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At least 19 recordsLinked to original sources
PubMed2002

Stimulation of B lymphocytes, macrophages, and dendritic cells by protozoan DNA.

This review summarizes recent work from our laboratory demonstrating the activation of B lymphocytes, macrophages and dendritic cells by DNA from three different protozoan parasites of cattle and humans that is qualitatively similar to the now well-described effects of CpG-containing bacterial DNA. This novel mechanism of protozoan parasite recognition by the innate immune system could facilitate recovery from acute infection or contribute to infection-related pathology.

Animals↗
PubMed1999

The modified base J is the target for a novel DNA-binding protein in kinetoplastid protozoans.

DNA from Kinetoplastida contains the unusual modified base beta-D-glucosyl(hydroxymethyl)uracil, called J. Base J is found predominantly in repetitive DNA and correlates with epigenetic silencing of telomeric variant surface glycoprotein genes in Trypanosoma brucei. We have now identified a protein in nuclear extracts of bloodstream stage T.brucei that binds specifically to J-containing duplex DNA. J-specific DNA binding was also observed with extracts from the kinetoplastids Crithidia fasciculata and Leishmania tarentolae. We purified the 90 kDa C.fasciculata J-binding protein 50 000-fold and cloned the corresponding gene from C.fasciculata, T.brucei and L.tarentolae. Recombinant proteins expressed in Escherichia coli demonstrated J-specific DNA binding. The J-binding proteins show 43-63% identity and are unlike any known protein. The discovery of a J-binding protein suggests that J, like methylated cytosine in higher eukaryotes, functions via a protein intermediate.

Amino Acid Sequence↗
PubMed1999

Fast and reliable extraction of protozoan parasite DNA from fecal specimens.

BACKGROUND: Polymerase chain reaction (PCR) detection of intestinal protozoa in fecal specimens is hampered by poor recovery of DNA and by the presence of PCR inhibitors. In this study we describe a novel method for DNA extraction from such specimens containing spores and oocysts of Enterocytozoon bieneusi and Cryptosporidium parvum, respectively. METHODS AND RESULTS: Extraction was done using commercial kits modified to maximize the recovery and purity of extracted DNA. In comparison with a procedure we previously reported, we estimate that this method may increase the sensitivity of parasite DNA detection in fecal specimens up to tenfold. An additional advantage of this method is that up to 12 samples may be processed simultaneously within 2 hours. CONCLUSIONS: By using this method, we were able to increase reproducibility of PCR amplification on fecal specimens and significantly reduce the hands-on time required to process the samples.

Animals↗
PubMed2001

DNA from protozoan parasites Babesia bovis, Trypanosoma cruzi, and T. brucei is mitogenic for B lymphocytes and stimulates macrophage expression of interleukin-12, tumor necrosis factor alpha, and nitric oxide.

The activation of innate immune responses by genomic DNA from bacteria and several nonvertebrate organisms represents a novel mechanism of pathogen recognition. We recently demonstrated the CpG-dependent mitogenic activity of DNA from the protozoan parasite Babesia bovis for bovine B lymphocytes (W. C. Brown, D. M. Estes, S. E. Chantler, K. A. Kegerreis, and C. E. Suarez, Infect. Immun. 66:5423-5432, 1998). However, activation of macrophages by DNA from protozoan parasites has not been demonstrated. The present study was therefore conducted to determine whether DNA from the protozan parasites B. bovis, Trypanosoma cruzi, and T. brucei activates macrophages to secrete inflammatory mediators associated with protective immunity. DNA from Escherichia coli and all three parasites stimulated B-lymphocyte proliferation and increased macrophage production of interleukin-12 (IL-12), tumor necrosis factor alpha (TNF-alpha), and nitric oxide (NO). Regulation of IL-12 and NO production occurred at the level of transcription. The amounts of IL-12, TNF-alpha, and NO induced by E. coli and protozoal DNA were strongly correlated (r2 > 0.9) with the frequency of CG dinucleotides in the genome, and immunostimulation by DNA occurred in the order E. coli > or = T. cruzi > T. brucei > B. bovis. Induction of inflammatory mediators by E. coli, T. brucei, and B. bovis DNA was dependent on the presence of unmethylated CpG dinucleotides. However, at high concentrations, E. coli and T. cruzi DNA-mediated macrophage activation was not inhibited following methylation. The recognition of protozoal DNA by B lymphocytes and macrophages may provide an important innate defense mechanism to control parasite replication and promote persistent infection.

Animals↗
PubMedDate not supplied

Use of hybridoma antibodies and recombinant DNA technology in protozoan vaccine development.

The use of hybridoma antibodies developed against the sporozoite stage of avian coccidia, coupled with genetic-engineering techniques, has made it possible to begin bird-immunization studies utilizing an Escherichia coli-elicited coccidial protein. The coccidia are currently controlled in the poultry industry by use of anticoccidial compounds, but it now may be possible to use the bird's own immune system for defense against the parasitic infection. Since the sporozoite stage, which initiates the infection in poultry, is quite complex and is made up of hundreds of proteins or antigens, hybridoma antibodies were produced to identify specific antigens. These antigens, once identified, were found in such minute amounts that it became necessary to utilize genetic engineering in order to produce enough protein for immunization studies. One such protein, designated 5401, has been shown to stimulate an antibody response in immunized birds and to impart partial protection against a coccidial challenge infection. The results of these studies indicate that development of a vaccine against coccidial parasites may someday be possible.

Animals↗
PubMed1972

Evidence of gene diminution during the formation of the macronucleus in the protozoan, Stylonychia (DNA density-melting curves-micronuclear DNA-polytene chromosomes).

The course of events by which a macronucleus is formed from a micronucleus after conjugation in the ciliated protozoan, Stylonychia, suggests that genetic diminution may occur. This idea is supported by determinations of the density profiles and melting curves for micro- and macronuclear DNAs. Macronuclear DNA consists of a single density component and melts as if it were a single component. Micronuclear DNA consists of four or more density components and melts as if it were a mixture of several DNAs of different base compositions. These data indicate that at least 60% of the micronuclear DNA components are absent from the macronuclear DNA.

Cell Nucleus↗
PubMed1999

Telomeres of polytene chromosomes in a ciliated protozoan terminate in duplex DNA loops.

The end of a telomeric DNA sequence isolated from a polytene chromosome of a hypotrichous ciliate folds back and hybridizes with downstream telomeric sequence to form a t loop that is stable in the absence of protein and DNA cross-linking. The single-stranded, telomeric DNA sequence at the end of a macronuclear molecule does not form a t loop but, instead, is complexed with a heterodimeric, telomere-binding protein. Thus, two mechanisms for capping the ends of DNA molecules are used in the same cell.

Animals↗
PubMed2003

Thelohania parastaci sp. nov. (Microspora: Thelohaniidae), a parasite of the Australian freshwater crayfish, Cherax destructor (Decapoda: Parastacidae).

Thelohania parastaci sp. nov. infects the Australian freshwater crayfish, Cherax destructor. Data on morphology, developmental patterns and sequences from the small subunit (SSU) and internal transcribed spacer (ITS) regions of the ribosomal DNA (rDNA) of T. parastaci sp. nov. are described. The ultrastructural features of different life cycle stages are very similar to those of the European crayfish parasite Thelohania contejeani. T. parastaci sp. nov. exhibits simultaneous dimorphic sporogony in muscle tissue. Meronts, sporonts and spores are found in muscle tissue, within haemocytes in the hepatopancreas, and in the intestinal wall of infected crayfish. T. parastaci sp. nov. shows 92% sequence identity with T. contejeani and only 67% sequence identity with the fire ant pathogen T. solenopsae, when SSU rDNA sequences are compared. Analysis of SSU rDNA and ITS sequences of T. parastaci sp. nov. from crayfish from Victoria, Western Australia, and New South Wales indicate that the parasite has a wide geographical distribution in Australia.

Animals↗
PubMed2004

Spore morphotypes of Thelohania solenopsae (microsporidia) described microscopically and confirmed by PCR of individual spores microdissected from smears by position ablative laser microbeam microscopy.

Development of Thelohania solenopsae, a parasite of the red imported fire ant (Solenopsis invicta), until recently was thought to include formation of two types of spores: unicellular meiospores, maturing inside sporophorous vesicles in sets of eight (octospores); and Nosema-like binuclear free spores. Megaspores, discovered in 2001, develop primarily in alates and are morphologically distinct from the two previously known types of spores. The role of megaspores in the T. solenopsae life cycle, as well as their existence, has been questioned. The current research includes light and electron microscopic descriptions of the three major spore morphotypes characteristic of T. solenopsae development. In addition, individual octospores and megaspores were isolated into groups of 8-20 from methanol-fixed and Calcofluor-stained smears of the infected ants for subsequent PCR analysis by the laser pressure catapulting function of a position ablative laser microbeam microscope, a technique applied for the first time to research of microsporidia. The PCR-amplified SSU rDNA nucleotide sequences from octospores and megaspores were identical. This, along with the consistency with which megaspores are detected in infected ants, demonstrates that megaspores are integral to the life cycle of T. solenopsae.

Animals↗
PubMedDate not supplied

higher-order phylogeny of plasmodial slime molds (Myxogastria) based on elongation factor 1-A and small subunit rRNA gene sequences.

The Myxogastria are common soil microorganisms with a life cycle comprised of a plasmodial trophic stage and large fruiting bodies generally visible with the unaided eye. Until now, their classification has been based exclusively on a combination of morphological, ultrastructural, and developmental characters. Our study is the first attempt to examine phylogenetic relationships among these taxa using molecular data. Partial small-subunit ribosomal RNA and/or elongation factor 1-alpha gene sequences were obtained from eleven, mostly field-collected species representing the five orders of Myxogastria. Nineteen sequences were obtained and subjected to phylogenetic analysis together with 10 sequences available from GenBank. Separate and combined analyses of the two data sets support the division of Myxogastria into three distinct groups. The most basal clade consists of the Echinosteliales, an order considered to have affinities with Protostelia. The three species examined possess unpigmented or slightly pigmented spores. The second group consists of Liceales and Trichiales, taxa characterized by the presence of clear, but pigmented, spores. The third group consists of the two remaining orders, Physarales and Stemonitales, both possessing dark spores. This suggests that spore pigmentation is an evolutionarily conservative character in myxogastrians, and that the simple morphology of echinostelids is not a derived feature.

DNA, Protozoan↗
PubMed1994

Trypanosoma cruzi ribosomal DNA: mapping of a putative distal promoter.

The nucleotide (nt) sequence (2106 bp) of a cloned rDNA (encoding ribosomal RNA) spacer region from Trypanosoma cruzi was determined and a putative transcription start point (tsp) was mapped. The assigned length for the transcribed spacer is 1768 bp and its tsp is present 270-bp upstream from an alternative tsp published for the equivalent gene from another T. cruzi strain [Dietrich et al., Gene 125 (1993) 103-107]. Sequence comparisons of the nt flanking both T. cruzi tsp with the homologous regions from both other trypanosomatids, and other eukaryotes, indicate that these sequences are poorly conserved within the family Trypanosomatidae. This finding reinforces the proposal that the speciation of trypanosomatids may have occurred early in evolution.

Animals↗
PubMedDate not supplied

Two new small-subunit ribosomal RNA gene lineages within the subclass gymnamoebia.

Phylogenetic analysis of small-subunit ribosomal RNA gene sequences for gymnamoebae of the families Vexilliferidae, Paramoebidae, and Vannellidae identified two distinct lineages that are supported by gross morphological characters. This analysis indicates that paramoebids and vexilliferids are part of one lineage and that vannellids belong to another. A shared morphological character unique to the paramoebid/vexilliferid lineage members is the presence of dactylopodiate subpseudopodia. However, cell surface structures, normally used for taxonomic discrimination, range from simple hair-like filaments without any apparent organization (Neoparamoeba), to hexagonal glycostyles (Vexillifera) or more elaborate surface scales (Korotnevella). Taxa within the vannellid lineage all lack subpseudopodia and appear flabellate, spatulate or linguiform while in locomotion. Cell surface structures of taxa within the vannellid lineage range from filaments organized into hexagonal arrays (Lingulamoeba, Platyamoeba) to pentagonal glycostyles (Clydonella, Vannella). Vannellid lineage members of the genera Clydonella and Lingulamoeba were studied at the level of electron microscopy. Unique cell surface features validate these as genera distinct from Vannella and Platyamoeba. Genetic and ultrastructural data are used to discuss the phylogenetic interrelationships for the taxa studied.

Animals↗
PubMedDate not supplied

Strombidinopsis jeokjo n. sp. (ciliophora: choreotrichida) from the coastal waters off western Korea: morphology and small subunit ribosomal DNA gene sequence.

The planktonic ciliate Strombidinopsis jeokjo n. sp. is described from Quantitative Protargol-Stained (QPS) preparations, and the sequence of the small subunit rDNA (SSU rDNA) from cultured cells is reported. This species is ovoid and bluntly tapered towards the posterior. The ranges (and mean +/- standard deviation, n = 31) of cell length, cell width, and oral diameter of the QPS-stained specimens were 100-190 microm (149 +/- 25), 60-105 microm (79 +/- 13), and 55-80 microm (64 +/- 5), respectively. Fifteen to seventeen external oral polykinetids had oral membranelle cilia 20-35 microm long. Twenty-six to twenty-eight somatic kineties were equally spaced around the cell body and extended from the oral to the posterior regions with 23-44 dikinetids per kinety. Both kinetosomes of each kinetid bore cilia 3-7 microm long. Strombidinopsis jeokjo had two ovoid macronuclei of 25-38 microm x 12-15 microm. When properly aligned, the sequence of the SSU rDNA of S. jeokjo (GenBank Accession No. AJ628250) was approximately 2% different from that of an unidentified Strombidinopsis species (GenBank Accession No. AF399132-AF399135), the closest species in the SSU rDNA sequence.

Animals↗
PubMed2004

Identification of Cryptosporidium isolates from cockatiels by direct sequencing of the PCR-amplified small subunit ribosomal RNA gene.

Cryptosporidium is a significant pathogen in humans and animals. Cases of infection by C. meleagridis or C. baileyi with zoonotic potential have also been reported in domestic birds; and recent studies indicate the presence of new host-adapted species or genotype in birds. Therefore, accurately identifying isolates is important for understanding the epizootiology of Cryptosporidium infection in birds and for the control of human cryptosporidiosis. Cryptosporidium has been detected in cockatiels, but the species or genotype of isolates remains unclear because identification was performed using conventional microscopy. We report herein the species or genotype of isolates from two cockatiels distinguished by a PCR-based diagnostic method. The isolates were found to be C. meleagridis and C. baileyi, respectively. This study documents the first discovery of C. meleagridis and C. baileyi in cockatiels and suggests that pet birds may play an important role in the epidemiology of cryptosporidiosis.

Animals↗
PubMedDate not supplied

Phylogenetic relationships of the subclass Peritrichia (Oligohymenophorea, Ciliophora) inferred from small subunit rRNA gene sequences.

The phylogenetic relationships among peritrichs remain unresolved. In this study, the complete small subunit rRNA (SSrRNA) gene sequences of seven species (Epistylis galea, Campanella umbellaria, Carchesium polypinum, Zoothamnium arbuscula, Vaginicola crystallina, Ophrydium versatile, and Opercularia microdiscum) were determined. Trees were constructed using distance-matrix, maximum-likelihood and maximum-parsimony methods, all of which strongly supported the monophyly of the subclass Peritrichia. Within the peritrichs, 1) E. galea grouped with Opercularia microdiscum and Campanella umbellaria but not the other Epistylis species, which indicates that the genus Epistylis might not be monophyletic; 2) the topological position of Carchesium and Campanella suggested that Carchesium should be placed in the family Zoothamniidae, or be elevated to a higher taxonomic rank, and that Campanella should be independent of the family Epistylididae, and probably be given a new rank; and 3) Opisthonecta grouped strongly with Astylozoon, which suggested that Opisthonecta species were not the ancestors of the stalked peritrichs.

Animals↗
PubMedDate not supplied

Description of Perkinsus andrewsi n. sp. isolated from the Baltic clam (Macoma balthica) by characterization of the ribosomal RNA locus, and development of a species-specific PCR-based diagnostic assay.

A Perkinsus species was isolated from the baltic clam Macoma balthica and an in vitro culture established under conditions described for P. marinus. As reported previously, morphological features remarkable enough to clearly indicate that this isolate is a distinct Perkinsus species were lacking. In this study, regions of the rRNA locus (NTS, 18S, ITS1, 5.8S, and ITS2) of this isolate were cloned, sequenced, and compared by alignment with those available for other Perkinsus species and isolates. Sequence data from the rRNA locus and species-specific PCR assays indicated not only that Perkinsus sp. from M. balthica was not P. marinus, but it was different from P. atlanticus and P. olseni. The degree of difference was comparable to or greater than differences between accepted Perkinsus species. In particular, NTS sequence and length were dramatically different from that of P. marinus and P. atlanticus. Therefore, we formally propose to designate the Perkinsus sp. from M. balthica as a separate species, P. andrewsi n. sp. Primers based on P. andrewsi NTS sequence were used to develop a PCR-based diagnostic assay that was validated for species-specificity and sensitivity. PCR-based assays specific for either P. andrewsi or P. marinus were used to test for their presence in bivalve species sympatric to M. balthica. Although isolated from M. balthica, P. andrewsi was also detected in the oyster Crassostrea virginica and clams Macoma mitchelli and Mercenaria mercenaria, and could coexist with P. marinus in all four bivalve species tested.

Animals↗