Fine structure and replication of the kinetoplast of Trypanosoma lewisi.

The kinetoplastic DNA of Trypanosoma lewisi is described as a filamentous body lying within a mitochondrion, with the filaments oriented parallel to the long axis of the cell. The manner of fixation, the replicative state, and perhaps the physiological state of the cell, may result in slight morphological differences among such bodies. The kinetoplastic DNA replicates to form "left" and "right" rather than "upper" and "lower" members, and both the kinetoplast and nucleus incorporate radiothymidine as shown by radioautography. Radioautographic analyses suggest a random incorporation of radiothymidine by kinetoplasts. Silver grains were occasionally observed over centriolar elements. Finally, the observations are discussed with respect to the sequential replication of the aforementioned organelles by T. lewisi.

Monoclonal antibodies to metacyclic stage antigens of Trypanosoma cruzi.

Hybridoma cell lines secreting monoclonal antibodies against the Tulahuén strain of Trypanosoma cruzi were produced by the fusion of SP2/O-Ag 14 myeloma cells with spleen cells from mice immunized with irradiated metacyclic trypomastigotes. Twenty of the monoclonals synthesized by the hybridomas were identified as IgM, 2 as IgG1, 10 as IgG2a, 3 as IgG2b, 4 as IgG3, 1 as IgE, and 1 as IgA. Twenty-three of these antibodies had kappa light chains and 18 showed delta chains. Twelve of the monoclonals agglutinated metacyclic trypomastigotes without additional concentration and 4 of these precipitated antigens in extracts of T. cruzi metacyclic or epimastigote stages. One monoclonal precipitated an epimastigote antigen, while another reacted with a metacyclic antigen, and 2 antibodies formed precipitin lines with antigens of both stages. Agglutinin assays performed to characterize surface antigenic specificities of the 12 monoclonal antibodies showed that 2 reacted only with the metacyclic stage of the Tulahuén strain. Two monoclonals agglutinated both metacyclic trypomastigotes and epimastigotes of the Tulahuén strains. Three antibodies caused clumping of metacyclics and epimastigotes of the Tulahuén, Raccoon V, and Corpus Christi strains of T. cruzi, while a fourth also reacted with bloodstream trypomastigotes. One monoclonal detected identical epitopes on metacyclics and epimastigotes of T. cruzi and epimastigotes of Trypanosoma musculi, while 2 antibodies reacted with metacyclics, epimastigotes, and bloodstream trypomastigotes of the Tulahuén and Raccoon V strains and the bloodstream stage of T. musculi. One antibody agglutinated all stages and strains of T. cruzi, T. musculi, and Trypanosoma lewisi which were tested.(ABSTRACT TRUNCATED AT 250 WORDS)

Antigenic analyses of two axenized strains of Entamoeba histolytica by two-dimensional immunoelectrophoresis.

Antigens in extracts of two strains of Entamoeba histolytica (HT-31 and HK-9) were studied by two-dimensional immunoelectrophoresis (2D-IEP) and 32 and 20 different antigenic components were detected, respectively. Cross-reactions between heterologous systems showed a large number of shared antigens between the two strains. Extracts were fractionated by Sephadex G-200 chromatography. Of the fractions obtained, fraction II of both strains was found to have the most reactive antigenic components. Precipitin patterns of fraction II from HT-31 and HK-9 with sera from patients showed similar 2D-IEP profiles and suggested that in this fraction antigens common to both strains exist which elicit antibody in individuals with E. histolytica infections.

Survival of Trypanosoma musculi in the kidneys of chronically infected mice: kidney form reproduction and immunological reactions.

Trypanosoma musculi are detected in the blood of the mouse host within 3-5 days after infection. Peak parasitemia is reached within 10 days and parasites persist at a plateau level for 2-3 wk. There are no intracellular stages and the flagellates are eliminated from the peripheral blood within 4 wk. However, trypanosomes persist in the vasa recta of the kidneys and may be present for the life of the host. Infection provides lifelong resistance to reinfection. Kidney forms (KF) of T. musculi were isolated and studied to define their morphology, reproductive activity, and serological reactivity. Dividing epimastigotes and trypomastigote stages were present in kidneys. Multinucleate and rosette forms were common. Measurements of the coefficient of variation of the KF confirmed that the trypanosomes were actively reproducing. Direct immunofluorescence reactions with rabbit antimouse IgG + IgA + IgM detected antibodies on bloodstream forms (BSF) but not on the KF. However, indirect immunofluorescence tests using antisera collected from mice during early and late phases of the infections showed that KF were capable of reacting with antibodies. The reproductive activity displayed by the KF appears to be responsible for the continuous replacement of the trypanosomes that are killed by the immune responses of the resistant mice when they leave the vasa recta. Solute concentrations of the blood within the vasa recta appear to prevent antibodies from complexing with surface antigens of the parasites. These capillaries provide nutrients that allow the trypanosomes to reproduce and persist unaffected by the host immune responses.

Trypanosoma musculi survival in the kidneys of chronically infected mice: kidney form ultrastructure, surface characteristics, and serological interactions.

After elimination of Trypanosoma musculi from the general circulation by the immune responses of infected mice, the animals are resistant to reinfection. Yet, parasites survive in the vasa recta of the kidneys for the life of these mice. These kidney forms (KF) actively reproduce in an environment that provides the necessary nutrients and appears to prevent their elimination from these capillaries by the hosts' immune responses. Comparative studies conducted with KF and the bloodstream forms (BSF) indicate that, although both forms appear to be similar morphologically at the ultrastructural level, they differ in their surface reactivities with lectins and tolerance to various pH and solute concentrations. Although antibodies are not detected on the surfaces of KF, urea levels approximating those in the vasa recta dissociate antibody from the surfaces of BSF. The data suggest that parasites found in the vasa recta of these chronically infected mice differ from the BSF and are protected from the humoral and cell-mediated immune responses of the murine hosts by the concentrated solutes present in these capillaries. The KF may be killed by these same immune effector mechanisms upon leaving the capillaries of the kidneys and, therefore, not be found in the general circulation of these chronically infected immune hosts.

Crossed-immunoelectrophoretic analyses of Trypanosoma cruzi epimastigote, metacyclic, and bloodstream forms.

Sonicated suspensions of epimastigote, metacyclic, or bloodstream forms of Trypanosoma cruzi were emulsified in Freund's complete adjuvant. Rabbits immunized with epimastigotes or metacyclics received five intramuscular (i.m.) injections of 1 x 10(9) sonicated trypanosomes at weekly intervals. Immunization with bloodstream forms included three i.m. injections of 5 x 10(7) and six injections of 2 x 10(8) sonicated trypanosomes. Selected antisera from these rabbits were employed in crossed immunoelectrophoretic studies against the homologous or heterologous extracts of sonicated trypanosomes. Extracts of epimastigote, metacyclic, and trypomastigotes produced 31, 29, and 11 precipitin peaks respectively against the homologous rabbit antisera. Tandem, crossed-immunoelectrophoresis of these extracts against antiepimastigote or antimetacyclic sera revealed that epimastigotes or metacyclics may each have at least four antigens that did not appear to be shared by the other, whereas each of these forms may have at least eight or nine antigens that were not detected with extracts from trypomastigotes. Cross-absorptions of antiepimastigote or antimetacyclic sera with live trypanosomes caused marked reductions in the numbers of precipitin peaks formed against the homologous extracts, but cross-absorptions with sonicated suspensions of epimastigotes or metacyclics showed that epimastigotes or metacyclics each have at least two antigens that were not detected in extracts of the other. Differentiation appeared to be accompanied by antigenic change. More antigens appear to be shared by epimastigotes and metacyclic forms than by trypomastigotes and epimastigotes or metacyclics.

Applications of biotechnological methods to studies of protozoan parasites.

Biotechnology applies and extends the concepts and techniques of molecular biology. An overview of the applications and potential uses of the technology is presented for selected protozoan parasites. The areas reviewed include the characterization of protozoa, the production of their antigens, and the uses of hybridomas for studies of the antigens and host responses. In addition to the traditional methods of classification, parasitic protozoa are identified and characterized according to stable molecular markers. Peptidemes, zymodemes, antigens, schizodemes, and chromosomal complements define isolates and correlate with biological activities of the parasites. While antigens are typically extracted from parasites obtained from infected hosts or grown in vitro, they may be produced with in vitro translation systems, recombinant procedures with bacteria, or more recently developed techniques such as co-transformation and electroporation with eucaryotic cells. Monoclonal antibodies produced by B-cell hybridomas are used to identify parasites, antigens, epitopes, and the locations and functions of specific antigens. T-cell hybridomas may provide insights into cell-mediated immunity and interactions with the parasites.

Survival of Vero, myeloma and hybridoma cells during cold storage.

Vero cells, SP2/O-Ag 14 myeloma cells and 4B87 hybridoma cells were stored either at refrigeration (5 degrees C) or freezing (-18 degrees C) temperatures. Cells were recovered every five days and percentages of viable cells were determined by the trypan blue exclusion staining method before the cells were incubated at 37 degrees C in a 5% CO2 atmosphere. SP2/0 cells grew after 30 days of storage at 5 degrees C. Hybridoma (4B87) cells survived 20 days of cold storage in HY medium and maintained antibody production. For each cell type, higher percentages of viable cells were observed among cells stored in HY medium than among cells stored in DMEM. Vero cells stored for 40 days at 5 degrees C grew when removed to optimal conditions of 37 degrees C and 5% CO2. There was no growth of cells recovered after storage at -18 degrees C.

Trypanocidal drugs and the role of kidney forms of Trypanosoma (Herpetosoma) musculi in immunity of mice against reinfection.

Adrenals, hearts, kidneys, livers, lungs, and spleens were removed from C3H/Anf mice which had been inoculated with Trypanosoma (Herpetosoma) musculi and no longer exhibited parasitemias. Imprints of each organ were examined microscopically, and each was homogenized and injected into recipient mice. It was confirmed that trypanosomes could be detected only in the donor kidneys. Lampit or Ethidium treatment eliminated bloodstream and kidney forms when administration was initiated after the development of patent parasitemias. However, mice treated with Lampit on the same day they were inoculated with T. musculi developed parasitemias later than animals injected with drug after parasites had appeared in their blood. Both Lampit and Ethidium depressed antibody production as detected in enzyme-linked immunosorbent assays of antisera from animals having parasitemias at the time of treatment. The elimination of kidney forms by Lampit or Ethidium treatment did not reduce the resistance of mice to reinfection by T. musculi 12 weeks or 15 and 22 weeks, respectively, after the initial inoculation of these animals with the parasites. Kidney forms were not required for the sustained protective immunity of the mice against reinfection during the intervals of these experiments.

Comparative studies of the isolation of metacyclic trypomastigotes of Trypanosoma cruzi by DEAE ion exchange chromatography.

Metacyclic trypomastigotes and epimastigotes of the Tulahuén strain of Trypanosoma cruzi were cultivated in a liquid metacyclic culture (LMC) medium. Trypanosome mixtures containing different percentages (7.5%, 15%, and 30%) of metacyclic stages obtained from these cultures were used to examine the effects of pH and anion exchange medium on the chromatographic isolation of the metacyclic stages. The isolations were performed on a DEAE-Sephacel, DEAE-cellulose DE 32, or DEAE-cellulose DE 52 columns at pH 4.0, 6.0, 8.0, or 10.0. All of the anion exchange media used exhibited the ability to enrich the metacyclic trypomastigote populations from trypanosome mixtures at all pH levels tested. However, the efficiencies varied: DEAE-cellulose DE 52 was about 15% less efficient than DEAE-Sephacel; DEAE-cellulose DE 32 was 10-20% less efficient than DEAE-cellulose DE 52. The greatest recovery of metacyclic stages was obtained by using DEAE-Sephacel at pH 8.0. This procedure was the most efficient for the isolation of metacyclic trypomastigotes from mixtures of stages of T. cruzi.

Trypanosoma cruzi: isolation and characterization of two clones derived from neonatal mice.

The Tulahuen strain of Trypanosoma cruzi was cloned in 15 C3H/Anf neonatal mice. Ten of these 15 neonates became parasitemic before the 12th day and died before the 19th day after the inoculation of a single bloodstream trypomastigote. Two clones were selected and maintained, while the other isolates which did not grow in a liquid metacyclic stage culture (LMC) medium were eventually discarded. The kinetics of in vitro growth and transformation from epimastigote to metacyclic trypomastigote of these two clones were characterized in LMC medium at 27 degrees C. Infectivities for vertebrate cells in vitro were retained by these two clones during the period of cultivation. The tropism for brain, heart, lungs, esophagus, stomach, large intestine, liver, pancreas, spleen, lymph nodes, kidneys, bladder, and skeletal muscles was also examined in the mice. The communication describes the establishment and characterization of T. cruzi clones. The utilization of these cloned parasites should produce some advantages in generating reproducible data in investigations.

Humoral immune responses to metacyclic stages of Trypanosoma cruzi in experimentally infected rats and mice.

Holzman rats and C3H/Anf mice were infected with the Tulahuén strain of Trypanosoma cruzi. Infected rats had a 16% cumulative mortality during a 49-day observation period. The parasitemias increased to a peak on the 21st day, and then decreased abruptly. In the inoculated mice, maximum numbers of parasites were detected after 15 days and an 87% cumulative mortality was observed during the infection. Three serological techniques using antigens of the metacyclic stages of T. cruzi were employed to compare the levels of humoral antibodies during the early phase of infection. These included: the direct agglutination test (DAT), the indirect immunofluorescent antibody test (IFAT), the enzyme-linked immunosorbent assay (ELISA) which detected IgG (ELISA-IgG) and the ELISA which detected IgM (ELISA-IgM). Antibody titers were first found in antisera from rats after 7 days by the DAT and the ELISA-IgM and after 14 days by the IFAT and the ELISA-IgG. Peak titers were measured on day 21 by the ELISA-IgM. The DAT, the IFAT, and the ELISA-IgG titers increased through 49 days. Antisera collected from mice first reacted with T. cruzi antigens on day 10 in the DAT, the ELISA-IgG, and the ELISA-IgM, and on day 15 in the IFAT. Peak titers were recorded on day 20 with the DAT and the ELISA-IgM. The IFAT and the ELISA-IgG titers continued to rise through 30 days. Alterations in production of antibodies during infections may in part reflect responses to different parasite antigens. The variations in titers of the antisera from infected rodents indicated that metacyclic trypomastigotes share antigens with other stages of the parasite. These metacyclic stage antigens showed a potential for use as serodiagnostic reagents.