Biomedical subjects
Publications and source records attributed to M Cinco.
To evaluate the diffusion of Borrelia burgdorferi in Tuscani (Italy) 720 ticks were collected and subsequently cultured for Borrelia burgdorferi. A strain of Borrelia burgdoferi was isolated from one tick; this being the first such reported identification in Central-Southern Italy.
A leptospira-like spirochete was isolated from a lymphocyte culture from a HIV-I and HTLV-I/II-positive patient. On the basis of serological, biological and morphological characteristics of the cells of the isolated strain (strain Lisboa), we conclude that it is a member of the genus Leptonema.
Peptidoglycan (PG) has been isolated from some species of spirochaetes, including Leptospira interrogans. Although leptospiral PG has been chemically characterized, no study has been carried out on its potential biological activity. Since PG of Treponema and Borrelia is biologically active both in vivo and in vitro, we investigated the capacity of a leptospiral PG preparation to induce relevant biological effects. PG extracted from L. interrogans strain Teramo was mitogenic at 0.1 microgram ml-1 for human peripheral blood mononuclear cells (PBMC) since it increased the PBMC fraction positive for Ki-67, an antigen expressed by human proliferating cells; at 4 micrograms ml-1, PG was able to induce complement consumption and to stimulate leucocyte phagocytosis and the metabolic burst of resting as well as phagocytosing leucocytes. These findings indicate that Leptospira PG may play a role in modulating the immunocompetent cell functions and suggest that PG can contribute to the host response during Leptospira infection.
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.
In this study we investigated the genotypic characteristics of some locally isolated strains of B. burgdorferi by three different methodologies: restriction endonuclease analysis (REA), Southern blot hybridization with whole DNAs from Borrelia strains and Southern blot hybridization with rRNA 16 + 23S genes derived from E. coli. REA fingerprintings were evaluated by cluster analysis, according to the principles of numerical taxonomy. The genomas of the locally isolated strains were compared with borreliae originating from different countries of Europe, including Sweden and with the American reference strain B31. Among the European strains, some already described by Baranton (Baranton et al., 1992) as representatives of different genomic groups Borrelia sensu stricto and Borrelia garinii were used. By the different techniques the isolates were included in three genomic groups which could correspond to the three genospecies identified by Baranton, namely B. burgdorferi sensu stricto, B. garinii and B. group VS461: in fact two strains were included in a homogeneous group, probably corresponding to the VS461 genomic group, together with other European borreliae; one isolate was included in a group consisting of B31 and some other European strains already described as belonging to Borrelia burgdorferi in sensu stricto. Finally two isolates were ascribed to a third genomic group probably corresponding to the genospecies indicated as Borrelia garinii. These findings indicate that a small number of Borrelia strains isolated from a very restricted area can be genetically heterogeneous.
A recombinant probe derived from a genomic library of serovar hardjo strain Hardjoprajitno, and a panel of serovar specific Monoclonal Antibodies (MAbs) were used for the characterization of 31 Leptospira isolates from cattle and swine. The two methods performed equally well in serovar identification except for the distinction of the genotypes hardjoprajitno and hardjobovis within serovar hardjo which could only be obtained by genomic analysis. The combination of immunological and genetic information was also useful to evaluate the degree of variability of Leptospira strains. The quality of the patterns and the sensitivity provided by a digoxigenin labelled probe were comparable to those obtained with a radioactive reagent.
This investigation was undertaken to select antigenic variants of a Borrelia burgdorferi strain in vitro. The original strain BITS was cultivated in BSK medium supplemented with increasing concentrations of homologous hyperimmune serum raised in rabbits. After a few serial passages starting from a subinhibitory serum dilution of 1:800 in BSK up to 1:200, a variant named BITSv was obtained; it grew abundantly like the control culture in the presence of hyperimmune serum. Analysis of the antigenic pattern of the original and derived variants by Western blotting revealed that BITSv, compared to the original strain BITS, had lost the reactivity with the immune serum at the level of the oligosaccharide moiety. These experiments, designed to mimic the possible action of antibodies that arise during a Borrelia infection, suggest that lipopolysaccharides are surface located and that they play a role in the integrity of the outer membrane during the multiplication of Borrelia burgdorferi.
An enzyme-linked immunosorbent assay (ELISA) was assessed to detect in humans IgM and IgG against the main serogroups/serovars of Leptospira present in Italy. Sonicated antigens from strain Wijnberg, serogroups Icterohaemorrhagiae and Riccio 2, serogroup Australis, were used in ELISA and the results were compared to microagglutination test (MAT). IgM were confirmed to be the predominant class of antibodies; the total sensitivity obtained by Wijnberg/ELISA with the sera of patients infected by different serogroups was 81.5% in relation to MAT, better sensitivity was achieved by the use of Riccio 2/ELISA; no reaction occurred with the control sera. Cross-reactivity was noted with some sera of patients with Lyme disease (IgM) and with few sera of patients with autoimmune disease (IgM and IgG). To improve the sensitivity of the ELISA both antigens Wijnberg and Riccio 2 must be employed in the immunoenzymatic assay, since serogroup Australis must be considered as a new emerging serogroup causing human leptospirosis in our country.
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Serum samples from patients infected by organisms of Leptospira interrogans serogroup Australis were tested by Western blot to determine the nature of major antigens that are involved in the immune response. Although there was some patient-to-patient variability, immunodominant genus-specific antigens were found to be proteins of apparent molecular ratio 68, 46 and 35-kDa, and lipopolysaccharide (LPS) sub-units in the 35-14-kDa region. Serogroup epitopes specific for Australis were exclusively saccharides of about 32 and 24 kDa: a serovar-specific antigen for serovar lora was of 38-40 kDa and behaved like a protein. Antibodies to the LPS serogroup-specific antigens and to the 38-40 kDa protein were long-lasting and consequently suggest that these immunodominant epitopes are important in resistance to re-infection.
This paper describes the antigenic characteristics of a strain of Borrelia burgdorferi isolated from a patient seronegative for Lyme borreliosis, in the early stage of the illness. The strain was not recognized by a late serum sample from the patient; the isolate reacted in immunoblotting with some of the monoclonal antibodies directed against the immunodominant antigens of Borrelia burgdorferi. In addition to the OspA antigen this strain carries also the epitopes of OspB proteins, unlike the majority of European Borrelia burgdorferi strains.
Seven strains of Borrelia burgdorferi isolated from ticks and from human beings in Europe and U.S.A. were analyzed for DNA restriction patterns with several enzymes and for DNA homology in Southern blot hybridizations. The restriction patterns showed a moderately high variability. In Southern blot hybridization, strain B31 (U.S.A.) DNA gave a strong signal with itself, strain Bsf (U.S.A.) and Alcaide (isolated in Italy but presumably contracted in Venezuela). Strain B45 (F.R.G.) hybridized to itself, strain BITS (Italy) and to strain D.A. (Italy). Strain Nancy (Italy) gave a signal only when hybridized to itself, although it was classified as Borrelia on the basis of the clinical manifestations, SDS-PAGE protein pattern and antigenic determinants. No hybridization differences were observed for strains isolated from different hosts in the same continental geographical area.
SDS-PAGE and Western immunoblot profiles have been determined for different strains of Borrelia burgdorferi. Major proteins of 60 kDa, 41 kDa corresponding to flagellin, 34-36 kDa and 30-31 kDa corresponding to OspB and OspA respectively, and 18-20 kDa corresponding to 'pC' fractions were detected. A "rough" lipopolysaccharide which we called lipooligosaccharide (LOS) of 8-11 kDa appeared to be present, being detected by specific silver staining, as in crude Borrelia lysates as in proteinase K digested Borrelia strains, quite similar in shape among the different strains examined. The LOS reacted in Western blotting with immune anti-B. burgdorferi rabbit serum and also with sera collected from humans affected by Lyme borreliosis. The LOS did not react with sera positive for syphilis or leptospirosis, and their immunological specificity is discussed.
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.
After preliminary in vitro screening of 17 newly synthesized compounds belonging to the chemical class of N1-(aryliden)-2-pyridinecarboxyamidrazones, active against Mycobacterium tuberculosis H37Rv, the minimum inhibitory concentrations (MICs) of the ten most promising agents against three clinical isolates were determined by agar dilution. Compounds 12 and 14 were the most active, each inhibiting strain H37Rv at concentrations of 8 microg/ml and having a MIC of 16 microg/ml against the human isolates. The results obtained in this preliminary study confirmed the interesting antitubercular properties of these newly synthesized compounds and allowed us to carry out our investigations over a large number of isolated clinical strains.
The slot blot method of DNA hybridization was used to study 38 strains of Leptospira biflexa belonging to 38 serovars. Fifteen of these serovars were placed into six groups. The remaining 23 serovars were generally too diverse to show significant DNA relatedness either to these groups or to one another. Serovar thracia was related to Group 5, but it was not included in this group because its percent relatedness was too low. We found that genetically related organisms were antigenically dissimilar. The absence of any significant genetic relationship between Leptonema illini and the Leptospira biflexa serovars tested supports the placement of the former species in a separate genus.