The role of humoral immune responses in determining susceptibility of A/J and C57BL/6 mice to infection with Trypanosoma congolense

The relative resistance of C57BL/6 mice to infection with Trypanosoma congolense as compared to A/J mice was found to be independent of the infective dose of trypanosomes and required an intact immune system, as sublethal levels of gamma irradiation abolished the differences in susceptibility between the two strains. C57BL/6 mice produced earlier and quantitatively superior antibody responses both to the variable surface glycoprotein and to common membrane antigens on the trypanosome than A/J mice. No difference was observed in the class of antibody produced. In parallel with the specific response, C57BL/6 mice also generated higher levels of antibody to an unrelated antigen (TNP) and developed higher levels of total serum IgM. However, despite the low levels of both specific antibody and antibody to TNP in A/J mice, these animals developed massive increases in total serum IgG2a. The role of this selective activation of IgG2a producing cells in the susceptibility of the A/J mice was unclear. Although susceptibility was closely correlated with specific antibody responses during infection, the two strains of mice showed a similar capacity to respond to fixed doses of irradiated trypanosomes. This indicates that an inherent difference in immune responsiveness to the trypanosomal antigens is not the major factor determining susceptibility. Moreover, the finding that a proportion of A/J mice which received infective and irradiated trypanosomes simultaneously showed depressed antibody responses to the trypanosome, suggests that active infection of A/J mice with T. congolense impairs their ability to initiate an appropriate immune response to the trypanosome.     

Immunosuppression mediated by adult worms in chronic schistosomiasis mansoni.

A marked reduction in the number of plaque-forming cells from spleens of mice infected with Schistosoma mansoni to sheep erythrocytes (SRBC) and lipopolysaccharide from Escherichia coli was observed. This reduction coincided with the late stages of the infection and was also observed in unisexual infection with male worms. Treatment of the animals with a schistosomicidal compound (oxamniquine) almost completely abolished the immunosuppression. The suppression could be induced by administration of 60 microgramg protein from worm membrane preparations (24 h before SRBC injection), but not by egg-extract injection. When the crude membrane preparation was injected 48 h before or 0 to 24 h after the SRBC challenge, the immunosuppression was not observed. Significant reduction of footpad swelling was also noted in infected mice when injected with SRBC.     

Giardia muris-induced depression of the primary immune response in spleen and mesenteric lymph node cell cultures to sheep red blood cells

Primary in vitro plaque-forming cell (PFC) responses to sheep red blood cells (SRBC) were examined for spleen and mesenteric lymph node (MLN) cell populations from susceptible (A/J) and resistant (B10.A) mice during the infection with Giardia muris. Spleen and MLN cells isolated from mice during the acute phase of the infection were less responsive to SRBC in vitro than those from uninfected mice. Depressed anti-SRBC PFC response was detected earlier and was more pronounced in MLN cell cultures when compared to the response of spleen cell cultures. Spleen and MLN cells from donors infected with G. muris for 15 days had the capacity of depressing PFC response to SRBC of cells isolated from uninfected mice. This suppressor activity was localized in the plastic-adherent fraction of spleen cell populations isolated from A/J and B10.A mice. Since G. muris is a gastro-intestinal infection of mice, lower capacity of the MLN cells to respond to an antigenic stimulation in vitro may explain, in part, the proliferation of the trophozoites during the acute phase of the infection.     

Trypanosome variant surface glycoprotein transfer to target membranes: a model for the pathogenesis of trypanosomiasis.

The variant surface glycoprotein (VSG) of trypanosomes is attached to the cell surface by means of a phosphatidylinositol-containing glycolipid membrane anchor. The studies presented in this paper support the hypothesis that the transfer of VSG from trypanosomes to erythrocytes could lead to one of the pathological features associated with trypanosome infection--i.e., anemia. Migration of trypanosome VSG from live trypanosomes to target cells (sheep erythrocytes) could be shown by preincubating erythrocytes with trypanosomes and subsequently testing the washed erythrocytes for insertion of VSG by their susceptibility to lysis by complement in the presence of an anti-VSG antibody. Complement-mediated lysis was found to depend on the strain-specific anti-VSG antibody used. Extent of erythrocyte lysis increased with time of cell exposure to trypanosomes and with trypanosome concentration. No erythrocyte lysis was observed when trypanosomes were preincubated with anti-VSG antibody before adding erythrocytes. Purified membrane-form VSG (which retains the glycolipid anchor), but not soluble VSG (which no longer has the terminal diacylglycerol moiety), could sensitize erythrocytes to anti-VSG antibody-mediated complement lysis. The intermembrane transfer of VSG from trypanosomes to cells of the infected host could provide a molecular mechanism for the pathogenesis of trypanosomiasis.     

Polyamine oxidase-mediated killing of African trypanosomes

African trypanosomes, Trypanosoma brucei brucei, T. vivax and T. congolense , were killed when incubated in vitro with ruminant sera in the presence of exogeneous spermidine, and were non-infective for mice. Purified polyamine oxidase in the presence of spermidine-mediated similar killing of trypanosomes. The abundance of polyamine oxidase activity in ruminant sera which can react with polyamines to produce products with cytotoxic properties may explain the trypanosome killing. This system may contribute to non-specific parasite killing in vivo.     

Shifting from wild to domestic hosts: The effect on the transmission of Trypanosoma congolense to tsetse flies

The epidemiology and impact of animal African trypanosomosis are influenced by the transmissibility and the pathogenicity of the circulating trypanosome strains in a particular biotope. The transmissibility of 22 Trypanosoma congolense strains isolated from domestic and wild animals was evaluated in a total of 1213 flies. Multivariate mixed models were used to compare infection and maturation rates in function of trypanosome origin (domestic or sylvatic) and pathogenicity. Both trypanosome pathogenicity and origin significantly affected the ability to establish a midgut infection in tsetse flies but not the maturation rates. The interaction between pathogenicity and origin was not significant. Since being pathogenic and having a domestic origin both increased transmissibility, dominant lowly pathogenic trypanosomes from domestic environments and highly pathogenic trypanosomes from sylvatic environments presented similar levels of transmissibility: 12% and 15%, respectively.Blood meals with parasite concentration ranging from 0.05 to 50 trypanosomes/μl blood for 3 strains of T. congolense were provided to different batches of tsetse flies to evaluate the relationship between the parasite load in blood meals and the likelihood for a fly to become infected. A linear relationship between parasite load and transmissibility was observed at low parasitaemia and a plateau was observed for meals containing more than 5 trypanosomes/μl. Maximum transmission was reached with 12.5 trypanosomes/μl blood. About 50% of the flies were refractory to T. congolense, whatever their concentration in the blood meal. The results suggest that the dose–transmissibility relationship presents a similar profile for different T. congolense isolates.     

Toward an understanding of the mechanisms of classical conditioning of antibody responses

Mice of different ages were subjected to repeated exposure to cyclophosphamide:saccharin (conditioned), or cyclophosphamide:saccharin followed by saccharin only (extinguished). Only young animals in the former group showed a decreased IgG antibody-forming-cell (AFC) response after challenge with sheep erythrocytes (SRBC) in the presence of saccharin. When irradiated conditioned young animals were used as recipients of antigen-challenged spleen cells from nonconditioned mice they were found to support a greater immune response than nonconditioned recipients. Similarly, cells from conditioned young mice gave a greater immune response in naive recipients than did cells from nonconditioned mice. Only when cells from conditioned young mice rather than conditioned "aged" mice were studied in irradiated conditioned young recipients was immunosuppression observed. These data are most consistent with a specific host cell:environment interaction being responsible for the conditioned immunosuppression observed in young mice. A deficit in both cells/environment apparently occurs during aging. At least one of these deficiencies seems related to loss of a suppressor T-cell population with age in conditioned mice.     

Murine tumour necrosis factor plays a protective role during the initial phase of the experimental infection with Trypanosoma brucei brucei

Soluble extracts from salivarian trypanosomes (Trypanosoma brucei brucei, T. evansi and T. congolense) were shown to be capable of inducing murine tumour necrosis factor (mTNF) secretion, both in vivo and in vitro, whereas the soluble extract of an intracellular trypanosome (T. cruzi) failed to do so. Furthermore, the role of mTNF during the initial phase of experimental infections with T. brucei was studied by treating infected mice with mTNF-inducing trypanosoma soluble extract and with neutralizing monoclonal anti-mTNF antibodies. Treatment of the infected animals with different doses of T. brucei soluble extract resulted in a lower first parasitaemia peak (low lysate dose) and in a longer survival time or in a nearly total inhibition of parasite development (high lysate dose). Cotreatment of the infected mice with both anti-mTNF antibodies and a high dose of soluble extract completely restored the parasite development in both trypanosusceptible C3H/He mice and trypanosubtolerant CBA/Ca mice, indicating a protective role of mTNF during the parasitaemia. Collectively these results suggest a negative influence of mTNF on T. brucei development in vivo.     

Thymic influence of proliferation and interaction of fractionated spleen cells of mice.

Cells from the spleens of mice were separated into seven subpopulations by centrifugation on a discontinuous density gradient of dextran. The lightest fraction (containing stem cells), the three heaviest fractions (containing small lymphocytes), or a mixture of these, were injected into thymectomized or sham-thymectomized irradiated mice together with sheep red blood cells as antigen. After 8 days, the spleens of treated mice were tested for their content of cells lysing sheep red blood cells in agar (PFC) or cells mounting graft-versus-host reaction in newborn mice (GVHR). Heavy fractions were depleted of progenitors of PFC, but contained progenitors of cells mounting GVHR. The light fraction was depleted of both. If light cells were given together with heavy ones, the potential to generate PFC was restored, but only if the recipients were without their thymus; by contrast, the potential to generate GVH-reactive cells was strongly suppressed in the absence of a thymus. It is concluded that, in this model, stem cells probably interact with immunocompetent cells, and that the interaction depends on the thymus.     

Cloning of African trypanosomes in mice immunosuppressed by cyclophosphamide treatment

The use of cyclophosphamide-treated mice for purpose of cloning the African trypanosomes was assessed. C3HeB/FeJ mice were injected with 200 mg/kg cyclophosphamide (CY) 24-72 hours prior to infection with a single trypanosome isolated from Trypanosoma rhodesiense infected blood samples. All CY-treated mice exhibited depressed parasite-exposure, which was a period of time sufficient to grow trypanosomes from a single organism to a fulminating parasitemia. Cloning efficiency was routinely 45% in these animals. Thus, our study demonstrates that CT-treated mice are a convenient and efficient vehicle for cloning African trypanosomes. Techniques which facilitate the selection of single trypanosomes from infected blood are also described in this report.     

Filaments of Trypanosoma brucei: some notes on differences in origin and structure in two strains of Trypanosoma (Trypanozoon) brucei rhodesiense.

Filaments attached to trypanosomes of two strains of T. (T.) brucei were studied by electron microscopy and two distinct types identified: short-thick and long-thin. The former are associated with stumpy trypanosomes and are secretions, via the flagellar pocket, which originate in the area of the Golgi complex, during the infection of the host. They are referred to as 'secretory filaments'. Their diameter is 0.09 to 0.14 mum. The long-thin filaments are associated with slender forms of trypanosome in various artificial situations; those shown by negative staining are believed to be cytoplasmic extrusions from the anatomically weak extremities of the parasite and are referred to as 'plasmanemes'. Their diameter is 0.06 mum. Both types appear to maintain their structure without the aid of the normal type of unit membrane as myelin formations.     

Trypanosoma brucei CTP synthetase: a target for the treatment of African sleeping sickness

The drugs in clinical use against African sleeping sickness are toxic, costly, or inefficient. We show that Trypanosoma brucei, which causes this disease, has very low levels of CTP, which are due to a limited capacity for de novo synthesis and the lack of salvage pathways. The CTP synthetase inhibitors 6-diazo-5-oxo-l-norleucine (DON) and alpha-amino-3-chloro-4,5-dihydro-5-isoxazoleacetic acid (acivicin) reduced the parasite CTP levels even further and inhibited trypanosome proliferation in vitro and in T. brucei-infected mice. In mammalian cells, DON mainly inhibits de novo purine biosynthesis, a pathway lacking in trypanosomes. We could rescue DON-treated human and mouse fibroblasts by the addition of the purine base hypoxanthine to the growth medium. For treatment of sleeping sickness, we propose the use of CTP synthetase inhibitors alone or in combination with appropriate nucleosides or bases.     

The role of natural agglutinins and trypanolytic activity in host specificity of Trypanosoma musculi

Trypanolytic activity and agglutinins for T. musculi were demonstrated in sera from refractory hosts. The agglutinins in human and bovine serum were specific antibodies. The trypanolytic activity was a result of the ability of the trypanosomes to activate complement in these normal sera. The results suggested that Trypanosoma musculi activates human complement by the alternative pathway. The activity was inhibited by EDTA but not EGTA, and trypanosome lysis occurred in the absence of C2. In addition, conversion of C3 occurred in the presence of EGTA. The trypanolytic activity of bovine serum was similarly inhibited by EDTA but not EGTA. Trypanosome lysis failed to occur in C6 deficient rabbit serum, showing that the late components of complement are required for parasite lysis. Trypanosome lysis by human or bovine serum was inhibited by the addition of mouse serum but not rat serum. These observations suggest that the presence of trypanolytic activity and antibodies to this trypanosome in sera of normal mammals may be responsible for the restricted host range of the trypanosome, and that the absence of these antibodies and the ability of this parasite to evade the trypanolytic activity enables T. musculi to establish infections in the mouse.     

Course of Trypanosoma musculi infections in NMRI mice (author's transl)]

The trypanosomes multiply during prepatency after minimal infection by a factor of 2.2-3.6 per day. During patency, increase of trypanosome number in the peripheral blood is basically non-logarithmic as the actual proliferating forms remain hidden in special vascular areas (kidney, placenta). The mean increase during patency is approximately linear, typically by 1-10 trypanosomes per 10(4) erythrocytes per day, and depends on the number of infective organisms introduced. - Length of parasitemia is 13-40 days, it is partly determined genetically. Prolonged parasitemias, very low in the later stages, were seen in mice preinfected with Trypanosoma congolense. - In 1 mouse with minimal infection, parasitemia only became patent 63 days after infection under the stress of an added infection with Trypanosoma brucei. - Parasitemia can be terminated by antiserum. Application of antiserum 3 or 4 days after infection may even suppress the development of immunity and priming. - Massive reinfection leads to parasitemia of up to 8 days duration without parasite multiplication.     

Hydrodynamic gene delivery of baboon trypanosome lytic factor eliminates both animal and human-infective African trypanosomes

Several species of African trypanosomes cause fatal disease in livestock, but most cannot infect humans due to innate trypanosome lytic factors (TLFs). Human TLFs are pore forming high-density lipoprotein (HDL) particles that contain apolipoprotein L-I (apoL-I) the trypanolytic component, and haptoglobin-related protein (Hpr), which binds free hemoglobin (Hb) in blood and facilitates the uptake of TLF via a trypanosome haptoglobin-hemoglobin receptor. The human-infective Trypanosoma brucei rhodesiense escapes lysis by TLF by expression of serum resistance-associated (SRA) protein, which binds and neutralizes apoL-I. Unlike humans, baboons are not susceptible to infection by T. b. rhodesiense due to previously unidentified serum factors. Here, we show that baboons have a TLF complex that contains orthologs of Hpr and apoL-I and that full-length baboon apoL-I confers trypanolytic activity to mice and when expressed together with baboon Hpr and human apoA-I, provides protection against both animal infective and the human-infective T. brucei rhodesiense in vivo. We further define two critical lysines near the C terminus of baboon apoL-1 that are necessary and sufficient to prevent binding to SRA and thereby confer resistance to human-infective trypanosomes. These findings form the basis for the creation of TLF transgenic livestock that would be resistant to animal and human-infective trypanosomes, which would result in the reduction of disease and the zoonotic transmission of human infective trypanosomes.     

Efflux of 3H-thymidine by erythrocytes from mice infected with Trypanosoma brucei brucei

Erythrocytes from mice infected with Trypanosoma brucei brucei showed a higher rate of efflux of labelled thymidine than did control erythrocytes from uninfected mice (0.56 +/- 0.10 and 0.38 +/- 0.06 mumole min-1 ml-1 packed cells respectively). Efflux of the nucleoside from erythrocytes of normal and infected mice were inhibited to the same extent by a specific nucleoside transport inhibitor, nitrobenzylthioinosine. Enumeration of nitrobenzylthioinosine binding sites on the erythrocytes showed that both have similar numbers of sites (6.2-6.6 X 10(3) sites/erythrocyte). It is concluded that the membrane permeability of the erythrocytes from infected mice was affected by the trypanosome in such a way as to enhance the purine nucleoside transport capacity. This may result in an increased supply of vital purine bases and nucleosides to trypanosomes which depend on their hosts for these nutrients.     

Regulation of parasite-specific antibody responses in resistant (C57BL/6) and susceptible (C3H/HE) mice infected with Trypanosoma (trypanozoon) brucei brucei

After infection with 10(3) T. brucei GUTat 3.1, C57BL/6 mice produced antibody responses and controlled the first parasitaemic wave whereas C3H/He mice did not. The inability of C3H/He mice to control parasitaemia resulted from an impaired ability of parasite-induced antibody-containing cells to secrete immunoglobulin. Antibody-containing cells in infected C3H/He mice regained the ability to secrete antibody within 24 h after trypanosome elimination by treatment with Berenil, suggesting that the block in antibody secretion was maintained by living parasites or short-lived components of degenerating parasites. Infected C3H/He mice also had an impaired ability to produce a rabbit erythrocyte-specific antibody response on challenge with rabbit erythrocytes and this response recovered when parasites were eliminated from the blood 24 h before analysis. It was not possible to inhibit secretion of antibody by rabbit erythrocyte-induced plasma cells either by incubating them with serum from infected C3H/He mice or by injecting large numbers of living trypanosomes into C3H/He mice already responding to rabbit erythrocytes. The process leading to failure of parasite and rabbit erythrocyte-induced antibody-containing cells to become high rate antibody-secreting cells was not identified but did not appear to correlate with any obvious change in the intra-cellular morphology of the antibody-containing cells.     

Behavior of a Trypanosoma brucei strain (STIB 348C) in mice. 3. Histopathological findings in the terminal stage of infection

Histopathological findings in the terminal stage of the infection of mice and rats with different variants of the Trypanosoma brucei brucei stock are described. Mice infected with mild variants showed after 12-149 days intense trypanosome colonization of the interstitial connective tissue, especially of the heart muscle, pancreas and choroid plexus, with severe tissue destructions, especially in the pancreas. With longer duration of the infection, round cell infiltrations of the leptomeninx and around intracerebral vessels developed. The alterations were the same in animals dying with high and low parasitemias. Some animals died with continuous bleeding from the tip of the tail, with serious effusions and massive edema or with generalized bacterial infection. The lymphatic organs showed intense reactive alterations, trypanosomes were only rarely found in lymph nodes. In the liver large fields of lymphatic and myeloid cells were seen, sometimes necroses developed. The kidneys showed marked deposition of eosinophilic material in the glomeruli and precipitation of proteinaceous material in dilated tubules. - Rats infected with mild trypanosomes exhibited intense colonization of the heart by trypanosomes, in some places many trypanosomes in lymph nodes, few trypanosomes in the pancreas. They died after a long phase of high parasitemia.--After infection with virulent parasites which killed the animals with high parasitemia in a few days, mice and rats had single small foci of trypanosome colonization of the interstitial connective tissue of heart, pancreas and choroid plexus as well as of the loose connective tissue in the hilum of the kidney and around lymph nodes.(ABSTRACT TRUNCATED AT 250 WORDS)