Molecular biological comparison of different Besnoitia species and stages from different countries

Besnoitia besnoiti tissue cysts from a recent outbreak in cattle in Germany were characterized with respect to their internal transcribed spacer regions 1, 2, and 18S rDNA gene sequences. These results were compared with own sequences of an Israelian isolate of B. besnoiti and of Besnoitia jellisoni cystozoites stored for years in liquid nitrogen. Furthermore, material was studied that was obtained from white mice (Balb/C) that had been successfully infected by intraperitoneal infection of fresh cystozoites from the German outbreak. All results were then compared and discussed with respect to databank sequences of other Besnoitia species. Comprehensive phylogenetic studies of B. besnoiti isolates from Germany revealed almost identical sequence alignments when compared to previously sequenced B. besnoiti isolates from Israel and Spain. More importantly, phylogenetic analysis revealed two distant clusters of Besnoitia species: the first one includes Besnoitia akodoni, Besnoitia darlingi, and Besnoitia oryctofelisi, while the second cluster includes B. besnoiti, Besnoitia bennetti, Besnoitia tarandi, and the Besnoitia species of rodents (B. jellisoni). The also B. jellisoni named species of the GenBank (AF 076860) must be another one, since our strain derives directly from Frenkel. These findings give strong hints that B. besnoiti has a cycle between rodents and a predator and that cattle and other are only accidental hosts.     

Characterization of <Emphasis Type="Italic">Oesophagostomum</Emphasis> spp. from pigs in China by PCR-based approaches using genetic markers in the internal transcribed spacers of ribosomal DNA

In the present study, samples of Oesophagostomum spp. collected from pigs from different geographical localities in mainland China were characterized genetically by polymerase chain reaction-linked single-strand conformation polymorphism (PCR-SSCP) and restriction fragment length polymorphism (PCR-RFLP) techniques using genetic markers in the internal transcribed spacers (ITS) of nuclear ribosomal DNA (rDNA). The second internal transcribed spacer (ITS-2) was amplified from 51 individual nodule worms by PCR, and the amplicons were analyzed by SSCP. With the exception of slight microheterogeneity, SSCP analyses displayed two distinct banding profiles that allowed the identification of all Oesophagostomum spp. samples examined into two groups, the first one represented O. dentatum, and the second one may represent O. quadrispinulatum. Then, the entire ITS was amplified from individual samples, and the amplicons were digested with restriction endonuclease Pst I. The results of RFLP analyses were consistent with that of SSCP. Sequence analysis of ITS rDNA supported the identification and differentiation of Chinese Oesophagostomum spp. samples into two species, namely, O. dentatum and O. quadrispinulatum. These PCR-based approaches provide useful complementary tools to traditional methods for the accurate identification of Oesophagostomum spp. (irrespective of developmental stage) and have implications for studying the ecology and population genetic structures of these parasites and for the prevention and control of the diseases they cause.     

Genetic characterization of <Emphasis Type="Italic">Fasciola hepatica</Emphasis> from Tunisia and Algeria based on mitochondrial and nuclear DNA sequences

Fasciolosis caused by Fasciola spp. (Platyhelminthes: Trematoda: Digenea) is considered the most important helminth infection of ruminants in tropical countries, causing considerable socioeconomic problems. In the present study, samples identified morphologically as Fasciola hepatica from sheep and cattle from different geographical locations of Tunisia and Algeria were genetically characterised by sequences of the first (ITS-1), the 5.8S and second (ITS-2) Internal Transcribed Spacers (ITS) of nuclear ribosomal DNA (rDNA) and mitochondrial Cytochrome c Oxidase subunit I (COI) gene. Comparison of the ITS and COI sequences of the North African samples with sequences of Fasciola spp. from GenBank confirmed that all samples from Tunisia and Algeria samples belong to a single species, namely F. hepatica. Several specimens from Tunisia and Algeria showed a substitution C/T in position 859 in the ITS-2 sequences, previously reported from Spain, suggesting that the above mentioned variant may have a common origin and spread recently throughout the three countries because of movement of infected animals. This is the first molecular characterization of F. hepatica in North Africa which provides a foundation for further studies on Fasciola spp. in Tunisia and Algeria.     

Molecular characterization of human Fasciola samples in Gilan province, Northern Iran on the basis of DNA sequences of ribosomal and mitochondrial DNA genes

The two species of Fasciola hepatica and Fasciola gigantica are the causative agents of fasciolosis in domestic animals and humans. Based on the morphometric data, differential diagnosis between these flukes is problematic. In addition, intermediate forms of Fasciola have recently been found in the Northern Iran, based on morphometrical analysis. The aim of the present study was to indicate the phenotypic and genetic characterization of the causative agent of human fasciolosis in the endemic area of Gilan province, Northern Iran. Sequence analysis of ribosomal (ITS1) and mitochondrial (CO1 and ND1) genes were used for the genetic characterization of two fasciolid adult specimens directly obtained from humans. Comparison of the morphometric and sequencing data of the Iranian Fasciola samples obtained in the present study with those previously reported for F. hepatica, F. gigantica, and the “intermediate Fasciola” revealed that both of our human Fasciola samples represent F. hepatica. This is the first demonstration of the existence of F. hepatica in the Iranian population by a genetic approach. Also, the results of the present study showed the occurrence of a similar sequence polymorphism for CO1 and ND1 in both Iranian human F. hepatica isolates, which exhibit?100% identity in ITS1 and CO1, to those of F. hepatica previously reported from a Japanese man.     

Molecular variability and genetic structure of the population of <Emphasis Type="Italic">Onion yellow dwarf virus</Emphasis> infecting garlic in Iran

Onion yellow dwarf virus (OYDV) is one of the most important viral diseases of garlic crops worldwide. This study surveyed the occurrence of OYDV in 26 garlic ecotypes collected from different regions in Iran during 2008–2009. Using an electron microscope, we detected filamentous particles with about 700–800 nm in length and 12 nm in width in five samples. These features are typical of the genus Potyvirus. The coat protein (CP) gene from 26 samples was PCR amplified, cloned, sequenced, and compared with the sequences available in GenBank. Phylogenetic analysis using 235 deduced amino acids of the CP gene showed that virus isolates fell into two groups, group A and group B. Members of group A were divided into two subgroups: A-I and A-II. The subgroup A-I appears to be a new subgroup comprising 17 Iranian isolates. The identity levels among the amino acid of 26 Iranian isolates ranged between 90 and 100%. The results indicated that the genetic diversity found in Iran is due to local OYDV populations rather than introduction from other geographical regions. This study is the first report about the molecular structure and geographically diverse range of OYDV populations in this country.     

Genetic characterization and phylogenetic analysis of Trypanosoma evansi in Iranian dromedary camels

Whole blood samples were collected from 117 male clinically healthy Camelus dromedarius aged between 6 months to 18 years from several farms in Yazd Province of Iran. Trypanosoma evansi-affected camels were detected by Giemsa-stained blood smears, and the positive blood samples (4 out of 117) were submitted to PCR examination and phylogenetic analysis. Basic Local Alignment Search Tool data of the obtained complete internal transcribed spacer (ITS) sequences revealed that they corresponded to those of T. evansi, Thailand cattle isolate (AY912276) with the homology of 99 %. Both phylogenetic trees generated by ITS1 and complete ITS were unable to clearly show inter- and intraspecific genetic diversity of Trypanosoma spp. isolates. The phylogenetic tree inferred from the ITS2 nucleotide sequences (569 bp) clearly showed the genetic diversity of the parasites. Phylogenetic and molecular analyses of this region showed that two distinct genotypes of T. evansi in Iranian dromedary camels are present. In contrast to the ITS1 and ITS2 regions, multiple alignment of the nucleotide sequence of the 5.8S rRNA showed a high degree of sequence conservation during evolution in various Trypanosoma spp.     

Genetic characterization and phylogenetic analysis of Eimeria arloingi in Iranian native kids

Among the 16 species of Eimeria from goats, Eimeria arloingi and Eimeria ninakohlyakimovae are regarded as the most pathogenic species in the world and cause clinical caprine coccidiosis. E. arloingi is known to be an important cause of coccidiosis in Iranian kids. Molecular analyses of two portions of nuclear ribosomal DNA (internal transcribed spacer1 (ITS1) and 18S rDNA) were used for the genetic characterization of the E. arloingi. Comparison of the sequencing data of E. arloingi obtained in the present study (ITS1: KC507793 and 18S rDNA: KC507792) with other Eimeria species in the GenBank database revealed a particularly close relationship between E. arloingi and Eimeria spp. from the cattle and sheep. The phylogram based on the ITS1 sequences shows that the E. arloingi, Eimeria bovis, and Eimeria zuernii formed a distinct group separate from the other remaining Eimeria spp. in cattle and poultry. In pairwise alignment, 18S rDNA sequence derived from E. arloingi showed 99 % similarity to Eimeria ahsata with differences observed at only three nucleotides. This study showed that the ITS1 and 18S rDNA gene are useful genetic markers for the specific identification and differentiation of Eimeria spp. in ruminants.     

Heterobasidion annosum 5.8s ribosomal DNA and internal transcribed spacer sequence: rapid identification of European intersterility groups by ribosomal DNA restriction polymorphism

Using conserved fungal ribosomal gene sequences the internal transcribed spacer (ITS) regions one and two (ITS1, ITS2) and the 5.8s ribosomal RNA gene (rRNA) of Heterobasidion annosum were amplified by the polymerase chain reaction (PCR). The nucleotide sequence was determined in three European intersterility groups (ISG-S,-F and-P). Three sequence variants of the ITS were found in ISG-S isolates. The sequence of the ITS of ISG-F differed by two residues from the major ISG-S sequence variant. The ISG-P sequence differed from ISG-S and ISG-F at 15–16 and 16 residues, respectively. Amplified intergenic spacer elements were informative for ISG fingerprinting following digestion with various 4-cutter restriction endonucleases. All differences in the restriction fragments between the ISGs were because of sequence differences in the ITS regions. The fingerprint patterns of isolates from the same intersterility group but from different European localities were identical. These results show that ribosomal DNA finger-printing is a rapid technique to identify ISGs in Heterobasidion annosum.     

Genetic structure and molecular variability of potato virus M populations

To investigate the genetic diversity of potato virus M (PVM; genus Carlavirus, family Betaflexiviridae), the complete nucleotide sequence of the coat protein gene of 30 PVM isolates from a major potato-growing region in Iran were determined. Phylogenetic analysis of these Iranian PVM isolates together with those available in the GenBank database suggested two divergent evolutionary lineages that did not reflect the origin of the isolates, and these were designated as PVM-o and PVM-d. Examination of the genetic variability of the coat protein of Iranian isolates and their counterparts whose sequences are available in the Genbank database revealed 16 genotype groups in the PVM population. Analysis of the synonymous-to-nonsynonymous ratio showed strong purifying selection in the CP gene in the genotype groups of divergent clades.     

Prevalence and molecular characteristics of <Emphasis Type="Italic">Enterocytozoon bieneusi</Emphasis> in cattle in Korea

Enterocytozoon bieneusi is the most common microsporidium associated with AIDS patients. Moreover, its detection in increasing numbers of immunocompetent patients has made it an emerging pathogen. This organism was also identified in a wide range of animals, and the zoonotic potential of human infections is of particular interest. In this study, 538 fecal samples from cattle in Korea were analyzed for the presence of E. bieneusi by PCR. Approximately 15% were found to be positive, with higher rates being detected over the summer months. The internal transcribed spacer (ITS) regions of the rRNA gene of ten E. bieneusi positive samples were amplified using nested PCR and sequenced. Genetic polymorphisms, which were represented by six distinct genotypes (CEbA–CEbF), were found among the E. bieneusi isolates. Five isolates from this study had identical ribosomal ITS to the previously known E. bieneusi genotype ITSs in cattle and other animals. Four isolates were previously unreported but were quite similar to the previously known genotypes of E. bieneusi from cattle and other animals. One isolate was identical to the human E. bieneusi type D, which indicated some E. bieneusi isolates from cattle in the country may be of public health importance. To the best of my knowledge, this is the first report of E. bieneusi study in cattle in Asia.     

No detection of Besnoitia besnoiti DNA in the semen of chronically infected bulls

Bovine besnoitiosis is a chronic and debilitating disease observed in many European countries that may cause important economic losses in cattle. The recent widespread of the parasite in Europe had led the European Food Safety Authority to declare bovine besnoitiosis as a re-emerging disease in Europe. Many aspects of the epidemiology of bovine besnoitiosis such as the main routes of transmission are still unclear and need to be further studied. Among the different hypotheses, a sexual transmission has not yet been investigated. Therefore, the aim of this study was to evaluate the presence of Besnoitia besnoiti DNA in the semen of naturally infected bulls by using a highly sensitive method (real-time qPCR). Both pre-sperm and sperm fractions of 40 bulls, including seronegative (n?=?11), seropositive subclinically (n?=?17), and seropositive clinically (n?=?12) infected animals, were collected by electroejaculation and analyzed by real-time qPCR. No B. besnoiti DNA was detected in 27 pre-sperm and 28 sperm fractions of the 40 examined bulls, suggesting that the transmission of B. besnoiti infection by the semen of chronically infected bulls is very unlikely.     

Differentiating betweenBesnoitia besnoiti from cattle andSarcocystis hoarensis from rodents

To provide a biological basis for studies designed to establish the mode of transmission of the veterinary pathogenBesnoitia besnoiti, we compared salient features of this pathogen in cattle with those ofSarcocystis hoarensis in rodents. The cysts and cystozoites of these organisms can readily be distinguished morphologically. In contrast toS. hoarensis which is well adapted to rodents,B. besnoiti fails to mature in jirds or mice and generally is lethal in jirds. Serological reagents discriminately detect these pathogens.B. besnoiti, therefore, can unambiguously be differentiated fromS. hoarensis either by morphological or serological methods or on the basis of experimental comparisons of virulence in laboratory rodents.     

Analysis of Iranian <Emphasis Type="Italic">Potato virus S</Emphasis> isolates

Two hundred forty potato samples with one or more symptoms of leaf mosaic, distortion, mottling and yellowing were collected between 2005 and 2008 from seven Iranian provinces. Forty-four of these samples tested positive with double-antibody sandwich enzyme-linked immunosorbent assays (DAS-ELISA) using a Potato virus S (PVS) polyclonal antibody. Of these 12 isolates of PVS were selected based on the geographical location for biological and molecular characterization. The full coat protein (CP) and 11K genes from 12 PVS isolates were PCR amplified, cloned and sequenced. All 12 PVS isolates showed mosaic symptoms on Nicotiana debneyii and N. tabacum cv. Whiteburly and local lesion on Chenopodium amaranticolor, C. quinoa and C. album. The Iranian isolates share between 93 and 100% pairwise nucleotide identity with other PVS^O isolates. Based on maximum likelihood phylogenetic analysis coupled with pairwise identity analysis, we propose 15 genotypes for the PVS^O strain and 3 genotypes for the PVS^A strain.     

The phylogeny of the tomato leaf mould fungus Cladosporium fulvum syn. Fulvia fulva by analysis of rDNA sequences

The nucleotide sequence of part of the ribosomal DNA from races of the fungal tomato pathogen Cladosporium fulvum and other Cladosporium species have been determined. Comparisons of the internal transcribed spacer regions (ITS1 and ITS2) of several C. fulvum races showed complete sequence homology suggesting a recent evolutionary divergence. Comparisons of these nucleotide sequences in the ITS region with those of other Cladosporium species showed the close relationship within the Cladosporium genus. Using the nucleotide sequence of part of the 18s ribosomal subunit from these isolates and comparing them with sequences of some Ascomycetes, Basidiomycetes and Chytridiomycetes, obtained from GenBank, we infer the phylogeny of the Cladosporium species studied here. Our analysis shows that the Cladosporia form a monophyletic group which falls within the order Ascomycotina.     

Molecular characterization of <Emphasis Type="Italic">Trichomonas vaginalis</Emphasis> isolates from the Philippines

Trichomonas vaginalis is a human urogenital pathogen that causes trichomoniasis, the most common nonviral parasitic sexually transmitted infection in the world. Presently, there are no reports on comparative sequence analysis as well as on the identification of phylogenetic positions of T. vaginalis isolates from the Philippines relative to known trichomonads. In this study, 5.8S rDNA and the flanking internal transcribed spacer (ITS) regions of 57 T. vaginalis isolates were sequenced. The phylogenetic positions of the isolates relative to known trichomonads were determined using the model-based (GTR+Γ+I) neighbor-joining, maximum likelihood, and Bayesian-inference analyses and the nonmodel-based maximum parsimony analysis. Construction of a phylogenetic tree showed the clustering of all the sequences in one branch together with other T. vaginalis strains obtained through basic local alignment search tool search. Sequencing of the 5.8S rDNA gene and the flanking ITS1and ITS2 regions of T. vaginalis isolates from the Philippines demonstrated low genetic polymorphism. However, comparison of the ribosomal DNA sequences may have implications on some phenotypic characteristics of T. vaginalis.     

Genetic characterization of Trichomonas gallinae (Rivolta, 1878) in companion birds in Japan and the genotypical relationship in the Asia region

Background/purposeAvian trichomonosis is a parasitic infection that affects a wide range of avian species, including free-ranging and pet birds worldwide, and Trichomonas gallinae has been considered as the only causative agent for decades. The sequence of the 5.8S ribosomal RNA with internal transcribed spacer (ITS) regions was widely used for identifying genotypes and determining inter-specific and intra-specific diversity. Moreover, the sequence of Fe-hydrogenase (FeHyd) was proposed as the second genetic marker for providing improved resolution of strain subtyping discrimination. Though the correlation between genetic variability and strain virulence is controversial, FeHyd analyses seemed to be useful to investigate the host or geographic origin of isolates. This study aimed to investigate the genetic characteristics of avian Trichomonas spp.MethodsForty-seven oral swabs and crop lavage fluids were collected from 9 avian genera, which were diagnosed as Trichomonas-positive by microscopy in animal hospitals in Japan, were analyzed.ResultsGenetic analysis of clonal isolates revealed the prevalence of the single genotype, ITS-OBT-Tg-1, by ITS region analysis, while two different subtypes, A2 and novel A3, were suggested by FeHyd gene analysis among Japanese companion birds. Phylogenetic analyses of available ITS sequences obtained from the Asia region (China, Iran, Iraq, and Saudi Arabia) were also preformed, revealing endemic ITS-OBT-Tg-1, ITS-OBT-Tg-2, ITS-OBT-Ttl-1, genotype III, and Saudi Arabia's unique lineages. Furthermore, ITS-OBT-Tg-2 predominance in these countries indicates different strains origination from Japan.ConclusionThis is the first report of the genetic characterization of T. gallinae in Japan with discovery of novel subtype A3.     

Molecular characterization of <Emphasis Type="Italic">Atractolytocestus sagittatus</Emphasis> (Cestoda: Caryophyllidea), monozoic parasite of common carp,and its differentiation from the invasive species <Emphasis Type="Italic">Atractolytocestus huronensis</Emphasis>

Sequence structure of complete internal transcribed spacer 1 and 2 (ITS1 and ITS2) of the ribosomal DNA region and partial mitochondrial cytochrome c oxidase subunit I (cox1) gene sequences were studied in the monozoic tapeworm Atractolytocestus sagittatus (Kulakovskaya et Akhmerov, 1965) (Cestoda: Caryophyllidea), a parasite of common carp (Cyprinus carpio carpio L.). Intraindividual sequence diversity was observed in both ribosomal spacers. In ITS1, a total number of 19 recombinant clones yielded eight different sequence types (pairwise sequence identity, 99.7–100%) which, however, did not resemble the structure typical for divergent intragenomic ITS copies (paralogues). Polymorphism was displayed by several single nucleotide mutations present exclusively in single clones, but variation in the number of short repetitive motifs was not observed. In ITS2, a total of 21 recombinant clones yielded ten different sequence types (pairwise sequence identity, 97.5–100%). They were mostly characterized by a varying number of (TCGT)_n repeats resulting in assortment of ITS2 sequences into two sequence variants, which reflected the structure specific for ITS paralogues. The third DNA region analysed, mitochondrial cox1 gene (669 bp) was detected to be 100% identical in all studied A. sagittatus individuals. Comparison of molecular data on A. sagittatus with those on Atractolytocestus huronensis Anthony, 1958, an invasive parasite of common carp, has shown that interspecific differences significantly exceeded intraspecific variation in both ribosomal spacers (81.4–82.5% in ITS1, 74.4–75.2% in ITS2) as well as in mitochondrial cox1, which confirms validity of both congeneric tapeworms parasitic in the same fish host.     

Genetic characterisation of <Emphasis Type="Italic">Fasciola</Emphasis> samples from different host species and geographical localities revealed the existence of <Emphasis Type="Italic">F. hepatica</Emphasis> and <Emphasis Type="Italic">F. gigantica</Emphasis> in Niger

In the present study, 16 samples representing Fasciola (Platyhelminthes: Trematoda: Digenea) from sheep and cattle from seven geographical locations in Niger were characterized genetically by sequences of the first (ITS-1) and second (ITS-2) internal transcribed spacers (ITS) of nuclear ribosomal DNA (rDNA). The ITS rDNA was amplified from individual liver flukes by polymerase chain reaction (PCR), and the amplicons were sequenced directly. The lengths of the ITS-1 and ITS-2 sequences were 422 and 361/362 bp, respectively, for all liver fluke samples sequenced. Comparison of the ITS sequences of the Niger Fasciola samples examined in the present study with that of Fasciola hepatica, Fasciola gigantica, and the “intermediate Fasciola” from elsewhere revealed that the Niger Fasciola samples examined represent two species, namely F. hepatica and F. gigantica. This is the first demonstration of the existence of both F. hepatica and F. gigantica in Niger by a genetic approach, which provides foundation for further studies on F. hepatica and F. gigantica in Niger and has implications for studying the population genetic structure of the Niger Fasciola and for the diagnosis and control of the disease they cause. H. Ali and L. Ai contributed equally to this work.     

Molecular characterization of <Emphasis Type="Italic">Cryptosporidium</Emphasis> from animal sources in Qinghai province of China

The presence of Cryptosporidium oocysts in 20 zoo animals of the Xining Zoo, 16 farm yaks and 42 farm goats in Qinghai province, China was investigated by an immunofluorescence test (IFT). The species and/or genotypes were determined by nested polymerase chain reaction (PCR) and sequence analysis of a fragment of the small subunit (SSU) rRNA gene. Cryptosporidium oocysts were found in 16 zoo animals, 2 yaks, and 15 goats by IFT. The IFT positive samples were further investigated by PCR, and 16 of them were found to be positive by that method also. Sequence analysis of the PCR products derived from Cryptosporidium oocysts from Black leopard (Panthera pardus), Heijing He (Grus nigricollis), Barbary sheep (Ammotragus lervia), Takin (Budorcas taxicolor), Lesser panda (Ailurus fulgens), and White-eared pheasant (Crossoptilon crossoptilon) fecal samples matched that of Cryptosporidium parvum mouse genotype. Sequence analyses of other PCR products were consistent with cervine genotype Cryptosporidium from Ibex (Capra ibex), a novel Cryptosporidium genotype from a wild yak (Bos mutus), C. bovis–like genotype from one goat sample and also a novel Cryptosporidium genotype from one other separate goat sample. The present work reports the first data on Cryptosporidium infections in animals from the Qinghai province of mountainous central western China and the first findings of the ‘cervine’ genotype in Capra ibex, C. bovis–like genotype and the new Cryptosporidium spp. in farm goat and in wild yak.     

Determination of the activity of sulfadiazine against<Emphasis Type="Italic"> Besnoitia darlingi</Emphasis> tachyzoites in cultured cells

The present study examined the efficacy of sulfadiazine in inhibiting tachyzoite production of Besnoitia darlingi in bovine turbinate cell cultures. A host cell lesion based assay was used to determine the effect of sulfadiazine on developing tachyzoites in 24-well plate format. Also, a cell culture flask assay was used to determine if the selected concentrations of sulfadiazine killed or only suppressed the development of B. darlingi. Sulfadiazine inhibited B. darlingi tachyzoite production by 50%, 66%, 84%, and 92% in cultures treated with 1.0 µg ml^–1, 2.5 µg ml^–1, 5.0 µg ml^–1, and 7.5 µg ml^–1, respectively. At least 99% inhibition of tachyzoite production was observed when infected cultures were treated with 10 µg ml^–1 sulfadiazine. Cultured host cells exposed to 10 µg ml^–1 sulfadiazine showed some signs of toxic changes. Sulfadiazine may have promise as a therapeutic agent in the treatment of other Besnoitia spp. The methods described for drug evaluation were rapid, inexpensive and allowed the concurrent assessment of the drug against the parasite and its cytotoxic effect.