Analysis of the Antigenic Composition of Trypanosoma brucei brucei Bloodstream and Culture Forms by the Quantitative Direct Fluorescent Antibody Methods*

SYNOPSIS The quantitative direct fluorescent antibody (QDFA) methods were employed for the antigenic analysis of bloodstream forms and culture procyclics of 2 variants, TRUM (Trypanosome Research University of Massachusetts) 106 and TRUM 107, of Trypanosoma brucei brucei. Intact and trypsinized trypanosomes were studied. It was demonstrated that: (A) The specific variant antigens are localized in the surface coat of bloodstream trypomastigotes. (B) In addition to the common antigens shared by bloodstream forms and culture procyclics, there are also certain antigens unique to these latter stages. (C) Still another group of antigens. not found in the culture procyclics, appears to be shared by the bloodstream forms, irrespective of their variant-specific antigens. These antigens may be present in part in the coat or on the cell membrane and in part within the cytoplasm. (D) Irrespective of the bloodstream-form variant from which they are derived, the procyclics are antigenically the same. The QDFA results are analyzed statistically and discussed in the light of the available literature.     

Trypanosoma cruzi: Role of macrophage membrane components in the phagocytosis of bloodstream forms

The phagocytosis of Trypanosoma cruzi bloodstream forms is mediated by macrophage Pronase-sensitive membrane components. Trypsin and chymotrypsin treatment of macrophages, which prevents the uptake of T. cruzi culture forms, does not inhibit the phagocytosis of bloodstream parasites. The phagocytosis activity of the macrophages was recovered within 6–8 hr after the removal of Pronase. Inhibition of protein synthesis after Pronase treatment prevents the recovery of the endocytic activity of the macrophages. Fc and C3b receptors are not apparently essential for the phagocytosis of T. cruzi bloodstream forms. The described membrane components may help to explain the tropism of some T. cruzi strains for cells of the mononuclear phagocytic system in the living host.     

Analysis of the Antigenic Relationships Among Trichomonas,Histomonas, Dientamoeba,and Entamoeba. I. Quantitative Fluorescent Antibody Methods*

SYNOPSIS. Antigens were prepared from axenic Entamoeba histolytica, Entamoeba invadens, and Trichomonas gallinae; dixenic Dientamoeba fragilis; and agnotobiotic Histomonas meleagridis cultures. Antisera were developed in rabbits against each of these species by subcutaneous inoculations of homogenized organisms with complete Freund's adjuvant. The globulin fraction of each serum was conjugated with fluorescein isothiocyanate (FITC) and then processed on Sephadex G-25 and DEAE-cellulose columns. Fluorescein/protein ratios were determined for the several DEAE fractions obtained from each of the 5 conjugated globulins, and those with ratios of approximately 3.0 were selected for use in all experiments. Conjugated anti-Dientamoeba and anti-Histomonas fractions were absorbed with the bacterial flora present in the respective cultures before being used for staining. Intact, formalin-fixed organisms of each of the species were subjected to direct staining, inhibition staining, and staining with cross-absorbed conjugated fractions. The emitted fluorescence was measured in an ultramicrofluorimeter. Cross reactions among the 5 antigens and 5 conjugated antisera suggested that very few, if any, common antigens were shared by Trichomonas and Entamoeba. They indicated also a close antigenic relationship between Trichomonas and Histomonas on the one hand and between Histomonas and Dientamoeba on the other. Trichomonas and Dientamoeba appeared to be less closely related, and still less relationship was noted between Dientamoeba and Entamoeba. Only very weak reactions were recorded between Histomonas and Entamoeba. Entamoeba invadens emitted much fluorescence after being stained with anti-Entamoeba histolytica conjugate and similar results were obtained by reciprocal staining. The phylogenetic implications of the immunologic findings are discussed.     

Trypanosoma lewisi: Avidity and adsorbability of ablastin,the rat antibody inhibiting parasite reproduction

The relation of naturally acquired host IgG in the surface coat of bloodstream forms of Trypanosoma lewisi to ablastin was studied to determine whether, contrary to a long-held conclusion, the antibody is avid and adsorbable. It was found by immunofluorescence and agglutination tests with monospecific antisera to rat IgG that bloodstream forms collected from immunosuppressed hosts, in contrast to those from immunocompetent hosts, have little or no detectable surface IgO. Specificity of adsorption was also demonstrated in other immunofluorescence experiments in which bloodstream forms from immunosuppressed hosts adsorbed IgG from immune serum with ablastic activity only (previously adsorbed with trypanosomes from immunocompetent hosts to remove the trypanocidal antibodies), but did not adsorb IgG from normal rat serum. To determine whether this specific adsorption of IgG by the parasite could be correlated with a reduction in ablastic activity, immune sera were adsorbed with bloodstream forms from immunosuppressed hosts at packed cell/serum ratios of either 1.2 or 2.0, and the adsorbed sera were then tested for ablastic activity in vitro. With both cell/serum ratios, ablastic activity was reduced by 50%. In comparison, similar adsorptions of immune sera with trypanosomes from immunocompetent hosts resulted in reductions of ablastic activity of only about 9 and 27% with the low and high cell/serum ratios, respectively. It is concluded that the failure to effect significant adsorption of ablastin in earlier studies resulted from the use of ablastinsensitized trypanosomes from immunocompetent hosts.     

Trypanosoma cruzi: Correlation of resistance and susceptibility in infected inbred mice with the in vivo primary antibody response to sheep red blood cells

Nonspecific immune responses during the course of murine Trypanosoma cruzi infection were examined in mouse strains genetically resistant or susceptible to the Brazil strain of T. cruzi. Spleen cells from infected susceptible (C3H) or resistant [C57 B1/10 and FI (C3H × C57)]mice at various points during the course of infection exhibited a reduced response to concanavalin A and lipopolysaccharide in vitro. Since this reduced response occurred in both susceptible and resistant mice, it was not predictive of resistance or susceptibility in vivo. We next examined the kinetics of in vivo primary antibody response to sheep red blood cells (SRBC) in infected C3H and C57 mice. C3H mice exhibited inhibition of the direct plaque-forming cell assay (d-PFC) which persisted until death. In contrast C57 mice exhibited no inhibition of the response at Day 5 and subsequently a markedly augmented response was observed. Other strains of mice were similarly investigated: all the susceptible mice examined (A/J, BALB/c) showed inhibition or depression of the primary antibody response and resistant mice [B10Br, C57B1/10, SJL, F1 (C3H × C57)]demonstrated either no inhibition or considerable augmentation of this response. CS7 mice resistant to the Brazil strain were susceptible to the Tulahuén strain. The mice in this latter group exhibited a markedly significant inhibition of the in vivo primary antibody response to SRBC. Culture forms of the Brazil strain protected C3H mice from a virulent challenge. This immunization resulted in a markedly augmented antibody response. The data reported herein are consistent with the notion that inhibition of the primary antibody response to SRBC correlates with susceptibility whereas no inhibition or, indeed, augmentation of the response correlates with natural as well as acquired resistance.     

Plasmodium yoelii: the effect of second blood meal and anti-sporozoite antibodies on development and gene expression in the mosquito vector, Anopheles stephensi

The sporogonic development of the malaria parasite takes place in the mosquito and a wide range of factors modulates it. Among those, the contents of the blood meal can influence the parasite development directly or indirectly through the mosquito response to the infection. We have studied the effect of a second blood meal in previously infected mosquitoes and the effect of anti-sporozoite immune serum on parasite development and mosquito response to the infection. The prevalence and intensity of infection and gene expression of both Plasmodium yoelii and Anopheles stephensi was analyzed. We verified that a second blood meal and its immune status interfere with parasite development and with Plasmodium and mosquito gene expression.