The kidney form of Trypanosoma musculi: A distinct stage in the life cycle?

Trypanosoma musculi is a parasite specific to mice, which resides in the blood and lacks intracellular stages. After immune clearance of the flagellates from the general circulation, mice are resistant to reinfection. Yet, long after parasites are no longer detected in the peripheral blood, they persist in the vasa recta of the kidneys and it has been proposed that this is an immunologically privileged site for T. musculi. This relationship provides a useful model for studies of latent or chronic infections in immune hosts. Here, Fernando Monroy and Donald Dusanic consider the immune responses of mice to T. musculi and compare characteristics of the parasites from the vasa recta (kidney forms, KFs) of mice with latent infections to trypanosomes from the peripheral blood (bloodstream forms, BSFs) of animals during active infections. They consider how KFs evade immune destruction and suggest that these sequestered parasites represent a distinct stage in the life cycle.

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.

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.

Survey of antitrypanosome antibodies and studies of their specificity and autoimmunity.

As a part of investigations to characterize trypanosome infections in Taiwan, sera collected from patients admitted to Veterans General Hospital, Taipei were tested for antitrypanosome antibodies. A Trypanosoma cruzi extract-based enzyme-linked immunosorbent assay (ELISA) was used to screen and titrate 1,297 patient sera. High antitrypanosome titers were detected in 166 (12.8%) of these sera. Retitration of random samples of the high titer (HT) sera indicated a 5.4% false positive. Thirteen donors with high antitrypanosome ELISA titers were followed up. Twelve of then remained high serum titers also showed high ELISA titers against an extract of Trypanosoma conorhini. Hemocultures conducted on freshly drawn blood specimens of the 13 subjects did not provide any evidence of trypanosome infections. Electrophoretic analyses of sera from HT and low titer (LT) patients suggested differences between serum proteins of the subjects in each of the groups. Atypical reactions were observed in immunodiffusion tests performed with HT and LT sera and trypanosome extracts, while western blot analyses revealed a complex pattern of binding by both sera. The qualitative and quantitative differences in these tests suggested interactions of T. cruzi antigens with donor antibodies against unrelated antigens and/or with autoantibodies. Subsequent analyses did not indicate any association between rheumatoid factor and the reactivities of the HT sera with the parasites. However, antinuclear antibodies were detected with an indirect fluorescent antibody test (IFAT) in 50% of the HT sera and 22% of the LT sera. No differences were found between the levels of antilaminin activity of the two groups. The IFAT employing T. cruzi epimastigotes was positive for 100% of the HT sera and 22% of the LT sera. The data indicate that the high seropositivity recognized in this study is due in part to the activities of cross-reacting antibodies and/or autoantibodies in the sample population.

Biological and immunological studies on a low virulence isolate of the Tulahuén strain of Trypanosoma cruzi.

Reduced virulence for mice was characterized in an isolate (LV1) of a clone of the Tulahuén strain of Trypanosoma cruzi. LV1 caused long term chronic parasitemias which were measured for 140 days in both C3H/He and BALB/c mice inoculated with 1 x 10(5) trypanosomes/mouse. In contrast to the acute and rapidly lethal Tulahuén strain infections in both strains of mice, all of the animals survived the LV1 infections. Sera of C3H/He mice infected with the Tulahuén strain or LV1 isolate displayed similar titers in an enzyme-linked immunosorbent assay (ELISA) when reacted against homologous or heterologous extracts of epimastigote stages of the trypanosomes. Western blot reactions of the Tulahuén, Raccoon V, LV1 isolates, and the closely related European bat parasite, Trypanosoma dionisii defined shared antigens between the strains and species, while some appeared to be strain- and species-specific. The studies indicate a mutational event(s) resulted in reduced virulence and suggest that survival of the mice infected with T. cruzi is not correlated with high ELISA antibody titers. Since lower antibody titers are exhibited by mice infected with LV1 than mice infected with the Tulahuén strain, survival may be dependent on the specificities of the antibodies synthesized during the infections, cell mediated immune responses, and/or biochemical factors of the LV1 isolate which control virulence and differ from those of the original Tulahuén strain.

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.

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.

Trypanosoma lewisi: stage-specific surface antigens and exoantigens recognized by monoclonal antibodies.

The stage-specific expression of surface antigens by Trypanosoma lewisi was investigated using monoclonal antibodies directed against this parasite. Hybridomas secreting monoclonal antibodies were produced by the fusion of SP2/0-Ag 14 mouse plasmacytomas with spleen cells from rats infected previously with the Taliaferro strain of T. lewisi. Additivity enzyme-linked immunosorbent assays and indirect immunofluorescent antibody tests indicated the determinant recognized by monoclonal antibody TL40.3 (IgM) was different from those recognized by monoclonal antibodies TL40.1 (IgA), TL40.2 (IgM), and TL40.6 (IgG2 alpha). Monoclonal antibody TL40.3 agglutinated trypanosomes collected 3 days after parasite inoculation while monoclonal antibodies TL40.1, TL40.2, and TL40.6 agglutinated trypanosomes collected 6 days after inoculation. Since agglutinin titers against trypanosomes from irradiated (700 rad from a 60Co source) and nonirradiated rats were similar, expression of the antigens recognized by the monoclonal antibodies appeared to be independent of the immunological state of the host and the morphology of the parasite. The reproduction of T. lewisi in in vitro trypanostatic assays was inhibited only when the monoclonal antibodies were present in concentrations greater than or equal to those needed to agglutinate the trypanosomes. Monoclonal antibodies TL40.1 and TL40.3, but not TL40.2 and TL40.6, agglutinated erythrocytes collected later in the infection from irradiated, infected rats. None of the monoclonal antibodies agglutinated erythrocytes from nonirradiated, infected rats, from irradiated, noninfected rats or from nonirradiated, noninfected rats. This suggests that immunocompetent rats may make blocking antibodies against the exoantigens recognized by monoclonal antibodies TL40.1 and TL40.3.

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)

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.

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.

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.

The activity of nifluridide on reduviids and rodent and human trypanosomes.

The ability of nifluridide to kill reduviids was assayed in mice fed 7 ppm in diet and on cattle injected subcutaneously at 5 mg/kg body weight. Nifluridide was systemically active against Triatoma infestans on mice and Rhodnius prolixus on cattle. No effects on Trypanosoma (Schizotrypanum) cruzi could be detected in the intestinal contents of Triatoma infestans killed by the compound. In vitro and in vivo studies were conducted to determine the effects of nifluridide on trypanosomes growing in medium and in experimentally infected mice. Culture forms of Trypanosoma cruzi grown at 27 degrees C that are morphologically similar to epimastigotes found in infected bugs were affected by 2.5 to 10 ppm in the medium. Mice fed nifluridide in the diet simultaneous with infection of Trypanosoma cruzi or Trypanosoma (Herpetosoma) musculi exhibited parasitemias and tissue infections similar to nontreated infected mice. At the concentration tested, bloodstream trypomastigotes and culture epimastigotes of Trypanosoma musculi were unaffected by nifluridide. Only the culture epimastigotes of Trypanosoma cruzi were affected by the drug but not the bloodstream and tissue forms.