different Borrelia genospecies, in one case detecting as few as a single copy of Borrelia DNA.

Using multilocus sequence analyses (MLSA), we investigated the phylogenetic relationship of spirochaete strains from North America previously assigned to the genospecies Borrelia bissettii. We amplified internal fragments of 8 housekeeping genes (clpA, clpX, nifS, pepX, pyrG, recG, rplB, and uvrA) located on the main linear chromosome by polymerase chain reaction. Phylogenetic analysis of concatenated sequences of the 8 loci showed that the B. bissettii clade consisted of 4 closely related clusters which included strains from California (including the type strain DN127-Cl9-2/p7) and Colorado that were isolated from Ixodes pacificus, I. spinipalpis, or infected reservoir hosts. Several strains isolated from I. scapularis clustered distantly from B. bissettii. Genetic distance analyses confirmed that these strains are more distant to B. bissettii than they are to B. carolinensis, a recently described Borrelia species, which suggests that they constitute a new Borrelia genospecies. We propose that it be named Borrelia kurtenbachii sp. nov. in honour of the late Klaus Kurtenbach. The data suggest that ecological differences between B. bissettii and the new Borrelia genospecies reflect different transmission cycles. In view of these findings, the distinct vertebrate host-tick vector associations and the distributions of B. bissettii and B. kurtenbachii require further investigation.

Salp15 is a multifunctional protein, vital to the tick in its need to obtain vertebrate host blood without stimulating a host inflammatory and immune response. The Salp15 protein from both Ixodes scapularis Say and Ixodes ricinus (L.), the principal vectors of the Lyme disease spirochete in eastern North America and Europe, respectively, have been well characterized and found to bind the murine CD4 receptor, DC-SIGN, and the OspC protein of Borrelia burgdorferi. In the current study, we characterized the full salp15 gene in Ixodes pacificus Cooley & Kohls and Ixodes persulcatus Schulze, the principal vectors of Lyme disease spirochetes in western North America and Asia, respectively. In comparing the Salp15 protein of all four principal vector ticks of public health importance for the transmission of Lyme disease spirochetes, we find the 53 C-terminal amino acids to have a high degree of similarity. There are at least three clades in the tree of Salp15 and its homologues, probably representing a multigene family.

Northern and southern Ixodes scapularis Say populations differ greatly in density, host utilization, and especially questing behavior of the immatures. Haplotypes of I. scapularis in North America can be divided into two major clades-the All American Clade (haplotypes A through J) and the Southern Clade (M through O). This genetic variation may affect feeding success and vector competence. This study compared feeding success of larval I. scapularis measured by time-to-drop-off and subsequent transmissibility success of Borrelia burgdorferi to mice using ticks from Mississippi, Connecticut (both F haplotype), and Louisiana (haplotype O). Northern ticks (CT) fed to repletion much faster than MS and LA ticks: overall, 73.6% of CT ticks had dropped off mice at Day 3 compared to only 1.7% and 6.6% of ticks dropped off for MS and LA ticks at that same time point. As for vector competence, 4 of the 4 mice in each case (MS or CT) that had been fed on by infected nymphs tested positive for B. burgdorferi. In a second experiment, 5 of the 6 mice tested positive for B. burgdorferi after exposure to infected LA ticks as compared with 3 of the 4 mice exposed to infected CT ticks. These data demonstrate that there is no difference in northern and southern populations of I. scapularis in their ability to transmit B. burgdorferi, but the ability of the northern populations to feed rapidly on rodents exceeds that of southern populations.

Ixodes scapularis, the tick vector of the Lyme disease spirochete, is distributed over most of the eastern United States, but >80% of all Lyme disease cases occur in the northeast. The role that genetic differences between northern and southern tick populations play in explaining this disparate distribution of Lyme disease cases is unclear. The present study was conducted with 1,155 SNP markers in eight nuclear genes; the 16S mitochondrial gene was examined for comparison with earlier studies. We examined 350 I. scapularis from 7 states covering a representative area of the species. A demographic analysis using Bayesian Extended Skyline Analysis suggested that I. scapularis populations in Mississippi and Georgia began expanding 500,000 years ago, those in Florida and North Carolina 200,000 years ago and those from Maryland and New Jersey only during the past 50,000 years with an accompanying bottleneck. Wisconsin populations only began expanding in the last 20,000 years. Analysis of current migration patterns suggests large amounts of gene flow in northern collections and equally high rates of gene flow among southern collections. In contrast there is restricted and unidirectional gene flow between northern and southern collections, mostly occurring from northern into southern populations. Northern populations are characterized by nymphs that quest above the leaf litter, are easy to collect by flagging, frequently feed on mammals such as rodents and shrews, commonly attach to people, and about 25% of which are infected with B. burgdorferi. If there is a genetic basis for these behaviors, then the patterns detected in this study are of concern because they suggest that northern I. scapularis populations with a greater ability to vector B. burgdorferi to humans are expanding south.

Borrelia burgdorferi synthesizes an HtrA protease (BbHtrA) which is a surface-exposed, conserved protein within Lyme disease spirochetes with activity toward CheX and BmpD of Borrelia spp, as well as aggrecan, fibronectin and proteoglycans found in skin, joints and neural tissues of vertebrates. An antibody response against BbHtrA is observed in Lyme disease patients and in experimentally infected laboratory mice and rabbits. Given the surface location of BbHtrA on B. burgdorferi and its ability to elicit an antibody response in infected hosts, we explored recombinant BbHtrA as a potential vaccine candidate in a mouse model of tick-transmitted Lyme disease. We immunized mice with two forms of BbHtrA: the proteolytically active native form and BbHtrA ablated of activity by a serine to alanine mutation at amino acid 226 (BbHtrA(S226A)). Although inoculation with either BbHtrA or BbHtrA(S226A) produced high-titer antibody responses in C3H/HeJ mice, neither antigen was successful in protecting mice from B. burgdorferi challenge. These results indicate that the search for novel vaccine candidates against Lyme borreliosis remains a challenge.

A field trial was conducted on residential properties in a Lyme disease endemic area of New Jersey to determine the efficacy of Maxforce Tick Management System (TMS) bait boxes modified with doxycycline hyclate-laden bait to reduce the acarological risk of Lyme disease and the utility of galvanized steel shrouds to protect the bait boxes from squirrel depredation and ability to routinely service these devices. The strategy began with a 9-wk deployment against larvae followed by a 17-wk deployment against nymphs and larvae the second year. Passive application of fipronil reduced nymphal and larval tick burdens on small mammals by 76 and 77%, respectively, and nymphal tick abundance by 81% on treated properties. In addition, the percentage of infected small mammals recovered from intervention areas following treatment was reduced by 96% for Borrelia burgdorferi and 93% for Anaplasma phagocytophilum. Infection prevalence in host-seeking nymphal ticks for both B. burgdorferi and A. phagocytophilum were reduced by 93 and 61%, respectively. Results indicate that Maxforce TMS bait boxes fitted with doxycycline-impregnated bait is an effective means of reducing ticks and infection prevalence for B. burgdorferi and A. phagocytophilum in both rodent reservoirs and questing Ixodes scapularis Say ticks. The protective shroud allows the device to be routinely serviced and protect against squirrel depredation.

As an alternative to oral prophylaxis for the prevention of tick transmission of Borrelia burgdorferi , we tested antibiotic cream prophylactic formulations in a murine model of spirochete infection. A 4% preparation of doxycycline cream afforded no protection, but a single application of 4% azithromycin cream was 100% protective when applied directly to the tick bite site at the time of tick removal. Indeed, the azithromycin cream was 100% effective when applied at up to 3 days after tick removal and protected 74% of mice exposed to tick bite when applied at up to 2 weeks after tick removal. Azithromycin cream was also protective when applied at a site distal to the tick bite site, suggesting that it was having a systemic effect in addition to a local transdermal effect. Mice that were protected from tick-transmitted infection did not seroconvert and did not infect larval ticks on xenodiagnosis. Azithromycin cream formulations appear to hold promise for Lyme disease prophylaxis.

A field trial was conducted in a Lyme disease-endemic area of New Jersey to determine the efficacy of a doxycyline hyclate rodent bait to prophylactically protect and cure small-mammal reservoirs and reduce infection rates in questing Ixodes scapularis ticks for Borrelia burgdorferi and Anaplasma phagocytophilum. The doxycycline-laden bait was formulated at a concentration of 500 mg/kg and delivered during the immature tick feeding season in rodent-targeted bait boxes. The percentage of infected small mammals recovered from treated areas after 2 years of treatment was reduced by 86.9% for B. burgdorferi and 74% for A. phagocytophilum. Infection rates in questing nymphal ticks for both B. burgdorferi and A. phagocytophilum were reduced by 94.3% and 92%, respectively. Results from this study indicate that doxycycline-impregnated bait is an effective means of reducing infection rates for B. burgdorferi and A. phagocytophilum in both rodent reservoirs and questing I. scapularis ticks.

Clinical studies have demonstrated that prophylactic antibiotic treatment of tick bites by Ixodes scapularis in Lyme disease hyperendemic regions in the northeastern United States can be effective in preventing infection with Borrelia burgdorferi sensu stricto, the Lyme disease spirochete. A large clinical trial in Westchester County, NY (USA), demonstrated that treatment of tick bite with 200mg of oral doxycycline was 87% effective in preventing Lyme disease in tick-bite victims (Nadelman, R.B., Nowakowski, J., Fish, D., Falco, R.C., Freeman, K., McKenna, D., Welch, P., Marcus, R., Aguero-Rosenfeld, M.E., Dennis, D.T., Wormser, G.P., 2001. Prophylaxis with single-dose doxycycline for the prevention of Lyme disease after an Ixodes scapularis tick bite. N. Engl. J. Med. 345, 79-84.). Although this excellent clinical trial provided much needed information, the authors enrolled subjects if the tick bite occurred within 3 days of their clinical visit, but did not analyze the data based on the exact time between tick removal and delivery of prophylaxis. An animal model allows for controlled experiments designed to determine the point in time after tick bite when delivery of oral antibiotics would be too late to prevent infection with B. burgdorferi. Accordingly, we developed a tick-bite prophylaxis model in mice that gave a level of prophylactic protection similar to what had been observed in clinical trials and then varied the time post tick bite of antibiotic delivery. We found that two treatments of doxycycline delivered by oral gavage to mice on the day of removal of a single potentially infectious nymphal I. scapularis protected 74% of test mice compared to controls. When treatment was delayed until 24 h after tick removal, only 47% of mice were protected; prophylactic treatment was totally ineffective when delivered ≥2 days after tick removal. Although the dynamics of antibiotic treatment in mice may differ from humans, and translation of animal studies to patient management must be approached with caution, we believe our results emphasize the point that antibiotic prophylactic treatment of tick bite to prevent Lyme disease is more likely to be efficacious if delivered promptly after potentially infectious ticks are removed from patients. There is only a very narrow window for prophylactic treatment to be effective post tick removal.

A total of 196 small mammals were collected in Portugal and tested for Borrelia burgdorferi sensu lato. Tissue samples were taken from each animal and cultured in Barbour-Stoenner-Kelly (BSK)-II medium. The single strain of spirochete isolated was confirmed as Borrelia lusitaniae by genetic analyses. This is the first report of B. lusitaniae isolated from Apodemus sylvaticus.

Nymphal Ixodes scapularis Say are the principal vectors of Lyme disease spirochetes (Borrelia burgdorferi sensu stricto) in the eastern United States. Physicians frequently face the decision of whether or not to administer prophylactic antibiotics to human tick bite victims in Lyme disease endemic regions, based on the overall probability that such bites will result in infection with B. burgdorferi s.s. We evaluated the transmission dynamics of B. burgdorferi s.s. during the key third day of nymphal I. scapularis feeding, when the risk of transmission rapidly increases. The cumulative probability that 50% of infected ticks transmitted B. burgdorferi s.s. occurred at 68 h of tick attachment and our overall estimate that a human tick bite would result in transmission of B. burgdorferi s.s. was 2.4%.

The mouse Pactolus gene is an evolutionary paralogue to the CD18/beta2 integrin subunit and is preferentially expressed by neutrophils. When first identified, it was assumed Pactolus would function as an adhesion receptor similar to other beta integrin subunits. The analysis of mice genetically deficient in Pactolus, however, did not define any lesion in neutrophil migration, adhesion or phagocytosis. Therefore a wider analysis of the Pactolus deficiency was initiated using transcriptional profiling during an inflammatory insult. This screen identified a single transcript, CXCL13, that was elevated in cells from a peritoneal lavage of the wild type animal compared to the Pactolus-deficient animal. Our analyses confirmed resident macrophages as being responsible for the chemokine using intracellular CXCL13 staining and additional cell markers to phenotypically characterize such cells. The resident CXCL13-expressing cells (which do not express Pactolus) are functionally distinct from the macrophages recruited into the peritoneal cavity following the inflammatory stimulation since the recruited macrophages do not express detectable levels of the chemokine. The numbers and expression patterns of these resident CXCL13-expressing cells do not vary in naïve animals of wild type or Pactolus-deficient origin. Additionally, Pactolus-deficient neutrophils do not preferentially kill (compared to wild type) CXC13-expressing macrophages. These data suggest that during an inflammatory response, Pactolus may help retain CXCL13-expressing cells within the peritoneal environment.

Pactolus is a cell surface protein expressed by murine neutrophils. Pactolus is similar to the β integrins, except it lacks a functional metal ion-dependent adhesion site domain and is expressed without an α-chain partner. The majority of the Pactolus protein is held within the cell in dense granules in a highly glycosylated form. This intracellular form of Pactolus can be released to the cell surface following inflammatory activation or ligation of Pactolus on the cell surface. In addition, intracellular Pactolus translocates to the neutrophil surface following induction of apoptosis. Neutrophil activation studies suggest that Pactolus does not serve as an activating or phagocytic receptor for the neutrophil. To further define the function of Pactolus, a Pactolus-null mouse was generated. Pactolus-deficient animals mature appropriately and possess normal numbers of neutrophils, display appropriate migration into sites of inflammation, and combat introduced infections efficiently. These data suggest that Pactolus does not function as a neutrophil phagocytic or adhesion receptor, but may instead serve as a sugar-bearing ligand for lectin recognition by other cells.

Murine Pactolus is a beta-integrin-like molecule expressed exclusively on the surface of granulocytes. Cell surface expression of Pactolus is dramatically increased following activation of bone marrow neutrophils with known agonists, and cross-linking of cell surface Pactolus, suggesting the bulk of the protein is in intracellular stores. The mature protein is found in two forms depending upon the extent of N-linked glycosylation. There is no evidence to suggest that Pactolus requires an associated alpha chain for expression. In some mouse strains, a truncated form of the protein is predicted based upon alternative splicing: this form, however, is unstable and rapidly degraded after synthesis. Differences in the quantities of these Pactolus mRNA isoforms have defined two alleles. BALB/c and C3H/HeJ mice possess allele B and preferentially express the truncated, unstable product, whereas C57BL/6 mice possess allele A and only produce the membrane-bound form. Sequence analysis has shown the difference between these two alleles is due to a single base pair difference at the splice acceptor site for the truncated product. The increased expression of the membrane form of Pactolus by granulocytes of C57BL/6 mice suggests a compensatory adhesion function that is reduced in cells from the low producing strains.

Indomethacin inhibits prostaglandin synthesis and causes gastric mucosal damage. The correlation between the gastric mucosal function and gastric blood flow was investigated. The intragastric liquid junction corrected potential difference (PD) across the stomach wall was used to characterize the gastric mucosal function. Mucosal blood flow was determined by the radiolabelled microsphere technique. Seven dogs were anaesthetized, intragastric PD was measured continuously and blood flow determined at basal condition during sympathetic activation, and after IV indomethacin (7.5 mg/kg). PD was unchanged during the basal period and during mild sympathetic activation. After indomethacin PD was reduced significantly (-40 +/- 6 mV to -20 +/- 5 mV, measured with gastric lumen negative, mean and SD, P less than 0.05). Mucosal blood flow decreased during sympathetic activation and a further significant reduction was seen after indomethacin. PD and flow reductions correlated (R = 0.92). As indomethacin given IV caused a parallel impairment of gastric mucosal function and blood flow, part of the indomethacin-related mucosal damaging effects might be due to the flow reduction.