Showing papers in "Journal of Eukaryotic Microbiology in 1995"
TL;DR: Phylogenetic analysis of the small subunit ribosomal DNA of a broad range of representative microsporidia including five species from humans, reveals that human microspora are polyphyletic in origin and it is suggested that S. intestinalis be designated Encephalitozoon intestinalis and N. corneum be designated N. cuniculi.
Abstract: Phylogenetic analysis of the small subunit ribosomal DNA of a broad range of representative microsporidia including five species from humans (Enterocytozoon bieneusi, Nosema corneum, Septata intestinalis, Encephalitozoon hellem and Encephalitozoon cuniculi), reveals that human microsporidia are polyphyletic in origin. Septata intestinalis and E. hellem are very similar to the mammalian parasite E. cuniculi. Based on the results of our phylogenetic analysis, we suggest that S. intestinalis be designated Encephalitozoon intestinalis. Furthermore, analysis of our data indicates that N. corneum is much more closely related to the insect parasite Endoreticulatus schubergi than it is to other Nosema species. This finding is supported by recent studies which have shown a similarity between E. schubergi and N. corneum based on the origin and development of the parasitophorous vacuole. Thus these opportunistic microsporidian parasites can originate from hosts closely or distantly related to humans. Finally, the phylogeny based on small subunit ribosomal DNA sequences is highly inconsistent with traditional classifications based on morphological characters. Many ofthe important morphological characters (diplokaryon, sporophorous vesicle, and meiosis) appear to have multiple origins.
205 citations
TL;DR: Bradyzoite development in vitro was confirmed by immuno‐electron microscopy and immunofluorescence in human fibroblasts infected with ME49 strain T. gondii and it is possible that the induction of an acute phase response in the host cell may be important for bradyzoites differentiation.
Abstract: Toxoplasma gondii is a ubiquitous apicomplexan parasite and a major opportunistic pathogen under AIDS-induced conditions, where it causes encephalitis when the bradyzoite (cyst) stage is reactivated. A bradyzoite-specific Mab, 74.1.8, reacting with a 28 kDa antigen, was used to study bradyzoite development in vitro by immuno-electron microscopy and immunofluorescence in human fibroblasts infected with ME49 strain T. gondii. Bradyzoites were detected in tissue culture within 3 days of infection. Free floating cyst-like structures were also identified. Western blotting demonstrated the expression of bradyzoite antigens in these free-floating cysts as well as in the monolayer. Bradyzoite development was increased by using media adjusted to pH 6.8 or 8.2. The addition of gamma-interferon at day 3 of culture while decreasing the total number of cysts formed prevented tachyzoite overgrowth and enabled study of in vitro bradyzoites for up to 25 days. The addition of IL-6 increased the number of cysts released into the medium and increased the number of cysts formed at pH 7.2. Confirmation of bradyzoite development in vitro was provided by electron microscopy. It is possible that the induction of an acute phase response in the host cell may be important for bradyzoite differentiation. This system should allow further studies on the effect of various agents on the development of bradyzoites.
162 citations
TL;DR: The results provide further evidence for the polyphyly of the Rhizopoda and support the creation of a new amoeboid lineage which includes the Euglyphina and the chlorarachniophyte algae; taxa with tubular mitochondrial cristae and filose or reticulate pseudopodia.
Abstract: The Rhizopoda comprise a diverse assemblage of protists which depend on lobose or filose pseudopodia for locomotion. The biochemical and morphological diversity of rhizopods has led to an uncertain taxonomy. Ribosomal RNA sequence comparisons offer a measure of evolutionary relatedness that is independent of morphology and has been used to demonstrate a polyphyletic origin of the Lobosea. We sequenced complete small subunit ribosomal RNA coding regions from the filose amoebae, Euglypha rotunda and Paulinella chromatophora (Euglyphina) to position these taxa in the eukaryote phylogeny. The neighbor-joining analyses show that E. rotunda and P. chromatophora share a monophyletic origin and are not closely related to any lobose amoebae in our analyses. Instead, the Euglyphina form a robust sister group to the Chlorarachniophyta. These results provide further evidence for the polyphyly of the Rhizopoda and support the creation of a new amoeboid lineage which includes the Euglyphina and the chlorarachniophyte algae; taxa with tubular mitochondrial cristae and filose or reticulate pseudopodia.
147 citations
TL;DR: A casein‐free medium is developed, YI‐S, consisting of a nutrient broth, vitamin mixture and serum, which is recommended as a replacement for the casein-dependent medium TYi‐S‐33, currently the most widely used for axenic culture of Entamoeba histolytica and other lumen‐dwellers.
Abstract: Pancreatic digests of casein are major ingredients of media used in the axenic cultivation of lumen-dwelling parasitic protozoa, especially Entamoeba, Giardia, and trichomonads. The digest used almost exclusively in the development of these media, Medo-Peptone (Trypticase BBL), has not been available since 1981. Moreover, none of dozens of similar type digests tested since then in our laboratory has proved equal to Medo-Peptone, and in the last two years it has become increasingly difficult to obtain new batches which will support even modest growth of Entamoeba histolytica. In response to this problem we have developed a casein-free medium, YI-S, consisting of a nutrient broth, vitamin mixture and serum. We recommend it as a replacement for the casein-dependent medium TYI-S-33, currently the most widely used for axenic culture of Entamoeba histolytica and other lumen-dwellers.
120 citations
TL;DR: Molecular trees inferred by distance, parsimony and likelihood techniques consistently show the Devescovinidae and Calonymphidae are the earliest diverging lineages within the Trichomonadida, however bootstrap values do not strongly support a particular branching order.
Abstract: Small subunit (16S-like) ribosomal RNA sequences were obtained from representatives of all four families constituting the order Trichomonadida. Comparative sequence analysis revealed that the Trichomonadida are a monophyletic lineage and a deep branch of the eukaryotic tree. Relative to the early divergent eukaryotic assemblages the branching pattern within the Trichomonadida is very shallow. This pattern suggests the Trichomonadida radiated recently, perhaps in conjunction with their animal hosts. From a morphological perspective the Devescovinidae and Calonymphidae are considered more derived than the Monocercomonadidae and Trichomonadidae. Molecular trees inferred by distance, parsimony and likelihood techniques consistently show the Devescovinidae and Calonymphidae are the earliest diverging lineages within the Trichomonadida, however bootstrap values do not strongly support a particular branching order. In an analysis of all known 16S-like ribosomal RNA sequences, the Trichomonadida share most recent common ancestry with unidentified protists from the hindgut of the termite Reticulitermes flavipes. The position of two putative free-living trichomonads in the tree is indicative of derivation from symbionts rather than direct descent from some free-living ancestral trichomonad.
117 citations
TL;DR: Comparison of N. corneum with established genera revealed that there were none with the same combination of characters and it is proposed that Nosema corneum be placed in a new genus as Vittaforma corneae n.
Abstract: A new genus, Vittaforma n.g. is proposed for the human microsporidium Nosema corneum Shadduck, Meccoli, Davis & Font, 1990, based on the ultrastructure of developmental stages in the liver of experimentally infected athymic mice. The diplokaryotic arrangement of the nuclei is the only character that conforms with the description of the genus Nosema. Sporogony is polysporoblastic, sporonts are ribbon-shaped, constricting to give rise to linear arrays of sporoblasts and each parasite is enveloped by a complete cisterna of host endoplasmic reticulum. Comparison of N. corneum, with established genera revealed that there were none with the same combination of characters. Consequently it is proposed that Nosema corneum be placed in a new genus as Vittaforma corneae n. comb.
109 citations
TL;DR: It is shown that naked amoebae and flagellates are numerically the dominant interstitial fauna in sediments of the Clyde Sea Area and clearly need to be considered in future models of benthic carbon flow.
Abstract: . The abundances of benthic naked amoebae in the sediments of the Clyde Sea Area, Scotland, were studied throughout 1991. Four sites differing in sediment grain size and organic carbon content were chosen for study. Amoebae were enumerated by enrichment cultivation methods and found to be numerically important, attaining densities of up to 14,883 amoebae/cm3 on one occasion. They were most abundant, and temporally stable, in finer sediments where they averaged 2,224 cells/cm3 and lower and more variable in the sandy sediments, averaging 874 cells/cm3 throughout the year. In general, amoebae were most abundant in the surface sediment layers. Around 70 different morphotypes were recorded, and 61% of all amoebae counted were less than 10 μm in length. This is the first detailed quantitative study of benthic marine gymanamoebae and shows that naked amoebae and flagellates are numerically the dominant interstitial fauna in sediments of this area. Moreover, the gymnamoebae comprised the largest proportion of total protozoan biomass (excluding foraminiferans) and clearly need to be considered in future models of benthic carbon flow.
95 citations
TL;DR: The appearance of large multivesicular bodies, especially after ketoconazole treatment, increased amount of lipid inclusions as well as numerous, polymorphic volutin granules, particularly in terbinafine‐treated cells may indicate the existence of an unusual autophagic process in cells treated with ergosterol biosynthesis inhibitors.
Abstract: The antiproliferative effects and ultrastructural alterations induced in vitro by two antifungal compounds, the azole ketoconazole and the allylamine terbinafine on Leishmania amazonensis are reported. Promastigotes treatment with ketoconazole and terbinafine induced growth arrest and cell lysis in 72 hours. Combination of the two agents produced additive effects on promastigote axenic growth and synergistic effects on intracellular amastigote proliferation. The amastigotes, either axenically grown or infecting murine macrophages, were about 100-fold more sensitive to the drugs. These compounds induced the appearance of large multivesicular bodies, especially after ketoconazole treatment, increased amount of lipid inclusions as well as numerous, polymorphic volutin granules, particularly in terbinafine-treated cells. Multivesicular bodies were observed in close apposition with organelles such as mitochondria, which also showed alterations in the distribution and appearance of cristae, and the formation of paracrystalline arrays within the matrix. Some cells presented large portions of cytoplasm wrapped by endoplasmic reticulum and many parasites also presented myelin-like endoplasmic reticulum profiles. Such alterations together with the strong acid phosphatase activity observed in the multivesicular bodies and volutin granules may indicate the existence of an unusual autophagic process in cells treated with ergosterol biosynthesis inhibitors.
87 citations
TL;DR: A review of variability of ciliation and nuclei among the 14 genera suggests that lines of evolution may have involved both the loss of cirri and nuclear simplification in the order Euplotida.
Abstract: The order Euplotida represents a monophyletic order of five families of hypotrich ciliates united by morphology, stomatogenesis, ultrastructure, cyst structure, and behavior. A review of variability of ciliation and nuclei among the 14 genera suggests that lines of evolution may have involved both the loss of cirri and nuclear simplification. We present a binary key to genera in the families Aspidiscidae (Aspidisca and Euplotaspis), Certesiidae n. fam. (Certesia), Gastrocirrhidae (Cytharoides, Euplotidium, and Gastrocirrhus), Uronychiidae (Diophryopsis, Diophrys, Paradiophrys, and Uronychia), and Euplotidae. The latter family contains species formerly in the genus Euplotes. Based primarily on cortical structure, endosymbionts, data from morphometric analysis, and ecology, we recognize four different groups. The first group of species remains in Euplotes with Euplotes charon as type. We place a second group of species into the genus Moneuplotes Jankowski 1979 with Moneuplotes vannus (Miller, 1786) as type. We erect two new genera: Euplotoides n. g. and Euplotopsis n. g. with Euplotoides patella (Miller, 1773) n. comb. and Euplotopsis affinis (Dujardin, 1841) n. comb. as type species respectively. We discuss possible phylogenetic relationships within the order.
84 citations
TL;DR: The historical burden placed on the genus Haemogregarina Danilewsky, 1885 as a repository for poorly known and inadequately described species is partially relieved through taxonomic revisions involving the genera Haemogenarina, and Desseria n.
Abstract: . The phylogenetic relationships of taxa representative of blood parasitic adeleids were investigated in a cladistic analysis. Two phylogenetic analyses were performed. Monophyly of species of Haemogregarina (sensu lato) of some marine fishes with species of Haemogregarina (sensu stricto) was not supported in either analysis. A new genus, Desseria n. g., was created to accommodate these species. The historical burden placed on the genus Haemogregarina Danilewsky, 1885 as a repository for poorly known and inadequately described species is partially relieved through taxonomic revisions involving the genera Haemogregarina, and Desseria n. g.
83 citations
TL;DR: DNA sequences of a portion of the 5‐enolpyruvyl shikimate phosphate synthase domain of the arom gene, encoding the pentafunctional AROM protein, were determined from isolates of Pneumocystis carinii from five mammalian host species.
Abstract: The DNA sequences of a portion of the 5-enolpyruvyl shikimate phosphate synthase domain of the arom gene, encoding the pentafunctional AROM protein, were determined from isolates of Pneumocystis carinii from five mammalian host species (rat, human, ferret, rabbit and mouse). High levels of genetic divergence were found among P. carinii derived from different host species, 7-22% at the DNA sequence level, and 7-26% at the derived amino acid sequence level. Two separate and distinct sequences were isolated from infected ferret lungs. Low levels of divergence were seen in human-derived organisms.
TL;DR: Phylogenetic reconstruction based on nonconservative amino acid differences with maximum parsimony and distance based methods showed that the T. vaginalis enzyme is the most divergent of all eukaryotic malic enzymes, indicating its long independent evolutionary history.
Abstract: The complete nucleotide sequence has been established for two genes (maeA and maeB) coding for different subunits of the hydrogenosomal malic enzyme [malate dehydrogenase (decarboxylating) EC 1.1.1.39] of Trichomonas vaginalis. Two further genes (maeC and maeD) of this enzyme have been partially sequenced. The complete open reading frames code for polypeptides of 567 amino acids in length. These two open reading frames are similar with less than 12 percent pairwise nucleotide differences and less than 9 percent pairwise amino acid differences. The open reading frames of the two partially sequenced genes correspond to the amino-terminal part of the polypeptides coded and are similar to the corresponding parts of the completely sequenced ones. The deduced translation products of the two complete genes differ in their calculated pI values by 1.5 pH unit. The genes code for polypeptides which contain 12 or 11 amino-terminal amino-acyl residues not present in the proteins isolated from the cell. Other hydrogenosomal enzymes also have similar amino-terminal extensions which probably play a role in organellar targeting and translocation of the newly synthesized polypeptides. A comparison of 19 related enzymes from bacteria and eukaryotes with the maeA product revealed 34-45 percent amino acid identity. Phylogenetic reconstruction based on nonconservative amino acid differences with maximum parsimony (phylogenetic analysis using parsimony, PAUP) and distance based (neighbor-joining, NJ) methods showed that the T. vaginalis enzyme is the most divergent of all eukaryotic malic enzymes, indicating its long independent evolutionary history.
TL;DR: An intronless gene encoding a protein of 674 amino acid residues with a molecular mass of 73,403 Da showing homology to the cytoplasmic form of the 70 kDa heat shock proteins has been cloned and sequenced from the intestinal pathogen Cryptosporidium parvum.
Abstract: An intronless gene encoding a protein of 674 amino acid residues with a molecular mass of 73,403 Da showing homology to the cytoplasmic form of the 70 kDa heat shock proteins has been cloned and sequenced from the intestinal pathogen Cryptosporidium parvum. Monospecific polyclonal antibodies obtained to recombinant protein recognized a single band with an approximate molecular mass of 70 kDa on a Western blot of C. parvum proteins, as well as the 70 kDa heat shock protein from bovine brain. Southern blot analysis suggested the gene was single copy in the C. parvum genome. Eleven perfect repeats of the sequence GGMP were found in the predicted protein near the carboxyl terminus.
TL;DR: The probe hybridized well with H. nelsoni plasmodia and immature spores, but poorly with mature spores, and did not hybridize with oyster tissue, with other common oyster parasites, or with the haplosporidians Haplosporidium louisiana from mud crabs or shipworms.
Abstract: Haplosporidium nelsoni is a pathogen of the eastern oyster, Crassostrea virginica, along the middle Atlantic coast of the U.S. Genomic DNA was extracted from H. nelsoni plasmodia and small subunit (SSU) rDNA was amplified by PCR, cloned and sequenced. The sequence of H. nelsoni SSU rDNA was aligned with that of another haplosporidian, Minchinia teredinis, and with SSU rDNA data of C. virginica and various protists in GenBank. A 21-base oligonucleotide unique to H. nelsoni, designated MSX1347, was commercially synthesized and tested for sensitivity and specificity. In dot blot hybridizations the probe detected 100 pg of cloned H. nelsoni rDNA and the presence of H. nelsoni in 1 microgram of genomic DNA from an infected oyster. It did not hybridize with 1 microgram of genomic DNA from uninfected C. virginica or with cloned SSU rDNA of M. teredinis. The probe was further tested for specificity with in situ hybridizations on AFA-fixed, paraffin-embedded tissue sections. The probe hybridized well with H. nelsoni plasmodia and immature spores, but poorly with mature spores. The probe did not hybridize with oyster tissue, with other common oyster parasites such as P. marinus or Nematopsis sp., or with the haplosporidians Haplosporidium louisiana from mud crabs (Panopeus spp.), Haplosporidium costale from C. virginica or M. teredinis from shipworms (Teredo spp.).
TL;DR: It is shown that a commercial, tetrazolium‐based cell proliferation assay yields a soluble formazan chromophore upon intracellular reduction by P. marinus at a rate proportional to cell population biovolume.
Abstract: . Because the in vitro cell cycle of the apicomplexan oyster pathogen Perkinsus marinus generates cell populations heterogeneous for size and typified by aggregation, both turbidimetric and counting methods for determining population densities and proliferation rates are inaccurate or cumbersome. We show that a commercial, tetrazolium-based cell proliferation assay yields a soluble formazan chromophore upon intracellular reduction by P. marinus. at a rate proportional to cell population biovolume. Using this assay system, we have 1) defined selected culture system parameters which maximize P. marinus in vitro proliferation, 2) assessed selected chemosensitivities, and 3) standardized the assay system for quantification of densities and doubling times of populations propagated with our optimized system. Growth was supported by four tested base media and was maximized in 1:1 DME/Ham's F-12. Temperatures of 10–40° C permitted growth, which was maximized at 35° C. pH 6.0–8.5 permitted growth, which was maximized at 7.0–7.5. Osmolalities of 340–1,930 mOsm supported growth, which was maximized at 790 mOsm. Serum supplements from 1–10% (v/v) did not enhance log phase growth, but enhanced stationary phase metabolic activity in proportion to concentration. Our isolate (ATCC 50439) has a 13 h log phase doubling time when propagated under optimized conditions: 28° C, 800 mOsm, pH 7.0, 1:1 DME/Ham's F-12 medium, 5% (v/v) FBS. It is tolerant of antibacterial agents at concentrations commonly used in vertebrate tissue culture, but is inhibited by several antimycotics at similar concentrations.
TL;DR: It was concluded that the P. marinus proteases in cell‐free culture supernatants are serine proteases.
Abstract: Analysis of the cell-free supernatants of Perkinsus marinus cultures by sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and silver staining revealed the presence of as many as 17 bands ranging in molecular weight from 239 to 32 kDa These bands were not present in un-inoculated medium Moreover, P marinus produces extracellular proteins that possess proteolytic activities; the cell-free supernatants of P marinus cultures could digest a variety of proteins including gelatin, casein, fibronectin and laminin Oyster plasma was also digested by cell-free culture supernatants The proteolytic activity in cell-free culture supernatants was detected 24 h post-inoculation, while no proteolytic activity could be detected in cell lysates The proteolytic activities were characterized using substrate-impregnated sodium dodecylsulfate-polyacrylamide gels and had approximate molecular weights ranging from 55 to 35 kDa The proteolytic activity of cell-free culture supernatants was inhibited by the serine protease inhibitors phenylmethylsulphonyl fluoride, 3,4-dichloroisocoumarin and soybean trypsin inhibitor In contrast, inhibitors (ie trans-epoxysuccinyll-leucylamido(4-guanidino)-butane, 1, 10-phenanthroline, captopril, ethylenediaminetetracetic acid, pepstatin A or diazoacetyl-DL-norleucine methyl ester) from the other three classes of proteases had no effect It was concluded that the P marinus proteases in cell-free culture supernatants are serine proteases
TL;DR: This study implies that the net biosynthesis of p33, an essential subunit of TAO, is decreased during differentiation from bloodstream to procyclic trypomastigotes.
Abstract: The glycerophosphate oxidase (GPO), the unique terminal oxidase of bloodstream trypanosome (TAO), appears to be functionally similar to the alternative oxidases of some plants and higher fungi. Immunoblotting of mitochondrial proteins of bloodstream trypomastigotes of Trypanosoma brucei with monoclonal or polyclonal antibodies to Sauromatum guttatum (voodoo lily) and Symplocarpus foetidus (skunk cabbage) alternative oxidases respectively revealed two proteins of about 33 kDa (p33) and 68 kDa (p68). These proteins are not present in procyclic trypomastigotes. Electrophoresis under rigorous denaturing conditions indicated p68 to be the dimer of p33. Indirect immunofluorescent studies of bloodstream and procyclic trypomastigotes with monoclonal antibody to plant alternative oxidase also showed the localization of 33 kDa protein in the mitochondria of the bloodstream trypomastigotes. The functional TAO activity could be solubilized efficiently from the mitochondrial membrane of the bloodstream trypomastigotes by 1% NP-40 or 10 mM lauryl maltoside. When fractionated by Superose 12 gel filtration chromatography, p33 was co-purified with the TAO enzymatic activity. The apparent molecular size of the active enzyme complex was found to be 160 kDa. Gradual disappearance of the 33 kDa protein and the TAO enzymatic activity were well correlated during in vitro differentiation of the bloodstream to procyclic trypomastigotes. This study implies that the net biosynthesis of p33, an essential subunit of TAO, is decreased during differentiation from bloodstream to procyclic trypomastigotes.
TL;DR: New understanding is provided of P. carinii transmission and the effect of possible reservoirs of Pneumocystis in the various species, as well as the sequence differences of the P.Carinii TS gene appeared as host‐species specific.
Abstract: Pneumocystis carinii is an opportunistic agent found in the lung of various mammals which often causes severe pneumonia in immunocompromised humans, especially in AIDS patients. In the past several years significant additions have been made to the collection of knowledge we have concerning the genetic diversity of P. carinii. These additions provide new understanding of Pneumocystis transmission and the effect of possible reservoirs of Pneumocystis in the various species. In this study, a 400-bp fragment of the thymidylate synthase (TS) gene of P. carinii has been amplified by PCR from 43 parasite isolates obtained from 4 mammalian host species: rat, mouse, rabbit and human. A probe selected from the TS gene sequence of rat-derived P. carinii was hybridized with the amplified products from rat- and mouse-derived P. carinii, but not with rabbit or human P. carinii DNA. Restriction profiles were performed on amplified fragments from all isolates, and the 4 nucleotide sequences of the TS gene fragment amplifed from rat, mouse, rabbit and human P. carinii were determined. Differences were detected in the gene fragment in P. carinii isolates from the 4 host species; however no difference was revealed in P. carinii isolates within a single host species, whatever the host strain or its geographic origin. Thus, the sequence differences of the P. carinii TS gene appeared as host-species specific. A specific probe which recognized all human P. carinii isolates was defined.
TL;DR: Experimental evidence has been gathered to show that the life cycle of the myxozoan gallbladder parasite Zschokkella nova Klokacewa, 1914, which infects the fish Carassius carassius, has a complex life cycle with alternation of two hosts (fish and Oligochaeta) and two developmental phases (myxosporean and actinosporean).
Abstract: Experimental evidence has been gathered to show that the life cycle of the myxozoan gallbladder parasite Zschokkella nova Klokacewa, 1914, which infects the fish Carassius carassius, has a complex life cycle with alternation of two hosts (fish and Oligochaeta) and two developmental phases (myxosporean and actinosporean). The gut epithelium of the oligochaete, Tubifex tubifex, exposed experimentally to Z. nova, obtained from C. carassius, became infected with organisms resembling Actinosporea. The spore structure and cube-like network of the interconnected spores is reminiscent of Siedleckiella silesica Janiszewska, 1952, although the spores are very different in size and number of sporoplasm nuclei. The life cycle of Z. nova resembles that of the whirling disease agent Myxosoma cerebralis described by Wolf and Markiw, which also alternates between fish and oligochaete hosts.
TL;DR: Three species of Entamoeba have been grown in axenic culture for the first time, in two cases, novel methods for adapting the organisms to growth without bacteria were employed.
Abstract: Three species of Entamoeba have been grown in axenic culture for the first time. In two cases, novel methods for adapting the organisms to growth without bacteria were employed. While E. ranarum was axenized by the classic technique of Diamond, from a monoxenic culture with Trypanosoma cruzi as the associate, both E. dispar and E. insolita were first grown in axenic culture medium supplemented with lethally irradiated bacteria. From there, E. insolita was axenized directly, but E. dispar initially required the presence of fixed bacteria. After prolonged culture under this technically axenic but unwieldy culture system, E. dispar was eventually adapted to growth in the absence of added bacteria.
TL;DR: Preliminary studies indicate that a short term in vitro culture of this parasite, Enterocytozoon bieneusi, may be established by inoculating human lung fibroblasts and/or monkey kidney cell cultures with duodenal aspirates and or biopsy from infected patients.
Abstract: The microsporidium, Enterocytozoon bieneusi, causes a severe, debilitating, chronic diarrhea in patients with the acquired immunodeficiency syndrome. Specific diagnosis of intestinal microsporidiosis, especially due to Enterocytozoon, is difficult and there is no known therapy that can completely eradicate this parasite. Preliminary studies indicate that a short term (about 6 months) in vitro culture of this parasite yielding low numbers of spores, may be established by inoculating human lung fibroblasts and/or monkey kidney cell cultures with duodenal aspirates and or biopsy from infected patients. The cultures may subsequently be used for the isolation and molecular analysis of parasite DNA.
TL;DR: The characterization of a number of polypeptide pheromones secreted by Euplotes raikovi and E. octocarinatus has now established that structural relationships of homology usually link these molecules, which is consistent with the genetic basis of the mating type systems evolved by these species.
Abstract: For long, our knowledge of the biology of ciliate pheromones has long relied solely upon the study of the two structurally unrelated "gamones" identified in culture filtrates of a Blepharisma species. However, the characterization of a number of polypeptide pheromones secreted by Euplotes raikovi and E. octocarinatus has now established that structural relationships of homology usually link these molecules, which is consistent with the genetic basis of the mating type systems evolved by these species. In this context, our growing appreciation of the conserved and variable elements of the pheromone architecture should foster progress in the understanding of pheromone-receptor interactions and thus, provide important clues into pheromone mechanisms of action.
TL;DR: It is shown that amoebae are uniquely suited as model cells with which to study these phenomena, including cell motility, nucleocytoplasmic interactions, membrane function, and symbiosis.
Abstract: The large, free-living amoebae have been widely used as model cells for studying a variety of biological phenomena, including cell motility, nucleocytoplasmic interactions, membrane function, and symbiosis. Results of studies by our group on amoebae as moving cells, as material for micrurgical manipulations, and as hosts for intracellular symbionts are summarized here. In particular, our recent studies of the amoeba as a microcosm, in which spontaneously infecting foreign microbes have become integrated as necessary cell components, are described in some detail. These processes have involved an initial microbial infection, mutual adaptation by the host and symbionts, and development of obligatory symbiosis. Evidence is presented to show that symbiont-derived macromolecules are involved in the protection of symbionts from digestion, the symbionts have acquired regulatory elements on their chromosomal genes to enhance production of beneficial gene products, and symbionts apparently utilize host-derived macromolecules to their benefit. These studies involved morphological observations both at light and electron microscopic levels, physiological and genetic studies, production and use of poly- and monoclonal antibodies, and molecular-biological approaches including gene cloning and sequencing. It is shown that amoebae are uniquely suited as model cells with which to study these phenomena.
TL;DR: It is demonstrated that when the linearized plasmid was injected into the macronucleus, the prokaryotic sequence conferred an antibiotic resistance to Paramecium despite codon‐usage differences.
Abstract: We have generated a transformation marker for Paramecium using a Paramecium expression vector (pPXV) and the open reading frame (ORF) of the bacterial antibiotic resistance gene aminoglycoside 3'-phosphotransferase-II (APH-3'-II or neor) from the transposon Tn5. The expression vector contained a small multiple cloning site between the 5' and 3' non-coding regions of the calmodulin gene, and Tetrahymena telomere sequences for the stability of the plasmid in Paramecium. After the neor ORF was inserted, the plasmid was referred to as pPXV-NEO. Delivery of approximately 10-20 picoliters of linearized PXV-NEO at > or = 2000 copies/pl into the macronucleus effected 100% transformation. Southern and Northern blot hybridization showed the presence of neor-specific DNA and RNA, respectively, in all of the transformed clones but not in the untransformed clones. The degree of resistance to G-418, and the concentrations of neor-specific DNA and neor-specific RNA in the clones were proportional to the concentration of the vector injected. We have demonstrated that when the linearized plasmid was injected into the macronucleus, the prokaryotic sequence conferred an antibiotic resistance to Paramecium despite codon-usage differences.
TL;DR: A new species of myxosporean from the gill filaments of the freshwater teleost fish, Acestrorhynchus falcatus collected in the Amazon river is described from light and transmission electron microscope observations.
Abstract: A new species of myxosporean from the gill filaments of the freshwater teleost fish, Acestrorhynchus falcatus collected in the Amazon river is described from light and transmission electron microscope observations. The mature spores (total length 32.3 [30.7-35.1] μm) and all developmental stages were found in the same sporogonic plasmodium. The ellipsoidal spore body consists of 2 unequal shell valves adhering together along the suture lines. Each valve, tapering as a caudal projection, forms a long tail (length 20.5 [18.0-21.7] μm). The tail was surrounded by a homogeneous sheath on its length. The polar capsules measuring 3.1 x 1.2 μm contain 3-4 coils of the polar filament. All surfaces ofthe immature and mature spores were surrounded by a closely adherent homogenous structural sheath, mainly thicker around the tails. The taxonomic affinities of this parasite to other species are discussed.
TL;DR: These two sequences, present in an otherwise conserved region of the 16S rRNA, are “signature” sequences, which divide Giardia isolates into two different groups.
Abstract: . The nucleotide sequence of the 16S rRNA gene, part of the 23S rRNA gene and the spacer DNA region was determined for Giardia duodenalis, obtained from humans in The Netherlands (AMC-4) and Washington State (CM). These rDNA sequences differ from other G. duodenalis isolates (Portland-1 and BRIS/83/HEPU/106) both of which have virtually identical rDNA sequences. The most characteristic feature was found close to the 5’end of the 16S rRNA. The Portland-1 - Bris/83/HEPU/106 type has GCG in position 22–24, while AMC-4 and CM have AUC in this position. These two sequences, present in an otherwise conserved region of the 16S rRNA, are “signature” sequences, which divide Giardia isolates into two different groups.
TL;DR: The study has revealed that the species Encephalitozoon cuniculi is not a homogeneous entity and more closely resembled the canine than the murine isolate.
Abstract: . A species of Encephalitozoon has been isolated from the urine of a patient with the acquired immunodeficiency syndrome and maintained in vitro in Madin Darby Canine Kidney cells. When examined by random amplified polymoprhic DNA polymerase chain reaction the new isolate was found to differ from E. hellem and to have amplified products in common with murine and canine E. cuniculi. However, it more closely resembled the canine than the murine isolate. Sodium dodecylsulphate polyacrylamide gel electrophoresis differentiated between all three isolates of E. cuniculi, with a band at 42–45 kDa present in the murine isolate only, bands at 52 kDa present in the canine and human isolates but not the murine, and a single band at 60 kDa (murine) and 65 kDa (canine) replaced by two bands at 55 and 70 kDa in the human isolate. The 55 kDa and 70 kDa antigens were also revealed as characteristic bands of the human isolate by Western blotting. The study has thus revealed that the species Encephalitozoon cuniculi is not a homogeneous entity.
TL;DR: The serendipitous growth of S. vortens in a culture system with lip tumor tissue, facilitated axenic cultivation in a modified TYM medium (trypticase, yeast extract, maltose), and the flagellate is now routinely maintained in an axenic TYI‐S‐33 medium, and is stabilized in the cryopreserved state.
Abstract: . A new diplomonad flagellate, Spironucleus vortens n. sp., is described from the intestinal lumen of the freshwater angelfish, (Pterophyllum scalare), bred in Florida. Live organisms are pyriform, and measure 12.5–20.5 μm long by 5.0–11.2 um wide. Scanning electron microscopy shows that the trophozoite bears two compound lateral longitudinal ridges, each originating posterior to three emerging anterior flagella, and continuing posteriorly to the emergence of the posterior flagellum. Each ridge comprises a broad central part, surrounded by a peripheral ridge. At the opening of the flagellar pocket, the broader right peripheral ridge crosses to the other side of the body, and then back again. The posterior end of the body bears two papillae. Transmission electron microscopy shows that the compound lateral ridges are supported by microtubules, and bear microfibrillar structures in discrete longitudinal plaques. The serendipitous growth of S. vortens in a culture system with lip tumor tissue, facilitated axenic cultivation in a modified TYM medium (trypticase, yeast extract, maltose). The flagellate is now routinely maintained in an axenic TYI-S-33 medium (trypticase, yeast extract, iron serum), and is stabilized in the cryopreserved state. Spironucleus vortens is an aerotolerant anaerobe that can be cultured at 25° C, 28° C and 30° C.
TL;DR: Electron microscopy demonstrated a peculiar form of adhesion that appears to be specific for trichomonas, in which the basal surface of T. vaginalis formed slender channels through which microvilli and cytoplasmic fragments of epithelial cells were internalized.
Abstract: The in vitro cytopathic effect of Trichomonas vaginalis on epithelial cells was explored through the interaction of trophozoites of the virulent strain GT-10 with MDCK monolayers The interaction was analyzed through electrophysiology, video microscopy, and transmission and scanning electron microscopy Electrical measurements revealed that living parasites produced severe damage to the cell monolayers within 30 min, manifested as a rapid decrease in transepithelial resistance Microscopic observations demonstrated that when placed in contact with epithelial cells, trichomonas formed clumps through interdigitations and transient plasma membrane junctions between adjacent parasites Also, attached trophozoites adopted an ameboid shape The in vitro cytopathic action of T vaginalis on MDCK cells was initially evident by modifications of the plasma membrane, resulting in opening of tight junctions, membrane blebbing, and monolayer disruption After 15 min of interaction the damage was focal, concentrating at sites where parasite clumps adhered to the monolayer At 30 min practically all MDCK cells were dead, whether or not trichomonas were attached to them These events were followed by detachment of lysed cells and complete disruption of the monolayer at 60 min Electron microscopy demonstrated a peculiar form of adhesion that appears to be specific for trichomonas, in which the basal surface of T vaginalis formed slender channels through which microvilli and cytoplasmic fragments of epithelial cells were internalized The same sequence of lytic events was found with the less virulent GT-3 strain However, the time course of cytolysis with GT-3 parasites was much slower, and lysis was limited to areas of attachment of T vaginalis
TL;DR: In this article, the phylogenetic position of the Nassulida (Class Nassophorea) within the Ciliophora was inferred using distance matrix and parsimony methods, which is largely congruent with recent analyses of 23S-like rRNA gene sequences and morphogenetic features.
Abstract: . Using comparisons of complete small subunit rRNA sequences from the ciliated protozoans Furgasonia blochmanni, Obertrumia georgiana, and Pseudomicrothorax dubius we inferred the phylogenetic position of the Nassulida (Class Nassophorea) within the Ciliophora. In distance matrix analyses the Nassulida share a common ancestry with the colpodean ciliate Colpoda inflata. Distance matrix and parsimony methods convincingly demonstrate that the Nassulida plus Colpodida are members of a complex ciliate assemblage that also includes the oligohymenophorans and phyllopharyngeans. These phylogenetic inferences are largely congruent with recent analyses of 23S-like rRNA gene sequences and morphogenetic features. Groups traditionally thought to represent ancestral lineages now appear as highly derived ciliates. In contrast, heterotrichs which were considered to represent a highly evolved group, diverge at the base of the ciliates.