OspA immunization decreases transmission of Borrelia burgdorferi spirochetes from infected Peromyscus leucopus mice to larval Ixodes scapularis ticks

Recombinant outer surface protein A (OspA) vaccination of wild animal reservoirs has potential application for reducing Borrelia burgdorferi transmission in nature and subsequent risk of human infection. As a major reservoir host, the white-footed mouse (Peromyscus leucopus) is a candidate for a vaccination program designed to reduce infection prevalence in vector ticks. In this study we characterized the effect of various levels of immunization with recombinant OspA-glutathione transferase fusion protein on transmission dynamics from infected P. leucopus to larval ticks. Control mice were vaccinated with glutathione transferase alone. All mice were experimentally infected with B. burgdorferi before vaccination. The immune responses of the immunized mice were assessed by enzyme-linked immunosorbent assay for antibodies to OspA. Transmission of B. burgdorferi from infected mice was determined by xenodiagnosis with uninfected larval ticks. Spirochetes in ticks were counted by direct immunofluorescence assay. The concentration of antibody to OspA increased with each OspA vaccination but most markedly after the first and second vaccinations. In comparison with control mice, there was reduced transmission by OspA-vaccinated mice to uninfected ticks. One, two, or three doses of OspA reduced infection prevalence in xenodiagnostic ticks by 48%, 92%, or 99% and the numbers of spirochetes per tick by 84%, 98%, or 99%, respectively. This study suggests that vaccination of P. leucopus with OspA could reduce transmission to the tick vector in nature despite prior infection of the reservoir host.     

Investigations on the mode and dynamics of transmission and infectivity of Borrelia burgdorferi sensu stricto and Borrelia afzelii in Ixodes ricinus ticks

Borrelia burgdorferi sensu lato (sl), the agent of Lyme disease, is transmitted to the host during the blood meal of Ixodes ticks. In most unfed ticks, spirochetes are present in the midgut and migrate during blood feeding to the salivary glands, from which they are transmitted to the host via saliva. In the present study, the efficiency of Ixodes ricinus ticks to transmit B. afzelii and B. burgdorferi sensu stricto (ss) and their infectivity for mice were examined in relation to the duration of the blood meal. In addition, we investigated whether these two Borrelia species can penetrate intact skin. Three modes of infection of mice were studied: tick-bite infection, inoculation of tick homogenates, and transcutaneous infection by topical application of tick homogenates on mouse skin. Transmission of B. burgdorferi sl from I. ricinus nymphs to mouse increased with duration of tick attachment. B. afzelii-infected ticks start to transmit infection earlier (< or = 48 h) than B. burgdorferi ss-infected ticks. As previously shown for B. burgdorferi ss in Ixodes scapularis, B. burgdorferi ss and B. afzelii in unfed I. ricinus were noninfectious for mice when tick homogenates were inoculated. However, the inoculation of homogenates of ticks fed for 24 h readily produced infection in mice. Therefore, B. burgdorferi ss and B. afzelii spirochetes are potentially infectious in the tick before natural transmission can occur. None of the mice (n = 33) became infected by transcutaneous transmission when tick homogenates were applied on mouse skin, showing that B. burgdorferi ss and B. afzelii are unable to penetrate intact skin, in contrast to relapsing fever spirochetes. This study also shows that B. afzelii is transmitted by I. ricinus to the host earlier than B. burgdorferi ss and that I. ricinus seems to be a more efficient vector of B. afzelii than B. burgdorferi ss.     

Detection of Borrelia burgdorferi DNA in tick feces provides evidence for organism shedding during vector feeding

Borrelia burgdorferi, the bacterium that causes Lyme disease, is transmitted to a susceptible host by Ixodes spp. tick bites. However, there is uncertainty whether B. burgdorferi are shed from ticks by the fecal route. In this study, B. burgdorferi-infected ticks were fed on mice while confined to a certain area of the skin by a capsule. During and after feeding, tick feces were collected and placed in Barbour-Stoenner-Kelley (BSK)-II media for cultivation and in sterile water for polymerase chain reaction (PCR) analysis. Although none of the tested samples were culture positive for B. burgdorferi, all but one of the fecal DNA samples from infected ticks were PCR positive. These results indicated that B. burgdorferi were shed from feeding ticks during defecation and suggest that the spirochetes did not remain viable once exposed to the outside environment. This finding has important ramifications for investigators interpreting B. burgdorferi-specific PCR results when conducting tick transmission experiments.     

Clethrionomys glareolus, but not Apodemus flavicollis, acquires resistance to lxodes ricinus L, the main European vector of Borrelia burgdorferi

The European rodents Clethrionomys glareolus (bank vole) and Apodemus flavicollis (yellow-necked-mouse) are important hosts of the tick species Ixodes ricinus, the main European vector of Borrelia burgdorferi. We have addressed the question whether or not these tick hosts develop resistance to I. ricinus larvae. C. glareolus and A. flavicollis were exposed to 40 I. ricinus larvae for five consecutive times at two week intervals. Resistance was tested by the following parameters: percentage of ticks fully engorged, time of attachment, engorgement index, percentage of recovered ticks and proportion of larvae moulting to nymphs. Repeated infestation ofC. glareolus resulted in progressive and significant reductions in the percentage of fully engorged ticks, the time of attachment of partially engorged ticks, the scutal index of partially engorged ticks and the moulting success. In contrast, repeatedly infested A. flavicollis did not acquire resistance to larval I. ricinus. Effects of resistance in C glareolus could be partially disrupted by treatment with the immunosuppressive agent, cyclosporin A (CsA), indicating that T helper cells participate in the immune responses to tick bites. The data suggest that acquired immunity to I. ricinus larvae in C. glareolus is a density-dependent factor regulating natural tick burdens and that it may have an impact on the transmission cycle of B. burgdorferi in Central Europe.     

An ecological approach to preventing human infection: vaccinating wild mouse reservoirs intervenes in the Lyme disease cycle

Many pathogens, such as the agents of West Nile encephalitis and plague, are maintained in nature by animal reservoirs and transmitted to humans by arthropod vectors. Efforts to reduce disease incidence usually rely on vector control or immunization of humans. Lyme disease, for which no human vaccine is currently available, is a commonly reported vector-borne disease in North America and Europe. In a recently developed, ecological approach to disease prevention, we intervened in the natural cycle of the Lyme disease agent (Borrelia burgdorferi) by immunizing wild white-footed mice (Peromyscus leucopus), a reservoir host species, with either a recombinant antigen of the pathogen, outer surface protein A, or a negative control antigen in a repeated field experiment with paired experimental and control grids stratified by site. Outer surface protein A vaccination significantly reduced the prevalence of B. burgdorferi in nymphal blacklegged ticks (Ixodes scapularis) collected at the sites the following year in both experiments. The magnitude of the vaccine's effect at a given site correlated with the tick infection prevalence found on the control grid, which in turn correlated with mouse density. These data, as well as differences in the population structures of B. burgdorferi in sympatric ticks and mice, indicated that nonmouse hosts contributed more to infecting ticks than previously expected. Thus, where nonmouse hosts play a large role in infection dynamics, vaccination should be directed at additional species.     

Field studies on Lyme disease in North America

The primary tick vector of Borrelia burgdorferi in eastern and central North America is Ixodes dammini; in western North America, Ixodes pacificus. Searching for the appropriate vector is the first step in determining whether a region is endemic and enzootic for the spirochete B burgdorferi, the etiological agent of Lyme disease, followed by examination of the ticks (questing or already attached to hosts) and wildlife for the spirochete. Questing ticks can be collected through a variety of methods. The two major animal hosts for I dammini are the white-footed mouse Peromyscus leucopus and the white-tailed deer Odocoileus virginianus. Sampling strategies should consider habitat and season. All three life stages of the vector tick should be located, indicating a self-sustaining population. Although B burgdorferi can be detected in many ways, there is no substitute for isolating the spirochete in Barbour-Stoenner-Kelly II medium for definitive proof of the presence of the Lyme disease spirochete.     

Borrelia burgdorferi intercepts host hormonal signals to regulate expression of outer surface protein A

The Borrelia burgdorferi infectious cycle requires that the organism adapt to vast differences in environmental conditions found in its tick and mammalian hosts. Previous studies have shown that B. burgdorferi accomplishes this accommodation in part by regulating expression of its surface proteins. Outer surface protein A (OspA) is a borrelial protein important in colonization of the tick midgut. OspA is up-regulated when the organism is in its tick host and down-regulated when it is in a mammalian host. However, little is known about how it is up-regulated again in a mammalian host in preparation for entry into a feeding tick. Here, we report that the host neuroendocrine stress hormones, epinephrine and norepinephrine, are specifically bound by B. burgdorferi and result in increased expression of OspA. This recognition is specific and blocked by competitive inhibitors of human adrenergic receptors. To determine whether recognition of catecholamines, which are likely to be present at the site of a tick bite, may play a role in preparing the organism for reentry into a tick from a mammalian host, we administered a beta-adrenergic blocker, propranolol, to infected mice. Propranolol significantly reduced uptake of B. burgdorferi by feeding ticks and decreased expression of OspA in B. burgdorferi recovered from ticks that fed on propranolol-treated mice. Our studies suggest that B. burgdorferi may co-opt host neuroendocrine signals to inform the organism of local changes that predict the presence of its next host and allow it to prepare for transition to a new environment.     

An enzootic transmission cycle of Lyme borreliosis spirochetes in the southeastern United States

Lyme borreliosis, or Lyme disease (LD), is a tick-borne zoonotic infection of biomedical significance, caused by Borrelia burgdorferi sensu lato (s.l.) spirochetes and transmitted by Ixodes species ticks. It usually circulates among wildlife vertebrate reservoirs and vector ticks but may infect humans, causing multisystem problems. In far western and northern North America, the host reservoirs, tick vectors, and genospecies of Borrelia are well known but not so in the southern U.S., where there is controversy as to the presence of "true" LD. Here we report the presence of the LD spirochete B. burgdorferi sensu stricto (s.s.) and Borrelia bissettii, three main reservoir hosts, and two enzootic tick vectors in the southeastern U.S. The two enzootic tick vectors, Ixodes affinis and Ixodes minor, rarely bite humans but are more important than the human biting "bridge" vector, Ixodes scapularis, in maintaining the enzootic spirochete cycle in nature. We also report extraordinary longevities and infections in the reservoir rodents Peromyscus gossypinus, Sigmodon hispidus, and Neotoma floridana.     

Significance of red deer (Cervus elaphus) in the ecology of Borrelia burgdorferi sensu lato

BACKGROUND: Red deer (Cervus elaphus) is one of the most important host of the adult tick (Ixodes ricinus) which is the basic vector of the Lyme disease causative agent--Borrelia burgdorferi sensu lato in Europe. The aim of the present study was to establish the role of red deer in the transmission of B. burgdorferi s.1. Material and methods. Tissues from 74 red deers were evaluated and the presence of B. burgdorferi s.1 DNA was identified using nested PCR technique based on fla gene. The identification of species belonging to B. burgdorferi s.1 complex was performed after restriction digestion of nested PCR product with Ddel enzyme and sequencing of nested PCR product. The study included also 55 isolates of I. ricinus females removed from red deer and 466 ticks (73 adult and 393 nymphs) collected from the vegetation in the area where the red deer lives. RESULTS: There were no DNA of B. burgdorferi s.1 complex in the red deer tissues and in ticks removed from deer, however in one tick removed from deer the DNA of other Borrelia species--B. miyamotoi was identified. In ticks collected from vegetation 3 species belonging to B. burgdorferi s.1. complex were identified: B. garinii (3.2% ticks studied), B. afzelii (6.9%) and B. valaisiana (3.6%), however DNA of B. miyamotoi was absent. These results confirm inability of survival of B. burgdorferi s.1. species in tick I. ricinus feeding on red deer blood. However there is a possibility of survival of B. miyamotoi in presence of deer blood at least in ticks feeding on red deer. The main role of red deer in keeping the constant infection level of B. burgdorferi s.1. in the whole population of I. ricinus ticks does not concern B. miyamotoi.     

Co-infection of Borrelia garinii and B. afzelii in a population of wild rodents from woodland

The maintenance of Borrelia burgdorferi s.l. in the environment is dependent on the zoonotic cycle involving tick vectors and certain reservoir hosts. It is well known, that the same species of wild rodents, as well as the vector Ixodes ricinus, are often co-infected with at least two genomospecies of B. burgdorferi s.l.: B. afzelii and B. garinii. The ticks collected from two rodent species: Clethrionomys glareolus and Apodemus flavicollis were examined for the presence of B. burgdorferi s.l., as well as for B. garinii and B. afzelii. In this study, an immunofluorescent antibody assay (IFA) and polymerase chain reaction (PCR) protocols were used. The high level of infestation in rodents (90% for C. glareolus and nearly 100% for A. flavicollis) shows that wild rodents are important hosts of the immature stages of I. ricinus. A high percent of Borrelia positive ticks collected from bank voles and yellow necked mice; above 7% determined by IFA and 2% determined by PCR, clearly revealed that these species of animals are competent zoonotic reservoirs of B. burgdorferi s.l.     

Ticks infesting wild and domestic animals and humans of Sri Lanka with new host records

An island-wide collection of tick species infesting humans, domesticated and wild animals and questing ticks in domestic and peridomestic environments was carried out during 2009–2011. A total of 30,461 ticks were collected from 30 different hosts and free living stages from the ground. The collection consisted of 22 tick species from 30 different hosts recording 12 tick species from humans, 19 from domesticated animals and 21 from wild animals, with a total of 97 new host records. The most common tick species on humans were Dermacentor auratus and Amblyomma testudinairum, while Haemaphysalis intermedia, Rhipicephalus microplus and Rhipicephalus sanguineus were common in domesticated and wild animals sharing 20 host species. Among the questing ticks, immature D. auratus was the most abundant. Humans and domesticated animals were mostly infested by the nymphal stages while adult ticks were found on wild animals. High number of new host records could be due to domestic animals picking tick species from wildlife and vise versa at the human/animal interface. Habitat destruction due to forest fragmentation has lead to wild animals roaming in urban and semi-urban neighbourhoods increasing the interactions of wild animals with domesticated animals. Wild animals play a significant role as a reservoir of many tick borne infections which can easily be spread to domesticated animals and then to humans via tick infestations. Data in this paper are useful for those interested in tick infesting wild and domestic animals and humans in describing the zoonotic potential of tick borne infections.     

Antigenic and genetic heterogeneity of Borrelia burgdorferi populations transmitted by ticks

The genome of Borrelia burgdorferi encodes a large number of lipoproteins, many of which are expressed only at certain stages of the spirochete's life cycle. In the current study we describe the B. burgdorferi population structure with respect to the production of two lipoproteins [outer surface protein A (OspA) and outer surface protein C (OspC)] during transmission from the tick vector to the mammalian host. Before the blood meal, the bacteria in the tick were a homogeneous population that mainly produced OspA only. During the blood meal, the population became more heterogeneous; many bacteria produced both OspA and OspC, whereas others produced only a single Osp and a few produced neither Osp. From the heterogeneous spirochetal population in the gut, a subset depleted of OspA entered the salivary glands and stably infected the host at time points >53 hr into the blood meal. We also examined genetic heterogeneity at the B. burgdorferi vlsE locus before and during the blood meal. In unfed ticks, the vlsE locus was stable and one predominant and two minor alleles were detected. During the blood meal, multiple vlsE alleles were observed in the tick. Tick feeding may increase recombination at the vlsE locus or selectively amplify rare vlsE alleles present in unfed ticks. On the basis of our data we propose a model, which is different from the established model for B. burgdorferi transmission. Implicit in our model is the concept that tick transmission converts a homogeneous spirochete population into a heterogeneous population that is poised to infect the mammalian host.     

Capacity of European animals as reservoir hosts for the Lyme disease spirochete.

The abundance of spirochete-infected Ixodes ricinus ticks naturally derived from endemic rodents was compared to identify the reservoir hosts of the agent of Lyme disease at a series of enzootic sites in Central Europe. Black-striped mice appear to be the most important rodent host for the tick and infect more ticks with Borrelia burgdorferi than do other mice and voles tested. Although rodents infect about half of the ticks that feed on them, lizards infect none. These spirochete-incompetent hosts dilute the force of transmission because many nymphal ticks feed on them. Older male hosts are more heavily infested by subadult ticks than are younger or female hosts. The intensity of transmission of the agent of Lyme disease at particular Central European sites appears to correlate directly with the presence of black-striped mice and inversely with that of lizards.     

甘肃、湖南和广东三省蜱体内嗜吞噬细胞无形体与伯氏疏螺旋体共感染研究

目的 嗜吞噬细胞无形体和伯氏疏螺旋体是由媒介蜱传播的引起人粒细胞无形体病和莱姆病的病原。目前,对两种病原在蜱体内共感染流行情况的研究相对较少。方法 以甘肃、湖南和广东三个省采集到3个蜱种、共543份样品为研究对象,检测嗜吞噬细胞无形体和伯氏疏螺旋体在蜱体内的感染情况。结果 嗜吞噬细胞无形体和伯氏疏螺旋体在不同地区采集到的蜱体内的感染率不同,分别为3.2%~20.0%和2.3%~19.3%。在检测的样品中,共发现有7份样品中同时感染嗜吞噬细胞无形体和伯氏疏螺旋体。青海血蜱、血红扇头蜱和微小牛蜱中均检测到这两种病原。结论 青海血蜱、血红扇头蜱和微小牛蜱均能够携带嗜吞噬细胞无形体和伯氏疏螺旋体,可能为这两种病原在自然界的持续存在和循环提供了条件。此研究结果丰富了人粒细胞无形体病和莱姆病的流行病学信息,有利于提高这两种病的防控策略。     

Distribution of borreliae among ticks collected from eastern states

Lyme disease is the most commonly reported vector-borne disease in the United States and is transmitted by Borrelia burgdorferi-infected Ixodes species. The disease is typically characterized by an erythema migrans (EM) rash at the site of tick feeding. EM rashes have also been associated with feeding by Amblyomma americanum ticks despite evidence suggesting that they are incompetent vectors for Lyme disease. In 1996, a Borrelia organism only recently cultivated in the laboratory was described in A. americanum ticks and designated B. lonestari. This Borrelia is believed to be the etiologic agent of a novel Lyme-like disease, southern tick associated rash illness (STARI). This study examined ticks collected from eight eastern states to evaluate the epidemiology of B. lonestari, B. burgdorferi, and their tick hosts. Three hundred individual or small pool samples were evaluated from tick genera that included Amblyomma, Ixodes, and Dermacentor. DNA was extracted following tick homogenization and the polymerase chain reaction (PCR) was performed using primers derived from the flagellin gene that amplify sequences from both B. burgdorferi and B. lonestari. Species specific digoxigenin labeled probes were designed and used to differentiate between B. burgdorferi and B. lonestari. Borrelia DNA was detected in approximately 10% of the A. americanum and I. scapularis tick samples, but none was present in any of the Dermacentor samples tested. Positive samples were detected in ticks collected from Kentucky, Maryland, Massachusetts, New Jersey, New York, and Virginia. This is the first known report of B. lonestari from Massachusetts and New York and the first detection in I. scapularis. This suggests that B. lonestari and its putative association with STARI may be more widespread than previously thought.     

Spatiotemporal variation in a Lyme disease host and vector: black-legged ticks on white-footed mice

We monitored population density of white-footed mice (Peromyscus leucopus), burdens of immature black-legged ticks (Ixodes scapularis) on mice, and infection prevalence of host-seeking ticks on six forest plots in southeastern New York State from 1995 through 1999. Despite densities of mice that fluctuated two orders of magnitude, average larval and nymphal tick burdens per mouse remained remarkably constant. Spatial variability in mouse density and tick burdens was modest. The total number of larval and nymphal ticks that fed on the mouse population each year depended strongly on population density of mice; a steady increase was observed in both mouse density and total tick meals on mice from 1996 through 1999. The result was a steady increase in the infection prevalence of nymphal and adult ticks with the etiological agent of Lyme disease, Borrelia burgdorferi, over this time. We suggest that fluctuations in population density of mice, combined with possible regulation of tick burdens on mice, may influence risk of human exposure to Lyme disease.     

Detection of Borrelia burgdorferi in ticks by species-specific amplification of the flagellin gene.

We developed a polymerase chain reaction (PCR) that specifically amplifies a fragment of the flagellin gene (fla) of Borrelia burgdorferi, the causative agent of Lyme disease. This fla target, amplified with nested primers, was conserved among all 80 strains of B. burgdorferi tested. Strains examined included cultures from ticks, humans, and rodents from major B. burgdorferi-endemic regions of the United States and parts of Europe and Asia. Templates from B. hermsii, B. parkeri, B. turicatae, and B. coriaceae were not amplified, nor were eukaryotic DNAs from three tick genera. Several host DNAs potentially present in a tick blood meal also were not amplified. Approximately six B. burgdorferi per PCR reaction could be detected by ethidium bromide staining of amplified DNA. Colony-raised Ixodes dammini were used to evaluate the method. One infected nymph in a pool of 40 ticks was routinely detected. The specificity of the assay for detecting B. burgdorferi-infected ticks in pools was 94% (29 of 31). This protocol should prove useful for assessing infection rates in other putative arthropod vectors.     

Characterization of Borrelia burgdorferi isolated from Ixodes persulcatus and Ixodes ovatus ticks in Japan.

The Lyme disease spirochete Borrelia burgdorferi was detected in the adult stage of the ixodid ticks Ixodes persulcatus and I. ovatus collected in various localities in Japan. The total prevalence values were 16.6% of 777 I. persulcatus and 23.6% of 687 I. ovatus. Sixty-two (63.9%) of 97 isolates from I. persulcatus contained the 30-34-kD heterogeneous outer surface protein A (OspA) that reacted with monoclonal antibody H5332. In contrast, all 128 isolates from I. ovatus were homogeneous with regard to the type of 30-kD OspA protein, and were different from the I. persulcatus isolates in their protein composition. These data suggest that the transmission cycle of B. burgdorferi in I. ovatus is not related to that in I. persulcatus. No cases of Lyme disease transmitted by I. ovatus have been confirmed in Japan, in spite of the abundance of human tick bites by this species. Accordingly, the spirochetes of I. ovatus are thought to be a variant of B. burgdorferi with low virulence.