Use of western-immunoblotting to determine serum IgG-antibodies to antigens of different genotypes of Borrelia--Borrelia burgdorferi sensu stricto,Borrelia afzelii,and Borrelia garinii

The reaction of the sera from 86 patients with Lyme borreliosis was evaluated in the immunoblotting using three genotypes of pathogenic Borrelia strains. The Russian isolates of Borrelia afzelii (strain IP3) and B. garinii 20047T (strain IP90) were compared with the USA typical strain B31--B. burgdorferi sensu stricto. The results were assessed by the criteria recommended for the USA and developed for B. burgdorferi sensu stricto. Certain differences were shown in the reactions of serum IgG with major proteins of three Borrelia genotypes. The sera interacted with p37 of an I-90 isolate and with p39 of both Russian isolates significantly more frequently. The rate of positive results of a serum test using the strain B31 was 18.6% (16 patients); 13 patients were additionally identified when the Russian isolates were applied. It is expedient to use the genotypes circulating in Russia as an antigenic material for immunoblotting. Criteria for positive serum test results may be individual for each genotype of Borreliae.     

Borrelia burgdorferi (strain B. afzelii) antibodies among Malaysian blood donors and patients

In this study, the presence of IgG and IgM antibodies against Borrelia burgdorferi (strain B. afzelii) among Malaysian blood donors and patients admitted to hospital with various infectious diseases was determined. Sera were screened using enzyme-linked immunosorbent assay (ELISA); positive sera were then subjected to Western blot testing. All but one of the blood donors were negative for borrelial antibodies. Of 121 patients' sera, IgM antibodies were detected in 24 (19.8%) and IgG antibodies were detected in 5 (4.1%) sera. Only one of two patients with skin manifestations suggestive of Lyme disease had IgM antibody against B. afzelii. Of 30 patients with exposure to tick typhus, 4 (13.3%) were IgM positive and 1 (3.3%) was IgG positive. Based on the detection of antigenic bands by Western blot, 6 patients' sera showed positive reactions. Antigenic bands of p39, p41 and p59/62 kDa were the commonest findings of Western blotting. This study provides serological evidence of B. afzelii infections in Malaysia; further investigation is needed to correlate serological and clinical findings.     

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.     

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.     

Isolation of Borrelia burgdorferi sensu lato in ticks Ixodes ricinus by polymerase chain reaction (PCR)

Attempts were made to identify the causative orgamsm of Lyme disease in Szczecin from tick Ixodes ricinus as a vector. Ticks were collected in 1997 year in forest areas of Szczecin, from localites associated with numerous attendance of people. The method used in this study was the polymerase chain reaction (PCR) on the flagellin structural gene fla of Borrelia burgdorferi sensu stricto. The flagellin PCR primer set reaction was conservative for B. burgdorferi sensu stricto, B. afzelii and B. garinii. The overall prevalence of B. burgdorferi sensu lato, in tick population studied was 8.8%. The female, nymphs and larves of Ixodes ricinus were infected almost just the some--about 10%, when the male 2.5% only.     

Antibodies to recombinant decorin-binding proteins A and B in the cerebrospinal fluid of patients with Lyme neuroborreliosis

Cerebrospinal fluid (CSF) and serum samples from 34 patients with proven neuroborreliosis (NB) and 22 patients with suspected neuroborreliosis (SNB) from Finland were analysed for antibodies to decorin-binding proteins A (DbpA) and B (DbpB). Antibodies to recombinant protein antigens originating from Borrelia burgdorferi sensu stricto, B. afzelii, or B. garinii species were studied by enzyme-linked immunosorbent assay (ELISA). Of the 34 patients with NB, 100% of the CSF and 88% of the serum samples had IgG antibodies to 1 to 3 variants of DbpA and 79% of the CSF and 70% of the serum samples were positive for 1 to 3 DbpB variants. Antibodies to DbpB seemed to be associated with lymphocytic pleocytosis in the CSF and short duration of the disease, whereas antibodies to DbpA in the CSF were observed irrespective of the duration of the disease and lymphocytic pleocytosis. Among the variant antigens, CSF reactivity was mainly with the DbpB from B. garinii, whereas positivity with the DbpA from B. afzelii or B. garinii predominated. The results suggest that CSF antibodies to DbpB might be useful as a marker of active infection whereas antibodies to DbpA seem to persist a long time after acute phases of NB.     

Increased cerebrospinal fluid levels of glial fibrillary acidic protein (GFAp) in Lyme neuroborreliosis

Summary Glial fibrillary acidic protein (GFAp), the main protein constituent of the intermediate filaments of astrocytes, was analysed in the cerebrospinal fluid (CSF) of 20 patients with Lyme neuroborreliosis as a marker of the astroglial reaction. The mean GFAp level before antibiotic treatment in the study group was significantly elevated (592 pg/ml ±596 [SD]) compared to that in 24 healthy controls (121±87 [SD]) (p<0.01). The highest CSF-GFAp levels were seen in the patients with the most severe disease, but the levels were also increased in patients with peripheral paresis, such as facial palsy with no or only minor encephalitic symptoms. This implies that the infection was not limited to radix dorsalis or the meningeal tissues, but affected the central nervous system as well. Furthermore, the astroglial reaction seemed to occur early in Lyme neuroborreliosis since CSF-GFAp levels were elevated also in patients with recent (<3 weeks) onset of disease. After antibiotic treatment, the GFAp levels decreased. It is suggested that CSF-GFAp concentrations might be useful for monitoring CNS involvement in Lyme neuroborreliosis.

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Erhöhte Spiegel an saurem Glia-Fibrillen-Protein im Liquor bei Patienten mit Lyme Neuroborreliose

Zusammenfassung Saures Glia-Fibrillenprotein (GFAp), der Haupt-Proteinanteil der Intermediärfilamente der Astrozyten, wurde im Liquor von 20 Patienten mit Lyme Borreliose bestimmt, um seine Bedeutung als Marker für eine Astroglia-Reaktion zu prüfen. In der Patientengruppe wurden vor Antibiotikatherapie mittlere GFAp-Spiegel (*/- SD) von 592±596 pg/ml gemessen, die signifikant über den Werten bei 24 gesunden Kontrollpersonen lagen (121±87 pg/ml). Die höchsten GFAp-Liquorspiegel fanden sich bei den Patienten mit dem schwersten Krankheitsverlauf; erhöhte Werte wurden aber auch bei Patienten mit peripherer Parese wie Facialis-Parese oder mit nur leichten oder ohne enzephalitische Symptome gefunden. Dies läßt darauf schließen, daß sich die Infektion nicht auf die Dorsalwurzeln oder die Meningen beschränkte, sondern auch das Zentralnervensystem erreichte. Die Astroglia-Reaktion schien darüber hinaus schon in einem frühen Stadium der Lyme Borreliose aufzutreten, da auch bei Patienten mit einem erst kurze Zeit (<3 Wochen) zurückliegenden Krankheitsbeginn erhöhte GF-Ap-Liquorspiegel zu finden waren. Nach Antibiotikatherapie nahmen die GFAp-Spiegel ab. Wir halten die Liquor-Spiegel an GFAp für einen brauchbaren Parameter zur Überwachung eines ZNS-Befalls bei Lyme Neuroborreliose.

     

Fibroblasts protect the Lyme disease spirochete, Borrelia burgdorferi, from ceftriaxone in vitro.

The Lyme disease spirochete, Borrelia burgdorferi, can be recovered long after initial infection, even from antibiotic-treated patients, indicating that it resists eradication by host defense mechanisms and antibiotics. Since B. burgdorferi first infects skin, the possible protective effect of skin fibroblasts from an antibiotic commonly used to treat Lyme disease, ceftriaxone, was examined. Human foreskin fibroblasts protected B. burgdorferi from the lethal action of a 2-day exposure to ceftriaxone at 1 microgram/mL, 10-20 x MBC. In the absence of fibroblasts, organisms did not survive. Spirochetes were not protected from ceftriaxone by glutaraldehyde-fixed fibroblasts or fibroblast lysate, suggesting that a living cell was required. The ability of the organism to survive in the presence of fibroblasts was not related to its infectivity. Fibroblasts protected B. burgdorferi for at least 14 days of exposure to ceftriaxone. Mouse keratinocytes, HEp-2 cells, and Vero cells but not Caco-2 cells showed the same protective effect. Thus, several eukaryotic cell types provide the Lyme disease spirochete with a protective environment contributing to its long-term survival.     

Detection of Borrelia burgdorferi by polymerase chain reaction in synovial membrane, but not in synovial fluid from patients with persisting Lyme arthritis after antibiotic therapy

OBJECTIVES—To identify possible sites of bacterial persistence in patients with treatment resistant Lyme arthritis. It was determined whether Borrelia burgdorferi DNA may be detectable by polymerase chain reaction (PCR) in synovial membrane (SM) when PCR results from synovial fluid (SF) had become negative after antibiotic therapy.
METHODS—Paired SF and SM specimens and urine samples from four patients with ongoing or recurring Lyme arthritis despite previous antibiotic therapy were investigated. A PCR for the detection of B burgdorferi DNA was carried out using primer sets specific for the ospA gene and a p66 gene of B burgdorferi.
RESULTS—In all four cases, PCR with either primer set was negative in SF and urine, but was positive with at least one primer pair in the SM specimens. In all patients arthritis completely resolved after additional antibiotic treatment.
CONCLUSIONS—These data suggest that in patients with treatment resistant Lyme arthritis negative PCR results in SF after antibiotic therapy do not rule out the intraarticular persistence of B burgdorferi DNA. Therefore, in these patients both SF and SM should be analysed for borrelial DNA by PCR as positive results in SM are strongly suggestive of ongoing infection.

Keywords: Lyme arthritis; polymerase chain reaction; synovial membrane; synovial fluid     

Isolation of Borrelia burgdorferi sensu lato from the skin of the European badger (Meles meles) in Switzerland

No data are available on the role of badgers in the ecology of Lyme borreliosis spirochetes in Europe. In a recent study describing validation of a molecular method allowing host DNA identification and Borrelia burgdorferi sensu lato detection in Ixodes ricinus, the simultaneous presence of B. afzelii DNA and of European badger (Meles meles) DNA was detected in I. ricinus ticks in Switzerland. This suggested that badgers might be reservoir hosts for B. afzelii. Here, we present results obtained in a study on badgers conducted in 1996-1997. Thirty-one tissue samples (ear biopsy: n = 25, aspiration fluid: n = 6) from 8 badgers were placed in BSK medium to isolate B. burgdorferi sensu lato and were then examined by polymerase chain reaction/restriction fragment length polymorphism (PCR/RFLP). Globally, six Borrelia isolates (6/31, 19.4%) were obtained from 3/8 (37.5%) badgers. These isolates were identified as B. afzelii (n = 3) and B. valaisiana (n = 3).     

Isolation of <Emphasis Type="Italic">Borrelia burgdorferi</Emphasis> sensu lato from blood of adult patients with borrelial lymphocytoma,Lyme neuroborreliosis,Lyme arthritis and acrodermatitis chronica atrophicans

Background  

Reports on patients with European Lyme borreliosis in whom borreliae were isolated from the blood are rare and nearly exclusively limited to those with solitary or multiple erythema migrans. Here we report on patients with other manifestations of Lyme borreliosis in whom borreliae were isolated from their blood.     

T cell proliferation induced by Borrelia burgdorferi in patients with Lyme borreliosis. Autologous serum required for optimum stimulation

The cellular immune response to Borrelia burgdorferi was studied in 24 patients with seropositive and seronegative Lyme borreliosis, 30 patients with arthritides of different origin (non-Lyme arthritides), and 20 normal blood donors. By far, the strongest T cell stimulation was induced by incubation with autologous serum; there was a significantly lower response or no response after incubation with allogeneic or heterologous sera. In patients with Lyme borreliosis, including seronegative patients, there was a strikingly elevated proliferation in response to whole B burgdorferi bacteria (mean 64,750 dpm) compared with that of normal donors (mean 19,700 dpm; P less than 0.0001) and especially that of non-Lyme arthritis patients (mean 11,600 dpm; P less than 0.0001). Levels of proliferation declined significantly in patients with Lyme borreliosis after successful antibiotic treatment. Parallel cultures using B burgdorferi and Treponema phagedenis as antigens showed that cells from patients with Lyme borreliosis responded significantly more to B burgdorferi than to T phagedenis, but this did not occur with cells from individuals with non-Lyme arthritides. There was no correlation between disease stages and proliferation values. These data indicate that lymphocyte proliferation assays may provide an important tool for the diagnosis of Lyme borreliosis, most notably in patients with arthritides and in those who are seronegative. Conversely, the lack of reactivity appears to be a strong indicator of the absence of active Lyme disease. It seems to be crucial, however, to use autologous sera in these assays.     

Evaluation of occurrence of spirochetes Borrelia burgdorferi sensu lato in Ixodes ricinus ticks in selected areas of the Lublin region by polymerase chain reaction method (PCR)

During the period 2001-2002, 1098 Ixodes ricinus ticks were collected at forest sampling sites and the degree of their infection with Borrelia burgdorferi spirochetes was determined by means of polimerase chain reaction (PCR). The presence of Borrelia burgdorferi genetic material was noted in 69 cases (6.3%). It was confirmed that the frequency of infection of adult forms of ticks (males and females) was nearly twice as high as nymphs. The highest degree of infection was observed in females--9.5%. The degree of infection among males and nymphs was smaller--5.9% and 4.4% respectively in individual provinces. The percentage of infected females ranged from 7.9% in the Zamo?? Province to 13.6% in the W?odawa Province. In males, the percentage of infected ticks remained within the range from 3.1% in the Lublin Province to 13.3% in the Lubartów Province.     

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.     

A comparison of two DNA extraction approaches in the detection of Borrelia burgdorferi sensu lato from live Ixodes ricinus ticks by PCR and reverse line blotting

We tested two approaches to extract Borrelia DNA from live Ixodes ricinus ticks before polymerase chain reaction (PCR) and reverse line blotting (RLB): DNA extraction of one half of the tick after incubation in BSK medium and DNA extraction of the other half of the tick directly, using ammonium hydroxide. Among 2079 ticks, 31.2% (n=649) were found to be Borrelia-infected by PCR-RLB test using at least one of the DNA extraction methods. Five hundred four ticks (24.2%) were found infected after incubation in BSK and 481 (23.1%) after direct DNA extraction from the tick. The difference was not significant. However, these prevalences were significantly lower when only one method was applied (23.1% and 24.2%) compared to the prevalence obtained by the use of both methods (31.2%). In 313 infected ticks discordant results were obtained, i.e., one half of the tick was found to be infected whereas the other half was uninfected. Among these ticks, B. garinii and B. burgdorferi sensu stricto (ss) were significantly more frequently identified in the half tick incubated in BSK. No significant differences were observed for B. burgdorferi ss, B. valaisiana, and for undetermined Borrelia species.     

Seroprevalence and geographic distribution of Dirofilaria immitis and tick-borne infections (Anaplasma phagocytophilum, Borrelia burgdorferi sensu lato, and Ehrlichia canis) in dogs from Romania

Tick-borne diseases are of great concern worldwide. Despite this, in Romania there is only limited information regarding the prevalence of vector-borne pathogens in dogs. In all, 1146 serum samples were tested by SNAP(?) 4Dx(?) (IDEXX Laboratories, Inc., Westbrook, ME) for Anaplasma phagocytophilum, Borrelia burgdorferi, and Ehrlichia canis antibodies, and for Dirofilaria immitis antigen. The correlation between positive cases and their geographic distribution, as well as potential risk factors (age, sex, breed, type of dog, habitat, and prophylactic treatments) were evaluated. Overall, 129 dogs (11.3%) were serologically-positive to one or more of the tested pathogens. The seroprevalence for the four infectious agents were: A. phagocytophilum 5.5% (63/1146), D. immitis 3.3% (38/1146), E. canis 2.1% (24/1146), and B. burgdorferi 0.5% (6/1146). Co-infection with E. canis and A. phagocytophilum was registered in 2 dogs (0.2%). The geographical distribution of the seropositive cases suggests clustered foci in southern regions and in the western part of the country for D. immitis, and in the southeastern region (Constan?a County) for E. canis. A. phagocytophilum and B. burgdorferi showed a homogenous distribution, with a tendency for Lyme-positive samples to concentrate in central Romania. For D. immitis, A. phagocytophilum, and E. canis, administering prophylactic treatments was a risk factor associated with infection. Another associated risk factor was the type of dog (stray dogs were at risk being positive for D. immitis, shelter dogs for E. canis, and hunting dogs for B. burgdorferi). The prevalence of D. immitis was significantly higher in males and in dogs older than 2 years. This survey represents the first data detailing A. phagocytophilum and E. canis seroprevalence in Romanian dogs, and the most comprehensive epidemiological study on vector-borne infections in dogs from this country.