Non-infective Form Research Articles

VIP: An Agent with License to Kill Infective Parasites

Antimicrobial peptides are small, cationic, and amphipathic peptides of variable length, sequence, and structure. They are effector molecules of innate immunity with microbicidal and both pro- or anti-inflammatory activities. Vasoactive intestinal polypeptide (VIP) and the structurally related pituitary adenylate cyclase-activating polypeptide (PACAP) are well-known immunomodulators. On the basis of their cationic and amphipathic structures, resembling antimicrobial peptides, we propose that their immune role could also include a direct lethal effect against pathogens. We thus investigated the potential antiparasitic activities of VIP and PACAP against the African trypanosome Trypanosoma brucei (T. brucei). Both peptides killed the bloodstream (infective) form but not the insect (noninfective) form of the parasite. VIP and PACAP caused complete destruction of the parasite integrity through a mechanism involving their entry and accumulation into the cytosol. These results provide the basis for further studies of these and other structurally related peptides as alternative treatments for parasitic diseases mainly with associated drug resistances.

Amino Acid Metabolic Routes in Trypanosoma cruzi: Possible Therapeutic Targets Against Chagas; Disease

Chagas' disease is a zoonosis caused by the parasite Trypanosoma cruzi, a haematic protozoan, transmitted by insects from the Reduviidae family. This constitutes a relevant health and socio-economic problem in the Americas, with 11 - 18 million people infected, and approximately 100 million people at risk. The therapeutic possibilities rely into two drugs, nifurtimox and benznidazole, that were discovered more than thirty years ago, and are mainly successful during the acute phase of the disease. In the majority of the cases the disease is diagnosed in the chronic phase, when the therapy is inefficient and the probability of cure is low. In addition, these drugs are highly toxic, with systemic side effects on patients. Trypanosoma cruzi has a metabolism largely based on the consumption of amino acids, mainly proline, aspartate and glutamate, which constitute the main carbon and energy sources in the insect stage of the parasite life cycle. These amino acids also participate in the differentiation process of the replicative non-infective form (epimastigote) to the non-replicative infective form (trypomastigote). In particular, the participation of proline in the intracellular differentiation cycle, which occurs in the mammalian host, was recently demonstrated. In addition, an arginine kinase has been described in T. cruzi and T. brucei, which converts free arginine to phosphoarginine, a phosphagen with a role as an energy reservoir. Arginine kinase seems to be an essential component of energy management during stress conditions. Taken together, these data indicate that amino acid metabolism may provide multiple as yet unexplored targets for therapeutic drugs.

Post genomic analysis of permeases from the amino acid/auxin family in protozoan parasites

The “amino acid/auxin permeases” is probably the most represented family of transporters in the Trypanosoma cruzi genome. Using a high-throughput searching routine and preliminary data from the T. cruzi genome project, more than 15,000 sequences were iteratively assembled into contigs, and 60 open reading frames corresponding to different putative amino acid transporters, clustered in 12 groups, were detected and characterized in silico. T. cruzi genomic organization of such sequences showed that these putative amino acid transporter genes are in an unusually large number and arranged in repeat clusters comprising about 0.2% of the genome. These data suggest that the family has evolved following tandem duplication events and constitutes a novel family of variable proteins in protozoan organisms. The mRNA expression of the predicted genes was demonstrated in infective and non-infective parasite forms. Orthologous sequences were also identified in other unicellular parasites such as Leishmania spp., Plasmodium spp., and Trypanosoma brucei.

Characterization and immunolocalization of an NTP diphosphohydrolase of Trypanosoma cruzi

An ecto-NTP diphosphohydrolase (NTPDase) activity, insensitive to inhibitors of ATPases and phosphatases, was characterized on the surface of live Trypanosoma cruzi intact parasites. The enzyme exhibits broad substrate specificity, typical of NTPDases, and a high hydrolysis rate for GTP. A 2282 bp message encoding a full-length NTPDase was cloned by RT-PCR using epimastigote mRNA. A single protein was immunoprecipitated from [ 35S]methionine-labeled parasites using antibodies against Toxoplasma gondii NTPase I. This antibody localized an NTPDase on the external surface of all forms of T. cruzi, as seen by confocal immuno-fluorescence microscopy. The NTPDase could be part of the parasite’s purine salvage pathway. Additionally, trypomastigotes (infective form) presented a 2:1 ATP/ADP hydrolysis ratio, while epimastigotes (non-infective form) presented a 1:1 ratio, suggesting a possible role for the NTPDase in the parasite’s virulence mechanisms.

Trypanosoma cruzi: cloning and characterization of two genes whose expression is up-regulated in metacyclic trypomastigotes

The differentiation of epimastigotes into metacyclic trypomastigotes (metacyclogenesis) involves the transformation of a replicative non-infective form of Trypanosoma cruzi into a non-replicative infective stage. The study of genes with stage-specific expression may provide insight into the mechanisms involved in the regulation of gene expression in this parasite. We cloned and characterized two genes whose expression is up-regulated in metacyclic trypomastigote, those encoding metacyclin-II (Met-II) and metacyclin-III (Met-III). Nucleotide sequence analysis identified no sequence similarity with sequences available from genetic databases. The deduced amino acid sequences of the genes indicated that Met-III encodes a basic polypeptide whereas Met-II encodes an acidic polypeptide. Northern and Western blot analyses showed that Met-II and Met-III were expressed by metacyclic trypomastigotes, but not by epimastigotes. Antisera directed against the recombinant Met-II and Met-III proteins recognized two polypeptides on Western blots: a 16-kDa and a 24-kDa polypeptide. Immunocytochemistry analysis using electron microscopy showed that metacyclin-II is localized mainly at the kinetoplast whereas metacyclin-III is localized at the nucleus of the parasite. Southern blot analysis, using genomic DNA and T. cruzi chromosomes separated by pulsed-field gel electrophoresis, indicated that these genes were present as single copies on different chromosomes of T. cruzi Dm28c.

Monitoring the developmental status of Trypanosoma brucei gambiense in the tsetse fly by means of PCR analysis of anal and saliva drops

Teneral Glossina palpalis gambiensis ( Diptera: Glossinidae) were infected with a culture of procyclic forms of Trypanosoma brucei gambiense using a single-bloodmeal membrane feeding technique. The infection was monitored by analysing the saliva (mature infection) and anal drop (midgut infection) of each fly at different post-infection times both by microscopic observation and polymerase chain reaction (PCR). Amplification revealed many more positive anal drops than microscopy. The monitoring showed that the installation of T. b. gambiense in Glossina took place at least 11 days after the infection and that maturation occurred after 29 days. It also reflected precisely the parasitic status of each tsetse fly as determined by the dissection, microscopic examination and PCR amplification of the midguts and salivary glands 47 days post-infection. Twice as many tsetse flies with mature salivary glands infection were revealed by PCR than by microscopic examination, but the two techniques gave exactly the same results regarding the proportion of flies with midgut infection. This study also demonstrated the ability of natural non-infective procyclic forms of T. b. gambiense, to colonise the midgut and subsequently establish in the salivary glands of G. p. gambiensis.

Biochemical Characterization of Proline Racemases from the Human Protozoan Parasite Trypanosoma cruzi and Definition of Putative Protein Signatures

Proline racemase catalyzes the interconversion of L- and D-proline enantiomers and has to date been described in only two species. Originally found in the bacterium Clostridium sticklandii, it contains cysteine residues in the active site and does not require co-factors or other known coenzymes. We recently described the first eukaryotic amino acid (proline) racemase, after isolation and cloning of a gene from the pathogenic human parasite Trypanosoma cruzi. Although this enzyme is intracellularly located in replicative non-infective forms of T. cruzi, membrane-bound and secreted forms of the enzyme are present upon differentiation of the parasite into non-dividing infective forms. The secreted form of proline racemase is a potent host B-cell mitogen supporting parasite evasion of specific immune responses. Here we describe that the TcPRAC genes in T. cruzi encode functional intracellular or secreted versions of the enzyme exhibiting distinct kinetic properties that may be relevant for their relative catalytic efficiency. Although the Km of the enzyme isoforms were of a similar order of magnitude (29-75 mM), Vmax varied between 2 x 10(-4 )and 5.3 x 10(-5) mol of L-proline/s/0.125 microM of homodimeric recombinant protein. Studies with the enzyme-specific inhibitor and abrogation of enzymatic activity by site-directed mutagenesis of the active site Cys330 residue reinforced the potential of proline racemase as a critical target for drug development against Chagas' disease. Finally, we propose a protein signature for proline racemases and suggest that the enzyme is present in several other pathogenic and non-pathogenic bacterial genomes of medical and agricultural interest, yet absent in mammalian host, suggesting that inhibition of proline racemases may have therapeutic potential.

Flagellar elongation induced by glucose limitation is preadaptive for Trypanosoma cruzi differentiation.

Trypanosomes must sense and respond to environmental change in order to progress through their life cycles. The American trypanosome, Trypanosoma cruzi, differentiates from the noninfective epimastigote form to the infective metacyclic form spontaneously in axenic culture. Here, we investigate the initial stimulus for that change and demonstrate that T. cruzi epimastigotes sense limitation of glucose in the medium and respond by undergoing significant morphological and biochemical change. As part of this change, the mean flagellar length of the population triples, which is correlated with an increased ability to maintain interactions with hydrophobic substrates, a requirement for differentiation to the next life cycle stage.

CAMP Regulates Ca2+-dependent Exocytosis of Lysosomes and Lysosome-mediated Cell Invasion by Trypanosomes

Ca2+-regulated exocytosis, previously believed to be restricted to specialized cells, was recently recognized as a ubiquitous process. In mammalian fibroblasts and epithelial cells, exocytic vesicles mobilized by Ca2+ were identified as lysosomes. Here we show that elevation in intracellular cAMP potentiates Ca2+-dependent exocytosis of lysosomes in normal rat kidney fibroblasts. The process can be modulated by the heterotrimeric G proteins Gs and Gi, consistent with activation or inhibition of adenylyl cyclase. Normal rat kidney cell stimulation with isoproterenol, a beta-adrenergic agonist that activates adenylyl cyclase, enhances Ca2+-dependent lysosome exocytosis and cell invasion by Trypanosoma cruzi, a process that involves parasite-induced [Ca2+]i transients and fusion of host cell lysosomes with the plasma membrane. Similarly to what is observed for T. cruzi invasion, the actin cytoskeleton acts as a barrier for Ca2+-induced lysosomal exocytosis. In addition, infective stages of T. cruzi trigger elevation in host cell cAMP levels, whereas no effect is observed with noninfective forms of the parasite. These findings demonstrate that cAMP regulates lysosomal exocytosis triggered by Ca2+ and a parasite/host cell interaction known to involve Ca2+-dependent lysosomal fusion.

Infectivity and Attenuation of Leishmania donovani Promastigotes: Association of Galactosyl Transferase with Loss of Parasite Virulence

During the course of long-term in vitro cultivation of Leishmania donovani parasites, the promastigotes were found to lose their infectivity. Lectin agglutination studies revealed that there is an up-regulation of terminally exposed galactose residues on the noninfective promastigotes. The enzyme galactosyl transferase was absent in the infective form but present in the non-infective form of the parasites. An association between galactosylation and loss of infectivity was also observed. We propose that the enzyme galactosyl transferase is developmentally regulated and induces galactosylation of surface carbohydrates.

Taenia solium cysticercosis: immunity in pigs induced by primary infection

The present study demonstrates that pigs experimentally infected with Taenia solium eggs develop resistance to reinfection that lasts at least five months. Thirteen 2-month-old piglets were infected with eggs of Taenia solium. After 5 months, two pigs were euthanized and five were challenged with eggs from a second tapeworm. Nine months after the first infection, six pigs were challenged with a third tapeworm. All 11 challenged pigs were euthanized 2 months after reinfection. In order to confirm the infectivity of the eggs, several piglets were inoculated with each taenia. Two of the five pigs reinfected after 5 months did not develop metacestodes, two showed few caseous non-infective forms and in the fifth pig, 14% of the metacestodes were vesicular and 86% colloidal and caseous. In the six animals challenged 9 months after the first infection, three were heavily infected with vesicular metacestodes and the other three showed only colloid and caseous forms in muscles. All parasites found in brains were vesicular. We conclude that immunity due to primary infection lasts at least 5 months. At 2 months of infection antigens of 24 and 39–42 kDa were the most frequently recognised. In those pigs with only a few caseous cysts in muscles and/or vesicular ones in brains no antibodies were detected.

Trypanosoma cruzi:Nitrogenous-Base-Containing Phosphatides in Trypomastigote Forms—Isolation and Chemical Analysis

In trypanosomatids, little is known about the biosynthetic pathways involved in the metabolism of ethanolamine. In an attempt to clarify this point, an exhaustive analysis of the chloroform:methanol extract ofT. cruzitrypomastigotes metabolically labeled with [14C]ethanolamine, in comparison with the lipids from [3H]palmitic acid-incorporated parasites, was performed. In both cases, phosphatidylethanolamine and lysophosphatidylethanolamine were detected, while phosphatidylcholine and lysophosphatidylcholine were only labeled with the fatty acid precursor. However, dimethylphosphatidylethanolamine was isolated from parasites labeled with the base precursor, indicating the ability of trypanosomes to methylate phosphatidylethanolamine to dimethylphosphatidylethanolamine. Fatty acids of the labeled phospholipids were analyzed by reverse-phase thin-layer chromatography and fluorography. Interestingly, phospholipids from the trypomastigote stage show palmitic acid (C16:0) and stearic acid (C18:0) as the only labeled components. The same saturated fatty acids were found free and as components of the radioactive triglycerides. No unsaturated fatty acids were detected, in accordance with the results obtained with inositolphospholipids. Conversely, when the fatty acids of phospholipids purified from nonlabeled parasites were analyzed by gas–liquid chromatography and gas–liquid chromatography–mass spectrometry, C18:1 was also detected. A striking finding was the presence of a considerable amount of free lignoceric acid (C24:0). Also, the C24:0 fatty acid was identified in the triglyceride fraction and as a component of phosphatidylcholine. The limited capacity of trypomastigote forms to elongate fatty acids was determined. In contrast with the results reported for other noninfective forms of the parasite, the absence of unsaturated fatty acids due to a low activity of desaturases was observed.

The Lipid Structure of the Glycosylphosphatidylinositol-anchored Mucin-like Sialic Acid Acceptors of Trypanosoma cruzi Changes during Parasite Differentiation from Epimastigotes to Infective Metacyclic Trypomastigote Forms

The major acceptors of sialic acid on the surface of metacyclic trypomastigotes, which are the infective forms of Trypanosoma cruzi found in the insect vector, are mucin-like glycoproteins linked to the parasite membrane via glycosylphosphatidylinositol anchors. Here we have compared the lipid and the carbohydrate structure of the glycosylphosphatidylinositol anchors and the O-linked oligosaccharides of the mucins isolated from metacyclic trypomastigotes and noninfective epimastigote forms obtained in culture. The single difference found was in the lipid structure. While the phosphatidylinositol moiety of the epimastigote mucins contains mainly 1-O-hexadecyl-2-O-hexadecanoylphosphatidylinositol, the phosphatidylinositol moiety of the metacyclic trypomastigote mucins contains mostly (approximately 70%) inositol phosphoceramides, consisting of a C18:0 sphinganine long chain base and mainly C24:0 and C16:0 fatty acids. The remaining 30% of the metacyclic phosphatidylinositol moieties are the same alkylacylphosphatidylinositol species found in epimastigotes. In contrast, the glycosylphosphatidylinositol glycan cores of both molecules are very similar, mainly Man alpha 1-2Man alpha 1-2Man alpha 1- 6Man alpha 1-4GlcN. The glycans are substituted at the GlcN residue and at the third alpha Man distal to the GlcN residue by ethanolamine phosphate or 2-aminoethylphosphonate groups. The structures of the desialylated O-linked oligosaccharides of the metacyclic trypomastigote mucin-like molecules, released by beta-elimination with concomitant reduction, are identical to the structures reported for the epimastigote mucins (Previato, J. O., Jones, C., Gonçalves, L. P. B., Wait, R., Travassos, L. R., and Mendoça-Previato, L. (1994) Biochem. J. 301, 151-159). In addition, a significant amount of nonsubstituted N-acetylglucosaminitol was released from the mucins of both forms of the parasite. Taken together, these results indicate that when epimastigotes transform into infective metacyclic trypomastigotes, the phosphatidylinositol moiety of the glycosylphosphatidylinositol anchor of the major acceptor of sialic acid is modified, while the glycosylphosphatidylinositol anchor and O-linked sugar chains remain essentially unchanged.

Immunocytochemical localization of neuraminidase in Trypanosoma cruzi

A polyclonal antibody obtained against neuraminidase purified from Trypanosoma cruzi was used for the localization of the protein in whole cells by immunofluorescence microscopy and in thin sections of parasites (epimastigote, amastigote, and trypomastigote forms) embedded at a low temperature in Lowicryl K4M resin. The intensity of labeling, as evaluated by the number of gold particles associated with the parasite, varied according to the protozoan developmental stage. In the noninfective epimastigote forms, labeling of the cell surface was very weak. However, an intense labeling of some cytoplasmic vacuoles was observed. Labeling of the surfaces of most of the trypomastigote forms was weak, while gold particles were seen in association with the flagellar pockets of these forms, which suggests that the enzyme is secreted through this region. Intense labeling of the surfaces of many, but not all, transition forms between trypomastigote and amastigote forms was observed. Amastigote forms found in the supernatant of infected cell cultures had their surfaces intensely labeled, while few particles were seen on the surfaces of intracellular amastigotes. The results obtained are discussed in relation to the role played by T. cruzi neuraminidase in the process of parasite-host cell interaction.

Observations on the development of Leishmania (L.) chagasi Cunha and Chagas in the midgut of the sandfly vector Lutzomyia longipalpis (Lutz and Neiva).

Previous observations have shown that forms of Leishmania, infective to hamsters, are present in the midgut of experimentally infected sandflies from 15-221 hours after the infective bloodmeal. In a continuation of these studies, stained smears of the midgut contents of Lutzomyia longipalpis infected with Leishmania (L.) chagasi, made at intervals between 15-120 hours, revealed two lines of parasite development. One of these is the direct transformation of small, non-dividing amastigotes into very small promastigotes, which are considered to represent the "infective" or "metacyclic" flagellates involved in subsequent transmission of the parasite. The other stems from enlarged, highly vacuolated amastigotes which undergo at least two divisions before giving rise to large, elongate and non-dividing promastigotes. These are thought to represent the non-infective forms of the parasite seen in in vitro cultures, and their function remains speculative.

In vitro Translation of Trypanosoma cruzi Antigens Recognized by Human Chagasic Sera

Several reports indicate that infective (trypomastigotes) and noninfective (epimastigotes) forms of Trypanosoma cruzi display relatively simple antigenic patterns when analyzed by immunoprecipitation with hyperimmune sera from experimental animals. Thus, epimastigotes show, as their major antigen, an Mr 72,000 glycoprotein, whereas trypomastigotes (both blood forms and tissue culture-derived forms) display instead an Mr 90,000 surface glycoprotein (Nogueira et al., 1981, Journal of Experimental Medicine 153: 629-639; Snary et al., 1981, Molecular and Biochemical Parasitology 3: 343-356). These patterns become more complex when sera from chagasic patients are used in either immunoprecipitation techniques (Zingales et al., 1982, Molecular and Biochemical Parasitology 11: 111-124) or immunoblotting (Grogl and Kuhn, 1984, Journal of Parasitology 70: 822824). We have extended these studies and found that lysates of epimastigotes and tissue culturederived trypomastigotes subjected to immunoblotting with human chagasic sera, display reactive bands over a wide range of molecular weights (Mr 150,000 to Mr 12,000) (Rangel-Aldao et al., 1986, Molecular and Biochemical Parasitology 20: 25-32). Most of these antigenic bands were shared by both parasite forms and the differences in their antigenic patterns were only manifested upon antigen dilution. Trypomastigotes showed, predominantly, a group of bands in the range of Mr 150,000 to Mr 75,000, whereas epimastigotes displayed a cluster of bands in the range of Mr 72,000 to Mr 36,000 (Rangel-Aldao et al., 1986, loc. cit.). everal reports indicate that infective (trypoThis qualitative homology of epimastigotes and trypomastigotes could be exploited to produce in vitro, by the techniques of molecular biology, most of the common antigens of the infective trypomastigotes from the mRNA of epimastigotes that are not infectious and can be easily cultured in mass quantities. These antigens could then be used to develop sensitive tools for diagnosis and vaccination tests for Chagas' disease. As a prerequisite to achieve this goal, we have isolated polyadenylated (poly A+) RNA from epimastigotes, translated it in vitro in a wheat germ cell-free system, immunoblotted the translated products with a pool of sera from 10 patients with Chagas' disease, and showed that it is possible to translate in vitro antigens produced n vivo by epimastigotes. These antigens are also shared by tissue culture-derived trypomastigotes (Rangel-Aldao et al., 1986, loc. cit.). Poly A+ RNA was isolated from 1 x 1010 cultured epimastigotes (Rangel-Aldao et al., 1983, Journal of Biological Chemistry 258: 6979-6983), washed twice with 50 ml of 1% w/v NaCI, 0.6 t,g/ml cycloheximide, and 20% glycerol. The cells were resuspended in lysis buffer (50 mM TrisHC1, pH 7.4, 0.5% Triton X-100, 50% glycerol, and 1,000 units of human placental ribonuclease inhibitor) and lysed at -20 C with a Dounce homogenizer. The supernatant obtained from the lysis step was mixed with 3 volumes of a buffer solution containing 10 mM Tris-HCl, pH 7.4, 0.1 M NaCl, 1 mM EDTA, and 0.5% sodium dodecyl sulfate (SDS) and the total cytoplasmic RNA extracted with phenol-chloroform (Perry et al., 1972, Biochimica et Biophysica Acta 262: qualitative hom logy of epimastigotes and astigotes could b exploited to produce itro, by the techniques of molecular biology, of the common antigens of the infective astigotes from the mRNA of epimastis that are not infectious and can be easily red in ma s quan ties. Th se antigens could e used to develop sensitive tools for disis and va cination te ts for Chagas' di ase. rerequisite to achieve this goal, we have

Age Susceptibility of Molluscan Intermediate Hosts to Protostrongylid Nematodes

genic bands may correspond in vivo to glycoproteins with additional epitopes, and with a carbohydrate moiety that does not significantly affect their electrophoretic migration. The strong signal given by the in vitro-translated antigenic bands of Mr's 51,000, 47,000, and 30,000, when confronted with sera from chagasic patients, and their presence in infective and noninfective forms of T. cruzi offers hope for the design of a diagnostic test for Chagas' disease based on antigen molecules produced in vitro. We appreciated the help of Dr. O. Valbuena on the isolation of poly A+ RNA from epimastigotes. This investigation received financial support from the UNDP/WB/WHO Special Programme for Research in Tropical Diseases, and CONICIT, CODECIH-UC (Venezuela). st for Chag s' disease based on antigen mol les produced in vitro. e ap reciated the lp of Dr. O. Valbuen the isolation of poly A+ RNA from epimasigotes. This investigation receiv d financial sup-

A neuraminidase from Trypanosoma cruzi removes sialic acid from the surface of mammalian myocardial and endothelial cells.

Trypanosoma cruzi causes Chagasic heart disease, a major public health problem in Latin America. The mechanism of interaction of this protozooan parasite with host cells is poorly understood. We recently found that the infective trypomastigote form a T. cruzi exhibits neuraminidase activity and can desialylate mammalian erythrocytes. However, it is not known if T. cruzi can also modify the surfaces of cardiovascular cells that are directly involved in the most important clinical manifestations of this disease. Accordingly, this study determined whether T. cruzi can remove sialic acid from cultured rat myocardial or human vascular endothelial cells. Sialic acid was labeled metabolically with the precursor 3H-N-acetyl-D-mannosamine. Soluble neuraminidase, isolated from intact T. cruzi trypomastigotes, caused significant release of labeled material from myocardial cells (e.g., 2,174 +/- 27 dpm/h vs. spontaneous release of 306 +/- 30 dpm/h, n = 4, P less than 0.001). Chromatographic analysis showed that the bulk of the radioactivity released by T. cruzi neuraminidase was sialic acid. Intact T. cruzi trypomastigotes also released sialic acid from metabolically labeled myocardial cells in a concentration-dependent manner. In contrast, a noninfective form of T. cruzi, the amastigote, did not desialylate these cells. Galactose oxidase labeling demonstrated newly desialylated glycoproteins on the surface of myocardial cells treated with T. cruzi neuraminidase. Desialylation of myocardial cells was confirmed histochemically by the appearance of binding sites for peanut agglutinin, a lectin that binds to complex oligosaccharide moieties after removal of the terminal sialyl residue. T. cruzi neuraminidase also removed sialic acid from adult human saphenous vein endothelial cells, as determined by both histochemical and metabolic labeling studies. Thus, infective forms of T. cruzi can chemically modify the surfaces of myocardial and vascular endothelial cells by desialylation. This alteration may play a role in the initial interaction of this parasite with these important target cells of the host cardiovascular system.

Infectivity of Monomorphic and Pleomorphic Trypanosoma brucei Stocks Cultivated at 28 C with Various Tsetse Fly Tissues

Noninfective procyclic forms of Trypanosoma brucei stocks derived from the pleomorphic EVE 10 were cultivated at 28 C in Cunningham's liquid medium in the presence of head-salivary gland, alimentary tract, and abdominal body wall explants of Glossina morsitans morsitans. After 8 to 10 days of cultivation some of the procyclic forms transformed into metacyclic stages infective for mice. Infectivity persisted for varying periods up to 66 days, when the experiments were terminated. Only 10 explants of alimentary tract or abdominal body wall tissues were required in the flasks to render the culture infective for most of the mice inoculated. Similar trypanosome suspensions grown with 10 head-salivary gland explants produced an infection on only one occasion. Cultures of procyclic organisms derived from the monomorphic stock 427 grown inthe presence of all three types of tsetse fly explants produced only sporadic infections in mice. Metacyclic forms failed to develop in trypanosome populations of stock EVE 10 cultivated at 28 C in the liquid medium alone or supplemented with mouse embryo tissues.

The separation and structure of infective trypanosomes from cultures of Trypanosomas brucei grown in association with tsetse fly salivary glands.

Infective trypanosomes developed when Trypanosoma brucei was cultivated at 28 C in a liquid medium containing tsetse fly head-salivary gland explants. They were separated from the noninfective culture forms using DEAE-cellulose column chromatography. It was demonstrated by light and electron microscopy that the separated organisms were morphologically similar to metacyclic stages found in a tsetse fly and that they had a characteristic surface coat. Single metacyclic trypanosomes isolated from the cultures gave rise to infections in mice.