Immunization with adenoviral-vectored tick salivary gland proteins (SALPs) in a murine model of Lyme borreliosis.

Prior exposure of vertebrate hosts to tick salivary proteins can induce specific immunity to tick infestation, as well as affording protection against tick-transmitted Borrelia burgdorferi infection in the mammalian host. Vaccination using an adenovirus expression system to deliver 4 tick salivary proteins (Ad-Salps) derived from Ixodes scapularis, Salp15, Salp25A, Salp25D, and Isac, was explored. Results indicate that vaccination with tick salivary proteins in an adenoviral vector can be used to modulate a Th1 response in the host and partially control spirochete load in immunized mice after infected tick challenge.

Midichloria mitochondrii is widespread in hard ticks (Ixodidae) and resides in the mitochondria of phylogenetically diverse species.

The hard tick Ixodes ricinus (Ixodidae) is the sole animal thus far shown to harbour an intra-mitochondrial bacterium, which has recently been named Midichloria mitochondrii. The objectives of this work were (i) to screen ixodid ticks for Midichloria-related bacteria and (ii) to determine whether these bacteria exploit the intra-mitochondrial niche in other tick species. Our main goal was to discover further models of this peculiar form of symbiosis. We have thus performed a PCR screening for Midichloria-related bacteria in samples of ixodid ticks collected in Italy, North America and Iceland. A total of 7 newly examined species from 5 genera were found positive for bacteria closely related to M. mitochondrii. Samples of the tick species Rhipicephalus bursa, found positive in the PCR screening, were analysed with transmission electron microscopy, which revealed the presence of bacteria both in the cytoplasm and in the mitochondria of the oocytes. There is thus evidence that bacteria invade mitochondria in at least 2 tick species. Phylogenetic analysis on the bacterial 16S rRNA gene sequences generated from positive specimens revealed that the bacteria form a monophyletic group within the order Rickettsiales. The phylogeny of Midichloria symbionts and related bacteria does not appear completely congruent with the phylogeny of the hosts.

Spatiotemporal patterns of host-seeking Ixodes scapularis nymphs (Acari: Ixodidae) in the United States.

The risk of Lyme disease for humans in the eastern United States is dependent on the density of host-seeking Ixodes scapularis Say nymphal stage ticks infected with Borrelia burgdorferi. Although many local and regional studies have estimated Lyme disease risk using these parameters, this is the first large-scale study using a standardized methodology. Density of host-seeking I. scapularis nymphs was measured by drag sampling of closed canopy deciduous forest habitats in 95 locations spaced among 2 degrees quadrants covering the entire United States east of the 100th meridian. Sampling was done in five standardized transects at each site and repeated three to six times during the summer of 2004. The total number of adults and nymphs of the seven tick species collected was 17,972, with 1,405 nymphal I. scapularis collected in 31 of the 95 sites. Peak global spatial autocorrelation values were found at the smallest lag distance (300 km) and decreased significantly after 1,000 km. Local auto-correlation statistics identified two significant high-density clusters around endemic areas in the northeast and upper Midwest and a low-density cluster in sites south of the 39th parallel, where only 21 nymphs were collected. Peak nymphal host-seeking density occurred earlier in the southern than in the most northern sites. Spatiotemporal density patterns will be combined with Borrelia prevalence data as part of a 4-yr survey to generate a nationwide spatial risk model for I. scapularis-borne Borrelia, which will improve targeting of disease prevention efforts.

Kinetics of Borrelia burgdorferi infection in larvae of refractory and competent tick vectors.

The acquisition of Borrelia burgdorferi by the larvae of competent and refractory ixodid ticks was assessed by quantitative polymerase chain reaction (PCR). Larvae were fed on infected mice, and the spirochete loads were determined during feeding and up to 93 d postfeeding. Amblyomma americanum (L.) was refractory to B. burgdorferi infection, with almost no detection of spirochete DNA during or postfeeding. In contrast, Ixodes scapularis Say supported high loads of spirochetes (10(3)-10(4) per larva). In Dermacentor variabilis (Say), B. burgdorferi uptake was reduced, with an average of 16 spirochetes per larvae acquired after 4 d of feeding, representing 1/195 of the counts in I. scapularis. However, during the first day postfeeding, the spirochete growth rate in D. variabilis reached 0.076 generations per hour, 7.7 times greater than the highest growth rate detected in I. scapularis. D. variabilis supported intense spirochete growth up to the fourth day postinfection, when the counts increased to an average of 282 spirochetes per larvae or 1/8.5 of the I. scapularis counts 4 d postfeeding. The kinetics of spirochete growth was unstable in D. variabilis compared with I. scapularis, and transmission of B. burgdorferi by D. variabilis could not be demonstrated. A cofeeding experiment indicated that I. scapularis feeding increased A. americanum spirochete uptake. These collective results indicate suboptimal conditions for B. burgdorferi uptake and colonization within A. americanum or the presence of anti-Borrelia factor(s) in this nonpermissive tick species.

Capillary feeding of specific dsRNA induces silencing of the isac gene in nymphal Ixodes scapularis ticks.

Ixodes scapularis transmits several pathogens including Borrelia burgdorferi. Bioactive compounds in tick saliva support tick feeding and influence pathogen transmission to the mammalian host. These studies utilized oral delivery of dsRNA to silence an anticomplement gene (isac) in I. scapularis nymphs. Silencing of isac significantly reduced fed-tick weight compared to delivery of control lacZ dsRNA, and immunoblots specific for FlaB protein indicated a reduction in spirochete load in isac-silenced infected nymphs. SDS-PAGE demonstrated that isac gene silencing affected expression of a number of salivary and non-salivary gland proteins in ticks. Finally, multiple isac cDNA homologues were cloned, and these may represent a new gene family coexpressed during tick feeding. This work presents a novel oral delivery approach for specific gene silencing in I. scapularis nymphs and characterizes the effect of isac on blood-feeding in an attempt to block transmission of B. burgdorferi.

Differential infectivity of the Lyme disease spirochete Borrelia burgdorferi derived from Ixodes scapularis salivary glands and midgut.

Blood fed nymphal Ixodes scapularis Say infected with Borrelia burgdorferi were dissected to obtain salivary gland and midgut extracts. Extracts were inoculated into C3H/HeJ mice, and ear, heart, and bladder were cultured to determine comparative infectivity. Aliquots of extracts were then analyzed by quantitative polymerase chain reaction to determine the number of spirochetes inoculated into mice. A comparative median infectious dose (ID50) was determined for both salivary gland and midgut extract inoculations. Our data demonstrated a statistically significant difference (P < 0.002) in the ID50 derived from salivary gland (average = 18) versus midgut (average = 251) extracts needed to infect susceptible mice. A rationale for the differential infectivity of salivary and midgut derived spirochetes is discussed.

Genome size and organization in the blacklegged tick, Ixodes scapularis and the Southern cattle tick, Boophilus microplus.

Genome sizes and the organization of repetitive DNA were determined in the hard ticks Ixodes scapularis and Boophilus microplus using reassociation kinetics. The I. scapularis genome contains approximately 2.15 pg (2.1x10(3) Mbp) of DNA and consists of no foldback (FB), 27% highly repetitive (HR), 39% moderately repetitive (MR), and 34% unique DNA. The B. microplus genome contains 7.5 pg (7.1x10(3) Mbp) DNA, and consists of 0.82% FB, 31% HR, 38% MR, and 30% unique DNA. In both species, repetitive sequences occur in a mixture of long and short period interspersion but most (65-80%) of the DNA follows a pattern of short period interspersion. Genome size and organization in the three tick species so far examined are distinct from other arthropods in having a greater proportion of MR, a lower proportion of unique and HR DNA of very low sequence complexity.

Borreliacidal activity of saliva of the tick Amblyomma americanum.

Amblyomma americanum (Linneaus) (Acari: Ixodidae), an important tick vector of human and animal disease, is not a competent vector of the bacterial agent of Lyme disease, Borrelia burgdorferi, although its range overlaps the geographical distribution of Lyme disease within the United States. A possible mechanism that could prevent acquisition of B. burgdorferi spirochetes from infected hosts is the toxic effect of A. americanum saliva on B. burgdorferi. The data presented here indicate that after 24 and 48 h of exposure to A. americanum saliva, significantly fewer B. burgdorferi were alive compared to treatment controls as assessed by spirochete motility under dark-field microscopy and resistance to the dead stain, propidium iodide. After 48 h, fewer than 13% of saliva-exposed B. burgdorferi were alive. In contrast, significantly more B. burgdorferi exposed to Ixodes scapularis (Acari: Ixodidae) saliva survived after 24 or 48 h compared to A. americanum saliva or treatment controls.

Three multiplex assays for detection of Borrelia burgdorferi sensu lato and Borrelia miyamotoi sensu lato in field-collected Ixodes nymphs in North America.

Two hundred fifty New Jersey field-collected Ixodes scapularis Say ticks and 17 Colorado Ixodes spinipalpis Hadwen & Nuttall ticks were tested using three separate multiplex real-time polymerase chain reaction (PCR) assays. One assay targets the rrs-rrlA IGS region of Borrelia spp. to detect Borrelia burgdorferi sensu lato (s.l.) and Borrelia miyamotoi s.l. The second assay targets the ospA region of B. burgdorferi s.l. to detect B. burgdorferi sensu stricto (s.s.), Borrelia bissettii, and Borrelia andersonii. The final assay targets the glpQ region of B. miyamotoi s.l. to differentiate B. miyamotoi LB-2001 and Borrelia lonestari. A testing scheme combining these tests yielded 18% of tested I. scapularis ticks surveyed from New Jersey positive for B. burgdorferi s.s., 3.2% I. scapularis ticks positive for B. miyamotoi LB-2001, and 41.2% I. spinipalpis ticks positive for B. bissettii surveyed from Colorado.

Sustained-release formulation of doxycycline hyclate for prophylaxis of tick bite infection in a murine model of Lyme borreliosis.

The prophylactic potential of a single injection of sustained-release doxycycline hyclate (Atridox) was compared to that of a single oral dose of doxycycline hyclate in a murine model of Lyme borreliosis. Prophylaxis, as measured by the lack of cultivable spirochetes and demonstrable pathology, was noted for 43% of orally treated mice; in contrast, the sustained-release doxycycline hyclate completely protected mice from infection and resultant pathology.

Lyme borreliosis in Europe and North America.

Since the discovery of the Lyme disease spirochete in North America in 1982 and in Europe in 1983, a plethora of studies on this unique group of spirochetes that comprise Borrelia burgdorferi sensu lato has been accumulated. In an attempt to compare and contrast Lyme borreliosis in Europe and North America we have reviewed the biology of the aetiologic agents, as well as the clinical aspects, diagnosis and treatment of this disease on both continents. Moreover, we have detailed the ecology of the Ixodes ticks that transmit this infection and the reservoir hosts that maintain the spirochete cycle in nature. Finally, we have examined the transmission dynamics of the spirochete on both continents, as well as the available prevention strategies. Although it has been over two decades since the discovery of the Lyme disease spirochete, Lyme borreliosis is an expanding public health problem that has defied our attempts to control it. By comparing the accumulated experience of investigators in North America and Europe, where the disease is most frequently reported, we hope to advance the cause of developing novel approaches to combat Lyme borreliosis.

A preliminary linkage map of the hard tick, Ixodes scapularis.

A linkage map of the Ixodes scapularis genome was constructed, based upon segregation amongst 127 loci. These included 84 random amplified polymorphic DNA (RAPD) markers, 32 Sequence-Tagged RAPD (STAR) markers, 5 cDNAs, and 5 microsatellites in 232 F1 intercross progeny from a single, field-collected P1 female. A preliminary linkage map of 616 cM was generated across 14 linkage groups with one marker every 10.8 cM. Assuming a genome size of approximately 109 bp, the relationship of physical to genetic distance was found to be approximately 300 kb/cM in the I. scapularis genome.

Coinoculation of Borrelia spp. with tick salivary gland lysate enhances spirochete load in mice and is tick species-specific.

C3H/HeN mice were inoculated with 10(6) spirochetes, either Borrelia burgdorferi strain N40 or the Portuguese strain of B. lusitaniae, PotiB2. Mice receiving spirochetes coinoculated with salivary gland lysate (SGL) demonstrated significantly higher spirochete loads in target organs as measured by quantitative real-time polymerase chain reaction. This effect was tick dependent, in that Ixodes ricinus SGL specifically enhanced B. lusitaniae load, whereas I. scapularis SGL specifically increased B. burgdorferi N40 load, but did not significantly affect the dissemination of B. lusitaniae. Protein profile analysis indicated at least 5 major protein differences between I. scapularis and I. ricinus SGL, which can possibly account for this specific tick-spirochete interaction.

Isolation and characterization of Borrelia parkeri in Ornithodoros parkeri (Ixodida: Argasidae) collected in Colorado.

This study describes the identification of Borrelia parkeri spirochetes in Colorado. Two isolates of B. parkeri (6230 and 6232) were recovered from Ornithodoros parkeri Cooley ticks collected at an inactive prairie dog town in Moffat County. Both isolates were partially characterized by sequencing and subsequent parsimony and neighbor-joining analyses of appropriate regions of the 16S ribosomal RNA, flagellin and P66 genes. Analyses of the 16S gene sequences from the Colorado isolates indicated that they were more closely related to B. parkeri and B. tucatae than to B. hermsii or the other species of Borrelia investigated in this study. Additional analyses of amino acid sequences for flagellin and P66, however, clearly demonstrated that isolates 6230 and 6232 were most closely related to B. parkeri. The possible significance of B. parkeri as an agent of human disease is discussed.

Use of quantitative PCR to measure density of Borrelia burgdorferi in the midgut and salivary glands of feeding tick vectors.

Quantitative real-time PCR was used to assay spirochetes in feeding ticks. Spirochetes in tick midguts increased sixfold, from 998 per tick before attachment to 5,884 at 48 h of attachment. Spirochetes in tick salivary glands increased >17-fold, from 1.2 per salivary gland pair before feeding to 20.8 at 72 h postattachment. The period of the most rapid increase in the number of spirochetes in the salivary glands occurred from 48 to 60 h postattachment; this time period coincides with the maximal increase in transmission risk during nymphal tick feeding.

Major histocompatibility complex class II-independent generation of neutralizing antibodies against T-cell-dependent Borrelia burgdorferi antigens presented by dendritic cells: regulation by NK and gammadelta T cells.

We previously showed that adoptive transfer of Borrelia burgdorferi-pulsed dendritic cells (DCs) into syngeneic mice protects animals from challenge with tick-transmitted spirochetes. Here, we demonstrate that the protective immune response is antibody (Ab) dependent and does not require the presence of major histocompatibility complex (MHC) class II molecules on DCs. Mice sensitized with B. burgdorferi-pulsed MHC class II-deficient (MHC class II(-/-)) DCs mounted a humoral response against protective antigens, including B. burgdorferi outer surface protein A (OspA) and OspC. B-cell help for the generation of neutralizing anti-OspC immunoglobulin G Abs could be provided by gammadelta T cells. In contrast, anti-OspA Ab production required the presence of alphabeta T cells, although this pathway could be independent of MHC class II molecules on antigen-presenting cells. Moreover, depletion of NK cells prior to transfer of antigen-pulsed MHC class II(-/-) DCs resulted in significant increases in the levels of neutralizing Abs induced by DCs. Altogether, these data suggest that the initial interactions between DCs and innate immune cells, such as gammadelta and NK cells, can influence the generation of a protective humoral response against B. burgdorferi antigens.

Identification of an IL-2 binding protein in the saliva of the Lyme disease vector tick, Ixodes scapularis.

A potent inhibitor of mitogen-stimulated T cell proliferation exists in the saliva of several species of hard ticks, including the Lyme disease vector tick, Ixodes scapularis. Our characterization of this phenomenon has led to the identification of a possible mechanism for the T cell inhibitory activity of I. scapularis saliva. The T cell inhibitor can overcome stimulation of mouse spleen cells with anti-CD3 mAb; however, a direct and avid interaction with T cells does not appear to be necessary. Tick saliva inhibits a mouse IL-2 capture ELISA, suggesting that a soluble IL-2 binding factor is present in the saliva. This hypothesis was verified by using a direct binding assay in which plate-immobilized tick saliva was shown to bind both mouse and human IL-2. Elimination of the IL-2 binding capacity of saliva in the in vitro assays by trypsin digestion demonstrated that the IL-2 binding factor is a protein. These experiments comprise the first demonstration of the existence of such a secreted IL-2 binding protein from any parasite or pathogen. This arthropod salivary IL-2 binding capacity provides a simple mechanism for the suppression of T cell proliferation as well as for the activity of other immune effector cells that are responsive to IL-2 stimulation. Relevance of the tick T cell inhibitory activity to the human immune system is demonstrated by the ability of tick saliva to inhibit proliferation of human T cells and CTLL-2 cells grown in the presence of human IL-2.

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.

Effect of tick removal on transmission of Borrelia burgdorferi and Ehrlichia phagocytophila by Ixodes scapularis nymphs.

The effect of feeding duration on pathogen transmission was studied for individual ticks infected with either laboratory or field strains of the Lyme disease spirochete Borrelia burgdorferi and field strains of Ehrlichia phagocytophila, an agent of human granulocytic ehrlichiosis. Infected nymphal Ixodes scapularis were allowed to feed individually on mice, and equal numbers were removed at 24-h intervals for < or =96 h. Mice were assayed for infection by culture, serologic testing, and polymerase chain reaction (PCR) analysis. Fed ticks were assayed by culture or PCR analysis. Transmission of B. burgdorferi did not occur during the first 24 h among 66 attempts, with maximum transmission occurring between 48 and 72 h. A model estimating the probability of infection from individual ticks removed by patients in a Lyme disease-endemic area yielded an overall probability of 4.6%. Infected I. scapularis nymphs transmitted E. phagocytophila within 24 h in 2 of 3 attempts, which indicates that daily tick removal may not be adequate to prevent human infection with this agent.

An analysis of spirochete load, strain, and pathology in a model of tick-transmitted Lyme borreliosis.

Four laboratory-grown, low-passage isolates of Borrelia burgdorferi sensu stricto, B31, JD-1, 910255, and N40, were incorporated into Ixodes scapularis ticks to examine the pathogenesis of these isolates in mice after tick transmission. All isolates induced multifocal, lymphoid nodular cystitis, subacute, multifocal, necrotizing myocarditis, and a localized periostitis and arthritis of the femorotibial joint 6-18 weeks after tick infestation. In terms of the number of mice that demonstrated pathology in bladder, heart, and joint, the highest incidence of lesions occurred 12 weeks after tick bite. Utilizing the Taqman quantitative polymerase chain reaction (q-PCR) fluorogenic detection technology to amplify a conserved region of the flagellin gene, a trend was demonstrated between the number of spirochetes in tissue with duration of pathology. The q-PCR assay developed for this study was sensitive and could reliably measure as few as 1 to 10 spirochetes in the target tissues tested. A higher percentage of B31- and N40-infected mice (92 and 100%, respectively) developed myocarditis than JD-1- or 910255-infected mice (67 and 46%, respectively) 12 weeks after tick bite. The amount of spirochetal DNA that could be amplified for heart at this time point was not statistically different between isolates, indicating a difference in virulence between B31 and N40 relative to JD-1 and 910225. N40-infected mice demonstrated a significantly higher spirochete load (an average of 1.23 spirochetes/mg of tissue, p = 0.045) in femorotibial joints 18 weeks after infection, with 60% of these mice maintaining lesions compared with those infected with B31 (13%), JD-1 (25%), or 910255 (50%), which averaged <0.5 spirochetes/mg of tissue. This mouse model of Lyme borreliosis, including the ability to monitor lesion development and spirochete load, can facilitate the testing of therapeutic regimens for the later stages of tick-transmitted Lyme disease and help investigate aspects of the immunopathogenesis of lesion development.