Identification and characterization of a novel <Emphasis Type="Italic">Tritimovirus</Emphasis> species isolated from wild <Emphasis Type="Italic">Trisetum flavescens</Emphasis> L., family <Emphasis Type="Italic">Poaceae</Emphasis>

Yellow oat-grass plants (Trisetum flavescens L.) with mild mosaic and pronounced dwarfing symptoms were observed at different locations in the Czech Republic. Electron microscope observations of symptomatic plants revealed the presence of filamentous particles and inclusion bodies characteristic of the family Potyviridae. The virus was readily mechanically transmitted to its original host plus a narrow host range of monocot species. Serological assays of infected plant extracts using antiserum specific to the closest species in the family Potyviridae were negative. The 3′ end of the viral genome was cloned, sequenced and compared to sequences of species in the family Potyviridae. The virus is more closely related to viruses in the genus Tritimovirus than to other genera within the Potyviridae. Based on phylogenetic analyses of the coat protein cistron and flanking genomic regions, we propose this is a distinct viral species of the genus Tritimovirus, tentatively named Yellow oat-grass mosaic virus (YOgMV).     

Repeat induced point mutation in two asexual fungi, <Emphasis Type="Italic">Aspergillus niger</Emphasis> and <Emphasis Type="Italic">Penicillium </Emphasis><Emphasis Type="Italic">chrysogenum</Emphasis>

Repeat induced point mutation (RIP) is a gene silencing mechanism present in fungal genomes. During RIP, duplicated sequences are efficiently and irreversibly mutated by transitions from C:G to T:A. For the first time, we have identified traces of RIP in transposable elements of Aspergillus niger and Penicillium chrysogenum, two biotechnologically relevant fungi. We found that RIP in P. chrysogenum has affected a large set of sequences, which also contain other mutations. On the other hand, RIP in A. niger is limited to only few sequences, but literally all mutations are RIP-like. Surprisingly, RIP occurred only in transposon sequences that have disrupted open reading frames in A. niger, a phenomenon not yet reported for other fungi. In both fungal species, we identified two sequences with strong sequence similarity to Neurospora crassa RID. RID is a putative DNA methyltransferase and the only known enzyme involved in the RIP process. Our findings suggest that both A. niger and P. chrysogenum either had a sexual past or have a sexual potential. These findings have important implications for future strain development of these fungi.     

Morphological study of <Emphasis Type="Italic">Tetratrichomonas didelphidis</Emphasis> isolated from opossum <Emphasis Type="Italic">Lutreolina crassicaudata</Emphasis> by scanning electron microscopy

Tetratrichomonas didelphidis is a flagellate protozoan found in the intestine of opossums Didelphis marsupialis, Didelphis albiventris, and Lutreolina crassicaudata. The isolate used in this study was from L. crassicaudata and it was cultivated in monoxenic culture with Escherichia coli in Diamond (TYM) medium without maltose and with starch solution (trypticase–yeast extract–starch), pH 7.5 at 28°C. Scanning electron microscopy showed the fine morphological features of the trophozoites: the emergence of the anterior flagella, the structure of the undulating membrane, the axostyle and posterior flagellum. In addition, we described spherical forms that are probably pseudocysts. Our data will contribute to a better understanding of surface structures in T. didelphidis.     

Purification and biochemical characterization of cytosolic glutathione-<Emphasis Type="Italic">S</Emphasis>-transferase from malarial parasites <Emphasis Type="Italic">Plasmodium yoelii</Emphasis>

Glutathione (GSH) metabolism represents a potential target for antiparasitic drug design. Glutathione-S-transferase (GST), an important enzyme of the GSH cycle, is considered to be an essential detoxification enzyme in parasitic species. Soluble GST from rodent malarial parasites Plasmodium yoelii was purified to homogeneity using a combination of salt precipitation, affinity chromatography on GSH–sepharose 6B and ultrafiltration. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis revealed a single band and activity staining was also detected on PAGE gels. Kinetic studies on the purified enzyme revealed significant differences between the parasitic and mammalian enzymes. The purified enzyme exhibited an optimum pH of 8.2 and K _m values of 0.2±0.213 and 3.3±0.056 mM with respect to co-substrate GSH and substrate 1-chloro-2, 4-dinitrobenzene (CDNB), respectively. Hemin, the known mammalian GST inhibitor was found to be a potent inhibitor of P. yoelii GST, with a K _i of 4.0 μM.     

Cercarial shedding from <Emphasis Type="Italic">Galba</Emphasis><Emphasis Type="Italic">truncatula</Emphasis> infected with <Emphasis Type="Italic">Fasciola</Emphasis><Emphasis Type="Italic">gigantica</Emphasis> of distinct geographic origins

Galba truncatula snails were experimentally infected with either of two different isolates of Fasciola gigantica, originating from Egypt or China, to determine the influence of these isolates on the characteristics of snail infections. The survival rates of G. truncatula on day 30 post-exposure were 90.0% and 60.2% in the Egyptian and Chinese groups, respectively. The frequency of cercaria-shedding snails within the Egyptian group was 79.8%, whereas in the Chinese group it was 22.4%. The parasite origin had a significant effect on the durations of the prepatent and patent periods. The mean number of cercariae shed from the Egyptian group was significantly greater than that shed from the Chinese group (a mean of 275.5 per cercaria-shedding snail compared with 29.0). These results could be explained by the fact that G. truncatula might be a natural intermediate host for F. gigantica in Egypt, and the greater adaptability of the Egyptian miracidia of F. gigantica to unusual snail hosts. These results demonstrate the influence of the geographic origin of the parasite on the success of trematodes infecting snails.     

Cysteine proteinases from promastigotes of <Emphasis Type="Italic">Leishmania</Emphasis> (<Emphasis Type="Italic">Viannia</Emphasis>) <Emphasis Type="Italic">braziliensis</Emphasis>

Leishmania (Viannia) braziliensis is the major causative agent of American tegumentary leishmaniasis, a disease that has a wide geographical distribution and is a severe public health problem. The cysteine proteinase B (CPB) from Leishmania spp. represents an important virulence factor. In this study, we characterized and localized cysteine proteinases in L. (V.) braziliensis promastigotes. By a combination of triton X-114 extraction, concanavalin A-affinity, and ion exchange chromatographies, we obtained an enriched fraction of hydrophobic proteins rich in mannose residues. This fraction contained two proteinases of 63 and 43 kDa, which were recognized by a CPB antiserum, and were partially sensitive to E-64 in enzymatic assays with the peptide Glu-Phe-Leu. In confocal microscopy, the CPB homologues localized in the peripheral region of the parasite. This data together with direct agglutination and flow cytometry assays suggest a surface localization of the CPB homologues. The incubation of intact promastigotes with phospholipase C reduced the number of CPB-positive cells, while anti-cross-reacting determinant and anti-CPB antisera recognized two polypeptides (63 and 43 kDa) derived from phospholipase C treatment, suggesting that some CPB isoforms may be glycosylphosphatidylinositol-anchored. Collectively, our results suggest the presence of CPB homologues in L. braziliensis surface and highlight the need for further studies on L. braziliensis cysteine proteinases, which require enrichment methods for enzymatic detection.     

Growth-inhibitory effect of a fucoidan from brown seaweed <Emphasis Type="Italic">Undaria pinnatifida</Emphasis> on <Emphasis Type="Italic">Plasmodium</Emphasis> parasites

The present study was undertaken to investigate the inhibitory effects of fucoidan, a sulfated polysaccharide isolated from the edible brown seaweed Undaria pinnatifida, on the growth of Plasmodium parasites. In order to assess the anti-malarial activity of fucoidan, growth inhibition activities were evaluated using cultured Plasmodium falciparum parasites in vitro and on Plasmodium berghei-infected mice in vivo. Fucoidan significantly inhibited the invasion of erythrocytes by P. falciparum merozoites, and its 50% inhibition concentration was similar to those for the chloroquine-sensitive P. falciparum 3D7 strain and the chloroquine-resistant K1 strain. Four-day suppressive testing in P. berghei-infected mice with fucoidan resulted in a 37% suppressive effect versus the control group and a delay in death associated with anemia (P < 0.05). In addition, fucoidans had no toxic effect on RAW 264.7 cells. These findings indicate that fucoidans from the Korean brown algae U. pinnatifida inhibits the invasion of Plasmodium merozoites into erythrocytes in vitro and in vivo. J.-H. Chen and J.-D. Lim contributed equally to this work.     

Redescription of <Emphasis Type="Italic">Lemuricola</Emphasis> (<Emphasis Type="Italic">Madoxyuris</Emphasis>) <Emphasis Type="Italic">bauchoti</Emphasis> (Nematoda,Oxyuridae) from <Emphasis Type="Italic">Lemur catta</Emphasis> in Madagascar

Lemuricola (Madoxyuris) bauchoti Chabaud, Brygoo et Petter, 1965 is redescribed from material collected from the ring-tailed lemur, Lemur catta, from the Beza Mahafaly Special Reserve in Madagascar using the scanning electron microscope. This is a new host record and the first oxyurid reported from the ring-tailed lemur. Previously, records of each species of the subgenus Madoxyuris have been restricted to a single host species, but the close relationship between these nematodes and their Strepsirrhini hosts will only be proven when additional records fill in the gaps in their distribution.     

Multilocus phylogenetic analysis of <Emphasis Type="Italic">Cryptosporidium andersoni</Emphasis> (Apicomplexa) isolated from a bactrian camel (<Emphasis Type="Italic">Camelus bactrianus</Emphasis>) in China

This is the first report of cryptosporidiosis in a bactrian camel (Camelus bactrianus) in China. Two Cryptosporidium isolates derived from the same bactrian camel (3-year-old) in November 2005 and April 2006 were characterized using sequence and phylogenetic analysis of the small-subunit rRNA (18S rRNA), 70-kDa heat shock protein (HSP70), actin and Cryptosporidium oocyst wall protein (COWP) genes. The sequences of the 18S rRNA and COWP were identical to all other Cryptosporidium andersoni isolates although minor differences were noticed between the isolates and the USA isolate at the actin locus (99.2% of similarity). The sequence of the HSP70 was identical to the Japanese C. andersoni isolate, with a minor difference from the Australian C. andersoni isolate (99.7% of similarity). Cross-transmission studies demonstrated that the C. andersoni isolates did not infect immunosuppressed or immunocompetent Kun-ming mice, severe combined immunodeficiency mice, and immunosuppressed or immunocompetent calves. Among the C. andersoni isolates reported so far, only isolates from Japan could infect SCID mice. Thus, the C. andersoni isolates from the bactrian camel were biologically similar to most bovine C. andersoni isolates characterized so far, but are different from bovine isolates from Japan.     

Antiplasmodial activity of two marine polyherbal preparations from <Emphasis Type="Italic">Chaetomorpha antennina</Emphasis> and <Emphasis Type="Italic">Aegiceras corniculatum</Emphasis> against <Emphasis Type="Italic">Plasmodium falciparum</Emphasis>

The ocean covers more than 70% of earth surface and hosts most 300,000 described species of plants and animals to use, which have been virtually unexploited for the development of medicines. Marine plants are the good source of biologically active entities which exhibit therapeutic properties, when applied single or in combination of different plant extracts (polyherbal). Polyherbal preparations are always a complex mixture of different forms and thus different compounds, which might act as agonistic, synergistic, complementary, antagonistic or toxic way. The present study was initially carried out to test the antiplasmodial activity of 13 mangrove plants and eight seaweeds species distributed along the coast of south India. Of these, mangrove species Aegiceras corniculatum and the seaweed species Chaetomorpha antennina have shown maximum antiplasmodial activity. Hence, the present study was mooted out to increase the percentage of antiplasmodial activity when applied as polyherbal preparations. The effect of marine polyherbal preparations from the methanolic extracts of two marine plants A. corniculatum and C. antennina for their antiplasmodial activity was tested. It shows that the polyherbal extract showed 63.50 ± 0.408% suppression of parasitaemia against Plasmodium falciparum at 1.5 mg ml⁻¹ concentration. In vivo test was carried out with rat animal model to find out the effectiveness of the polyherbal extracts in the live system, which reveals that polyherbal extracts have exhibited remarkable antiplasmodial activity (50.57 ± 0.465%) against Plasmodium berghei at 120 mg kg⁻¹ bw. This study shows that combinations of mangrove plants and seaweeds extracts had a source of lead compounds for the development of new drugs for the treatment of malaria.     

Molecular identification of <Emphasis Type="Italic">Trichuris vulpis</Emphasis> and <Emphasis Type="Italic">Trichuris suis</Emphasis> isolated from different hosts

Trichuris suis was isolated from the cecum of two different hosts (Sus scrofa domestica—swine and Sus scrofa scrofa—wild boar) and Trichuris vulpis from dogs in Sevilla, Spain. Genomic DNA was isolated and internal transcribed spacers (ITS)1-5.8S-ITS2 segment from the ribosomal DNA (rDNA) was amplified and sequenced using polymerase chain reaction techniques. The sequence of T. suis from both hosts was 1,396 bp in length while that of T. vulpis was 1,044 bp. ITS1 of both populations isolated of T. suis was 661 nucleotides in length, while the ITS2 was 534 nucleotides in length. Furthermore, the ITS1 of T. vulpis was 410 nucleotides in length, while the ITS2 was 433 nucleotides in length. One hundred fifty-four nucleotides were observed along the 5.8S gene of T. suis and T. vulpis. Intraindividual and intraspecific variations were detected in the rDNA of both species. The presence of microsatellites was observed in all the individuals assayed. Sequence analysis of the ITSs and the 5.8S gene has demonstrated no sequence differences between T. suis isolated from both hosts (S. scrofa domestica—swine and S. scrofa scrofa—wild boar). Nevertheless, clear differences were detected between the ITS1 and ITS2 of T. suis and T. vulpis. Furthermore, a comparative molecular analysis between both species and the previously published ITS1-5.8S-ITS2 sequence data of Trichuris ovis, Trichuris leporis, Trichuris muris, Trichuris arvicolae, and Trichuris skrjabini was carried out. A common homology zone was detected in the ITS1 sequence of all species of trichurids.     

Surface features of the monogenean gill parasites <Emphasis Type="Italic">Pseudodactylogyrus anguillae</Emphasis> and <Emphasis Type="Italic">Pseudodactylogyrus bini</Emphasis> from the European eel <Emphasis Type="Italic">Anguilla anguilla</Emphasis> in Egypt

In the present study, scanning electron microscopy is used to demonstrate the surface features of Pseudodactylogyrus anguillae Yin and Sproston (1948) Gussev, 1965 and Pseudodactylogyrus bini Kikuchi (1929) Gussev, 1965 from the European eel Anguilla anguilla. Specimens of the eel A. anguilla were collected from the River Nile, near Mansoura, Dakahlia Province, Egypt. Morphologically, P. anguillae could be distinguished from P. bini based on the total length, the shape and size of the haptor, and the constriction between the haptor and body proper. The general body tegument and the haptoral tegument of both species are microvillous. Possible functions of the microvilli associated with the tegument covering the anterior adhesive areas are discussed. The region of the body proper just anterior to the haptor of P. anguillae, appears to be highly folded and provided with many projections forming scale-like structures. Many surface ciliary sensilla presumed to be sensory structures are found on the tegument of the anterior adhesive area of P. anguillae. Secretory bodies and mucus-like substances are recorded on the tegument covering the anterior adhesive areas of P. anguillae and the haptor of P. bini. The possible role of these secretions in the attachment of the anterior adhesive areas and the posterior attachment haptor to the host tissues are also discussed.     

Functional polymorphisms in <Emphasis Type="Italic">XRCC</Emphasis>-<Emphasis Type="Italic">1</Emphasis> and <Emphasis Type="Italic">APE</Emphasis>-<Emphasis Type="Italic">1</Emphasis> contribute to increased apoptosis and risk of ulcerative colitis

Objective  

The present study was designed to investigate the role of X-ray cross-complementing group 1 (XRCC1) and apurinic/apyrimidinic endonuclease 1 (APE1) polymorphisms in apoptosis and the risk of ulcerative colitis (UC).     

<Emphasis Type="Italic">Trichinella</Emphasis> T6 and <Emphasis Type="Italic">Trichinella nativa</Emphasis> in Wolverines (<Emphasis Type="Italic">Gulo gulo</Emphasis>) from Nunavut,Canada

Infection of Trichinella spp. is common among animals in the Canadian Arctic. We determined the prevalence of Trichinella spp. infection in wolverines (Gulo gulo) from Nunavut, Canada. Diaphragms from 41 wolverines were examined by artificial digestion. Trichinella spp. larvae were detected in 36 (87.8%) examined animals. Trichinella T6 was detected in 33 (91.7%), Trichinella nativa in only one (2.8%), and a mixed Trichinella T6 and T. nativa infections were detected in two (5.6%) wolverines. This is the first report of Trichinella spp. infection in wolverines from Nunavut and the first report of sympatric Trichinella T6 and T. nativa in any host. The high prevalence of Trichinella spp. infection in combination with the natural history of wolverines suggests that the mustelid may be a key species in the natural cycle of these parasites in Arctic and Subarctic areas.     

Transmission of <Emphasis Type="Italic">Yersinia pestis</Emphasis> cultures with different plasmid content from <Emphasis Type="Italic">Xenopsylla cheopis</Emphasis> to <Emphasis Type="Italic">Calomys callosus</Emphasis>

Most Brazilian Yersinia pestis isolates display a typical plasmid profile composed of the three classical plasmids: pYV, pPst and pFra. However, some cultures lack at least one of these plasmids, while a few of them harbour atypical DNA bands of molecular weight ranging from 147 to 11.5 kb. To investigate whether Y. pestis displaying atypical plasmid content could be propagated among rodents in nature through flea bites, we carried out studies with fleas ( Xenopsylla cheopis) and rodents ( Calomys callosus) reared in the laboratory and five Y. pestis cultures differing in plasmid content. The results suggest that: (1) the single presence of pYV is not sufficient for the transmission of Y. pestis by fleas, (2) pPst is not essential for transmission, (3) two atypical DNA bands of molecular weight of 30 kb and >90 kb have no biological role, and (4) pFra is required for the transmission of Y. pestis by flea bites. Other studies are needed to determine whether this plasmid alone is sufficient for transmission.     

Uncommonly isolated clinical <Emphasis Type="Italic">Pseudomonas</Emphasis>: identification and phylogenetic assignation

Fifty-two Pseudomonas strains that were difficult to identify at the species level in the phenotypic routine characterizations employed by clinical microbiology laboratories were selected for genotypic-based analysis. Species level identifications were done initially by partial sequencing of the DNA dependent RNA polymerase sub-unit D gene (rpoD). Two other gene sequences, for the small sub-unit ribosonal RNA (16S rRNA) and for DNA gyrase sub-unit B (gyrB) were added in a multilocus sequence analysis (MLSA) study to confirm the species identifications. These sequences were analyzed with a collection of reference sequences from the type strains of 161 Pseudomonas species within an in-house multi-locus sequence analysis database. Whole-cell matrix-assisted laser-desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) analyses of these strains complemented the DNA sequenced-based phylogenetic analyses and were observed to be in accordance with the results of the sequence data. Twenty-three out of 52 strains were assigned to 12 recognized species not commonly detected in clinical specimens and 29 (56 %) were considered representatives of at least ten putative new species. Most strains were distributed within the P. fluorescens and P. aeruginosa lineages. The value of rpoD sequences in species-level identifications for Pseudomonas is emphasized. The correct species identifications of clinical strains is essential for establishing the intrinsic antibiotic resistance patterns and improved treatment plans.