Infected Rabbit Testes Research Articles

Immunization of guinea pigs with recombinant TmpB antigen induces protection against challenge infection with Treponema pallidum Nichols

Treponema pallidum-susceptible guinea pigs of strain C4D were immunized with recombinant T. pallidum antigens TmpA, TmpB, TmpC, and TmpA plus TmpB plus TmpC; with Escherichia coli membranes; or with adjuvant alone. Animals in groups of five received six immunizing injections, each of 100 micrograms of antigen incorporated in RIBI adjuvant. After the sixth immunization, all experimental and nonimmunized controls were intradermally challenged with 3 x 10(6) T. pallidum Nichols freshly extracted from infected rabbit testes. Although high titers of antitreponemal antibodies in the fluorescent-treponemal-antibody test or an enzyme-linked immunosorbent assay were evoked in all animals immunized with recombinant antigens, only guinea pigs receiving TmpB antigen demonstrated protection expressed by the development of significantly (P less than 0.01) smaller, atypical lesions of significantly (P less than 0.01) shorter duration and devoid of or containing fewer T. pallidum organisms than lesions in the remaining immunized and control animals.

In-vitro activity of azithromycin and CP-63,956 against Treponema pallidum

The ability of the macrolide antibiotic azithromycin and its epimer CP-63,956 to inhibit protein synthesis in two pathogenic strains of Treponema pallidum was investigated. Using an in-vitro radiolabelling procedure employing treponemes freshly extracted from infected rabbit testes, our results indicate that both antibiotics were comparable with erythromycin in their ability to inhibit protein synthesis in T. pallidum Nichols strain. An erythromycin-resistant clinical isolate of T. pallidum, designated Street strain 14, exhibited only partial inhibition of protein synthesis at the highest antibiotic concentration tested.

In vitro assay to demonstrate high-level erythromycin resistance of a clinical isolate of Treponema pallidum.

We have previously demonstrated that cells of Treponema pallidum freshly extracted from infected rabbit testes can be intrinsically radiolabeled with [35 S]methionine to very high specific activities. In this study we used the inhibition of [35 S]methionine incorporation into trichloroacetic acid-precipitable protein in vitro as an assay to test the susceptibilities of three different pathogenic treponemal strains to various antibiotics. In general, the results correlated very well with the known efficacies of these antibiotics in treating human patients with syphilis. One of the strains tested, however, a clinical isolate of T. pallidum designated street strain 14, was found to exhibit high-level resistance to erythromycin and a closely related macrolide, roxithromycin (RU 965). Street strain 14 was originally isolated from a human patient with active secondary syphilis who failed to respond to erythromycin therapy. Thus, our results indicate that an erythromycin-resistant strain of T. pallidum can be responsible for erythromycin treatment failure. In addition, street strain 14 treponemes were found to be generally less susceptible by this assay to a variety of antibiotics than were treponemes of the T. pallidum Nichols strain. These findings suggest that the outer envelope of street strain 14 treponemes may be generally less permeable to antibiotics than is that of Nichols strain treponemes.

Mucopolysaccharides in suspensions of Treponema pallidum extracted from infected rabbit testes.

The amount and nature of mucopolysaccharides present in extraction fluids routinely obtained in the isolation procedure of Treponema pallidum from infected rabbit testes was investigated. The mean quantity of mucopolysaccharides extracted from both testes of groups of 10 rabbits was 3.09 mg after infection for seven days and 26.88 mg after infection for 12 days, while from the testes of uninfected rabbits a mean of 0.42 mg was obtained. On electrophoresis the isolated mucopolysaccharides showed only one single band with the migration characteristics of hyaluronic acid. This band disappeared completely after pretreatment with hyaluronidase from bovine testes, which showed that during infection with T pallidum increasing amounts of hyaluronic acid accumulate. They can, at least in part, be extracted by a gentle extraction procedure, suggesting that this material binds loosely. The amount of hyaluronic acid isolated 12 days after infection showed positive correlations with the wet weight of testes as well as the number of treponemes isolated; seven days after infection such correlations were not present.

Susceptibility of Treponema pallidum to the toxic products of oxygen reduction and the non-treponemal nature of its catalase.

We examined the sensitivity of Treponema pallidum (Nichols strain) to toxic products of oxygen reduction. T pallidum was sensitive to hydrogen peroxide at concentrations similar to those to which obligate anaerobes are sensitive. Accelerated death of T pallidum occurred at hydrogen peroxide concentrations below 50 mumol/l. Agents protective against hydrogen peroxide and the hydroxyl free radical (catalase, peroxidase, and mannitol) significantly enhanced treponemal survival in vitro under all three conditions of aerobiosis tested--that is, air, 3% oxygen, and 3% oxygen in conjunction with a reduced medium. Superoxide dismutase (which provides protection against superoxide radicals) did not enhance treponemal survival in normal media. When superoxide radicals were generated in the medium by means of a xanthine and xanthine oxidase system, however, the enzyme did protect T pallidum. A possible toxic involvement of singlet oxygen was also indicated by enhanced treponemal survival in air in the presence of histidine. Extracts of T pallidum from infected rabbit testes showed catalase activity which, on polyacrylamide gel electrophoresis, had the same relative mobility as purified rabbit catalase. The treponemal catalase activity was neutralised by anti rabbit catalase antiserum (raised in guinea pigs). This confirmed that the catalase was of rabbit origin and not an endogenous enzyme of T pallidum. We discuss the relation of these results to the obligate parasitism of T pallidum.

Avoidance of host defences by Treponema pallidum in situ and on extraction from infected rabbit testes.

Electron microscopic observations of acutely infected rabbit testes showed that the majority of Treponema pallidum were extracellular, and confined to interstitial regions of the tissue. Organisms were often adjacent to small blood vessels, where they should be freely accessible to non-specific humoral and cellular defence mechanisms. However, there was no accumulation of leucocytes in blood vessels or infiltration of inflammatory cells into infected areas. Inoculation of live treponemes into perforated plastic chambers which had been implanted subcutaneously in rabbits or guinea-pigs did not incite significant infiltration into the chamber fluid of inflammatory cells, in contrast to that seen after inoculation of Neisseria gonorrhoeae. Interaction of antibody from infected rabbits with live treponemes freshly extracted from rabbit testes could not be detected by an indirect fluorescent antibody method. These observations suggest that T., pallidum escapes recognition by host defences.

Preparation of T. pallidum extracts from infected rabbit testes.

Preparation of T. pallidum extracts from infected rabbit testes.

Parasitism by Virulent Treponema pallidum of Host Cell Surfaces

The interaction between virulent Treponema pallidum extracted from infected rabbit testes and animal cells in culture was examined. The extent of treponemal attachment to monolayers of normal rabbit testicular and HEp-2 cells was dependent upon the incubation temperature and retained motility of the spirochetes. The specific orientation of treponemes to host cell surfaces was demonstrated by dark-field microscopic examination of wet-mount preparations and scanning and transmission electron microscopy. Once attached, T. pallidum organisms remained actively motile yet anchored in place by their terminal tapered structures. After several hours of co-incubation, maximal attachment was attained, and the degree of parasitism seemed regulated not only by available surface sites on individual host cells but also by the proposed membrane response of parasitized cells to continued exposure to treponemes. The avirulent strain, Treponema phagedenis biotype Reiter, did not adhere to monolayer cultures. Characterization of host cell determinants that permitted surface colonization by T. pallidum was attempted. Also, properties of virulent treponemes that enabled surface parasitism were monitored by measuring the effects of enzymes, detergents, and metabolic inhibitors on the host-parasite interaction. Results reinforced the specific nature of the treponemal attachment mechanism. Furthermore, the ability of convalescent rabbit sera to reduce attachment of treponemes to host cells suggested that surface structures on T. pallidum could be masked or inactivated by host components, thus providing a potentially effective research approach for investigating the pathogenesis of syphilis and screening appropriate vaccine candidates.

Brief report. separation of pathogenic Treponema pallidum from host rabbit tissue to obtain virulent and viable microorganisms.

T. pallida extracted from infected rabbit testes were purified from different tissue particles by equilibrium density centrifugation at 25 degrees C after layering on a column of Ficoll-sodium metrizoate solution. After centrifugation, 32 per cent of the treponemes were present in a visible band and about 57 per cent were concentrated in the three ml suspension just above the interface between the Ficoll-Na metrizoate cushion and treponeme-containing medium. The treponemes were viable, virulent and retained their morphological integrity, infectivity, and antigenicity. Purity of the different fractions was studied by immunodiffusion experiments. The purified preparations of virulent, viable treponemes will provide a better antigen in the FTA-ABS tests and possibly in the TPI and TPHA tests; in cellular immunity studies, electron microscopic investigations of the ultrastructure of treponemes, vaccination studies and in tissue culture investigations.

Protein synthesis by Treponema pallidum extracted from infected rabbit tissue.

Virulent Treponema pallidum organisms, extracted from infected rabbit testes, incorporated amino acids into protein. A temperature of 34 C and a pH of 7.6 were optimal for protein synthesis, which was linear during in vitro incubation for 24 h. Selective inhibition of protein synthesis by erythromycin as judged by the incorporation of radiolabeled amino acids and radioautography demonstrated that treponemes were actively synthesizing proteins. Since addition of various sera and ultrafiltrates to the basal incubation mixture did not stimulate the level of protein synthesis, it was uncertain whether treponemes synthesized protein at a maximal or endogenous rate. Based upon the size of the unlabeled amino acid pool contained in infected testicular extract, it appeared that virulent treponemes utilized the majority of amino acids for protein synthesis but at varying efficiency.

Purification of Treponema pallidum from Infected Rabbit Tissue: Resolution into Two Treponemal Populations

Virulent Treponema pallidum organisms, extracted from infected rabbit testes, were subjected to velocity sedimentation in discontinuous gradients of Hypaque, a high density, low viscosity material. After centrifugation of extracts at 20 C for 45 min at 100,000 x g, treponemes separated into two distinct bands based upon their relative velocities, although some variation was observed in the densities of the two bands and the number of treponemes per band. Rabbit tissue components sedimented more rapidly. Dark field and electron microscopy of preparations after velocity sedimentation indicated that treponemes retained general structural characteristics and no tissue contamination occurred in the treponemal fractions. Purification of treponemes in Hypaque resulted in their loss of motility and infectivity based upon animal inoculation. Antigenicity with respect to reactivity with antibody was preserved as shown by the high fluorescence intensity of treponemes in the fluorescent treponemal antibody adsorption test.