Resistance to normal human serum reveals Trypanosoma lewisi as an underestimated human pathogen.

Human-infectious trypanosomes such as Trypanosoma cruzi, T. brucei rhodesiense, and T. b. gambiense can be discriminated from those only infecting animals by their resistance to normal human serum (NHS). These parasites are naturally resistant to trypanolysis induced by the human-specific pore-forming serum protein apolipoprotein L1 (ApoL-1). T. lewisi, a worldwide distributed parasite, has been considered as rat-specific and non-pathogenic to the natural hosts. Here we provide evidence that 19 tested T. lewisi isolates from Thailand and China share resistance to NHS. Further investigation on one selected isolate CPO02 showed that it could resist at least 90% NHS or 30 µg/ml recombinant human ApoL-1 (rhApoL-1) in vitro, in contrast to T. b. brucei which could not survive in 0.0001% NHS and 0.1 µg/ml rhApoL-1. In vivo tests in rats also demonstrated that this parasite is fully resistant to lysis by NHS. Together with recent reports of atypical human infection by T. lewisi, these data allow the conclusion that T. lewisi is potentially an underestimated and thus a neglected human pathogen.

[The immunosuppressant effect of T. lewisi (Kinetoplastidae) infection on the multiplication of Toxoplasma gondii (Sarcocystidae) on alveolar and peritoneal macrophages of the white rat]. / Efecto inmunosupresor de Trypanosoma lewisi (Kinetoplastidae) sobre la multiplicación de Toxoplasma gondii (Sarcocystidae) en macrófagos alveolares y peritoneales de rata blanca.

The immunosuppressant effect of T. lewisi infection on the multiplication of T. gondii was compared in peritoneal (MP) and alveolar macrophages (MA) of white rat. Two animal groups were infected with T. lewisi and sacrificed after four days and seven days post infection. A group without infection was maintained as a control. The number of intracellular parasites (tachyzoites) (IT) was counted by light microscopy, calculating the rate infection rate per 100 total cells (TC) and per infected cells (IC) for each group of phagocyte cells. The relation quotient IT, TC or IC multiplied percent, provided a statistical ratio (RE) of the relative number of parasites in both cellular types for each time interval. MA as well as MP obtained after 4 days showed a significant increase in the multiplication of T. gondii with respect to the control. Unlike the MP (which had an increase in the multiplication of T. gondii the fourth day of infection with T. lewisi diminishing towards the seventh day), the MA had an increase in the multiplication of the parasite from the fourth to the seventh day. This difference can be related to the route of infection used for the experiments, that affect the MP directly with a greater effect in comparison with the MA of the lungs. Lung compartment will be affected later, when the infection becomes systemic between the fourth and sixth day of infection. The immunity against T. gondii is similar between both phagocytes, but the time of infection and the compartment where the cells are located, makes the difference in the response time against T. gondii. Supernatants from macrophage cultures or T. lewisi by rat did not induced any immunosuppression.

Efecto inmunosupresor de Trypanosoma lewisi (Kinetoplastidae) sobre la multiplicación de Toxoplasma gondii (Sarcocystidae) en macrófagos alveolares y peritoneales de rata blanca

The immunosuppressant effect of T. lewisi (Kinetoplastidae) infection on the multiplication of Toxoplasma gondii (Sarcocystidae) on alveolar and peritoneal macrophages of the white rat. The immunosuppressant effect of T. lewisi infection on the multiplication of T. gondii was compared in peritoneal (MP) and alveolar macrophages (MA) of white rat. Two animal groups were infected with T. lewisi and sacrificed after four days and seven days post infection. A group without infection was maintained as a control. The number of intracellular parasites (tachyzoites) (IT) was counted by light microscopy, calculating the rate infection rate per 100 total cells (TC) and per infected cells (IC) for each group of phagocyte cells. The relation quotient IT, TC or IC multiplied percent, provided a statistical ratio (RE) of the relative number of parasites in both cellular types for each time interval. MA as well as MP obtained after 4 days showed a significant increase in the multiplication of T. gondii with respect to the control. Unlike the MP (which had an increase in the multiplication of T. gondii the fourth day of infection with T. lewisi diminishing towards the seventh day), the MA had an increase in the multiplication of the parasite from the fourth to the seventh day. This difference can be related to the route of infection used for the experiments, that affect the MP directly with a greater effect in comparison with the MA of the lungs. Lung compartment will be affected later, when the infection becomes systemic between the fourth and sixth day of infection. The immunity against T. gondii is similar between both phagocytes, but the time of infection and the compartment where the cells are located, makes the difference in the response time against T. gondii. Supernatants from macrophage cultures or T. lewisi by rat did not induced any immunosuppression. Rev. Biol. Trop. 57 (1-2): 13-22. Epub 2009 June 30.

El efecto inmunosupresor de la infección de T. lewisi sobre la multiplicación de T. gondii fue comparado en macrófagos peritoneales (MP) y alveolares (MA) de rata. El número de parásitos (taquizoitos) intracelulares (TI) fue contado por microscopía de luz. Los macrófagos alveolares y peritoneales (MP) de animales con 4 días de infección con T. lewisi muestran un aumento significativo en la multiplicación de T. gondii. A diferencia de los MP (que muestran un aumento en la multiplicación de T. gondii al cuarto día de infección con T. lewisi disminuyendo hacia el séptimo día), los MA mantienen un aumento en la multiplicación del parásito desde el cuarto, aumentando hacia el séptimo día de infección. Esta diferencia se puede deber a la ruta de infección utilizada para los experimentos que afectan directamente los MP donde se observa un efecto mayor y más temprano en comparación con los MA aislados de los pulmones, compartimiento afectado cuando la infección se vuelve sistémica entre el cuarto y sexto día de infección. La inmunidad contra T. gondii es similar entre ambas células fagocíticas, pero el tiempo de infección y el compartimiento donde se encuentren las células hace la diferencia en el tiempo de respuesta contra un parásito dado, en nuestro caso T. gondii. No hubo evidencia de que los sobrenadantes de cultivos de macrófagos provenientes de ratas infectadas ni el lisado de tripanosomas indujeran el efecto inmunosupresor.

Alpha 2 macroglobulin activity in rats infected with Typanosoma lewisi and treated with cyclophosphamide and its effect on the malignancy of the disease.

BACKGROUND & OBJECTIVES: Trypanosoma lewisi is a common, flagellated parasite of the rat. Our previous study showed that rabbits injected with serum collected from rats infected with Trypanosoma lewisi and treated with cyclophosphamide (CyI) produced high levels of antibodies against a new protein in the CyI rat serum. RESULTS: In the present study, this protein was characterised as alpha2 macroglobulin (alpha2M) and the kinetics of its production and its influence on the malignancy of the disease were determined. In rats infected with T. lewisi, alpha2M was first demonstrated and peaked on the second day post-infection (972 microg/ml) and then reduced gradually, reaching a level of 32 microg/ml on the eighth day post-infection. However, in the CyI rats the level of alpha2M was gradually increased as the disease progressed, reaching a level of 890 microg/ml on the eighth day post-infection. Injection of both crude and purified alpha2M into rats infected with T. lewisi led to increased parasitaemia. INTERPRETATION & CONCLUSION: The present study suggests that increased levels of alpha2M in the CyI rats contribute to the malignancy of the disease.

Trypanosoma lewisi-induced immunosuppression: the effects on alveolar macrophage activities against Cryptococcus neoformans.

The immunosuppressive effect of Trypanosoma lewisi infection on alveolar macrophage (AM) activities against Cryptococcus neoformans was studied in an animal model. Two groups of rats were treated with T. lewisi and killed after 4 (4d-rats) and 7 days (7d-rats), respectively. A third group not given T. lewisi, served as control. AM were challenged in vitro with C. neoformans. Phagocytosis was assessed with a fluorescence method. Superoxide anion production was evaluated with the nitroblue tetrazolium (NBT) test. The survival of cryptococci was estimated by counting colony-forming units. The numbers of detached AM from culture plates were determined using a Bürker chamber. The NBT response, adhesion to plate surface and killing activity, but not the phagocytosis of AM from 4d-rats were significantly impaired compared to control or 7d-rats. Thus, T. lewisi causes transitory immunosuppressive effects on AM activities. This rapid T. lewisi immunosuppression model may be useful to study new approaches to anticryptococcal therapy.

Infection of white rat peritoneal macrophages with Toxoplasma gondii, (Coccidia: Sarcocystidae) after Trypanosoma lewisi (Kinetoplastida: Trypanosomatidae) infection.

Peritoneal macrophages from Wistar rats, inoculated and non-inoculated with 10(6) T. lewisi trypomastigotes, were cultured and infected with 10(6) T. gondii tachyzoites. Multiplication rates of this parasite were studied after 1, 24 and 48 h of infection but there were not significant differences between the number of parasites found inside of macrophages coming, either from T. lewisi infected or non infected rats. On the other hand, in vivo studies of Toxoplasma multiplication inside peritoneal macrophages, showed that there is an increase of parasite number in cells from T. lewisi infected rats, as compared with those macrophages from non infected rats. This effect was statistically significant and was more evident after four days of infection. Therefore, it has been demonstrated that in vivo, but not in vitro T. lewisi infections, causes an important decrease of the natural resistance to T. gondii of the white rats, which is manifested by the major invasion and multiplication of the parasite inside of peritoneal macrophages.

Effect of Trypanosoma lewisi (Kinetoplastida: Trypanosomatidae) on the infection of white rats with Toxoplasma gondii (Eucoccidia: Sarcocystidae) oocysts.

White rats were inoculated with 10(6) trypomastigotes of Trypanosoma lewisi, simultaneously or two days before and after inoculation with 10(5) oocysts of T. gondii. A greater number of cysts was found in the brain of the animals having concomitant inoculations, as compared with rats inoculated with either one of the two parasites. An apparent immunosuppressive effect is likely. Since both organisms can be found in rats, it is possible that infections with T. lewisi, could make this rodent another intermediate host for Toxoplasma infections.

Trypanocidal drugs and their effect on parasitaemia, specific IgG production and protective immunity in rats infected with Trypanosoma lewisi.

In rats infected with Trypanosoma lewisi, parasitaemia normally resolves by day 32; thereafter, the rodents become solidly immune to re-infection. Rats treated on day 5 of infection with a single i.m. dose of 35 mg/kg of the trypanocidal drug ethidium bromide (EB) had recovered from parasitaemia by day 12, whereas berenil (BE) given at 100 mg/kg, more than twice the recommended dose, had no effect on parasitaemia. However, rats treated for 4 consecutive days beginning on day 5 of infection with Lampit (LA) and Radanil (RA) at 350 mg/kg showed no parasitaemia on days 16 and 20, respectively. EB was the most effective drug in lowering the total IgG antibody as compared with the control animals, whose specific IgG titres remained elevated for over 200 days after the parasitaemia had been cleared. LA also significantly reduced the antibody levels through day 240, whereas RA only transitorily depressed the antibody levels on days 20 and 30. BE, which had no effect on parasitaemia, correspondingly failed to depress the total IgG levels. Re-challenge infection of the drug-treated, recovered animals on day 240 (208 days after the normal resolution of the infection) revealed that except for the EB group, which displayed transitory parasitaemia for 4 days, other treated and control rats completely resisted the challenge; pre-challenge antibody titres were lower than 1:160 in EB-treated animals in contrast to the levels of 1:320 or higher measured in the other drug-treated and control animals, which resisted the infection.(ABSTRACT TRUNCATED AT 250 WORDS)

Cellular responses to culture-derived soluble exoantigens of Trypanosoma lewisi.

Soluble exoantigens of Trypanosoma lewisi were obtained from short-culture systems of dividing epimastigotes and non-dividing trypomastigotes incubated in phosphate-buffered saline glucose solution (PBSG) for 3 h at 37 degrees C. Suppression of normal rat-spleen cell responses to mitogen concanavalin A (Con A) occurred at high exoantigens concentrations. The epimastigote-derived exoantigens were more suppressive than the trypomastigote-derived exoantigens. The suppression of Con A stimulation was ablated by the addition of exogenous interleukin-2 (IL-2) or washing of spleen cells that had been incubated with suppressing concentrations of the epimastigote exoantigens for 24 h prior to Con A stimulation. These results strongly suggest that exoantigens of T. lewisi play an important immunoregulatory role during the course of infection and that they achieve this by inhibiting the production of IL-2 by T-helper cells.

Cellular responses to phase fractions of Trypanosoma lewisi.

Infections by Trypanosoma lewisi are characterized by hyporesponsiveness of the immune system during the early phase of parasitaemia. Blastogenic response of normal rat spleen cells to amphiphilic and hydrophilic components of Triton X-114 solubilized epimastigote forms of T. lewisi which characterizes the early phase of infection showed that suppression of responses to mitogens Concanavalin A (Con-A) and lipopolysaccharide (LPS) occurred exclusively with the amphiphilic fraction that consists of integral surface membrane constituents. The Con-A-induced suppression by the amphiphilic constituents was ablated by addition of exogenous IL-2 or by the removal of the adherent cell population in the cultures. This suggests that the integral surface membrane components play an important regulatory role in infections with Trypanosoma lewisi, through complex mechanisms that probably involve the B cells and suppressor macrophages; the suppressor macrophages probably produce a suppressor factor that inhibits the proliferation of T helper cells and subsequently the production of interleukin-2.

Suppressor and protector factors derived from Trypanosoma lewisi.

Trypanosoma lewisi is a specific protozoan blood parasite of rats. Normal rats infected 2 h after treatment with plasma from day 8 irradiated (8.5 Gy) infected rats had significantly higher parasitaemia; in contrast, animals infected 7 days post-plasma treatment were significantly protected. Trypanolytic and ablastic antibodies could be demonstrated in the serum of normal rats treated with the plasma; the trypanolytic antibodies were stage-specific. Suppression of normal rat splenocyte responses to Con A and lipopolysaccharide (LPS) stimulation were also observed in the presence of different protein concentrations of whole lysate from epimastigote forms. The suppression by mitogen Con A was ablated by the addition of exogenous IL 2, or by washing cells incubated with the lysate prior to mitogen stimulation. These results indicate that immunoregulatory factors are present in the plasma of rats infected with T. lewisi, and the effect of these factors can be demonstrated in vitro with whole parasite lysate. The restoration of normal splenocyte responses to Con A by addition of exogenous IL 2 or by washing cells suggests that the suppressor factor(s) act(s) on the T cells by inhibiting their proliferation and IL 2 production, and the continued presence of these products is essential in the maintenance of suppression.

Immune responses of germfree mice to experimental infection with Trypanosoma cruzi.

1. The immune responses to Trypanosoma cruzi infection of germfree mice were compared to the responses of infected conventional mice. Two groups (40 animals in each group) of 2-month old female CFW germfree and conventional mice were used. The IgM and IgG which bound to the surface of T. cruzi epimastigotes determined by ELISA were significantly lower in germfree than in conventional mice (1/3 and 1/5 for IgM and IgG, respectively). 2. After infection there was a three-fold increase in the circulating levels of these immunoglobulins in germfree but not in conventional mice. Twenty-one days after T. cruzi inoculation, both IgG and IgM levels were similar in germfree and conventional animals. 3. Footpad swelling after T. cruzi-antigen inoculation was initially four-fold more intense in germfree than in conventional mice. 4. These results suggest that the reduced humoral immune response of germfree mice during the initiation of experimental Chagas' disease may be responsible for the more severe parasitism when compared to conventional mice.

Immune responses of germfree mice to experimental infection with trypanosoma cruzi

1. The immune responses to Trypanosoma cruzi infection of germfree mice were compared to the reponses of infected conventional mice. Two groups (40 animals in each group) of 2-month old female CFW germfree and vonventional mice were used. The IgM and IgG which bound to the surface of T. cruzi epimastigotes determined by ELISA were significantly lower in germfree than in conventional mice (1/3 and 1/5 for IgM and IgG, respectively). 2. After infection there was a three-fold increase in the circulating levels of these immunoglobulins in germfree but not in conventional mice. twenty-one days after T. cruzi inoculation, both IgG and IgM levels were similar in germfree and conventional animals. 3. Footpad swelling after T. cruzi-antigen inoculation was initially four-fold more intense in germfree than in conventional mice. 4. These results suggest that the reduced humoral immune response of germfree mice during ythe initiation of experimental Chagas' disease may be responsible for the more severe parasitism when compared to conventional mice

A medium for the continuous cultivation of bloodstream forms of Trypanosoma lewisi at 37 C.

Trypanosoma lewisi has been maintained continuously at 37 C for more than 2 yr in Iscove's modified Dulbecco's medium with 10% fetal calf serum and a feeder layer of rat fibroblasts. In this medium the continuously reproducing hematozoic culture forms resemble bloodstream forms of T. lewisi in that they appear morphologically similar in Giemsa-stained preparations examined by light microscopy and have a surface coat that is absent in culture forms grown at ambient temperatures, when examined by electron microscopy. To determine whether these hematozoic culture forms also are similar functionally to bloodstream forms, comparative tests of the 2 forms were made of infectivity for the natural rat host, growth in vitro in the described culture medium, sensitivity to inhibition of reproduction by the rat antibody ablastin, and agglutinability by the 2 trypanocidal antibodies produced during a natural course of infection in the rat. Initially, differences between the 2 forms were minor, but after 16 mo in vitro greater differences began to emerge. Most marked was a reduction in infectivity by 22 mo, although sensitivity to ablastin, the single most important characteristic of bloodstream forms of T. lewisi, was still appreciable at this time. Nevertheless, despite this limitation, the culture system described supports hematozoic culture forms of T. lewisi for a considerably longer time than has been reported thus far.

Trypanosoma lewisi: effects of trace metal contaminants on immunological responses.

The effects of trace metal contaminants (lead, cadmium, and mercury) on the immunologic responses of rats infected with Trypanosoma lewisi were investigated to determine whether chronic exposures to subclinical levels of these metals have adverse effects on the host's ability to respond to an infective agent.Earlier and higher levels of parasitemia were detected in animals exposed to cadmium, lead, and mercury. The variability in length of trypanosome cells in metal-exposed animals became constant at a later time and persisted for a longer time, indicating a delay in antibody synthesis. Serum levels of IgG and IgM were increased in infected animals exposed to trace metals; however, lower levels were observed when compared with infected control animals. Animals exposed only to trace metals showed decreases in serum levels of IgG and IgM. In contact sensitivity to dinitrofluorobenzene (DNFB), animals exposed to trace metals demonstrated no significant difference in sensitization when compared with those infected and exposed to trace metals.

Trypanosoma lewisi: restriction of alternative complement pathway C3/C5 convertase activity.

The rat parasite Trypanosoma lewisi was incubated in vitro with rat or human serum, washed, and extracted in detergent. Extracts were fractionated by electrophoresis in denaturing gels, transferred to nitrocellulose, allowed to renature, then immunoblotted with polyclonal antibodies to rat complement component C3 and human complement components C3, C5, and factor B. Molecules that reacted with these antibodies were detected in the extracts. Fragments of rat C3 were detected in extracts of parasites that had not been exposed to serum in vitro. Additional complement deposition occurred during in vitro incubations; human complement components deposited in vitro could be distinguished from rat components deposited in vivo. Complement deposition in vitro required magnesium ions and did not occur when heat inactivated serum was used. Components reacting with antibodies to human C3 included a group of bands with molecular weights higher than C3 alpha or beta chains. Blotting with affinity purified, chain specific antibodies demonstrated that a 68 kDa component on parasites is C3 beta and that a 44 kDa molecule is derived from C3 alpha. A 73 kDa component that was difficult to resolve from C3 beta is probably also a C3 alpha fragment. This suggests that an inactive iC3b-like molecule is present on parasites. Kinetic studies showed that cleavage of C3 alpha is rapid and that the amount of C3 alpha fragments and C3 beta on intact parasites reached a steady state after 15 min. When parasites were trypsinized prior to incubation in C5 or C6 deficient serum, the rate and extent of C3 and C5 deposition increased. Unprocessed C3 alpha' and C5 alpha' chains were detected. Trypsinized parasites were lysed by the alternative complement pathway in normal serum. Intact parasites could be lysed by complement in the presence of antibody. The data support our previous suggestion that trypsin sensitive surface proteins on intact T. lewisi limit alternative pathway activity by restricting C3/C5 convertase activity.

Cooperative action of complement component C3 and phagocytic effector cells in innate murine resistance to Trypanosoma lewisi.

Mice display strong natural resistance to the rat-specific Trypanosoma lewisi. Intravenously injected intact T. lewisi parasites were eliminated by mice within 18 to 24 h. In comparison, "nude" T. lewisi organisms lacking the surface coat were rapidly and totally cleared from the bloodstream within 2 h postinoculation. Similarly, in vitro-cultivated trypanosomes were readily eliminated by mice with the conspicuous absence of a lag phase. Elimination of nude T. lewisi, like that of intact trypanosomes, required murine complement component C3. Splenectomy of mice did not affect their ability to eliminate T. lewisi. However, C3 depletion with cobra venom factor rendered splenectomized mice susceptible to this rat trypanosome; in these mice, T. lewisi established prolonged and frequently fatal infections. Beige mice were able to efficiently eliminate T. lewisi. But combined treatments of beige (bg/bg) and heterozygous (bg/+) mice with cobra venom factor and silica dust, or normal rat serum and silica dust, incapacitated the natural resistance of these mice to T. lewisi. Such combined treatments of beige and control mice resulted in fulminating parasitemias and death of the animals. Altogether, the results of the present studies indicate that T. lewisi elimination by mice requires the following: exposure of C3 acceptors on the surface of the parasites; activation of murine C3, probably via the alternative complement pathway; and destruction of the C3b-coated parasites by their interaction with C3b receptor-bearing, phagocytic effector cells that are abundant in the spleen and sensitive to silica dust.

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.

A surface glycoprotein of Trypanosoma lewisi binds immunoglobulin G from the serum of uninfected rats.

Detergent extracts of whole Trypanosoma lewisi were fractionated by polyacrylamide gel electrophoresis and transferred to nitrocellulose for immunoblotting analysis. Antibody probes to rat immunoglobulin G (IgG) and IgM detected intact gamma chains, mu chains, and light chains in extracts. The amount of immunoglobulin detected increased as the infection progressed. Transfers were also incubated in serum from conventionally reared (CRS), specific-pathogen-free (SPFS), or germ-free rats before being probed with anti-rat IgG or anti-rat IgM. Components of 200, 175, and 120 kilodaltons (kDa) bound IgM from all sera tested and were present in extracts of trypanosomes isolated from lethally irradiated or intact rats on different days during infection. No parasite components bound IgG from serum of germ-free rats. However, 145-, 175-, and 200-kDa components bound IgG from CRS and SPFS. A 74-kDa protein was the major IgG-binding component in extracts of reproducing parasites. This component bound much more IgG from CRS than it bound from SPFS. The 74-kDa protein was removed from parasites by mild trypsinization and corresponded to a major surface glycoprotein detected when intact cells were radioiodinated. These results indicate that natural antibodies to T. lewisi exist in rats or that these parasites have surface proteins that bind immunoglobulins without regard to antigenic specificity.

Flow cytometric analysis of immunoglobulin and complement component C3 on the surface of Trypanosoma lewisi.

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 gamma-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 parasite. 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.