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.     

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.     

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.     

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).     

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.     

Anurans as paratenic hosts in the transmission of <Emphasis Type="Italic">Hepatozoon caimani</Emphasis> to caimans <Emphasis Type="Italic">Caiman yacare</Emphasis> and <Emphasis Type="Italic">Caiman latirostris</Emphasis>

Prevalence of Hepatozoon caimani has been reported in 76% of caimans Caiman yacare from the Pantanal region. Culex (Melanoconion) spp. mosquitoes were recently identified as natural vectors of this parasite. However, culicids are not typically eaten by crocodilians, suggesting that the main transmission route is through ingestion of insectivorous vertebrates, such as anurans. The susceptibility of wild frogs Leptodactylus chaquensis, Leptodactylus podicipinus and Scinax nasicus to infection by H. caimani was verified. Wild-caught anurans were force fed with sporulated oocysts from laboratory-bred Culex (Melanoconion) mosquitoes. Frogs were killed 30 days postinfection, and their internal organs were fed to caimans C. yacare and Caiman latirostris. Cystozoites were identified in fresh liver impression smears of L. chaquensis. C. yacare fed on anuran organ presented gametocytes in peripheral blood circulation between 74 and 80 days postinoculation (dpi). Gametocytes were also verified in C. latirostris fed on the internal organs of L. podicipinus and S. nasicus between 60–70 and 69–75 dpi, respectively. Since frogs used in experiment are sympatric with C. yacare and C. latirostris and may occur in the diet of these caimans, the results suggest these amphibians are paratenic hosts in the natural transmission cycle of H. caimani in Pantanal.     

Antibodies to <Emphasis Type="Italic">Neospora caninum</Emphasis> and <Emphasis Type="Italic">Toxoplasma gondii</Emphasis> in domestic cats from Brazil

The occurrence of antibodies to Neospora caninum and Toxoplasma gondii was determined in 400 domestic cats (Siamese, Persian, and undetermined breeds) from the Municipality of Araçatuba, Sao Paulo, Brazil, through the indirect fluorescence antibody test (IFAT). Of the 400 cats, 100 were seropositive to T. gondii (25%, titer ≥64) and 98 to N. caninum (24.5%, titer ≥16). The rate of seropositive cats for T. gondii was correlated with age (χ ²=35.7; p<0.001), with a higher number of infected animals at older ages. Of the 219 cats younger than 1-year-old, 13.2% were seropositive for T. gondii, while 39.2% were positive in the 181 older animals. The presence of N. caninum was also correlated with age (÷²=8.8; p<0.01), with 18.7% (41/219) and 31.5% (57/181) of positive animals at ages below and above 12-month, respectively. An association between the occurrences of both protozoa in the felines was also observed (χ ²=19.6; p<0.001).     

A member of a new <Emphasis Type="Italic">Tospovirus</Emphasis> species isolated in Italy from wild buckwheat (<Emphasis Type="Italic">Polygonum convolvulus)</Emphasis>

Electron microscopy of extracts from diseased Polygonum convolvulus plants from Piedmont (Italy) revealed particles with the morphological features of a tospovirus. Sequencing of the full-length small (S) and medium (M) genome segments indicated that the virus is a member of a new Tospovirus species provisionally named Polygonum ringspot virus. A feature distinguishing it from members of other Tospovirus species was the presence of a very short intergenic region on the S segment lacking the potential for formation of the predicted hairpin structure involved in subgenomic expression. Antibodies made against purified nucleocapsids allowed serological comparison with other tospovirus isolates and revealed a relationship with tomato yellow ring virus, and to a lesser extent, to iris yellow spot virus. Serological tests detected the virus in various locations in northern and central Italy. The experimental host range was wide, although in nature the virus appeared restricted to two Polygonum species. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Genotyping <Emphasis Type="Italic">Borrelia burgdorferi sensu lato</Emphasis> isolates from <Emphasis Type="Italic">Ixodes ricinus</Emphasis> ticks in Russia and Ukraine

The four Borrelia burgdorferi sensu lato isolates obtained from I. ricinus ticks collected from the natural loci in Russia and Ukraine that had an unusual RFLP MseI pattern were studied using the sequencing of the rrfA-rrlB spacer and the rrs gene. Isolate Ir-5215 from a tick collected in southern Ukraine represented the recently described genospecies B. spielmanii, which is pathogenic for humans. The three atypical isolates Ir-3519, Ir-4721, and Ir-4812 had 100% identity with the sequence of atypical European B. burgdorferi sensu stricto strains; they constituted a subgroup of B. burgdorferi sensu stricto based on multilocus sequence analysis (MLSA). These data are indicative of the genetic heterogeneity of the current group B. burgdorferi sensu stricto.     

<Emphasis Type="Italic">Hepatozoon canis</Emphasis> infection in ticks during spring and summer in Italy

Hepatozoon canis is a common protozoan of dogs, being among the most prevalent tick-borne pathogens infecting dogs around the world. It is primarily transmitted by Rhipicephalus sanguineus, the brown dog tick. In this study we tested ticks collected from dogs and from the environment in order to track the origin of an outbreak of H. canis infection detected in October 2009 in a private dog shelter in southern Italy. Ticks from dogs (n = 267) were collected during the spring of 2009, whereas ticks from environment (n = 300) were found on sticky traps placed in the same shelter during the summer of 2009. All ticks were tested by PCR for the detection of a H. canis 18S ribosomal RNA gene fragment. Four (1.5%, one female and three males) ticks collected from dogs were PCR positive. None of the larvae collected from the environment were positive, but a relatively high infection rate (8.0%) was detected in nymphs. These findings point out that dogs became infected during the summer, when ticks were abundant and highly infected by H. canis. Moreover, this study suggests that castor oil sticky traps might be useful to collect engorged immature ticks in highly infested environments (e.g., dog shelters). This might be particularly interesting to evaluate the level of infection by certain pathogens in free-ranging ticks R. sanguineus, as done in the present study.     

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.     

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).     

Occurrence of <Emphasis Type="Italic">Cryptosporidium parvum</Emphasis> and <Emphasis Type="Italic">Giardia duodenalis</Emphasis> in healthy adult domestic ruminants

To determine the prevalence and intensity of infection of Cryptosporidium spp. and Giardia duodenalis in healthy adult domestic ruminants, faecal samples were collected from 379 cattle of between 3 and 13 years old, 446 sheep and 116 goats selected at random from 60 dairy farms and 38 and 20 herds, respectively, in Galicia (NW Spain). Cryptosporidium spp. oocysts were detected in 32 cows (8.4%), 24 sheep (5.3%) and in nine goats (7.7%) from, respectively, 48.3% of the farms and 34.2 and 30.0% of the herds. The intensity of infection in cows ranged between 25 and 5,924 oocysts per gram of faeces (OPG), whereas in sheep and goats, the number of oocysts shed ranged from 8–515 OPG and from 17–782 OPG, respectively. Parasitization by Cryptosporidium spp. was significantly higher (P < 0.05) in cows than in sheep and goats. G. duodenalis cysts were identified in 101 cows (26.6%), 86 sheep (19.2%) and 23 goats (19.8%) from, respectively, 96.6% of the farms and 92.1 and 90% of the herds. The number of cysts shed by cows ranged between 15 and 3,042 cyst per gram of faeces (CPG), whereas the intensity of infection in sheep and goats ranged from 16–3010 CPG and from 15–1845 CPG, respectively, and was significantly lower (P < 0.05) than in cows and sheep. The number of Cryptosporidium spp. oocysts isolated from sheep and goats was insufficient for successful polymerase chain reaction analysis. Nevertheless, gene sequence analysis of the hsp70 and 18SrRNA genes of Cryptosporidium revealed the presence of only C. parvum in faecal samples from cows. Genotyping studies of the β-giardin and glutamate dehydrogenase genes of G. duodenalis revealed mainly assemblage E of Giardia in cows, sheep and goat faecal samples. Assemblage B of G. duodenalis was also detected in one sheep sample. These animals should be considered as a possible source of cryptosporidiosis and giardiosis, thereby maintaining the infections on farms and in herds.     

<Emphasis Type="Italic">Helicobacter pylori</Emphasis> and <Emphasis Type="Italic">cagA</Emphasis> and <Emphasis Type="Italic">vacA</Emphasis> gene status in children from Brazil with chronic gastritis

Helicobacter pylori has been shown to be strongly associated with chronic gastritis, gastric and duodenal ulceration, and is a risk factor for gastric carcinoma. Histology, urease, culture, and polymerase chain reaction have been employed as for H. pylori diagnostic methods, pre and post treatment or during follow-up of dyspeptic adult individuals referred for endoscopy. In order to obtain a more-sensitive and specific method for H. pylori detection, we evaluated gastric body and antrum biopsies of 134 consecutive Brazilian consecutive dyspeptic children aged 1-16 years by rapid urease test, histology and polymerase chain reaction using two pairs of oligonucleotides. Our results indicated that polymerase chain reaction with Southern blotting and hybridization with specific chemiluminescent probes increased the number of positive H. pylori patients by 35%. The genotyping of H. pylori strains directly from gastric biopsy using the same nucleic acid methodology revealed that there is no association of chronic gastritis in our infant patients with vacA s1 and the presence of the cagA gene. These data suggest an initial infection of children with normal mucosa and probably others factors than vacA s1 genotype or the presence of the cagA gene are associated with the onset of gastric disease. Altogether, our results reinforce the need for using more sensitive diagnostic methods in order to understand the role of H. pylori in the genesis of gastric disease in children and its progression in adults.     

<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.