Purification of the Borrelia burgdorferi flagellum by use of a monoclonal antibody.

The flagellum associated polypeptide p41 is an immunodominant antigen of Borrelia burgdorferi in the early and late stages of Lyme borreliosis. p41 was prepared by affinity chromatography using monoclonal antibody specific for p41. An immunoglobulin class specific ELISA (IgM-, IgG-ELISA) was established with purified p41 as antigen and compared to the conventional ELISA with whole cell ultrasonic antigen. Whereas the sensitivity of IgM- and IgG-ELISA was comparable in both antigen preparations, crossreactivity of sera from syphilitic patients was reduced in the p41 IgG-ELISA. Discrepant results obtained by use of ultrasonic antigen or p41 antigen, were controlled by Western blots. A correlation between the results of p41-ELISA and Western blot was shown.

Comparative antimicrobial activity of the new macrolides against Borrelia burgdorferi.

The in vitro and in vivo activity of the new macrolides azithromycin, clarithromycin and roxythromycin was compared with that of erythromycin against Borrelia burgdorferi. In in vitro tests using ten clinical isolates all macrolides were highly active against Borrelia burgdorferi (MIC90 0.015-0.06 micrograms/ml). Azithromycin was more potent than the other macrolides in experimental animal infection, eradicating the organism in all animals tested at a dosage of 8 mg/kg.

[Immunochemical analysis of the immune response in late manifestations of Lyme borreliosis]. / Immunochemische Analyse der Immunantwort bei Spätmanifestationen der Lyme Borreliose.

Compared to American strains, European Borrelia burgdorferi strains revealed considerable heterogeneity of major proteins. Four strains isolated from ticks, human skin and human CSF were selected from our 23 Borrelia burgdorferi isolates. These strains and the American type strain B31 were characterized by SDS-PAGE (Coomassie Blue staining) and Western blots (using rabbit immune sera against two of the strains and two monoclonal antibodies (H5323 and H3TS) against a major outer surface protein (OspA]. The strains showed considerable differences in SDS-PAGE pattern. Corroborating the results of a previous study, we could demonstrate that the OspA (31/32K) can change from a minor to a major protein and in reverse the pC (21/22K) from a major to a minor protein during subculturing. Moreover, European strains can antigenically differ in OspA, pC and also in a further low molecular weight protein of 17/18K. To examine whether the antigenic heterogeneity of European isolates is reflected in the immune response of European patients we examined sera from patients with late manifestations of Lyme Borreliosis by Western blot using the five strains as antigens. Sera from seven patients with acrodermatitis chronica atrophicans (ACA) showed a surprisingly strong reactivity with the skin isolate. All sera had antibodies against the 17/18K protein of the skin isolate, but none was reactive with the analogous 17/18K of the other strains. On the other hand a comparable predominance of one strain was not found testing sera from patients with Lyme arthritis. One patient even had antibodies against OspA and OspB proteins of strain B31. Contrary to findings in American Lyme Disease antibodies against the OspA were rarely observed in the sera of our patients (only one patient had such antibodies) although we tested the patients sera with five different strains. Only two patients had stronger reactions with the skin isolate. These findings suggest that ACA is caused by antigenically closely related Borreliae. This could explain the finding that ACA is rarely observed in the US (US strains are antigenically closely related to strain B31). Our findings in patients with Lyme Arthritis--on the other hand--suggest that "different serotypes" can cause Lyme Arthritis. This does not exclude the possibility that Borrelia proteins are an important factor in the pathogenesis of Lyme arthritis. Finally the differences in reactivity of sera with different strains in the Western blot led us to examine whether such differences are also found in serodiagnostic tests using different strains as antigens.(ABSTRACT TRUNCATED AT 400 WORDS)

Antibiotic therapy of early European Lyme borreliosis and acrodermatitis chronica atrophicans.

In a study on 121 consecutive patients with erythema migrans, 65 patients obtained oral penicillin, 36 tetracyclines, and 20 amoxicillin-clavulanic-acid. Follow-up was carried out for a median of 29, 17, and 7 months, respectively. In another limited trial on 29 patients with acrodermatitis chronica atrophicans (ACA), 14 patients received oral penicillin, 9 parenteral penicillin, and 6 tetracyclines. There was no statistically significant difference among treatment groups in both therapeutic trials, with the exception of different follow-ups due to the nonrandomized study design and different occurrence of the Jarisch-Herxheimer reaction in patients with erythema migrans. Later extracutaneous manifestations developed in 27% of the patients with erythema migrans and in 47% of the patients with ACA despite antibiotic therapy. We could not prove the superiority of any antibiotic tested in either early or late European Lyme borreliosis.

Antigenic variability of Borrelia burgdorferi.

Borrelia burgdorferi strains (six isolates from North America and 28 isolates from Europe) were analyzed by physicochemical and immunological methods. By SDS-PAGE, all Borrelia burgdorferi strains tested had two major proteins with constant molecular weights of 60 and 41 kDa and one, two, or three variable low molecular weight proteins (OspA = 30-32 kDa, OspB = 34-36 kDa, pC = 21-22 kDa). All combinations--except OspB alone or OspB/pC--were observed. Borrelia burgdorferi strains were different from relapsing fever borreliae by strong reactivity with OspA- and/or pC-specific polyclonal antibodies, whereas relapsing fever borreliae were only weakly reactive. Among 25 Borrelia burgdorferi isolates, seven different serotypes of Borrelia burgdorferi were defined according to their reactivity in the Western blot with three monoclonal OspA-specific antibodies (H5332, H3TS, and LA5), four OspA- or OspB-specific polyclonal antibodies, and 12 polyclonal antibodies against whole borreliae. Antigenic differences between European CSF and skin isolates were observed, all skin isolates (n = 11) belonging to serotype 2 in contrast to only two out of seven CSF isolates. CSF isolates were antigenically heterogenous (serotypes 1, 2, 3, 4, and 5). Serotypes 6 and 7 were represented by two tick isolates, and the other European tick isolates are not yet fully characterized. Antigenic differences between European and North American strains may play a role in differences in the clinical picture of Lyme borreliosis.

[Lyme borreliosis in South Germany. Epidemiologic data on the incidence of cases and on the epidemiology of ticks (Ixodes ricinus) carrying Borrelia burgdorferi]. / Lyme-Borreliose in Süddeutschland. Epidemiologische Daten zum Auftreten von Erkrankungsfällen sowie zur Durchseuchung von Zecken (Ixodes ricinus) mit Borrelia burgdorferi.

Among 2403 ticks (Ixodes ricinus) tested in 1985 for Borrelia 328 (13.6%) were carriers (adults about 20%, nymphs about 10%, larvae about 1%). The highest prevalence of infected ticks was among adult ticks in the Isar region north of Munich (33.8%). Among 9383 persons whose serum had been examined by fluorescence serology in 1985 and 1986, 1035 (11%) had raised Borrelia-specific IgG and/or IgM antibodies greater than or equal to 1:64. In 18.7% only IgM antibodies were demonstrated. Among 375 proven cases there were 78 with erythema migrans, 211 with neurological signs, 48 with Lyme arthritis and 36 with acrodermatitis. Seasonal incidence peaks were in June-August for erythema migrans, July-September for neurological signs, with no clear-cut seasonal peaks with Lyme arthritis and acrodermatitis. The incubation time for 80% of cases of each abnormality was 5-29 days for erythema migrans, 20-59 for neurological signs and 2-8 months for Lyme arthritis. Erythema migrans was most frequent among those aged 30-60 years, neurological signs among children and juveniles up to 20 years and those aged 40 to 70 years, Lyme arthritis among those aged 30-60 years, and acrodermatitis among those aged 40-80 years. Significantly more women than men developed acrodermatitis.

In vitro and in vivo susceptibility of Borrelia burgdorferi.

The antispirochetal activity in vitro and in vivo of several antibiotics against ten isolates of Borrelia burgdorferi from human spinal fluids and skin biopsies was determined. Borrelia burgdorferi was most susceptible in vitro to erythromycin, ceftriaxone and cefotaxime (MIC90: 0.06, 0.06, 0.12 mcg/ml respectively). Less activity was observed with tetracycline, amoxycillin and lincomycin (MIC90: 0.50 mcg/ml), imipenem and augmentin (MIC90: 0.25 mcg/ml), oxacillin (MIC90: 1 mcg/ml), ciprofloxacin (MIC90: 2 mcg/ml) and ofloxacin (MIC90: 4 mcg/ml). Penicillin G, normally regarded as appropriate treatment for Lyme disease, had an MIC90 of only 4 mcg/ml. With the exception of erythromycin, activity in vitro corresponded to the activity in vivo. Erythromycin, however, was less active in vivo, and penicillin G showed poor activity both in vitro and in vivo.

Antibodies against Borrelia burgdorferi in Bavarian forest workers.

The sera of 496 forest workers (fw), 44 customs officials and 20 other persons in outdoor professions from different areas of Bavaria were tested for antibodies against Borrelia burgdorferi (strain B 31) by indirect immunofluorescence. In sera previously absorbed with Treponema phagedenis ultrasonicate, significantly elevated IgG antibody titers were found in 69 cases, in 68 fw (13.7%) and 1 custom agent. In accordance with previous observations the distribution of positive results showed a distinct age dependance: under 24 years (y) 1.6% (n = 64), 25-34 y 0 (n = 30), 35-44 y 10.1% (n = 79), 45-54 y 17.4% (n = 190). Infections without manifest illness seem to predominate. In 1 area the number of positive persons was significantly higher than in the other: In Donauries near Illertissen 14 (45%) of 31 probands had elevated IgG titers. This area and the high level regions excepted, no significant differences were demonstrable amongst the 13 regions of Bavaria included in this investigation.

European Borrelia burgdorferi isolated from humans and ticks culture conditions and antibiotic susceptibility.

Growth of Borrelia burgdorferi in a modified Kelly-medium is described. Borrelia strains were isolated from patients (n = 11) and ticks I. ricinus (n = 19). The modified medium which contained Co-trimoxazole is a very effective medium for isolating and culturing of Borrelia sp. The susceptibility of 7 strains of B. burgdorferi to antibiotics was studied by the macrodilution and microdilution test. After preliminary testing for optimal conditions, we determined MICs in modified Kelly medium. The MIC concentration of each antibiotic was determined as the lowest concentration which completely inhibited growth of the tested organism. The B. burgdorferi was most susceptible to Erythromycin with MIC of less than or equal to 0.15 microgram/ml. Of the Penicillins tested, Ampicillin and Mezlocillin were more active than Penicillin G. The use of Tetracycline-HCl is recommended because of its low MIC in vitro its extra- and intracellular efficiency.

Immunochemical and immunological analysis of European Borrelia burgdorferi strains.

Borrelia burgdorferi strains (n = 23), isolated from patients (n = 8) and ticks (n = 14), were analyzed by SDS PAGE and Western blotting with monoclonal antibodies and polyclonal sera (rabbit immune serum and sera from patients). Testing the 23 strains by SDS PAGE 9 different patterns of major protein bands were observed. In contrast to US strains some of our strains showed only weak or negative reactivity with the OspA specific monoclonal antibody H5332. Analysis with polyclonal sera gave further support that the OspA proteins of our strains have specific epitopes (recognized by patients sera) besides common ones (recognized by rabbit immune serum). Fifteen of the strains had another major protein in the 22K range without detectable antigenic variability. Patients with erythema migrans and lymphocytic meningitis had a good IgM- and IgG-immunoresponse to this protein. In contrast antibodies to the OspA were very rarely observed, probably due to the antigenetic heterogeneity of the causative agent on the one hand but also due to low response of the human immune system to the OspA on the other hand.

Lyme arthritis: clinical features, serological, and radiographic findings of cases in Germany.

The clinical manifestations, serological data, and radiographic findings of ten cases of Lyme arthritis in Germany are summarized. Qualitative assessment shows that the characteristics of the disease in Germany do not differ fundamentally from those reported in the USA. However, since a serological test for antibodies to the causative spirochete is now available, the great variety of the clinical features of Lyme arthritis can be described more precisely. The cases of chronic Lyme arthritis without prior erythema chronicum migrans, hitherto the most important diagnostic hallmark of the disease, may have been underestimated. One of the cases reported provides evidence that the disease was transmitted via a fly bite. Radiographic abnormalities consisting of marked juxta-articular osteoporosis and osseous erosions were found in two patients with chronic arthritis. Three patients were treated with high-dose intravenous penicillin, two did not respond to the therapy.

Intrathecal production of specific antibodies against Borrelia burgdorferi in patients with lymphocytic meningoradiculitis (Bannwarth's syndrome).

Central nervous system infection with Borrelia burgdorferi, the causative agent of Lyme disease in the USA, manifests itself most frequently in Europe as lymphocytic meningoradiculitis (Bannwarth's syndrome). We examined 12 patients with lymphocytic meningoradiculitis with cerebrospinal fluid (CSF) antibodies against B. burgdorferi to establish whether these antibodies were produced intrathecally or were serum derived. By comparison of titers of antibody to B. burgdorferi with total IgG and antibodies against tetanus toxoid in CSF and serum, we demonstrated intrathecal production of antibodies to B. burgdorferi in 11 patients. Reactions of IgG antibodies in CSF with a panel of borrelial proteins (molecular weights, 22k, 23k, 39k, 41k, 42k, 48k, 60k, 66k, and 75k) were stronger than those of the serum antibodies; patterns of reactivity varied considerably between patients. However, CSF reactions (in comparison to serum reactions) to the 41k protein were stronger in all patients. Examination of CSF for intrathecal production of antibodies to B. burgdorferi is helpful in diagnosing neurological manifestations caused by B. burgdorferi infections.

Reinfection in erythema migrans disease.

Two patients received oral penicillin for erythema migrans disease (EMD) in 1973 and 1977. Five and seven years later, respectively, they developed EMD for a second time after being bitten by ticks. The first and second erythema migrans lesions appeared in different places. Tests of sera evaluated for antibodies to Borrelia burgdorferi before (in one case), during and after the second episode of EMD remained negative (patient 1) or yielded an at least four-fold rise of IgG antibody titers, but did not become positive (patient 2). These cases provide evidence that reinfection in EMD may occur without a significant elevation of specific antibody titers.

[Acrodermatitis chronica atrophicans--a borreliosis!]. / Acrodermatitis chronica atrophicans--eine Borreliose!

Spirochetes were recovered from the skin lesions of two patients with acrodermatitis chronica atrophicans. Both patients assume that they had a tick bite. Sera from both patients showed increased IgG-antibody titers to a Borrelia burgdorferi strain isolated from the skin.

[Lymphocytoma--a borreliosis?]. / Das Lymphozytom--eine Borreliose?

4 patients suffering from lymphocytoma had been observed for a mean of 1.75 years (13 to 21 month). The lesions were located on the ears; all patients developed regional lymphadenopathy. Two patients experienced headaches of short duration, one child showed six erythema migrans lesions, and another child had an elevated IgM level of 322 mg/dl. A tick bite was noted in one child. Indirect immunofluorescence tests revealed significantly elevated IgG or IgM antibody titers against Ixodes dammini and Ixodes ricinus spirochetes in all patients. Serological evaluation may be helpful in differentiating borrelial lymphocytoma from other pseudolymphomas.

[Clinical aspects and etiology of acrodermatitis chronica atrophicans]. / Zur Klinik und Atiologie der Acrodermatitis chronica atrophicans.

We followed up nine patients with acrodermatitis chronica atrophicans (ACA) for an average of 2 years (6-44 months). Extradermal symptoms were observed in five patients, three of whom developed systemic signs such as fatigue or general sensitivity to cold; three suffered from cardiac symptoms; and two had joint and/or nervous system involvement. In one patient, erythema migrans and oligoarthralgia preceded the ACA. In most patients, the laboratory tests showed changes often seen in inflammatory diseases; we found an elevation of IgA, IgG and/or IgM in five patients. Using an indirect immunofluorescence test with Ixodes dammini spirochetes as antigen, the patients had more or less elevated IgG antibody titers and insignificant IgM antibody titers; similar results were obtained in some patients when an Ixodes ricinus spirochete isolated in the area of Munich was used as antigen. We believe that ACA is a chronic spirochetosis with manifestations present in the skin, joints, nervous system, and presumably in the heart.

Serological diagnosis of erythema migrans disease and related disorders.

An indirect immunofluorescence technique for the determination of antibodies against ixodid tick spirochetes is described. Differences in the reactivity between Ixodes ricinus spirochete and Ixodes dammini spirochete antigens were not observed. Cross-reacting antibodies against Treponema pallidum and Treponema phagedenis can be eliminated by quantitative absorption with T. phagedenis. Cross-reactions with leptospira were not observed by immunofluorescence. In the IgM test, false negative reactions caused by high-titered specific IgG antibodies or false positive reactions caused by rheumatoid factor occur. This can be avoided by testing the IgM fraction (19S-IgM-test) or using sera previously treated with anti-IgG serum. Significantly elevated antibody titers against ixodid tick spirochetes were observed in 45% of 44 cases with erythema migrans disease, in 72% of 29 cases of lymphocytic meningoradiculitis, in all of nine patients with acrodermatitis chronica atrophicans and in all of four investigated patients with lymphocytoma (lymphadenosis benigna cutis).