The role of internal transcribed spacer 2 secondary structures in classifying mycoparasitic Ampelomyces.

Many fungi require specific growth conditions before they can be identified. Direct environmental DNA sequencing is advantageous, although for some taxa, specific primers need to be used for successful amplification of molecular markers. The internal transcribed spacer region is the preferred DNA barcode for fungi. However, inter- and intra-specific distances in ITS sequences highly vary among some fungal groups; consequently, it is not a solely reliable tool for species delineation. Ampelomyces, mycoparasites of the fungal phytopathogen order Erysiphales, can have ITS genetic differences up to 15%; this may lead to misidentification with other closely related unknown fungi. Indeed, Ampelomyces were initially misidentified as other pycnidial mycoparasites, but subsequent research showed that they differ in pycnidia morphology and culture characteristics. We investigated whether the ITS2 nucleotide content and secondary structure was different between Ampelomyces ITS2 sequences and those unrelated to this genus. To this end, we retrieved all ITS sequences referred to as Ampelomyces from the GenBank database. This analysis revealed that fungal ITS environmental DNA sequences are still being deposited in the database under the name Ampelomyces, but they do not belong to this genus. We also detected variations in the conserved hybridization model of the ITS2 proximal 5.8S and 28S stem from two Ampelomyces strains. Moreover, we suggested for the first time that pseudogenes form in the ITS region of this mycoparasite. A phylogenetic analysis based on ITS2 sequences-structures grouped the environmental sequences of putative Ampelomyces into a different clade from the Ampelomyces-containing clades. Indeed, when conducting ITS2 analysis, resolution of genetic distances between Ampelomyces and those putative Ampelomyces improved. Each clade represented a distinct consensus ITS2 S2, which suggested that different pre-ribosomal RNA (pre-rRNA) processes occur across different lineages. This study recommends the use of ITS2 S2s as an important tool to analyse environmental sequencing and unveiling the underlying evolutionary processes.

Genetic identification of medicinally used Salacia species by nrDNA ITS sequences and a PCR-RFLP assay for authentication of Salacia-related health foods.

ETHNOPHARMACOLOGICAL RELEVANCE: The roots and stems of several Salacia species have been used as traditional medicines, especially in Ayurvedic medical system for the treatment of diabetes, rheumatism, gonorrhea, amenorrhea, skin diseases, etc. Due to reported evidence supporting Salacia's beneficial effects in early-stage diabetes and other lifestyle-related diseases, Salacia-based dietary supplements and health foods have been gaining popularity in Japan and other countries in recent years. However, due to the morphological similarities between Salacia plants, particularly in the medicinally used parts (roots and stems), the authentication of the botanical identities of Salacia-derived products is challenging. AIM OF THIS STUDY: This study aims to develop a genetic approach to authenticate the medicinally used Salacia species and to determine the botanical sources of the commercially available Salacia-derived products. MATERIALS AND METHODS: The sequences of nuclear DNA internal transcribed spacer (ITS) and chloroplast trnK-rps16 region were determined and compared between 10 plant specimens from three medicinally used Salacia species as well as 48 samples of commercial crude drugs. Moreover, a PCR-restriction fragment length polymorphism (RFLP) assay was developed for rapid identification based on the ITS sequences. RESULTS: The plant specimens from the three medicinally used Salacia species showed three main types of sequences in both ITS (types I, II, III) and trnK-rps16 (i, ii, iii) regions. Combined the sequences of ITS and trnK-rps16 regions, S. reticulata and S. oblonga had type I-i and type III-iii or similar sequences, respectively. S. chinensis had type II-ii or II(536M)-i sequences. Forty-eight samples of commercial crude drugs were identified based on ITS and trnK-rps16 DNA barcode. A convenient PCR-RFLP assay using Cac8I restriction enzyme was established and applied to identify the botanical sources of health food products purchased from online retailers. All the twelve samples were identified as S. chinensis. CONCLUSION: The nrDNA ITS sequences provided useful information to authenticate Salacia species and to elucidate the phylogenetic relationship within the Salacia genus. Genetic identification results revealed that S. chinensis and S. reticulata are the major sources of commercially available Salacia-products. Based on the ITS sequences, a convenient PCR-RFLP assay was established for the identification of the medicinally used Salacia species as well as their derived health food products.

Environmental DNA can act as a biodiversity barometer of anthropogenic pressures in coastal ecosystems.

Loss of biodiversity from lower to upper trophic levels reduces overall productivity and stability of coastal ecosystems in our oceans, but rarely are these changes documented across both time and space. The characterisation of environmental DNA (eDNA) from sediment and seawater using metabarcoding offers a powerful molecular lens to observe marine biota and provides a series of 'snapshots' across a broad spectrum of eukaryotic organisms. Using these next-generation tools and downstream analytical innovations including machine learning sequence assignment algorithms and co-occurrence network analyses, we examined how anthropogenic pressures may have impacted marine biodiversity on subtropical coral reefs in Okinawa, Japan. Based on 18 S ribosomal RNA, but not ITS2 sequence data due to inconsistent amplification for this marker, as well as proxies for anthropogenic disturbance, we show that eukaryotic richness at the family level significantly increases with medium and high levels of disturbance. This change in richness coincides with compositional changes, a decrease in connectedness among taxa, an increase in fragmentation of taxon co-occurrence networks, and a shift in indicator taxa. Taken together, these findings demonstrate the ability of eDNA to act as a barometer of disturbance and provide an exemplar of how biotic networks and coral reefs may be impacted by anthropogenic activities.

Identification of opportunistic and nonopportunistic Exophiala species using high resolution melting analysis.

Exophiala is a genus comprising several species of opportunistic black yeasts. Exophiala species identification by morphological, physiological, and biochemical characteristics is challenging because of the low degree of phenotypic differences between species and its polyphyletic nature. We aimed to develop a high-resolution melting (HRM) assay based on the internal transcribed spacer (ITS) region to differentiate between pairs of clinical and environmental Exophiala species. HRM primers were designed based on the conserved ITS region of five Exophiala species (E. dermatitidis, E. phaeomuriformis, E. heteromorpha, E. xenobiotica, and E. crusticola). Environmental and clinical Exophiala isolates representing these five species (n = 109) were analyzed. The HRM assay was optimized using clinical and environmental reference isolates (n = 22), and then the results were compared with those obtained with nonreference isolates of Exophiala (n = 87) using two designed primer sets. The designed HRM assay was based on the normalized melting peak approach and two primer sets, and successfully distinguished between the five Exophiala species. The HRM1 primer set provided sufficient resolution, with a melting temperature (Tm) difference of approximately 2.5°C among the analyzed species and of approximately 1°C between E. dermatitidis and E. phaeomuriformis. HRM typing results were in agreement with those of ITS-sequence typing (100% sensitivity and specificity). The developed HRM assay can be used to ascertain the identity of Exophiala species, which may differ in clinical significance, with high accuracy. Its application to identify species directly in clinical samples and/or environmental niches may be possible in the future.

Life Cycle of the Trout Cecal Nematode, Truttaedacnitis truttae (Nematoda: Cucullanidae): Experimental and Field Observations.

Truttaedacnitis truttae is a cucullanid nematode of primarily salmonine fishes. Brown trout (Salmo trutta) in Europe reportedly become parasitized by ingesting lampreys (Lampetra planeri) carrying infective larvae. However, our field and laboratory observations suggested that North American specimens of T. truttae have an alternative life cycle. High abundances and potential impact of T. truttae in rainbow trout, Oncorhynchus mykiss, in the Colorado River drainage in Grand Canyon, where there are no lampreys, prompted a study on the transmission dynamics of this nematode. Eggs of T. truttae, collected from live gravid females, were incubated in the laboratory. Snails, Physa gyrina and Lymnaea sp., were exposed to T. truttae larvae 3-4 wk later. Active larvae of T. truttae were observed penetrating the intestinal wall of exposed snails, and worm larvae were found in the visceral tissues when examined 1 wk after exposure. Larvae in snails showed little growth and development 2 wk later and corresponded to L3 larvae. Infected snails were fed to hatchery-reared juvenile rainbow trout. Developing stages were subsequently found in the mucosal lining and lumen of trout intestines. Adult male and female (gravid) worms were found in the ceca of trout examined 5-6 mo after consuming infected snails. Larvae found in pepsin/trypsin digests and mucosal scrapings from wild, naturally infected, trout corroborate laboratory findings. Screening of Physa sp. and gammarids collected from Colorado River, Grand Canyon, for natural infections with T. truttae using the ITS1 rDNA marker gave positive results. Truttaedacnitis truttae is the second species, after Truttaedacnitis clitellarius of lake sturgeon, capable of using a snail first intermediate/paratenic host and is similar to several other cucullanids in having a histotropic phase of development in the definitive fish host.

Internal transcribed spacer (ITS) sequencing reveals considerable fungal diversity in dairy products.

Fungi are important spoilage organisms in dairy products. However, little is known about the diversity of naturally occurring spoilage fungi in raw milk and processed dairy products, due at least in part to the fact that classical fungal identification methods require considerable expertise. To gain further insight into the fungal diversity in the dairy system, we isolated fungi from raw milk, raw and pasteurized milk cheese, and yogurt using the selective dichloran rose bengal chloramphenicol agar. In total, 361 fungal isolates were obtained and further characterized by DNA sequencing of the internal transcribed spacer (ITS) region and the nuclear ribosomal large subunit (LSU) rRNA gene if needed. We conducted BLAST (https://blast.ncbi.nlm.nih.gov/Blast.cgi) searches of the ITS region sequences against the UNITE Database (https://unite.ut.ee/analysis.php), and selected other databases if needed, which allowed identification to the species level of 183 isolates and to the genus level of 107 of the 346 isolates that were successfully ITS sequenced. The isolates characterized represented 3 phyla and 19 genera; the most common genera isolated were Penicillium (25% of isolates), Debaryomyces (18%), and Candida (9%). This study not only provides, by using modern molecular tools, a baseline understanding of the types of fungi in dairy products, but also confirms that ITS sequencing is a useful approach for identification of fungal organisms found in the dairy food chain.

A systematic overview of Descolea (Agaricales) in the Nothofagaceae forests of Patagonia.

The descolea clade includes species of ectomycorrhizal basidiomycetes in the genera Descolea, Setchelliogaster, Descomyces, and Timgrovea that are known primarily from the Southern Hemisphere. Taxa in this group produce basidiomes that range in morphology from typical epigeous mushrooms (Descolea) and secotioid taxa (Setchelliogaster) to fully gasteroid species (Descomyces and Timgrovea). High intraspecific morphological variation has been reported in several species within this clade, suggesting that careful morphological and molecular studies are needed to refine species concepts. Molecular analyses of fresh Patagonian collections in conjunction with taxonomic studies have confirmed high variability in key morphological features, including overall sporocarp form, spore shape and dimensions, universal veil remnants, and cuticle configuration. Based on our synthesis, we emend the genus Descolea to include sequestrate species. We describe the new sequestrate taxon Descolea inferna sp. nov. from Nothofagaceae forests in Patagonia and we propose Cortinarius squamatus as a synonym of our new combination Descolea brunnea. We also formalize the identity of Descolea pallida as a synonym of Descolea antarctica and provide new specimens of Cortinarius archeuretus, a species that has not been encountered since the original discovery during the expeditions of Roland Thaxter in 1905-1906. Here we re-describe and transfer this species to Descolea as D. archeureta. We also discuss diagnostic features that can be used to delimitate the four known South American taxa in the descolea clade.

Comparison of Different Drying Methods for Recovery of Mushroom DNA.

Several methods have been reported for drying mushroom specimens for population genetic, taxonomic, and phylogenetic studies. However, most methods have not been directly compared for their effectiveness in preserving mushroom DNA. In this study, we compared silica gel drying at ambient temperature and oven drying at seven different temperatures. Two mushroom species representing two types of fruiting bodies were examined: the fleshy button mushroom Agaricus bisporus and the leathery shelf fungus Trametes versicolor. For each species dried with the eight methods, we assessed the mushroom water loss rate, the quality and quantity of extracted DNA, and the effectiveness of using the extracted DNA as a template for PCR amplification of two DNA fragments (ITS and a single copy gene). Dried specimens from all tested methods yielded sufficient DNA for PCR amplification of the two genes in both species. However, differences among the methods for the two species were found in: (i) the time required by different drying methods for the fresh mushroom tissue to reach a stable weight; and (ii) the relative quality and quantity of the extracted genomic DNA. Among these methods, oven drying at 70 °C for 3-4 h seemed the most efficient for preserving field mushroom samples for subsequent molecular work.

Patulin and patulin producing Penicillium spp. occurrence in apples and apple-based products including baby food.

INTRODUCTION: Patulin has raised the international attention because of its health risk. In fact, it has mutagenic, neurotoxic, immunotoxic, genotoxic and gastrointestinal effects in animals. In the present work, patulin and patulin-producing Penicillium spp. in apple and apple-based products marketed in Qatar were analysed. METHODOLOGY: Sampling was carried out using apple fruits and apple-based products. Fungi were isolated from undamaged apples, apple juice and baby apple food. DNA extraction was carried out with DNeasy Plant Mini Kit (QIAGEN, Valencia, USA). The molecular identification of fungal isolates was carried out using ITS1-ITS4 PCR. PCR products were sequenced and blasted. Patulin was extracted and analyzed by LC/MS/MS, then quantified using Agilent 1290UHPLC coupled to 6460 triple quadruple mass spectrometer. RESULTS: Forty-five samples of undamaged fresh apple fruits, apple juice and apple-based baby food products sold in different markets in Qatar were surveyed for both fungal and patulin contamination using Liquid Chromatography Tandem Mass Spectrometery (LC/MS/MS). Twenty-five Penicillium spp. isolates were selected, including 23 P. expansum and one isolate each of P. brevicompactum and P. commune. All the tested Penicillium spp. isolates produced patulin in vitro (from 40 to 100 µg/g on Malt Yeast Extract agar medium). Patulin was detected in 100% of apple juice samples at levels ranging from 5.27 to 82.21 µg/kg. Only 5 samples contained patulin levels higher than European Union recommended limit (50 µg/kg). The average patulin contamination was 30.67 µg/kg and 10.92 µg/kg in baby apple juice and in baby apple compote, respectively.

16S-23S Internal Transcribed Spacer Region PCR and Sequencer-Based Capillary Gel Electrophoresis has Potential as an Alternative to High Performance Liquid Chromatography for Identification of Slowly Growing Nontuberculous Mycobacteria.

Accurate identification of slowly growing nontuberculous mycobacteria (SG-NTM) of clinical significance remains problematic. This study evaluated a novel method of SG-NTM identification by amplification of the mycobacterial 16S-23S rRNA internal transcribed spacer (ITS) region followed by resolution of amplified fragments by sequencer-based capillary gel electrophoresis (SCGE). Fourteen American Type Culture Collection (ATCC) strains and 103 clinical/environmental isolates (total n = 24 species) of SG-NTM were included. Identification was compared with that achieved by high performance liquid chromatography (HPLC), in-house PCR and 16S/ITS sequencing. Isolates of all species yielded a SCGE profile comprising a single fragment length (or peak) except for M. scrofulaceum (two peaks). SCGE peaks of ATCC strains were distinct except for peak overlap between Mycobacterium kansasii and M. marinum. Of clinical/environmental strains, unique peaks were seen for 7/17 (41%) species (M. haemophilum, M. kubicae, M. lentiflavum, M. terrae, M. kansasii, M. asiaticum and M. triplex); 3/17 (18%) species were identified by HPLC. There were five SCGE fragment length types (I-V) each of M. avium, M. intracellulare and M. gordonae. Overlap of fragment lengths was seen between M. marinum and M. ulcerans; for M. gordonae SCGE type III and M. paragordonae; M. avium SCGE types III and IV, and M. intracellulare SCGE type I; M. chimaera, M. parascrofulaceum and M. intracellulare SCGE types III and IV; M. branderi and M. avium type V; and M. vulneris and M. intracellulare type V. The ITS-SCGE method was able to provide the first line rapid and reproducible species identification/screening of SG-NTM and was more discriminatory than HPLC.

Species identification of dermatophytes in paraffin-embedded biopsies with a new polymerase chain reaction assay targeting the internal transcribed spacer 2 region and comparison with histopathological features.

BACKGROUND: Dermatophytosis is a very common skin infection with a broad clinical spectrum. Biopsies are often used to confirm the diagnosis, especially when the clinical presentation is unusual. Not uncommonly, organisms are hard to find even with periodic acid-Schiff stains. Polymerase chain reaction (PCR) for dermatophytes can be used in such cases. OBJECTIVES: To test a new PCR assay allowing species identification of dermatophytes on paraffin-embedded biopsies, and to reassess histopathological criteria for diagnosis of dermatophytosis. METHODS: In total, 121 biopsies of 92 patients with clinical suspicion of tinea were included. In 42 samples the clinical diagnosis had been confirmed histopathologically, and in 79 no fungal elements had been identified. PCRs targeting the internal transcribed spacer (ITS)2 region of dermatophytes were performed on the biopsies with subsequent sequencing. Sections were reassessed for the presence/absence of hyphae/spores, pattern and composition of infiltrate, and epidermal/follicular changes. Patient charts were reviewed for clinical data. RESULTS: The new ITS2 PCR assay detected 94% of the dermatophyte infections (compared with 79% identified by microscopy). Trichophyton rubrum was the dominant species (89%), and other species identified were Trichophyton verrucosum (2%), Microsporum canis (4%), Epidermophyton floccosum (2%) and Trichophyton interdigitale (4%). In particular, infections with T. interdigitale and manifestations with prominent spongiosis were not diagnosed histologically. Intracorneal neutrophils, which have been emphasized as a histopathological clue to dermatophytosis, were present in only 46% of PCR-positive samples. CONCLUSIONS: Molecular species identification of dermatophytes via ITS2 PCR can easily be implemented in a routine dermatopathology setting. It is fast and highly specific and improves the sensitivity of histopathological diagnosis of dermatophytosis.

Development and Evaluation of a Novel Real-Time PCR for Pan-Dermatophyte Detection in Nail Specimens.

An accurate diagnosis of tinea unguium is necessary for the selection of antimycotics and successful treatment. To rapidly and accurately identify the aetiological agents causing tinea unguium, we improved upon the conventional boiling method for DNA extraction and developed a novel real-time PCR detection system that includes two assays. The two assays, based on the amplification of ribosomal internal transcribed spacer regions and 28S rDNA, were designed to detect pan-dermatophyte and Trichophyton rubrum, respectively. The analytical sensitivities of both assays permitted the detection of ten copies of plasmid DNA template. The analytical specificity of the detection system was confirmed using 11 dermatophyte strains and 25 non-dermatophyte strains. In total, 165 nail specimens were examined by microscopy, culture, conventional PCR, and the novel real-time PCR method. Real-time PCR gave positive results in 47.3 % of the specimens (78), a rate exceeding those obtained using microscopy (72, 43.6 %), conventional PCR (69, 41.8 %), and culture (49, 29.7 %). All conventional PCR-positive specimens were detected by real-time PCR, and real-time PCR detected nine specimens that were missed by conventional PCR. The results from latent class analysis, and further calculations, showed that real-time PCR was the most sensitive method, but the diagnostic specificity of the four approaches was equivalent. In particular, molecular approaches may be more effective than microscopy and culture when the clinical symptoms of tinea unguium are not evident.

High negative predictive value diagnostic strategies for the reevaluation of early antifungal treatment: A multicenter prospective trial in patients at risk for invasive fungal infections.

Early antifungal therapeutic strategies are proposed during invasive fungal infection (IFI), but antifungal stewardship programs should institute a systematic reevaluation of prescriptions, particularly in the context of empirical treatment. Here, we aimed to evaluate the performances and particularly the negative predictive value (NPV) of diagnostic strategies, including a whole blood panfungal quantitative PCR assay (PF-qPCR) in a high risk population for IFI. The first step was to standardize and optimize a new PF-qPCR targeting ITS2 region. Then, this method was evaluated in a multicenter prospective study including 313 patients with suspected IFI for whom an early antifungal treatment was prescribed. All patients enrolled at day 0 of their treatment benefited from serum Aspergillus galactomannan (GM) antigen detection twice a week, weekly PF-qPCR assay, and when indicated and feasible, CT-scan and mycological sampling. In total, 125 of 313 patients were diagnosed with IFI: 68 invasive aspergillosis (eight proven, 48 probable and 12 possible), one fusariosis, 47 candidemia, three disseminated candidiasis and six cryptococcosis. Globally, the sensitivity of the PF-qPCR assay was only 40%, but the specificity, PPV and NPV were 96%, 88% and 69%, respectively. In the population of patients at high risk for invasive aspergillosis who also benefited from Aspergillus GM detection, the sensitivity and the NPV of the combined detection reached to 78% and 84%, respectively. Even higher NPV were obtained when combining negative PF-qPCR and CT scan (95%) as well as negative GM and CT scan (93%), thus allowing to rationalize and re-evaluate the prescription of empirical treatment in such highly selected population.

A procedure to evaluate the efficiency of surface sterilization methods in culture-independent fungal endophyte studies.

Extraneous DNA interferes with PCR studies of endophytic fungi. A procedure was developed with which to evaluate the removal of extraneous DNA. Wheat (Triticum aestivum) leaves were sprayed with Saccharomyces cerevisiae and then subjected to physical and chemical surface treatments. The fungal ITS1 products were amplified from whole tissue DNA extractions. ANOVA was performed on the DNA bands representing S. cerevisiae on the agarose gel. Band profile comparisons using permutational multivariate ANOVA (PERMANOVA) and non-metric multidimensional scaling (NMDS) were performed on DGGE gel data, and band numbers were compared between treatments. Leaf surfaces were viewed under variable pressure scanning electron microscopy (VPSEM). Yeast band analysis of the agarose gel showed that there was no significant difference in the mean band DNA quantity after physical and chemical treatments, but they both differed significantly (p < 0.05) from the untreated control. PERMANOVA revealed a significant difference between all treatments (p < 0.05). The mean similarity matrix showed that the physical treatment results were more reproducible than those from the chemical treatment results. The NMDS showed that the physical treatment was the most consistent. VPSEM indicated that the physical treatment was the most effective treatment to remove surface microbes and debris. The use of molecular and microscopy methods for the post-treatment detection of yeast inoculated onto wheat leaf surfaces demonstrated the effectiveness of the surface treatment employed, and this can assist researchers in optimizing their surface sterilization techniques in DNA-based fungal endophyte studies.

A procedure to evaluate the efficiency of surface sterilization methods in culture-independent fungal endophyte studies

Extraneous DNA interferes with PCR studies of endophytic fungi. A procedure was developed with which to evaluate the removal of extraneous DNA. Wheat (Triticum aestivum) leaves were sprayed with Saccharomyces cerevisiae and then subjected to physical and chemical surface treatments. The fungal ITS1 products were amplified from whole tissue DNA extractions. ANOVA was performed on the DNA bands representing S. cerevisiae on the agarose gel. Band profile comparisons using permutational multivariate ANOVA (PERMANOVA) and non-metric multidimensional scaling (NMDS) were performed on DGGE gel data, and band numbers were compared between treatments. Leaf surfaces were viewed under variable pressure scanning electron microscopy (VPSEM). Yeast band analysis of the agarose gel showed that there was no significant difference in the mean band DNA quantity after physical and chemical treatments, but they both differed significantly (p < 0.05) from the untreated control. PERMANOVA revealed a significant difference between all treatments (p < 0.05). The mean similarity matrix showed that the physical treatment results were more reproducible than those from the chemical treatment results. The NMDS showed that the physical treatment was the most consistent. VPSEM indicated that the physical treatment was the most effective treatment to remove surface microbes and debris. The use of molecular and microscopy methods for the post-treatment detection of yeast inoculated onto wheat leaf surfaces demonstrated the effectiveness of the surface treatment employed, and this can assist researchers in optimizing their surface sterilization techniques in DNA-based fungal endophyte studies.

Towards the molecular characterisation of parasitic nematode assemblages: an evaluation of terminal-restriction fragment length polymorphism (T-RFLP) analysis.

Identifying factors which regulate temporal and regional structuring within parasite assemblages requires the development of non-invasive techniques which facilitate both the rapid discrimination of individual parasites and the capacity to monitor entire parasite communities across time and space. To this end, we have developed and evaluated a rapid fluorescence-based method, terminal restriction fragment length polymorphism (T-RFLP) analysis, for the characterisation of parasitic nematode assemblages in macropodid marsupials. The accuracy with which T-RFLP was capable of distinguishing between the constituent taxa of a parasite community was assessed by comparing sequence data from two loci (the ITS+ region of nuclear ribosomal DNA and the mitochondrial CO1) across ∼20 species of nematodes (suborder Strongylida). Our results demonstrate that with fluorescent labelling of the forward and reverse terminal restriction fragments (T-RFs) of the ITS+ region, the restriction enzyme Hinf1 was capable of generating species specific T-RFLP profiles. A notable exception was within the genus Cloacina, in which closely related species often shared identical T-RFs. This may be a consequence of the group's comparatively recent evolutionary radiation. While the CO1 displayed higher sequence diversity than the ITS+, the subsequent T-RFLP profiles were taxonomically inconsistent and could not be used to further differentiate species within Cloacina. Additionally, several of the ITS+ derived T-RFLP profiles exhibited unexpected secondary peaks, possibly as a consequence of the restriction enzymes inability to cleave partially single stranded amplicons. These data suggest that the question of T-RFLPs utility in monitoring parasite communities cannot be addressed without considering the ecology and unique evolutionary history of the constituent taxa.

Population genetic structure of the acanthocephalan Acanthosentis cheni in anadromous, freshwater, and landlocked stocks of its fish host, Coilia nasus.

The acanthocephalan Acanthosentis cheni was found in anadromous, freshwater, and landlocked stocks of its fish host, Coilia nasus. To examine the genetic variations of the acanthocephalan among the 3 populations with the adaptation of the host to the freshwater, the genetic structure of the helminth was investigated in anadromous (Zhoushan and Chongming islands, and Anqing), freshwater (Anqing, Ezhou, and Poyang Lake), and landlocked (Tian'ezhou Reserve) populations by sequencing intergenic transcribed spacers (ITS) of the ribosomal RNA coding genes. Low Fst values and high gene flow were found among the 7 populations (Fst = 0.0135, P = 0.2723; Nm = 36.48) and the 3 ecotypes of Acanthosentis cheni (Fst = 0.0178, P = 0.1044; Nm = 27.67). On the other hand, significant genetic differentiation of the C. nasus host populations was detected between the upstream and downstream areas of Xiaogu Mountain (Fst = 0.1961, P = 0.0030; Nm = 2.05), which is the farthest location of spawning migration for C. nasus . However, the migration break of the fish host appeared not to cause significant genetic differentiation of A. cheni populations between the upper and lower reaches of Xiaogu Mountain. Other factors might promote genetic exchange of A. cheni populations such as dispersal of the intermediate host by flooding or other fish species serving as the definitive or paratenic hosts. In Anqing, nucleotide diversity of the acanthocephalan was highest in the freshwater population (0.0038) and lower in the anadromous population (0.0026). This suggested that new mutations may have occurred in the freshwater A. cheni population in Anqing when adapting to a freshwater environment.

Detection of Clostridium tyrobutyricum in milk to prevent late blowing in cheese by automated ribosomal intergenic spacer analysis.

Clostridium tyrobutyricum has been identified as the main causal agent of the late blowing defect in cheese, with major effects on quality and commercial value. In this work, for the first time, we applied automated ribosomal intergenic spacer analysis (ARISA) approach to diagnose the presence of C. tyrobutyricum in raw milk before cheesemaking. A species-specific primer set was designed and used for this original application of the ARISA. Sensitivity of detection, reproducibility of the fluorescent PCR assay, and repeatability of the capillary electrophoretic analysis of amplicons were evaluated using DNA extracted from milk added with known amounts of C. tyrobutyricum genome copies, ranging from 3 × 10(6) to 3. Results indicated that the sensitivity of the technique permits to detect the bacterium in all the samples. The reproducibility, evaluated by analyzing 3 sets of serial dilutions, resulted satisfactory, with little deviation within PCR reactions amplifying the same starting amount of template (standard deviations ≤ 0.1, coefficients of variation ≤ 3%). The peaks' fluorescence displayed an evident correspondence with the number of genome copies contained in each dilution. The capillary electrophoretic analysis, tested by running a single PCR product per dilution point in 10 repeats, resulted efficient and highly repeatable, with excellent coefficients of variation ≤ 2% and standard deviations ≤ 0.1 in all the sample sets. This application of ARISA gives good estimates of the total C. tyrobutyricum DNA content allowing a specific, fine-scale resolution of this pollutant species in a complex system as milk. A further advantage linked to the automatization of the process.

Borrelia burgdorferi sensu lato prevalence in tick populations in Estonia.

BACKGROUND: Estonia is located in a unique area of co-distribution of Ixodes ricinus and I. persulcatus, which are the main tick vectors of Borrelia burgdorferi sensu lato. In the last decade, the incidence rate of Lyme borreliosis in Estonia has increased dramatically up to 115.4 per 100,000 in 2012. Here we present the first survey of the presence, the prevalence and genetic characteristics of B. burgdorferi s.l. complex spirochetes in the tick population in Estonia. METHODS: During the years 2006-2009, 2833 unfed Ixodes ricinus and I. persulcatus were collected from 43 sites in 7 counties in mainland Estonia as well as in 10 sites on the Saaremaa Island. DNA samples from ticks were analyzed individually using nested PCR of the ribosomal 5S-23S spacer region followed by bidirectional sequencing. RESULTS: The overall estimated prevalence of B. burgdorferi s.l was 9.7% and varied from 4.9% to 24.2% on the mainland and to 10.7% in Saaremaa Island. Ixodes persulcatus ticks showed significantly higher prevalence rates compared to that in I. ricinus-16.3% and 8.2%, respectively. The most prevalent genospecies was B. afzelii which was detected in 53.5% of Borrelia-positive ticks, followed by B. garinii and B. valaisiana with 26.2% and 5.5%, respectively. Also, B. bavariensis and B. burgdorferi s.s. DNA in single I. ricinus ticks were detected. Borrelia afzelii, B. garinii and B. valaisiana were detected in both tick species. Two genetic subgroups of B. garinii (NT29 and 20047) and two genetic subgroups of B. afzelii (NT28 and VS461) were found to be circulating in all studied regions as well as in both tick species, except B. garinii subgroup NT29, which was found only in I. persulcatus ticks. CONCLUSIONS: In the current study we detected the circulation of five B. burgdorferi s.l. genospecies and estimated the prevalence in ticks in different regions of Estonia. Detection and genetic characterization of Borrelia genospecies, especially those of public health importance, in the natural foci may help assessing high risk areas of human exposure to B. burgdorferi s.l.

Natural infection of wild caught Phlebotomus tobbi to Leishmania infantum in East Azerbaijan province, northwestern Iran.

BACKGROUND & OBJECTIVES: Zoonotic visceral leishmaniasis is caused by Leishmania infantum, which is transmitted to humans by bites of phlebotomine sandflies and is one of the most important public health problems in Iran. To detect and identify the Leishmania parasites and their corresponding vector(s), an investigation was carried out in Azarshahr County, a new and important focus of the disease in East Azerbaijan province in northwestern Iran during late April to late October 2010. METHODS: Sandflies were sampled using sticky papers (A4 white paper soaked in castor oil) from inside and outside of the houses and animal shelters, close to the vegetation and crevices. The head and three last abdomen segments of the specimens were removed and mounted in Puri's medium for species identification. The rest of body was subjected to molecular methods for detection of leishmanial parasites. RESULTS: Among 400 female sandflies tested by polymerase chain reaction (PCR) of kDNA, ITS1-rDNA, and CPB genes of the parasite followed by restriction fragment length polymorphism (RFLP), only 2 out of 8 Phlebotomus tobbi were positive to L. infantum parasites. CONCLUSION: The results indicated that, P. tobbi was the only species found infected by L.infantum and the principal vector of the disease agent to human.