Identification of Immunogenic Epitopes of the 170-kDa Subunit Adhesin of Entamoeba histolytica in Patients with Invasive Amebiasis

ABSTRACT. Entamoeba histolytica causes amebic dysentery (AD) and liver abscess (ALA). Little is known about protective immunity to amebiasis, and studies in this area have been complicated by the paucity of defined ameba antigens. We examined the proliferative responses of peripheral blood mononuclear cells (PBMC) from patients with AD and ALA to a recombinant protein containing a portion of the 170 kDa adhesin of E. histolytica (170CR), and to two synthetic peptides (1 and 2) derived from the 170 kDa sequence that were predicted to contain T cell epitopes. A significant number of patients with AD and ALA had PBMC that proliferated to 170CR molecule, and several individuals with ALA and AD had T cells that recognized one or both peptides. Contrarily, individuals from a non-endemic region for amebiasis did not respond to 170CR protein, or to both peptides. In regard to antibody response, nine of fifteen patients with ALA showed antibodies to 170CR protein. These same patients had antibodies to peptide 2. We identified peptides from 170-kDa adhesin that may contain both T and B cell epitopes recognized by some patients with invasive amebiasis. These peptides may be valuable reagents in studies of the immune response to amebiasis.     

Fine Structure and Cytochemistry of Tritrichomonas foetus and Rat Neutrophil Interaction

ABSTRACT. The fine structure of normal and antibody-coated Tritrichomonas foetus cells and their interaction with rat peritoneal neutrophils was studied. Peritoneal neutrophils were obtained by glycogen stimulation. The neutrophils readily associated with and killed the parasites, which were subsequently ingested. The process involved activation of the respiratory burst, as demonstrated by the use of cytochemical methods. Images were obtained indicating that binding of parasites to the neutrophil surface triggers an exocytic response with release of oxygen-derived products. Cytochemical localization of acid phosphatase and peroxidase activities showed that leukocyte granules fused with the parasite-containing phagocytic vacuoles. We also showed the cytochemical localization of alkaline phosphatase in the parasite-neutrophil interaction.     

In Vitro Excystation of Spironucleus muris

ABSTRACT. In vitro excystation of Spironucleus muris cysts, purified by sequential sucrose and Percoll gradients from mouse feces, was studied. Three in vitro excystation procedures, used for Giardia , were assessed to determine the most useful method. Excystation was monitored by light microscopy and subsequently characterized by transmission and scanning electron microscopy. Spironucleus muris excysted routinely at a level greater than 90% when induced in Hanks' balanced salt solution containing sodium bicarbonate at pH 2.0 and transferred to Tyrodes' salt solution as an excystation medium. Similarly, high rates of excystation were recorded after induction of S. muris cysts in 0.1 M potassium phosphate buffer (pH 7.0) with sodium bicarbonate and excystation in trypticase-yeast extract-iron medium (TYI medium) or phosphate-buffered saline. A lower rate and percentage of excystation were observed after induction of S. muris cysts in an aqueous hydrochloric acid solution (pH 2.0) followed by excystation in TYI medium. All excystation methods produced extremely active S. muris trophozoites with normal morphology. Nonexcysting S. muris cysts have a wall composed of an outer fibrous and an inner membranous portion. Following induction, numerous vesicles appeared in the peritrophic space. Excystation began by the cyst wall opening at one pole, and the anterior part of the trophozoite protruding from the cyst wall. The trophozoite emerged progressively from the cyst wall and the empty cyst wall appeared to collapse. Excysted trophozoites exhibited normal morphological features of S. muris trophozoites isolated from the mouse intestine.     

Giardia lamblia: isolation and axenic cultivation.

Giardia lamblia trophozoites have been axenically cultured for more than a year. Initially, organisms were established in a complex liquid medium in the presence of the host's intestinal fungi; subcultures were made of these protozoa-fungus mixtures. G. lamblia trophozoites, free of yeast, were obtained by inoculating a protozoafungus culture in one arm of a U-tube, then later removing, from the other arm of the tube, Giardia trophozoites that had migrated across the base. Medium was changed at 2- or 3-day intervals; numerous subcultures were made. Tests for the possible presence of other organisms in these axenic cultures were negative. Trophozoite cultures remained viable, after freezing in the presence of glycerol, for 14 months. This is the first reported axenic culture of this common human intestinal parasite and pathogen; its study in pure culture is now possible.     

Conjugation is necessary for a bacterial plasmid to survive under protozoan predation

Horizontal gene transfer by conjugative plasmids plays a critical role in the evolution of antibiotic resistance. Interactions between bacteria and other organisms can affect the persistence and spread of conjugative plasmids. Here we show that protozoan predation increased the persistence and spread of the antibiotic resistance plasmid RP4 in populations of the opportunist bacterial pathogen Serratia marcescens. A conjugation-defective mutant plasmid was unable to survive under predation, suggesting that conjugative transfer is required for plasmid persistence under the realistic condition of predation. These results indicate that multi-trophic interactions can affect the maintenance of conjugative plasmids with implications for bacterial evolution and the spread of antibiotic resistance genes.     

Exuviotrophic apostome ciliates from crustaceans of St. Andrew Bay, Florida, and a description of Gymnodinioides kozloffi n. sp

Gymnodinioides kozloffi n. sp. is described from the eelgrass broken-back shrimp Hippolyte zostericola. The species is distinct from others in the apostome genus Gymnodinioides in that the trophont ciliature has a small group of kinetosomes located to the right of Kinety 9a, and Kinety 1 and 2 are divided. Other apostome morphologies are described from many decapod crustaceans from St. Andrew Bay, Florida, including Gymnodinioides inkystans, Hyalophysa chattoni, and variants of both H. chattoni and G. kozloffi. All of these apostome ciliates are exuviotrophic, found feeding on exuvial fluid within the exoskeleton of the host after ecdysis. The hosts surveyed for this study are the following: Callinectes sapidus, Eurypanopeus depressus, Hippolyte zostericola, Farfantepenaeus spp., Palaemonetes intermedius, Palaemon floridanus, Portunus spp., Tozeuma carolinense, and Sicyonia laevigata, which revealed a number of new host-apostome records.     

Ancient weapons for attack and defense: the pore-forming polypeptides of pathogenic enteric and free-living amoeboid protozoa

Pore-forming polypeptides have been purified from several amoeboid protozoans that are well-known human pathogens. Obligate enteric parasites, such as Entamoeba histolytica, and free-living but potentially highly pathogenic species, such as Naegleria fowleri, contain these cytolytic molecules inside cytoplasmic granules. Comprehensive functional and structural studies have been conducted that include isolation of the proteins from their natural sources, monitoring of their biological activity towards different targets, and molecular cloning of the genes of their precursors. In the case of the most prominent member of the protein family, with respect to protozoans, the three-dimensional structure of amoebapore A was solved recently. The amoebic pore-forming polypeptides can rapidly perforate human cells. The antibacterial activity of amoebapores and of related polypetides from free-living protozoa points to a more vital function of these molecules: inside the digestive vacuoles they combat growth of phagocytosed bacteria which are killed when their cytoplasmic membranes are permeabilized. The concommitant activity of these proteins towards host cells may be due to a coincidental selection for an efficient effector molecule. Nonetheless, several lines of evidence indicate that these factors are involved in pathogenesis of fatal diseases induced by amoeboid protozoa.     

A role for a galactose lectin and its ligands during encystment of Entamoeba

In the life cycle of Entamoeba parasites alternate between the colon-dwelling trophozoite and the infectious cyst forms. The physiologic stimuli that trigger differentiation of trophozoites into cysts remain undefined. On the surface of the human-infecting Entamoeba, parasites express a galactose/N-acetylgatactosamine (gal/galNAc)-binding lectin, which plays demonstrated roles in contact-dependent lysis of target cells and resistance to host complement. Using a reptilian parasite, Entamoeba invadens, to study cyst formation in vitro, we found that efficient encystation was dependent on the presence of gal-terminated ligands in the induction medium. Precise concentration ranges of several gal-terminated ligands, such as asialofetuin, gal-bovine serum albumin (gal-BSA), and mucin, functioned in encystation medium to stimulate differentiation. Greater than 10 mM levels of free gal inhibited the amoeba aggregation that precedes encystation and prevented formation of mature cysts. Inhibitory levels of gal also prevented the up-regulation of genes which normally occurs at 24 h of encystation. The surface of Entamoeba invadens was found to express a gal lectin which has a heterodimeric structure similar to that of Entamoeba histolytica. The 30 kDa light subunit (LGL) of the E. invadens lectin is similar in overall size and sequence to the LGL of E. histolytica. The heavy subunits, however, differ in size, have an identical spacing of cysteines in their extracellular domains, and have highly conserved C-terminal transmembrane and cytoplasmic domains. These results suggest a new role for the Entamoeba gal lectins in monitoring the concentrations of gal ligands in the colon and contributing to stimuli that induce encystment.     

Impact of solar radiation in disinfecting drinking water contaminated with Giardia duodenalis and Entamoeba histolytica/dispar at a point-of-use water treatment

Aims:  To determine the impact of natural sunlight in disinfecting water contaminated with cysts of Giardia duodenalis and Entamoeba histolytica/dispar using plastic containers.
Methods and Results:  Known quantities of Giardia duodenalis and Entamoeba histolytica/dispar cysts in sterile water were exposed to the sun. Containers were made of polyethylene terephthalate, eight painted black on one side, one not painted and another cut open at the top and the last was a high density polypropylene container. Viability testing was performed using vital and fluorescent dyes. The same assays were conducted under cloudy conditions. Thermal control tests were also performed using heat without ultra violet light from the sun. Results show that 99·9% of parasites was inactivated when water temperatures reached 56°C after sunlight exposure.
Conclusion:  Both solar radiation and heat produced by the sun have a synergistic effect in killing cysts of Giardia duodenalis and Entamoeba histolytica/dispar when temperatures rise above 50°C, with complete death at 56°C, using painted 2-l PET containers.
Significance and Impact of the Study:  Solar disinfection system using PET containers painted black on one side can be used to disinfect water against Giardia duodenalis and Entamoeba histolytica/dispar using natural sunlight.     

Comparison of the effect of hormones on the hormone synthesis of Tetrahymena in medium or salt solution

Tetrahymena pyriformis was maintained in TYM (tryptone‐yeast medium) as well as in Losina salt solution. One hour treatment of 10^?15 M histamine, serotonin or insulin was given before the histamine, serotonin, triiodothyronine and adrenocorticotropin contents of the cells were measured by flow cytometry after immunocytochemical staining. Maintenance in salt solution increased the hormone level in the cells, and use of the treatment hormone treatments further increased the endogenous hormone content relative to that in medium. The cells in salt mimic better the natural conditions, which means that the effects of hormones under more natural conditions are expressed to a greater extent than the exogenously given hormones in TYM typically used under laboratory conditions. Intercellular hormonal communication between the cells of a Tetrahymena population might assist in the survival of the individual cells.