Genospecies identification and characterization of Lyme disease spirochetes of genospecies Borrelia burgdorferi sensu lato isolated from rodents in Taiwan

Lyme disease spirochetes of the genospecies Borrelia burgdorferi sensu lato were identified and characterized for the first time in Taiwan. Seven isolates, designated TWKM1 to TWKM7, were purified from the ear tissues of three species of rodents captured from seven localities of Taiwan. The immunological characteristics of these Taiwan isolates were compared with those of other genospecies of Lyme disease spirochetes by analyzing the protein profiles and reactivities with B. burgdorferi-specific monoclonal antibodies (MAbs). The genospecies of these Taiwan isolates were also identified by the similarities in their plasmid profiles and differential reactivities with genospecies-specific PCR primers. Although two distinct protein profiles were observed among the seven Taiwan isolates, the MAb reactivities against the outer surface proteins of B. burgdorferi of all of these isolates were consistent with those of B. burgdorferi sensu lato. The similarities of the plasmid profiles also confirmed the identities of these Taiwan isolates. PCR analysis indicated that all of these Taiwan isolates were genetically related to the genospecies B. burgdorferi sensu stricto. These results demonstrate the first identification of Lyme disease spirochetes in Taiwan and also highlight the increasing demand for defining the reservoirs and vector ticks of B. burgdorferi. A serosurvey for Lyme disease infection in the human population of Taiwan may also be required.     

Temporal correlations between tick abundance and prevalence of ticks infected with Borrelia burgdorferi and increasing incidence of Lyme disease

The abundance of host-seeking Ixodes scapularis nymphs, the principal vector for the Lyme disease agent, Borrelia burgdorferi, in Old Lyme, Lyme, and East Haddam, Connecticut, was compared with the incidence of reported human Lyme disease in the 12-town area around the Connecticut River and the State of Connecticut for the period 1989 to 1996. Ticks were sampled from lawns and woodlands by dragging flannel over the vegetation and examined for the presence of B. burgdorferi by indirect fluorescent antibody staining. The infection rate of the nymphal ticks by B. burgdorferi during the 9-year period was 14.3% (of 3,866), ranging from 8.6% (1993) to 24.4% (1996). The incidence of Lyme disease was positively correlated with tick abundance in the 12 town area (r = 0.828) and the State of Connecticut (r = 0.741). An entomological risk index based upon the number of I. scapularis ticks infected by B. burgdorferi was highest in 1992, 1994, and 1996 and was highly correlated with the incidence of Lyme disease in Connecticut (r = 0.944). The number of Lyme disease cases has been influenced, in part, by annual changes in population densities of I. scapularis and, presumably, a corresponding change in the risk of contact with infected ticks. Based upon tick activity and spirochetal infection rates, epidemiologically based Lyme disease case reports on a regional scale appear to reflect real trends in disease.     

Plasminogen is required for efficient dissemination of B. burgdorferi in ticks and for enhancement of spirochetemia in mice

The role of the host plasminogen activation system in transmission of and invasion by Borrelia burgdorferi, the tick-borne spirochetal agent of Lyme disease, was investigated using plasminogen (Plg)-knockout mice. PLG was not detected in spirochetes from unfed ticks, but binding occurred as ticks fed on the host's blood. Plasminogen activators were derived from the host blood meal. PLG was required for efficient dissemination of B. burgdorferi within the tick and for enhancement of spirochetemia in mice but was not critical for transmission and infection. These results provide evidence for a bacterium using a vertebrate protease to disseminate in an invertebrate vector and underscores the interplay among vector, pathogen, and host in promoting the life cycle and disease.     

Neuropsychiatric manifestations of Lyme disease

Lyme disease is a multisystem illness that may affect the central nervous system and subsequently produce mild to severe psychiatric disorders. Physicians who treat patient with Lyme disease need to be aware of its neuropsychiatric symptoms, which may emerge months to years after the initial infection. Prompt diagnosis and effective treatment are needed to avoid the debilitating and possibly irreversible mental illness associated with the neurologic involvement of this spirochetal infection. The author reviews the neuropsychiatric manifestations of Lyme disease and provides diagnostic and therapeutic approaches for the management of the central nervous system disease that may cause them.     

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.     

A simple technical modification for abdominal cardiac transplantation in rats

We report herein the first laboratory-diagnosed case of Lyme disease in a human in Taiwan. A 45-year-old Taiwanese man living in Taipei, in northern Taiwan, had an expanding skin lesion (measuring 23 by 15 cm) on his abdomen for 2 to 3 weeks and recurrent attacks of pain and swelling of the knee joint. Serologic tests indicated a significantly elevated titer of antibody to Borrelia burgdorferi. After appropriate antibiotic treatment for 3 weeks, the skin lesion was cured and the joint swelling was improved. Although several strains of Borrelia spirochetes had been isolated from rodents (Rattus losea) in Taiwan, the tick vector responsible for the transmission remains to be identified.     

Surface exposure and species specificity of an immunoreactive domain of a 66-kilodalton outer membrane protein (P66) of the Borrelia spp. that cause Lyme disease

A chromosomally encoded 66-kDa protein (P66) of Borrelia spp. that cause Lyme disease has previously been shown to be associated with the spirochetal outer membrane. A topological model of P66 predicts a surface-exposed fragment which links the N- and C-terminal intramembranous domains of the protein (J. Bunikis, L. Noppa, and S. Bergstr?m, FEMS Microbiol. Lett. 131:139-145, 1995). In the present study, an immunogenic determinant of P66 was identified by a comparison of the immunoreactivities of different fragments of P66 generated either by proteolytic treatment of intact spirochetes or as recombinant proteins expressed in Escherichia coli. The immune response to P66 during natural infection was found to be directed against the predicted surface domain which comprises amino acids at positions 454 through 491. A sequence comparison revealed considerable polymorphism of the surface domains of P66 proteins of different Lyme disease-causing Borrelia species. Five sequence patterns of this domain were observed in the B. garinii strains studied. In contrast, sequences of the relevant part of P66 of the B. afzelii and B. burgdorferi sensu stricto isolates studied were identical within the respective species. In immunoblotting, 5 of 17 (29.4%) sera from North American patients with early disseminated or persistent Lyme disease reacted against P66 of B. burgdorferi sensu stricto B31. These sera, however, failed to recognize P66 of B. afzelii and B. garinii, as well as an analog of P66 in the relapsing fever agent, B. hermsii. In conclusion, the topological model of P66 is supported by the demonstration of an apparent surface localization of an immunoreactive domain of this protein. Furthermore, analogous to the plasmid-encoded borrelial outer surface proteins, the predicted surface-exposed portion of chromosomally encoded P66 appears to be antigenically heterogenous.     

Physician-diagnosed erythema migrans and erythema migrans-like rashes following Lone Star tick bites

OBJECTIVE: To differentiate cases of physician-diagnosed erythema migrans and erythema migrans-like rashes associated with Lone Star tick (Amblyomma americanum) bites. DESIGN: Retrospective case series. SETTING: Private primary care clinic in rural Missouri. PATIENTS: Seventeen patients with physician-diagnosed erythema migrans following a definite Lone Star tick bite at the rash site. INTERVENTIONS: A biopsy was performed on all rash sites. All patients were treated with oral antibiotics. MAIN OUTCOME MEASURES: Rash appearance, size, body location, multiple lesions, incubation times, associated symptoms, seasonal occurrence, histopathological features, tick stage and sex, patient age and sex, treatment response, growth in BSK II culture media, and serologic evaluation. RESULTS: Rashes associated with Lone Star ticks were similar to erythema migrans vectored by other Ixodes ticks. Differences were noted in Lyme disease serology results, especially flagellin-based enzyme immunoassays, and failure to yield spirochetes in BSK II cultures. Lyme serology results were often negative, but were also frequently inconsistent with results of controls without Lyme disease. CONCLUSIONS: Lone Star ticks are associated with rashes that are similar, if not identical, to erythema migrans associated with borrelial infection. The recent isolation and cultivation of Borrelia burgdorferi from ticks (including 1 Lone Star tick) from the farm of a patient included in this report has raised the possibility that Lone Star ticks are "bridge vectors" for human borrelial infection. Although further investigation is needed, these rashes may be secondary to spirochetal infection.     

Macrophages exposed to Borrelia burgdorferi induce Lyme arthritis in hamsters

The mechanism(s) by which Lyme arthritis is induced has not been elucidated. In this study, we showed that macrophages have a direct, effector role in the pathogenesis of Lyme arthritis. Severe destructive arthritis was induced in recipients of macrophages obtained from Borrelia burgdorferi-vaccinated and nonvaccinated hamsters exposed to Formalin-inactivated B. burgdorferi in vitro and then challenged with the Lyme spirochete. Swelling of the hind paws was detected within 8 h of infection, increased rapidly, and peaked at 21 h. This initial swelling decreased, and by day 4 only slight swelling was detected. Severe swelling of the hind paws was detected 8 days after infection and increased rapidly, with peak swelling occurring on day 11. Histopathologic examination affirmed that macrophages exposed to Formalin-inactivated spirochetes induced a severe destructive Lyme arthritis. The onset and severity of the severe destructive arthritis were dependent on the number of macrophages transferred. By contrast, macrophages not exposed to Formalin-inactivated B. burgdorferi failed to induce severe destructive arthritis in recipients after challenge with B. burgdorferi. Similarly, severe destructive arthritis was not detected in recipients of macrophages injected with spirochetal growth medium. Our results also showed that transferred macrophages could not protect hamsters from infection with B. burgdorferi, as spirochetes were readily recovered from their tissues when cultured. These findings demonstrate that macrophages exposed to B. burgdorferi are directly involved in the induction of Lyme arthritis.     

Psychiatric manifestations of Lyme borreliosis

BACKGROUND: Lyme borreliosis (Lyme disease), a tick-borne spirochetal illness, has later manifestations that may include arthritic, neurologic, ophthalmologic, and cardiac symptoms. Recent reports suggest psychiatric symptoms may also be part of the clinical picture. METHOD: Using a structured interview (SCID), we interviewed three patients who had developed a psychiatric disorder for the first time after infection with Borrelia burgdorferi. RESULTS: During Lyme borreliosis, one patient had major depression and panic disorder, one patient had an organic mood syndrome with both depression and mania, and the third patient had panic disorder. These disorders remitted after adequate antibiotic treatment. CONCLUSION: While depression has been previously linked to neuroborreliosis, this is the first report to link panic disorder and mania with borrelial infection. Because of the rapid rise of Lyme borreliosis nationwide and the need for antibiotic treatment to prevent severe neurologic damage, mental health professionals need to be aware of its possible psychiatric presentations.     

Mycobacterium tuberculosis reactive T cell clones from naturally converted PPD-positive healthy subjects: recognition of the M. tuberculosis 16-kDa antigen

The methods of spatial statistics were applied to assess the geographical pattern of risk of Lyme borreliosis in Central Bohemia, the Czech Republic, based on retrospective data on disease contractions. The statistical risk was then compared at 15 selected localities with the infection challenge presented by ticks and insects carrying borreliae. Over 5,000 Ixodes ricinus (L.) ticks and 390 haematophagous dipterans were screened by direct immunofluorescence method, and the spatial and seasonal variance of infection rates were studied. Infected ticks were found at each locality throughout the warm season; in nymphs, sample infection rates ranged from 4.9% to 23.1% with a mean of 14.5% in spring, from 7.7% to 28.7% with a mean of 16.1% in summer, and from 7% to 20.6% with a mean of 13.6% in autumn. The statistical risk was found to correlate well with an average nymphal infection challenge, i.e. I. ricinus nymphal abundance x infection rate, at a given locality. Statistically significant cumulation of insect-history recalling patients into several, generally wetland, areas was ascertained; borreliae were revealed in 0.5% of the dipterans examined.     

Molecular typing of Borrelia burgdorferi from Lyme disease patients by PCR-restriction fragment length polymorphism analysis

Ninety-three Borrelia burgdorferi isolates obtained from erythema migrans lesions or blood of Lyme disease patients in Westchester County, N.Y., between 1991 and 1994 were characterized by PCR-restriction fragment length polymorphism (PCR-RFLP) analysis of the 16S-23S rRNA gene spacer. All isolates could be classified into three distinct RFLP types. Among the 82 skin biopsy isolates studied, 21 (25.6%) were type 1, 37 (45.1%) were type 2, and 21 (25.6%) were type 3. Three (3.7%) cultures contained a mixture of two isolates with distinct RFLP types. The 11 isolates cultured from blood showed a similar predominance of RFLP type 2 (6 of 11; 54.5%) relative to types 1 (2 of 11; 18.2%) and 3 (3 of 11; 27.3%). For one patient both skin and blood isolates were cultured, and RFLP analysis revealed that these isolates differed from one another. This study demonstrates that there is genotypic heterogeneity in B. burgdorferi strains infecting Lyme disease patients, and this typing approach may allow differentiation of isolates with various degrees of pathogenic potential.     

Identification of quantitative trait loci governing arthritis severity and humoral responses in the murine model of Lyme disease

A spectrum of disease severity has been observed in patients with Lyme disease, with approximately 60% of untreated individuals developing arthritis. The murine model of Lyme disease has provided strong evidence that the genetic composition of the host influences the severity of arthritis following infection with Borrelia burgdorferi: infected C3H mice develop severe arthritis while infected C57BL/6N mice develop mild arthritis. Regions of the mouse genome controlling arthritis severity and humoral responses during B. burgdorferi infection were identified in the F2 intercross generation of C3H/HeNCr and C57BL/6NCr mice. Rear ankle swelling measurements identified quantitative trait loci (QTL) on chromosomes 4 and 5, while histopathological scoring identified QTL on a unique region of chromosome 5 and on chromosome 11. The identification of QTL unique for ankle swelling or histopathological severity suggests that processes under distinct genetic control are responsible for these two manifestations of Lyme arthritis. Additional QTL that control the levels of circulating Igs induced by B. burgdorferi infection were identified on chromosomes 6, 9, 11, 12, and 17. Interestingly, the magnitude of the humoral response was not correlated with the severity of arthritis in infected F2 mice. This work defines several genetic loci that regulate either the severity of arthritis or the magnitude of humoral responses to B. burgdorferi infection in mice, with implications toward understanding the host-pathogen interactions involved in disease development.     

The risk of acquiring Lyme disease or babesiosis from a blood transfusion

To determine the risk of acquiring Lyme disease or babesiosis from blood transfusion, serum was collected before and 6 weeks after patients received multiple transfusions during cardiothoracic surgery and antibodies to Borrelia burgdorferi and Babesia microti were measured. Of 155 subjects, 149 received 601 total units of packed red blood cells (PRBC) and 48 received 371 total units of platelets. No patient developed clinical or serologic evidence of Lyme disease; 1 (who received 5 units of PRBC) developed clinical and serologic evidence of babesiosis. The risk of acquiring Lyme disease from a transfused unit of PRBC was 0 (95% confidence interval [CI], 0-0.5%) and from a transfused unit of platelets was 0 (95% CI, 0-0.8%); the same risks for babesiosis were 0.17% (95% CI, 0.004%-0.9%) and 0 (95% CI, 0-0.8%), respectively. The risk of acquiring either Lyme disease or babesiosis from a blood transfusion in Connecticut is very low.     

FlaA, a putative flagellar outer sheath protein, is not an immunodominant antigen associated with Lyme disease

FlaA was recently found to be associated with flagellar filaments of Borrelia burgdorferi. We tested whether antibodies to this protein are a good indicator of infection, as antibodies to FlaA proteins in other spirochetal infections show an increase in titer. Although overproduction of intact FlaA was highly toxic to Escherichia coli, truncated proteins which lacked the N-terminal signal sequence could be successfully overexpressed. Immunoblotting with sera from mammalian hosts infected with B. burgdorferi indicated that FlaA is not an immunodominant antigen in Lyme disease. However, sera from two patients reacted with both recombinant and native FlaA protein, suggesting that B. burgdorferi FlaA was antigenic and expressed in vivo.     

Nucleotide sequence and phylogenetic analysis of Newcastle disease virus isolates from recent outbreaks in Taiwan

Portions of the hemagglutinin neuraminidase (HN) gene of Newcastle disease virus (NDV) isolates from two recent outbreaks were sequenced to investigate epidemiology of this disease in Taiwan. These NDV isolates were all viscerotropic velogenic according to the clinical lesions produced in chickens. Sequence data were obtained from 14 NDV isolates (12 from 1995 and 2 from 1984). All isolates differed in their nucleotide sequences (from 0.3 to 15.3%), and represented potentially different strains of NDV. Phylogenetic analysis revealed that these isolates are closely related to viruses isolated from Japan and Malaysia. Some viruses isolated in 1995 appeared to evolve from viruses isolated in 1984. The results suggest that the 1995 outbreak of Newcastle disease (ND) in Taiwan may have been caused by multiple strains of velogenic NDV that have cocirculated in Taiwan for some time. Moreover, NDV isolates from racing pigeons were very similar to isolates from chickens in the same period, suggesting that both domestic and free-living birds were involved in the spread of ND in Taiwan.     

Borrelia burgdorferi and interleukin-1 promote the transendothelial migration of monocytes in vitro by different mechanisms

A prominent feature of Lyme disease is the perivascular accumulation of mononuclear leukocytes. Incubation of human umbilical vein endothelial cells (HUVEC) cultured on amniotic tissue with either interleukin-1 (IL-1) or Borrelia burgdorferi, the spirochetal agent of Lyme disease, increased the rate at which human monocytes migrated across the endothelial monolayers. Very late antigen 4 (VLA-4) and CD11/CD18 integrins mediated migration of monocytes across HUVEC exposed to either B. burgdorferi or IL-1 in similar manners. Neutralizing antibodies to the chemokine monocyte chemoattractant protein 1 (MCP-1) inhibited the migration of monocytes across unstimulated, IL-1-treated, or B. burgdorferi-stimulated HUVEC by 91% +/- 3%, 65% +/- 2%, or 25% +/- 22%, respectively. Stimulation of HUVEC with B. burgdorferi also promoted a 6-fold +/- 2-fold increase in the migration of human CD4(+) T lymphocytes. Although MCP-1 played only a limited role in the migration of monocytes across B. burgdorferi-treated HUVEC, migration of CD4(+) T lymphocytes across HUVEC exposed to spirochetes was highly dependent on this chemokine. The anti-inflammatory cytokine IL-10 reduced both migration of monocytes and endothelial production of MCP-1 in response to B. burgdorferi by approximately 50%, yet IL-10 inhibited neither migration nor secretion of MCP-1 when HUVEC were stimulated with IL-1. Our results suggest that activation of endothelium by B. burgdorferi may contribute to formation of the chronic inflammatory infiltrates associated with Lyme disease. The transendothelial migration of monocytes that is induced by B. burgdorferi is significantly less dependent on MCP-1 than is migration induced by IL-1. Selective inhibition by IL-10 further indicates that B. burgdorferi and IL-1 employ distinct mechanisms to activate endothelial cells.     

An avian reservoir (Turdus merula) of the Lyme borreliosis spirochetes

The reservoir competence of passerine birds for the Lyme borreliosis spirochetes was studied in an enzootic focus in Switzerland. Skin aspirates and skin biopsies were used to isolate Borrelia spirochetes from Turdus species. B. burgdorferi sensu lato was isolated and/or PCR-detected in BSK medium containing skin biopsy or skin aspirate from 5 blackbirds (T. merula) and one song thrush (T. philomelos). Seven isolates were obtained from 3 different blackbirds. Either B. garinii or Borrelia from the genomic group VS116 was found in bird skin samples. Mixed infection occurred in 2 cases. Tick xenodiagnosis was used to determine whether blackbirds transmitted Borrelia to ticks. Five xenodiagnoses were performed on 3 different blackbirds. Borrelia DNA was detected in BSK medium inoculated with xenodiagnostic ticks from all the passerines tested. Isolates cultured from xenodiagnostic ticks were obtained from 2 blackbirds. Isolates belonged to group VS116 (n = 10) and to B. garinii (n = 1). Our study has shown that Turdus sp. are infected by B. garinii and by Borrelia from group VS116 and that blackbirds are implicated as reservoirs for these 2 genomic groups of Borrelia, as they transmit living borreliae to ticks. An association seems to exist between birds and Borrelia VS116, and to a lesser extent, B. garinii, similar to the association existing between small rodents and B. afzelii. Our observations emphasize the fact that different enzootic cycles maintain Lyme borreliosis spirochetes in nature.     

Influence of autologous serum on in vitro reactivity of peripheral lymphocytes of patients with breast cancer

Data pertaining to 149 strains belonging to genus Yersinia are summarized in this paper. Yersinia enterocolitica (Y.e.) was isolated from the faeces of 31 of 305 small rodents and from 5 of 31 shrews (Soricidae) trapped at five localities in Norway and one locality in Denmark. Isolations were obtained from 9 of 29 water samples collected within the trapping areas. Three of 25 red foxes (Vulpes vulpes) from one locality in Norway harbored Y.e. in their faeces. Y.e. serotype 16 was isolated from one zoologist suffering from diarrhoea. A total of 85 strains from small rodents, shrews, water and foxes showed biochemical properties intermediate to Y.e. and Y. pseudotuberculosis. Another three strains were classified as Y. pseudotuberculosis on biochemical basis. They were obtained from small rodents in Denmark. Serological examination of 59 small rodents naturally infected with Y.e. and related microbes, revealed two cases of low antibody titres (80) against homologous isolates. Pathological examination of 44 of these animals gave a negative result. Strains antigenically related to the same serotypes were frequently isolated from both terrestrial ecosystems and adjacent freshwater. Strains related to serotype 6 dominated both in red fox and in their small rodent prey. No evidence was found for a dynamical significance of Y.e. in populations of small rodents. The results indicate that Y.e. and Yersinia like microbes have a broad occurrence in terrestial ecosystems.     

Genetic diversity of Borrelia burgdorferi in lyme disease patients as determined by culture versus direct PCR with clinical specimens

Two hundred seventeen isolates of Borrelia burgdorferi originally cultured from skin biopsy samples or blood of early Lyme disease patients were genetically characterized by PCR-restriction fragment length polymorphism (RFLP) typing of the 16S-23S ribosomal DNA intergenic spacer. Three major RFLP types were observed. Of the cultured isolates, 63 of 217 (29.0%) were type 1, 85 of 217 (39.2%) were type 2, and 58 of 217 (26.7%) were type 3; mixtures of two RFLP types were obtained in 6.0% (13 of 217) of the cultures. Comparison of typing of B. burgdorferi performed directly on 51 patient skin specimens with typing of cultures originally isolated from the same tissue revealed that a much larger proportion of direct tissue samples had mixtures of RFLP types (43.1% by direct typing versus 5.9% by culture [P < 0.001). In addition, identical RFLP types were observed in only 35.5% (11 of 31) of the paired samples. RFLP type 3 organisms were recovered from blood at a significantly lower rate than were either type 1 or type 2 strains. These studies demonstrate that the genetic diversity of B. burgdorferi patient isolates as determined by cultivation differs from that assessed by PCR performed directly on patient tissue.