Characterization of concanavalin A-binding glycoproteins from procyclic culture forms ofTrypanosoma congolense,T. simiae andT. brucei brucei

Concanavalin A-binding glycoproteins were obtained from procyclic culture forms (PCFs) ofTrypanosoma congolense, T. simiae, andT. b. brucei strains. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis revealed that glycoproteins of 38.5, 30.5, and 27 kDa were conserved between the different species and strains of the procyclic parasites. There were few similarities in the profiles of the high-molecular-weight glycoconjugates between the parasites. Monoclonal antibody analysis revealed that the 38.5- and 27-kDa glycoproteins were intracellular molecules and that they contained cross-reactive antigenic determinants. Surface biotinylation of PCFT. congolense K45/1 identified surface-accessible glycoproteins of 81.5, 59, and 38–42 kDa. By use of lectin blots and enzymatic deglycosylation studies, we demonstrated that the 81.5-, 59-, 38.5-, and 27-kDa glycoproteins contained N-linked oligosaccharide chains with both high-mannose-type and complex-type oligosaccharides, and the 81.5- and 59-kDa surface glycoproteins contained sialic acid residues. The glycoproteins identified in this study provide a starting point for further structure and function studies.     

Human serum resistance of metacyclic forms ofTrypanosoma brucei brucei,T. brucei rhodesiense andT. brucei gambiense

Resistance against the lytic action of human serum has been tested among metacyclic and bloodstream forms ofTrypanosoma brucei brucei, T. b. rhodesiense andT. b. gambiense stocks and clones. The resistance was determined by applying an in vitro human serum resistance test. Whereas the majority ofT. b. gambiense metacyclic forms exhibited stable human serum resistance,T. b. rhodesiense metacyclics showed inconsistent resistance within a minority of parasites, which tended to diminish completely with prolonged passages in rodents. Infection of tsetse flies with in vivo or in vitro selected human serum resistant forms did not significantly increase the proportion of resistant parasites among extruded metacyclic forms. In aT. b. rhodesiense stock which never showed human serum resistance in the metacyclic forms, human serum resistance reappeared after a 2-day cultivation period in the presence of a mammalian serum. These results reflect important phenotypic dynamics and may lead to a better understanding of the epidemiology of African human sleeping sickness.     

Vectorial competence of non-teneral Glossina morsitans morsitans (Mall strain) flies infected by Trypanosoma (Nannomonas) congolense IL 1180

Non-teneral tsetse flies of Glossina morsitans morsitans (strain Mall) about 16 days old were fed, once, on a rat infected by Trypanosoma congolense IL 1180. The global vectorial competence (VC) of these flies was appraised at 0.1035. VC in males was more important than for females. Infection by mesoprocyclic index was greater in female flies than in male ones, whereas for metacyclic index the reverse was true. This work shows that the age limits, but does not impede metacyclogenesis of non-teneral tsetse flies of G. m. morsitans (strain Mall).     

Cyclical transmission of in vitro cultivated bloodstream forms and procyclic trypomastigotes ofTrypanosoma brucei brucei byGlossina morsitans morsitans

In vitro cultivated bloodstream and procyclic forms ofTrypanosoma b. brucei STIB 247 were cyclically transmitted byGlossina m. morsitans. The tsetse flies were infected artificially on a silicon membrane. Metacyclic trypanosomes from mature salivary gland infections were used to initiate bloodstream form cultures. They transformed into slender bloodstream forms and gave rise to established cultures that proved to be infective for the vector. The metacyclic forms retained the strain-specific basic set of variable antigen types.     

Comparison of the longevity of tsetse flies (Glossina morsitans morsitans Westwood, 1850) infected with trypanosomes (Trypanosoma nannomonas congolense Broden, 1904) and uninfected tsetse flies

This preliminary note results from a comparative study on susceptibility to pyrethroid insecticides of tsetse flies infected and not with trypanosomes. Trypanosoma infection increases the susceptibility to insecticides as previous study showed it (Golder et al., 1982, 1984). Moreover, infected control flies showed a significant lower longevity than uninfected ones.     

Surface coat variant antigen of Trypanosoma brucei brucei: its clearance from blood and concentration in organs of normal, infected, and immune mice.

Experiments were conducted to determine the fate of variant antigen once it was shed from the surface of Trypanosoma brucei brucei. Radioiodinated variant antigen was administered intravenously to normal mice, mice immunized to the homologous variant antigen, and mice infected with an antigenically dissimilar (heterologous) T. brucei brucei variant. The variant antigen was cleared slightly faster in infected and immune animals. Though over 80% of the variant antigen was cleared in all animals within 4 h, traces of radioactivity could be detected in the peripheral blood at 48 h postinjection. At 1 h postinjection, most of the variant antigen had collected in the liver, kidneys, bone marrow, spleen, lungs, thymus, and lymph nodes (listed in the order of decreasing radioactivity). At 24 h, the kidneys, liver, and spleen retained the most radioactivity. The kidneys had 10 to 20 times, the liver had 8 to 10 times, and the spleen had 5 to 7 times more variant antigen than was present in the organs' blood supply. At 48 h postinjection, two-thirds of the radioactivity present at 24 h remained in these tissues. The livers and spleens from infected animals had, on a per gram of tissue weight basis, reduced uptake of the soluble antigen whereas their lungs had an increased uptake. Radioactivity in blood and organs was proved to be associated with protein by electrophoresis/autoradiography and precipitation of radioactive protein of tissue extracts.     

Comparative structural analysis of calmodulins from Trypanosoma brucei, T. congolense, T. vivax, Tetrahymena thermophila and bovine brain

Calmodulin is an intracellular calcium receptor protein utilized extensively by eukaryotic cells to mediate responsiveness to calcium signals. The present study evaluates the effects on protein structure of amino acid substitutions in trypanosome calmodulin. Calmodulin conformation, hydrophobicity and antigenic determinants are compared among Trypanosoma brucei, Trypanosoma congolense, Trypanosoma vivax, Tetrahymena thermophila and bovine brain. Trypanosome calmodulin differs from brain and Tetrahymena calmodulins based upon isoelectric point, retention time on a C-2/C-18 reverse phase column and interaction with polyclonal antibodies against trypanosome calmodulin by radioimmunoassay or Western procedures. These same analyses do not distinguish trypanosome calmodulins from each other. Polyclonal antibodies against Tetrahymena calmodulin are equally specific and do not recognize the trypanosome or brain calmodulins. Calcium-induced exposure of hydrophobic binding sites are quantitated using the fluorescent probe, N-phenyl-1-naphthylamine. All calmodulins, regardless of source, enhance the fluorescence of N-phenyl-1-naphthylamine 3-4 fold in the presence of calcium. These data demonstrate the extent to which functional calmodulins vary in their structures. We conclude that African trypanosomes share a common calmodulin that is structurally distinct from calmodulin of vertebrates or Tetrahymena.     

Detection of Trypanosoma congolense and T. brucei subspecies in cattle in Zambia by polymerase chain reaction from blood collected on a filter paper

To facilitate epidemiology studies of African trypanosomiasis in cattle in Zambia, we adapted a polymerase chain reaction (PCR) method using blood spotted on filter papers. For easy preparation of template DNA from the dried blood, we adapted a simple DNA extraction method using Chelex-100, an anion-exchange resin. Using primers directed for repetitive nuclear DNA sequences, species-specific DNA amplifications were detected from the blood of rats infected with Zambian isolates of T. congolense and T. brucei subspecies. The method was sensitive enough to detect a single trypanosome for both species. In the Eastern Province of Zambia, 240 cattle were examined for motile flagellates in the buffy coat by the microhematocrit method, and 100 of them were positive for the test. These 100 animals were further examined by thin blood smears and PCR for species identification. The thin blood smear revealed 62 and 14 animals with T. congolense and T. brucei subspecies infection, respectively, whereas the PCR detected 73 of the former and 38 of the latter species. These results indicate that dried blood spots on filter papers are a useful source of DNA for detection of African trypanosomes by PCR.     

Modulation of the calcium pump of the kidney and testes of rats infected with Trypanosoma congolense.

The activity of the CaMgATPase (Ca-pump) of the kidney and testes of Wistar rats infected with Trypanosoma congolense was studied during the course of infection. The activity of the enzyme in both organs was found to decrease with increase in parasitaemia. The transition temperature (Tc) decreased and activation energy (Ea) of the enzyme increased with increase in parasitaemia. The relevance of the Ca-pump in the pathogenesis of trypanosomiasis is discussed.     

Suppressor cells and loss of B-cell potential in mice infected with Trypanosoma brucei.

The functional changes in splenic lymphoid populations from mice infected with T. brucei strain S42 were studied throughout the 3 weeks of infection. Within a week of infection, proliferation of B and T cells profoundly increased as shown by 3H-labelled thymidine incorporation and fluorescent staining of surface Ig; the spleen cells secreted high levels of both IgM and IgG immediately cells were put into culture; but with progressing infection this Ig production declined. The early effect on T cells was reflected by lack of responsiveness to PHA. B-cell potential was studied in low-density cultures treated with lipopolysaccharide (E. coli). Normal spleen cells proliferate extensively in these cultures with subsequent secretion of IgG as well as IgM. The ability to proliferate and produce Ig in response to LPS was severely depressed by day 7 and almost totally absent by day 12 of infection. Removal of T cells from the spleen cells obtained early in infection partly restored the response to LPS but as the infection neared its fatal end, B-cell potential appeared to become exhausted. Macrophages obtained from infected mice even early in infection profoundly depressed the ability of normal spleen cells to proliferate and secrete immunoglobulin in LPS cultures. The general immunodepressing effect of trypanosomes can be attributed to clonal exhaustion of B-cell potential caused by an undefined blastogenic stimulus from the parasites which may operate at least in part by the generation of suppressive T cells and macrophages.     

Erythrocytic profile of rats infected with T. brucei brucei and treated with a combination of Azadirachta indica leaf extract and diminazene diaceturate

This study investigated the erythrocytic profile of rats experimentally infected with Trypanosoma brucei brucei and treated with a combination of methanolic leaf extract of Azadirachta indica and diminazene diaceturate (DDA). Acute toxicity study of the drug and extract combinations was carried; selection of the best drug and extract combinations was carried out using 54 rats of both sexes separated into nine groups. Three dose combinations were derived from the selection of the best drug and extract combinations used for the final study, viz: 7?mg/kg body weight (bw) DDA plus 125?mg/kg bw extract (group B), 3.5?mg/kg bw DDA plus 250?mg/kg bw extract (group C) and 1.8?mg/kg bw DDA plus 500?mg/kg bw extract (group D). The final study had, in addition to the three groups derived from the dose–response study, four other groups, viz: uninfected untreated negative control (group F), infected and treated with 3,000?mg/kg bw extract alone (group E), infected and treated with 7?mg/kg bw DDA alone (group A) and infected untreated positive control (group G). The parameters assessed were onset of parasitaemia (OP), level of parasitaemia (LOP), clearance of parasites posttreatment (COPPT), relapse of infection period (RIP), red blood cell counts (RBC) and packed cell volume (PCV). There was no significant difference (p?<?0.05) in OP between the groups. A day following treatment, the LOP of groups A, B and C was found to be significantly lower (p?<?0.05) than that of group D (p?<?0.05) which in turn was lower (p?<?0.05) than that of group E and G, respectively. The mean LOP of group E was significantly (p?<?0.05) lower than group G (p?<?0.05) 2?days posttreatment, and this trend continued throughout the experimental period. Mean COPPT of group D was significantly (p?<?0.05) longer than that of groups A, C and B. There was no significant difference (p?<?0.05) in the mean COPPT among groups B, C and A. The mean RIP of group D was significantly shorter (p?<?0.05) than group C, and that of group C was significantly shorter (p?<?0.05) than group A. There was no relapse of infection in group B rats. Group B rats had significantly higher (p?<?0.05) PCV and RBC counts when compared to other infected groups. Group E rats had significantly higher (p?<?0.05) PCV and RBC counts when compared to group G rats. It was concluded that dose combination of 125?mg/kg bw extract plus 7?mg/kg bw DDA led to significant enhancement of erythrocytic profile and potentiation of diminazene in its trypanocidal activity. This combination therapy proved to be better than single therapy of DDA.     

Immunohistology of lymph nodes draining local skin reactions (chancres) in sheep infected with Trypanosoma congolense.

Marked enlargement of lymph nodes draining local skin reactions (chancres) occurred in sheep following intradermal inoculation of cultured metacyclic forms of Trypanosoma congolense. Histologically, these lymph nodes were characterized by follicular hypertrophy and hyperplasia, compression and relative reduction of the paracortical areas and expansion of the medullary regions. Immunohistochemical staining with monoclonal antibodies to ovine lymphocyte subsets and Fc receptor (FcR) bearing macrophages, revealed increased expression of B cells (CD45R+), major histocompatibility complex (MHC) Class II, FcR+ macrophages, and CD1+ cells in the cortical and paracortical areas. The paracortical areas were found to be sparsely populated by CD5+, CD4+ and CD8+ cells, while the medullary areas contained numerous CD8+ cells and FcR+ macrophages. FcR+ macrophages were also present in cortical trabecular and subcapsular sinuses. As the chancre regressed, lymph node reactivity also subsided and fewer B cell follicles were observed and there was decreased expression of CD45R+ and MHC Class II+ cells.     

Bovine cysticercosis: demonstration in experimentally infected calves of serum IgG antibodies reactive with neutral glycolipids ofTaenia saginata andT. crassiceps metacestodes

The immunoreactivity ofTaenia saginata andT. crassiceps metacestode neutral glyco(sphingo)lipids towards IgG antibodies derived from the sera of calves with experimental cysticercosis has been established. The glyco(sphingo)lipids are separable by normal-phase HPTLC (high-performance thin-layer chromatography) into groups of increasing sugar-chain length (lipid/ceramide mono-, di-, tri-, tetra- and >tetrasaccharides), with those corresponding to three and four hexoses being the main immunoreactive components (HPTLC immunostaining). In ELISA (enzyme-linked immunosorbent assay), reverse-phase HPTLC-isolatedT. crassiceps metacestode glyco(sphingo)lipids equivalent to tri- and tetrahexoside allowed a discrimination between non-infected and infected calves (at least 80 metacestodes recovered). The formation of IgG antibodies was correlated with the infection, not with other non-specific inducing factors, as seen by the differential humoral response detected in experimentally infected (T. saginata) calves before and after Praziquantel treatment (HPTLC immunostaining and ELISA).     

Characterization of permeation pathways appearing in the host membrane of Plasmodium falciparum infected red blood cells

The host cell membrane of Plasmodium falciparum infected cells becomes permeabilized at the trophozoite stage. A variety of otherwise impermeant substances such as carbohydrates, polyols, amino acids and anions easily gain access to the cytosol of infected cells. Using the isotonic-hemolysis method or uptake of labeled substances, we characterized the new permeation pathways as pores of approximately 0.7 nm equivalent radius. The pores bear a positively charged character which facilitates movement of small anions and excludes cations, so that the ionic composition and osmotic properties of infected cells are not drastically altered. Substances of a molecular size similar to that of disaccharides are fully excluded. Substances of limiting size might be accommodated in the pore, provided they bear a side group of hydrophobic character. The new permeation pathways may provide a vital route for acquisition or release of essential nutrients or catabolites.     

Phagocytosis of antibody-sensitized Trypanosoma brucei in vitro by bovine peripheral blood monocytes.

Cells separated by the fluorescence activated cell sorter on the basis of their surface IgD (sIgD) phenotype have been examined for responsiveness to thymus-dependent and thymus-independent antigens. The ability of monoclonal anti-IgD alloantibodies to inhibit responses in vitro to the various classes of antigen has also been investigated. Evidence is presented indicating that both sIgD positive and sIgD negative cells can respond to all types of antigen tested. However, although the presence of sIgD was necessary for the response of sIgD positive cells to thymus-dependent antigens, the presence of this isotype was not obligatory for the response of the sIgD positive population to thymus independent antigens. The possible role of sIgD as the obligatory purveyor of a B-cell activation signal is discussed in the light of these findings.     

In vivo effects of alpha-DL-difluoromethylornithine on the metabolism and morphology of Trypanosoma brucei brucei

The EATRO 110 isolate of Trypanosoma brucei brucei was grown in rats for 60 h and the animals treated with the ornithine decarboxylase inhibitor alpha-DL-difluoromethylornithine 12 h or 36 h prior to sacrifice. Control untreated animals died 72-80 h after infection. Treated parasites were shorter and broader than the predominantly long slender forms found in untreated controls and many had two or more nuclei and kinetoplasts. Trypanosomes were purified from blood and examined for disruption of polyamine metabolism. ODC activity decreased by more than 99% after 12 h treatment and putrescine and spermidine levels also decreased dramatically. Spermine, not normally present in control cells, increased to detectable, low levels (less than 1 nmol mg-1 protein) after 36 h treatment. alpha-DL-Difluoromethylornithine-treated cells were unable to synthesize putrescine from [3H]ornithine but were able to convert [3H]putrescine + methionine to spermidine. 12-h treated parasites responded to polyamine depletion by assimilating radiolabeled polyamines in vitro at 2- to 4-times the rate of untreated cells. The metabolism of S-adenosylmethionine was also altered in treated parasites: decarboxylated S-adenosylmethionine increased more than 1000-fold over untreated cells while S-adenosylmethionine decarboxylase activity, associated with the formation of spermidine and spermine in other eukaryotes, paradoxically declined in treated cells. Synthesis of macromolecules was perturbed in treated parasites: rates of DNA and RNA synthesis declined 50-100%, while protein synthesis increased up to 4-fold in 36-h treated cells. alpha-DL-Difluoromethylornithine treatment progressively limits the parasites' ability to synthesize nucleic acids and blocks cytokinesis while inducing morphological changes resembling long slender leads to short stumpy transformation.     

Detection of Trypanosoma brucei spp. in human blood by a nonradioactive branched DNA-based technique.

We have developed a nonradioactive branched DNA (bDNA)-based assay for the diagnosis of the African trypanosomiases in simple buffy coat preparations of human blood. Two repetitive DNA sequences specific to the Trypanosoma brucei complex were chosen as targets of the bDNA assay, a technique which amplifies the signal from a target molecule rather than the target itself. Comparable sensitivities were observed with cloned target sequences, purified T. brucei DNA, procyclic trypanosomes, and bloodstream trypomastigotes. The results of bDNA analysis of human blood samples from Côte d'Ivoire (n = 50) showed excellent agreement with those of buffy coat microscopy. The bDNA technology offers certain advantages over alternative molecular biological techniques, including the simplicity of sample preparation and of the procedure itself, the stability of the reagents, the ability to process large numbers of samples simultaneously, and freedom from crosscontamination artifacts. We have successfully applied the bDNA technique to the detection of T. brucei in clinical samples from regions where T. brucei infection is endemic; to our knowledge, this is the first report of the molecular detection of T. brucei in human blood.     

Isolation of Francisella tularensis from infected frozen human blood.

Francisella tularensis was isolated from human blood that was frozen for 3 months before it was examined. Before he became ill, the patient operated a "bush-hog" in an area thickly populated with rabbits. His illness was undiagnosed and untreated before his death. Portions of blood and tissue homogenates from necropsy were injected intraperitoneally into mice and inoculated onto glucose-cysteine-blood agar plates. F. tularensis did not grow from the culture plates, but mice inoculated with the blood died in 48 to 72 h. Fluorescent-antibody stains of mouse liver and spleen impression smears showed clumps of cells or amorphous masses of brightly staining envelope material around the cells. Tissue impressions of liver, spleen, and heart blood inoculated onto glucose-cysteine-blood agar yielded pure cultures of F. tularensis.