A comparison of horizontal and transovarial transmission efficiency of Borrelia miyamotoi by Ixodes scapularis.

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.

Experimental Infection of Amblyomma americanum (Acari: Ixodidae) With Bourbon Virus (Orthomyxoviridae: Thogotovirus).

Following the recent discovery of Bourbon virus (BRBV) as a human pathogen, and the isolation of the virus from Amblyomma americanum (L.) collected near the location of a fatal human case, we undertook a series of experiments to assess the laboratory vector competence of this tick species for BRBV. Larval ticks were infected using an immersion technique, and transstadial transmission of virus to the nymphal and then to the adult stages was demonstrated. Transstadially infected nymphs transmitted virus to adult ticks at very high rates during cofeeding, indicating the presence of infectious virus in the saliva of engorging ticks. Vertical transmission by transstadially infected females to their progeny occurred, but at a low rate. Rabbits fed on by infected ticks of all active life stages developed high titers of antibody to the virus, demonstrating host exposure to BRBV antigens/live virus during tick blood feeding. These results demonstrate that A. americanum is a competent vector of BRBV and indicate that cofeeding could be critical for enzootic maintenance.

Experimental Demonstration of Reservoir Competence of the White-Footed Mouse, Peromyscus leucopus (Rodentia: Cricetidae), for the Lyme Disease Spirochete, Borrelia mayonii (Spirochaetales: Spirochaetaceae).

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.

Failure of the Asian longhorned tick, Haemaphysalis longicornis, to serve as an experimental vector of the Lyme disease spirochete, Borrelia burgdorferi sensu stricto.

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 (<0.5%) when larvae were placed freely on the fur of the mice. We conclude that H. longicornis is unlikely to contribute more than minimally, if at all, to transmission of Lyme disease spirochetes in the United States.

Immunoproteomic analysis of Borrelia miyamotoi for the identification of serodiagnostic antigens.

The tick-borne spirochete, Borrelia miyamotoi, is an emerging pathogen of public health significance. Current B. miyamotoi serodiagnostic testing depends on reactivity against GlpQ which is not highly sensitive on acute phase serum samples. Additionally, anti-B. miyamotoi antibodies can cross-react with C6 antigen testing for B. burgdorferi, the causative agent of Lyme disease, underscoring the need for improved serological assays that produce accurate diagnostic results. We performed an immunoproteomics analysis of B. miyamotoi proteins to identify novel serodiagnostic antigens. Sera from mice infected with B. miyamotoi by subcutaneous inoculation or tick bite were collected for immunoblotting against B. miyamotoi membrane-associated proteins separated by 2-dimensional electrophoresis (2DE). In total, 88 proteins in 40 2DE immunoreactive spots were identified via mass spectrometry. Multiple variable large proteins (Vlps) and a putative lipoprotein were among those identified and analyzed. Reactivity of anti-B. miyamotoi sera against recombinant Vlps and the putative lipoprotein confirmed their immunogenicity. Mouse anti-B. burgdorferi serum was cross-reactive to all recombinant Vlps, but not against the putative lipoprotein by IgG. Furthermore, antibodies against the recombinant putative lipoprotein were present in serum from a B. miyamotoi-infected human patient, but not a Lyme disease patient. Results presented here provide a comprehensive profile of B. miyamotoi antigens that induce the host immune response and identify a putative lipoprotein as a potentially specific antigen for B. miyamotoi serodetection.

Impact of Wearing and Washing/Drying of Permethrin-Treated Clothing on Their Contact Irritancy and Toxicity for Nymphal Ixodes scapularis (Acari: Ixodidae) Ticks.

Permethrin-treated clothing is available as consumer products to prevent bites by tick and insect pests. We used bioassays to examine the impact of wearing and washing/drying of permethrin-treated shirts, pants, and socks, and wearing of treated shoes, on their contact irritancy and toxicity for nymphal Ixodes scapularis Say (Acari: Ixodidae) ticks, the primary vectors in the eastern United States of the causative agents of Lyme disease, human anaplasmosis, and human babesiosis. Pristine permethrin-treated clothing displayed strong contact irritancy and toxicity toward I. scapularis nymphs, with 0-30% of ticks across clothing types and tick sources displaying normal movement 1 h after forced contact for 30-120 s with treated textile. Following 16 d of wear and 16 rounds of machine washing and drying, we recorded reduced concentrations (by 50-90%) of permethrin, compared with pristine treated clothing, from shirts, pants, and socks. This loss of permethrin was associated with reduced contact irritancy and toxicity for ticks after forced contact with worn and washed/dried treated clothing: 31-67% of ticks displayed normal movement 1 h after contact. Nevertheless, the worn and washed/dried treated clothing was still superior to nontreated textile, for which 90-100% of ticks displayed normal movement. Treated shoes, which were worn but not washed, remained as toxic to the ticks as pristine treated shoes. We caution that these laboratory bioassay results should not be interpreted as being directly indicative of the outcome of using washed/worn permethrin-treated clothing in daily life. Although wear and washing/drying did reduce the irritancy and toxicity of permethrin-treated clothing for I. scapularis nymphs more than we had expected, the remaining effect might still reduce the risk of tick bites in a real-life scenario.

An immunocompromised mouse model to infect Ixodes scapularis ticks with the relapsing fever spirochete, Borrelia miyamotoi.

The hard tick-borne relapsing fever spirochete, Borrelia miyamotoi, has recently gained attention as a cause of human illness, but fundamental aspects of its enzootic maintenance are still poorly understood. Challenges to experimental studies with B. miyamotoi-infected vector ticks include low prevalence of infection in field-collected ticks and seemingly inefficient horizontal transmission from infected immunocompetent rodents to feeding ticks. To reliably produce large numbers of B. miyamotoi-infected ticks in support of experimental studies, we developed an animal model where immunocompromised Mus musculus SCID mice were used as a source of B. miyamotoi-infection for larval and nymphal Ixodes scapularis ticks. Following needle inoculation with 1 × 105 spirochetes, the SCID mice developed a high spirochetemia (greater than 1 × 107 copies of B. miyamotoi purB per mL of blood) that persisted for at least 30 d after inoculation. In comparison, immunocompetent M. musculus CD-1 mice developed transient infections, detectable for only 2-8 d within the first 16 d after needle inoculation, with a brief, lower peak spirochetemia (8.5 × 104 - 5.6 × 105purB copies per mL of blood). All larval or nymphal ticks fed on infected SCID mice acquired B. miyamotoi, but frequent loss of infection during the molt led to the proportion infected ticks of the resulting nymphal or adult stages declining to 22-29%. The ticks that remained infected after the molt had well-disseminated infections which then persisted through successive life stages, including transmission to larval offspring.

Transmission of the relapsing fever spirochete, Borrelia miyamotoi, by single transovarially-infected larval Ixodes scapularis ticks.

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.

Contact Irritancy and Toxicity of Permethrin-Treated Clothing for Ixodes scapularis, Amblyomma americanum, and Dermacentor variabilis Ticks (Acari: Ixodidae).

Clothing treated with the pyrethroid permethrin is available in the United States as consumer products to prevent tick bites. We used tick bioassays to quantify contact irritancy and toxicity of permethrin-treated clothing for three important tick vectors of human pathogens: the blacklegged tick, Ixodes scapularis Say (Acari: Ixodidae); the lone star tick, Amblyomma americanum (L.) (Acari: Ixodidae); and the American dog tick, Dermacentor variabilis (Say) (Acari: Ixodidae). We first demonstrated that field-collected I. scapularis nymphs from Minnesota were as susceptible as laboratory-reared nymphs to a permethrin-treated textile. Field ticks examined in bioassays on the same day they were collected displayed contact irritancy by actively dislodging from a vertically oriented permethrin-treated textile, and a forced 1-min exposure resulted in all ticks being unable to move normally, thus posing no more than minimal risk of biting, 1 h after contact with the treated textile. Moreover, we documented lack of normal movement for laboratory-reared I. scapularis nymphs by 1 h after contact for 1 min with a wide range of permethrin-treated clothing, including garments made from cotton, synthetic materials, and blends. A comparison of the impact of a permethrin-treated textile across tick species and life stages revealed the strongest effect on I. scapularis nymphs (0% with normal movement 1 h after a 1-min exposure), followed by A. americanum nymphs (14.0%), I. scapularis females (38.0%), D. variabilis females (82.0%), and A. americanum females (98.0%). Loss of normal movement for all ticks 1 h after contact with the permethrin-treated textile required exposures of 1 min for I. scapularis nymphs, 2 min for A. americanum nymphs, and 5 min for female I. scapularis, D. variabilis, and A. americanum ticks. We conclude that use of permethrin-treated clothing shows promise to prevent bites by medically important ticks. Further research needs are discussed.

Lack of Evidence for Transovarial Transmission of the Lyme Disease Spirochete Borrelia mayonii by Infected Female Ixodes scapularis (Acari: Ixodidae) Ticks.

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.

Transmission of the Lyme Disease Spirochete Borrelia mayonii in Relation to Duration of Attachment by Nymphal Ixodes scapularis (Acari: Ixodidae).

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.

Bioassays to evaluate non-contact spatial repellency, contact irritancy, and acute toxicity of permethrin-treated clothing against nymphal Ixodes scapularis ticks.

Summer-weight clothing articles impregnated with permethrin are available as a personal protective measure against human-biting ticks in the United States. However, very few studies have addressed the impact of contact with summer-weight permethrin-treated textiles on tick vigor and behavior. Our aim was to generate new knowledge of how permethrin-treated textiles impact nymphal Ixodes scapularis ticks, the primary vectors in the eastern United States of the causative agents of Lyme disease, human anaplasmosis, and human babesiosis. We developed a series of bioassays designed to: (i) clarify whether permethrin-treated textiles impact ticks through non-contact spatial repellency or contact irritancy; (ii) evaluate the ability of ticks to remain in contact with vertically oriented permethrin-treated textiles, mimicking contact with treated clothing on arms or legs; and (iii) determine the impact of timed exposure to permethrin-treated textiles on the ability of ticks to move and orient toward a human finger stimulus, thus demonstrating normal behavior. Our results indicate that permethrin-treated textiles provide minimal non-contact spatial repellency but strong contact irritancy against ticks, manifesting as a "hot-foot" effect and resulting in ticks actively dislodging from contact with vertically oriented treated textile. Preliminary data suggest that the contact irritancy hot-foot response may be weaker for field-collected nymphs as compared with laboratory-reared nymphs placed upon permethrin-treated textile. We also demonstrate that contact with permethrin-treated textiles negatively impacts the vigor and behavior of nymphal ticks for >24h, with outcomes ranging from complete lack of movement to impaired movement and unwillingness of ticks displaying normal movement to ascend onto a human finger. The protective effect of summer-weight permethrin-treated clothing against tick bites merits further study.

Transmission of Borrelia miyamotoi sensu lato relapsing fever group spirochetes in relation to duration of attachment by Ixodes scapularis nymphs.

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 72h) and for a complete feed (>72-96h). 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 24h of attachment (evidence of infection in 3/30 mice) to 31% by 48h (11/35 mice), 63% by 72h (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 24h of nymphal attachment, and (iii) the probability of transmission increases with the duration of nymphal attachment.

Isolation of the Lyme Disease Spirochete Borrelia mayonii From Naturally Infected Rodents in Minnesota.

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.

Comparison of Vector Efficiency of Ixodes scapularis (Acari: Ixodidae) From the Northeast and Upper Midwest of the United States for the Lyme Disease Spirochete Borrelia mayonii.

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.

Duration of Borrelia mayonii infectivity in an experimental mouse model for feeding Ixodes scapularis larvae.

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.