Protection against lyme disease spirochete transmission provided by prompt removal of nymphal Ixodes scapularis (Acari: Ixodidae)

Public health recommendations for Lyme disease prevention generally include daily tick checks and prompt removal of attached ticks as a means of decreasing the risk of acquiring Lyme disease in highly endemic regions. In the current study, we determined whether crushing nymphal ticks during removal with forceps increased the risk of B. burgdorferi transmission, what degree of protection from transmission of B. burgdorferi was provided by removal of nymphal Ixodes scapularis Say at specific intervals, and whether commercial devices marketed for tick removal worked when tested against nymphal I. scapularis. Both removal via gentle pressure (26% transmission) or crushing the tick (30% transmission) caused a significant decrease in transmission as compared with the sham control (70% transmission). The degree of protection provided via tick removal decreased steadily up to 60 h of attachment; between 60 and 66 h, a dramatic falloff in protection occurred to the point where no protection was observed at 66 h. Finally, commercial tick removal devices varied widely in their efficacy for the removal of attached nymphal I. scapudaris.     

LuxS-mediated quorum sensing in Borrelia burgdorferi,the lyme disease spirochete

The establishment of Borrelia burgdorferi infection involves numerous interactions between the bacteria and a variety of vertebrate host and arthropod vector tissues. This complex process requires regulated synthesis of many bacterial proteins. We now demonstrate that these spirochetes utilize a LuxS/autoinducer-2 (AI-2)-based quorum-sensing mechanism to regulate protein expression, the first system of cell-cell communication to be described in a spirochete. The luxS gene of B. burgdorferi was identified and demonstrated to encode a functional enzyme by complementation of an Escherichia coli luxS mutant. Cultured B. burgdorferi responded to AI-2 by altering the expression levels of a large number of proteins, including the complement regulator factor H-binding Erp proteins. Through this mechanism, a population of Lyme disease spirochetes may synchronize production of specific proteins needed for infection processes.     

Reservoir competence of native North American birds for the lyme disease spirochete, Borrelia burgdorfieri

Reservoir competence for the Lyme disease spirochete, Borrelia burgdorferi, was tested for six species of native North American birds: American robin, gray catbird, brown thrasher, eastern towhee, song sparrow, and northern cardinal. Wild birds collected by mist netting on Fire Island, NY, were held in a field laboratory in cages over water and locally collected larval ticks were placed on the birds, harvested from the water after engorgement, and tested for infection by direct fluorescentantibody staining after molting to the nymphal stage. American robins were competent reservoirs, infecting 16.1% of larvae applied to wild-caught birds, compared with 0% of control ticks placed on uninfected laboratory mice. Robins that were previously infected in the laboratory by nymphal feeding infected 81.8% of applied larvae. Wild-caught song sparrows infected 4.8% of applied larvae and 21.1% when infected by nymphal feeding. Results suggest moderate levels of reservoir competence for northern cardinals, lower levels for gray catbirds, and little evidence of reservoir competence for eastern towhees or brown thrashers. Lower infection rates in larvae applied to wild-caught birds compared with birds infected in the laboratory suggest that infected birds display temporal variability in infectiousness to larval ticks. Engorged larvae drop from birds abundantly during daylight, so the abundance of these bird species in the peridomestic environment suggests that they might contribute infected ticks to lawns and gardens.     

The OspE-related proteins inhibit complement deposition and enhance serum resistance of Borrelia burgdorferi, the lyme disease spirochete

Borrelia burgdorferi, the Lyme disease spirochete, binds the host complement inhibitors factor H (FH) and FH-like protein 1 (FHL-1). Binding of FH/FHL-1 by the B. burgdorferi proteins CspA and the OspE-related proteins is thought to enhance resistance to serum-mediated killing. While previous reports have shown that CspA confers serum resistance in B. burgdorferi, it is unclear whether the OspE-related proteins are relevant in B. burgdorferi serum resistance when OspE is expressed on the borrelial surface. To assess the role of the OspE-related proteins, we overexpressed them in a serum-sensitive CspA mutant strain. OspE overexpression enhanced serum resistance of the CspA-deficient organisms. Furthermore, FH was more efficiently bound to the B. burgdorferi surface when OspE was overexpressed. Deposition of complement components C3 and C5b-9 (the membrane attack complex), however, was reduced on the surface of the OspE-overexpressing strain compared to that on the CspA mutant strain. These data demonstrate that OspE proteins expressed on the surface of B. burgdorferi bind FH and protect the organism from complement deposition and subsequent serum-mediated destruction.     

Intrinsic competence of three ixodid ticks (Acari) as vectors of the Lyme disease spirochete

We compared the intrinsic vector competence of Ixodes dammini Spielman et al., Dermacentor variabilis (Say), and Amblyomma americanum (L.) for the Lyme disease spirochete (Borrelia burgdorferi Johnson et al.) on Prudence Island, Rhode Island, a Lyme disease-endemic site where all three ticks occur together. Natural and experimental spirochete infection rates were determined in those ticks and their degree of contact with white-footed mice (Peromyscus leucopus), the principal reservoir host, was compared. Host-seeking nymphal and adult I. dammini and A. americanum, collected by flagging, were nearly equally abundant, as were adult D. variabilis, but only I. dammini were infected with B. burgdorferi. Larvae and nymphs of both I. dammini and D. variabilis were found infesting mice, but A. americanum never were found on this host. Furthermore, although larvae of all three tick species became infected by ingesting spirochetes while feeding on experimentally infected mice, only I. dammini remained infected following the transstadial molt. These findings suggest that of these three tick species, only I. dammini is competent as a vector of the Lyme disease spirochete.     

Temporal changes in outer surface proteins A and C of the lyme disease-associated spirochete, Borrelia burgdorferi, during the chain of infection in ticks and mice

The Lyme disease-associated spirochete, Borrelia burgdorferi, is maintained in enzootic cycles involving Ixodes ticks and small mammals. Previous studies demonstrated that B. burgdorferi expresses outer surface protein A (OspA) but not OspC when residing in the midgut of unfed ticks. However, after ticks feed on blood, some spirochetes stop making OspA and express OspC. Our current work examined the timing and frequency of OspA and OspC expression by B. burgdorferi in infected Ixodes scapularis nymphs as they fed on uninfected mice and in uninfected I. scapularis larvae and nymphs as they first acquired spirochetes from infected mice. Smears of midguts from previously infected ticks were prepared at 12- or 24-h intervals following attachment through repletion at 96 h, and spirochetes were stained for immunofluorescence for detection of antibodies to OspA and OspC. As shown previously, prior to feeding spirochetes in nymphs expressed OspA but not OspC. During nymphal feeding, however, the proportion of spirochetes expressing OspA decreased, while spirochetes expressing OspC became detectable. In fact, spirochetes rapidly began to express OspC, with the greatest proportion of spirochetes having this protein at 48 h of attachment and then with the proportion decreasing significantly by the time that the ticks had completed feeding. In vitro cultivation of the spirochete at different temperatures showed OspC to be most abundant when the spirochetes were grown at 37 degrees C. Yet, the synthesis of this protein waned with continuous passage at this temperature. Immunofluorescence staining of spirochetes in smears of midguts from larvae and nymphs still attached or having completed feeding on infected mice demonstrated that OspA but not OspC was produced by these spirochetes recently acquired from mice. Therefore, the temporal synthesis of OspC by spirochetes only in feeding ticks that were infected prior to the blood meal suggests that this surface protein is involved in transmission from tick to mammal but not from mammal to tick.     

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.     

"Candidatus Neoehrlichia mikurensis," Anaplasma phagocytophilum, and lyme disease spirochetes in questing european vector ticks and in feeding ticks removed from people

To estimate the likelihood of people coming into contact with the recently described tick-borne agent "Candidatus Neoehrlichia mikurensis," we compared its prevalence to those of Lyme disease spirochetes and Anaplasma phagocytophilum in questing adult Ixodes ricinus ticks collected in various Central European sites and examined ticks, which had been removed from people, for the presence of these pathogens. Whereas spirochetes infected questing adult ticks most frequently (22.3%), fewer than a third as many ticks were infected by "Ca. Neoehrlichia mikurensis" (6.2%), and about a sixth harbored A. phagocytophilum (3.9%). On average, every twelfth encounter of a person with an I. ricinus tick (8.1%) may bear the risk of acquiring "Ca. Neoehrlichia mikurensis." Although a fifth of the people (20%) had removed at least one tick infected by "Ca. Neoehrlichia mikurensis," none displayed symptoms described for this pathogen, suggesting that its transmission may not be immediate and/or that immunocompetent individuals may not be affected. Because immunosuppressed patients may be at a particular risk of developing symptoms, it should be considered that "Ca. Neoehrlichia mikurensis" appears to be the second most common pathogen in I. ricinus ticks. In our survey, only Borrelia afzelii appears to infect Central European vector ticks more frequently.     

Glass capillary tube feeding: a method for infecting nymphal Ixodes scapularis (Acari: Ixodidae) with the lyme disease spirochete Borrelia burgdorferi

We evaluated an artificial capillary feeding method to infect nymphal Ixodes scapularis (Say) ticks with Borrelia burgdoeferi, the causative agent of Lyme disease. Thirty to 70% of the nymphs were infected after feeding for 2.5 h from glass capillary tubes filled with a solution of spirochetes. Capillary infection was stable and persisted in the nymphs for at least 10 d after feeding. Capillary feeding also maintained natural vector competence patterns because I. scapularis ticks acquired infection unlike Dermacentor variablis (Say), which did not become infected. Capillary infected I. scapularis nymphs were capable of transmitting the infection to naive mice although not as efficiently as naturally infected nymphs. The capillary infection method is convenient and is a better alternative to syringe inoculation as a means of infecting animals with B. burgdorferi.     

Infectivity of the highly transformable BBE02- lp56- mutant of Borrelia burgdorferi, the Lyme disease spirochete, via ticks

Infectious Borrelia burgdorferi strains that have increased transformability with the shuttle vector pBSV2 were recently constructed by inactivating the gene encoding BBE02, a putative restriction-modification gene product expressed by the linear plasmid lp25 (Kawabata et al., Infect. Immun. 72:7147-7154, 2004). The absence of the linear plasmid lp56, which carries another putative restriction-modification gene, further enhanced transformation rates. The infectivity of these mutants was assessed previously in mice that were inoculated with needle and syringe and was found to be equivalent to that of wild-type spirochetes. Here we examined the infectivity of spirochetes to ticks after capillary inoculation of Ixodes scapularis nymphs and the subsequent spirochetal infectivity to mice via ticks by using B. burgdorferi B31 clonal isolates lacking lp56 and/or BBE02. The absence of lp56 (but not BBE02) correlated with a lower number of spirochetes in ticks after feeding on mice; this plasmid thus may play a role, albeit not an essential one, in supporting spirochetal survival in the feeding tick. Importantly, however, the absence of lp56 and BBE02 did not detectably influence infectivity to mice via ticks.     

Histopathological studies of experimental lyme disease in the dog

Experimental borrelia infection was induced in 62 specific--pathogen-free beagle dogs by exposure to Ixodes scapularis ticks harbouring the spirochaete Borrelia burgdorferi. Clinical signs of Lyme disease occurred in 39/62 dogs, the remaining 23 being subclinically infected. Clinical signs consisted of one to six episodes of transitory lameness with joint swelling and pain, most commonly affecting the elbow or shoulder joints. The polymerase chain reaction and culture demonstrated that the dogs remained infected for up to 581 days. At necropsy, gross findings consisted of lymphadenopathy in the area of tick attachment. Microscopical changes consisted of effusive fibrinosuppurative inflammation or nonsuppurative inflammation, or both, affecting synovial membranes, joint capsules and associated tendon sheaths. Plasma cells dominated areas of chronic inflammation, with CD3(+) T cells being present in lesser numbers. Microscopical signs of arthritis were polyarticular and more widespread than indicated by clinical signs, and most of the subclinically affected animals also had synovitis. In areas of tick attachment to the skin, hyperkeratosis and a mixture of suppurative and nonsuppurative dermatitis were encountered. Lymphadenopathy in superficial lymph nodes resulted from follicular and parafollicular hyperplasia. In 14/62 dogs, lymphoplasmacytic periarteritis and perineuritis were noted, resembling lesions found in human Lyme disease and syphilis, in which an underlying microangiopathy has been proposed.     

DNA characterization of the spirochete that causes Lyme disease

Lyme disease, a tick-borne disease long recognized in Europe but only recently recognized in the United States, was shown in 1982-1983 to be caused by a spirochete, the Lyme disease spirochete. Whether one or more species of the spirochete exists is unknown, as is its taxonomic status. To answer these questions, we determined (i) the DNA base (guanidine-plus-cytosine) content for five strains; (ii) the DNA relatedness of 10 strains from Europe or the United States (isolated from ticks, humans, and a mouse) by DNA hybridization (hydroxyapatite assay at 50 and 65 degrees C); and (iii) the DNA relatedness to other pathogenic spirochetes. The guanine-plus-cytosine content of the Lyme disease spirochete strains was 27.5 to 29.0 mol%, most similar to those of Borrelia hermsii (30.6 mol%) and Treponema hyodysenteriae (25.6 mol%) among the other spirochetes tested. DNA hybridization studies with 32P-labeled DNA from Lyme disease spirochete strain TLO-005, a human blood isolate, revealed divergence (unpaired bases) within related nucleotide sequences of only 0.0 to 1.0% for all nine Lyme disease spirochete strains tested for relatedness to TLO-005. Relatedness values of seven strains to TLO-005 were 58 to 98% (mean, 71%) in 50 degrees C reactions and 50 to 93% (mean, 69%) in 65 degrees C reactions. Two other strains, from which very low yields of DNA were obtained, showed less relatedness (36 to 50 degrees C, 38 to 47% at 65 degrees C). These were nonetheless considered to belong to the same species because of the low amount of divergence in the sequences related to TLO-005 and the absence of decreased relatedness in reactions done at 65 degrees Celsius compared with those done at 50 degrees Celsius. DNA from strain TLO-005 showed relatedness of 1% to DNAs of two leptospires and 16% relatedness to DNA from T. hyodysenteriae. B. hermsii DNA was 30 to 40% related to three Lyme disease spirochete strains in 50 degrees Celsius reactions. Divergence in these reactions was 16.5 to 18.5%, and relatedness in 65 degree Celsius reactions was 8 to 10%. On the bases of phenotypic similarity, guanine-plus-cytosine content, and DNA relatedness to B. hermsii, we conclude the Lyme disease spirochete is single previously undescribed species which belongs in the genus Borrelia.     

Experimental transmission of Theileria lestoquardi by developmental stages of Hyalomma anatolicum ticks

Theileria-free Hyalomma anatolicum larvae were fed on a naturally infected sheep with Theileria lestoquardi. Resulting flat nymphs of the tick were able to transmit T. lestoquardi infection upon feeding to 3/3 susceptible sheep. Adults emerging from the same batch of larvae were also infective to 3/3 susceptible sheep when they had the infection during their larval feeding. Transmission of T. lestoquardi to sheep was confirmed through clinical monitoring, examination of blood and lymph node biopsy smears, serology using indirect immunoflourescent test, and molecular using polymerase chain reaction technique.     

Antibody-mediated disease remission in the mouse model of lyme borreliosis

In the mouse model of Lyme borreliosis, the host immune response during infection with Borrelia burgdorferi results in the remission of carditis and arthritis, as well as global reduction of spirochete numbers in tissues, without elimination of infection. These events were recapitulated by passive transfer of immune serum from infected immunocompetent mice or T-cell-deficient mice to severe combined immunodeficient (SCID) mice. Previous studies have shown that immune serum is reactive against arthritis-related protein (Arp) and that Arp antiserum induces arthritis remission. However, although immune serum from T-cell-deficient mice induced disease remission, it was not reactive against Arp, suggesting that antibody to another antigen may be responsible. T-cell-deficient mouse immune serum was reactive to decorin binding protein A (DbpA). Therefore, DbpA antiserum was tested to determine its ability to induce disease remission in SCID mice. Antisera to Arp or DbpA induced both carditis and arthritis remission but did not significantly reduce spirochete numbers in tissues, based upon quantitative flaB DNA analysis, nor did treatment affect RNA levels of several genes, including arp and dbpA. Immunohistochemical labeling of spirochetes in hearts and joints during disease remission induced by adoptive transfer of lymphocytes, passive transfer of immune serum, or passive transfer of DbpA antiserum revealed that such treatment resulted in elimination of spirochetes from heart base and synovium but not vascular walls, tendons, or ligaments. These results suggest that Arp and DbpA antibodies may be active as disease-resolving components in immune serum but antibody against other antigens may be involved in reductions of spirochetes in tissues.     

Hemagglutination and proteoglycan binding by the Lyme disease spirochete, Borrelia burgdorferi.

The ability of the Lyme disease spirochete to attach to host components may contribute to its ability to infect diverse tissues. We present evidence that the Lyme disease spirochete expresses a lectin activity that promotes agglutination of erythrocytes and bacterial attachment to glycosaminoglycans. Among a diverse collection of 21 strains of Lyme disease spirochete, hemagglutinating activity was easily detected in all but 3 strains, and these three strains were noninfectious. The ability to agglutinate erythrocytes was associated with the ability of the spirochete to bind to the sulfated polysaccharide dextran sulfate and to mammalian cells. Soluble dextran sulfate was a potent inhibitor of both hemagglutination and attachment to mammalian cells, while dextran had no effect on either activity, suggesting that dextran sulfate may inhibit attachment by mimicking host cell glycosaminoglycans. Consistent with this, the spirochete bound to immobilized heparin, and soluble heparin inhibited bacterial adhesion to mammalian cells. The bacterium did not bind efficiently to Vero cells treated with heparinase or heparitinase or to mutant CHO cell lines that are deficient in proteoglycan synthesis. Sulfation of glycosaminoglycans was critical for efficient bacterial recognition, as Vero cells treated with an inhibitor of sulfation, or a mutant CHO cell line that produces undersulfated heparan sulfate, did not mediate maximal spirochetal binding. Binding of the spirochete to extracellular matrix also appeared to be dependent upon this attachment pathway. These findings suggest that a glycosaminoglycan-binding activity which can be detected by hemagglutination contributes to the attachment of the Lyme disease spirochete to host cells and matrix.     

Human phagocyte interactions with the Lyme disease spirochete.

The interaction between human polymorphonuclear leukocytes and monocytes and the Lyme disease spirochete was investigated by incubating phagocytes with microorganisms adherent to plastic or glass surfaces. Both cell populations readily phagocytized and killed spirochetes, and antibodies facilitated but were not essential for phagocytosis.     

Strain variation in glycosaminoglycan recognition influences cell-type-specific binding by lyme disease spirochetes

Lyme disease, a chronic multisystemic disorder that can affect the skin, heart, joints, and nervous system is caused by Borrelia burgdorferi sensu lato. Lyme disease spirochetes were previously shown to bind glycosaminoglycans (GAGs). In the current study, the GAG-binding properties of eight Lyme disease strains were determined. Binding by two high-passage HB19 derivatives to Vero cells could not be inhibited by enzymatic removal of GAGs or by the addition of exogenous GAG. The other six strains, which included a different high-passage HB19 derivative (HB19 clone 1), were shown to recognize both heparan sulfate and dermatan sulfate in cell-binding assays, but the relative efficiency of binding to these two GAGs varied among the strains. Strains N40, CA20-2A, and PBi bound predominantly to heparan sulfate, PBo bound both heparan sulfate and dermatan sulfate roughly equally, and VS461 and HB19 clone 1 recognized primarily dermatan sulfate. Cell binding by strain HB19 clone 1 was inhibited better by exogenous dermatan sulfate than by heparin, whereas heparin was the better inhibitor of binding by strain N40. The GAG-binding preference of a Lyme disease strain was reflected in its cell-type-specific binding. Strains that recognized predominantly heparan sulfate bound efficiently to both C6 glioma cells and EA-Hy926 cells, whereas strains that recognized predominantly dermatan sulfate bound well only to the glial cells. The effect of lyase treatment of these cells on bacterial binding was consistent with the model that cell-type-specific binding was a reflection of the GAG-binding preference. We conclude that the GAG-binding preference varies with the strain of Lyme disease spirochete and that this variation influences cell-type-specific binding in vitro.     

Satellite data and disease transmission by vectors: the creation of maps for risk prediction

The complex set of criteria determining the reproduction rate of infection (distribution, density, dynamics of arthropod populations, temperature, humidity...) can be estimated with far greater precision by satellite than by conventional meteorological stations. By linking a) distribution modes and type of environment and b) satellite data and rates of underlying biological processes, we have been able to elaborate two super-imposed models: one for vector populations and one for pathogenic agents. We have used such models to look at, for example, trypanosomiasis, malaria and Lyme disease.     

Absence of lipopolysaccharide in the Lyme disease spirochete, Borrelia burgdorferi

We were unable to demonstrate the presence of the classic enterobacterium-type lipopolysaccharide in the cells of the Lyme disease spirochete, Borrelia burgdorferi B31. This finding was primarily based on chemical analysis and the absence of free lipid A upon mild acid hydrolysis of the appropriate cell extracts. These results do not preclude the possible existence of an unusual lipopolysaccharide-like compound(s) in B. burgdorferi.