Transmission Of Lyme Disease Spirochetes Research Articles

Tick saliva interference with LTβR signaling facilitates the transmission of Lyme disease spirochetes

Tick saliva interference with LTβR signaling facilitates the transmission of Lyme disease spirochetes

Interference with LTβR signaling by tick saliva facilitates transmission of Lyme disease spirochetes

Lyme spirochetes have coevolved with ticks to optimize transmission to hosts using tick salivary molecules (TSMs) to counteract host defenses. TSMs modulate various molecular events at the tick-host interface. Lymphotoxin-beta receptor (LTβR) is a vital immune receptor and plays protective roles in host immunity against microbial infections. We found that Ltbr knockout mice were more susceptible to Lyme disease spirochetes, suggesting the involvement of LTβR signaling in tick-borne Borrelia infection. Further investigation showed that a 15-kDa TSM protein from Ixodes persulcatus (I. persulcatus salivary protein; IpSAP) functioned as an immunosuppressant to facilitate the transmission and infection of Lyme disease spirochetes. IpSAP directly interacts with LTβR to block its activation, thus inhibiting the downstream signaling and consequently suppressing immunity. IpSAP immunization provided mice with significant protection against I. persulcatus-mediated Borrelia garinii infection. Notably, the immunization showed considerable cross-protection against other Borrelia infections mediated by other ixodid ticks. One of the IpSAP homologs from other ixodid ticks showed similar effects on Lyme spirochete transmission. Together, our findings suggest that LTβR signaling plays an important role in blocking the transmission and pathogenesis of tick-borne Lyme disease spirochetes, and that IpSAP and its homologs are promising candidates for broad-spectrum vaccine development.

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.

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.

An investigation of binding ability of Ixodes persulcatus Schulze Salp15 with Lyme disease spirochetes

Salp15, a 15-kDa tick salivary gland protein, has several suppressive modes of activity against host immunity and plays a critical role in the transmission of Lyme disease spirochetes in Ixodes scapularis and Ixodes ricinus, major vectors of Lyme disease in North America and Western Europe. Salp15 adheres to Borrelia burgdorferi and specifically interacts with its outer surface protein C (OspC), protecting the spirochete from antibody-mediated cytotoxicity and facilitating infection in the mice. Recently, we identified two Salp15 homologues, IperSalp15-1 and IperSalp15-2, in Ixodes persulcatus, a vector for Lyme disease in Japan. Here we describe the function of IperSalp15 in the transmission of Lyme borreliosis. To investigate the function of IperSalp15, recombinant IperSalp15-1 and IperSalp15-2 were prepared in bacterial and insect cells. Both were identified in the sera of tick-immunized hamsters, indicating that these are secretory proteins in exposed host animals. Solid-phase overlay and indirect fluorescence assays showed that IperSalp15 binds to OspC from B. burgdorferi, Borrelia garinii, and Borrelia afzelii. Importantly, this binding likely protected the spirochete from antibody-mediated cytotoxicity in vitro. In addition, IperSalp15 tended to facilitate infection in mice. Thus, further characterization of tick molecules, including IperSalp15, could lead to the development of new strategies to prevent the transmission of tick-borne diseases.

Absence of Lyme Disease Spirochetes in LarvalIxodes ricinusTicks

To determine which kind of spirochete infects larval Ixodes ricinus, we examined questing larvae and larvae derived from engorged females for the presence of particular spirochetal DNA that permitted species differentiation. Borrelia miyamotoi was the sole spirochete detected in larval ticks sampled while questing on vegetation. Questing nymphal and adult ticks were infected mainly by Borrelia afzelii, whereas larval ticks resulting from engorged females of the same population were solely infected by B. miyamotoi. Since larvae acquire Lyme disease spirochetes within a few hours of attachment to an infected rodent, questing larvae in nature may have acquired Lyme disease spirochetes from an interrupted host contact. Even if transovarial transmission of Lyme disease spirochetes may occasionally occur, it seems to be an exceedingly rare event. No undisputable proof exists for vertical transmission of Lyme disease spirochetes, whereas B. miyamotoi appears to be readily passed between generations of vector ticks.

Molecular Identification of Salp15, a Key Salivary Gland Protein in the Transmission of Lyme Disease Spirochetes, FromIxodes persulcatusandIxodes pacificus(Acari: Ixodidae)

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 Salpl5 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.

A Doxycycline Hyclate Rodent Bait Formulation for Prophylaxis and Treatment of Tick-transmitted Borrelia burgdorferi

The prophylactic and curative potential of doxycycline hyclate formulated in a rodent bait at concentrations of 250 and 500 mg/Kg was evaluated in a murine model of Lyme borreliosis. Both bait formulations prevented tick-transmitted Borrelia burgdorferi infection in 100% of C3H/HeJ mice (N = 16), as well as cured acute, established infection in mice (N = 8) exposed to bait for 14 days. Spirochete infection was cleared in 88.9% to 100% of infected nymphs feeding on mice fed 250 and 500 mg/Kg antibiotic bait formulations, respectively. These data provide evidence for exploring alternative techniques to prevent transmission of Lyme disease spirochetes.

Response of nymphal Ixodes scapularis, the primary tick vector of Lyme disease spirochetes in North America, to barriers derived from wood products or related home and garden items

Forest products were tested to see if they functioned as a barrier to nymphal Ixodes scapularis. These products could potentially be used to define a border between high density and low density tick zones on residential properties in Lyme disease endemic regions of North America. Common home and garden items were also tested. Three wood products effectively acted as barriers to nymphal I. scapularis: Alaska Yellow Cedar sawdust, Alaska Yellow Cedar woodchips, and cellulose. These three products were then weathered to determine how long they remained active. Cellulose and Alaska Yellow Cedar woodchips lost their activity almost immediately (within three days); in contrast, Alaska Yellow Cedar sawdust impeded crossing by nymphal ticks for up to one month. Creating barriers at the woods-lawn interface may someday play a role in integrated campaigns to prevent Lyme disease but will not serve as a stand-alone measure to block transmission of Lyme disease spirochetes.

Co-feeding Transmission and Its Contribution to the Perpetuation of the Lyme Disease SpirocheteBorrelia afzelii(In Reply to Randolph and Gern)

To the Editor: Although transmission between co-feeding vector ticks may perpetuate particular tick-borne viruses, this mode of transmission plays no role in the epizootiology of Lyme disease spirochetes (1,2). In their letter, Randolph and Gern defend their suggestion that tick-borne pathogens perpetuate effectively by direct passage from one feeding tick to another by criticizing our analysis (3). These researchers mainly address our comparison of the transmission efficiency between simultaneously feeding ticks with that between ticks feeding sequentially on a persistently infected rodent. Our experiments demonstrate that approximately six times as many larvae (85.4%) acquire Borrelia afzelii spirochetes from a systemically infected mouse than from a mouse on which an infected nymph is feeding simultaneously (13.6%) (1). In nature, however, larval ticks rarely co-feed with nymphs on mice or voles; only approximately one fifth (18.8%) of these hosts harbor both subadult stages simultaneously. And of the nymphs, only approximately one quarter (26.4%) are infected by Lyme disease spirochetes. As a result, the natural transmission efficiency between simultaneously feeding ticks would be only one twentieth (5%) of that observed in the laboratory. Multiplying the experimentally observed efficiency of co-feeding transmission (13.6%) by the likelihood of larval and nymphal ticks co-infesting small rodents, as well as by the prevalence of infected nymphal ticks, reduces the efficiency of co-feeding transmission in nature to <1%. Although Randolph and Gern commit several minor mathematical errors, their calculations support our argument that few larval vector ticks would acquire spirochetal infection directly from an infected nymph (3). Randolph and Gern err, however, by applying the same mathematical modifications to the transmission efficiency by which larvae acquire spirochetes from a persistently infected host (3). Whereas the efficiency of co-feeding transmission observed in the laboratory must be modified to pay tribute to the rare event when larvae co-feed with an infected nymph in nature, the efficiency by which larvae acquire infection from a persistently infected host is independent of such limiting parameters. Because a competent rodent host remains infectious to larval ticks throughout its life, the proportion of hosts infested by particular subadult stages of the vector is irrelevant. Thus, the transmission efficiency on a persistently infected host is unchanged in the laboratory and the field. Almost 85.4% of larvae feeding on mice or voles in nature would, therefore, acquire spirochetal infection—far more than by co-feeding. We are correct in stating that natural transmission by sequentially feeding ticks is more efficient than transmission between co-feeding ticks. Randolph and Gern suggest that we could have recorded the distance between the feeding ticks to clarify whether the increase from a 13.6% transmission efficiency between co-feeding ticks to a transmission efficiency of 85.4% from a persistently infected host is associated with the development of a systemic infection. Our experimental observation (Table 1 in our article [1]), as well as a study on the movement of spirochetes through their host’s skin (4), conclusively demonstrates that the increase in transmission efficiency is due to the progressive dissemination of spirochetes from the site of inoculation. The likelihood of a larva’s acquiring spirochetes from any site of its host’s skin increases with the passage of time since the infected nymph attached. To compare the two modes of transmission in terms of efficiency (Table 2 in our article [1]), we permitted the larvae to attach randomly to their rodent hosts, just as they would do in nature. Table 1 Spirochetal infection in larval Ixodes ricinus ticks that fed on mice during the period of attachment of a single Borrelia afzelii–infected nymph and that fed at specified distances from the infecting nympha In the epizootiology of Lyme disease spirochetes, “simultaneous” transmission between co-feeding ticks (<1%) is some two orders of magnitude less efficient than sequential transmission between ticks feeding on persistently infected reservoir rodents (85.4%). The two studies that relied on natural infestation densities and refrained from using artificial feeding capsules conclusively demonstrated that transmission of Lyme disease spirochetes between ticks feeding simultaneously and in close proximity contributes little to the perpetuation of this pathogen, either in North America or in Europe (1,2). We are correct in concluding that Lyme disease spirochetes are maintained in nature mainly by sequential attachment of ticks to persistently infected reservoir hosts. Table 2. Spirochetal infection in larval Ixodes ricinus ticks that fed randomly on bodies of mice beginning at 3 days and 14 days after a single Borrelia afzelii–infected nymph had begun to feed

Chemotactic migration of the Lyme disease spirochete (Borrelia burgdorferi) to salivary gland extracts of vector ticks.

The migration of Lyme disease spirochetes (Borrelia burgdorferi) toward salivary gland extracts (SGEs) of vector ticks was determined by a modified U-tube chemotaxis assay. Responses of cultured spirochetes to the SGEs were measured by dark-field microscopy at intervals after the initial inoculation. The average numbers of spirochetes that migrated were compared between U-tubes containing either SGEs or phosphate-buffered saline (PBS). Spirochetes showed increased migration in tubes containing SGE, and migration was approximately 10-20-fold higher than that for spirochetes observed in tubes containing PBS. In addition, the span of migration showed no significant difference relative to the origin of spirochete isolation. These results demonstrate that spirochete migration can be enhanced by SGEs from fed vector ticks. This mechanism may contribute to the novel transmission of Lyme disease spirochetes between cofeeding infected and uninfected ticks in nature.

Refeeding Activity of Immature Ticks of Ixodes persulcatus and Transmission of Lyme Disease Spirochete by Partially Fed Larvae

The refeeding activity of immature ticks of Ixodes persulcatus was studied under laboratory conditions. BALB/c mice were used as blood sources. Feeding larvae and nymphs spontaneously detached themselves from parasitized mice when the mice were killed 1 or 2 days after tick exposure. Partially fed ticks were reexposed on other mice. Both larvae and nymphs were able to refeed on mice. The resultant engorged ticks normally molted to the next developmental stages. We also examined the transmission of the Lyme disease spirochete, Borrelia garinii, by bites of partially fed ticks. Larvae that had fed on infected mice for 1 or 2 days transmitted the spirochete to mice by refeeding.

201 シュルツェマダニ幼若虫の再吸血性とライム病ボレリア伝播

201 シュルツェマダニ幼若虫の再吸血性とライム病ボレリア伝播

Accelerated transmission of Lyme disease spirochetes by partially fed vector ticks

To determine how rapidly Lyme disease spirochetes (Borrelia burgdorferi) can be transmitted by partially fed vector ticks (Ixodes dammini), attached nymphs were removed from their hosts at various intervals post-attachment and subsequently permitted to re-feed to repletion on noninfected mice. We confirm previous reports that ticks deposit Lyme disease spirochetes in the skin of their hosts mainly after 2 days of attachment. Those that have been removed from a host within this interval can reattach and commence feeding. Spirochete-infected nymphs that have previously been attached to a host for 1 day become infectious to other hosts within another day. Noninfected nymphs acquire infection from spirochete-infected hosts within a day of attachment and become infectious to other hosts 3 to 5 days later. Virtually all ticks transmitted infection when reattaching after first feeding for 2 days. We conclude that partially fed nymphal ticks transmit spirochetal infection more rapidly than do ticks that have never been attached to a host and that infected ticks become infectious before they molt.

Diversionary Role of Hoofed Game in the Transmission of Lyme Disease Spirochetes

To determine whether the presence of ungulates may inhibit transmission of the agent of Lyme disease (Borrelia burgdorferi) while promoting the abundance of its European vector tick (Ixodes ricinus), we compared the feeding density of subadult ticks on roe deer (Capreolus capreolus), red deer (Cervus elaphus), fallow deer (Dama dama), and wild sheep (Ovis ammon) near Berlin and in Brandenburg State, Germany. The prevalence of spirochetal infection in these ticks was compared with that in ticks swept from nearby vegetation. Spirochetes are present in nearly one-fifth of nonfed, questing nymphal and adult wood ticks in the region. Many ungulates in this intensely enzootic region fail to mount a detectable humoral response against the agent of Lyme disease, even when exposed to numerous infected ticks. During the height of the summer, each ungulate may support the feeding of hundreds of subadult ticks. Larvae feed lower on the bodies of hoofed game than do nymphs. Few ticks retain infection by the Lyme disease spirochete after feeding on hoofed game animals. We conclude that numerous I. ricinus ticks feed on ungulates, but that such host-contact fails to infect these ticks while eliminating pre-existing spirochetal infection.

Absence of Transplacental Transmission of Lyme Disease Spirochetes from Reservoir Mice (Peromyscus leucopus) to their Offspring

Lyme disease spirochetes (Borrelia burgdorferi) are naturally maintained in an enzootic cycle mainly by vector ticks (Ixodes dammini) infesting white-footed mice (Peromyscus leucopus). Suggestions that mice may become infected without exposure to ticks prompted a study to evaluate whether mice could transmit spirochetes transplacentally to their offspring. Mice were live-captured in two Massachusetts sites where Lyme disease spirochetes are intensely enzootic. Pregnant females were housed separately in the laboratory through delivery, and mothers and their offspring were caged together until weaning. Each female and two offspring were then examined for evidence of infection serologically and by tick xenodiagnosis. All 14 mother mice examined produced infected ticks and exhibited serum antibodies to B. burgdorferi. However, none of 28 offspring tested produced infected ticks and only a few had evidence of circulating antibody. In a separate experiment, no young CD-1 mice, born of infected mothers, had IgM antibody to B. burgdorferi. It would appear that immature mice are not transplacentally infected with spirochetes and must be exposed to infected ticks before becoming infected and infective themselves.

Reducing Transmission of Lyme Disease Spirochetes in a Suburban Setting

Reducing Transmission of Lyme Disease Spirochetes in a Suburban Setting