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.     

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 cruzi: Isolation and characterization of a proteinase

Lysates of Trypanosoma cruzi epimastigotes were able to hydrolyze casein (K_m = 2.5 mg/ml) as well as bovine and human hemoglobins (K_m = 12.2 mg/ml); there was optimum activity was around pH 7.0. The proteinase activity detected with these substrates was enhanced by sodium diaminotetraacetate (EDTA) and reducing agents (SO^2?₃, mercaptoethanol, cysteine) and was inhibited by sulfhydryl reagents, thus suggesting an SH-dependent enzyme. Purification (60×) of the proteinase was carried out as follows: (1) precipitation at ?20 C, pH 4.5, with 80% acetone, (2) gel filtration on Sephadex G-200, (3) affinity chromatography on Sepharose 4B covalently linked to p-aminophenyl mercuric acetate. Only a single component (with an estimated molecular weight of 60,000) was detected in purified preparations by polyacrylamide gel electrophoresis. However, in addition to the major component identified as a proteinase, crossed immunoelectrophoresis experiments indicated the presence of at least three other antigens that apparently were devoid of proteinase activity. Optimum pH activity of the purified preparations was around pH 6.0 for casein and pH 3.0 for hemoglobins, but these activities probably are due to the one enzyme since they were altered identically by the same agents.     

Trypanosoma musculi: Passive hemagglutination technique to measure antibody in mice

A passive hemagglutination assay was developed to measure Trypanosoma musculi-specific antibody in mice. Indicator-erythrocyte donor mice received 550 rad ⁶⁰Co 24 hr before intraperitoneal injection of 3 × 10⁴T. musculi. T. musculi antigen-coated erythrocytes were obtained from these mice on Day 9 postinfection. T. musculi antigen-coated erythrocytes obtained in this manner were used as indicator erythrocytes in a passive hemagglutination procedure. Serum from hyperimmunized mice (three consecutive infections at 21-day intervals) gave titers as high as 1:1024. Titers of 1:256 and 1:512 were obtained from singly infected mice on Days 18 and 28 postinfection, respectively. In marked contrast, nude mice infected with T. musculi did not produce a detectable agglutinating antibody response. Erythrocytes obtained from either irradiated (550 rad ⁶⁰Co) uninfected mice, nonirradiated infected mice, or normal mice did not agglutinate when combined with any of the sera tested. These data suggest the usefulness of this passive hemagglutination assay for the measurement of antibody to T. musculi in the serum of infected mice.     

Trypanosoma lewisi: alterations in membrane function in the rat.

DEAE-cellulose-purified Trypanosoma lewisi from 4-day (dividing trypanosomes) and 7-day (non-dividing trypanosomes) infections in rats were compared for initial uptake of glucose, leucine, and potassium. Glucose entered the parasitic cells by mediated (saturable) processes, whereas leucine and K⁺ entered by mediated processes and diffusion. Glucose entry was significantly elevated in 4-day cells (V_max 4.00 ± 1.02 nmoles/ 1 × 10⁸ cells/min) with respect to 7-day cells (V_max 1.83 ± 0.62 nmoles 1 × 10⁸ cells/min). Likewise, the affinity of the glucose carrier was significantly greater in 4-day cells (K_m = 0.30 ± 0.02 mM) than in 7-day cells (K_m = 0.59 ± 0.11 mM). When leucine and K⁺ transport were compared in 4- and 7-day populations, significant elevations in the rate of entry (V_max) of both substrates were observed for 4-day cells; K_m values for leucine and K⁺ were not altered by the stage of infection. For leucine, the V_max and K_m for 4-day cells were 2.40 ± 0.50 nmoles/1 × 10⁸ cells/30 sec and 78 ± 7 μM, respectively; corresponding values in 7-day cells were 1.06 ± 0.02 nmoles/1 × 10⁸ cells/30 sec and 66 ± 11 μM. For K⁺, the V_max and K_m for 4-day cells were 15.97 ± 0.38 nmoles/1 × 10⁸ cells/min and 1.2 mM, respectively; corresponding values in 7-day cells were 4.76 ± 1.82 nmoles/1 × 10⁸ cells/min and 1.05 mM. The observed increase in the rate of K⁺ entry into 4-day cells was attributable to enhanced influx; no significant difference in the rate of K⁺ efflux was noted when 4- and 7-day cells were compared ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ of K⁺ leak for 4- and 7-day cells were 68.1 ± 9.3 and 67.9 ± 15.2 min, respectively). Potassium influx was ouabain insensitive. Membrane function in 7-day cells was not uniformly inhibited. No significant difference in the activity of the membrane-bound enzyme, 5′-nucleotidase, was observed when 4- and 7-day cells were compared.     

Trypanosoma brucei: Biochemical and morphological changes during in vitro transformation of bloodstreamto procyclic-trypomastigotes

Trypanosoma brucei brucei in whole rat blood inoculated into a semidefined medium undergoes complete morphological transformation (observed by light microscopy) in 72 hr. This reproducible system permits the biochemical and physiological study of transformation from bloodstream to procyclic trypomastigotes and mitochondrial biogenesis in these organisms. Infectivity for mice is lost after 6 days. Proline stimulates cell growth after transformation. High levels of glucose adversely affect the growth of transforming cells. Respiration during transformation is by an α-glycerophosphate oxidase although a cyanide-sensitive pathway is present after 24–48 hr but does not become fully functional with respect to procyclic trypomastigotes until 20–24 days in culture. The success of this system will permit the biochemical characterization of African trypanosomes as the development of the cytochrome system occurs.     

Trypanosoma equiperdum: Movement from the dermis

Twelve dogs were injected intradermally with 352,770 to 14,391,660 Trypanosoma equiperdum and afferent and efferent lymph, lymph nodes, and blood examined by mouse inoculation at minute, hourly, and daily intervals following inoculation. The log dosage of trypanosomes given each dog was closely related to their body weight (P < 0.01). Afferent lymph contained trypanosomes as soon as 5 and 27 min after inoculation. Lymph nodes on the side of injection became positive within 5 min of injection, while those on the contralateral side remained negative for at least 120 min after injection. Blood contained trypanosomes as soon as 5 min after injection, although the average time for all dogs, before trypanosomes were demonstrated in the blood, was 40 min postinjection. Efferent lymph did not contain organisms until 25–76 hr after inoculation. We consider this sequence to indicate that T. equiperdum can leave the dermis in afferent lymphatics, reach the local lymph node, invade the blood stream from this site, and only after a day or longer do they leave the node via the efferent lymphatics.     

Trypanosoma congolense: Natural and acquired resistance in the bovine

A total of 42 animals of various ages were infected with Trypanosoma congolense to investigate age resistance. Ten of eleven animals between 4 months and 1 year of age survived the infection without treatment. Two of eleven animals in the age range of 1 to 2 years also survived the infection whereas all 20 animals between 2 and 5 years of age died or needed treatment to survive. Young animals which needed no treatment to survive were refractive to challenge for at least 1 year after their last patent parasitemia. Older animals which required treatment to survive were also challenged at intervals after therapy. Three animals infected for 49 to 75 days before treatment were rechallenged 198 to 296 days later. Extensions in prepatent periods ranged from 5 to 13 days when compared to controls and the resulting infections were of a relapsing nature followed by self-cure. Effects of this disease on clinical parameters of previously infected animals were minimal. One animal infected for 196 days and rechallenged 501 days later had a prepatent period of 14 days as compared to 5 days for controls. This animal developed a brief relapsing infection followed by self-cure. Animals which were infected for periods of 41 to 77 days, received treatment, and were then rechallenged from 600 to 900 days later, showed some resistance to infection. Prepatent periods were extended from 1 to 3 days over those of control animals and although the resulting disease was severe, one of four animals self-cured without treatment. When animals which had self-cured primary challenges were rechallenged at periods up to 2 years later, they were completely refractory. When 12 animals which were presumed to be immune to syringe-passaged T. congolense were challenged by tsetse fly bite with the same strain of trypanosome, an appreciable immunity was evident. Five of twelve immune animals did not become patent while the other seven developed mild infections without severe clinical signs. All nine controls developed severe infections with eight requiring treatment to survive. When animals immune to the Trans-Mara I strain of T. congolense were challenged either by syringe or tsetse fly bite with a heterologous strain of T. congolense obtained from a different geographical area, no evidence of immunity was detected.     

Trypanosoma cruzi: Role of different antibody classes in protection against infection in the mouse

Immune serum obtained from mice with a chronic infection of Trypanosoma cruzi was fractionated on Sephadex G-200 or on protein ASepharose 4B. Mice were infected with a standard infective dose of T. cruzi 24 hr after injection with either IgM, IgG, IgG1, or IgG2 + IgG3 fractions. Mice were also pretreated with immune serum depleted by affinity chromatography of either IgG2a, IgG2b, or both subclasses before infection with T. cruzi. Control mice were pretreated with normal mouse serum or immune serum depleted of IgG. The parasitemia and survival of the animals were determined and used as parameters of protection. The results of these experiments demonstrated that the protective antibodies were mostly IgG2 and seem to be preferentially located in IgG2b subclass. IgM and IgG1 fractions were very little, if any, protection.     

Trypanosoma cruzi: Nucleotide and vitamin requirements of growing epimastigotes

Using a defined culture medium it was shown that Trypanosoma cruzi epimastigotes (strains Y, Ma, and F1) do not require exogeneous nucleotides for continuous cultivation. Biochemical determinations carried out on parasites grown in the presence or absence of exogenous nucleotides revealed no differences in intracellular nucleotide concentrations. This suggests that T. cruzi epimastigotes have the capacity for de novo nucleotide synthesis. Choline and folic acid were necessary only for high yields of T. cruzi, suggesting that epimastigotes can partially satisfy their vitamin requirements.     

Trypanosoma vivax: Courses of infection with three stabilates in inbred mouse strains

The courses of infection in inbred mouse strains were compared following infection with three Stabilates of high, intermediate, and low virulence of Trypanosoma vivax stock Zaria Y486. Mouse strains could only be shown to differ in their resistance to T. vivax infections as judged by the height of the initial parasitemia and survival times when a trypanosome population of low or intermediate virulence was used. A T. vivax population of high virulence was uniformly lethal. Comparison of lytic antibody titers between groups of resistant ($\text{C}\text{57}\text{B}\text{1}\text{6}$) and susceptible ($\text{Balb}\text{c}$) mice did not show any significant differences in titers of the surviving mice but the mice in either group which did not control the initial parasitemia had lower lytic antibody titers than those which did. A significantly larger number of $\text{Balb}\text{c}$ mice failed to control the initial infection as compared to the $\text{C}\text{57}\text{B}\text{1}\text{6}$. Treatment with cyclophosphamide did not ablate differences in susceptibility between the two strains. The use of congenic mice showed that these differences in susceptibility were not related to differences in the major histocompatibility complex between these strains.     

Trypanosoma cruzi: Effect on B-cell-responsive and -responding clones

During the course of Trypanosoma cruzi infection in C57BL/6 mice, which are relatively resistant to the parasite, the hosts developed antibody activity against previously unencountered antigens. The anti-sheep erythrocyte and antitrinitrophenyl antibody levels increased rapidly from Day 7 of infection, reached a peak by the 21st day, and were maintained at this level through 120 days postinfection in these mice. In contrast, highly susceptible C3H(He) mice did not have demonstrable antibody responses to SRBC or TNP during the 24-day infection period. Autoantibody activity against the selfantigens presented on isologous erythrocytes or thymocytes, however, were reduced in infected C57BL/6 mice. No significant reduction in autoreactivity to the self-antigens on erythrocytes or thymocytes was observed in C3H(He) mice infected with T. cruzi although a trend of reduced autoresponsiveness toward erythrocytes appeared to be developing by the time of death. C57BL/6 mice immunized with sheep erythrocytes as neonates and infected with T. cruzi as adults, or adult mice primed with low doses of sheep erythrocytes prior to infection, had elevated antibody responses to sheep erythrocytes unless the mice were immunized with sheep erythrocytes during the course of infection, in which case suppression of the response against sheep erythrocytes resulted. The nonspecific synthesis of immunoglobulins in infected C57BL/6 mice was, in part, a result of the lymphocyteactivating properties of T. cruzi-associated antigens. The T. cruzi-associated antigens induced proliferative and differentiative responses in spleen cells in vitro. It is proposed that the T. cruzi-associated antigens differentially affect lymphocytes capable of responding to antigen and those lymphocytes previously stimulated by antigen.     

Trypanosoma congolense: Thrombocyte survival in infected steers

Charolais steers infected with Trypanosoma congolense developed a thrombocytopenia that was first demonstrated shortly before the onset of parasitemia. The thrombocyte count progressively decreased from a level of 6 × 10⁵/mm³ on the 3rd day postinfection to l × 10⁵/mm³, its most depressed level, on the 11th day postinfection. The mean of the thrombocyte level of five infected bovines at the time of thrombocyte survival analysis was 195.6 ± 83.5 × 10³(± 2 SE) as compared to 998 ± 245.9 × 10³ in the control group. In parallel with depressed thrombocyte levels, the mean of the apparent half-lives of ⁵¹Cr-labeled thrombocytes was 1.3 ± 0.5 days as compared to 3.7 ± 0.5 days in control animals. A similar survival was noted in the apparent half-lives of ⁵¹Cr-labeled autologous and heterologous thrombocytes transfused to normal recipients.     

Trypanosoma lewisi: Enhanced resistance in naive lactating rats and their suckling pups

Lactating rats and their suckling offspring were shown to have a naturally occurring resistance to Trypansoma lewisi which was greater than that seen in normal adult rats and was not dependent on previous exposure to the parasite. In the case of the pups maximum protection was dependent on suckling being the sole source of nutriment and also on the peak of the parasitemia falling within the nursing period. Supplementary solid food reduced survival rates in concurrent Hemobartonella muris—T. lewisi infections: pups infected at 10 days of age and totally dependent on nursing showed 82.5% survival but only 6.6% survival when solid food was allowed to supplement milk. However, supplementary food did not reduce survival in pups infected with only T. lewisi. When pups were totally dependent upon nursing until the normal time of weaning (21 days of age), infection with H. muris—T. lewisi at the following days of age allowed the indicated mean survival rates: 10 days (82.5%), 20 (32.5%), 30 (14.3%), and 40 (100%). Infection with T. lewisi alone at the following days of age allowed the indicated mean survival rates: 10 days (100%), 15 (76%), 17 (52%), 20 (100%), and 30 (100%). Lactating rat serum agglutinated “adult forms” of T. lewisi, which correlated well with the observed sudden resolution of parasitemia in lactating animals at the time of the antigenic transition from “juvenile” to “adult” type. The “adult” stage parasitemia in suckling pups was selectively reduced when compared to that of nonlactating adult rats. The lactating rat serum factor could be passively transferred with lactating rat serum to animals already weaned.     

Trypanosoma brucei: The peptidases of bloodstream trypanosomes

Peptidases (EC.3.4.11 and EC.3.4.13.9) from bloodstream Trypanosoma brucei were examined by starch gel electrophoresis and substrate specificities and relative activities of six peptidases (S,B,E,A,F,D,) determined. The substrate specificities corresponded closely to those of the peptidases of human blood cells and tissues although human peptidase C appeared not to have a T. brucei equivalent.     

Trypanosoma congolense: Specific transformation in vitro of leukocytes from infected or immunized cattle

An assay to measure the specific proliferation in vitro of peripheral blood leukocytes (PBL) in response to ultrasonicated trypanosomes was adapted for use in cattle. The kinetics of mitosis exhibited by PBL from cattle which had been treated following infection with Trypanosoma congolense paralleled the development of a delayed-type skin reaction elicited with ultrasonicated and Formalin-fixed T. congolense. Responses in both tests were maximal on the fourth day after initiation. Specific proliferation of PBL harvested from cattle which had been immunized with intact, nonviable trypanosomes was also elicited in vitro by trypanosomal antigen. Peripheral blood leukocytes taken from cattle immunized with 50 μg of variant-specific surface antigen (VSSA) from T. brucei and from cattle infected with T. congolense were not stimulated to divide in vitro by ultrasonicated trypanosomes.     

Eimeria nieschulzi and Trichinella spiralis: Analysis of concurrent infection in the rat

The effects of concurrent primary infection of the rat with Eimeria nieschulzi and Trichinella spiralis on the number of oocysts of E. nieschulzi shed by the host and on the number, distribution, and fecundity of adult T. spiralis were analyzed. When rats were initially infected with E. nieschulzi followed 9 days later by infection with T. spiralis there occurred a significant decrease in the total numbers of adult worms in the small intestine, a significant shift in the position of these worms along the length of the small gut, a decrease in the fecundity of adult female worms, and a decrease in muscle parasitism when compared with rats infected with T. spiralis alone. When rats were initially infected with T. spiralis, followed 9 days later by infection with E. nieschulzi, there occurred a significant decrease in the numbers of oocysts shed over 24 hr on Days 7, 9, and 11 postinfection below that seen with rats infected only with Eimeria. These changes are discussed in terms of the enteropathophysiologic lesions and enteric inflammation known to occur during infections with these two parasites.     

Trypanosoma equiperdum: Active immunization of rabbits with actinomycin D-inactivated parasites

Trypanosoma equiperdum was inactivated with actinomycin D. Rabbits inoculated with inactivated parasites were completely protected from a challenge inoculation of viable T. equiperdum of the same stabilate. Immune serum from the inoculated rabbits, as well as the 7 S and 19 S serum fractions, were tested for their ability to neutralize trypanosomes. During the first 6 weeks of immunization the neutralizing activity of the 19 S fraction was stronger than that of the 7 S fraction. After 9 weeks, the situation was reversed.     

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.     

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.