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It was attempted to isolate antigens from Treponema Reiter, relevant to syphilis serology, by immunoadsorption with patients' antibodies coupled to CNBr Sepharose 4B. One antigen was desorbed by 2 M KSCN in 0.05 M Tris barbital buffer, pH 8.6. The recovery was 3% and 7% in two experiments. A small amount of human antibodies in the isolate was removed on an immunoadsorbent column with insolubilized rabbit antibodies against normal human scrum proteins. The antigen thus obtained was immunologically pure when analysed by crossed immunoelectrophoresis. By electron microscopy of immunoprecipitates and by tandem crossed immunoprectrophoresis it was shown that the antigen differed from the flagellar antigen of T. Reiter, but was identical to antigen d previously described in T. Reiler. Antigen d could also be isolated from supernatants of 7% Reiler cultures. The d antigen was not denatured at pH 2.8. by 8 M urea or by 3 M KSCN, and it resisted heating to too C for 30 min. No protein could be detected in a concentrated preparation, and the antigen might be a polysaccharide. Antigen d is probably present in the sorbent used in the fluorescence treponemal antibody absorption (FTA-ABS) test and may constitute the active substance of this reagent. [ABSTRACT FROM AUTHOR]

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.

An increasing number of phenotypically heterogeneous borrelia isolates have been obtained by culture. Genetic analysis of some of them suggest that a reorganisation in delineation of the different species within the genus Borrelia may be necessary. Specifically increasing numbers of variants phenotypically different from the American type strain of B. burgdorferi have been described. In this workshop experts have discussed phenotypic and genotypic characters for (a) differentiation of B. burgdorferi strains from other borrelia and from treponemes, (b) characterization of B. burgdorferi isolates at the subspecies level. Ultrastructure was discussed by K. Hovind-Hougen, antigen structure by B. Wilske and genotypic characters by G. Baranton, A. G. Barbour, R. C. Johnson and V. Preac-Mursic. J. F. Anderson discussed differences between strains isolated from a wide range of vectors and hosts in North America. The outcome of the workshop was that B. burgdorferi may comprise different genomic species which however share common epitopes recognized by certain monoclonal antibodies. At the subspecies level a broad heterogeneity was demonstrated using methods as restriction endonuclease analysis, hybridization with whole B. burgdorferi-DNA or specific probes as well as plasmid analysis. A serotyping system based on monoclonal antibody reactivity against the outer surface protein OspA was proposed. A comparison of the different typing methods is impaired by use of different strains for analysis. In the future a broad variety of phenotypically different and defined strains need to be analysed for genotypic clusters and later on reexamined for phenotypic characters. To accomplish this close cooperation of different research groups is necessary.

Pathogenic and non-pathogenic spirochetes isolated from the intestines of pigs were examined by electron microscopy using the negative staining and ultrathin sectioning techniques. Morphological differences were observed among cells of different strains. The cells differed in length as well as in width and in the number of flagella inserted at each end. In addition, the cells from different strains also varied in their resistance to the action of the detergents, Teepol and sodium deoxycholate. Three of the strains studied contained weakly haemolytic spirochetes, two of which differed markedly in their morphology from the cells of the other strains. These spirochetes had fewer flagella inserted at each end than those from other isolates and showed a distinct lattice-like substructure covering the ends of the cells. The spirochetes examined were found to be morphologically more similar to those of the genus Borrelia than to those of the genus Treponema but were clearly different from the cells of both of these genera. The taxonomic implications of the observations are discussed in brief.

Previous studies on cells of strains B31 isolated in the U.S.A. from Ixodes dammini and strain G25 isolated in Sweden from Ixodes ricinus, showed that their ultrastructure was similar, but not identical. For this reason the studies were extended to spirochetes isolated directly from patients with Lyme disease and related disorders. Included in the present study were three strains isolated from skin, blood and spinal fluid, respectively, from patients with Lyme disease, two strains from patients with erythema chronicum migrans and one strain from a patient with acrodermatitis chronica atrophicans. Three additional strains isolated in Sweden from Ixodes ricinus were also studied. All spirochetes were examined after negative straining with 1% ammonium molybdate. The cells of each individual strain were identical except for one strain isolated from a tick. This isolate was found to consist of two morphologically different spirochetes. Comparison of morphological features of cells from various isolates revealed certain differences. The cells of the different strains could be divided into at least four groups for which cell size and shape as well as number of flagella varied. By morphological criteria, all cells were found to belong to the genus Borrelia.

Mucosal biopsy specimens from 300 consecutive patients with symptoms requiring sigmoidoscopy were examined for spirochetosis by light and electron microscopy. Colorectal spirochetosis was detected in 15 of the patients (5%). Apart from the spirochetal infestation, the mucosa appeared normal with no inflammatory reaction. The spirochetes were eliminated upon treatment with neomycin and bacitracin, but the symptoms of the patients remained unchanged. It is concluded that in the present material, colorectal spirochetosis was without clinical significance.

It was attempted to isolate antigens from Treponema Reiter, relevant to syphilis serology, by immunoadsorption with patients' antibodies coupled to CNBr--Sepharose 4B. One antigen was desorbed by 2 M KSCN in 0.05 M Tris--barbital buffer, pH 8.6. The recovery was 3% and 7% in two experiments. A small amount of human antibodies in the isolate was removed on an immunoadsorbent column with insolubilized rabbit antibodies against normal human serum proteins. The antigen thus obtained was immunologically pure when analysed by crossed immunoelectrophoresis. By electron microscopy of immunoprecipitates and by tandem crossed immunoelectrophoresis it was shown that the antigen differed from the flagellar antigen of T. Reiter, but was identical to antigen d previously described in T. Reiter. Antigen d could also be isolated from supernatants of T. Reiter cultures. The d antigen was not denatured at pH 2.8, by 8 M urea or by 3 M KSCN, and it resisted heating to 100 degrees C for 30 min. No protein could be detected in a concentrated preparation, and the antigen might be a polysaccharide. Antigen d is probably present in the sorbent used in the fluorescence treponemal antibody absorption (FTA-ABS) test and may constitute the active substance of this reagent.

Two kinds of spirochetes were observed by dark-field microscopy in the urine of a leptospirosis patient on day 26 after the onset of the disease. The spirochetes were identified as treponemes and leptospires. A classification of the treponemes on basis of their morphology was attempted by means of electron microscopy with no conclusive result. Cultivation of the spirochetes was unsuccessful. Sera obtained prior to, during and after the excretion of spirochetes were tested for antibodies in routine serology tests for leptospirosis and syphilis. The diagnosis leptospirosis was confirmed by a rise and subsequent fall in antibody titres. The sera were nonreactive in syphilis serology tests, but showed a weak fluorescence in a fluorescent treponemal antibody absorption (FTA-ABS) test when Treponema calligyrum was used as antigen. It could not be determined whether this reaction was due to antibodies raised in response to antigens of the excreted treponemes.

The morphology of freshly isolated strains of Leptospira interrogans serovar hardjo was studied by electron microscopy and was compared to that of established laboratory strains of the same serovar and to some other serovars within the Hebdomadis serogroup. Changes in the morphology of freshly isolated strains of serovar hardjo were observed when the isolates were subcultured in the laboratory. Cells of strains subcultured more than 20 times were longer and had longer wavelengths, and electron lucent inclusions were observed less frequently than in cells of strains subcultured less than 10 times. Upon hamster passage, cells subcultured 40 and 50 times became more similar to cells of low passage number than to those of the inoculate. The results are discussed and the importance of environmental factors for the growth of leptospires is emphasized.

Cells of Treponema pertenue Gauthier obtained by elution from skin lesions and lymph nodes of experimentally infected hamsters were studied in the electron microscope by means of negative staining. The cells were also examined in thin secions of skin biopsies and lymph nodes. Fimbriae were observed on the negatively stained cells. Until now fimbriae have not been demonstrated on negatively stained cells of other species of Treponema, but at present only one straon of T. pertenue has been studied in our laboratory. Otherwise, the ultrastructure of the T. pertenue cells was found to be very similar, if not identical to, the substructural details observed in cells of T. pallidum and T. cuniculi. In thin sections of skin biopsies, treponemes were observed in the intercellular spaces between cells of the stratum basale and the stratum spinosum layers of the epidermis.

Two lines of a spirochete were isolated as contaminants in a culture of a leptospire and from a bottle of uninoculated medium. Studies on morphological and biological characters led to the conclusion that these lines represent a new species of the genes Leptospira, Leptospira parva sp. nov. The morphology of the cells was rather similar to that of previously examined leptospires, but the cells were shorter and had a shorter wavelength (more tightly wound). Furthermore, the surface layer of cells of L. parva formed numerous small blebs with no apparent substructure when detached during preparation for negative staining, while the surface layer of previously studied leptospires formed cross-striated tubules under similar conditions. L. parva shows biological characteristics of both parasitic and saprophytic leptospires. The deoxyribonucleic acid base composition of cells of this organism differed from that found in leptospires, as well as from that found in cells of Leptonema illini.

The experiments were carried out in order to investigate whether human IgG globulin is absorbed on to the surface of T. pallidum cells during incubation with human syphilis serum in the Treponema pallidum immobilization (TPI) test. Cells of T. pallidum Nichols subjected to the TPI test were further incubated with ferritin conjugated rabbit antihuman IgG globulin. Human IgG globulin could only be demonstrated on immunoimmobilized cells, i.e. cells incubated with human syphilis serum and unheated guinea pig serum (GPS). The surface of the swollen cells was completely covered by a fuzzy layer on to which the ferritin molecules appeared to be attached. In ultrathin sections of cells obtained from the same suspensions the surface of the outer cell membrane was outlined by ferritin molecules. In these cells a rather wide gap was observed between the outer membrane and the cytoplasmic membrane. The ribosomes seemed to have disappeared from the cytoplasm of the immunoimmobilized treponemes, but were present in motile cells obtained from control incubations.

Three groups of Copenhagen municipality male employees-77 sewer workers, 81 gardeners, and 79 clerks-matched for age and duration of employment, were studied for clinical and serological evidence of infection with viral hepatitis types A and B and pathogenic leptospires. "Antibody against hepatitis A virus" (anti-HAV) was found significantly more often among sewer workers (80.5%), than among gardeners (60.5%) or clerks (48.1%). The anti-HAV prevalence rates correlated with age rather than duration of employment. Of all the 11 cases of jaundice reported, only 3 cases (sewer workers) occurred while employed for the city. One case of the 11 resulted from leptospirosis. Anti-HAV was detected in the other 10 subjects and was assumed to be of etiological importance. Hepatitis B serological markers were similar in each group. It is concluded that exposure to metropolitan sewage provides a limited risk of enteric infections, such as hepatitis A, while the hepatitis B virus apparently is not successfully transmitted by this route.

The ultrastructure of cells of T. pallidum Nichols subjected to the TPI test was studied in negatively stained specimens. Cells incubated in basal medium to which was added either human syphilis serum or heated guineapig serum (GPS) showed a normal morphology. This was also the cause for cells incubated with basal medium to which was added either human syphilis serum and heated GPS or normal human serum and unheated GPS. By dark-field microscopy cells obtained from these different incubation mixtures were found to be motile. In contrast, cells incubated in basal medium to which was added human syphilis serum and unheated GPS were all immobilized, and in the electron microscope they presented a morphology strikingly different from that of normal cells. The immunoimmobilized cells were swollen and their surface was completely covered with a layer of fuzzy material. The nature of this material and its possible role in rendering the treponemes immobile is discussed.

The ultrastructure of cells of Borrelia persica was compared with that of cells of a new isolate of Barrelia (Borrelia "x"). This organism (B. "x") was isolated in a region of Iran where B. persica is already known to exist. The morphology of the cells of the two strains differed in mean cell length and cell diameter. Cells of B. persica were covered by a regularly structured surface layer, while those of B. "x" possessed an amorphous surface coat. Furthermore, the strains differed in their experimental pathogenicity for animals. It is concluded that the new isolate represents a new species of the genus Borrelia, and Borrelia baltazardi sp. nov. is proposed.