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A representative sample of the population of two villages located in an area of North Baden where borreliosis is endemic was studied to ascertain the prevalence of antibodies against Borrelia burgdorferi and of clinical abnormalities. The aim of the study was to determine what clinical symptoms and signs were correlated with positive antibody status. Out of the 2928 inhabitants 1228 were investigated, and 207 of these (16.9%) were seropositive. Certain clinical abnormalities showed highly significant statistical correlations with positive antibody status. Arthritis (defined in terms of heat, redness and effusion) was noted in 34.3% of the seropositive patients, but in only 9.3% of seronegative patients (P less than 0.0001). Complaints of arthralgia were noted in 23.4% of seropositive patients and 13.3% of seronegative patients (P less than 0.001). Motor neuropathies showed similar correlation with seropositive status (12.0% vs 4.0%; P less than 0.001), as did sensory neuropathies (25.4% vs 6.7%; P less than 0.001). Differences were also noted in the prevalence of cardiac arrhythmias (ventricular extrasystoles, conduction abnormalities and intermittent tachyarrhythmias), which were found in 19.8% of the seropositive as against 3.0% of the seronegative subjects (P less than 0.001). The findings are evidence of a causal link between the listed clinical abnormalities and the presence of antibodies against Borrelia burgdorferi.

A 54-year-old patient with an intact immune system developed Lyme disease three times within 4 years. The first time an erythema migrans occurred, which was successfully treated with oral doxycycline (100 mg twice daily for 20 days). Specific antibodies were subsequently demonstrated. Re-infection nonetheless occurred a year later, again as erythema migrans. Oral doxycycline in higher dosage (three times 100 mg daily for 20 days) failed to prevent generalization of the infection with rigor, head and neck aches, myalgia, fatigue and subfebrile temperatures. There was a marked increase in Borrelia-specific antibody titre. Parenteral treatment with cefotaxime (twice daily 3 g for 12 days) was curative. But 2 years later yet another re-infection occurred with classic erythema migrans, which regressed under doxycycline. The course of the disease in this case demonstrates that Borrelia-specific antibodies do not always protect against re-infection. This may have consequences for the possible development of a vaccine.

Many approaches were made in recent years to establish urine PCR as a diagnostic tool for Lyme borreliosis, but results are contradictory. In the present study, a standardized protocol spiking urine from healthy donors with a defined amount of whole Borrelia or Borrelia DNA was established. The development of a nested real-time PCR targeting ospA enabled a highly sensitive and quantitative analysis of these samples. We show the following. (i) Storage of spiked urine samples for up to 6 months at −20°C had no negative effect on spike recovery. (ii) Centrifugation of 10 ml of urine at 40,000 × g for 30 min resulted in a concentration of both spikes, i.e., whole Borrelia and DNA. (iii) The inhibition of DNA spike recovery in 48% (11 of 23 samples) of urine samples tested could be attributed to nuclease activity. This was abrogated by alkalizing the urine or by working with the samples on ice. Despite optimized conditions, analysis of urine samples of 12 patients with erythema migrans, the clinical stage considered to be associated with the highest bacterial load, revealed a positive result in only one sample. All 12 samples were negative by an alternative PCR targeting flagellin. The results of our study support doubts that urine is a suitable material for diagnosis of Lyme borreliosis.

In lipopolysaccharide-stimulated blood from 71 late-stage borreliosis patients, the ex vivo cytokine release capacity of tumor necrosis factor alpha (TNF-α) and gamma interferon (IFN-γ) was reduced to 28% ± 5% and to 31% ± 5% (P≤ 0.001), respectively, compared to that of 24 healthy controls. White blood cell counts were normal in both groups. To investigate direct interactions between the pathogen and the immune cells, blood from healthy controls was exposed in vitro to live or heat-killedBorreliaor toBorrelialysate. Compared to the pattern induced by bacterial endotoxins, a reduced release of TNF-α and IFN-γ and an enhanced secretion of interleukin-10 and granulocyte colony-stimulating factor was found. In blood from 10 borreliosis patients stimulated withBorrelialysate, TNF-α formation was decreased to 31% ± 14% and IFN-γ formation was decreased to 8% ± 3% (P≤ 0.001) compared to the cytokine response of blood from healthy controls (n= 24). We propose to consider anti-inflammatory changes in the blood cytokine response capacity elicited byBorreliaas a condition that might favor the persistence of the spirochete.

The phage surface display technique was used to identify Borrelia burgdorferi antigens. By affinity selection with immunoglobulin G from pooled sera of six Lyme borreliosis (LB) patients, the ribosomal protein L25 was identified. The diagnostic value of L25 was investigated by an enzyme-linked immunosorbent assay, using sera from 80 LB patients and 75 controls, and the use of the protein resulted in a specificity of 99% and a 23% sensitivity, which qualify L25 as a useful antigen when combined with others.

A total of 4803 domestic and wild animals which were presented for examination at a veterinary clinic in north Baden, Germany over a period of 1 year were examined for tick infestation. A total of 434 nymphal and adult ticks were collected from 175 hosts. Ticks found belonged to the species Ixodes ricinus (385), Ixodes hexagonus (48), and Ixodes ventalloi (one). The polymerase chain reaction was used to examine 132. I. ricinus and 21 I. hexagonus for the presence of Borrelia burgdorferi . Twenty-two per cent of adult I. ricinus were infected as were one female and one larval I. hexagonus .

Lyme disease (LD) is caused by Borrelia burgdorferi and displays different stages, including localized, early disseminated, and persistent infection, all of which are associated with profound inflammatory reactions in the host. Induction of proinflammatory cytokines by B. burgdorferi is mainly mediated by outer surface proteins interacting with TLR-2/TLR-1 heterodimers. In this study, we show that TNF-α induction by Borrelia lysate was impaired in heterozygous TLR-2 knockout mice, while reactivity to lipoteichoic acid, another TLR-2 ligand signaling via TLR-2/TLR-6 heterodimers, was unaffected. Blood from individuals heterozygous for the TLR-2 polymorphism Arg753Gln was tested for cytokine release upon stimulation with Borrelia lysate, and induction of TNF-α and IFN-γ was significantly lower as compared with individuals not exhibiting this variation. Overexpression of TLR-2 carrying the Arg753Gln polymorphism in HEK 293 cells led to a significantly stronger impairment of activation by TLR-2/TLR-1 ligands as compared with TLR-2/TLR-6 ligands. To study whether heterozygosity for the Arg753Gln variant of TLR-2 influenced susceptibility for LD, we analyzed 155 patients for this polymorphism. The Arg753Gln variant occurs at a significantly lower frequency in LD patients as compared with matched controls (5.8 vs 13.5%, odds ratio 0.393, 95% confidence interval 0.17–0.89, p = 0.033), with an even more pronounced difference when late stage disease was observed (2.3 vs 12.5%, odds ratio 0.163, 95% confidence interval 0.04–0.76, p = 0.018). These data suggest that Arg753Gln may protect from the development of late stage LD due to a reduced signaling via TLR-2/TLR-1.

512 consecutive patients suffering from chronic Lyme borreliosis have been treated according to a standardized therapy regimen which was developed using the results of a controlled trial (Hassler et al., 1990; Hassler, 1992). Follow up was performed for at least 6 (maximum 16) years. In the first two years after initial therapy clinical and serological data were collected every six months including Western blot testing, later once a year.

Tick-transmitted diseases like tick-borne encephalitis and Lyme borreliosis have been well known in Germany for decades. Ongoing research now gives an additional focus to a broad range of other bacteria and parasites in ticks like Anaplasma phagocytophilum, former Ehrlichia sp., Rickettsia sp. and Babesia sp. Knowledge about the prevalence of these infectious agents in ticks is an important prerequisite for risk assessment of human diseases. Therefore nymphs and adult Ixodes ricinus ticks were collected and examined for Anaplasma phagocytophilum (n = 5424 ticks), Rickettsia sp. (n = 1187), and Babesia sp. (n = 3113). For the detection of Anaplasma phagocytophilum, DNA from the 16S rDNA gene was amplified by nested PCR and hybridized with a DIG-labeled oligonucleotide probe. The examination of Rickettsia sp. was performed by single PCR. A partial sequence of the citrate synthase gene was amplified. As a target for the detection of Babesia sp., DNA from the 18S rDNA gene was amplified, also by single PCR. All positive PCR products were sequenced to control specificity. Anaplasma phagocytophilum was detected by PCR in n = 103 (1.9%) out of 5,424 examined ticks from 11 investigation areas. However, not all positive PCR products hybridized using DIG-labeled oligonucleotide probe. Thus, the result of sequencing indicated that only 1.0% (n = 54) belonged to Anaplasma phagocytophilum and nearly half of these PCR products (0.9%) were identified as Wolbachia sp. Rickettsia sp. in Ixodes ricinus ticks from 3 areas were found in n = 105 (8.9%) out of 1,187 ticks examined (range from 13.3% to 5.6%). Sequencing showed Rickettsia helvetica exclusively. In about 2.6% of Rickettsia-positive ticks, double infection with Anaplasma phagocytophilum was found. Babesia sp. was detected in n= 31 (1.0%) out of 3,113 ticks examined, which originated from 4 different areas. By sequencing, n = 28 (90.0%) were identified as Babesia divergens. Three of all Babesia-positive ticks were identified as harboring Babesia microti. The detection of Anaplasma phagocytophilum, Rickettsia sp. and Babesia sp. demonstrates their possible role as a source of human infection in Germany.

In lipopolysaccharide-stimulated blood from 71 late-stage borreliosis patients, the ex vivo cytokine release capacity of tumor necrosis factor alpha (TNF-alpha) and gamma interferon (IFN-gamma) was reduced to 28% +/- 5% and to 31% +/- 5% (P < or = 0.001), respectively, compared to that of 24 healthy controls. White blood cell counts were normal in both groups. To investigate direct interactions between the pathogen and the immune cells, blood from healthy controls was exposed in vitro to live or heat-killed Borrelia or to Borrelia lysate. Compared to the pattern induced by bacterial endotoxins, a reduced release of TNF-alpha and IFN-gamma and an enhanced secretion of interleukin-10 and granulocyte colony-stimulating factor was found. In blood from 10 borreliosis patients stimulated with Borrelia lysate, TNF-alpha formation was decreased to 31% +/- 14% and IFN-gamma formation was decreased to 8% +/- 3% (P < or = 0.001) compared to the cytokine response of blood from healthy controls (n = 24). We propose to consider anti-inflammatory changes in the blood cytokine response capacity elicited by Borrelia as a condition that might favor the persistence of the spirochete.