Effect of ambient temperature on competence of deer ticks as hosts for Lyme disease spirochetes.

We determined whether the temperature of extrinsic incubation affects the competence of vector ticks as hosts for Lyme disease spirochetes (Borrelia burgdorferi). Larval Ixodes dammini ticks that had engorged on spirochete-infected C3H mice were incubated continuously at various temperatures, and the gut contents of the resulting nymphs were examined for spirochetes by direct immunofluorescence microscopy. Spirochetes were present in virtually all nymphs kept at 27 degrees C or less for 6 months, in only 10% of those kept at 33 degrees C, and in none kept at 37 degrees C. Spirochetes became undetectable within 8 weeks when nymphs were warmed from 27 to 33 degrees C beginning at the time of molting. Nymphs became virtually noninfective for mice after incubation at temperatures higher than 27 degrees C for 2 weeks or longer. We conclude that ambient temperatures in excess of 27 degrees C are not permissive for transmission of the agent of Lyme disease.     

Transhemispheric exchange of Lyme disease spirochetes by seabirds.

Lyme disease is a zoonosis transmitted by ticks and caused by the spirochete Borrelia burgdorferi sensu lato. Epidemiological and ecological investigations to date have focused on the terrestrial forms of Lyme disease. Here we show a significant role for seabirds in a global transmission cycle by demonstrating the presence of Lyme disease Borrelia spirochetes in Ixodes uriae ticks from several seabird colonies in both the Southern and Northern Hemispheres. Borrelia DNA was isolated from I. uriae ticks and from cultured spirochetes. Sequence analysis of a conserved region of the flagellin (fla) gene revealed that the DNA obtained was from B. garinii regardless of the geographical origin of the sample. Identical fla gene fragments in ticks obtained from different hemispheres indicate a transhemispheric exchange of Lyme disease spirochetes. A marine ecological niche and a marine epidemiological route for Lyme disease borreliae are proposed.     

Accelerated infectivity of tick-transmitted Lyme disease spirochetes to vector ticks.

We determined whether the span of infectivity of Lyme disease spirochetes (Borrelia burgdorferi) to vector ticks varies with the mode of infection in laboratory mice. Noninfected larval deer ticks were permitted to feed on two strains of spirochete-infected mice that had been naturally (via tick bite) and parenterally (via needle injection) infected with B. burgdorferi 2, 4, or 8 weeks earlier, and engorged ticks were dissected and examined for spirochetes by direct immunofluorescence microscopy. After initial infection, spirochetal infectivity to ticks was less efficient in needle-infected mice than in mice infected via tick bites. Tick-transmitted spirochetes develop more rapidly from the skin of infected mice and do not induce a strong antispirochete antibody response during the early stage of infection.     

Experimental infection of Ixodes scapularis larvae (Acari: Ixodidae) by immersion in low passage cultures of Borrelia burgdorferi

We describe a procedure for the introduction of Borrelia burgdorferi, the spirochetal agent of Lyme disease, into larvae of the tick vector Ixodes scapularis. Internalized spirochetes were observed in larvae examined after 15 or 45 min immersion at 32 degrees C in liquid culture suspensions of low passage B. burgdorferi strain B31. Larval ticks immersed in low passage strain B31 were able to feed to repletion on white-footed mice. Midguts of larvae contained many spirochetes 1 wk postengorgement, while larvae incubated with high passage strain B31 were free of detectable spirochetes at the same interval. Larvae incubated with low passage strain B31 were competent to transmit the pathogen to mice, as shown by serology, reisolation of B. burgdorferi from mice, and xenodiagnosis. Ticks maintained the infection transstadially to the nymphal stage and transmitted the infection to naive mice, replicating an essential aspect of natural infection. This method requires no special equipment and allows artificial infection of large numbers of ticks at the larval stage. It will facilitate studies of the contribution of specific B. burgdorferi genetic loci to tick colonization.     

Partial destruction of Borrelia burgdorferi within ticks that engorged on OspE- or OspF-immunized mice.

We determined whether Borrelia burgdorferi outer surface proteins (Osps) E and F could elicit immune responses useful for a Lyme disease vaccine. Thirty days after challenge with B. burgdorferi, mice produced antibodies to OspE but not OspF, whereas antibodies to OspF were present in sera of mice obtained 90 days after infection. Examination of sera from patients with Lyme disease revealed antibodies to OspF in a small number (14%) of early-stage disease patients but in a majority (58%) of patients with late-stage disease, while antibodies to OspE were rarely detected in patients. Mice immunized with recombinant OspE or OspF produced high titers of antibodies to OspE or OspF, respectively. OspF-immunized mice were partially protected from both intradermal syringe challenge and tick-mediated transmission of B. burgdorferi while vaccination with OspE did not confer immunity. B. burgdorferi organisms were, however, substantially destroyed within ticks that engorged on either OspE- (75% reduction in the number of spirochetes within the ticks, compared with controls) or OspF (90% reduction in the number of spirochetes within the ticks)-immunized mice.     

Cystitis induced by infection with the Lyme disease spirochete, Borrelia burgdorferi, in mice.

Previous studies have demonstrated that the urinary bladder is a consistent source for isolating the Lyme disease spirochete, Borrelia burgdorferi, from both experimentally infected and naturally exposed rodents. We examined histopathologic changes in the urinary bladder of different types of rodents experimentally infected with Lyme spirochetes, including BALB/c mice (Mus musculus), nude mice (M. musculus), white-footed mice (Peromyscus leucopus), and grasshopper mice (Onychomys leucogaster). Animals were inoculated intraperitoneally, subcutaneously, or intranasally with low-passaged spirochetes, high-passaged spirochetes, or phosphate-buffered saline. At various times after inoculation, animals were killed and approximately one-half of each urinary bladder and kidney were cultured separately in BSK-II medium while the other half of each organ was prepared for histologic examination. Spirochetes were cultured from the urinary bladder of all 35 mice inoculated with low-passaged spirochetes while we were unable to isolate spirochetes from any kidneys of the same mice. The pathologic changes observed most frequently in the urinary bladder of the infected mice were the presence of lymphoid aggregates, vascular changes, including an increase in the number of vessels and thickening of the vessel walls, and perivascular infiltrates. Our results demonstrate that nearly all individuals (93%) of the four types of mice examined had a cystitis associated with spirochetal infection.     

Identification of endemic foci of Lyme disease: isolation of Borrelia burgdorferi from feral rodents and ticks (Dermacentor variabilis).

Borrelia burgdorferi, the etiological agent of Lyme disease, was isolated from the blood, kidneys, spleens, eyes, or livers of white-footed mice (Peromyscus leucopus) (n = 19 of 22) and from the blood, kidneys, or spleens of eastern chipmunks (Tamias striatus) (n = 2 of 2) captured at three foci for Lyme disease in eastern Connecticut. These bacteria were cultured most frequently from spleens (n = 19) and kidneys (n = 15). B. burgdorferi persisted in one mouse for at least 60 days. One spirochetemic mouse had infected Ixodes dammini and Dermacentor variabilis larvae attached, suggesting that these ticks may have acquired spirochetes from the host. Spirochetes isolated from P. leucopus, T. striatus, and D. variabilis larvae were serologically and genetically indistinguishable from reference B. burgdorferi isolates. We conclude that isolation of spirochetes from feral rodents is a method for identifying endemic areas of Lyme disease.     

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.     

Lack of detectable variation at Borrelia burgdorferi vlsE locus in ticks

VlsE is a surface exposed lipoprotein of the Lyme disease spirochete, Borrelia burgdorferi. Spirochetes are able to generate many antigenic variants of VlsE by DNA recombination at the vlsE locus. Novel VlsE antigenic variants are readily observed in mice infected with B. burgdorferi. We followed a clonal population of spirochetes through a tick transmission cycle and report that unlike in vertebrates, the vlsE locus is stable in ticks.     

Decorin-binding protein A (DbpA) of Borrelia burgdorferi is not protective when immunized mice are challenged via tick infestation and correlates with the lack of DbpA expression by B. burgdorferi in ticks

Previous studies showed that decorin-binding protein A (DbpA) of Borrelia burgdorferi was a protective immunogen in the murine model of Lyme borreliosis when mice were challenged (needle inoculated) intradermally with in vitro-cultivated spirochetes. In the present study, DbpA-immunized C3H/HeJ mice were not protected from infection when infested with Ixodes scapularis nymphs harboring virulent B. burgdorferi 297. This lack of protection correlated with the failure to detect DbpA on B. burgdorferi in ticks, suggesting that DbpA is not available as a target for bactericidal antibodies in serum when B. burgdorferi-infected ticks take their blood meal from an immunized host. The failure of DbpA immunization to protect tick-challenged mice contradicts the results of earlier needle inoculation vaccination experiments and suggests that DbpA may not be suitable as a Lyme disease vaccine.     

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.     

Prevalence of Borrelia burgdorferi in Ixodes ricinus ticks in urban recreational areas of Helsinki

Lyme borreliosis, an infection caused by the tick-borne spirochete Borrelia burgdorferi, is a major health problem for populations in areas of endemicity in the Northern Hemisphere. In the present study we assessed the density of ticks and the prevalence of B. burgdorferi sensu lato among ticks in popular urban recreational areas of Helsinki, Finland. Altogether 1,688 Ixodes ricinus ticks were collected from five areas located within 5 km of the downtown section of Helsinki, and 726 of them (303 nymphs, 189 females, and 234 males) were randomly chosen for laboratory analysis. The midguts of the ticks were divided into three pieces, one for dark-field microscopy, one for cultivation in BSK-II medium, and one for PCR analysis. Ticks were found in all the study areas; their densities varied from 1 to 36 per 100 m along which a cloth was dragged. The rate of tick infection with B. burgdorferi sensu lato varied from 19 to 55%, with the average being 32%. Borellia afzelii was the most predominant genospecies in all the areas, and no B. burgdorferi sensu stricto isolates were detected. Only two ticks were concurrently infected with both B. afzelii and Borrelia garinii. Dark-field microscopy gave more positive results for B. burgdorferi than did cultivation or PCR analysis. However, the agreement between all three methods was fairly good. We conclude that Lyme borreliosis can be contracted even in urban environments not populated with large mammals like deer or elk. The disease should be taken into account in the differential diagnosis of certain symptoms of patients from these areas, and the use of measures to improve the awareness of the general population and health care officials of the risk of contracting the disease is warranted.     

Borrelia burgdorferi OspC protein required exclusively in a crucial early stage of mammalian infection

This study demonstrates a strict temporal requirement for a virulence determinant of the Lyme disease spirochete Borrelia burgdorferi during a unique point in its natural infection cycle, which alternates between ticks and small mammals. OspC is a major surface protein produced by B. burgdorferi when infected ticks feed but whose synthesis decreases after transmission to a mammalian host. We have previously shown that spirochetes lacking OspC are competent to replicate in and migrate to the salivary glands of the tick vector but do not infect mice. Here we assessed the timing of the requirement for OspC by using an ospC mutant complemented with an unstable copy of the ospC gene and show that B. burgdorferi's requirement for OspC is specific to the mammal and limited to a critical early stage of mammalian infection. By using this unique system, we found that most bacterial reisolates from mice persistently infected with the initially complemented ospC mutant strain no longer carried the wild-type copy of ospC. Such spirochetes were acquired by feeding ticks and migrated to the tick salivary glands during subsequent feeding. Despite normal behavior in ticks, these ospC mutant spirochetes did not infect naive mice. ospC mutant spirochetes from persistently infected mice also failed to infect naive mice by tissue transplantation. We conclude that OspC is indispensable for establishing infection by B. burgdorferi in mammals but is not required at any other point of the mouse-tick infection cycle.     

Lyme disease spirochete,Borrelia burgdorferi endemic at epicenter in Rondeau Provincial Park,Ontario

The Lyme disease spirochete, Borrelia burgdorferi Johnson, Schmidt, Hyde, Steigerwalt, and Brenner was discovered in blacklegged ticks, Ixodes scapularis Say at Rondeau Provincial Park, Ontario, Canada During this 2-yr study, spirochetes were found in B. burgdorferi-positive I. scapularis larvae attached to B. burgdorferi-infected white-footed mice, Peromyscus leucopus Rafinesque. Isolates of B. burgdorferi were cultured from blacklegged tick adults, and confirmed positive with polymerase chain reaction by targeting OspA and rrf (5S)-rrl (23S) genes. These findings show an endemic area for B. burgdorferi within an established population of L. scapularis at Rondeau Provincial Park.     

Cultivation at 37°C enhances Borrelia burgdorferi sensu stricto infectivity for hamsters

Borrelia burgdorferi sensu stricto (s.s.), the causative agent of Lyme disease in North America is transmitted to the mammalian host by ticks belonging to the genus, Ixodes. Antibodies to several spirochetal proteins, most notably outer surface protein C (OspC), have been observed in early infection in both humans and laboratory animals. Thus, the expression of these proteins have been postulated to play a role in tick transmission and spirochetal infectivity for the mammalian host. B. burgdorferi strain JMNT was induced to produce increased levels of OspC by cultivation in BSK medium at 37 degrees C. To diminish expression of OspC, spirochetes were cultivated at 31 degrees C. Spirochetes shifted down from 37 degrees C to 31 degrees C or up from 31 degrees C to 37 degrees C for 1 week contained equivalent amounts of OspC. To evaluate spirochetal infectivity, hamsters were inoculated subcutaneously with 1 x 10(4) or 1 x 10(6) spirochetes grown at the above-mentioned temperatures. Hamsters inoculated with spirochetes expressing high amounts of OspC all became infected, irrespective of the inoculum size. None of the hamsters inoculated with 1 x 10(4) spirochetes grown at 31 degrees C or in cultures shifted down from 37 degrees C to 31 degrees C were infected. All infected hamsters, confirmed by isolation of spirochetes in ear and/or bladder cultures, had an antibody response to OspC. In contrast, all non-infected hamsters lacked antibodies to OspC. We conclude that cultivation of spirochetes at 37 degrees C enhances their infectivity for hamsters. This study also suggests there is a correlation between enhancement of OspC expression and spirochetal infectivity for hamsters.     

Culturing selects for specific genotypes of Borrelia burgdorferi in an enzootic cycle in Colorado.

In Colorado, Borrelia burgdorferi sensu stricto, the etiologic agent of Lyme disease, is maintained in an enzootic cycle between Ixodes spinipalpis ticks and Neotoma mexicana rats (27). The frequencies of flagellin (fla), 66-kDa protein (p66), and outer surface protein A (ospA) alleles were examined in 71 B. burgdorferi isolates from samples from Colorado. Approximately two-thirds of these samples were isolates from I. spinipalpis ticks that had been cultured in BSK-H medium prior to DNA extraction. The remaining samples were from total DNA extracted directly from infected I. spinipalpis ticks. A portion of each gene was amplified by PCR and screened for genetic variability by single-strand conformation polymorphism (SSCP) analysis. We identified three alleles in the fla gene, seven in the p66 gene, and seven in the ospA gene. Sequencing verified that the amplified products originated from B. burgdorferi template DNA and indicated 100% sensitivity and specificity of the SSCP analysis. The frequencies of the p66 and ospA alleles were significantly different between cultured and uncultured spirochetes. The number of three-locus genotypes and the genetic diversity of alleles at all loci were consistently lower in cultured spirochetes, suggesting that culturing of B. burgdorferi in BSK-H medium may select for specific genotypes.     

Differential spirochetal infectivities to vector ticks of mice chronically infected by the agent of Lyme disease.

We determined whether the infectivity of the Lyme disease spirochete (Borrelia burgdorferi) to vector ticks varies with the duration of infection in laboratory mice. Thus, noninfected nymphal deer ticks were permitted to feed on two strains of early (2 months after infection) and late (8 months after infection) spirochete-infected mice. The attached ticks were removed from their hosts at specified time intervals and were thereafter examined for spirochetes by direct immunofluorescence microscopy. Spirochetes can be acquired by nymphal ticks as fast as 8 h after attachment. More than 80% of the attached ticks acquired spirochetal infection within 48 h after feeding on early spirochete-infected mice. In contrast, spirochetal infectivity to ticks was less than 50% after feeding on late spirochete-infected mice. The overall infectivity of spirochete-infected mice to ticks correlated with the duration of tick attachment. In addition, there was no adverse effect on the spirochetal infectivity to ticks by high levels of host antibody against spirochetes, and no obvious differences in infectivity to ticks was observed by the site of tick feeding. We conclude that the span of spirochetal infectivity to ticks varies with the duration of infection in mice and suggest that spirochetes may persist and may be evenly distributed in the skin of infected hosts, regardless of prominent host immunity.     

Comparison of growth of Borrelia afzelii, B. garinii, and B. burgdorferi sensu stricto in MKP and BSK-II medium

Lyme borreliosis is a tick-borne disease caused by genetically diverse Borrelia strains including B. afzelii, B. garinii, and B. burgdorferi sensu stricto (s.s.). The aim of the present study was to assess and compare the growth of one strain per species of B. afzelii, B. garinii, and B. burgdorferi s.s. in modified Kelly-Pettenkofer (MKP) and Barbour-Stonner-Kelly-II (BSK-II) medium, and to check for the presence of the overgrowth after inoculating the media with a mixture of two different Borrelia species. All three Borrelia strains grew well in both media. In the majority of the experiments the number of B. afzelii cells was higher in MKP than in BSK-II medium while for B. garinii and B. burgdorferi s.s. a tendency for better growth in BSK-II than MKP was established. In a mixture of equivalent amounts of two species, B. burgdorferi s.s. as a rule overgrew the other two species while in the mixture of B. afzelii and B. garinii the latter was a "dominant" strain. Comparing the performance of the two media, B. burgdorferi s.s. usually overgrew either B. afzelii or B. garinii in MKP as well as in BSK-II medium, however, the results were found to be statistically significant only for MKP medium. In the mixture of B. afzelii and B. garinii the latter was the predominant species but significant differences were established only for BSK-II medium. It seems that the overgrowth is predominantly the result of the characteristics of the individual Borrelia species and most probably not a consequence of growth differences in the two culture media. Further work with a larger number of strains is needed to confirm these findings.     

Rapid dissemination by the agent of Lyme disease in hosts that permit fulminating infection.

We determined whether the agent of Lyme disease (Borrelia burgdorferi) disseminates more rapidly following deposition in hosts that permit fulminating infection than in hosts in which infection is relatively benign. Thus, individual infected nymphal deer ticks (Ixodes dammini) were permitted to engorge on the ears of C3H mice, and the site of attachment was excised at intervals thereafter. Infection in each mouse was determined by serology and by examining previously noninfected ticks that had engorged on these mice. These results were compared with data obtained similarly by using the CD-1 strain of mice in which the agent is relatively nonpathogenic. When the site of inoculation was ablated within 2 days after the infected tick became replete, dissemination was aborted. Spirochetemia could not be demonstrated in any of these mice. We conclude that Lyme disease spirochetes disseminate from the feeding lesion of an infecting tick more rapidly in certain highly spirochete-susceptible mice than in others in which pathogenesis is less severe.     

Differential survival of Lyme borreliosis spirochetes in ticks that feed on birds

The abilities of the most common European genospecies of Borrelia burgdorferi sensu lato to survive blood meals taken by ticks feeding on birds were analyzed. A pattern of differential survival of the spirochetes in feeding ticks was observed. The result is consistent with the concept of selective transmission of Lyme borreliosis spirochetes.