A method for the assay of ablastin in the serum of rats infected with Trypanosoma lewisi.

This paper describes a simple quantitative assay for albastin, a factor present in the serum from rats infected with Trypanosoma lewisi, which prevents the division of the parasite. The assay measures in vitro the inhibition of the incorporation of 3H-TdR into the DNA of T. lewisi in the presence of serum from infected animals. Utilising this method, one can measure the titre of albastin in a particular serum sample and the time and duration of its appearance in the circulation of infected rats.     

Pathological developments mediated by cyclophosphamide in rats infected with Trypanosoma lewisi

Trypanosoma lewisi is an obligatory, flagellated parasite of the rat. Despite the fact that naturally the rats overcome the disease, a lethal infection can be induced by the administration of an immunosuppressive agent, i.e. cyclophosphamide (Cy). In the Cy treated infected rats (CyI) the severity of the trypanosome infection was demonstrated in the internal organs, in the following order: lungs > liver > heart > spleen > kidney. The parasites were not detected in the brain. The accumulation of the parasites in the lungs led to the development of hemorrhagic inflammatory foci. The rupture of blood vessels was accompanied by lymphocyte infiltrations into the damaged tissues and multiple foci of edema around the blood vessels. In most cases the lungs were dark brown in color due to intra-alveolar hemorrhages. The spleen of the CyI rats showed general deformation of the tissue's architecture, migration of macrophages and cell depletion due to the Cy action. The liver showed inflammatory hemorrhagic foci associated with massive destruction of the parenchyma. In spite of the heavy parasitemia (> 50%) developed in the CyI rats the brain remained free of parasites, which might explain the non-virulent character of this parasite compared to the African trypanosomes.     

The specific anti-parasite immune responses of germ-free and conventional rats infected with Trypanosoma lewisi

To test the hypothesis that the rapid immune response of rats to Trypanosoma lewisi is elicited by prior exposure to cross-reacting environmental antigens, the early immune response to infection with this nonpathogenic protozoan was studied in germ-free and conventional rats. In germ-free rats, initial levels of both IgG and IgM were significantly lower than those of conventional rats. After infection, the germ-free rats made more immunoglobulins of both classes, and made them more quickly, than did conventional rats. Trypanosome-specific antibodies appeared earlier and in higher titers in the germ-free rats. Because they lacked intestinal microflora, it is unlikely that the germ-free rats' responses had been primed; thus, these observations indicated that the conventional rats' responses to some trypanosome antigens had been down-regulated by their prior exposure to environmental antigens. However, protective antibodies that inhibited parasite reproduction (ablastin) may have been primed, because these appeared in sera 2 days earlier in conventional rats. Despite much lower rates of production of trypanosome-specific antibodies, the conventional rats had the same peak parasitemias and times to crisis as germ-free rats. Thus it is apparent that protective immunity to this nonpathogenic parasite is not down-regulated by prior exposure to environmental antigens, as would be predicted.     

Trypanosoma lewisi: electron microscopy of the infected spleen

The spleens of Lewis rats, both normal and infected with Trypanosoma lewisi were examined by electron microscopy. Special attention was directed to clusters of splenic cells which occur in the course of the infection. The reticular cells first showed alterations of their structure by the second day of infection, with considerable surface membrane activity. By the fourth day and thereafter various cells were found gathered around the reticular cells. These cell clusters mainly contained lymphocytes, plasma cells, and erythropoietic elements in many stages of differentiation. It was not unusual that several cell types were found adjacent to the same central reticular cell. These arrays, similar in geometry to the erythropoietic island of the bone marrow, became more predominantly “plasma cell” islands as the infection progressed. Parasites were recognizable within the reticular cells, and were noted to be in regions where the cellular membranes of adjacent cells demonstrated vesiculations resembling rhopheocytosis. A further observation was the pinching off of neighboring plasma cell cytoplasm into the reticular cells.     

Trypanosoma lewisi: characteristics of an antigen which induced ablastic antibody

The antigen-inducing ablastic antibody was found in the plasma of Trypanosoma letwsi-infected rats which were treated with high doses of hydrocortisone acetate.The antigen (ablastinogen) was demonstrated by immunizing normal rats with hydrocortisone treated, infected rat plasma (TIRP) and testing their antiserum for ablastic antibody. Plasma from either hydrocortisone treated, uninfected rats or from untreated, uninfected rats did not induce detectable ablastic antibody.Ablastinogen in TIRP was stable to ether treatment, to heating at 37 C for 4 hr, and to heating at 56 C for 45 min, but was inactivated by heating at 100 C for 15 min. Pronase treatment (2 mg/ml TIRP) for 4 hr at 37 C inactivated the antigen in 2 out of 3 samples.Ablastinogen was not dialyzable, and gel filtration of TIRP on Sephadex G-200 in aqueous buffer (1.0 M NaCl, 0.1 M Tris-hydrochloride, pH 8.0) separated the antigen into at least 2 components. The separate components did not induce ablastic antibody, however, recombinations of the components did induce ablastic antibody in immunized rats.     

Elicitation of the reproduction-inhibiting antibody ablastin by serum exoantigens of Trypanosoma lewisi

Serum exoantigens of Trypanosoma lewisi were collected 5 days after infection from immunocompetent (untreated) rats and rats immunosuppressed by treatment with either hydrocortisone acetate or dexamethasone. Normal rats were then immunized with pooled, whole exoantigen-containing serum from 1 of these 3 sources plus alum as an adjuvant, and the immune sera produced were tested individually. All contained agglutinating (trypanocidal) antibodies to both antigenic variants of T. lewisi, but only about two-thirds showed precipitating activity with exoantigens in gels. More importantly, however, when these antisera were thoroughly adsorbed with living trypanosomes (from immunocompetent hosts) to remove agglutinating antibody only and then tested for ablastic activity in vitro, all showed significant (P less than 0.01) reproduction-inhibiting activity, comparable to that shown by ablastic serum collected from rats that experienced a natural infection. Antisera from control rats similarly immunized with normal rat serum were negative in all antibody tests. The exoantigens of T. lewisi are, therefore, a complex mixture of immunogens that are related to the known immune responses to the parasite and can elicit the formation of ablastic antibody with the same biological properties as that produced during a natural infection.     

Cytokines in rats experimentally infected with Trypanosoma evansi

The aim of this study was to measure the levels of interferon-gamma (IFN-γ), tumor necrosis factor-alpha (TNF-α), interleukin 1 (IL-1) and interleukin 6 (IL-6) in the serum of rats experimentally infected with Trypanosoma evansi and to correlate these levels with hematological parameters. Initially, 48 rats (group T) were intraperitoneally inoculated with cryopreserved blood containing 1 × 10⁶ trypomastigotes per animal. Twenty-eight animals (group C) were used as negative controls and received 0.2 mL of saline by the same route. The experimental groups were formed according to the time after infection and the degree of parasitemia as follows: four control subgroups (C3, C5, C10 and C20) with seven non-inoculated animals each and four test subgroups (T3, T5, T10 and T20) with 10 animals each inoculated with T. evansi. The blood samples were collected by cardiac puncture at days 3 (C3, T3), 5 (C5, T5), 10 (C10, T10) and 20 (C20, T20) post-infection (PI) to perform the complete blood count and the determination of IFN-γ, TNF-α, IL-1 and IL-6 levels using an ELISA quantitative sandwich. Infected rats showed normocytic normochromic anemia during the experimental period. T. evansi infection in rats caused a serum increase (P < 0.01) of IFN-γ, TNF-α, IL-1 and IL-6 levels at days 3, 5, 10 and 20 PI compared to the controls. The multiple linear regressions showed a reduction of 24% in the hematocrit as a consequence of the increased IFN-γ, TNF-α and IL-1. Therefore, we conclude that the infection caused by T. evansi causes an increase in the pro-inflammatory cytokines. These results suggest a synergism among IL-1, TNF-α and IFN-γ contributing to the development of anemia. This increase is associated with the regulation of immune responses against the parasite.     

Iron metabolism in Trypanosoma lewisi infection: serum iron and serum iron-binding capacity.

Progressive changes in iron levels, total iron binding capacity and hematocrit values in sera of rats infected with Trypanosoma lewisi are described. The host dietary group were: (1) complete or full complement; (2) iron-deficient, and (3) pair-fed or calorically restricted. The hematocrit values of T. lewisi-infected rats given the various diets were not significantly different from those of the controls. The decrease in total iron binding capacity (TIBC) of rats inoculated with T. lewisi and fed complete and pair-fed diets ranged up to 15% over uninfected controls. TIBC levels in rats fed an iron-deficient diet and inoculated with T. lewisi ranged up to 32% over uninfected controls. TIBC levels of deficient infected rats were significantly different from the controls from day 90 to infection to the end of the observation period. Serum iron (SI) values of non-infected rats regardless of dietary regimen showed significantly higher values than T. lewisi-infected animals between days 95 and 120. The average SI value, for this period, in adequately fed control rats was 204 +/- 7 microgram/100 ml as compared to 172 +/- 5 microgram/100 for trypanosome-infected rats. SI levels of rats on a pair-fed diet and infected with T. lewisi decreased to 17% over uninfected controls. SI levels of animals on an iron-deficient diet and infected with T. lewisi decreased up to 76% over uninfected controls.