Whence comes Trypanosoma lewisi antigen which induces ablastic antibody: Studies in the occult?

I propose that trypanosomes have three antigen compartments: the intracellular, plasma membrane, and extracellular compartments. The intracellular antigens are released when trypanosomes lyse; the plasma membrane antigens are the structural or transport components of the plasma membrane and the adhering surface coat; the extracellular antigens are secreted by the trypanosomes. I further suggest that ablastinogen, the Trypanosoma lewisi antigen which induces ablastic antibody, is a plasma membrane antigen, and that the T. lewisi trypanocidal antigens are in the surface coat. The T. lewisi exoantigens described by D'Alesandro (1972) are, as he stated, different from ablastinogen and trypanocidal antigens. I suggest that the exoantigens are secreted extracellular antigens. Data from the literature are presented to support the hypothesis, and an experimental protocol to test the hypothesis is outlined.     

Trypanosoma lewisi: effects of immunosuppression on the serological reactivities of exoantigens and cellular antigens of bloodstream parasites.

Groups of rats were immunosuppressed with antithymocyte serum (ATS) and infected with Trypanosoma lewisi. Immunodiffusion studies were performed which demonstrated that trypanosome exoantigens, present in the plasma of these animals, were precipitated by antibodies in the sera of rats undergoing a typical primary T. lewisi infection; extracts of trypanosomes which had been collected from ATS-treated rats contained antigens which also were precipitated by antibodies in these sera. These precipitating antibodies could not be detected using either the plasma of untreated infected rats or extracts of trypanosomes which had been collected from untreated rats. With the exoantigens, precipitating antibodies were detected in serum samples collected from rats 14 to 250 days after infection. With the extract, precipitating antibodies were found as early as 5 days after infection and could be detected as late as 90 days after infection. Antigens of trypanosome extracts partially blocked the precipitin reactions between antisera and exoantigens, suggesting the presence of common antigens in the two preparations. Intact trypanosomes were serologically more reactive when collected from immunosuppressed rats. Trypanosomes collected from ATS-treated rats were agglutinated by antisera at titers fourfold higher than trypanosomes collected from untreated hosts. Absorption with exoantigens from immunosuppressed infected rats blocked trypanosome agglutination, indicating that these antigens are of cell surface origin. The experiments suggest that a likely result of immunosuppressing the host is a trypanosome antigen preparation that is a more reactive serodiagnostic reagent.     

Trypanosoma lewisi: production of exoantigens during infection in the rat

Exoantigens are produced by Trypanosoma lewisi during infections in the rat. They were detected in rat serum and plasma by gel-diffusion techniques with hyperimmune rat sera and with rabbit antiserum to washed, living trypanosomes. Their parasite origin is indicated by their presence in trypanosome homogenates, which also contain bound antigens, the continued reactivity of rabbit antisera after absorption with normal rat serum, and the reactions of identity obtained with rat and rabbit antisera. Moreover, by immunoelectrophoresis, the exontigens are revealed as new components in infected rat serum with a mobility slightly anodal to the origin. The results also show that the exoantigens are continuously released in vivo and that the trypanosomes avidly bind non-antibody rat serum proteins to their surface. Unlike the complete qualitative changes in exoantigens that accompany antigenic variation of pathogenic species of trypanosomes, at least one exoantigen remains unchanged when antigenic variation occurs with T. lewisi although additional exoantigens may appear and disappear. The relation of the exoantigens to the known ablastic and trypanocidal antibodies is difficult to determine since these antibodies and the exoantigens occur simultaneously in the blood during and after the infection. Although it cannot yet be ruled out that the exoantigens elicit the formation of these antibodies, a review of all the available evidence suggests that the exoantigens of T. lewisi may not be immunogenic during a natural course of infection. Possibly they are hemolysins with a nutritive function.     

Further Studies on Antigenic Changes in Trypanosoma lewisi*

SYNOPSIS. An investigation of day-to-day changes in adult form antigens of T. lewisi in the rat shows that adult antigens, 2 of which are present in trace amounts in young forms, suddenly appear in large amounts in 9-day-old trypanosomes. An additional antigen is formed on the 13th day. In addition, 2-day-old trypanosomes contain some antigens not present in 6-day-old forms. The combined results of the present paper and a previous one indicate that there are antigenically 3 distinct stages of T. lewisi in the rat: 1- to 3-day-old, 4- to 8-day-old, and 9- to 14-day-old forms. From 9- to 11-day-old trypanosomes, there is a form containing both young and adult antigens.     

Immunosuppression and ablastin

Ablastin formed by animals in response to infections by rodent trypanosomes possesses the characteristics of an antibody. Partial resistance to Trypanosoma lewisi is demonstrable in animals previously injected with live Trypanosoma musculi. Antisera from T. musculi infected mice do not inhibit reproduction by T. lewisi bloodstream forms in vitro as efficiently as homologous antisera collected at similar times during infections, indicating a degree of specificity. Ablastin activity in antisera is not altered by treatment with 2-mercaptoethanol or by heatng at 60 °C for 3 hr. Sephadex G-200 gel filtration of early and late antisera from T. lewisi infected rats and assays with bloodstream forms cultured at 37 °C detect ablastin activity in the second major fraction eluted from the columns. Ablastin appears to be an antibody of the immunoglobulin G species.Immunosuppressant procedures utilized in studies of the host responses to rodent trypanosomes are reviewed and include: chemical agents, irradiation, splenectomy, reticuloendothelia blockade and thymectomy, and treatment with antilymphocyte and antithymocyte sera. Evaluation of the results of the application of these procedures to rodent parasite systems indicates ablastin is an antibody and supports the concept that the inhibition of trypanosome reproduction is separate and distinct from the first trypanocidal event responsible for the decreasing parasitemias observed during the infections. Recent studies concur and suggest that the first crisis in the infections is mediated by the combined actions of a thymus-dependent ablastin and a thymus-independent trypanocidal antibody.     

Flow Cytometric Analysis of Immunoglobulin and Complement Component C3 on the Surface of Trypanosoma lewisi1

ABSTRACT. We have reinvestigated whether surface immunoglobulin (sIg) on Trypanosoma lewisi is antibody directed toward parasite antigen by using flow cytometry to analyze parasites stained with fluoresceinated F(ab′)₂ fragments of antibodies to rat IgG and IgM. We have confirmed that IgG antibody to the parasites is present both in the serum of rats and on the surface of parasites between the fourth and twentieth days of infection, that the amount of sIg per cell increases as the infection progresses, and that considerably more IgG is present on parasites harvested from intact rats than on those from rats that had been immunosuppressed by whole body γ-irradiation. In addition sIgM was detected on trypanosomes from intact, but not on parasites from irradiated rats. We have also made two observations suggesting that not all sIg is specific antibody made in response to T. lewisi. First, a low but significant amount of sIgG was detected on parasites throughout infection in irradiated rats; no sIgM was detected on these parasites. Second, when parasites harvested from immunosuppressed rats were incubated in normal rat serum, the amount of both sIgG and sIgM detected by flow immunofluorescence increased. Parasites harvested from intact animals bound IgM but not IgG from normal rat serum. These results suggest either that natural antibody to the trypanosomes is present in the serum of uninfected rats or that some rat immunoglobulins bind to structures on the trypanosome surface in ways that do not depend on usual antigen-antibody interactions. Finally, flow immunofluorescence was also used to detect complement component C3 on the surface of both intact and trypsinized bloodstream forms harvested from intact or immunosuppressed rats. The amount of sC3 per cell did not increase until late in the infection and consequently did not correlate with the increase of sIgG. Therefore, T. lewisi avoids destruction by the immune system although immune effector molecules, IgG, IgM, and C3, are on its surface.     

Trypanosoma cruzi: location of a specific antigen on the surface of bloodstream trypomastigote and culture epimastigote forms.

The nature of surface antigens of culture epimastigote and bloodstream trypomastigote forms of Trypanosoma cruzi was investigated by light and electron microscopy using indirect immunofluorescence and peroxidase labeling techniques and antisera against unique, common, and contaminant antigens. A specific antigen, identified by monospecific rabbit antiserum (anti-component 5 antiserum), is the major constituent of the cell surface and flagellar membrane of both the culture epimastigote and bloodstream trypomastigote forms. Antigens of heterologous stercorarian trypanosomes (Trypanosoma rangeli) and of culture medium proteins could not be detected on the cell surface of culture epimastigote forms and bloodstream trypomastigote forms.     

A culture and biochemical assay system for Trypanosoma lewisi

Trypanosoma lewisi was cultivated as forms which appeared to be physiologically similar to those found in vivo. The medium consisted of 1.0 g peptone, 1.0 g glucose, 10 ml rat serum, 10,000 units penicillin G, 10,000 μg streptomycin and 90 ml Hank's Balanced Salt Solution. It was supplemented with 8.0 × 10⁸ rat erythrocytes per milliliter. In the complete medium trypanosomes multiplied for 48–72 hr. Cultured forms were lethal to newborn rats and infective to adults.Adsorbed early immune serum inhibited the growth of the trypanosomes in vitro and the percentage of reproductives declined from 66 to 45%. The cultured trypanosomes were also susceptible to both trypanocidal antibodies.     

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.     

Unexpectedly high diversity of trypanosomes in small sub-Saharan mammals

The extremely species-rich genus Trypanosoma has recently been divided into 16 subgenera, most of which show fairly high host specificity, including the subgenus Herpetosoma parasitizing mainly rodents. Although most Herpetosoma spp. are highly host-specific, the best-known representative, Trypanosoma lewisi, has a cosmopolitan distribution and low host specificity. The present study investigates the general diversity of small mammal trypanosomes in East and Central Africa and the penetration of invasive T. lewisi into communities of native rodents. An extensive study of blood and tissue samples from Afrotropical micromammals (1528 rodents, 135 shrews, and five sengis belonging to 37 genera and 133 species) captured in the Central African Republic, Ethiopia, Kenya, Malawi, Mozambique, Tanzania, and Zambia revealed 187 (11.2%) trypanosome-positive individuals. The prevalence of trypanosomes in host genera ranged from 2.1% in Aethomys to 37.1% in Lemniscomys. The only previously known trypanosome detected in our dataset was T. lewisi, newly found in Ethiopia, Kenya, and Tanzania in a wide range of native rodent hosts. Besides T. lewisi, 18S rRNA sequencing revealed 48 additional unique Herpetosoma genotypes representing at least 15 putative new species, which doubles the known sequence-based diversity of this subgenus, and approaches the true species richness in the study area. The other two genotypes represent two new species belonging to the subgenera Ornithotrypanum and Squamatrypanum. The trypanosomes of white-toothed shrews (Crocidura spp.) form a new phylogroup of Herpetosoma, unrelated to flagellates previously detected in insectivores. With 13 documented species, Ethiopia was the richest region for trypanosome diversity, which corresponds to the very diverse environments and generally high biodiversity of this country. We conclude that besides T. lewisi, the subgenus Herpetosoma is highly host-specific (e.g., species parasitizing the rodent genera Acomys and Gerbilliscus). Furthermore, several newly detected trypanosome species are specific to their endemic hosts, such as brush-furred mice (Lophuromys), dormice (Graphiurus), and white-toothed shrews (Crocidura).     

ANIONIC SITES OF HUMAN ERYTHROCYTE MEMBRANES : II. Antispectrin-Induced Transmembrane Aggregation of the Binding Sites for Positively Charged Colloidal Particles

The effects of affinity-purified antispectrin γ-globulins on the topographic distribution of anionic residues on human erythrocytes membranes was investigated using collo ida iron hydroxide labeling of mounted, fixed, ghost membranes. Antispectrin γ-globulins were sequestered inside ghosts by hemolysis and the ghosts were incubated for 30 min at 37°C and then fixed with glutaraldehyde. The topographic distribution of colloidal iron hydroxide clusters on ghosts incubated with low (<0.05 mg/ml) or high (>5–10 mg/ml concentrations of sequestered antispectrin was dispersed, but the distribution at intermediate concentrations (0.1–5 mg/ml) was highly aggregated. The aggregation of colloidal iron hydroxide binding sites was time and temperature dependent and required the sequestering of cross-linking antibodies (antispectrin Fab could not substitute for γ-globulin antibodies) inside the ghosts. Prior glutaraldehyde fixation or fixation at the time of hemolysis in antispectrin solutions prevented the antispectrin-induced colloidal iron site aggregation. The antispectrin reacted exclusively at the inner ghost membrane surface and the colloidal iron hydroxide bound to N-acetylneuraminic acid residues on the outer membrane surface which are overwhelming on the sialoglycoprotein glycophorin. These results were interpreted as evidence for a structural transmembrane linkage between the inner surface peripheral protein spectrin and the integral membrane component glycophorin.     

Trypanosoma cruzi: Antibody-induced mobility of surface antigens

Antibody induces mobility of surface antigens of live blood forms of Trypanosoma cruzi when these cells are incubated with human or animal antisera. Parasites of one strain (Y) showed aggregation of surface antigens to form an anterior or more frequently a posterior cap. The proportion of cap forming trypomastigotes increased with time and was dependent on temperature and amount of antiserum. Cap formation was inhibited by sodium azide or iodoacetamide. Crosslinking of surface antigens by antibodies causing agglutination of trypomastigotes decreased antigenic mobility and capping. Capping was not affected by treatment of the parasites with colchicine or cytochalasin B. Antigen mobility was not related to the presence of antibodies against parasite and host tissue cross-reacting antigens. Aggregation of surface membrane antigens was also observed in parasites which survive after immune lysis. Results of light and electron microscopic studies suggested that at least part of the aggregated antigens was eliminated from the trypomastigote's surface. Cap formation was strain and stage dependent. It was not observed when Y-strain epimastigotes were used and it occurred less frequently in CL than in Y-strain trypomastigotes.     

Grafting of different glycosides on the surface of liposomes and its effect on the tissue distribution of 125I-labelled γ-globulin encapsulated in liposomes

Different glycosides were grafted on the surface of liposomes containing ¹²⁵I-labelled γ-globulin by two ways: (1) by using glycolipid and (2) by covalent coupling of $\text{p-}\text{aminophenyl-}\text{d}\text{-glycosides}$ to phosphatidylethanolamine liposomes using glutaraldehyde. The distribution of ¹²⁵I-labelled γ-globulin was determined in mouse tissues from 5–60 min after a single injection of these liposomes. The liver uptake of encapsulated ¹²⁵I-labelled γ-globulin was highest from liposomes having galactose and mannose on the surface. Competition experiments and cross-inhibition studies indicate that this uptake are mediated by specific recognition of the surface galactose and mannose residues of liposomes by the receptors present on the plasma membrane of liver cells. Stearylamine-containing liposomes were found to be more efficient in mediating the uptake of ¹²⁵I-labelled γ-globulin by the lung, whereas in the case of spleen, phosphatidylethanolamine liposomes were more efficient. The extent of uptake of ¹²⁵I-labelled γ-globulin from all types of liposome decreases as the amount of given liposomes increases. The uptake of ¹²⁵I-labelled γ-globulin from liposomes containing asialogangliosides depends upon the phospholipid/ glycolipid ratio. These experiments clearly demonstrate that enhanced liposome uptake by liver cells could be achieved by grafting galactose and mannose on the liposomal surface.     

Antigen-specific and nonspecific mediators of T-cell/B-cell cooperation. VI. Distinct patterns of cross-reactivity by two human gamma-globulin-primed helper T-cell subpopulations.

We have previously characterized the activities, in vitro, of two different helper T-cell subpopulations, primed with human γ-globulin (HGG). One T-cell subpopulation helps the response of B cells to determinants (e.g., haptens) bound to the same antigen to which the T cells are primed (specific help); the other helper T-cell subpopulation responds to the same priming antigen by secreting a nonspecific molecule which helps B-cell responses to erythrocyte antigens co-cultured with the priming antigen (nonspecific help). These subpopulations also differ in their frequency and dose response to antigen, both in vivo and in vitro. They are similarly susceptible to the induction of unresponsiveness to HGG. In order to determine whether these T-cell subpopulations share or differ in their ranges of antigen recognition, we have compared the reaction of these two HGG-primed helper T-cell subpopulations to a number of γ-globulins (γG's) from other species. Plaque-forming cells generated in response to HGG shared little or no cross-reactivity with any of the heterologous (γG's) tested. In contrast, HGG-primed nonspecific helper T cells responded with significant cross-reactivity when challenged in vitro with dog γG, but HGG-primed specific helper T cells did not respond with any such cross-reactivity. No other heterologous γG tested stimulated any significant cross-reactivity from either HGG-primed T-cell subpopulation. Thus, these two T-cell subpopulations differ in their antigenic recognition. Possible explanations of these data include: (i) a difference in receptor specificity; (ii) a difference in the receptor affinity; (iii) a difference in Ia determinants of the two subpopulations.     

Growth and Immunologic Studies on the Culture Forms of Trypanosoma lewisi*

SYNOPSIS. Culture forms of Trypanosoma lewisi grown at 27 C in a diphasic blood agar medium resemble in structure the stage found in the invertebrate host. Cultures inoculated with approximately 1 × 10⁶ trypanosomes/ml attain maximum populations of 2–7 × 10⁷ organisms/ml after 5–6 days of incubation. The stationary phase persists 6–15 days. The decline of the population is of relatively long duration with approximately 1 × 10⁶ viable organisms/ml present after 90 days. Variations in growth were attributed to the preparation of defibrinated heated rabbit blood incorporated into the culture medium. With inocula of 3.0 × 10⁵ trypanosomes/ml there was a lag in growth not observed with larger inocula. Trypanosomes incubated at elevated temperatures had altered growth curves compared to organisms at 27 C. Agitation of cultures did not affect the growth or stationary phases, but hastened the population decline. Heated and unheated 5% (v/v) normal rat serum incorporated in the liquid phase of the medium altered the growth of the organisms. Heated serum caused a decrease in the population and an extended lag phase. The effects on growth were more marked with unheated serum suggesting that both heat-stable and labile components affect growth. Antisera from rats injected with live culture forms included in the liquid phase inhibited, while antisera from rats 24 days after infection with the blood stream forms had no effect on the growth of the culture forms. Antisera from rabbits immunized with sonicates of culture forms also altered the growth of the organisms in culture. Rabbit antisera prepared by immunization with sonicates of dividing and non-dividing blood stream forms had no effect on the in vitro growth. Antisera from animals immunized with rat blood and culture medium were also without effect. The immunologic implications of the data are considered and discussed.     

Trypanosoma brucei: Antigenic analysis of bloodstream,vector, and culture stages by the quantitative fluorescent antibody methods

Quantitative direct fluorescent antibody methods were used in antigenic analysis of developmental stages of Trypanosoma brucei brucei strains, most of them having the same variant antigen B, which were derived from a cyclically transmissible stabilate. Antigen-B trypanosomes were used for initiation of cultures in modified Tobie's (Tm) medium and in Glossina morsitans morsitans organ cultures, and for the infective feed of G. m. morsitans. Antisera against antigen-B bloodstream forms and against Tm-grown culture forms were developed in rabbits by inoculations of disrupted organisms mixed (1:1) with complete Freund's adjuvant. The globulin fractions of the antisera were conjugated with fluorescein isothiocyanate, and processed on Sephadex G-25 and DEAE-cellulose columns. The DEAE fractions with 2.0 and 4.7 or 4.8 molar fluorescein:protein ratios were pooled and concentrated twofold.Examination of 109 flies at 30 or 31 days after the infective feed revealed about 18.3% midgut, about 10.1% proventricular, and about 3.7% salivary-gland infections. A salivary gland suspension from one of the infected flies gave rise to a parasitemia in a mouse, and trypanosomes from the first parasitemia were transferred by two 3-day syringe passages into another mouse. Smears were prepared of trypanosomes (antigens B-164, B-167) from the first parasitemias from these two mice, of intact B-antigen trypanosomes, of culture forms (CT) from Tm medium, and of procyclics (CG) from Glossina cultures as well as of midgut (GM), proventricular (GP), and salivary-gland (GS) forms from tsetse flies. All these forms were fixed by one or more of the three following methods: complete fixation (CoFix) by the formalin-NH₄OH-Tween 80 procedure; fixation before affixation to slides (F+); fixation after affixation to slides (F?). The best results with regard to fluorescence intensity and specificity were obtained by using the CoFix technique.Statistical analyses of the fluorescence means of the antigens subjected to direct and inhibition staining gave the following results: (1) CT, CG, GM, and GP forms were antigenically the same. (2) GM and GP trypanosomes from different flies were antigenically indistinguishable. (3) The surface antigen of the variant-B bloodstream trypanosomes was different from these antigens of culture, midgut, and proventricular forms. It differed also from those of metacyclics from two flies and of B-164 and B-167 bloodstream forms. (4) No antigenic differences were found, in preparations fixed by the F? method, between B-164 and B-167 bloodstream trypanosomes and the metacyclics from two flies, one of which served as the source of the salivary-gland trypomastigotes (GS-98) that gave rise to these two bloodstream form antigens. (5) Closer antigenic relationships were noted between B forms and B-164 and B-167 trypanosomes than between B and CT organisms in smears fixed by the F+ technique, but no such differences were discernible in preparations fixed by the F? procedure.     

PCR identification of Trypanosoma lewisi,a common parasite of laboratory rats

Trypanosoma (Herpetosoma) lewisi is a trypanosome of the sub-genus Herpetosoma (Stercoraria section), parasite of rats (Rattus rattus and Rattus norvegicus) transmitted by fleas. T. lewisi has a stringent species specificity and cannot grow in other rodents such as mice. Rats are infected principally by oral route, through contamination by flea faeces or ingestion of fleas. Trypanosoma lewisi infections in rat colonies can interfere with research protocols and fleas of wild rats are often the source of such infections. Currently, diagnosis of T. lewisi in rats is performed by microscopic observation of stained blood smears. In the course of a research project at CIRDES, a T. lewisi infection was detected in the rat colony. In this study we evaluated PCR primer sets for their ability to diagnose multiple species of trypanosomes with a single amplification. We show that the use of ITS1 sequence of ribosomal DNA provides an efficient and sensitive assay for detection and identification of T. lewisi infection in rats and recommend the use of this assay for monitoring of T. lewisi infections in rat colonies.     

Trypanosoma lewisi: accumulation of antigen-specific host IgG as a component of the surface coat during the course of infection in the rat.

Naturally acquired host IgG, adsorbed to the surface of Trypanosoma lewisi during the course of infection in the rat, was labeled with fluorescein-conjugated rabbit IgG, or Fab fragments of this IgG, directed against rat IgG. The intensity of fluorescent labeling increases with time, concomitant with the increase in anti-T. lewisi activity of host plasma. Trypanosomes harvested from immunosuppressed hosts lack detectable surface IgG. Trypanosomes having little or no detectable surface IgG (harvested from immunosuppressed hosts or early in the infection from immunocompetent hosts) can adsorb IgG from serum with ablastic activity only (obtained from other infected rats between the first and second crises and adsorbed to remove trypanocidal antibodies), but not from normal serum. Therefore, the absence of detectable surface IgG on such cells is not caused by the parasites' inability to adsorb host IgG, but rather results from the immune state of the host. Hence surface IgG on T. lewisi is specific antibody. Host albumin is nonspecifically adsorbed, in contrast to IgG. Trypanosomes from immuno-suppressed and immunocompetent rats were positive and visually indistinguishable from each other when labeled with anti-rat albumin, and were equally agglutinable with anti-rat albumin serum.     

Trypanosoma lewisi: antibody classes which mediate precipitation, agglutination, and the inhibition of reproduction

Sera from Trypanosoma lewisi-infected and uninfected rats were applied to Protein A-Sepharose CL-4B columns. The absorbed fractions of antisera which contained only IgG molecules were reacted in microimmunodiffusion analyses with the exoantigens of T. lewisi in plasma collected from irradiated infected rats, and formed one precipitin line. These sera were also applied to T. lewisi extract immunoabsorbent columns and bound proteins were eluted and analyzed by immunodiffusion against antisera specific for rat immunoglobulins. IgG₁, IgG_2a, IgG_2b, IgG_2c, and IgM were absorbed by the immuno-absorbent columns. Absorption of the rat antisera with anti-rat IgG or anti-rat IgM removed one of the two precipitin lines against extracts prepared from parasites collected from irradiated infected animals. The absorbed IgG fractions and nonabsorbed fractions of antisera which were collected after Protein A-Sepharose CL-4B column chromatography agglutinated trypanosomes. After treatment of antisera with 2-mercaptoethanol, the agglutinin titers were lower than those of the control antisera suggesting both IgG and IgM are involved in the agglutination. The ablastic activity of the fractions eluted from Protein A-Sepharose CL-4B Chromatographic columns was assayed in cultures of bloodstream forms ofT. lewisi. Ablastic activity of proteins of antisera absorbed by the columns was demonstrated indicating they belonged to the IgG class of antibodies.