No evidence to implicate Borrelia burgdorferi in the pathogenesis of dilated cardiomyopathy in the United Kingdom.

OBJECTIVE--To determine whether Borrelia burgdorferi is implicated in the pathogenesis of dilated cardiomyopathy in the United Kingdom. DESIGN--A controlled prospective study. Patients' notes were reviewed for evidence of Lyme disease and serum samples were tested by enzyme linked immunoadsorbent assay (ELISA) for antibodies to B burgdorferi. Samples with raised antibody concentrations were subsequently analysed by immunoblotting to determine their antibody binding specificity. SETTING--Tertiary referral centre. PATIENTS--97 consecutive patients with dilated cardiomyopathy diagnosed according to World Health Organisation criteria were studied. Serum samples were taken from two matched control groups. The first group (n = 38) was age, sex, and geographically matched. The second control group (n = 39) was environmentally matched and consisted of members of the patients' own households. MAIN OUTCOME MEASURES--Clinical evidence of Lyme disease. Presence of raised antibody concentrations to B burgdorferi. RESULTS--No patients had a previous illness compatible with Lyme disease. Analysis of the ELISA data showed eight of 97 patients with dilated cardiomyopathy (8.2%) and two of 77 controls (3.9%) had raised antibody concentrations. Immunoblot analysis, however, did not show binding patterns consistent with the presence of IgG specific for B burgdorferi in any of these samples. CONCLUSIONS--There was no clinical or serological evidence to implicate B burgdorferi in the pathogenesis of idiopathic dilated cardiomyopathy in the United Kingdom. In the absence of specific symptoms or likely exposure to B burgdorferi routine serological testing for Lyme disease in this group of patients is not recommended. Furthermore, raised antibodies to B burgdorferi are not diagnostic of active infection and ELISA results should be interpreted with caution unless specific B burgdorferi antibody bands have been found by immunoblot analysis.     

Antibody response to IR6, a conserved immunodominant region of the VlsE lipoprotein, wanes rapidly after antibiotic treatment of Borrelia burgdorferi infection in experimental animals and in humans

Invariable region (IR)(6), an immunodominant conserved region of VlsE, the antigenic variation protein of Borrelia burgdorferi, is currently used for the serologic diagnosis of Lyme disease in humans and canines. A longitudinal assessment of anti-IR(6) antibody levels in B. burgdorferi-infected rhesus monkeys revealed that this level diminished sharply after antibiotic treatment (within 25 weeks). In contrast, antibody levels to P39 and to whole-cell antigen extracts of B. burgdorferi either remained unchanged or diminished less. A longitudinal analysis in dogs yielded similar results. In humans, the anti-IR(6) antibody titer diminished by a factor of > or =4 in successfully treated patients and by a factor of <4 in treatment-resistant patients. This result suggests that the quantification of anti-IR(6) antibody titer as a function of time should be investigated further as a test to assess response to Lyme disease therapy or to determine whether a B. burgdorferi infection has been eliminated.     

Borrelia bissettii isolates induce pathology in a murine model of disease

The spirochete Borrelia burgdorferi is a tick-borne pathogen that causes Lyme disease. Although B. burgdorferi sensu lato is a diverse group of bacteria, only three genospecies, B. burgdorferi sensu stricto, Borrelia afzelii, and Borrelia garinii, are known to be pathogenic and commonly recognized to cause human disease. To assess the potential of another common genospecies, Borrelia bissettii, to induce disease, a mouse model was employed. Two Colorado isolates of B. bissettii (CO-Bb) induced lesions of the bladder, heart, and femorotibial joint 8 weeks after inoculation into mice. In contrast, two British Columbia (BC-Bb) isolates, could not be cultured or amplified by PCR from target organs, and did not induce lesions. Consistent with pathology and culture results, the antibody response in mice to BC-Bb was minimal compared to CO-Bb, indicating either transient localized infection or rapid immune clearance of BC-Bb. Although sequence analysis of the rrf (5S)-rrl (23S) intergenic spacer region indicated 99% homology between CO-Bb and BC-Bb, polyacrylamide gel electrophoresis (PAGE) analysis indicated five distinct protein differences between these low-passage isolates. These studies support the prospect that B. bissettii may indeed be the causative agent of Lyme borreliosis cases in Eastern Europe, associated with the atypical Borrelia strain 25015, and in other regions. To our knowledge, this is the first evidence that B. bissettii can induce pathology in a vertebrate host.     

Enzyme-linked immunosorbent assays for Lyme disease: reactivity of subunits of Borrelia burgdorferi

We prepared fractions of Borrelia burgdorferi, the etiologic agent of Lyme disease, from cultured spirochetes and used them as antigen in an enzyme-linked immunosorbent assay (ELISA) for IgG antibody. Polystyrene plates coated with an extract containing major proteins with apparent molecular masses of 34, 39, 59, and 68 kilodaltons had comparable sensitivity but greater specificity than plates coated with whole cells. Of the 33 serum specimens from individuals with Lyme disease that reacted with whole cells of B. burgdorferi in the class-specific ELISA, 30 (91%) remained positive when this extract was used. Cross-reactivity was minimal with antibody to treponemes. Use of subunit antigens may improve serological diagnosis of Lyme disease.     

Detection of multiple reactive protein species by immunoblotting after recombinant outer surface protein A lyme disease vaccination.

Laboratory confirmation of the diagnosis of Lyme disease is based on the detection of an immune response to Borrelia burgdorferi. The serodiagnosis of B. burgdorferi infection is complex and may be further confounded by the immune response to the recombinant outer surface protein A (OspA) Lyme disease vaccine. To describe how the serological response to the recombinant OspA Lyme disease vaccine affects testing for antibody to B. burgdorferi, 240 specimens from 80 study subjects were obtained at defined intervals after recombinant OspA Lyme disease vaccination. Samples were tested by indirect enzyme-linked immunosorbent assay (ELISA), antibody capture enzyme immunoassay (EIA), and Western blotting (WB). After recombinant OspA Lyme disease vaccination, ELISA for 98% of the study subjects revealed reactivity. WB with use of OspA-containing B. burgdorferi strains as sources of antigens demonstrated multiple bands. Results of testing with a US Food and Drug Administration-approved WB kit showed homogeneous reactivity in the molecular weight region >30 kDa. Testing with OspA-free strains completely eliminated all vaccine-associated reactivity by both antibody capture EIA and WB.     

以急性脑膜炎为首发症状的莱姆病：附一例报告

为加强对莱姆病的认识，报道１例以急性脑膜炎为首发症状、经血清学检测（ＥＬＩＳＡ及Ｗｅｓｔｅｒｎｂｌｏｔｉｎｇ两种方法）证实为神经莱姆病的诊治经过。表明北京地区有莱姆病存在。如发现淋巴细胞增多性脑膜炎而原因不明时，神经莱姆病应作为鉴别诊断之一，及时送检血清及脑脊液莱姆病的抗体检测将有助于诊断。     

Serodiagnosis of Lyme disease by kinetic enzyme-linked immunosorbent assay using recombinant VlsE1 or peptide antigens of Borrelia burgdorferi compared with 2-tiered testing using whole-cell lysates

In a study of US patients with Lyme disease, immunoglobulin (Ig) G and IgM antibody responses to recombinant Borrelia burgdorferi antigen VlsE1 (rVlsE1), IgG responses to a synthetic peptide homologous to a conserved internal sequence of VlsE (C6), and IgM responses to a synthetic peptide comprising the C-terminal 10 amino acid residues of a B. burgdorferi outer-surface protein C (pepC10) were evaluated by kinetic enzyme-linked immunoassay. At 99% specificity, the overall sensitivities for detecting IgG antibody to rVlsE1 or C6 in samples from patients with diverse manifestations of Lyme disease were equivalent to that of 2-tiered testing. When data were considered in parallel, 2 combinations (IgG responses to either rVlsE1 or C6 in parallel with IgM responses to pepC10) maintained high specificity (98%) and were significantly more sensitive than 2-tiered analysis in detecting antibodies to B. burgdorferi in patients with acute erythema migrans. In later stages of Lyme disease, the sensitivities of the in parallel tests and 2-tiered testing were high and statistically equivalent.     

Growth-inhibiting antibody responses of humans vaccinated with recombinant outer surface protein A or infected with Borrelia burgdorferi or both

Serial serum samples from a 2-year human trial of outer surface protein (Osp) A vaccine were analyzed by Borrelia burgdorferi growth-inhibition assay (GIA) and anti-OspA ELISA to assess the antibody responses of vaccine recipients and subjects with Lyme disease. Although 74% of OspA recipients had a reciprocal GIA titer >/=64 after 3 vaccinations, none of the placebo recipients, even those with Lyme disease, had a GIA titer this high. The correlation between GIA and ELISA titers after 3 doses of vaccine was.84; however, more vaccine recipients had an elevated ELISA titer paired with low GIA titer than had a low ELISA titer with a high GIA titer. OspA-vaccine recipients who acquired Lyme disease had significantly lower serum GIA and ELISA titers after 3 immunizations than did age- and sex-matched OspA recipients without Lyme disease. Thus, vaccinated subjects had antibodies to native antigen on viable cells, and antibody assays with this specificity may predict protection of vaccinees against infection.     

莱姆病螺旋体鞭毛平截性蛋白的表达及其诊断潜力研究

目的原核表达伯氏疏螺旋体鞭毛蛋白Flagellin A基因特异性区段,获得重组鞭毛蛋白平截性蛋白作为诊断抗原,建立间接ELISA方法用于动物莱姆病的诊断。方法 PCR扩增获取伯氏疏螺旋体鞭毛蛋白基因的同源性较低的第394-798bp区段,构建重组质粒pGEX-4T-1/tFlaA,构建好的表达质粒转化到大肠杆菌BL21（DE3）中进行表达,并纯化重组蛋白,用纯化的表达蛋白作为莱姆病诊断的抗原,用于ELISA检测实验感染小鼠莱姆病。结果成功构建莱姆病螺旋体鞭毛平截性蛋白的表达载体,重组蛋白在宿主菌内高效、稳定表达,重组平截性蛋白显示了可作为ELISA诊断的抗原用于莱姆病的诊断价值。结论纯化的伯氏疏螺旋体鞭毛蛋白可作为莱姆病ELISA诊断抗原用于莱姆病的诊断,为莱姆病快速诊断试剂盒的开发打下基础。     

中国B.afzelii基因型莱姆病螺旋体GDsh1表面蛋白VlsE保守区段的克隆表达及抗原性分析

目的 克隆表达中国莱姆病螺旋体B.afzelii基因型菌株GDsh1的表面蛋白VlsE保守区段,并对其抗原性进行分析,为制备中国莱姆病重组抗原ELISA检测试剂盒提供依据。方法 结合文献,下载并比对PubMed上所有莱姆病螺旋体B.a型菌株的VlsE基因序列,确定保守区段,设计引物,扩增GDsh1 的VlsE基因片段。将扩增产物与载体PET-32a连接,转入大肠杆菌BL21(DE3)中表达。对重组载体进行序列测定,表达产物用SDS-PAGE和Western blot 分析。利用重组VlsE蛋白制备ELISA试剂盒,检测83份莱姆病阳性血清,90份阴性血清以及90份梅毒血清,计算重组试剂盒的灵敏度和特异性。并与科室已有的全菌蛋白ELISA试剂盒检测结果进行比较。结果 成功克隆表达了B.afzelii型VlsE保守区蛋白,Western blot结果显示VlsE保守区蛋白与免疫兔血清有较强的抗原抗体反应。ELISA结果表明:重组VlsE蛋白的灵敏度60.2%低于全菌蛋白的灵敏度92.8%(P<0.001);特异性分别为73.3%、68.9%,差异无统计学意义(P=0.511)。在检测梅毒血清上特异性分别为83.3%、18.9%,重组蛋白的特异性远远高于全菌蛋白(P<0.001)。结论 重组VlsE基因保守区段蛋白在莱姆病的检测中具有一定的灵敏度和特异性,且在区分梅毒血清与莱姆病血清上,其特异性远远高于全菌蛋白,在莱姆病血清学检测中具有不容忽视的重要意义。     

An OspA-based genospecies identification of Lyme disease spirochetes (Borrelia burgdorferi) isolated in Taiwan

To further identify the genospecies of Lyme disease spirochetes (Borrelia burgdorferi) isolated in Taiwan, we analyzed the genomic identities of these Taiwan isolates (TWKM1-7) by genospecies-specific polymerase chain reaction (PCR) assay, restriction fragment length polymorphism (RFLP) analysis, and gene sequencing based on the OspA gene sequences of B. burgdorferi sensu lato. PCR analysis indicates that all of these Taiwan isolates were genetically related to the genospecies of B. burgdorferi sensu stricto by their differential reactivities with genospecies-specific PCR primers. After cleavage by DraI, three different RFLP patterns in relation to three different genospecies of Lyme disease spirochetes were observed, and all of these Taiwan isolates were affiliated with the genospecies of B. burgdorferi sensu stricto. The phylogenetic analysis also reveals that the sequence similarity of PCR-amplified OspA gene of these Taiwan isolates is highly homogeneous, with a homogeneity of more than 99.8% within the genospecies of B. burgdorferi sensu stricto. These results confirm that the genomic identities of these Taiwan isolates belong to the genospecies of B. burgdorferi sensu stricto.     

Molecular characterization of Lyme disease spirochetes (Borrelia burgdorferi sensu lato) isolated in Taiwan by restriction fragment length polymorphism analysis of 5S(rrf)-23S(rrl) intergenic spacer amplicons

We analyzed the 5S (rrf)-23S (rrl) intergenic spacer amplicon gene of Lyme disease spirochetes (Borrelia burgdorferi sensu lato) for the first time in Taiwan. The genetic identities of these Taiwan isolates (TWKM1-7) were clarified by comparing their restriction fragment length polymorphism patterns and sequence similarities of the polymerase chain reaction-amplified intergenic spacer amplicon genes with 3 major genospecies of Lyme disease spirochetes. Amplified-spacer DNAs were purified further and subjected to the cleavage by nuclease DraI or MseI. Differential fragment patterns in relation to different genospecies of Lyme disease spirochetes were observed among tested Borrelia isolates, and all of these Taiwan isolates were closely related to the genospecies of B. burgdorferi sensu stricto. The phylogenetic analysis also revealed that the sequence similarity of polymerase chain reaction-amplified spacer genes of these Taiwan isolates was highly homogeneous (95.7-100%) within the genospecies of B. burgdorferi sensu stricto and can be distinguished clearly from other genospecies of Lyme disease spirochetes with a 4.1% sequence divergence. Based on the differential fragment patterns and sequence similarity among these Taiwan isolates, the genetic identity of these Taiwan isolates should be classified into the genospecies of B. burgdorferi sensu stricto.     

Evaluation of the intrathecal antibody response to Borrelia burgdorferi as a diagnostic test for Lyme neuroborreliosis

The intrathecal antibody response to Borrelia burgdorferi was evaluated in American and West German patients with Lyme neuroborreliosis. By an antibody capture enzyme immunoassay, 12 (92%) of 13 patients from the USA with Lyme meningitis were found to have intrathecal antibody production to B. burgdorferi, usually of multiple isotypes, most commonly IgA. Of 12 patients with putative late central nervous system manifestations of Lyme disease, 5 (42%) had local production of IgG or IgA spirochetal antibody, but cerebrospinal fluid (CSF) abnormalities could not be demonstrated in 6 patients with late peripheral nervous system manifestations of the disorder. Compared with American patients, 30 European patients with neuroborreliosis had significantly higher CSF:serum ratios of specific antibody both early and late in the illness. Intrathecal antibody determinations are the most specific diagnostic test currently available for Lyme neuroborreliosis, but local antibody production in CSF is an inconsistent finding in American patients with late neurologic manifestations of the disorder.     

Borrelia burgdorferi genes selectively expressed in the infected host.

An immunological screening strategy was used to select microbial genes expressed only in the host. Differential screening of a Borrelia burgdorferi (the Lyme disease agent) expression library identified a gene (p21) encoding a 20.7-kDa antigen that reacted with antibodies in serum from actively infected mice but not serum from mice immunized with heat-killed B. burgdorferi. Selective expression of p21 in the infected host was confirmed by Northern blot analysis and RNA PCR. Further differential screening of the expression library identified at least five additional B. burgdorferi genes are selectively expressed in vivo. This screening method can be used to identify genes induced in vivo in a wide variety of pathogenic microorganisms for which a gene transfer system is not currently available.     

中国莱姆病螺旋体特异性单克隆抗体的制备及初步鉴定

目的制备特异的莱姆病螺旋体单克隆抗体,为我国莱姆病的诊断和莱姆病螺旋体菌株鉴定提供基础。方法以中国莱姆病螺旋体伽氏疏螺旋体(Borreliagarinii)的代表菌株PD91的全菌蛋白为抗原,免疫BALB/c小鼠,取脾细胞与骨髓瘤细胞SP2/0融合,用间接酶联免疫吸附试验(ELISA)和蛋白免疫印迹方法(WB)筛选,并经过2次或3次克隆,以获得单克隆抗体。结果共制备出10株单克隆抗体,经鉴定为3种,分别针对中国莱姆病螺旋体的外膜蛋白OspA(4株)、OspB(3株)和OspC(3株)。结论成功制备出3种抗莱姆病螺旋体不同蛋白的单克隆抗体,可用于我国莱姆病的病原诊断和莱姆病螺旋体菌株鉴定。     

Immune complexes from serum of patients with lyme disease contain Borrelia burgdorferi antigen and antigen-specific antibodies: potential use for improved testing

We report sequestration of specific IgM anti-Borrelia burgdorferi (Bb) and Bb antigens within immune complexes (ICs) isolated from serum of patients with Lyme disease (LD). The relative enrichment in specific IgM measured by ELISA was apparent, even after correcting for differences in total IgM concentration in serum versus ICs. Immunoblot demonstrated that ICs contained antibodies against specific Bb proteins, whereas reactivity was absent or significantly lessened in unprocessed serum. This is the first study to show ICs containing Bb antigen identified by immunoblot with anti-Bb monoclonal antibody. ICs may be a useful source of antigen and antibody for development of more-accurate testing for LD.     

Lyme borreliosis in laboratory animals: effect of host species and in vitro passage of Borrelia burgdorferi

The susceptibility of several common laboratory animal species to a known pathogenic isolate of Borrelia burgdorferi (N40) was evaluated following intraperitoneal (ip) inoculation of 10(6-8) spirochetes into 3-day-old Lewis rats, CD-1 mice, Syrian hamsters, and 3-week-old American Dutch rabbits. At 30 days, tissues were cultured for spirochetes and examined histologically. All species developed multisystemic infection as well as arthritis and carditis, but disease was most severe in rats and mice. In order to evaluate the effect of in vitro passage on the pathogenicity of B. burgdorferi, 3-day-old Lewis rats were inoculated ip with borreliae passaged in culture 2, 5, 11, 17, 21, 26, and 31 times, and evaluated at 30 days by culture, histology, and ELISA antibody titers. Based upon these parameters, B. burgdorferi (N40) lost its virulence at 17-21 passages. This study demonstrated that B. burgdorferi was infectious for infant rats, mice, hamsters, and 3-week-old rabbits, although pathogenicity was modulated by host species and the in vitro passage history of the spirochete. Of the 4 laboratory animal species evaluated in this study, rats and mice appear to have the most potential for further use as animal models of Lyme disease.     

Monoclonal antibodies specific for the outer surface protein A (OspA) of Borrelia burgdorferi prevent Lyme borreliosis in severe combined immunodeficiency (scid) mice.

We have recently shown that viable Borrelia burgdorferi organisms induce a chronic infection associated with arthritis and carditis in severe combined immunodeficiency (scid) mice but not in immunocompetent mice. The disease is similar to that found in patients suffering from Lyme disease. We now show that B. burgdorferi-specific immune mouse sera as well as a monoclonal antibody to the spirochetal outer surface antigen A (31 kDa) but not monoclonal antibodies specific for the 41-kDa antigenic component of the periplasmic flagella are able to prevent (or mitigate) the development of the disease in scid mice when passively transferred at the time of the bacterial inoculation. The identification of a B. burgdorferi-associated protective antigen suggests that the corresponding spirochetal protein should be tested as a vaccine against Lyme disease.     

Three major Lyme Borrelia genospecies (Borrelia burgdorferi sensu stricto,B. afzelii and B. garinii) identified by PCR in cerebrospinal fluid from patients with neuroborreliosis in Sweden

The Lyme Borrelia genospecies Borrelia afzelii and B. garinii have previously been isolated using a culture method in Swedish patients with Lyme borreliosis (LB). There are reports suggesting that the genospecies distribution in human tissue specimens as determined by molecular methods is different from that obtained by culture. In the present study, we developed a nested PCR for detection of Lyme Borrelia-specific DNA in cerebrospinal fluid from Swedish patients with LB. The genospecies were subsequently identified by sequence analysis in a total of 7 PCR-positive patients. Two sequences were identified as B. burgdorferi sensu stricto (s. s.), 1 as B. afzelii and 4 as B. garinii. These are the first reported cases in which B. burgdorferi s. s. has been shown to be the causative agent of human LB in Sweden. The results of our study confirm that the use of direct molecular analytical methods for Borrelia genospecies identification in clinical specimens can provide epidemiological information additional to that obtained by culture.     

Lymphoproliferative responses to Borrelia burgdorferi in Lyme disease

OBJECTIVE: To compare lymphocyte proliferative responses to Borrelia burgdorferi in healthy controls and patients with Lyme disease. PATIENTS: Twelve patients fulfilling case-definition criteria for Lyme disease. Twelve healthy volunteers and two newborns served as controls. MEASUREMENTS: Antibodies to B. burgdorferi were measured by enzyme-linked immunosorbent assay (ELISA). Proliferation of peripheral blood lymphocytes cultured for 5 days with B. burgdorferi, recall antigens, or pokeweed mitogen was measured by radioactive thymidine uptake. RESULTS: Lymphocytes from 11 patients with Lyme disease, 8 healthy seronegative controls, and two newborns showed elevated responses when stimulated with B. burgdorferi. When a patient and a control were studied on the same day, the patient's lymphocyte response to B. burgdorferi exceeded the control's in only 5 of 12 cases. Lymphocytes from both patients and controls responded to B. burgdorferi isolates from three different sources. CONCLUSIONS: Heightened lymphocyte responses to B. burgdorferi are found in patients with Lyme disease but elevated responses also frequently occur in healthy controls. At present, the interpretation of a positive lymphocyte response to B. burgdorferi would be difficult in ambiguous clinical situations.