Trypanosoma lewisi and T. cruzi: Effect of infection on gestation in the rat

Termination of pregnancy occurred in the rat after infection with Trypanosoma lewisi early in gestation. Rats were inoculated on Day 2 of pregnancy and the uteri were excised and examined on Days 10 and 14 of gestation. There were no detectable differences in size in the fetuses of infected and control females at Day 10, but by Day 14 young of infected females were being reabsorbed, and their weight was markedly less than that of control young.A bioassay of estrogens and progestogens based on the decidual cell response indicated that there was a sufficient hormone level to maintain pregnancy beyond Day 10, so the mechanism of action by which fetal death was produced did not appear to be hormone depletion. The crucial changes in fetal weight occurred between Days 12 and 14 of gestation in rats infected with T. lewisi on Day 2 of gestation.Trypanosoma cruzi caused little or no pathologic change in the pregnant laboratory rat throghout the period of gestation and infected females gave birth to apparently normal young.     

Trypanosoma brucei: New radioisotope assay for quantitating cell lysis

A new radioisotope technique for quantitatively determining the extent of cell lysis of trypanosomes is described. Trypanosoma brucei was pulse-labeled with [³H]leucine and then incubated at 37 C in lytic (human serum) or nonlytic (rabbit serum) media. The release into the incubation medium of radioactively labeled intracellular macromolecules was then determined. A linear relationship was found between the release of TCA-precipitable radioactivity and lysis, as determined by conventional hemacytometer counting. This method is used to quantitate the trypanocidal activity in normal human serum.     

Trypanosoma lewisi: Avidity and adsorbability of ablastin,the rat antibody inhibiting parasite reproduction

The relation of naturally acquired host IgG in the surface coat of bloodstream forms of Trypanosoma lewisi to ablastin was studied to determine whether, contrary to a long-held conclusion, the antibody is avid and adsorbable. It was found by immunofluorescence and agglutination tests with monospecific antisera to rat IgG that bloodstream forms collected from immunosuppressed hosts, in contrast to those from immunocompetent hosts, have little or no detectable surface IgO. Specificity of adsorption was also demonstrated in other immunofluorescence experiments in which bloodstream forms from immunosuppressed hosts adsorbed IgG from immune serum with ablastic activity only (previously adsorbed with trypanosomes from immunocompetent hosts to remove the trypanocidal antibodies), but did not adsorb IgG from normal rat serum. To determine whether this specific adsorption of IgG by the parasite could be correlated with a reduction in ablastic activity, immune sera were adsorbed with bloodstream forms from immunosuppressed hosts at packed cell/serum ratios of either 1.2 or 2.0, and the adsorbed sera were then tested for ablastic activity in vitro. With both cell/serum ratios, ablastic activity was reduced by 50%. In comparison, similar adsorptions of immune sera with trypanosomes from immunocompetent hosts resulted in reductions of ablastic activity of only about 9 and 27% with the low and high cell/serum ratios, respectively. It is concluded that the failure to effect significant adsorption of ablastin in earlier studies resulted from the use of ablastinsensitized trypanosomes from immunocompetent hosts.     

Recombinant expression and biochemical characterisation of two alanyl aminopeptidases of Trypanosoma congolense

Trypanosoma congolense is a haemoprotozoan parasite that causes African animal trypanosomosis, a wasting disease of cattle and small ruminants. Current control methods are unsatisfactory and no conventional vaccine exists due to antigenic variation. An anti-disease vaccine approach to control T. congolense has been proposed requiring the identification of parasitic factors that cause disease. Immunoprecipitation of T. congolense antigens using sera from infected trypanotolerant cattle allowed the identification of several immunogenic antigens including two M1 type aminopeptidases (APs). The two APs were cloned and expressed in Escherichia coli. As the APs were expressed as insoluble inclusion bodies it was necessary to develop a method for solubilisation and subsequent refolding to restore conformation and activity. The refolded APs both showed a distinct substrate preference for H-Ala-AMC, an optimum pH of 8.0, puromycin-sensitivity, inhibition by bestatin and amastatin, and cytoplasmic localisation. The two APs are expressed in procyclic metacyclic and bloodstream form parasites. Down-regulation of both APs by RNAi resulted in a slightly reduced growth rate in procyclic parasites in vitro.     

Trypanosoma musculi: In vitro cultivation of blood forms in cell culture media

Blood forms of Trypanosoma musculi were grown at 37°C in media containing adherent peritoneal cells from normal or from T. musculi immune mice. Rat peritoneal cells and Hela cells supported growth equally well which was surprising in view of the strict mouse-host specificity of this parasite in vivo. Cultures showed a 12- to 46-fold increase in numbers over the inoculum during periods up to 23 days whereas control (cell-free) media did not promote growth, the inoculated organisms merely surviving for about 7 days. Sub-culture through several passages was successful and cultured trypanosomes remained fully infective to mice. Foetal calf serum was an essential constituent of the medium.     

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.     

Trypanosoma cruzi: Isolation and characterization of a protease

Ion-exchange chromatography and Sephadex gel filtration were used to isolate a soluble proteolytic enzyme from culture (epimastigote) forms of Trypanosoma cruzi. The enzyme had a molecular weight of ~200,000 and an isoelectric point of pH 5.5. The enzyme exhibited protease, esterase, and transamidase activity, with a Michaelis constant of 0.122 mmole/liter [substrate: α-N-benzoyl-dl-arginine-p-nitroanilide (BAPA)]. The enzyme was specific for peptide bonds, involving the carboxyl groups of arginine, tryptophan, or α-N-substituted lysine. Two percent of the enzyme molecule was carbohydrate; glucose, mannose, xylose, galactose, and glucosamine were detected. The enzyme was inhibited by several sulfhydryl inhibitors, and was highly susceptible to oxidation. We concluded that the enzyme possesses active sulfhydryl groups.     

Catabolic metabolism in Trypanosoma cruzi

Culture, blood and intracellular forms of Trypanosoma cruzi have a high rate of endogenous oxygen uptake and probably utilize amino acids and carbohydrates as their exogenous energy sources. It is likely that triglyceride is the main energy reserve. Oxidation of carbohydrate by all forms is probably via a glycolytic sequence and a complete tricarboxylic acid cycle. These data suggest that the substrates utilized and catabolic pathways present in mammalian forms of T. cruzi are similar to those of culture forms of the organism and are quite distinct from those of the bloodstream forms of African trypanosomes.     

In situ assay for ornithine decarboxylase in tissue culture

An in situ method for assaying ornithine decarboxylase of cells in tissue culture is described. Values obtained were higher than those derived from homogenates and sonicates. An added advantage of the in situ assay was the use of the cultures following assay to determine cell nuclei and protein.     

Purine metabolism in Trypanosoma brucei gambiense

The procyclic forms of Trypanosoma brucei gambiense do not incorporate glycine or serine into ribonucleotides. Although de novo purine synthesis does not occur, all purine bases and ribonucleotides are interconverted, indicating the presence of active salvage pathways. Guanine is actively deaminated to xanthine by guanase activity. Purine ribonucleosides are cleaved to their respective free bases. The order of salvage efficiency for purine bases and their respective ribonucleotides is: adenine > hypoxanthine > guanine > xanthine.     

Trypanosoma cruzi: Responses by cells from infected mice to alloantigens

In unidirectional mixed lymphocyte cultures containing (as responders, stimulators, or regulators) spleen cells from mice infected with Trypanosoma cruzi, alloantigen responses were less than in cultures containing normal spleen cells only. Depletion of plastic adherent cells from infected spleen cells (stimulators or regulators) reversed their inhibitory effect on normal spleen cells (responders); removal of adherent responder cells and/or B lymphocytes did not alter the low alloantigen responses of normal spleen cells (stimulated by infected spleen cells) or infected spleen cells (stimulated by normal spleen cells). Infected spleen cells were effective in regulating mixed lymphocyte cultures only when added at the initiation of the culture. Serum from infected mice suppressed mixed lymphocyte cultures containing responder spleen cells syngeneic to the serum donor if added up to 24 hr after initiation of cultures, whereas the “suppressor serum” had to be present at the initiation of cultures when responder cells were allogeneic to the serum donor. Cultures of infected spleen cells (whole or macrophage enriched) produced a factor which was suppressive when added to mixed lymphocyte cultures containing syngeneic responder cells at initiation. It is proposed that the serum suppressor substance regulates cell-mediated immune responses directly by suppressing the response-potential of cells and indirectly by triggering the release of a factor from adherent splenic cells which induces a hyporesponsive state in T lymphocytes.     

Trypanosoma musculi infections in complement-deficient mice

Strains of mice (CFW, C57B1/10Sn, B10.D2/nSn, and B10.D2/oSn) were infected with Trypanosoma musculi (Trypanosoma duttoni). The complement-deficient B10.D2/oSn mice showed typical parasitemias similar to those presented by the strains possessing hemolytic complement activity. Peak parasitemias occurred 12 days postinoculation. The highest parasitemias were measured in CFW mice (657 ± 82 T. musculi/30 hi-power fields), while infections in C56BL/10Sn (528 ±44 T. musculi/30 h.p.f.), B10.D2/oSn (502 ± 20 T. musculi/30 h.p.f.) and B10.D2/nSn (512 ± 35 T. musculi/30 h.p.f.) were less severe and quantitatively comparable. The percentages of dividing forms were similar during infections in each of the strains. While parasites were detected in peripheral blood until Day 22 of infection in three of 10 C57BL/10Sn mice, none could be found at this time in blood films of CFW, B10.D2/nSn or B10.D2/oSn mice. Giemsa stained kidney imprints indicated the presence of parasites in animals of each of the strains after 33 days, when trypanosomes could no longer be detected in the peripheral blood of the mice. The minor variations in the parasitemias appeared related to the mouse strain. Complement dependent, antibody mediated immune cytolysis was not indicated as a mechanism for the elimination of T. musculi by the infected mouse.     

Trypanosoma rangeli: A possible role for ecto-phosphatase activity on cell proliferation

Here we demonstrate for the first time that growth of Trypanosoma rangeli, a protozoa parasite, is strongly dependent on the presence of inorganic phosphate (Pi) in the culture medium and that the replacement of the inorganic phosphate in the culture medium by β-glycerophosphate, a substrate for phosphatases lead the cells to achieve its maximal growth. The ecto-phosphatase activity present on the external surface of T. rangeli decreased during the growth phase of the parasite, suggesting that this enzyme could be important for the development. Accordingly, the inhibition of this ecto-phosphatase activity by sodium orthovanadate also inhibited the proliferation of T. rangeli. Parasites maintained in a Pi-starved culture medium (2 mM Pi) had 4-fold more ecto-phosphatase activity as compared to parasites maintained in a Pi-supplemented culture medium (50 mM Pi). Altogether, these results presented here suggest that this ecto-phosphatase activity leads to hydrolysis of phosphorylated compounds present in the extracellular medium, which could contribute to the acquisition of inorganic phosphate during the development of T. rangeli epimastigotes.     

Probing for primary functions of prohibitin in Trypanosoma brucei

Prohibitins (PHBs) 1 and 2 are small conserved proteins implicated in a number of functions in the mitochondrion, as well as in the nucleus of eukaryotic cells. The current understanding of PHB functions comes from studies of model organisms such as yeast, worm and mouse, but considerable debate remains with regard to the primary functions of these ubiquitous proteins. We exploit the tractable reverse genetics of Trypanosoma brucei, the causative agent of African sleeping sickness, in order to specifically analyse the function of PHB in this highly divergent eukaryote. Using inducible RNA interference (RNAi) we show that PHB1 is essential in T. brucei, where it is confined to the cell’s single mitochondrion forming a high molecular weight complex. PHB1 and PHB2 appear to be indispensible for mitochondrial translation. Their ablation leads to a decrease in mitochondrial membrane potential, however no effect on the level of reactive oxygen species was observed. Flagellates lacking either PHB1 or both PHB1 and PHB2 exhibit significant morphological changes of their organelle, most notably its inflation. Even long after the loss of the PHB proteins, mtDNA was unaltered and mitochondrial cristae remained present, albeit displaced to the periphery of the mitochondrion, which is in contrast to other eukaryotes.     

S-Adenosylmethionine synthetase in bloodstream Trypanosoma brucei

S-adenosylmethionine synthetase was studied from bloodstream forms of Trypanosoma brucei brucei, the agent of African sleeping sickness. Two isoforms of the enzyme were evident from Eadie Hofstee and Hanes-Woolf plots of varying ATP or methionine concentrations. In the range 10–250 μM the K_m for methionine was 20 μM, and this changed to 200 μM for the range 0.5–5.0 mM. In the range 10–250 μM the K_m for ATP was 53 μM, and this changed to 1.75 mM for the range 0.5–5.0 mM. The trypanosome enzyme had a molecular weight of 145 kDa determined by agarose gel filtration. Methionine analogs including selenomethionine, L-2-amino-4-methoxy-cis but-3-enoic acid and ethionine acted as competitive inhibitors of methionine and as weak substrates when tested in the absence of methionine with [¹⁴C]ATP. The enzyme was not inducible in procyclic trypomastigotes in vitro, and the enzyme half-life was > 6 h. T. b. brucei AdoMet synthetase was inhibited by AdoMet (K_i 240 μM). The relative insensitivity of the trypanosome enzyme to control by product inhibition indicates it is markedly different from mammalian isoforms of the enzyme which are highly sensitive to AdoMet. Since trypanosomes treated with the ornithine decarboxylase antagonist DL-α-difluoromethylornithine accumulate AdoMet and dcAdoMet (final concentration ≈ 5 mM), this enzyme may be the critical drug target linking inhibition of polyamine synthesis to disruption of AdoMet metabolism.     

Trypanosoma gambiense: Blocking ability of parasite and macrophage homogenates on attachment during phagocytosis

This study deals with the ability of the parasite and macrophage homogenates, each fractionated into soluble and insoluble parts, to block the attachment of Trypanosoma gambiense on the macrophage in the process of phagocytosis. The soluble parasite fraction and the insoluble macrophage fraction both showed a high blocking ability. However, the ability of the parasite fraction was completely lost on trypsin digestion, while that of the macrophage was not changed at all. In addition, sonication of the macrophage did not change its blocking ability.     

Nested PCR assay for the rapid detection of Naegleria fowleri from swimming pools in Egypt

The free-living amoeboflagellate Naegleria fowleri is the only species infects humans world widely distributed. N. fowleri is the causative agent of very rare but severe brain infection called primary amoebic meningo-encephalitis (PAM), a rapidly fatal disease of the central nervous system mainly in immuno-compromised individuals. N. fowleri infects human through the entry of the nose, and it happens when human swimming or diving in warm freshwater, such as lakes, rivers and swimming pools. The disease is acute, and patients often die within 5–10 days and before the infectious agent can be diagnosed. Limited information is available about the existence of pathogenic N. fowleri, in Egypt, so the present of N. fowleri is an important public health. In the present study, we examined hundred water, dust and swap samples collected from 5 swimming pools in Cairo, Egypt. Based on morphological characteristics of trophozoite and cyst, flagellation test 56% of thermo-tolerant Naegleria like amoeba was detected. The incidence of thermo-tolerant free-living amoebae reached 84, 80and 70% from water, cotton swap and dust samples, respectively at cultivation temperature of 45 °C. The highest occurrence of thermo-tolerant amoebae were recorded in summer (100 & 87.5%) while the lowest one were recorded in winter (58 & 37.5%) in both water and dust samples, respectively. In swap samples, the highest occurrence of thermo-tolerant free-living amoeba was recorded in both summer and spring (100%), while the lowest one was recorded in winter (40%). N. fowleri was performed on 24 samples from a total of 56 (42.2%) samples which are positive by culture. Nested PCR using Mp2Cl5 gene primers that is unique to N. fowleri was carried out. The N. fowleri specific primer showed band at 166 bp against 24 of 56 (42.2%) samples. The majority of positive samples unique to N. fowleri was detected in water samples followed by swap samples and finally dust samples 14 of 24 (58%), 7 of 24 (29%), 3 of 24 (13%), respectively. In conclusion, swimming pools water may be the source of Naegleria invasion. The use of molecular methods to identify free-living amoebae N. fowleri could provide a more rapid means to diagnose infections caused by those amoebae.