Ability of transovarially and subsequent transstadially infected Ixodes hexagonus ticks to maintain and transmit Borrelia burgdorferi in the laboratory

In a previous study, transstadial and transovarial survival of Borrelia burgdorferi in Ixodes hexagonus and transmission to laboratory mice via the bite of infected females were demonstrated. Here, we report the ability of I. hexagonus progeny infected transovarially to maintain and transmit the spriochaete to the host.Ticks were examined for spirochaetes by direct immunofluorescence antibody test. I. hexagonus larvae derived from the parental transstadially infected females were fed on two white mice: 21/54 (38.9%) of these ticks examined as unfed nymphs were infected.I. hexagonus nymphs were fed on three white mice and examined for spirochaetes after moulting as adults: 7/25 (28%) were found to harbour the spirochaete. The success of B. burgdorferi transmission to the mice by larval and nymphal I. hexagonus was determined by xenodiagnosis using I. ricinus larvae: 20/50 (40%) and 30/99 (30.3%) of the I. ricinus larvae fed on the mice infected by I. hexagonus larvae and nymphs respectively became infected.This study shows that B. burgdorferi can be maintained through transovarial and subsequent transstadial transmissions in I. hexagonus.     

Ability of Ixodes persulcatus, Haemaphysalis concinna and Dermacentor silvarum ticks to acquire and transstadially transmit Borrelia garinii

In China, the ability of Ixodes persulcatus, Haemaphysalis concinna and Dermacentor silvarum to transmit Borrelia spirochetes was determined under laboratory conditions. Results showed that all three tick species can acquire spirochetes by feeding on infected mice. However, the capability of the three species to maintain spirochetes was different. Only I. persulcatus is able to maintain spirochetes alive during molting, and subsequent tick stages transmitted the infection to naive mice. In H. concinna and D. silvarum ticks, spirochetes could not survive after the digestion period of blood and after the molting process was finished, spirochetes were no longer detected. Contrary to H. concinna and D. silvarum, I. persulcatus should be considered the principal vector of Lyme disease in north China.     

Borrelia burgdorferi in ticks of the Canton Tessin (Switzerland)

In the northern Alps it is hard to find an Ixodes ricinus population which does not harbour Borrelia burgdorferi. The infection rates range from 5 to 34% for adults and nymphs to 3.1% for larvae. This study shows that the infection rates on the southern side of the Alps are similar, at 25% for adults, 36.2% for nymphs and 3.2% for larvae. With respect to tick species other than I. ricinus, we did not succeed in finding any spirochetes in Dermacentor marginatus, Haemaphysalis punctata, Ixodes hexagonus, Rhipicephalus sanguineus.     

Dynamics of Borrelia burgdorferi infection in nymphal Ixodes ricinus ticks during feeding

We report the sequential developmental events of Borrelia burgdorferi in histological sections of Ixodes ricinus nymphs before, during and after feeding. During the blood meal a decrease of approximately 50% in the number of infected ticks was recorded (eight out of 76, 11%) in comparison with the infection rate of unfed ticks (12 out of 56, 21%). Spirochetes were detected in tick salivary glands only after 2 days of attachment. From day 3 until drop-off, the number of infected ticks increased to 31% (15 out of 49). A quadratic logistic regression analysis showed that the variation in the number of infected ticks was significant, but only during the blood meal. The drop in the percentage of infected ticks during the first hours following attachment to the host is explained by our observation of spirochetes in the faeces of the ticks. The increase in the infection rate of replete ticks may be due to an uptake of spirochetes from the host skin at the feeding site.     

Investigation of venereal, transplacental, and contact transmission of the Lyme disease spirochete, Borrelia burgdorferi, in Syrian hamsters.

A hamster was inoculated with the SI-1 strain of Borrelia burgdorferi and subsequently served as a host to larval Ixodes scapularis Say. Approximately 68% of the nymphs resulting from the fed larvae were infected. Nymphs from this group were fed on uninfected hamsters, and 3 of 4 males and 6 of 6 females became infected. The infected hamsters were allowed to mate with uninfected partners to test for venereal transmission. Six infected females were mated with 6 uninfected males, whereas 3 infected males were mated with 6 uninfected females. None of the uninfected hamsters became infected after mating. Two protocols were used to determine if transplacental transmission of B. burgdorferi occurred. One group included 6 nonpregnant infected females that were subsequently mated and became pregnant. Three of the females were allowed to carry to full term, whereas the other 3 were killed prior to parturition. All fetuses and offspring were negative for B. burgdorferi based on cultures and monoclonal antibody assays. Another group of 6 females was infected via tick bite after becoming pregnant; those females were allowed to carry fetuses to birth and all were negative. Attempts at contact transmission of B. burgdorferi from 2 infected females to 2 uninfected male and 2 uninfected female hamsters and from 2 infected males to 2 uninfected male and uninfected female hamsters via urine or feces failed.     

Role of Borrelia burgdorferi linear plasmid 25 in infection of Ixodes scapularis ticks

The tick-borne bacterium Borrelia burgdorferi has over 20 different circular and linear plasmids. Some B. burgdorferi plasmids are readily lost during in vitro culture or genetic manipulation. Linear plasmid 25, which is often lost in laboratory strains, is required for the infection of mice. Strains missing linear plasmid 25 (lp25(-)) are able to infect mice if the BBE22 gene on lp25 is provided on a shuttle vector. In this study, we examined the role of lp25 and BBE22 in tick infections. We tested the hypothesis that complementation with BBE22 in spirochetes lacking lp25 would restore the ability of spirochetes to infect ticks. A natural tick infection cycle was performed by feeding larvae on mice injected with the parental, lp25(-), or lp25(-) BBE22-complemented spirochete strains. In addition, larvae and nymphs were artificially infected with different strains to study tick infections independent of mouse infections. B. burgdorferi missing lp25 was significantly impaired in its ability to infect larval and nymphal ticks. When an lp25(-) strain was complemented with BBE22, the ability to infect ticks was partially restored. Complementation with BBE22 allowed spirochetes lacking lp25 to establish short-term infections in ticks, but in most cases the infection prevalence was lower than that of the wild-type strain. In addition, the number of infected ticks decreased over time, suggesting that another gene(s) on lp25 is required for long-term persistence in ticks and completion of a natural infection cycle.     

Borrelia burgdorferi in ticks and dogs in the province of Vojvodina, Serbia

Lyme disease is a tick borne zoonotic infection, caused by Borrelia burgdorferi s.l. bacteria. For the transmission of the disease, the presence of ticks is a prerequisite. Lyme borreliosis mostly occurs in people and dogs, but it may occur in other animals. Ticks which carry B. burgdorferi s.l. in Serbia are of the Ixodes ricinus specis. In Serbia, Lyme disease was detected for the first time in the late '80-es. In dogs, clinical symptoms may occur even months after a tick bite, and include weakness, lymphadenopathy, fever, lameness, arthritis, etc. In our survey, we have observed tick and dog populations in the province of Vojvodina (northern part of Serbia). I. ricinus ticks were collected and examined for the presence of B. burgdorferi s.l. in several chosen locations. In addition, blood samples were collected from house dogs and pets from the same locations, and analyzed for the presence of antibodies specific for B. burgdorferi s.l. The results showed a mean infection of ticks of 22.12%, and a mean seroprevalence of Lyme disease in dogs of 25.81%. We conclude that in Vojvodina there is an actual risk of Lyme borreliosis for other animals and humans, because of the persistence of B. burgdorferi s.l. in both tick and dog populations.     

Prevalence of Borrelia burgdorferi sensu lato in ticks collected from migratory birds in Switzerland

The prevalence of ticks infected by Borrelia burgdorferi sensu lato on birds during their migrations was studied in Switzerland. A total of 1,270 birds captured at two sites were examined for tick infestation. Ixodes ricinus was the dominant tick species. Prevalences of tick infestation were 6% and 18.2% for birds migrating northward and southward, respectively. Borrelia valaisiana was the species detected most frequently in ticks, followed by Borrelia garinii and Borrelia lusitaniae. Among birds infested by infected ticks, 23% (6/26) were infested by B. lusitaniae-infected larvae. Migratory birds appear to be reservoir hosts for B. lusitaniae.     

Birds and their ticks in northwestern California: minimal contribution to Borrelia burgdorferi enzootiology

Birds and their attendant ticks were surveyed for infection with the Lyme disease spirochete Borrelia burgdorferi, in chaparral and woodland-grass habitats in northwestern California from March to July, 1998 to 1999. In total, 234 birds were captured and recaptured (15%); nearly 2.5 times more birds were captured in chaparral than in woodland-grass. Overall, 34 species representing 15 families were collected during this study; of these, 24 species were caught in chaparral, 19 in woodland-grass, and 9 in both vegetational types. The most frequently captured birds were sage sparrows (Amphispiza belli) in chaparral, and American robins (Turdus migratorius) and oak titmice (Baelophus inornatus) in woodland-grass. Birds hosted 35 Ixodes pacificus (15 larvae, 20 nymphs) and 9 Haemaphysalis leporispalustris (3 larvae, 5 nymphs, 1 adult) ticks, of which 32 were removed from chaparral birds and 12 from woodland birds. The prevalence of tick infestation was 13% (21/167) in chaparral and 5% (3/67) in woodland-grass, but the relative and mean tick intensities of 0.19 and 1.5 for chaparral birds, and 0.18 and 4.0 for woodland birds, respectively, did not differ significantly by habitat. Spirochetes were not detected in either bird-blood or tick-tissue samples when tested by culture, immunofluorescence, or Giemsa-staining. In contrast, over 90% (86/94) of western fence lizards (Sceloporus occidentalis) collected in June or July were infested with an average of 6.9 and 8.9 immature I. pacificus in chaparral and woodland-grass, respectively. We conclude that birds contribute little to the enzootiology of B. burgdorferi in chaparral and woodland-grass habitats in northwestern California because of their limited parasitism by tick vectors and lack of detectable spirochetemias.     

Geographic distribution of white-tailed deer with ticks and antibodies to Borrelia burgdorferi in Connecticut.

Ticks and blood specimens were collected from white-tailed deer (Odocoileus virginianus) in Connecticut and analyzed to identify foci for Lyme borreliosis. Males and females of Ixodes scapularis, the chief vector of Borrelia burgdorferi, were collected from deer in five of eight counties during 1989-1991. Analysis by indirect fluorescent antibody (IFA) staining of midgut tissues showed that prevalence of infection was highest (9.5% of 367 ticks) in south central and southeastern Connecticut. Infected I. scapularis also were collected from southwestern regions of the state (12.1% of 99 ticks), but prevalence of infection in northern counties was considerably lower (0.8% of 124 ticks). Deer sera, obtained in 1980 and 1989-1991, were analyzed by an enzyme-linked immunosorbent assay or by IFA staining methods. Antibodies to B. burgdorferi were detected in sera collected from all eight counties in Connecticut. Deer had been infected by this spirochete in at least 50 towns, 17 (34%) of which are in south central and southeastern parts of the state. Borrelia burgdorferi is widely distributed in I. scapularis populations in Connecticut.     

The histopathology of experimentally infected hamsters with the Lyme disease spirochete, Borrelia burgdorferi

Seven hamsters, experimentally infected with Borrelia burgdorferi, were examined by both cultural and histological techniques at 1 to 9 months postinfection. Spirochetes were detected in the spleen, kidney, or eye of all animals by culture and in the spleen, kidney, eye, liver, or heart blood of five of seven animals by histological examination. Two animals showed nonspecific hepatic portal lymphocytic infiltration, while five of the hamsters displayed no significant histologic signs of inflammation or granuloma formation in the major organ systems. Synovitis and arthropathy did not occur. All animals showed some degree of follicular lymphoid hyperplasia of the spleen. Spirochetes were predominantly extracellular with a rare organism appearing to be partially within a macrophage.     

Problems of isolating Borrelia burgdorferi from ticks collected in United Kingdom foci of Lyme disease

Abstract. Many isolates of Borrelia burgdorferi have been obtained from ticks and vertebrate tissues collected in North America and continental Europe but only one established culture of United Kingdom Borrelia burgdorferi has been recorded. In this paper we report the isolation of B.burgdorferi from one of 108 tick pools representing 733 ticks and eighty-four tissue samples from twenty-six rodents collected in the U.K., and the subsequent failure to establish the isolate (from ticks collected in Fordingbridge) in culture. In contrast, using identical techniques and culture medium, B.burgdorferi was isolated from one of seven tick pools collected in Switzerland, and from a single pool of ticks collected in Slovakia, and both isolates were successfully passaged. Analysis of questing I.ricinus collected from Fordingbridge by direct immunofluorescence showed 6/32 (19%) of adults and 8/108 (7%) of nymphs were positive for B. burgdorferi , although only one nymph contained ≥ 1000 spirochaetes. To examine further the problem of isolating U.K. B.burgdorferi , twelve Ixodes ricinus tick samples from Fordingbridge, a recognized focus of Lyme disease, were subjected to isolation and culturing techniques, and the procedures monitored by use of the polymerase chain reaction (PCR). Whereas 11/12 samples were PCR positive after 2 weeks in culture, only one was PCR positive after 4 weeks. Motile spirochaetes were not visible by dark-field microscopy in any of the cultures. The results indicate that the standard BSK II medium routinely used to isolate and culture B. burgdorferi does not readily support the replication of the Borrelia species endemic to the U.K.     

Comparison of Borrelia burgdorferi isolated from humans and ixodid ticks in Hokkaido, Japan.

Four spirochetal isolates (JEM1 to JEM4) were obtained from cutaneous lesions of patients manifesting erythema chronicum migrans in Hokkaido, Japan. In the protein profiles by SDS-PAGE and the reactivities with monoclonal antibodies (H5332 and H9724) by immunoblotting, all the human isolates were identical with the tick isolates from Ixodes persulcatus. These data indicate that I. persulcatus is an important vector of Lyme disease for humans in Japan.     

Chemotaxis in Borrelia burgdorferi

Borrelia burgdorferi is a motile spirochete which has been identified as the causative microorganism in Lyme disease. The physiological functions which govern the motility of this organism have not been elucidated. In this study, we found that motility of B. burgdorferi required an environment similar to interstitial fluid (e.g., pH 7.6 and 0.15 M NaCl). Several methods were used to detect and measure chemotaxis of B. burgdorferi. A number of chemical compounds and mixtures were surveyed for the ability to induce positive and negative chemotaxis of B. burgdorferi. Rabbit serum was found to be an attractant for B. burgdorferi, while ethanol and butanol were found to be repellents. Unlike some free-living spirochetes (e.g., Spirochaeta aurantia), B. burgdorferi did not exhibit any observable chemotaxis to common sugars or amino acids. A method was developed to produce spirochete cells with a self-entangled end. These cells enabled us to study the rotation of a single flagellar bundle in response to chemoattractants or repellents. The study shows that the frequency and duration for pausing of flagella are important for chemotaxis of B. burgdorferi.     

Host associations of ticks parasitizing rodents at Borrelia burgdorferi enzootic sites in South Carolina.

A total of 237 rodents was collected in 4 regions of South Carolina from July 1994 through December 1995. Eight species were collected, including cotton mouse, hispid cotton rat, eastern woodrat, marsh rice rat, white-footed mouse, eastern harvest mouse, golden mouse, and black rat. Of the 1,514 ticks recovered from these hosts, Ixodes minor Neumann, including larvae, nymphs, and adults, was the most abundant species, representing 54% of the total. Only immature stages of other tick species were found, including larvae and nymphs of Dermacentor variabilis (Say), Amblyomma maculatum Koch, Ixodes affinis Neumann, and Ixodes scapularis Say. All 5 tick species parasitized cotton mice, cotton rats, and woodrats, which were the most important small mammal hosts for ticks at the localities studied. Rice rats were hosts of A. maculatum, D. variabilis, and L. minor. Amblyomma maculatum was more strongly associated with cotton rats than other rodent species. Ixodes scapularis was most strongly associated with cotton mice, and I. minor was more strongly associated with both woodrats and cotton mice than other species of rodents. Ixodes minor parasitized hosts in the Coastal Zone only, where among spirochete-infected hosts, it was present in significantly greater numbers than other ticks. Furthermore, I. minor was the only tick species that showed a statistically significant positive association with spirochetal infection in rodents. More I. affinis parasitized spirochete-infected hosts than I. scapularis, but fewer than I. minor. The findings discussed herein provide evidence that implicates I. minor as the possible primary enzootic vector of the Lyme disease spirochete Borrelia burgdorferi Johnson, Schmid, Hyde, Steigerwalt and Brenner in the Coastal Zone of South Carolina. They also indicate that the high level of B. burgdorferi infection in rodents from this region may be a function of the combined involvement of I. minor, I. affinis, and I. scapularis in the enzootic transmission of the spirochete.     

Outer surface protein B is critical for Borrelia burgdorferi adherence and survival within Ixodes ticks

Survival of Borrelia burgdorferi in ticks and mammals is facilitated, at least in part, by the selective expression of lipoproteins. Outer surface protein (Osp) A participates in spirochete adherence to the tick gut. As ospB is expressed on a bicistronic operon with ospA, we have now investigated the role of OspB by generating an OspB-deficient B. burgdorferi and examining its phenotype throughout the spirochete life cycle. Similar to wild-type isolates, the OspB-deficient B. burgdorferi were able to readily infect and persist in mice. OspB-deficient B. burgdorferi were capable of migrating to the feeding ticks but had an impaired ability to adhere to the tick gut and survive within the vector. Furthermore, the OspB-deficient B. burgdorferi bound poorly to tick gut extracts. The complementation of the OspB-deficient spirochete in trans, with a wild-type copy of ospB gene, restored its ability to bind tick gut. Taken together, these data suggest that OspB has an important role within Ixodes scapularis and that B. burgdorferi relies upon multiple genes to efficiently persist in ticks.     

Investigation of genotypes of Borrelia burgdorferi in Ixodes scapularis ticks collected during surveillance in Canada

The genetic diversity of Borrelia burgdorferi sensu stricto, the agent of Lyme disease in North America, has consequences for the performance of serological diagnostic tests and disease severity. To investigate B. burgdorferi diversity in Canada, where Lyme disease is emerging, bacterial DNA in 309 infected adult Ixodes scapularis ticks collected in surveillance was characterized by multilocus sequence typing (MLST) and analysis of outer surface protein C gene (ospC) alleles. Six ticks carried Borrelia miyamotoi, and one tick carried the novel species Borrelia kurtenbachii. 142 ticks carried B. burgdorferi sequence types (STs) previously described from the United States. Fifty-eight ticks carried B. burgdorferi of 1 of 19 novel or undescribed STs, which were single-, double-, or triple-locus variants of STs first described in the United States. Clonal complexes with founder STs from the United States were identified. Seventeen ospC alleles were identified in 309 B. burgdorferi-infected ticks. Positive and negative associations in the occurrence of different alleles in the same tick supported a hypothesis of multiple-niche polymorphism for B. burgdorferi in North America. Geographic analysis of STs and ospC alleles were consistent with south-to-north dispersion of infected ticks from U.S. sources on migratory birds. These observations suggest that the genetic diversity of B. burgdorferi in eastern and central Canada corresponds to that in the United States, but there was evidence for founder events skewing the diversity in emerging tick populations. Further studies are needed to investigate the significance of these observations for the performance of diagnostic tests and clinical presentation of Lyme disease in Canada.     

Distinct combinations of Borrelia burgdorferi sensu lato genospecies found in individual questing ticks from Europe

The genetic diversity of Borrelia burgdorferi sensu lato was assessed in individual adult Ixodes ricinus ticks from Europe by direct PCR amplification of spirochetal DNA followed by genospecies-specific hybridization. Analysis of mixed infections in the ticks showed that B. garinii and B. valaisiana segregate from B. afzelii. This and previous findings suggest that host complement interacts with spirochetes in the tick, thereby playing an important role in the ecology of Lyme borreliosis.     

Longevity of forest and taiga ticks (Ixodidae) infected and non-infected with Borrelia burgdorferi groups

A life longevity of Ixodes ricinus and I. persulcatus have been examined in laboratory conditions. It was found that specimens infected with borreliae live slightly longer than non-infected ones (difference is not reliable); females live 1.5 times longer than males; I. ricinus lives 2-4 times longer than I. persulcatus. It is suggested that I. persulcatus has a higher rate of metabolism than I. ricinus and that the latter species obtains an ability to survive after the last moult two winters and keep an activity in two summer seasons not counting the year of moulting.