Characterization of an anti-Borrelia burgdorferi OspA conformational epitope by limited proteolysis of monoclonal antibody-bound antigen and mass spectrometric peptide mapping

Lyme borreliosis is a multisystem disorder caused by the spirochete Borrelia burgdorferi that is transmitted to humans by the tick Ixodes dammini. The immune response against the 31 kDa OspA, which is one of the most abundant B. burgdorferi proteins, appears to be critical in preventing infection and tissue inflammation. Detailed knowledge of the immunological and molecular characteristics of the OspA protein is important for the development of reliable diagnostic assays. In this study, we characterized a new conformational epitope present within the middle part of B. burgdorferi OspA. Our approach used enzymatic proteolyses of the immune complex followed by mass spectrometric identification of the peptides bound to the antibody. It appears to be one of the first reports on the characterization of a discontinuous epitope using mass spectrometry.     

Negative incidence of Lyme disease-related Borrelia spp. in Alishan, Taiwan

To investigate the prevalence of Lyme disease-related Borrelia species, wild rodents were captured around Yushan National Park and Alishan Forest Recreation Area Park in Taiwan 2,000 to 3,000 meters above sea level. Borrelia was not isolated from 67 small mammals of 7 species. Sera from rodents showed no positive reactivity against whole cell antigens of B. garinii, B. afzelii or B. valaisiana by ELISA. These results suggested that Lyme disease is not endemic to the Alishan area.     

Preliminary Observations on Ultrastructure of Borreliae in Tissues of Ixodes persulcatus

We observed Lyme borrelia by electron microscopy in the tissues of the ticks, Ixodes persulcatus, which were indicated positive for borreliae by BSK cultures of their internal organs. Borreliae (0.25 μm in diameter) were found only in the lumen of the midgut. They were closely associated with the microvilli on the midgut epithelium but never penetrated into the epithelial cells. Ultrastructural features common to Lyme borreliae., i.e., the three-layered membranes surrounding the cytoplasm and orientation of the flagella insertions, were obviously confirmed. The present results are useful to understand tick tissue-borrelia interface.     

The genetic structure of Borrelia afzelii varies with geographic but not ecological sampling scale

The genetic structure of a pathogen is an important determinant of its potential rate of adaptation and can thereby influence the dynamics of host-parasite interactions. We investigated how the genetic structure of Borrelia afzelii varies with geographic and ecological sampling scale. Genetic structure was measured as the degree of linkage disequilibrium (LD) across three loci. To test for the effects of geographic and ecological scale, we calculated LD across or within populations 4-82 km apart and across or within different mammal host species. There was highly significant LD across populations and host species. However, there was also evidence for genome-wide recombination, and the LD largely resulted from epidemic spread of certain haplotypes, rather than lack of recombination. Interestingly, the degree of LD was higher in each population than in the sample as a whole, i.e. LD increased with decreasing geographic scale. In contrast, there was no effect of ecological sampling scale on LD. Strong LD may impede the rate of adaptive evolution. Our results suggest this effect might be particularly strong at a small geographic scale.     

Complement-Mediated Killing of Borrelia garinii —Bactericidal Activity of Wild Deer Serum

The susceptibility of Borrelia garinii to fresh wild deer sera was determined by incubating strain SIKA2 at 10% serum concentration for 1 hr at 37 C in an in vitro bactericidal assay. Each serum showed bactericidal effects at various levels. The effect was dependent on the concentration of antibody to the spirochetes. Complement was essential in the bactericidal assay because the inactivated deer serum showed greatly decreased activity. Our results suggest that B. garinii is sensitive to deer serum, in the presence of antibody and the bactericidal effect is important for preventing Lyme disease in wild sika deer.     

Biopsy of canine ear tissue for detection of Borrelia burgdorferi following Ixodes dammini feeding

A punch biopsy method was developed to detect and cultivate Borrelia burgdorferi from canine ear tissue. The biopsy procedure was sensitive in that spirochetes were detected in 100% of the dogs upon which infected ticks had fed. B. burgdorferi could be cultured as early as 6 days after tick feeding at which time the organisms could not be concurrently recovered from peripheral blood. The ear punch biopsy provides a reliable method for sampling live canines and should prove useful for detecting early B. burgdorferi infection and for monitoring duration of non-disseminated infection. Additionally, the procedure will aid in the development of an animal model for evaluation of preventive and therapeutic treatment modalities.     

Borrelia tanukii Sp. Nov. and Borrelia turdae Sp. Nov. Found from Ixodid Ticks in Japan: Rapid Species Identification by 16S rRNA Gene-Targeted PCR Analysis

Based on the results of RFLP-ribotyping, whole DNA/DNA hybridization and phylogenetic analysis of the 16S rRNA gene, we previously defined two genomic groups of spirochetes closely related to Borrelia burgdorferi sensu lato: group Hk501 for strains isolated from Ixodes tanuki ticks and group Ya501 for strains isolated from Ixodes turdus ticks. In this report, we propose that group Hk501 should be classified as Borrelia tanukii sp. nov. and group Ya501 as Borrelia turdae sp. nov. The alignment of previously published Borrelia 16S rRNA gene sequences led us to design species-specific PCR primer sets. The primers allowed the rapid identification of B. tanukii and B. turdae.     

Simultaneous measurement by flow cytometry of phagocytosis and metabolic burst induced in phagocytic cells in whole blood by Borrelia burgdorferi

Abstract This paper describes the interactions between a strain of Borrelia burgdorferi and phagocytic cells, measured in whole blood, by a two-color flow cytometric method, which allowed the simultaneous quantification of both the phagocytosis rate and the oxidative burst activation. The data obtained indicated that: a) phagocytosis and metabolic activation increased as a function of spirochete concentration; b) the number of ingesting cells peaked within 10 min but activation followed later, and did not involve all the phagocytosing cells; c) opsonization of borreliae with a patient's serum enhanced the two cellular activities, mostly phagocytosis. The intensity of such functions was lower than those found for Staphylococcus aureus . The flow cytometric assay of phagocytosis interactions with Borrelia burgdorferi assessed in whole blood represents an experimental approach which simulates the physiological conditions in nature.     

Experimental Induction of Lyme Arthritis in Outbred Mice

Outbred ddY mice inoculated with live cells of Borrelia burgdorferi strain 297 into hind footpad displayed swelling of the footpad at days 7 to 11 after inoculation. Marked neutrophilic infiltration was observed in the subcutaneous tissue and the part of bone tissue which was partially destroyed, and synovial layer of articular capsule was thickened and protruded into the joint space in the histopathological examination of footpad inoculated with live Borrelia cells. The inflammation peaked at day 7 and B. burgdorferi was cultured from bladder and heart of the mice at day 14 after inoculation. The mice inoculated with heat-inactivated cells at 56 C for 30 min did not show any significant histopathological change. In this mice model, nontreated littermates were not infected in contact with infected littermates for 14 days of experimental period. The outbred ddY mice model is useful for evaluating the effectiveness of vaccination against Lyme disease.