吉林省林区家畜中斑点热群立克次体与莱姆病螺旋体复合感染的血清学调查

目的了解吉林珲春地区家畜中斑点热、莱姆病及其复合感染的情况。方法应用间接免疫荧光法检测家畜血清中斑点热群立克次体和伯氏疏螺旋体的IgG抗体。结果牛血清中斑点热感染率为18.0%,莱姆病感染率为27.5%,复合感染率为9.5%;羊血清中斑点热感染率为19.2%,莱姆病感染率为31.5%,复合感染率为12.8%。结论吉林珲春地区家畜中广泛存在斑点热和莱姆病的复合感染。     

Seroreactivity to Borrelia burgdorferi antigens in early rheumatoid arthritis: a case-control study

This study investigates the seroreactivity to Borrelia burgdorferi antigens of patients suffering from rheumatoid arthritis (RA) for < 5 yr. Subjects were matched with controls for age, sex and area of residence in order to minimize the risk of differential exposure to B. burgdorferi. A total of 57 pairs were tested by immunofluorescence assay (IFA) and Western blotting. Only two RA patients showed positive IFA results, and another was positive by Western blot analysis. No significant difference in Lyme seropositivity was detected between cases and controls using either serological test. Furthermore, no significant difference in antibody response was observed to specific or non-specific proteins (e.g. Osp C, 93 kDa protein, flagellin, heat shock proteins). These results do not support the previous suggestions that B. burgdorferi may be involved in the aetiology of RA, or that RA favours the production of antibodies that cross-react with B. burgdorferi proteins.      

Antibody response in Lyme disease: evaluation of diagnostic tests

The antibody response to the Ixodes dammini spirochete was determined in 41 serial serum samples from 12 patients with Lyme disease. By enzyme-linked immunosorbent assay (ELISA), 11 of the 12 patients had higher titers of specific IgM antibody (greater than 1:200) during early disease than did 40 control subjects. Specific IgM antibody titers, which correlated with total amounts of IgM antibody (P less than .001), sometimes remained elevated throughout the illness. During neuritis, nine of 10 patients had higher specific IgG antibody titers (greater than 1:200) than did controls, and when arthritis was present, all had such titers, which remained elevated after months of remission. In the ELISA, antibody responses determined by single or serial dilutions were similar, but the ELISA was more sensitive and specific than was immunofluorescence. Adsorption of sera with Borrelia hermsii generally resulted in a fourfold decrease in titers of cross-reactive antibodies, but the titers of sera from patients with Lyme disease were also reduced. Currently, the ELISA, without adsorption, is the best diagnostic test for Lyme disease.     

A specific and sensitive assay for the Lyme disease spirochete Borrelia burgdorferi using the polymerase chain reaction

A highly specific and sensitive assay for Borrelia burgdorferi, the causative agent of Lyme disease, was developed using the polymerase chain reaction (PCR). The target DNA sequence was of chromosomal origin and conserved, by hybridization analyses, among all strains of B. burgdorferi tested but was not present in the most closely related member of the genus, B. hermsii. The PCR assay developed from this sequence reacted with 17 of 18 strains of B. burgdorferi but not with any other Borrelia species tested. The assay was sensitive to fewer than five copies of the B. burgdorferi genome, even in the presence of a 10(6)-fold excess of eukaryotic DNA. This assay should greatly facilitate the accurate diagnosis of Lyme disease and provide a means with which to investigate the pathogenesis, transmission, and basic biology of B. burgdorferi.     

Prevalence of antibody to Borrelia burgdorferi by indirect fluorescent antibody assay, ELISA, and western immunoblot in healthy adults in Wisconsin and Arizona.

The prevalence of antibody to Borrelia burgdorferi in healthy adults from Wisconsin and Arizona was determined by indirect fluorescent antibody assay (IFA), ELISA, and Western immunoblotting. A total of 301 sera from adult volunteer blood donors were collected from three areas of Wisconsin and compared with 49 consecutive anonymous adult volunteer donor sera from Tucson, Arizona, an area without reported Lyme borreliosis. Regional differences in seropositivity were found for Western immunoblotting (34[11%] of 301 from Wisconsin and none of 49 from Tucson; P less than .01) but not IFA or ELISA. No correlation was found among Western immunoblotting and IFA or ELISA results. For persons living in Madison or Milwaukee, Wisconsin (cities not endemic for Lyme borreliosis), 19 (86%) of 22 with a positive Western blot, but only 12 (48%) of 25 with a positive IFA or ELISA, had a significant exposure risk to B. burgdorferi-infected Ixodes dammini (odds ratio, 6.9; 95% confidence interval, 1.4-43.5). Western blot results were consistent with epidemiologic exposure to B. burgdorferi and implied frequent asymptomatic infection among healthy adults living in or visiting areas endemic for Lyme borreliosis.     

Gonarthritis, lymphadenopathy and disseminated choroiditis as a primary manifestation of Lyme borreliosis. Case report and discussion of diagnostic possibilities in Lyme borreliosis

A 26-year-old female patient was admitted to the hospital because of bilateral gonarthritis, lymphadenopathy and disseminated chorioiditis as primary manifestation of Lyme borreliosis. Antibody titers against Borrelia burgdorferi did not reach diagnostic levels as determined by an indirect immunofluorescence assay. However, diagnosis was established by the detection of IgG and IgM antibodies in Western blot analysis, and by the demonstration of an enhanced T-cell proliferation to Borrelia burgdorferi in a lymphocyte proliferation assay. This case report indicates that arthritides may already occur in stage II (disseminated infection) of Lyme borreliosis. Therefore, Lyme borreliosis must be considered in patients with chorioiditis and pauciarticular arthritis. In the case of non-diagnostic antibody titers in indirect immunofluorescence tests (or ELISA), Western blot analysis and lymphocyte proliferation assays should be performed in addition.     

Molecular and serological evidence of Borrelia burgdorferi sensu lato in wild rodents in the Czech Republic

The aim of the present study was to determine the frequency and spatial distribution of the Borrelia species in wild rodents in the Czech Republic. In total, 293 muscle tissue samples and 106 sera from 293 wild rodents captured in North Bohemia and North-East and South Moravia were examined for the presence of Borrelia spp. and antibodies. Muscle samples were investigated with real-time polymerase chain reaction (PCR) with a recA primer set, with DNA quantification and melting curve analysis, and with restriction fragment length polymorphism (RFLP) analysis of the 5S-23S rDNA intergenic spacer. Infection with Borrelia burgdorferi sensu lato was found in 16.4% of the muscle samples. The most abundant genospecies was Borrelia afzelii (11.3%), followed by Borrelia burgdorferi sensu stricto (4.8%) and Borrelia garinii (0.7%). Borrelia infection was more frequently observed in Clethrionomys glareolus than in Apodemus spp. Sera were analyzed with an enzyme-linked immunosorbent assay (ELISA) test, yielding the total seropositivity rates of 24.5% for anti-Borrelia IgM antibodies and 25.5% for IgG antibodies. Total seroprevalence was higher in Apodemus spp. than in C. glareolus. In conclusion, our data indicate that in the Czech Republic small wild rodents can serve as hosts for B. burgdorferi s. s. as well as for B. afzelii.     

Antibody responses to Borrelia burgdorferi in patients with antibiotic-refractory, antibiotic-responsive, or non-antibiotic-treated Lyme arthritis

OBJECTIVE: To compare the pattern of antibody responses to Borrelia burgdorferi in patients with antibiotic-refractory, antibiotic-responsive, or non-antibiotic-treated Lyme arthritis as an indirect measure of spirochetal persistence or eradication. METHODS: At least 3 serial serum samples from 41 patients with antibiotic-refractory arthritis and 23 patients with antibiotic-responsive arthritis, and samples from 10 non-antibiotic-treated, historical control patients were tested for IgG reactivity with B burgdorferi sonicate and 4 differentially expressed outer surface lipoproteins of the spirochete, by enzyme-linked immunosorbent assay. RESULTS: Among non-antibiotic-treated patients, antibody titers to B burgdorferi antigens remained high throughout a 2-5-year period of arthritis. In contrast, in patients with antibiotic-responsive arthritis, in whom joint swelling usually resolved during a 1-month course of oral antibiotic therapy, the median antibody titers to most of the spirochetal antigens remained steady or decreased during the first 1-3 months after starting antibiotic therapy. In patients with antibiotic-refractory arthritis, who had persistent joint swelling for a median duration of 10 months despite 2-3 months of oral or intravenous antibiotics, the median titers to most antigens increased slightly during the first 1-3 months. However, by 4-6 months after starting antibiotic therapy, reactivity with all antigens declined similarly in both antibiotic-treated groups. CONCLUSION: Whereas the antibody titers to B burgdorferi remained high in non-antibiotic-treated patients, the titers declined similarly 4-6 months after starting therapy in patients with antibiotic-responsive or antibiotic-refractory arthritis, suggesting that synovial inflammation persisted in patients with antibiotic-refractory arthritis after the period of infection.     

Lyme borreliosis in Dutch forestry workers.

Serum samples from 127 Dutch forestry workers and 127 matched controls were tested for antibodies against Borrelia burgdorferi in an indirect immunofluorescence assay (IFA). Those of the forestry workers were also tested by Western blotting. The forestry workers were examined clinically for evidence of Lyme borreliosis without the examiner or the workers knowing the results of the laboratory tests. Seroprevalence of B. burgdorferi antibodies among forestry workers (25/127) was significantly higher than among controls matched for age and place of residence (8/127), odds ratio 3.7 (95% CI 1.5-9.7). Of the 25 sera of forestry workers positive in the IFA, 23 reacted with at least five bacterial polypeptides in the Western blot test. According to adapted CDC criteria, seven forestry workers (6%) were classified as being a case of Lyme borreliosis. In only one of them had the diagnosis been made before this investigation. Five persons had a history of erythema migrans, one of arthritis, and one of persistent infection. We conclude that Lyme borreliosis is an occupational disease among forestry workers in the Netherlands, with a three-fold higher seroprevalence than among matched controls. The disease, often not diagnosed among this high-risk group, warrants more attention to achieve early recognition and to prevent late complications.     

Immune responses to Borrelia burgdorferi in patients with reactive arthritis

In reactive arthritis (ReA), including Reiter's syndrome, a close relationship between chronic enteric and genitourinary infections and the clinical features of enthesitis has been described. In contrast, in Lyme arthritis, a distinct clinical entity, chronic infection with the tick-transmitted spirochete Borrelia burgdorferi has been associated with the disease. In a prospective study, 51 patients with ReA were tested for evidence of chlamydial and spirochetal infection. The presence of Chlamydia was determined by culture in 8 patients, and 7 additional patients had markedly elevated antibody titers. In 9 patients, antibodies specific to B burgdorferi were found. Purified peripheral blood T lymphocytes of all 9 patients proliferated specifically to stimulation with macrophages pre-pulsed with B burgdorferi antigens. Compared with other protein antigens, higher numbers of antigen-pulsed macrophages were necessary to activate B burgdorferi-specific T cells. Although antibody titers decreased in response to antibiotic treatment in 8 of 9 patients, second-line therapy with sulfasalazine or methotrexate was required to obtain clinical remission. These data suggest that chronic infection with B burgdorferi can cause ReA. In predisposed individuals, the arthritogenic immune response might be triggered by persisting infectious agents independent of their antigenic specificities.     

从全沟硬蜱分离的伯氏疏螺旋体的实验研究

本文报道了从全沟硬蜱分离的一株伯氏疏螺旋体(VL株)实验研究的结果。该株螺旋体同莱姆病螺旋体标准株在超微结构上相近,可以和高稀释度的抗伯氏疏螺旋体抗体发生间接免疫荧光反应。SDS-PAGE电泳结果显示其蛋白组成和标准株的蛋白图谱相同。热变性温度法测定其DNA的G+Cmol％含量为28.1％,和标准株的含量无明显区别,试验结果证实此株螺旋体属伯氏疏螺旋体。     

福建省北部林区人群人粒细胞无形体血清流行病学检测

目的了解福建省北部林区人群人粒细胞无形体病感染状况与分布特点,以及无形体与莱姆病、恙虫病重叠感染的情况,为林区人群无形体的预防提供科学依据。方法2007年7月开始在福建省武夷山林区采集林业工人、农民和干部等269份血清,用试剂盒采用间接免疫荧光法(IFA)检测人粒细胞无形体、莱姆病和恙虫病抗体,并进行不同地区、年龄、性别、职业分布比较。数据库建立和统计分析均采用SPSS14.0,χ2检验的显著性水平α=0.05。结果269份血清中检出46份人粒细胞无形体血清抗体阳性,阳性率17.10。人粒细胞无形体血清抗体除年龄外不存在地区、性别、职业差异。46份人粒细胞无形体抗体阳性的血清中检测到1份与莱姆病混合感染,9份混合感染了莱姆病和恙虫病病原体。结论福建省武夷山林区人群存在人粒细胞无形体感染,并且是莱姆病、恙虫病重叠流行区。在林区人粒细胞无形体病、莱姆病、恙虫病三者的防治应同时进行。     

The T-cell proliferative assay in the diagnosis of Lyme disease

OBJECTIVE: To determine the sensitivity and specificity of the T-cell proliferative assay as a diagnostic test in Lyme disease. DESIGN: Cross-sectional study of patients with Lyme arthritis or chronic neuroborreliosis who had a history of erythema migrans, positive antibody responses to Borrelia burgdorferi by enzyme-linked immunosorbent assay (ELISA), or both; patients with other diseases; and healthy subjects. SETTING: Diagnostic Lyme disease clinic in a university hospital. PATIENTS: Forty-two of the 67 patients with active Lyme arthritis or chronic neuroborreliosis who were seen during the study period; 16 patients with inactive late Lyme disease; 77 patients with other rheumatologic or neurologic diseases; 9 workers from the Borrelia laboratory; and 9 healthy subjects. MEASUREMENTS AND MAIN RESULTS: Nineteen of 42 patients with Lyme arthritis or chronic neuroborreliosis and 4 of 77 patients with other diseases had positive T-cell proliferative responses to B. burgdorferi antigens. The sensitivity of the proliferative assay was 45% (95% Cl, 30% to 60%) and the specificity was 95% (95% Cl, 87% to 99%). Twelve of 27 patients with active Lyme arthritis, 7 of 15 patients with chronic neuroborreliosis, 4 of 16 patients with inactive Lyme disease, 4 of 9 healthy Borrelia laboratory workers, and 0 of 9 healthy subjects had positive responses. Three of five patients with Lyme disease who had negative or indeterminant antibody responses by ELISA had positive T-cell proliferative responses. CONCLUSION: The T-cell proliferative assay may be a helpful diagnostic test in the small subset of patients with late Lyme disease who have negative or indeterminant antibody responses by ELISA.     

Clinical evaluation of commercial serological test for Bartonella infection

We evaluated the usefulness of a serological diagnostic kit (Bartonella IFA IgG, IgM; MRL Diagnostics) for Bartonella henselae infection. Of the 110 healthy individuals, 107 (97.3%) were with titers being less than 1:64 for IgG antibody to B. henselae, 2 were with titers being 1:64 and 1 with 1:128, IgM antibody to B. henselae was negative in all individuals. Serological diagnosis of cat scratch disease (CSD) using indirect fluorescence antibody (IFA) methods (in-house and diagnostic kit) was made in either elevated titers of IgM (> or = 1:20) or IgG (> or = 1:256) antibodies, or a four-fold rise in IgG titer between acute and convalescent sera. Of the 18 individuals with serological diagnosis of CSD by in-house IFA method in 26 CSD clinical diagnosed patients, 15 (83%) were compatible with the results of the diagnostic kit, whereas 3 (17%) were not compatible. Of the 8 without serological diagnosis, 1 (13%) was serologically diagnosed as CSD, and the others were negative. Overall, the serological diagnosis was made in 16 of 26 (62%). The specificity and sensitivity of this kit were 100% and 62%, respectively. The cross-reaction between B. henselae and Bartonella quintana was observed in sera from controls and patients. Our results show that the diagnostic kit as well as in-house method is an useful tool for the serological diagnosis of cat scratch disease.     

Co-infection of Borrelia garinii and B. afzelii in a population of wild rodents from woodland

The maintenance of Borrelia burgdorferi s.l. in the environment is dependent on the zoonotic cycle involving tick vectors and certain reservoir hosts. It is well known, that the same species of wild rodents, as well as the vector Ixodes ricinus, are often co-infected with at least two genomospecies of B. burgdorferi s.l.: B. afzelii and B. garinii. The ticks collected from two rodent species: Clethrionomys glareolus and Apodemus flavicollis were examined for the presence of B. burgdorferi s.l., as well as for B. garinii and B. afzelii. In this study, an immunofluorescent antibody assay (IFA) and polymerase chain reaction (PCR) protocols were used. The high level of infestation in rodents (90% for C. glareolus and nearly 100% for A. flavicollis) shows that wild rodents are important hosts of the immature stages of I. ricinus. A high percent of Borrelia positive ticks collected from bank voles and yellow necked mice; above 7% determined by IFA and 2% determined by PCR, clearly revealed that these species of animals are competent zoonotic reservoirs of B. burgdorferi s.l.     

伯氏疏螺旋体与苍白密螺旋体、钩端螺旋体的免疫交叉反应抗原研究

目的研究莱姆病螺旋体与梅毒、钩端螺旋体较常出现的交叉反应抗原，明确产生交叉反应的蛋白抗原成分，为精确蛋白免疫印迹法检测莱姆病的阳性判断标准提供参考依据。方法以中国莱姆病螺旋体伽氏疏螺旋体基因型代表菌株PD91作抗原，用蛋白免疫印迹法对梅毒病人和钩端螺旋体病人的血清进行抗体(IgG和IgM)检测。结果共检测梅毒病人血清196份和钩端螺旋体病人血清68份。伯氏疏螺旋体与苍白密螺旋体抗原在75kDa，60kDa，43kDa，41kDa处有交叉，交叉反应阳性率分别为IgG：8．2％、20．4％、9．2％、17．3％，IgM：7．7％、10．2％、8．7％、9．7％；伯氏疏螺旋体与钩端螺旋体抗原的交叉发生在75kDa，60kDa，41kDa，交叉反应阳性率分别为IgG：1．5％、1．5％和13．2％，IgM：8．8％、2．9％、17．6％。结论莱姆病螺旋体蛋白抗原中75kDa，60kDa，43kDa和41kDa蛋白成分与梅毒和钩端螺旋体存在交叉反应。     

Rational diagnosis and treatment in unclassified arthritis: how clinical data may guide requests for Lyme serology and antibiotic treatment.

To improve the appropriateness and efficiency of diagnostic serological tests and subsequent antibiotic treatment, clinical data from 102 patients with unclassified arthritis were analysed to investigate whether the presence of positive IgG antibodies to Borrelia burgdorferi could be predicted. The clinical data were blindly ranked from 1 to 4 (1, Lyme arthritis unlikely; 4, Lyme arthritis very likely). Antibodies to B burgdorferi were positive in nine of 102 patients (9%). Six of 15 (40%) patients with rank numbers 3 and 4 were positive for antibodies to B burgdorferi, in contrast with only three of 87 (3%) patients with rank numbers 1 and 2. The likelihood ratio of positive Lyme serology for patients ranked 3 and 4 was 12.0, for patients ranked 2 to 4, 4.5, and for patients with arthritis of the knee, 3.0. These likelihood ratios were associated with a post-test probability of 55, 30, and 20% respectively. The clinical history in patients with unclassified arthritis can largely predict the presence of antibodies to B burgdorferi. The absolute value of a likelihood ratio can be a contributing factor in deciding to request tests for antibodies to B burgdorferi in patients with unclassified arthritis.     

Cross-reactivity in serological tests for Lyme disease and other spirochetal infections

Serum specimens from 163 persons with Lyme disease, tick-borne or louse-borne relapsing fever, yaws, syphilis, leptospirosis, or Rocky Mountain spotted fever were analyzed to assess the specificity of indirect fluorescent antibody (IFA) tests, an enzyme-linked immunosorbent assay (ELISA), and microscopic agglutination (MA) procedures. Strong cross-reactivity occurred when sera from individuals with Lyme disease, tick-borne relapsing fever, and louse-borne relapsing fever were tested against heterologous Borrelia antigens. Antibodies to Borrelia burgdorferi bound to Treponema pallidum in immunofluorescence tests for syphilis. Sera from subjects with syphilis cross-reacted in IFA tests and the ELISA for Lyme disease. Immunoglobulin antibodies to Borrelia or Treponema spirochetes, however, did not react with serovars of Leptospira interrogans in MA or IFA tests, and the prevalence of false-positive results in the reciprocal analyses was negligible.     

Relapsing fever spirochetes retain infectivity after prolonged in vitro cultivation

Borrelia hermsii and Borrelia burgdorferi, two closely related spirochetes, are the etiological agents of tick-borne relapsing fever and Lyme disease, respectively. Previous studies have shown the loss of infectivity of B. burgdorferi is associated with in vitro cultivation. This diminished infectivity of B. burgdorferi has occurred as early as three in vitro passages, and the loss of plasmids have been observed with these less virulent to noninfective cultures. The effects of long-term in vitro cultivation on B. hermsii have not been investigated. However, understanding the degree of genomic degradation during in vitro cultivation is important for investigating pathogenic mechanisms of spirochetes. In this study, we analyzed the effects of continuous in vitro cultivation on the genomic composition and infectivity of B. hermsii and B. turicatae.We report that all seven isolates of B. hermsii and the one isolate of B. turicatae examined retained infectivity in mice after 1 year of continuous in vitro cultivation. Furthermore, there were few apparent differences in the plasmid profiles after long-term cultivation. Two isolates of B. hermsii remained infective after high passage despite losing a portion of the 200-kb linear plasmid containing the fhbA gene encoding the factor H binding protein. Also, sequence analysis of multiple B. hermsii isolates demonstrated two types of fhbA with complete congruence with the two genomic groups of B. hermsii spirochetes. Therefore, these results suggest that relapsing fever spirochetes are genetically stable during in vitro cultivation, and the fhbA-containing segment of DNA that is lost during cultivation is not required for infection.     

Seroprevalence of human granulocytic ehrlichiosis in high-risk groups in Denmark

The aim of this study was to evaluate the seroprevalence of human granulocytic ehrlichiosis (HGE) among 300 residents in the county of Funen, Denmark. All of these people had either suspected or confirmed borreliosis. Two hundred control sera were included in the study. Samples were submitted by general practitioners and by hospital departments. An indirect immunofluorescence assay was used to identify sera reactive to HGE and an enzyme-linked immunosorbent assay was used to analyse Borrelia burgdorferi antibodies. There were 63 (21%) HGE-positive sera, 53 of which came from Borrelia-seropositive patients. Among patients with negative Borrelia serology, but with clinical suspicion of borreliosis, 14.3% were HGE-positive (n = 70). Of the 200 control sera, 3.5% were HGE-positive and 10.5% were Borrelia-positive. No HGE-positive samples were found among subjects < 20 y of age, wheras 20.4% of Borrelia seropositive samples where from subjects < 20 y of age. No mortality was observed in the HGE-positive group and the percentage of serum samples positive for both Borrelia and HGE did not differ significantly between hospitalized and non-hospitalized patients. Our study indicates that HGE infection with or without concomitant or previous Borrelia burgdorferi infection is common in tick-exposed individuals > 20 y old in the county of Funen, Denmark.