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Persistence of borreliae within the vertebrate host depends on the fate of interactions between the spirochetes and target cells. The present work demonstrates the direct binding of the Borrelia burgdorferi outer surface proteins OspA and OspB to CR3 and that this binding is independent of iC3b.

This study reports for the first time that leptospires are killed by H(2)O(2) and by low-molecular-weight primary granule components, which are agents normally released by neutrophils upon stimulation. Although both pathogenic and nonpathogenic strains were sensitive to H(2)O(2)-mediated killing, nonpathogenic organisms were found to be more susceptible. In addition, the killing of leptospires by H(2)O(2) was found to be independent of the presence of the neutrophil primary granule component myeloperoxidase and therefore not a consequence of halogenation reactions. We have also determined that leptospires are significantly sensitive only to primary granule components and, among those, to proteins and/or peptides of less than 30 kDa.

Between 1993 and 1998, we isolated Borrelia burgdorferi sensu lato from 55 of the 119 patients with clinically diagnosed Lyme borreliosis who were admitted to "San Martino" Hospital in Belluno, Veneto, an Adriatic region in northeastern Italy where Lyme borreliosis is endemic. Upon hospitalization, all patients presented erythema migrans. Isolates were typed using ribosomal DNA PCR-restriction fragment length polymorphism (RFLP) analysis of the rrfA-rrlB intergenic spacer. Of the 41 isolates typed, 37 belonged to Borrelia afzelii, 2 to Borrelia garinii, and 2 to B. burgdorferi sensu stricto. Pulsed-field gel electrophoresis, performed on 21 strains (13 new isolates and 8 controls), revealed different RFLP patterns within the B. garinii and B. afzelii strains; among the five B. garinii strains and the 12 B. afzelii strains, three or two different RFLP patterns were identified, according to the restriction enzyme used. The protein patterns of the new isolates confirmed their genotypic classification and revealed the level of expression of some immunodominant proteins like OspA and other characteristic Osps. These findings constitute the first report of such a high recovery rate of B. burgdorferi from patients in a very restricted area in Italy; they also indicate the predominance of the genospecies B. afzelii in the study area and the heterogeneity of the circulating strains.

The mannose receptor (MR) plays an important role in the recognition of some pathogens in nonopsonic phagocytosis and in antigen presentation to T cells. We found that Borrelia burgdorferi, the agent of Lyme borreliosis, adheres to monocyte-derived macrophages and to rat MR-transfected cells but not to untransfected cells. Antibodies to MR and sugars such as mannose, mannan, fucose, and some lectins significantly lowered the adhesion, confirming participation of the MR in the binding.

The MIC and MSC (minimum spirocheticidal concentration) and killing rate for Borrelia burgdorferi, the etiological agent of Lyme disease, were assessed for cefodizime in comparison with ceftriaxone, minocycline, azithromycin, roxithromycin, and ciprofloxacin. The range of cefodizime MICs was greater than those of azithromycin and roxithromycin but comparable to those of ceftriaxone and minocycline. The MSCs were 1 to 2 dilutions higher than the MICs of all of the tested compounds. The killing curves of cefodizime and ceftriaxone showed parallel courses. In conclusion, cefodizime exerted an activity comparable to that of ceftriaxone against B. burgdorferi.

Phagocytosis of Borrelia burgdorferi by human polymorphonuclear leukocytes triggers oxygen-dependent and -independent mechanisms of potentially cidal outcome. Nevertheless, no factor or process has yet been singled out as being borreliacidal. We have studied the B. burgdorferi-killing ability of the myeloperoxidase-H2O2-chloride system and that of primary and secondary granule components in an in vitro assay. We found that neither secondary granule acid extracts nor the chlorinating system could kill these microorganisms, while primary granule extracts were effective. The Borrelia-killing factor was purified to homogeneity and demonstrated to be elastase. Its cidal activity was found to be independent of its proteolytic activity.

The mechanisms involved in coiling phagocytosis are not yet known, and it is not even clear whether this phenomenon is either an incidental event or a specific response. Therefore, the phagocytic uptake of Borrelia burgdorferi and other spirochetes by human monocytes in vitro was used to investigate the involvement of both sides--microbes and phagocytes--in coiling phagocytosis. As seen with electron microscopy, morphologically similar Borrelia, Leptospira and Treponema strains induced markedly different frequencies of coiling phagocytosis. The monocytes used coiling phagocytosis for both live (motile) and killed (nonmotile) B. burgdorferi, but pseudopod coils were observed neither with fragmented B. burgdorferi nor with cell-free supernatant from B. burgdorferi cultures. Investigation of the relationship of coiling phagocytosis with other pseudopod-based cellular mechanisms revealed that the use of bioreagents that inhibit conventional phagocytosis also inhibited coiling phagocytis but did not affect membrane ruffling. Bioreagents that increase membrane ruffling did not affect phagocytosis of B. burgdorferi, except for granulocyte-macrophage colony-stimulating factor and phorbol myristate acetate, which increased coiling phagocytosis selectively. These results demonstrate that coiling phagocytosis is not induced by microbial motility, viability, or a certain morphology and that it is not a random event. Rather, it is a selective uptake mechanism actively driven by the phagocytes. However, whether coiling phagocytosis represents an independent alternative to conventional phagocytosis or, alternatively, a fault in conventional phagocytosis remains to be determined.

Like other pathogens, the spirochete Borrelia burgdorferi, the agent of Lyme disease, possesses multiple pathways for cell binding; adhesion to phagocytic cells is of particular interest since it reportedly occurs even in the absence of specific antibodies. This study sets out to investigate how B. burgdorferi binds to human polymorphonuclear leukocytes (PMNs) when an exogenous complement is added and how the CR3 complement receptor, known as Mac-1 or alpha(m)beta2 integrin, is involved in the binding process. Experiments performed on PMNs and CHO Mac-1-expressing cells demonstrate that binding is inhibited by monoclonal anti-iC3b site antibodies, fibrinogen, and N-acetyl-D-glucosamine. These findings, which are not present with non-Mac-transfected CHO cells, indicate that the integrin alpha(m)beta2 acts as a receptor for spirochetes in nonimmune phagocytosis; furthermore, binding occurs on different domains of the CD11b subunit, involving the iC3b site and the lectin domain. The interaction of B. burgdorferi with alpha(m)beta2 integrin adds a novel pathway to Borrelia-phagocyte binding; not only does this binding affect the early stages of phagocytosis, but also it can influence the effector intracellular mechanisms which are activated by the beta2 integrin, as are the cytotoxic mechanisms.

We have examined the effect of the virulent Leptospira interrogans strain Teramo, serotype icterohemorrhagiae, on the adherence of human neutrophilic polymorphonuclear leukocytes (PMN) to cultured human umbilical vein endothelial cells (HEC). Selective pretreatment of HEC with intact or sonicated leptospires caused a dose- and time-dependent increase of HEC-PMN adhesion (13.2% +/- 2.5% adherence to untreated HEC versus 46.3% +/- 5.6% adherence to HEC pretreated for 4 h with 10(8) intact leptospires per ml [mean +/- standard error of six experiments; P < 0.001]). In contrast, selective leptospire pretreatment of PMN or the addition of leptospires during the adherence assay did not alter HEC-PMN adherence. Leptospire induction of endothelial-cell adhesiveness occurred without detectable HEC damage and was prevented by RNA and protein synthesis inhibitors and by monoclonal antibodies to the CD11/CD18 adhesion complex of neutrophils and to the endothelial-leukocyte adhesion molecule 1 (ELAM-1) of endothelial cells. Similar results were obtained with pretreatment of HEC with interleukin-1 or with the lipopolysaccharide (LPS) of the gram-negative bacterium Escherichia coli. The possibility that contamination by the LPS of gram-negative bacteria could be involved in the induction of HEC adhesiveness was ruled out by the observation that the LPS inhibitor polymyxin B, which abolished the proadhesive effect of E. coli LPS, was ineffective in inhibiting leptospire- as well as interleukin-1-induced adherence. Similarly, leptospire LPSs seemed to have no role in the increase of endothelial-cell adhesiveness, since pretreatment of HEC with a leptospire LPS extract (phenol-water method) or with a leptospire total lipid extract failed to induce the proadhesive phenotype for neutrophils. Instead, peptidoglycans extracted from our leptospires actively stimulated the endothelial proadhesive activity for neutrophils (16.5% +/- 2.1% adherence to untreated HEC versus 51.2% +/- 2.9% adherence to HEC pretreated for 4 h with 1 microgram of peptidoglycan per ml; [mean +/- standard error of four experiments; P < 0.001]). This peptidoglycan-induced activity was inhibited by monoclonal antibodies to the CD11/CD18 adhesion complex and to ELAM-1 but not by polymyxin B. We conclude that peptidoglycans from pathogenic leptospires are among the molecules that can directly activate vascular endothelial cells to increase their adhesiveness for neutrophilic granulocytes. These observations may contribute to a better understanding of the mechanisms whereby non-gram-negative bacteria modulate the local and systemic inflammatory reaction.

The activity of rokitamycin, a new macrolide with a 16-member ring, was tested against Borrelia burgdorferi in vitro. The antibiotic had a lower MIC at which 50% of the isolates are inhibited than erythromycin, the parent 14-member macrolide, but the same MIC at which 50% of the isolates are inhibited as the other recent 14- and 15-member macrolides, like clarithromycin and azithromycin. The MBC was equal to the MIC at which 50% of the isolates are inhibited, so rokitamycin can be considered bactericidal against B. burgdorferi. The sensitivity of the Borrelia strains tested was not correlated with the particular species Burgdorferi sensu stricto, B. garinii, and B. afzelii or with the number of subcultures of the isolates.

Bac5 and Bac7 are antimicrobial peptides of bovine neutrophils that act on enteric gram-negative bacteria. We report here that these two peptides immobilize and kill Leptospira interrogans and Leptospira biflexa with MBCs of 6 to 25 micrograms/ml. Conversely, although both peptides bind to Borrelia burgdorferi, the organism is resistant to their action.

Four Borrelia burgdorferi strains isolated in the same restricted geographic area share different protein patterns on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The use of polyclonal rabbit antisera and a battery of monoclonal antibodies directed toward the immunodominant proteins OspA, OspB, and pC also revealed different epitope distributions and specificities of these antigens on the strains examined. For the first time, a computerized analysis of these phenotypic characters was done mainly by cluster analysis. The computerized analysis revealed the levels of similarities among the strains and indicated, on a quantitative basis, that one of them is much closer to the American strain B31 than to the other strains.

Eighteen patients involved in a localized outbreak of leptospirosis were subjected to a serological follow-up study over a 5-year period. Four distinct sets of sera from all patients and a fifth sample obtained from 10 of them were examined by the microscopic agglutination test (MAT) for demonstration of leptospiral antibodies. The test was carried out by using live leptospires from reference strains of 17 Leptospira interrogans serovars known to occur in Italy. In all cases, the highest titers of agglutinins were recorded against one or more of the three Australis group serovars tested (australis, bratislava, and lora). The highest antibody levels were reached soon after the acute phase of infection in some patients but only after some months in others. Titers then tended to recede with varying rapidity, but titers against the Australis group serovars were still detectable in some patients after 5 years. Coagglutinins against serovars of other serogroups were detected, generally at low levels, in the early sets of sera of most patients, but tended to disappear in the late-set sera. Specific immunoglobulin M (IgM) and IgG against the three Australis group serovars were determined in most serum samples from 16 patients by solid-phase enzyme immunoassay (EIA). In general, EIA titers were considerably lower than MAT titers, but there was a certain patient-to-patient variability in both the IgM/IgG ratio and the evolution and persistence of the two immunoglobulin classes. Since all the evidence indicated that the initial outbreak from a single source, the observed patient-to-patient variability in the progress of both MAT and EIA titers appeared to be attributable to factors inherent in the individual patients. Cross agglutination absorption tests, aimed at retrospectively determining to which of the Australis group serovars the outbreak-specific infecting strain belonged, were performed with six serum samples from different patients. Most absorbed sera seemed to originate from an australis or lora infection, but it was not possible to discriminate conclusively between the two serovars.

I assayed the lack of reactivity of monoclonal antibody (MAb) F9-4 with nonpathogenic leptospires. Of 47 saprophytic strains tested, 46 did not react in an enzyme immunoassay and 1 was recognized by MAb F9-4, as usually reported with pathogenic Leptospira strains. On the other hand, the MAb did not react with one pathogenic strain, thus showing that the ability of MAb F9-4 to discriminate between pathogenic and nonpathogenic leptospires is not absolute.