Terminal Restriction Fragment Length Polymorphism for Identification of Cryptosporidium Species in Human Feces

Effective management of human cryptosporidiosis requires efficient methods for detection and identification of the species of Cryptosporidium isolates. Identification of isolates to the species level is not routine for diagnostic assessment of cryptosporidiosis, which leads to uncertainty about the epidemiology of the Cryptosporidium species that cause human disease. We developed a rapid and reliable method for species identification of Cryptosporidium oocysts from human fecal samples using terminal restriction fragment polymorphism (T-RFLP) analysis of the 18S rRNA gene. This method generated diagnostic fragments unique to the species of interest. A panel of previously identified isolates of species was blind tested to validate the method, which determined the correct species identity in every case. The T-RFLP profiles obtained for samples spiked with known amounts of Cryptosporidium hominis and Cryptosporidium parvum oocysts generated the two expected diagnostic peaks. The detection limit for an individual species was 1% of the total DNA. This is the first application of T-RFLP to protozoa, and the method which we developed is a rapid, repeatable, and cost-effective method for species identification.     

Ancylostoma ceylanicum infections in humans in Vietnam

Ancylostoma ceylanicum is recognized as the only zoonotic hookworm species that is able to mature into adult stage in the human intestine. While human infections caused by this hookworm species have been reported from neighboring countries and this hookworm is prevalent in dogs in Vietnam, human infection has never been reported in Vietnam. The present study, therefore, aimed to identify human infections with A. ceylanicum in Vietnam. A total of 526 fecal samples from the residents in Long An Province were collected and the presence of hookworm eggs was detected by the Kato-Katz method. The results indicated that the overall prevalence of human hookworm infection was 85/526 (16.2%). After filter paper culture, 3rd stage larvae were successfully obtained from 48 egg-positive samples. The larvae were identified for their species using semi-nested PCR-RLFP on the cox1 gene. As a result, two hookworm species were confirmed; single species infections with Necator americanus or A. ceylanicum, and mixed infections with both species were found in 47.9%, 31.3%, and 20.8% of the samples, respectively.     

Identification and differentiation of Fasciola hepatica and Fasciola gigantica using a simple PCR-restriction enzyme method

Accurate morphological differentiation between the liver fluke species Fasciola hepatica and Fasciola gigantica is difficult. We evaluated PCR-restriction enzyme profiles of internal transcribed spacer 1 (ITS1) that could aid in their identification. Fifty F. hepatica and 30 F. gigantica specimens were collected from different hosts in three provinces of Iran. For DNA extraction, we crushed fragments of the worms between two glass slides as a new method to break down the cells. DNA from the crushed materials was then extracted with a conventional phenol-chloroform method and with the newly developed technique, commercial FTA cards. A primer pair was selected to amplify a 463-bp region of the ITS1 sequence. After sequencing 14 samples and in silico analysis, cutting sites of all known enzymes were predicted and TasI was selected as the enzyme that yielded the most informative profile. Crushing produced enough DNA for PCR amplification with both the phenol-chloroform and commercial FTA card method. The DNA extracted from all samples was successfully amplified and yielded a single sharp band of the expected size. Digestion of PCR products with TasI allowed us to distinguish the two species. In all samples, molecular identification was consistent with morphological identification. Our PCR-restriction enzyme profile is a simple, rapid and reliable method for differentiating F. hepatica and F. gigantica, and can be used for diagnostic and epidemiological purposes.     

Comparison of two methods characterising the seed bank of amphibious plants in submerged sediments

We analysed the submerged soil seed bank of three fishponds in the Waldviertel region in Austria. We aimed our study at comparing the efficiency of two methods in detecting seeds quantitatively from soil samples of four characteristic mud-flat species, i.e. Carex bohemica, Coleanthus subtilis, Elatine hexandra, and Eleocharis ovata: the seedling-emergence and the rinsing method. Additionally, the actual vegetation was described based on 65 phytosociological relevés. Soil seed bank analyses were based on 31 samples representing the vegetation zonation. Mixed soil samples were portioned into 62 subsamples further treated with the two methods in parallel.     

Validation of High Resolution Melting Analysis (HRM) of the Amplified ITS2 Region for the Detection and Identification of Yeasts from Clinical Samples: Comparison with Culture and MALDI-TOF Based Identification

AimCandida species are known as opportunistic pathogens, and a possible cause of invasive infections. Because of their species-specific antimycotic resistance patterns, reliable techniques for their detection, quantification and identification are needed. We validated a DNA amplification method for direct detection of Candida spp. from clinical samples, namely the ITS2-High Resolution Melting Analysis (direct method), by comparing it with a culture and MALDI-TOF Mass Spectrometry based method (indirect method) to establish the presence of Candida species in three different types of clinical samples.ResultsFor 83.9% of the samples there was complete concordance between both techniques, i.e. the same Candida species were detected in 31.1% of the samples or no Candida species were detected in 52.8% of the samples. In 16.1% of the clinical samples, discrepant results were obtained, of which only 6.01% were considered as major discrepancies. Discrepancies occurred mostly when overall numbers of Candida cells in the samples were low and/or when multiple species were present in the sample.DiscussionMost of the discrepancies could be decided in the advantage of the direct method. This is due to samples in which no yeast could be cultured whereas low amounts could be detected by the direct method and to samples in which high quantities of Candida robusta according to ITS2-HRM were missed by culture on Candida ID agar. It remains to be decided whether the diagnostic advantages of the direct method compensate for its disadvantages.     

Cryptosporidium Source Tracking in the Potomac River Watershed

To better characterize Cryptosporidium in the Potomac River watershed, a PCR-based genotyping tool was used to analyze 64 base flow and 28 storm flow samples from five sites in the watershed. These sites included two water treatment plant intakes, as well as three upstream sites, each associated with a different type of land use. The uses, including urban wastewater, agricultural (cattle) wastewater, and wildlife, posed different risks in terms of the potential contribution of Cryptosporidium oocysts to the source water. Cryptosporidium was detected in 27 base flow water samples and 23 storm flow water samples. The most frequently detected species was C. andersoni (detected in 41 samples), while 14 other species or genotypes, almost all wildlife associated, were occasionally detected. The two common human-pathogenic species, C. hominis and C. parvum, were not detected. Although C. andersoni was common at all four sites influenced by agriculture, it was largely absent at the urban wastewater site. There were very few positive samples as determined by Environmental Protection Agency method 1623 at any site; only 8 of 90 samples analyzed (9%) were positive for Cryptosporidium as determined by microscopy. The genotyping results suggest that many of the Cryptosporidium oocysts in the water treatment plant source waters were from old calves and adult cattle and might not pose a significant risk to human health.     

Fingerprinting of cerrado species based on cork lipophilic constituents

Cerrado is a savanna ecosystem of central and southeastern Brazil. Many woody species of cerrado have thick layers of cork. The present work aimed to characterize, by GC/MS analysis, the constituents of n-hexane extracts from the cork of common species from cerrado. Cork samples from 31 individuals, corresponding to 14 species and six families, were analyzed. Similarities and differences were noticed between cork and cuticular waxes regarding profiles of lipophilic constituents. The distribution of cork constituents was analyzed using the UPGMA clustering method and DICE coefficient. All clusters in the dendrogram obtained comprise individuals from a same species, suggesting that the distribution of lipophilic cork constituents is useful for species characterization and possibly also for species identification, resembling results commonly obtained with molecular markers. Seven samples of Bignoniaceae, corresponding to two genera and seven species, emerged in a common cluster, in an arrangement in accordance with the recent segregation of Tabebuia species to a new genus Handroanthus. The markers analyzed were not efficient regarding characterization of other families.     

Diversity and Species Relationship in Pearl Millet (Pennisetum typhoides) and Related Species

Thirteen accessions of pearl millet (Pennisetum typhoides (L) Leeke) collected from different states of India and eight wild species of the genus Pennisetum across the world were analyzed for genetic diversity using AFLP markers. A combined analysis of eight primer combinations showed 35% polymorphism among P. typhoides accessions while analysis with five primer combinations showed 99% polymorphism among the wild species. The dendrogram constructed for the P. typhoides accessions based on the UPGMA method revealed two major clusters with samples from Gujarat forming a separate cluster from the rest of the samples. Principal component analysis of the same data set revealed similar results with the first principal component accounting for 65% of the total variation. The percentage of rare and common alleles contributing to the diversity in the sample was analyzed using the Shannon Weiner diversity index. The SW index revealed that the samples from Gujarat contributed significantly to the overall diversity among the accessions. Among accessions of each geographical region, considerable variation was revealed by SW index with samples from Tamil Nadu being most polymorphic. The genetic diversity in the accessions could be utilized for future breeding work. The dendrogram constructed for the wild species revealed the extent of genetic diversity among them. Analysis with one primer combination showed P. typhoides being closer to P. mollissimum than to the other analyzed species.     

Quantitative PCR Method for Enumeration of Cells of Cryptic Species of the Toxic Marine Dinoflagellate Ostreopsis spp. in Coastal Waters of Japan

Monitoring of harmful algal bloom (HAB) species in coastal waters is important for assessment of environmental impacts associated with HABs. Co-occurrence of multiple cryptic species such as toxic dinoflagellate Ostreopsis species make reliable microscopic identification difficult, so the employment of molecular tools is often necessary. Here we developed new qPCR method by which cells of cryptic species can be enumerated based on actual gene number of target species. The qPCR assay targets the LSU rDNA of Ostreopsis spp. from Japan. First, we constructed standard curves with a linearized plasmid containing the target rDNA. We then determined the number of rDNA copies per cell of target species from a single cell isolated from environmental samples using the qPCR assay. Differences in the DNA recovery efficiency was calculated by adding exogenous plasmid to a portion of the sample lysate before and after DNA extraction followed by qPCR. Then, the number of cells of each species was calculated by division of the total number of rDNA copies of each species in the samples by the number of rDNA copies per cell. To test our procedure, we determined the total number of rDNA copies using environmental samples containing no target cells but spiked with cultured cells of several species of Ostreopsis. The numbers estimated by the qPCR method closely approximated total numbers of cells added. Finally, the numbers of cells of target species in environmental samples containing cryptic species were enumerated by the qPCR method and the total numbers also closely approximated the microscopy cell counts. We developed a qPCR method that provides accurate enumeration of each cryptic species in environments. This method is expected to be a powerful tool for monitoring the various HAB species that occur as cryptic species in coastal waters.     

Weak divergence among African, Malagasy and Seychellois hinged terrapins (Pelusios castanoides, P. subniger) and evidence for human-mediated oversea dispersal

Using phylogenetic and haplotype network analyses of 2036 bp of mitochondrial DNA, we compare samples of the two hinged terrapin species Pelusios castanoides and P. subniger from continental Africa, Madagascar and the Seychelles to infer their biogeography. Owing to the long independent history of Madagascar and the Seychelles, the populations from those islands should be deeply divergent from their African conspecifics. Seychellois populations of the two species are currently recognized as Critically Endangered endemic subspecies. However, even though we found within P. subniger evidence for a cryptic species from the Democratic Republic of the Congo, all other samples assigned to this species were undifferentiated. This suggests that Malagasy and Seychellois populations of P. subniger were introduced by humans and that the Seychellois subspecies P. s. parietalis is invalid. This has implications for current conservation strategies for the Critically Endangered Seychellois populations and suggests that measures should rather focus on endemic species. The situation of P. castanoides could be different. Samples from Madagascar and the Seychelles are weakly, but consistently, differentiated from continental African samples, and Malagasy and Seychellois samples are reciprocally monophyletic in maximum likelihood analyses. However, due to a lack of samples from central and northern Mozambique and Tanzania, we cannot exclude that identical continental haplotypes exist there.     

Enzyme electrophoresis,genetic identity and description of a new genus and species of heteronemertean (Nemertea,Anopla) from northwestern Spain and North Wales

The anoplan order Heteronemertea, particularly the genera Cerebratulus, Lineus and Micrura, contains a very large number of nominate species, many of which are inadequately described. As a consequence, systematic difficulties are encountered with the identification of many taxa in this group, especially those originally established primarily on the basis of their external features. The present paper concerns heteronemerteans collected from two locations, the Foz Estuary (north-western Spain) and Llandudno (North Wales). The Spanish collection included specimens identified as Lineus longissimus (Gunnerus), whilst samples from Llandudno contained large numbers of Lineus viridis (Müller); samples of a third similar but apparently undescribed species were found at both locations. Starch gel electrophoresis showed that samples of the apparent third species were genetically almost identical from each of the two locations, but were clearly different from the two described Lineus species. Histological studies of the unknown specimens revealed anatomical characters, including the unique feature of a proboscis epithelium ciliated throughout its length, which exclude it from any known heteronemertean taxon; it is accordingly placed in a new genus and species, for which the name Riseriellus occultus is proposed.     

Molecular Method To Assess the Diversity of Burkholderia Species in Environmental Samples

In spite of the importance of many members of the genus Burkholderia in the soil microbial community, no direct method to assess the diversity of this genus has been developed so far. The aim of this work was the development of soil DNA-based PCR-denaturing gradient gel electrophoresis (DGGE), a powerful tool for studying the diversity of microbial communities, for detection and analysis of the Burkholderia diversity in soil samples. Primers specific for the genus Burkholderia were developed based on the 16S rRNA gene sequence and were evaluated in PCRs performed with genomic DNAs from Burkholderia and non-Burkholderia species as the templates. The primer system used exhibited good specificity and sensitivity for the majority of established species of the genus Burkholderia. DGGE analyses of the PCR products obtained showed that there were sufficient differences in migration behavior to distinguish the majority of the 14 Burkholderia species tested. Sequence analysis of amplicons generated with soil DNA exclusively revealed sequences affiliated with sequences of Burkholderia species, demonstrating that the PCR-DGGE method is suitable for studying the diversity of this genus in natural settings. A PCR-DGGE analysis of the Burkholderia communities in two grassland plots revealed differences in diversity mainly between bulk and rhizosphere soil samples; the communities in the latter samples produced more complex patterns.     

Ecological characterization of entomopathogenic nematodes isolated in stone fruit orchard soils of Mediterranean areas

Entomopathogenic nematodes in the families Steinernematidae and Heterorhabditidae were isolated from stone-fruit orchards in two Mediterranean regions of Spain. A total of 630 soil samples (210 sites) from Catalonia and 90 soil samples (30 sites) from Murcia were evaluated resulting in 5.2% and 20% of the soils testing positive for nematodes, respectively. Ten steinernematid isolates and three heterorhabditid isolates were recovered using the Galleria mellonella baiting method. Based on morphometric data, molecular data, and cross-breeding experiments the nematode species were identified as Steinernemafeltiae and Heterorhabditis bacteriophora. Environmental tolerance to heat, desiccation and hypoxia, the effect of temperature on infectivity and reproduction and nematode migration in sand columns were compared among isolates and one Steinernema carpocapsae strain. Results showed differences among species and a great variability within species. Beneficial traits for each strain were added up to identify a superior candidate to control Mediterranean flat-headed rootborer, Capnodis tenebrionis. When all analyzed factors were considered, three S. feltiae isolates (Bpa, Sor and M116) obtained the best scores, and when hypoxia was removed, two of the strains (Bpa and Sor) continued ranking superior to other strains.     

Detection of Diverse New Francisella-Like Bacteria in Environmental Samples

Following detection of putative Francisella species in aerosol samples from Houston, Texas, we surveyed soil and water samples from the area for the agent of tularemia, Francisella tularensis, and related species. The initial survey used 16S rRNA gene primers to detect Francisella species and related organisms by PCR amplification of DNA extracts from environmental samples. This analysis indicated that sequences related to Francisella were present in one water and seven soil samples. This is the first report of the detection of Francisella-related species in soil samples by DNA-based methods. Cloning and sequencing of PCR products indicated the presence of a wide variety of Francisella-related species. Sequences from two soil samples were 99.9% similar to previously reported sequences from F. tularensis isolates and may represent new subspecies. Additional analyses with primer sets developed for detection and differentiation of F. tularensis subspecies support the finding of very close relatives to known F. tularensis strains in some samples. While the pathogenicity of these organisms is unknown, they have the potential to be detected in F. tularensis-specific assays. Similarly, a potential new subspecies of Francisella philomiragia was identified. The majority of sequences obtained, while more similar to those of Francisella than to any other genus, were phylogenetically distinct from known species and formed several new clades potentially representing new species or genera. The results of this study revise our understanding of the diversity and distribution of Francisella and have implications for tularemia epidemiology and our ability to detect bioterrorist activities.     

Parallel Tagged Next-Generation Sequencing on Pooled Samples – A New Approach for Population Genetics in Ecology and Conservation

Next-generation sequencing (NGS) on pooled samples has already been broadly applied in human medical diagnostics and plant and animal breeding. However, thus far it has been only sparingly employed in ecology and conservation, where it may serve as a useful diagnostic tool for rapid assessment of species genetic diversity and structure at the population level. Here we undertake a comprehensive evaluation of the accuracy, practicality and limitations of parallel tagged amplicon NGS on pooled population samples for estimating species population diversity and structure. We obtained 16S and Cyt b data from 20 populations of Leiopelma hochstetteri, a frog species of conservation concern in New Zealand, using two approaches – parallel tagged NGS on pooled population samples and individual Sanger sequenced samples. Data from each approach were then used to estimate two standard population genetic parameters, nucleotide diversity (π) and population differentiation (F_ST), that enable population genetic inference in a species conservation context. We found a positive correlation between our two approaches for population genetic estimates, showing that the pooled population NGS approach is a reliable, rapid and appropriate method for population genetic inference in an ecological and conservation context. Our experimental design also allowed us to identify both the strengths and weaknesses of the pooled population NGS approach and outline some guidelines and suggestions that might be considered when planning future projects.     

Identification of Malassezia Species from Pityriasis Versicolor Lesions with a New Multiplex PCR Method

Despite the fact that a range of molecular methods have been developed as tools for the diagnosis of Malassezia species, there are several drawbacks associated with them, such as inefficiency of differentiating all the species, high cost, and questionable reproducibility. In addition, most of the molecular methods require cultivation to enhance sensitivity. Therefore, alternative methods eliminating cultivation and capable of identifying species with high accuracy and reliability are needed. Herein, a multiplex polymerase chain reaction (PCR)-based method was especially developed for the detection of eleven Malassezia species. The multiplex PCR was standardized by incorporating a consensus forward primer, along with Malassezia species-specific reverse primers considering the sizes of the PCR products. In the method, the multiplex-PCR primer content is divided into three parts to circumvent the problem of increased nonspecific background resulting from the use of a large number of primers. DNA extraction protocol described by Harju and colleagues was modified using liquid nitrogen instead of ?80 °C to break down the yeast membrane. By a modified extraction procedure followed by multiplex PCR and electrophoresis, the method enables identification and differentiation of Malassezia species from both of the samples obtained directly from skin and yeast colonies grown in culture. Fifty-five patients who were confirmed with pityriasis versicolor were enrolled in the study. Multiplex PCR detected and differentiated all 55 samples obtained directly from the patients’ skin. However, 50 out of 55 samples yielded Malassezia colony in the culture. In addition, eight of 50 colonies were misdiagnosed or not completely differentiated by conventional methods based on the sequence analysis of eight colonies. The method is capable of identifying species with high accuracy and reliability. In addition, it is simple, quick, and cost-effective. More importantly, the method works efficiently for the diagnosis of Malassezia species obtained directly from patient samples.     

Dynamics of lactic acid bacteria populations in Rioja wines by PCR-DGGE, comparison with culture-dependent methods

Lactic acid bacteria populations of red wine samples from industrial fermentations, including two different vinification methods were studied. For this investigation, polymerase chain reaction–denaturing gradient gel electrophoresis (PCR-DGGE) analysis was employed to supplement previous results that were obtained by culture-dependent methods. PCR-DGGE was aimed to study two targeted genes, 16S ribosomal DNA (rDNA) and rpoB, and the results were useful to evaluate the microbial populations in wine samples. Moreover, an improvement of a detection limit determined so far for DGGE analysis was obtained with the method described in this study, what made possible to identify lactic acid bacteria populations below 10¹ colony-forming unit/mL. The species Oenococcus oeni was the most frequently detected bacterium, but identifications close to species Oenococcus kitaharae and Lactococcus lactis that are not often found in wine were firstly identified in samples of this research. PCR-DGGE allowed to detect 9 out of 11 lactic acid bacteria species identified in this study (nine by PCR-16S rDNA/DGGE and four by PCR-rpoB/DGGE), while five species were detected using the modified de Man, Rogosa and Sharpe agar. Therefore, the two methods were demonstrated to be complementary. This finding suggests that analysis of the lactic acid bacteria population structure in wine should be carried out using both culture-dependent and culture-independent techniques with more than one primer pair.     

Next-Generation Sequencing for Rodent Barcoding: Species Identification from Fresh,Degraded and Environmental Samples

Rodentia is the most diverse order among mammals, with more than 2,000 species currently described. Most of the time, species assignation is so difficult based on morphological data solely that identifying rodents at the specific level corresponds to a real challenge. In this study, we compared the applicability of 100 bp mini-barcodes from cytochrome b and cytochrome c oxidase 1 genes to enable rodent species identification. Based on GenBank sequence datasets of 115 rodent species, a 136 bp fragment of cytochrome b was selected as the most discriminatory mini-barcode, and rodent universal primers surrounding this fragment were designed. The efficacy of this new molecular tool was assessed on 946 samples including rodent tissues, feces, museum samples and feces/pellets from predators known to ingest rodents. Utilizing next-generation sequencing technologies able to sequence mixes of DNA, 1,140 amplicons were tagged, multiplexed and sequenced together in one single 454 GS-FLX run. Our method was initially validated on a reference sample set including 265 clearly identified rodent tissues, corresponding to 103 different species. Following validation, 85.6% of 555 rodent samples from Europe, Asia and Africa whose species identity was unknown were able to be identified using the BLASTN program and GenBank reference sequences. In addition, our method proved effective even on degraded rodent DNA samples: 91.8% and 75.9% of samples from feces and museum specimens respectively were correctly identified. Finally, we succeeded in determining the diet of 66.7% of the investigated carnivores from their feces and 81.8% of owls from their pellets. Non-rodent species were also identified, suggesting that our method is sensitive enough to investigate complete predator diets. This study demonstrates how this molecular identification method combined with high-throughput sequencing can open new realms of possibilities in achieving fast, accurate and inexpensive species identification.     

Outcome of Colonization of Apis mellifera by Nosema ceranae

A multiplex PCR-based method, in which two small-subunit rRNA regions are simultaneously amplified in a single reaction, was designed for parallel detection of honeybee microsporidians (Nosema apis and Nosema ceranae). Each of two pairs of primers exclusively amplified the 16S rRNA targeted gene of a specific microsporidian. The multiplex PCR assay was useful for specific detection of the two species of microsporidians related to bee nosemosis, not only in purified spores but also in honeybee homogenates and in naturally infected bees. The multiplex PCR assay was also able to detect coinfections by the two species. Screening of bee samples from Spain, Switzerland, France, and Germany using the PCR technique revealed a greater presence of N. ceranae than of N. apis in Europe, although both species are widely distributed. From the year 2000 onward, statistically significant differences have been found in the proportions of Nosema spp. spore-positive samples collected between and within years. In the first period examined (1999 to 2002), the smallest number of samples diagnosed as Nosema positive was found during the summer months, showing clear seasonality in the diagnosis, which is characteristic of N. apis. From 2003 onward a change in the tendency resulted in an increase in Nosema-positive samples in all months until 2005, when a total absence of seasonality was detected. A significant causative association between the presence of N. ceranae and hive depopulation clearly indicates that the colonization of Apis mellifera by N. ceranae is related to bee losses.