Lyme disease in children and adolescents

148 children and adolescents with Lyme borreliosis and tick bite or suspection on tick bite were examined. The examined patients were aged from 14 months to 24 years and divided into four age groups. Skin lesions were discovered in 25 percent of patients with tick bite. Erythema migrans occurred in 91 percent, Lymphocytoma in 3 percent and sclerodermatous lesions (Lichen sclerosus et atrophicus and Morphea) in 6 percent of patients with Lyme disease. Serologic tests on the presence of antibodies to Borreliae burgdorferi were performed in 96 percent of cases with tick bite. Antibody titer 1:80 or higher in 8 percent of patients with tick bite, was discovered. We found positive serologic test results in 5 (29 percent) of 29 persons with Erythema migrans, in 4 (4 percent) of 110 patients with tick bite (without skin lesions), as well as, in 1 patient with Lymphocytoma. Antibiotic therapy was applied in all cases with Erythema migrans, in person with Lymphocytoma, as well as, in patients with asymptomatic infections (patients without skin lesion recalling a tick bite and with antibodies against Borrelia burgdorferi). A general sensitivity, to infection with Borrelia burgdorferi, is stressed, a fact based on appearance by Lyme borreliosis in all age groups even in the newborn children.     

Rheumatic manifestations in Lyme borreliosis: personal experience in patients with oligoarthritis of "unknown" origin

Lyme borreliosis is an infectious illness caused by the spirochete Borrelia burgdorferi and is transmitted by tick vectors. A prospective study was performed from January 1990, to investigate whether Lyme arthritis might have been undetected among patients with (unclassified arthritis) oligoarthritis of "unknown" aetiology. 210 patients were tested for antibodies to Borrelia burgdorferi: 82 patients with oligoarthritis of "unknown" aetiology; 52 patients with Reiter's syndrome; 20 patients with seronegative, B-27 positive oligoarthritis and 56 controls. Serological testing for Borrelia burgdorferi was performed by indirect immunofluorescence assay. The occurrence of positive antibodies (1:80) in 11 (13.4%) patients with arthritis of "unknown" aetiology was significantly different from the combined control group (1.6%) (p < 0.05). Four out of 11 patients remembered a tick bite, two out of 11 patients developed erythema migrans after 3 to 10 days. Six weeks later 2 patients developed oligoarthritis and one patient after a month. In the remaining 8 patients arthritis was the first sign of the disease. Knees were most commonly affected (90%). Radiographic abnormalities (osteoporosis, soft tissue swelling) were noted in 3 patients. The synovial fluid findings were typical for inflammatory arthritides in 6 patients. The diagnosis of Lyme borreliosis was made according to following data: origin from an area endemic for Lyme borreliosis, tick bite, erythema migrans, significant levels of the antibodies to the Borrelia burgdorferi and oligoarthritis. It can be concluded that arthritis may be the main manifestation of Borrelia burgdorferi infection.     

Borrelia burgdorferi erp proteins are immunogenic in mammals infected by tick bite, and their synthesis is inducible in cultured bacteria

Borrelia burgdorferi, the causative agent of Lyme disease, can contain multiple genes encoding different members of the Erp lipoprotein family. Some arthropod-borne bacteria increase the synthesis of proteins required for transmission or mammalian infection when cultures are shifted from cool, ambient air temperature to a warmer, blood temperature. We found that all of the erp genes known to be encoded by infectious isolate B31 were differentially expressed in culture after a change in temperature, with greater amounts of message being produced by bacteria shifted from 23 to 35 degrees C than in those maintained at 23 degrees C. Mice infected with B31 by tick bite produced antibodies that recognized each of the Erp proteins within 4 weeks of infection, suggesting that the Erp proteins are produced by the bacteria during the early stages of mammalian infection and may play roles in transmission from ticks to mammals. Several of the B31 Erp proteins were also recognized by antibodies from patients with Lyme disease and may prove to be useful antigens for diagnostic testing or as components of a protective vaccine.     

The Borrelia burgdorferi 37-kilodalton immunoblot band (P37) used in serodiagnosis of early lyme disease is the flaA gene product

The 37-kDa protein (P37) of Borrelia burgdorferi is an antigen that elicits an early immunoglobulin M (IgM) antibody response in Lyme disease patients. The P37 gene was cloned from a B. burgdorferi genomic library by screening with antibody from a Lyme disease patient who had developed a prominent humoral response to the P37 antigen. DNA sequence analysis of this clone revealed the identity of P37 to be FlaA, an outer sheath protein of the periplasmic flagella. Recombinant P37 expression was accomplished in Escherichia coli by using a gene construct with the leader peptide deleted and fused to a 38-kDa E. coli protein. The recombinant antigen was reactive in IgM immunoblots using serum samples from patients clinically diagnosed with early Lyme disease that had been scored positive for B. burgdorferi anti-P37 reactivity. Lyme disease patient samples serologically negative for the B. burgdorferi P37 protein did not react with the recombinant. Recombinant P37 may be a useful component of a set of defined antigens for the serodiagnosis of early Lyme disease. This protein can be utilized as a marker in diagnostic immunoblots, aiding in the standardization of the present generation of IgM serologic tests.     

A method and device for measuring force transfers between the deep flexors in the musician''s hand

The efficacy of passive immunisation against tick-transmitted Lyme disease spirochaetal infection was determined in relation to the duration of previous feeding of infected vector ticks. Thus, mice challenged with spirochaete-infected unfed or partially fed nymphal ticks were passively immunised with monoclonal and polyclonal antibodies against the Lyme disease spirochaete (Borrelia burgdorferi) at various intervals after tick attachment. Spirochaetal infection in challenged mice and engorged ticks was verified by xenodiagnosis and indirect immunofluorescent antibody assay, respectively. Although tick-transmitted spirochaetal infection could be aborted by anti-OspA antibodies and hyperimmune antiserum, nearly all immunised mice challenged with infected ticks that had previous 36-h attachment became infected. More than 72% of the nymphal ticks used in this challenge retained their B. burgdorferi infection after engorgement on mice immunised with anti-spirochaete antibodies, and their subsequent infectivity to mice remained effective. It is concluded that a higher efficiency of transmission by partially fed infected nymphs and a lower efficacy of passive immunisation against infection result from an effect of previous feeding of infected ticks that activates antigenic change and enables the spirochaetes to circumvent OspA-based humoral immunity.     

Diagnostic tools in Lyme borreliosis: clinical history compared with serology

The occurrence of a history of clinical Lyme borreliosis and the prevalence of positive antibodies to Borrelia burgdorferi were studied in 431 Dutch hunters. The majority of the hunters (336 or 78%) did not report any complaints and had no positive IgG antibodies to B. burgdorferi. Sixty-five hunters (15.1%) had no clinical manifestations but did not have positive antibodies to B. burgdorferi. Only 1.9% of the population studied had had past symptoms of definite or probable Lyme borreliosis. Likelihood ratios were high (21.3) for the recognition of erythema migrans, but much lower for tick bites (3.6) or positive IgG Lyme serology (3.5). Clinical history turned out to be a more powerful diagnostic tool than Lyme serology.     

A case of Lyme disease with parotitis

A-59-year-old Japanese woman presented a cellulitis-like erythematous skin rash, low-grade fever, and general fatigue, accompanied by a firm swelling of the right parotid gland. She had a history of tick bite on the right lateral neck 2 weeks before. Serum anti-Borrelia burgdorferi antibody was positive by Western blot analysis, and B. burgdorferi was isolated from the skin lesion. Serum amylase level was elevated with predominant salivary gland isozyme; the level returned to normal within 3 weeks following penicillin and tetracycline treatment. Parotitis might be included among the rare complications of Lyme disease affecting the head and neck region.     

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.     

Novel Borrelia burgdorferi isolates from Ixodes scapularis and Ixodes dentatus ticks feeding on humans

Seven cultures of Borrelia burgdorferi differing from strains B31 and ZS7 were identified from among 99 isolates from Ixodes scapularis ticks and from white-footed mice (Peromyscus leucopus) and 1 isolate from an Ixodes dentatus tick. Five of the six novel isolates from I. scapularis and the isolate from I. dentatus were from ticks feeding on humans. The six isolates from I. scapularis lacked OspA and OspB, four possessed an OspD band, and two reacted with an anti-OspC monoclonal antibody. Restriction fragment length polymorphisms of HindIII-digested DNAs from six OspA-negative isolates did not hybridize with radiolabeled ospA or LA88 DNA, and only isolate 46047 hybridized with the pG gene. Fragments similar to those recorded for the standard B. burgdorferi sensu stricto strains B31 and ZS7 were obtained with the fla and the HSP70 genes. Pulsed-field gel electrophoresis patterns of DNA digested with MluI included the specific B. burgdorferi sensu stricto band at 135 kbp for the five OspA-negative isolates from I. scapularis ticks. The six novel isolates apparently lack the 55-kbp plasmid encoding OspA. The pG-containing plasmid may be missing from all but isolate 46047. The isolate from the I. dentatus tick was similar to previous isolates from I. dentatus ticks feeding on rabbits. None of the isolates could be recovered from inoculated C3H/HeNCrlBR or white-footed mice. All isolates reacted with sera from humans with early or late Lyme disease. Our studies demonstrate that these borreliae occur in ticks feeding on humans, and therefore, at least some humans in the northeastern United States are likely being exposed to borreliae other than the classic B31-type strains that have thus far been isolated from humans.     

A population-based seroepidemiologic study of human granulocytic ehrlichiosis and Lyme borreliosis on the west coast of Sweden

Ehrlichioses are emerging infections in the United States. Human granulocytic ehrlichiosis (HGE) and Lyme borreliosis (LB) are acquired after Ixodes ricinus-complex tick bites. An ongoing seroepidemiologic study of the 185 of the 356 permanent residents of the Koster Islands in Sweden was expanded to include ehrlichioses. Ehrlichial antibodies were measured by IFA using Ehrlichia equi and Ehrlichia chaffeensis. Borrelia burgdorferi IgG ELISA-seropositive subjects were confirmed by Western blot. E. equi and E. chaffeensis antibodies (titer > or = 80) were found in 21 (11.4%) and 2 (1.1%) of 185 samples, respectively. Antibodies to B. burgdorferi were found in 25 (13.5%) of 185. Six persons were seropositive for both HGE and LB. Among data from questionnaires, clinical symptoms, antibiotic treatments, or tick bites were not more frequent in E. equi- or B. burgdorferi-seropositive than -seronegative persons. The seroprevalence of HGE was similar to that of Lyme borreliosis. Prospective studies of European HGE are needed.     

Humoral immunity to Borrelia burgdorferi N40 decorin binding proteins during infection of laboratory mice

A Borrelia burgdorferi N40 genomic expression library was screened with serum from actively infected mice to identify gene products that elicit protective immunity. A clone that contained a putative bicistronic operon containing two genes that encoded 20- and 22-kDa lipoproteins was identified and sequenced. These genes showed homology with the genes encoding decorin binding proteins DbpB and DbpA, respectively, of B. burgdorferi 297 and B31. N40-dbpA DNA hybridized with B. burgdorferi N40 DNA on a single 48-kb linear plasmid. Homologous genes could be amplified under various degrees of stringency by PCR or hybridized by Southern blotting from B. burgdorferi sensu stricto N40 and B31, and from B. burgdorferi sensu lato PBi and 25015, but not PKo. Recombinant N40-DbpB and N40-DbpA were reactive with antibody in serum from infected mice, and serum was more reactive against N40-DbpA than against B. burgdorferi N40 recombinant P39, OspC, or OspA. Sera from mice infected with B. burgdorferi sensu lato strains PKo and PBi were weakly reactive against N40-DbpB and N40-DbpA, and sera from mice infected with 25015 were moderately reactive, compared to sera from mice infected with B. burgdorferi N40. Hyperimmunization of mice with N40-DbpA, but not N40-DbpB, induced protective immunity against syringe challenge with cultured B. burgdorferi N40. DbpA may therefore be one of the antigens responsible for eliciting protective antibody known to exist in serum from infected mice. DNA amplification and serology suggest that DbpB and DbpA are likely to have homologs throughout the B. burgdorferi sensu lato family, but they are likely to be heterogeneous.     

Characterization of Borrelia burgdorferi isolated from different organs of Ixodes ricinus ticks collected in nature

Borrelia burgdorferi was isolated from 22 out of 133 adult Ixodes ricinus ticks collected from vegetation at two sites in Switzerland. From 17 ticks, spirochetes could be isolated from more than one organ. When the different isolates obtained from one tick were compared by SDS-PAGE analysis, differences in the protein profiles were observed in 8 cases. The isolates were further compared by immunological methods using mono- and polyclonal antibodies. Differences were observed in the proteins of 31-35 kDa and 18-25 kDa. Genetic divergence among isolates was evaluated by use of a B. burgdorferi specific gene probe for ospA. Correlation could be observed between immunological differences in OspA defined by monoclonal antibody LA31 and genetic variation of ospA as judged by restriction fragment length polymorphism (RFLP). Our findings indicate that systemic infection in unfed I. ricinus adults, as reflected by isolation of B. burgdorferi from multiple organs of one tick, is more frequent (8/22, 36%) than previously described (5%). Moreover, the presence of different B. burgdorferi phenotypes/genotypes in one tick is described for the first time. The findings may have bearings (i) on the time of tick attachment required for spirochete transmission since borreliae are already present in the salivary glands of systemically infected ticks at the beginning of the blood meal and (ii) perhaps also on the diversity of B. burgdorferi phenotypes inoculated by these ticks.     

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.     

Distribution and molecular analysis of Lyme disease spirochetes, Borrelia burgdorferi, isolated from ticks throughout California

Previous studies describing the occurrence and molecular characteristics of Lyme disease spirochetes, Borrelia burgdorferi, from California have been restricted primarily to isolates obtained from the north coastal region of this large and ecologically diverse state. Our objective was to look for and examine B. burdorferi organisms isolated from Ixodes pacificus ticks collected from numerous regions spanning most parts of California where this tick is found. Thirty-one isolates of B. burgdorferi were examined from individual or pooled I. pacificus ticks collected from 25 counties throughout the state. One isolate was obtained from ticks collected at Wawona Campground in Yosemite National Park, documenting the occurrence of the Lyme disease spirochete in an area of intensive human recreational use. One isolate from an Ixodes neotomae tick from an additional county was also examined. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis, immunoblot analysis, agarose gel electrophoresis, Southern blot analysis, and the polymerase chain reaction were used to examine the molecular and genetic determinants of these uncloned, low-passage-number isolates. All of the isolates were identified as B. burgdorferi by their protein profiles and reactivities with monoclonal and polyclonal antibodies, and all the isolates were typed by the polymerase chain reaction as North American-type spirochetes (B. burgdorferi sensu stricto). Although products of the ospAB locus were identified in protein analyses in all of the isolates, several isolates contained deleted forms of this locus that would result in the expression of chimeric OspA-OspB proteins. The analysis of OspC demonstrated that this protein was widely conserved among the isolates but was also quite variable in its molecular mass and the amount of it that was expressed.     

Erythema migrans-like rash illness at a camp in North Carolina: a new tick-borne disease?

BACKGROUND: Borrelia burgdorferi, the causative agent of Lyme disease, has never been isolated from a patient thought to have acquired Lyme disease in any southeastern state. OBJECTIVE: To investigate 14 cases of an erythema migrans (EM)-like rash illness that occurred during 2 summers at an outdoor camp in central North Carolina in an effort to determine the etiologic, epidemiological, and clinical aspects of this illness. METHODS: Using active surveillance, we identified cases of clinically diagnosed EM in residents and staff of the camp. We collected clinical and demographic information; history of exposure to ticks; acute and convalescent serum antibodies to B. burgdorferi, Rickettsia rickettsii, and Ehrlichia chaffeensis; and cultures for spirochetes from biopsy specimens of skin lesions. Serum samples from a group of residents and staff who did not develop rashes were tested for the same antibodies. We speciated ticks removed from people and collected from vegetation. RESULTS: We identified 14 cases of EM-like rash illness during the 2 summers. Of the 14 case-patients, 10 had associated mild systemic symptoms and 1 had documented fever. All 14 case-patients had removed attached ticks, and 8 remembered having removed a tick from the site where the rash developed a median of 12 days earlier (range, 2-21 days). One tick removed from the site where a rash later developed was identified as Amblyomma americanum, the Lone Star tick; 97% of ticks collected from vegetation and 95% of ticks removed from people were A. americanum. No spirochetes were isolated from skin biopsy specimens. Paired serum samples from 13 case-patients did not show diagnostic antibody responses to B. burgdorferi or other tick-borne pathogens. CONCLUSIONS: This investigation suggests the existence of a new tick-associated rash illness. We suspect that the disease agent is carried by A. americanum ticks. In the southern United States, EM-like rash illness should no longer be considered definitive evidence of early Lyme disease.     

Protection of C3H/He mice from experimental Borrelia burgdorferi infection by immunization with a 110-kilodalton fusion protein

A 110-kDa Borrelia burgdorferi fusion protein, Escherichia coli expressing the fusion protein, transformed E. coli lacking the fusion protein insert, and lyophilized whole B. burgdorferi bacteria were compared for immunogenicity in C3H/He mice. Immunized mice were challenged with a variety of isolates from the United States or the European isolate P/Gau 3 weeks following the last inoculation. An average of 76.7% of the mice immunized with 25 micrograms of lyophilized whole B. burgdorferi cells were protected from infection, while 60% of the mice immunized with the 110-kDa fusion protein were protected. Whole E. coli bacteria expressing the fusion protein protected 57.7% of immunized mice against experimental challenge. Lower levels of protection occurred in mice challenged with the European isolate than in those challenged with isolates originating from the United States. These results demonstrate the potential of the 110-kDa fusion protein for use as a component of a subunit vaccine for prevention of Lyme borreliosis.     

DbpA, but not OspA, is expressed by Borrelia burgdorferi during spirochetemia and is a target for protective antibodies

DbpA is a target for antibodies that protect mice against infection by cultured Borrelia burgdorferi. Infected mice exhibit early and sustained humoral responses to DbpA and DbpB, suggesting that these proteins are expressed in vivo. Many antigens expressed in mammals by B. burgdorferi are repressed in vitro at lower growth temperatures, and we have now extended these observations to include DbpA and DbpB. To confirm that the protective antigen DbpA is expressed in vivo and to address the question of its accessibility to antibodies during infection, we examined B. burgdorferi in blood samples from mice following cutaneous inoculation. B. burgdorferi was visualized by dark-field microscopy in plasma samples from spirochetemic mice, and an indirect immunofluorescence assay showed that these spirochetes were DbpA positive and OspA negative. We developed an ex vivo borreliacidal assay to show that hyperimmune antiserum against DbpA, but not OspA, killed these plasma-derived spirochetes, demonstrating that DbpA is accessible to antibodies during this phase of infection. Blood transferred from spirochetemic donor mice readily established B. burgdorferi infection in naive recipient mice or mice hyperimmunized with OspA, while mice hyperimmunized with DbpA showed significant protection against challenge with host-adapted spirochetes. Antiserum from persistently infected mice had borreliacidal activity against both cultured and plasma-derived spirochetes, and adsorption of this serum with DbpA substantially depleted this killing activity. Our observations show that immunization with DbpA blocks B. burgdorferi dissemination from the site of cutaneous inoculation and suggest that DbpA antibodies may contribute to control of persistent infection.