bluuebunny/crossref_metadata_2025 huggingface dataset:
Leishmania, Trypanosoma and Monoxenous Trypanosomatids as Emerging Opportunistic Agents1
Authors: DEDET JEAN‐PIERRE , PRATLONG FRANCINE
Published: July 2005

ABSTRACT. Immunosuppression is associated with the occurrence of a large variety of infections, several of them due to opportunistic protozoa. The parasitic protozoa of the family Trypanosomatidae vary greatly in their importance as potential opportunistic pathogens. African trypanosomiasis is no more common nor severe during AIDS. The situation with Chagas' disease, however, is much different. Although the process is not clearly understood, there appears to be a reactivation of Trypanosoma cruzi infection, which can lead to severe meningoencephalitis. In persons with AIDS, leishmaniasis is often exacerbated, particularly Leishmania infantum, which causes visceral leishmaniasis in southern Europe. Since 1990, 1,616 cases of visceral leishmaniasis/HIV co‐infection have been reported, mainly from southern Europe, and particularly from Spain, southern France, and Italy. The co‐infected patients are primarily young adults and belong to the risk group of intravenous drug users. Isoenzymatic identification of 272 isolates showed 18 different L. infantum zymo‐ demes, of which 10 represent new zymodemes hitherto found only during HIV co‐infection. New foci of co‐infection are emerging in various parts of the world, including Brazil and East Africa. Moreover, since 1995, non‐human monoxenous trypanosomatids have been found in AIDS patients, causing both diffuse cutaneous lesions and visceral infections. In countries where visceral leishmaniasis is endemic, particularly in southern Europe, immunosuppressive treatments for organ transplants or malignant diseases often result either in reactivation of asymptomatic visceral leishmaniasis or in facilitation of new infections.
Protein Kinase Activation and Protein Phosphorylation in Naegleria fowleri Amebae in Response to Normal Human Serum
Authors: CHU DAN‐MY T. , FERGUSON TAMMY J. , MARCIANO‐CABRAL FRANCINE
Published: July 2005

ABSTRACT. Activation of signal transduction pathways in response to serum complement in Naegleria fowleri amebae was investigated. We examined the activation of protein kinases and changes in the phosphorylation state of proteins in N. fowleri stimulated by normal human serum (NHS). To determine differences in phosphorylation of proteins when amebae were exposed to NHS or heat inactivated serum (HIS) lacking complement, amebae were labeled with [32P] orthophosphate. An increase in phosphorylation of relatively low molecular weight proteins was noted in N. fowleri incubated in NHS with a concomitant decrease in phosphory lation of high molecular mass polypeptides. To investigate whether serine/threonine or tyrosine kinases were stimulated by NHS, amebae were treated with protein kinase inhibitors H7, staurosporine or genistein, prior to serum exposure and examined for susceptibility to complement. Treatment with each of these inhibitors resulted in increased complement lysis. Incubation of N. fowleri with genistein specifically inhibited tyrosine phosphorylation of proteins stimulated by NHS. A tyrosine kinase activity assay using exogenous polyGlu‐Tyr substrate demonstrated differential activation of tyrosine kinases in amebae treated with NHS when compared to treatment with HIS. The results suggest that activation of protein kinases and subsequent protein phosphorylation are important in mediating complement resistance in N. fowleri.
Identification of Proteins in Encephalitozoon intestinalis, a Microsporidian Pathogen of Immunocompromised Humans: An Immunoblotting and Immunocytochemical Study
Authors: PRIGNEAU ODILE , ACHBAROU ABDERRAHIM , BOULADOUX NICOLAS , MAZIER DOMINIQUE , DESPORTES‐LIVAGE ISABELLE
Published: July 2005

ABSTRACT. Microsporidia are unicellular and obligate intracellular spore‐forming parasites. The spore inoculates the host cell with its non‐motile infectious content, the sporoplasm, by way of the polar tube—the typical invasive apparatus of the microsporidian spore. Molecules involved in host cell invasion were investigated in Encephalitozoon intestinalis. Mouse polyclonal and monoclonal antibodies were raised against spore proteins and their reactivity was tested by Western‐blotting and immunolocalization techniques, including electron and confocal microscopy. The antibodies thus generated could be divided into two major groups. One group reacted to the surface of the parasite at different developmental stages, mostly presporous stages and mature spores, whereas the other group recognized the polar tube. Of the antibodies reacting to the spore wall, one identified an exospore protein at 125 kDa while all others recognized a major doublet at 55–60 kDa, and minor proteins present at the surface of sporogonic stages and in the endospore. All antibodies recognizing spore wall proteins reacted also to the material forming septa in the parasitophorous vacuole. A major polar tube protein at 60 kDa was identified by another group of antibodies.
First Record of Lagenophrys dennisi (Ciliophora: Peritrichia) on the Exoskeleton of Crayfish Cambarellus patzcuarensis
Authors: MAYÉN‐ESTRADA ROSAURA , ALADRO‐LUBEL MA. ANTONIETA
Published: July 2005

ABSTRACT. Lagenophrys dennisi, a peritrich ciliate, was observed attached to the exoskeleton of the crayfish Cambarellus patzcuarensis in Lake Patzcuaro, Michoacan, Mexico. Lagenophrys dennisi presents a hemispheroidal, suboval or oval lorica in dorsal view, the distinctive lorica aperture consists of a pair of lips highly arched, unthickened, and smooth. Comparison of morphometric characters of the ciliate with previous records is made. Structures such as a “V” ‐shaped lorica suture, collar ridges, and myoneme are proposed for species identification. An anterior crescentic thickening on the dorsal surface of the lorica was observed under the scanning electron microscope. Lagenophryids were associated with 11 of 13 body parts with antennules and rostrum showing the highest prevalence. Lagenophrys dennisi was also found attached to submerged glass slides. This study represents the first record of L. dennisi on C. patzcuarensis and the first record of its presence in Mexico.
Comparison of Growth Efficiencies of Protozoa Growing on Bacteria Deposited on Surfaces and in Suspension
Authors: ZUBKOV MIKHAIL V. , SLEIGH MICHAEL A.
Published: July 2005

ABSTRACT. Bacteria were deposited in tubes as compact pellets by centrifuging suspensions of cultured Vibrio at stationary phase. Numbers and protein biomass of flagellates added to these tubes and of the Vibrio, were followed and compared with the growth of the same and other protists on identical, uncentrifuged Vibrio. The flagellates Bodo saliens and Caecitellus parvulus, which could not be seen to multiply in tubes of suspended bacteria, grazed deposited bacteria actively as did the more versatile flagellate Cafeteria roenbergensis. The growth of these flagellates and their consumption of deposited bacteria were very similar to those of the flagellate Pteridomonas danica or the ciliate Uronema marinum fed with suspended bacteria, although deposit‐feeders grew more slowly. Gross growth efficiencies (30–60%) of deposit‐feeding flagellates were similar to those of the suspension‐feeding protists. Caecitellus consumed 55 Vibrio to produce one flagellate, while 4,500 Vibrio were consumed to produce one Uronema. Surface‐feeding flagellates are shown to be efficient bacterivores, capable of restricting the numbers of bacteria deposited on surfaces just as other protozoa control numbers of suspended bacteria.
Molecular Phytogeny of Parabasalids Based on Small Subunit rRNA Sequences, with Emphasis on the Trichomonadinae Subfamily
Authors: DELGADO‐VISCOGLIOSI PILAR , VISCOGLIOSI ERIC , GERBOD DELPHINE , KULDA JAROSLAV , SOGIN MITCHELL L. , EDGCOMB VIRGINIA P.
Published: July 2005

ABSTRACT. We determined small subunit ribosomal DNA sequences from three parabasalid species, Trichomitus batrachorum strain R105, Tetratrichomonas gallinarum, and Pentatrichomonas hominis belonging to the Trichomonadinae subfamily. Unrooted molecular phylogenetic trees inferred by distance, parsimony, and likelihood methods reveal four discrete clades among the parabasalids. The Trichomonadinae form a robust monophyletic group. Within this subfamily T. gallinarum is closely related to Trichomonas species as supported by morphological data, with P. hominis and Pseudotrypanosoma giganteum occupying basal positions. Our analysis does not place T. batrachorum within the Trichomonadinae. Trichomitus batrachorum (strains R105 and BUB) and Hypotrichomonas acosta form a well‐separated cluster, suggesting the genus Trichomitus is polyphyletic. The emergence of T. batrachorum precedes the Trichomonadinae‐Tritrichomonadinae dichotomy, emphasizing its pivotal evolutionary position among the Trichomonadidae. A third cluster unites the Devescovinidae and the Calonymphidae. The fourth clade contains the three hypermastigid sequences from the genus Trichonympha, which exhibit the earliest emergence among the parabasalids. The addition of these three new parabasalid species did not however resolve ambiguities regarding the relative branching order of the parabasalid clades. The phylogenetic positions of Tritrichomonas foetus, Monocercomonas sp., Dientamoeba fragilis, and the unidentified Reticulitermes flavipes gut symbiont 1 remain unclear.
Oligonucleotide Probes for the Identification of Three Algal Groups by Dot Blot and Fluorescent Whole‐Cell Hybridization
Authors: SIMON NATHALIE , CAMPBELL LISA , ÖRNOLFSDOTTIR ERLA , GROBEN RENÉ , GUILLOU LAURE , LANGE MARTIN , MEDLIN LINDA K.
Published: July 2005

ABSTRACT. Photosynthetic pico‐ and nanoplankton dominate phytoplankton biomass and primary production in the oligotrophic open ocean. Species composition, community structure, and dynamics of the eukaryotic components of these size classes are poorly known primarily because of the difficulties associated with their preservation and identification. Molecular techniques utilizing 18S rRNA sequences offer a number of new and rapid means of identifying the picoplankton. From the available 18S rRNA sequence data for the algae, we designed new group‐specific oligonucleotide probes for the division Chlorophyta, the division Haptophyta, and the class Pelagophyceae (division Heterokonta). Dot blot hybridization with polymerase chain reaction amplified target rDNA and whole‐cell hybridization assays with fluorescence microscopy and flow cytometry were used to demonstrate probe specificity. Hybridization results with representatives from seven algal classes supported the phylogenetic affinities of the cells. Such group‐ or taxon‐specific probes will be useful in examining community structure, for identifying new algal isolates, and for in situ detection of these three groups, which are thought to be the dominant algal taxa in the oligotrophic regions of the ocean.
Molecular Characterization of Myxozoan Parasites from Lake Sasajewun, Algonquin Park, Ontario, by Riboprinting
Authors: XIAO CHONGXIE , DESSER SHERWIN S.
Published: July 2005

ABSTRACT. The small subunit‐rRNA genes of 18 myxozoans from Lake Sasajewun, Algonquin Park were amplified and digested with restriction endonucleases for riboprinting analysis. Identical riboprints were not found between the myxosporeans and the actino‐sporeans. The distinct riboprinting patterns observed among these myxozoans indicate considerable genetic diversity within this group. Identical riboprints were found between Myxobolus pendula and Myxobolus pellicides, and between triactinomyxon ‘C’ and Triactinomyxon ignotum. Parsimony analysis of the riboprints demonstrated that neither the myxosporeans nor the actinosporeans formed a monophyletic group. Some species of Myxobolus are more closely related to forms of triactinomyxon, echinactinomyxon or raabeia than to other Myxobolus species. These results are consistent with the two‐host life cycle hypothesis of myxozoans that myxosporeans and actinosporeans are alternating stages of the same organisms.
Cryptosporidium andersoni n. sp. (Apicomplexa: Cryptosporiidae) from Cattle, Bos taurus
Authors: LINDSAY DAVID S. , UPTON STEVE J. , OWENS DOUGLAS S. , MORGAN UNA M. , MEAD JAN R. , BLAGBURN BYRON L.
Published: July 2005

ABSTRACT. A new species of Cryptosporidium is described from the feces of domestic cattle, Bos taurus. Oocysts are structurally similar to those of Cryptosporidium muris described from mice but are larger than those of Cryptosporidium parvum. Oocysts of the new species are ellipsoidal, lack sporocysts, and measure 7.4 ± 5.5 μm (range, 6.0–8.1 by 5.0–6.5 μm). The length to width ratio is 1.35 (range, 1.07–1.50). The colorless oocyst wall is < 1 μm thick, lacks a micropyle, and possesses a longitudinal suture at one pole. A polar granule is absent, whereas an oocyst residuum is present. Oocysts were passed fully sporulated and are not infectious to outbred, inbred immunocompetent or immunodeficient mice, chickens or goats. Recent molecular analyses of the rDNA 18S and ITSI regions and heat‐shock protein 70 (HSP‐70) genes demonstrate this species to be distinct from C. muris infecting rodents. Based on transmission studies and molecular data, we consider the large form of Cryptosporidium infecting the abomasum of cattle to be a new species and have proposed the name Cryptosporidium andersoni n. sp. for this parasite.
conZA8 Encodes an Abundant Protein Targeted to the Developing Macronucleus in Euplotes crassus
Authors: JACOBS MARY ELLEN , LING ZHONG , KLOBUTCHER LAWRENCE A.
Published: July 2005

ABSTRACT. During macronuclear development in the ciliate Euplotes crassus, micronuclear‐derived chromosomes undergo a series of rearrangements that include polytenization, DNA splicing, chromosome fragmentation, and telomere addition and processing. Although cis‐acting signals that may function in the regulation of these events have been characterized, the proteins that mediate these events have not yet been identified. To identify development‐specific factors that may be involved in DNA rearrangement, we previously isolated clones of a number of genes that are expressed only during early macronuclear development. Here, we report the genomic and cDNA sequences of one of these genes, conZA8. The analysis indicates that the conZA8 gene encodes a novel, 468‐amino acid, prolinerich protein. Antibodies were raised against both a recombinant form of the conZA8 protein and an internal peptide. Immunoblotting and immunofluorescence analyses indicated that the conZA8 protein is highly abundant, expressed only during the polytene chromosome stage of macronuclear development, and localized to the developing macronucleus. Possible functions of the conZA8 protein are discussed.
1‐O‐Alkyl‐2‐Acetyl‐sn‐Glycero‐3‐Phosphate: Phosphohydrolase Activity in Tetrahymena pyriformis
Authors: TELLIS CONSTANTINOS , LEKKA MARILENA E.
Published: July 2005

ABSTRACT. Within the frame of the de novo formation of Platelet‐Activating Factor in Tetrahymena, the occurrence as well as the properties of a lipid phosphate phosphohydrolase activity catalyzing the dephosphorylation of l‐O‐alkyl‐2‐acetyl‐sn‐glycero‐3‐phosphate was investigated. The activity was distributed in all the membrane fractions of the cell and in the cytosol. It showed preference for acyl‐acetyl‐sn‐glycero‐phosphate as well, and at a much lower level, for dipalmitoyl‐glycero‐phosphate. Mg2+ and Ca2+ caused a dose‐dependent inhibition, while F, EDTA and EGTA had no effect. The enzymic activity was linear for at least up to 60 min incubation time and up to 150 μg protein. Microsomal activity exhibited two optimal pH areas, around 7.0 and 9.0, while mitochondrial activity showed one peak, at pH 7.0. Acyl‐GP, acyl‐acetyl‐GP and alkyl‐GP could replace alkyl‐acetyl‐GP in significant rates, while dipalmitoyl‐GP, β‐GP, fructose‐6‐GP, p‐nitrophenylphosphate, creatine phosphate or ATP had no effect. Side phospholipase A2 and C activities were also detected. Taking into account the presence of PAF and alkylacetylglycerol in the protozoan as well as the presence of a dithiothreitol‐ insensitive CDP‐choline: cholinephosphotransferase activity that converts alkylacetylglycerol to PAF, we suggest that the present phosphohydrolase activity may be involved in the de novo production of PAF within Tetrahymena.
Centrin Protein and Genes in Trichomonas vaginalis and Close Relatives
Authors: BRUGEROLLE GUY , BRICHEUX GENEVIEVE , COFFE GERARD
Published: July 2005

ABSTRACT. Anti‐centrin monoclonal antibodies 20H5 and 11B2 produced against Chlamydomonas centrin decorated the group of basal bodies as well as very closely attached structures in all trichomonads studied and in the devescovinids Foaina and Devescovina. Moreover, these antibodies decorated the undulating membrane in Trichomonas vaginalis, Trichomitus batrachorum, and Tritrichomonas foetus, and the cresta in Foaina. Centrin was not demonstrated in the dividing spindle and paradesmosis. Immunogold labeling, both in pre‐ and post‐embedding, confirmed that centrin is associated with the basal body cylinder and is a component of the nine anchoring arms between the terminal plate of flagellar bases and the plasma‐membrane. Centrin is also associated with the hook‐shaped fibers attached to basal bodies (F1, F3), the X‐fiber, and along sigmoid fibers (F2) at the pelta‐axostyle junction, which is the microtubule organizing center for pelta‐axostyle microtubules. There was no labeling on the striated costa and parabasal fibers nor on microtubular pelta‐axostyle, but the fibrous structure inside the undulating membrane was labeled in T. vaginalis. Two proteins of 22–20 kDa corresponding to the centrin molecular mass were recognized by immunoblotting using these antibodies in the three trichomonad species examined.

By screening a T. vaginalis cDNA library with 20H5 antibody, two genes encoding identical protein sequences were found. The sequence comprises the 4 typical EF‐hand Ca++ ‐binding domains present in every known centrin. Trichomonad centrin is closer to the green algal cluster (70% identity) than to the yeast Cdc31 cluster (55% identity) or the Alveolata cluster (46% identity).
Macronuclear Development in Conjugants of Tetrahymena thermophila, which Were Artificially Separated at Meiotic Prophase
Authors: KIERSNOWSKA MAURYLA , KACZANOWSKI ANDRZEJ , MORGA JOANNA
Published: July 2005

ABSTRACT. Conjugant pairs of Tetrahymena thermophila were mechanically separated by vigorous pipetting at the early stages of meiotic prophase. The complete sequence of conjugational nuclear events including the appearance of pronuclei, development of the new macronuclei (postzygotic development), and resorption of the old macronuclei was observed in the separated cells, without pronuclear exchange. The pronuclei in the separated cells were recognised by the presence of components of the extranuclear cytoskeleton, which were labelled with anti‐tubulin and anti‐fenestrin antibodies in the same way as in undisturbed conjugants. The apical region of the separated conjugants (the post‐junction area), corresponding to the junction area of conjugants was labelled with anti‐fenestrin antibody and maintained the properties required for the nuclear development. The results of the genetic study were consistent with a hypothesis that cytogamy (pronuclear fusion) was induced in the separated conjugants. Therefore, the lasting cell contact is not necessary for the successful completion of conjugational nuclear events.
Abundance of Terrestrial Gymnamoebae at a Northeastern U. S. Site: A Four‐year Study, Including the El Nino Winter of 1997–1998
Authors: ANDERSON O. ROGER
Published: July 2005

ABSTRACT. The abundance, sizes, and when appropriate, diversity of gymnamoebae were documented at approximately monthly intervals for four years (1995–1998) at a grassy, terrestrial site slightly upslope from a freshwater pond. Soil samples were analyzed for viable gymnamoebae using a standard laboratory culturing protocol. The mean density of gymnamoebae based on the total data set was ca. 1,600/g (s.e. ± 190). Minimum densities of gymnamoebae (156/g) occurred in January 1995, and a maximum for the sampling period (5,838/g) occurred in July 1997, when a rainy period followed an extended period of drought. Among the environmental variables monitored (precipitation, soil moisture, organic content, and temperature) only precipitation correlated significantly with abundance of gymnamoebae (r= 0.34, p = 0.02). During the mild, moist El Niño winter of 1997–1998, a larger than usual number of gymnamoebae was recorded at the site (∼3,800/g) compared to a mean density of ∼900/g for comparable periods in preceding years. The mean sizes were also larger. Since gymnamoebae are increasingly recognized as major members of soil microbial communities enhancing soil fertility through nutrient mineralization, it is important to document environmental variables that influence their abundance and activity in terrestrial ecosystems.
Rotifers Ingest Oocysts of Cryptosporidium parvum
Authors: FAYER RONALD , TROUT JAMES M. , WALSH ELIZABETH , COLE REBECCA
Published: July 2005

ABSTRACT. Six genera of rotifers including Philodina, Monostyla, Epiphanes, Euchlanis, Brachionus, and Asplanchna were exposed to oocysts of Cryptosporidium parvum cleaned of fecal debris. Unstained oocysts and those stained with fluorescein‐conjugated monoclonal antibody were added to suspensions of viable rotifers and were examined by phase‐contrast, differential interference contrast, and fluorescence microscopy. Rotifers of all six genera were observed ingesting oocysts. A maximum of 25 oocysts was observed in the stomachs of Euchlanis and Brachionus. Euchlanis and Epiphanes were observed excreting boluses containing up to eight oocysts. It was not determined whether rotifers digested or otherwise rendered oocysts nonviable.
Use of Micromanipulation and “Feeder Layers” to Clone the Oyster Pathogen Perkinsus marinus
Authors: BUSHEK DAVID , HOLLEY RUSSELL A. , REECE KIMBERLY S.
Published: July 2005

ABSTRACT. Genetic and biochemical characterization of microbes often requires the use of clonal cultures. A method to clone the oyster parasite Perkinsus marinus is described. Individual cells are isolated via micromanipulation and maintained above an actively proliferating “feeder layer” of P. marinus on a 0.45‐μm membrane. Extracellular products released from the proliferating feeder layer can diffuse across the membrane and bathe the isolated cell, stimulating it to proliferate. The method is relatively simple and should be applicable to most protists that can be cultured in the laboratory.
Long Branch Attraction Effects and the Status of “Basal Eukaryotes”: Phylogeny and Structural Analysis of the Ribosomal RNA Gene Cluster of the Free‐living Diplomonad Trepomonas agilis
Authors: MORIN LOÏC
Published: July 2005

ABSTRACT. The three taxa emerging at the base of the eukaryotic ribosomal RNA phylogenetic tree (Diplomonadida, Microspora, and Parabasalia) include a wide array of parasitic species, and some free‐living organisms that appear to be derived from a parasitic ancestry. The basal position of these taxa, which lack mitochondria, has recently been questioned. I sequenced most of the ribosomal RNA gene cluster of the free‐living diplomonad Trepomonas agilis and a secondary structure model was reconstructed for the SSU rRNA. I conducted a RASA matrix analysis to identify, independently from tree reconstruction, putative long branch attraction effects in the data matrix. The results show that each of the basal clades and the euglenozoan clade act, indeed, as long branches and may have been engaged in a process of accelerated rate of evolution. A nucleotide signature analysis was conducted in the conserved regions for positions defining the three great domains of life (Eubacteria, Archea, and Eukaryota). For the three basal taxa, this analysis showed the presence of a significant number of different non‐eukaryotic nucleotides. A precise study of the nature and location of these nucleotides led to conclusions supporting the results of the RASA analysis. Altogether, these findings suggest that the basal placement of these taxa in the SSU ribosomal RNA phylogenetic tree is artifactual, and flawed by long branch attraction effects.
Rumen Ciliated Protozoan Fauna of the Yak (Bos grunniens) in China with the Description of Entodinium monuo n. sp.
Authors: GUIRONG  , SU NA‐RI , XIANG HUA ZHAI , ZHU SE , IMAI SOICHI
Published: July 2005

ABSTRACT. Rumen ciliate species composition was surveyed in domestic yaks kept in Tibet, Sichuan, and Inner Mongolia, China. Twelve genera including 36 species with 18 formae were identified. The species compositions were slightly different among the three areas: yaks in Tibet had the simplest fauna, in contrast, the fauna of yaks in Inner Mongolia were the most abundant and similar to those found in the cattle kept in the same area. This suggests that the rumen ciliate composition of yaks is affected by that of cattle kept together or in proximity. A new species belonging to the genus Entodinium, Entodinium monuo n. sp., was recognized from the yaks in all areas. This new species was common in the yaks but was not detected in the cattle fed near yaks in Inner Mongolia. There was a similar generic ciliate composition in yaks kept in respective areas. Entodinium was the most predominate ciliate (51.9–61.0%) with total ciliate densities estimated as 105/ml per yak.
The Role of Unconventional Myosins in Dictyostelium Endocytosis
Authors: TITUS MARGARET A.
Published: July 2005

Dictyostelium discoideum is a simple eukaryote amenable to detailed molecular studies of the endocytic processes phagocytosis and macropinocytosis. Both the actin cytoskeleton and associated myosin motors are well‐described and a range of mutants arc now available that enable characterization of the role of the cytoskeleton in a range of cellular functions. Molecular genetic studies have uncovered roles for two different classes of Dictyostelium unconventional myosins in endocytosis. The class I myosins contribute to both macropinocytosis and phagocytosis by playing a general role in controlling actin‐dependent manipulations of the actin‐rich cortex. A class VII myosin has been shown to be important for phagocytosis. This brief review summarizes what is known about the role of these different myosins in both fluid and particle uptake in this system.
Is Leishmania (Viannia) peruviana a Distinct Species? A MLEE/RAPD Evolutionary Genetics Answer
Authors: BAÑULS ANNE‐LAURE , DUJARDIN JEAN‐CLAUDE , GUERRINI FRANCOISE , DE DONCKER SIMONNE , JACQUET DIANE , AREVALO JORGE , NOËL SEBASTIEN , LE RAY DOMINIQUE , TIBAYRENC MICHEL
Published: July 2005

A set of 38 Leishmania stocks from the Andean valleys of Peru was characterized by both Multilocus Enzyme Electrophoresis (MLEE) and Random Amplified Polymorphic DNA (RAPD). Data were analyzed in terms of taxonomy and evolutionary genetics. Synapomorphic MLEE and RAPD characters, clear‐cut clustering, and strong agreement between the phylogenies inferred from either MLEE or RAPD supported the view that Leishmania (Viannia) peruviana and Leishmania (Viannia) braziliensis correspond to two closely related, but distinct monophyletic lines (clades) and can therefore be considered as “discrete typing units” (DTUs). The question whether the L. (V.) peruviana DTU deserves species status is dependent upon the desirability of it, in terms of epidemiological and medical relevance. A previous Orthogonal Field Alternating Gel Electrophoresis (OFAGE) analysis of the same L. (V.) peruviana isolates was published by Dujardin et al. (1995b). The data from the different markers (i.e. MLEE, RAPD and OFAGE) were compared by population genetics analysis. RAPD and OFAGE provided divergent results, since RAPD showed a strong linkage disequilibrium whereas OFAGE revealed no apparent departure from panmictic expectation. MLEE showed no linkage disequilibrium. Nevertheless, contrary to OFAGE, this is most probably explainable by the limited variability revealed by this marker in L. (V.) peruviana (statistical type II error). RAPD data were consistent with the hypothesis that the present L. (V.) peruviana sample displays a basically clonal population structure with limited or no genetic exchange. Disagreement between RAPD and OFAGE can be explained either by accumulation of chromosomal rearrangements due to amplification/deletion of repeated sequences, or by pseudo‐recombinational events.
Ultrastructural and Biochemical Characterization of Promastigote and Cystic Forms of Leptomonas wallacei N. Sp. Isolated from the Intestine of its Natural Host Oncopeltus fasciatus (Hemiptera: Lygaeidae)
Authors: ROMEIRO ALEXANDRE , SOLÉ‐CAVA ANTôNIO , SOUSA MARIA A. , DE SOUZA WANDERLEY , ATTIAS MÁRCIA
Published: July 2005

ABSTRACT. Promastigote forms of a trypanosomatid were isolated from the third and fourth ventricles of the midgut and from the hindgut of the phytophagous hemipteran Oncopeltus fasciatus. Some individuals had adhered to its anterior region, close to the flagellar pocket, or to the flagellum up to four rounded aflagellated forms known as straphangers cysts. Scanning electron microscopy revealed that the flagellated forms presented a twisted cell body and a long flagellum, and the cysts, smaller than the parental promastigote, had a nascent flagellum. Transmission electron microscopy showed that promastigotes were typical, while cystic forms were ovoid dense cells devoid of a cyst wall, but presenting a cell coat, a special subpellicular region limited by a membrane unit, and a condensed cytoplasm. The kinetoplast‐DNA fibrils appeared as dense spots and the condensed chromatin was arranged in a labyrinthic structure. Desmosome‐like structures, observed in the region of adhesion of the precystic forms to the parental promastigote, could explain how cysts remain attached to the mother cell during the encystation process. Release of membranes from the surfacc of promastigotes and cyats seems to be correlated with the condensation of the cytoplasm during encystment. Morphological and isozymc analyses indicated that this trypanosomatid belongs to the genus Leptomonas. The molecular karyotype of this isolate was compared with that of a strain of Leptomonas oncopelti obtained from Oncopeltus varicolor by contour‐clamped homogeneous electric field (CHEF) electrophoresis and revealed similar DNA banding patterns between 2,200‐825 Kb, but not in lower hands (825‐225 Kb). This suggested that the isolate from O. fasciafus and that from O. varicolor were not identical. Based on our findings we are describing Leptomonas wallacei n. sp. for our isolate from O. fasciatus.
High Numbers of Naked Amoebae in the Planktonic Waters of a Mangrove Stand in Southern Florida, USA
Authors: ROGERSON ANDREW , GWALTNEY CHRISTINA
Published: July 2005

ABSTRACT. This is the first study to examine the abundance of naked amoebae in the water column of a mangrove stand. A total of 37 different morphotypes was noted and at least 13 of these are probably new species. Over a one‐year sampling interval, amoebae averaged 35,400 cells liter−1 (range 2,000–104,000) by an indirect enrichment cultivation method. Densities in the upper end of this range arc the highest ever reported for any planktonic habitat. Variation between samples was related to the quantity of suspended aggregates (floes) in the water column emphasizing that amoebae are usually floc‐associated. The study also showed that it is essential to disrupt floc material prior to withdrawing sample aliquots for the indirect counting method since several amoebae can occupy the interstices of aggregates. There is concern that indirect enumeration methods that require organisms to be cultured in the laboratory seriously underestimate the true count. A direct counting method using acridine orange staining and epifluorescence microscopy was attempted to assess the possible magnitude of the error associated with indirect counting. While this direct method had limitations, notably the difficulty of unambiguously differentiating between small amoebae and nanoflagellates, the results suggested that the indirect method gave estimates that were close to the true count (within a factor of two). Mangrove waters are rich in heterotrophic protozoa (∼3 × 106 liter1) and while the heterotrophic flagellates are by far the dominant group, naked amoebae outnumber ciliates some 20‐foid. The ecological consequences of high numbers of amoebae, particularly the common small forms less than 10 μm in length, need to be examined for these important coastal sites.
Analysis of the Conserved Cysteine Periodicity of Parameciumbi Variable Surface Antigens
Authors: THAI KWAN Y. , FORNEY JAMES D.
Published: July 2005

ABSTRACT. The major surface antigens expressed by free‐living and parasitic protozoa commonly contain repeating cysteine motifs. Despite the common occurrence of these repeats their functional significance remains largely unexplored. In this paper we investigate the conserved cysteine repeats within the variable surface antigens of Paramecium tetraurelia. We show that deletion of 2 entire repeating units or portions of repeats near the N‐terminus does not prevent expression of the A51 variable surface antigen. Alteration of a single cysteine to serine residue also has no effect on A51 expression. In contrast, deletions near the C‐terminus of the protein have identified a small segment within the repeats that is required for expression on the surface. The required region contains a number of conserved amino acid residues, yet site‐directed mutagenesis of two residues (serine and threonine to alanine) did not prevent expression. These studies demonstrate the feasibility of using deletion analysis to identify regions critical for the expression of cysteine‐rich surface antigens. The relationship of these results to the structure and expression of cysteine‐rich surface proteins in other protozoa is discussed.
Phylogenetic Identification of Hypermastigotes, Pseudotrichonympha, Spirotrichonympha, Holomastigotoides, and Parabasalian Symbionts in the Hindgut of Termites
Authors: OHKUMA MORIYA , OHTOKO KUNIYO , IIDA TOSHIYA , TOKURA MITSUNORI , MORIYA SHIGEHARU , USAMI RON , HORIKOSHI KOKI , KUDO TOSHIAKI
Published: July 2005

ABSTRACT. The phylogenetic diversity of parabasalian flagellates was examined based on the sequences of small subunit ribosomal RNA genes amplified directly from the mixed population of flagellates in the hindgut of lower termites. In total, 33 representative sequences of parabasalids were recovered from eight termite species. Fluorescent‐labeled oligonucleotide probes specific for certain sequences were designed and used for the in situ identification of parabasalian species by whole‐cell hybridization. The hypermastigotes, Pseudotrichonympha grassii, Spirotrichonympha leidyi, and Holomastigotoides mirabile in the hindgut of Coptotermes formosanus, and Spirotrichonympha sp. and Trichonympha spp. in Hodotermopsis sjoestedti were identified. In the phylogenetic tree constructed, the sequences from the termites were dispersed within the groups of known members of parabasalids, reflecting the presence of diverse parabasalids in the hindgut of termites. There were three paraphyletic lineages of hypermastigotes represented by Pseudotrichonympha, Trichonympha, and Spirotrichonympha, in agreement with the morphology‐based taxonomic groups. The analysis of the tree‐root suggested that the Pseudotrichonympha group is the most probable ancient lineage of parabasalids and that the Trichonympha group is the secondly deep‐branching lineage. The Spirotrichonympha group and the Trichomonadida may have emerged later.
Ultrastructural Distribution of Poly (A)+ RNA During Trypanosoma cruzi‐Cardiomyocyte Interaction in vitro: A Quantitative Analysis of the Total mRNA Content by in situ Hybridization
Authors: PEREIRA MIRIAN CLAUDIA S. , SINGER ROBERT H. , DE MEIRELLES MARIA DE NAZARETH S. L.
Published: July 2005

ABSTRACT. Ultrastructural in situ hybridization was used to visualize the spatial distribution of poly (A)+ RNA and quantitate its relative amount within different cellular compartments of cardiomyocytes after T. cruzi infection. The amount of poly (A) RNA remained about the same up to 24 h post‐infection. In contrast, its content was reduced 65% after 72 h of interaction, showing a marked decrease in the cell cytoplasm. This decline in poly (A)+ RNA level in host cell cytoplasm was concomitant with intracellular proliferation of T. cruzi amastigotes. Thus, T. cruzi may affect host cell cytoplasmic mRNA stability, associated with the parasite's intracellular multiplication.
Trypanosoma cruzi Infection Affects Actin mRNA Regulation in Heart Muscle Cells
Authors: PEREIRA MIRIAN CLAUDIA S. , SINGER ROBERT H. , DE MEIRELLES MARIA DE NAZARETH S. L.
Published: July 2005

ABSTRACT. We have previously described alterations in the cytoskeletal organization of heart muscle cells (HMC) infected with Trypanosoma cruzi in vitro. Our aim was to investigate whether these changes afso affect the regulation of the actin mRNAs during HMC differentiation. Northern blot analysis revealed that α‐cardiac actin mRNA levels increased during cell differentiation while β‐actin mRNA levels declined. Nonmuscle cells displayed β‐actin mRNA signal localized at the cell periphery, while a‐cardiac actin mRNA had a perinuclear distribution in myocytes. Trypanosoma cruzi‐infected cells showed 50% reduction in α‐cardiac actin mRNA expression after 72 h of infection. In contrast, β‐actin mRNA levels increased approximately 79% after 48 h of infection. In addition, in situ β‐actin mRNA was delocalized from the periphery into the perinuclear region. These observations support the hypothesis that Trypanosoma cruzi affects actin mRNA regulation and localization through its effect on the cytoskeleton of heart muscle cells.
Phylogenetic Relationships of Pleistophora‐like Microsporidia Based on Small Subunit Ribosomal DNA Sequences and Implications for the Source of Trachipleistophora hominis Infections
Authors: CHENEY SARAH A. , LAFRANCHI‐TRISTEM NATHALIE J. , CANNING ELIZABETH U.
Published: July 2005

ABSTRACT. The microsporidian Trachipleistophora hominis was isolated in vitro from the skeletal muscle of an AIDS patient. Since its discovery several more cases of myositis due to Trachipleistophora have been diagnosed but the source of infection is unknown. Morphologically, T. hominis most closely resembles Pleistophora and Vavraia, which undergo polysporous sporogony in sporophorous vesicles, but differs from these genera in the mode of formation of sporoblasts and in the morphology of the sporophorous vesicles. Alignment and analyses of the small subunit ribosomal DNA sequences of T. hominis and several other polysporoblastic genera indicated that its closest phylogenetic relationships were with species of the genera Pleistophora and Vavraia, in line with morphological predictions. The type species of the latter two genera are Pleistophora typicalis and Vavraia culicis; these are parasites of fish and mosquitoes, respectively. These results suggest two possible routes and sources of infection to AIDS patients, these being perorally by ingestion of inadequately cooked fish or crustaceans or percutaneously during a bloodmeal taken by a haematophagous insect. Support for an insect source has been provided by recent detection of a microsporidium from mosquitoes in human corneal tissue.
Inter‐laboratory Comparison of the CD‐I Neonatal Mouse Logistic Dose‐Response Model for Cryptosporidium parvum Oocysts
Authors: KORICH D. G. , MARSHALL M. M. , SMITH H. V. , O'GRADY J. , BUKHARI Z. , FRICKER C. R. , ROSEN J. P. , CLANCY J. L.
Published: July 2005

ABSTRACT. Cryptosporidium parvum oocyst viability can be determined by vital dyes, in vitro excystation, and cell culture; however, neonatal mouse infectivity assays are the reference method. Unfortunately, there have been few efforts to standardize methods for infectivity assays thus casting a veil of uncertainty over the significance and comparability of results. In order to address this issue, two laboratories proficient in measuring oocyst infectivity conducted independent dose titration studies with neonatal CD‐I mice using standardized protocols and a well‐characterized isolate of Cryptosporidium parvum. The resulting independent logistic dose‐response models derived by regression analysis were compared with each other and with a published model. The comparisons showed these dose‐response functions to be reproducible under standardized conditions. It is important to standardize mouse strain, age of mice at inoculation and necropsy, oocyst isolate, and age of oocysts. However, other factors, including methods used to detect infectivity and to count oocyst doses, appear less critical. Adopting a standardized assay for oocyst infectivity will provide both a basis for comparing data from various oocyst disinfection studies and a suitable platform for evaluating new or existing in vitro viability surrogates such as excystation, vital dyes or cell culture.
Fluorescent in situ Detection of Encephalitozoon hellem Spores with a 6‐Carboxyfluorescein‐Labeled Ribosomal RNA‐Targeted Oligonucleotide Probe
Authors: HESTER JEFF D. , ALAN LINDQUIST H. D. , BOBST ALBERT M. , SCHAEFER FRANK W.
Published: July 2005

ABSTRACT. A fluorescent in situ hybridization assay has been developed for the detection of the human‐pathogenic microsporidian, Encephalitozoon hellem in water samples using epifluorescence microscopy. The assay employs a 19‐nucleotide species‐specific 6‐carboxyfluorescein‐labeled oligonucleotide probe, HEL878F, designed to be complementary to the nucleic acid sequence 878–896, a highly variable segment of the 16S ribosomal RNA of E. hellem spores. The specificity of this probe for its ribosomal RNA target site was confirmed using RNA degradation, ribosomal RNA target site competition, and nucleotide base mismatch control probe assays. Furthermore, the specificity of the HEL878F oligonucleotide probe for E, hellem spores was established when it was evaluated on spores from all three species of the genus Encephalitozoon that had been seeded in reagent water and environmental water concentrates. The specificity of the HEL878F oligonucleotide probe was further corroborated when tested on algae, bacteria, and protozoa commonly found in environmental water. The study demonstrates the applicability of a fluorescent in situ hybridization assay using a species‐specific fluorescent‐labeled oligonucleotide probe for the detection of E. hellem spores in water samples.
Sequence Analysis of the Rhop‐3 Gene of Plasmodium yoelii1
Authors: ANTHONY ROBERT N. , YANG JIACHENG , KRALL JENNIFER A. , SAM‐YELLOWE TOBILI Y.
Published: July 2005

ABSTRACT. The 110 kDa/Rhop‐3 rhoptry protein of Plasmodium falciparum is non‐covalently associated with two other proteins, the 140 kDa Rhop‐1 and the 130 kDa Rhop‐2. cDNAs encoding Rhop‐3 from Plasmodium yoelii were isolated using rhoptry‐specific antisera from Plasmodium falciparum, P. yoelii, and Plasmodium chabaudi. The cDNAs encoded peptides with partial homology to the C‐terminal region (residues 541–861) of P. falciparum Rhop‐3. Core regions of homology to the P. falciparum gene will be useful in determining the biological role of Rhop‐3 and its potential as a vaccine candidate for malaria.
Toward Sequencing the Tetrahymena Genome: Exploiting the Gift of Nuclear Dimorphism
Authors: ORIAS EDUARDO
Published: July 2005

ABSTRACT. Important scientific discoveries have utilized the unique advantages of Tetrahymena thermophila as a research organism. Recently developed molecular genetic manipulations allow full exploitation of the many scientific dividends that would result from having its genome sequenced. As a typical ciliated protozoan, Tetrahymena exhibits “nuclear dimorphism”. It possesses two differentiated forms of its nuclear genome: a globally repressed, diploid germline or micronuclear genome, and a polyploid, site‐specifically fragmented somatic or macronuclear genome. The macronuclear genome is, in effect, a natural, large‐insert library of the micronuclear genome. This presentation describes how the gifts of nuclear dimorphism are being exploited in the experimental analysis of molecular and cell biology. Mechanisms present in humans that are either absent in other eukaryotic microbial model systems, or not as readily accessible in them as in Tetrahymena, are especially relevant. This presentation also reviews unique tools generated by nuclear dimorphism that are being used for genetically and physically mapping the Tetrahymena genome.
Toward the Functional Analysis of the Dictyostelium discoideum Genome1
Authors: SUCGANG RICHARD , SHAULSKY GAD , KUSPA ADAM
Published: July 2005

ABSTRACT. Dictyostelium discoideum is a useful model for molecular studies of cell biology and development. The 34‐megabase Dictyostelium genome is currently being sequenced through the efforts of an international consortium. The genome is expected to encode 8–10,000 genes, including all those required for a free‐living eukaryote capable of multicellular development. A complete description of the Dictyostelium genome will open the way toward the application of genome‐based experimental approaches to studies of cell biology and development in this organism, and allow detailed physiological and evolutionary comparisons to other species.
Chlamydomonas reinhardtii: Biological Rationale for Genomics1
Authors: DUTCHER SUSAN K.
Published: July 2005

ABSTRACT. Chlamydomonas reinhardtii has been the subject of genetic, biochemical, cytological, and molecular analyses for over 50 years. It is an ideal model system for the study of flagella and basal bodies as well as the study of photosynthesis and chloroplast biogenesis, cell‐cell recognition and fusion, phototaxis, and secretion. It is clear that many of the genes identified in Chlamydomonas have homologs in land plants as well as animals. Thus, a genomic approach in Chlamydomonas will provide another important avenue for the understanding of important biological processes.
Particle Handling during Interception Feeding by Four Species of Heterotrophic Nanoflagellates
Authors: BOENIGK JENS , ARNDT HARTMUT
Published: July 2005

ABSTRACT. High resolution video‐microscopy was used to observe grazing patterns of the heterotrophic nanoflagellates Cafeteria roenbergensis, Bodo sultans, Spumella sp., and Ochromonas sp. Spumella and Ochromonas enclose food particles with pseudopodia while Cafeteria and Bodo engulf particles by invagination of the cell surface. The following parameters of the feeding process were quantified: frequency of flagellar beating, speed of particles in different positions of the feeding current, food size selection, feeding rate, and the time budget for the handling of particles. The mean handling times differed between 94 s for Cafeteria and 4 s for Ochromonas for ingested particles. Handling times for ingested particles were significantly longer than for non‐captured particles. Long handling times were calculated to be disadvantageous only for flagellates which propel a high water volume per hour (esp. Ochromonas) or live in a bacteria‐rich environment. Our model calculations may provide a reasonable theoretical explanation for a concentration‐dependent behavioural variability of the feeding strategy of different heterotrophic nanoflagellates (HNF) species.