Species-specific Probes Research Articles

Method for absolute quantification of microbial communities by using both microarrays and competitive PCR

Methods for the robust quantification of bacterial communities are still under development. In this context, the present study aimed to evaluate a method combining competitive PCR (cPCR) and microarray assays for the determination of absolute content of total bacteria and individual bacterial species in samples. For this, a competitor DNA for cPCR and microarrays containing three types of DNA probes was prepared. A calibration curve was generated with genomic DNA samples as standards, which was then utilized for cPCR-based determination of the total number (in moles) of 16S rRNA genes in other bacterial samples. Moreover, scatter plots of species-specific probes versus total bacteria probe for each genomic DNA of known concentration was fit to the regression model, and the obtained slope value was defined as the hybridization affinity ratio.The cPCR assay was performed for both a commercially available mixed genomic DNA sample and human oral bacterial DNA samples, and the total number of moles of 16S rRNA genes was determined. These values were distributed among each species on the basis of the signal intensities of species-specific probes and the hybridization affinity ratio. The total number of bacterial genomes and those of individual species were determined by dividing the copy number of 16S rRNA genes per genome. The obtained results were confirmed by quantitative real-time PCR (qPCR). For values of >1 × 102 copies determined by qPCR, the ratio of the values measured by DNA chips to by qPCR was 1.53-fold on average and <2.6-fold for all data. These results show that the combined method of cPCR and microarray is useful to quantify the absolute numbers of several types of bacteria in a sample at one time.

Neosporosis: An Overview of Its Molecular Epidemiology and Pathogenesis

Abstract Neospora caninum (N. caninum), a cyst-forming protozoan parasite, is a major cause of bovine abortions and neonatal mortality worldwide. N. caninum has a broad intermediate host range, and its sexual cycle occurs exclusively in canids. Another species of Neospora, Neospora hughesi (N. hughesi), has been identified and causes myeloencephalitis in horses. Although molecular epidemiology studies are in their infancy, the 18S ribosomal RNA (rRNA) and ITS1 regions within the small subunit ribosomal RNA (ssuRNA) and an N. caninum species-specific DNA probe (pNc5) have been used extensively to differentiate Neospora from other closely related apicomplexan parasites. While these repetitive regions have higher sensitivity and specificity than housekeeping or antigen genes, they suffer from low discriminatory power and fail to capture intra-species diversity. Similarly, although multiple minisatellite or microsatellite marker studies have shown clear geographic substructures within Neospora, strains are often misclassified due to a convergence in the size of different alleles at microsatellite loci, known as homoplasy. Only one strain, N. caninum Liverpool (Nc-Liv), has been genome sequenced and compared with its closest relative, Toxoplasma gondii (T. gondii). Hence, detailed population genomics studies based on whole-genome sequences from multiple strains worldwide are needed in order to better understand the current population genetic structure of Neospora, and ultimately to determine more effective vaccine candidates against bovine neosporosis. The aim of this review is to outline our current understanding of the molecular epidemiology and genomics of Neospora in juxtaposition with the closely related apicomplexan parasites Hammondia hammondi and T. gondii.

A qPCR Assay for the Detection of Phytophthora cinnamomi Including an mRNA Protocol Designed to Establish Propagule Viability in Environmental Samples.

Phytophthora cinnamomi causes root and collar rot in many plant species in natural ecosystems and horticulture. A species-specific primer and probe PCIN5 were designed based on a mitochondrial locus encoding subunit 2 of cytochrome c oxidase (cox2). Eight PCR primers, including three forward and five reverse, were designed and tested in all possible combinations. Annealing temperatures were optimized for each primer pair set to maximize both specificity and sensitivity. Each set was tested against P. cinnamomi and two closely related clade 7 species, P. parvispora and P. niederhauseri. From these tests, five primer pairs were selected based on specificity and, with a species-specific P. cinnamomi probe, used to develop quantitative real-time PCR (qPCR) assays. The specificity of the two most sensitive qPCR assays was confirmed using the genomic DNA of 29 Phytophthora isolates, including 17 isolates of 11 species from clade 7, and representative species from nine other clades (all except clade 3). The assay was able to detect as little as 150 ag of P. cinnamomi DNA and showed no cross-reaction with other Phytophthora species, except for P. parvispora, a very closely related species to P. cinnamomi, which showed late amplification at high DNA concentrations. The efficiency of the qPCR protocol was evaluated with environmental samples including roots and associated soil from plants artificially infected with P. cinnamomi. Different RNA isolation kits were tested and evaluated for their performance in the isolation of RNA from environmental samples, followed by cDNA synthesis, and qPCR assay. Finally, a protocol was recommended for determining the presence of P. cinnamomi in recalcitrant environmental samples.

Tetraplex real-time PCR with TaqMan probes for discriminatory detection of cat, rabbit, rat and squirrel DNA in food products

Cat, rabbit, rat, and squirrel species are very sensitive in food products because most of them are potential carriers of zoonotic diseases and rejected in most religions and cultures. Since cats and rats are abundant in most parts of the world and their meats do not carry any value in legal markets, these meats could be considered as potential adulterants in halal, kosher, and other food markets. Rabbit and squirrel meats are also susceptible to adulteration. Therefore, both health and economic interests in rat, rabbit, cat and squirrel species are significant. In this work, a novel tetraplex real-time PCR assay with TaqMan probes was described to discriminate and identify all four species (cat, rabbit, rat, and squirrel) in a single assay platform. Species-specific primers and probes were developed against ATP6, and cytochrome b genes to amplify 108, 123, 161 and 176 bp DNA fragments from rat, rabbit, squirrel and cat meat products under various states. A 141-bp internal amplification control (IAC) of 18S rRNA was used to avoid any false-negative results. Specificity was evaluated against 22 species but no cross-reactivity was found. Efficiency of PCR assay as well as target quantification were determined based on a standard curve that was generated using tenfold serially diluted mixed DNA extract (1:1:1:1) from squirrel, rat, rabbit and cat species. The assay was valid under pure, processed and admixed states with 10–0.1% (w/w) adulterant from each species. The limit of detection was 0.1% under admixed samples and 0.003 ng DNA under pure states from each species. Analyses of 18 model burgers (9 chicken and 9 beef) and 18 frankfurters (9 chicken and 9 beef) revealed 91–122% target recovery at 0.1–10% adulteration. Finally, 72 commercial burgers (36 chicken and 36 beef) and 72 frankfurters (36 chicken and 36 beef) were screened but no target species was detected except IAC.

First molecular survey of piroplasm species in cattle from Kyrgyzstan.

Bovine piroplasmosis is a tick-borne disease caused by apicomplexan hemoparasites of the genera Theileria and Babesia. This study was carried out to assess the presence and frequency of piroplasm parasites in apparently healthy cattle in Kyrgyzstan. A total of 454 blood samples were collected from animals of various ages in eight villages located in the Chu valley and around the Lake Issyk Kul. The hypervariable V4 region of the 18S ribosomal RNA (rRNA) gene was amplified with a set of primers specific targeting members of the genera Theileria and Babesia. Amplified PCR products were hybridized onto a membrane to which generic and species-specific oligonucleotide probes were covalently linked. The results revealed the presence of three piroplasm species (Theileria orientalis, Babesia major, Theileria annulata). Theileria orientalis was the most prevalent species (32.8%; CI 28.5-37.3). Babesia major was the only species of Babesia found in any of the samples (1.3%; CI 0.5-2.8). The co-existence of Theileria annulata and T. orientalis was detected in nine animals (1.9%; CI 0.9-3.7). BLAST search revealed that the Theileria sequences shared 100% identity with the recently reported sequences for T. buffeli and T. annulata. The sequence of B. major was also 100% identical to an existing B. major sequence. This molecular survey provides important epidemiological data for control of bovine piroplasmosis caused by T. orientalis, B. major, and T. annulata in Kyrgyzstan.

Spatial and seasonal distributions of the phototrophic dinoflagellate Biecheleriopsis adriatica (Suessiaceae) in Korea: quantification using qPCR

The phototrophic dinoflagellate Biecheleriopsis adriatica is a small suessioid species characterized by a fragile thin wall. Although the morphology of this dinoflagellate is well established, there is currently little information available on its distribution and the environmental factors that influence this distribution. Thus, to investigate the spatial and seasonal distributions of the vegetative cells of B. adriatica in Korean waters, surface water samples were collected on a seasonal basis from 28 stations in the East, West, and South Sea of Korea and Jeju Island from April 2015 to October 2018, and abundances of the vegetative cells of B. adriatica were quantified using quantitative real-time polymerase chain reactions, for which we developed the species-specific primer and probe set. Simultaneously, major environmental parameters, including temperature, salinity, nutrient concentrations, and dissolved oxygen concentrations were measured. The vegetative cells of B. adriatica were detected at 20 of the 28 sampling stations: 19 stations in summer and 6 in autumn, although from no stations in either spring or winter. The ranges of water temperature and salinity at sites where this species was detected were 17.7–26.4°C and 9.9–34.3, respectively, whereas those of nitrate and phosphate concentrations were not detectable-96.2 and 0.18–2.66 μM, respectively. Thus, the sites at which this species is found are characterized by a narrow range of temperature, but wide ranges of salinity and concentrations of nitrate and phosphate. The highest abundance of the vegetative cells of B. adriatica was 41.7 cells mL−1, which was recorded in Jinhae Bay in July 2018. In Jinhae Bay, the abundance of vegetative cells was significantly positively correlated with the concentration of nitrate, but was negatively correlated with salinity. On the basis of these findings, it appears that the abundance of B. adriatica vegetative cells shows strong seasonality, and in Jinhae Bay, could be affected by the concentrations of nitrate. Key words: abundance; molecular method; nationwide distribution; protist; Suessiaceae

Molecular survey of Anaplasma and Ehrlichia species in cattle from Karaman of Turkey, including a novel tandem report of Anaplasma marginale msp1a gene

Tick-borne pathogens cause serious health problems and loss of productivity in domesticated and wild animals. A molecular study was performed to detect the frequency of infection with Anaplasma/Ehrlichia (A/E) in cattle from Karaman province of Turkey. Venous blood samples were taken from 150 apparently healthy cattle in 2016. After amplification the hypervariable V1 region of the 16S rRNA gene of A/E species, a reverse line blot (RLB) assay was performed using species-specific probes. Since some samples gave signal only to A/E catch-all probe, the samples analyzed in terms of major surface proteins (MSPs) of Anaplasma marginale. Genetic diversity and tandem repeat analysis were made for msp1α gene sequences of A. marginale. Anaplasma-like bodies were detected in four (2.66%) animals via microscopic examination. Anaplasma centrale was detected in eight (5.33%) animals via RLB. When the samples were examined in terms of A. marginale msp1a gene with semi-nested PCR, a total of nine (6.00%) animals [six of them (4.00%) were positive for A. centrale with RLB] were found to be infected with A. marginale. In addition, the sequences of MSP1a amplicons revealed one new tandem repeat (Tr70). According to these results, it was determined that A. marginale and A. centrale were found in cattle in Karaman province and this study provided the first evidence of genetic diversity of A. marginale with one new tandem repeat in cattle in the region.

Characterization of Weissella confusa DD_A7 isolated from kimchi

Foodborne pathogenic bacteria like, Escherichia coli 0157:H7, Bacillus cereus and Salmonella typhimurium are the biggest threat to food industries. Their growth in the environment and transmission of deadly diseases has become a challenge. The present study confirmed the genetic identity of DD_A7 is similar to the reported probiotic strains of Weissella confusa by analyzing the polymorphism in the conserved region of 16S-rRNA sequence. The Kimchi isolated, non-pathogenic W. confusa DD_A7 strain has shown the potential to control the growth of foodborne pathogens. Furthermore, the cell-free culture supernatant of DD_A7 shows the potential to rapture the membrane integrity of E. coli 0157:H7 by influencing the Ca++ ion channels. The safety concern associated with the usage of DD_A7 strain was also determined by testing its antibiotic susceptibility against different antibiotics and confirmed its non-toxic behavior on mammalian cells. Nevertheless, the strain has also encompassed several applications such as isolation of amylolytic and proteolytic enzymes. Collectively, the study will help to control the growth of foodborne pathogenic bacteria using the studied probiotic strain. In addition, the study will also help to construct a species-specific probe in future for the identification of the non-pathogenic strain of W. confusa for its application in food industries.

A fast and sensitive multiplex real-time PCR assay for simultaneous identification of Bursaphelenchus xylophilus, B. mucronatus and B. fraudulentus – three closely related species from the xylophilus group

The quarantine nematode Bursaphelenchus xylophilus is the causal agent of pine wilt disease in Asia and Europe. Differentiation between B. xylophilus and other related, non-pathogenic species can be ambiguous when based exclusively on morphological characters. The morphology of B. mucronatus and B. fraudulentus most closely resembles that of B. xylophilus. Moreover, all of these nematodes are found in both Asia and Europe and can colonise various species of pine. Therefore, for phytosanitary purposes it is necessary to identify the three species precisely and rapidly. In this paper we described the results of the optimized multiplex real-time PCR protocol that utilizes two universal primers and three specific TaqMan probes to identify and differentiate the three Bursaphelenchus species, simultaneously. Species-specific real-time PCR reactions gave expected products for B. xylophilus, B. mucronatus and B. fraudulentus templates. The optimized primer combination together with designed species-specific probes has produced reliable results in multiplex PCR assays with eleven geographically distant isolates of B. xylophilus, eleven of B. mucronatus, seven of B. fraudulentus, and no cross-reactions were observed between tested samples and with other species (i.e., B. piniperdae, B. pinophilus, B. populi and Parasitaphelenchus papillatus). All species-specific real-time PCR reactions performed on DNA extracted from a single nematode individual consistently gave specific amplicons, confirming the reproducibility of the method. Moreover, the conducted study revealed that 30 fg was the lowest DNA input still detected in multiplex real-time PCR analysis. The described approach is simple, time-saving, and reliable in comparison to conventional PCR, and it can be used for simultaneous identification of the above three species within the xylophilus group.

Character based identification system for Elasmobranchs for conservation and forensic applications

The term Elasmobranchs or Chondrichthyans refer to the group of marine organisms include sharks, skates, rays and chimeras. Due to high demand most of the Elasmobranch species depleted drastically in recent years. Being the apex predators in the marine environment most of the Chondrichthyan species are vulnerable with some risk of extinction. Accurate identification of species is prerequisite for conservation and management of these declining resources. In this context, character-based identification methods are of immense use which classify specimen to species using classification rules that compactly characterized species in terms of key diagnostic nucleotides in the selected gene sequences. In the present study, a total of 82 species of elasmobranches distributed in Indian waters is taken as the target group. A total of 20–30 mitochondrial CO1 sequences of each species were downloaded and modified. Using the BLOG2.0 software, species specific diagnostic nucleotides were identified for the selected group of species. Based on identified diagnostic nucleotide sites, species-specific probes with a length range of 23–30 bp were designed. These probes could be useful in detecting species of interest in customized microarrays (DNA chip). The technique is a powerful tool to obtain a significant amount of accurate information quickly and conveniently and could be used for forensic applications and conservation of elasmobranches in Indian waters.

Validated Identity Test Method for Ginkgo biloba NHPs Using DNA-Based Species-Specific Hydrolysis PCR Probe

Background: There is considerable risk of adulteration of Ginkgo biloba herbal products in the natural health product (NHP) industry. Authentication of G. biloba products is challenging because of the standard, complex, analytical chemistry methods that may be too costly and not appropriate for both raw and finished products. Objective: We sought to develop and validate an alternative method to authenticate G. biloba herbal dietary supplements, based on the use of a species-specific hydrolysis PCR probe assay. Methods: A species-specific hydrolysis probe assay was developed, validated, and evaluated for the performance of the assay in accurately identifying the species of interest using the following analytical validation criteria: (1) specificity (accuracy) in identifying the target species ingredient, while not identifying other nontarget species, (2) sensitivity in detecting the smallest amount of the target material, and (3) reliability (repeatability and reproducibility) in detecting the target species in raw materials on a real-time PCR platform. Results: The species-specific hydrolysis probe assay was successfully developed for raw materials of G. biloba. The specificity of the assay was 100% to the target species. Efficiency of the assay was observed to be 99%, and the reliability of the assay was 100% for the raw/starting materials. Conclusions and Highlights: The method developed in this study is simple, rapid, and easy for supplement manufacturers to perform in their laboratories to ensure that their G. biloba supplements are authentic.

A simultaneous triplex TaqMan real-time PCR approach for authentication of caprine and bovine meat, milk and cheese

Goat foodstuffs are considered as healthy foods with high nutritional value. This study demonstrated the development and validation of a triplex real-time PCR on the basis of species-specific and species-conservative TaqMan probes for the simultaneous identification of caprine and bovine DNA in meats, milk and cheeses with a prerequisite designed endogenous control. In this research, caprine and bovine meat, milk and cheese were specifically identified via developed primers and probes, and the limits of detection of this methodology were 0.005 and 0.01 ng DNA of milk and cheese from goat, and 0.01 and 0.05 ng DNA of milk and cheese from cow. Taken together, this approach was elaborated to address dairy adulteration issues to eliminate the fraud of economically motivated goat milk and cheese adulteration by adding cow milk.

Polymicrobial synergy within oral biofilm promotes invasion of dendritic cells and survival of consortia members

Years of human microbiome research have confirmed that microbes rarely live or function alone, favoring diverse communities. Yet most experimental host-pathogen studies employ single species models of infection. Here, the influence of three-species oral microbial consortium on growth, virulence, invasion and persistence in dendritic cells (DCs) was examined experimentally in human monocyte-derived dendritic cells (DCs) and in patients with periodontitis (PD). Cooperative biofilm formation by Streptococcus gordonii, Fusobacterium nucleatum and Porphyromonas gingivalis was documented in vitro using growth models and scanning electron microscopy. Analysis of growth rates by species-specific 16s rRNA probes revealed distinct, early advantages to consortium growth for S. gordonii and F. nucleatum with P. gingivalis, while P. gingivalis upregulated its short mfa1 fimbriae, leading to increased invasion of DCs. F. nucleatum was only taken up by DCs when in consortium with P. gingivalis. Mature consortium regressed DC maturation upon uptake, as determined by flow cytometry. Analysis of dental plaques of PD and healthy subjects by 16s rRNA confirmed oral colonization with consortium members, but DC hematogenous spread was limited to P. gingivalis and F. nucleatum. Expression of P. gingivalis mfa1 fimbriae was increased in dental plaques and hematogenous DCs of PD patients. P. gingivalis in the consortium correlated with an adverse clinical response in the gingiva of PD subjects. In conclusion, we have identified polymicrobial synergy in a three-species oral consortium that may have negative consequences for the host, including microbial dissemination and adverse peripheral inflammatory responses.

Development and validation of probe-based multiplex real-time PCR assays for the rapid and accurate detection of freshwater fish species.

Reliable species identification methods are important for industrial environmental monitoring programs. Probe based real-time quantitative polymerase chain reaction (qPCR) provides an accurate, cost-effective and high-throughput method for species identification. Here we present the development and validation of species-specific primers and probes for the cytochrome c oxidase (COI) gene for the identification of eight ecologically and economically important freshwater fish species: lake whitefish (Coregonus clupeaformis), yellow perch (Perca flavescens), rainbow smelt (Osmerus mordax), brook trout (Salvelinus fontinalis), smallmouth bass (Micropterus dolomieu), round whitefish (Prosopium cylindraceum), spottail shiner (Notropis hudsonius) and deepwater sculpin (Myoxocephalus thompsonii). In order to identify novel primer-probe sets with maximum species-specificity, two separate primer-probe design criteria were employed. Highest ranked primer-probe sets from both methods were assayed to identify sequences that demonstrated highest specificity. Specificity was determined using control species from same genus and non-target species from different genus. Selected primer-probe sets were optimized for annealing temperature and primer-probe concentrations to identify minimum reagent parameters. The selected primer-probe sets were highly sensitive, with DNA concentrations as low as 1 ng adequate for positive species identification. A decoder algorithm was developed based on the cumulative qPCR results that allowed for full automation of species identification. Blinded experiments revealed that the combination of the species-specific primer/probes sets with the automated species decoder resulted in target species identification with 100% accuracy. We also conducted a cost/time comparison analysis between the qPCR assays established in this study with other species identification methods. The qPCR technique was the most cost-effective and least time consuming method of species identification. In summary, probe-based multiplex qPCR assays provide a rapid and accurate method for freshwater fish species identification, and the methodology established in this study can be utilized for various other species identification initiatives.

Fluorescence in Situ Hybridization (FISH) for the Diagnosis of Periprosthetic Joint Infection in Formalin-Fixed Paraffin-Embedded Surgical Tissues.

As the number of arthroplasties performed increases, periprosthetic joint infection (PJI) represents a common and challenging problem. The Musculoskeletal Infection Society (MSIS) recommends diagnosing PJI according to its guidelines. The aim of the current study was to assess whether fluorescence in situ hybridization (FISH) analysis of formalin-fixed paraffin-embedded periprosthetic membranes can successfully improve the diagnosis of infection in patients with orthopaedic implants. We retrospectively analyzed 88 periprosthetic membranes of joint prostheses using FISH analysis according to a standard protocol, with a probe targeting a sequence found in most bacteria. We compared the results with routine clinical classification according to the guidelines of the MSIS, microbiological culture, and histopathological classification according to Morawietz and Krenn. We additionally performed FISH analysis using 2 species-specific probes for several culture-positive cases. FISH successfully detected bacteria in 38 (95%) of 40 periprosthetic membranes that were rated positive by clinical classification. FISH results compared with clinical classification demonstrated a sensitivity of 95% (95% confidence interval [CI], 83.08% to 99.39%), a specificity of 85.42% (95% CI, 72.24% to 93.93%), a positive predictive value of 84.44% (95% CI, 70.55% to 93.50%), and a negative predictive value of 95.35% (95% CI, 84.19% to 99.43%). FISH results compared with histopathological classification demonstrated a sensitivity of 95.12% (95% CI, 83.47% to 99.40%), a specificity of 87.23% (95% CI, 74.26% to 95.17%), a positive predictive value of 86.67% (95% CI, 73.21% to 94.95%), and a negative predictive value of 95.35% (95% CI, 84.19% to 99.43%). We successfully detected Pseudomonas aeruginosa and Staphylococcus aureus with species-specific FISH probes in all cases that were positive for these respective bacteria by microbiological culture. FISH-based diagnosis of PJI is feasible and can be used as an additional diagnostic criterion. FISH not only can detect bacteria in periprosthetic membranes but can also differentiate pathogens at the species level. FISH represents a fast and reliable tool for detecting PJI in periprosthetic membranes, especially in combination with clinical and histopathological classification. Diagnostic Level III. See Instructions for Authors for a complete description of levels of evidence.

Dispersion and degradation of environmental DNA from caged fish in a marine environment

Environmental DNA (eDNA) consists of DNA fragments shed from organisms into the environment, and can be used to identify species presence and abundance. This study aimed to reveal the dispersion and degradation processes of eDNA in the sea. Caged fish were set off the end of a pier in Maizuru Bay, the Sea of Japan, and their eDNA was traced at sampling stations located at the cage and 10, 30, 100, 300, 600 and 1000 m distances from the cage along two transect lines. Sea surface water was collected at each station at 0, 2, 4, 8, 24 and 48 h after setting the cage, and again after removing the cage. Quantitative PCR analyses using a species-specific primer and probe set revealed that the target DNA was detectable while the cage was present and for up to 1 h after removing the cage, but not at 2 h or later. Among the 57 amplified samples, 45 (79%) were collected within 30 m from the cage. These results suggest that eDNA can provide a snapshot of organisms present in a coastal marine environment.

Monospecies and polymicrobial biofilms differentially regulate the phenotype of genotype-specific oral cancer cells.

Microbial infection has been shown to involve in oral carcinogenesis; however, the underlying mechanisms remain poorly understood. The present study aimed to characterize the growth of oral microorganisms as both monospecies and polymicrobial biofilms and determine the effects of their products on oral keratinocytes. Candida albicans (ALC3), Actinomyces naeslundii (AN) and Streptococcus mutans (SM) biofilms or a combination of these (TRI) were grown in flow-cell system for 24 h. The biofilms were subjected to fluorescent in situ hybridization using species-specific probes and analysed using confocal laser scanning microscopy. The effluent derived from each biofilm was collected and incubated with malignant (H357) and normal (OKF6) oral keratinocytes to assess extracellular matrix adhesion, epithelial-mesenchymal transition (EMT) and cytokines expression. Incubation of OKF6 with ALC3 and TRI effluent significantly decreased adhesion of the oral keratinocyte to collagen I, whereas incubation of H357 with similar effluent increased adhesion of the oral keratinocyte to laminin I, significantly when compared with incubation with artificial saliva containing serum-free medium (NE; P < 0.05). In OKF6, changes in E-cadherin and vimentin expression were not consistent with EMT although there was evidence of a mesenchymal to epithelial transition in malignant oral keratinocytes incubated with AN and SM effluent. A significant increase of pro-inflammatory cytokines expression, particularly interleukin (IL)-6 and IL-8, was observed when H357 was incubated with all biofilm effluents after 2- and 24-h incubation when compared with NE (P < 0.05). In conclusion, C.albicans, A.naeslundii and S.mutans form polymicrobial biofilms which differentially modulate malignant phenotype of oral keratinocytes.

Visualization of the impatiens downy mildew pathogen using fluorescence in situ hybridization (FISH)

BackgroundPlasmopara obducens is the biotrophic oomycete responsible for impatiens downy mildew, a destructive disease of Impatiens that causes high crop loss. Currently, there are no available methods for the microscopic detection of P. obducens from leaves of impatiens, which may be contributing to the spread of the disease. Fluorescence in situ hybridization (FISH) is a sensitive and robust method that uses sequence-specific, fluorescence-labeled oligonucleotide probes to detect target organisms from the environment. To study this important pathogen, we developed and standardized a FISH technique for the visualization of P. obducens from Impatiens walleriana tissues using a species-specific 24-mer oligonucleotide probe designed to target a region of the rRNA internal transcribed spacer 2 (ITS2).ResultsSince P. obducens cannot be propagated in vitro, we developed a custom E. coli expression vector that transcribes the P. obducens rRNA-ITS target sequence (clone-FISH) for use as a control and to optimize hybridization conditions. The FISH assay could detect P. obducens sporangiophores, sporangia and oospores, and hyphae from naturally infected I. walleriana leaves and stems. Cross-reactivity was not observed from plant tissue, and the assay did not react when applied to E. coli with self-ligated plasmids and non-target oomycete species.ConclusionsThis FISH protocol may provide a valuable tool for the study of this disease and could potentially be used to improve early monitoring of P. obducens, substantially reducing the persistence and spread of this destructive plant pathogen.

Species-specific detection and quantification of environmental DNA from marine fishes in the Baltic Sea

Biomass assesment of fish stocks is a difficult task that often involves costly fisheries trawl surveys. Trawl surveys rarely return replicate samples, as trawling attempts are expensive and difficult to reproduce. Furthermore, traditional benthic trawling is often detrimental to habitats and the organisms associated with the sea bottom. The rapidly developing field of environmental DNA (eDNA) analysis offers a new approach to non-invasive monitoring of fish. In the present study, we develop and test species-specific primers and probes for qPCR detection of eDNA from Atlantic herring (Clupea harengus), Atlantic cod (Gadus morhua), European flounder (Platichthys flesus), European plaice (Pleuronectes platessa), and Atlantic mackerel (Scomber scombrus) in the Baltic Sea. A recently published qPCR system was applied for European eel (Anguilla anguilla). Filtered water samples were collected during a stratified benthic trawl survey, enabling parallel comparisons of eDNA concentrations with biomass caught by trawling. No significant correlation was found between eDNA concentrations and the biomass of fish caught by the trawl, although an association was observed between the measured concentrations of eDNA and the known distributions and main abundances of cod, herring, plaice and flounder. This indicates that while eDNA concentrations may not be directly comparable to results from existing methods – likely because aquatic eDNA concentrations are not controlled by the same combinations of factors as e.g. trawl biomass catch – eDNA analysis could prove a useful supplement for monitoring fish stocks in the future.

Diversity, distribution, and azaspiracids of Amphidomataceae (Dinophyceae) along the Norwegian coast

Azaspiracids (AZA) are a group of lipophilic polyether compounds which have been implicated in shellfish poisoning incidents around Europe. They are produced by a few species of the dinophycean genera Azadinium and Amphidoma (Amphidomataceae). The presence of AZA toxins in Norway is well documented, but knowledge of the distribution and diversity of Azadinium and other Amphidomataceae along the Norwegian coast is rather limited and poorly documented. On a research survey along the Norwegian coast in 2015 from the Skagerrak in the South to Trondheimsfjorden in the North, plankton samples from 67 stations were analysed for the presence of Azadinium and Amphidoma and their respective AZA by on-board live microscopy, real-time PCR assays specific for Amphidomataceae, and liquid chromatography-tandem mass spectrometry (LC–MS/MS).Microscopy using live samples and positive real-time PCR assays using a general family probe and two species specific probes revealed the presence of Amphidomataceae distributed throughout the sampling area. Overall abundance was low, however, and was in agreement with a lack of detectable AZA in plankton samples. Single cell isolation and morphological and molecular characterisation of established strains revealed the presence of 7 amphidomatacean species (Azadiniun spinosum, Az. poporum, Az. obesum, Az. dalianense, Az. trinitatum, Az. polongum, Amphidoma languida) in the area. Azaspiracids were produced by the known AZA producing species Az. spinosum, Az. poporum and Am. languida only. LC–MS/MS analysis further revealed that Norwegian strains produce previously unreported AZA for Norway (AZA-11 by Az. spinosum, AZA-37 by Az. poporum, AZA-38 and AZA-39 by Am. languida), and also four novel compounds (AZA-50, -51 by Az. spinosum, AZA-52, -53 by Am. languida), whose structural properties are described and which now can be included in existing analytical protocols. A maximum likelihood analysis of concatenated rDNA regions (SSU, ITS1-ITS2, partial LSU) showed that the strains of Az. spinosum fell in two well supported clades, where most but not all new Norwegian strains formed the new Ribotype B. Ribotype differentiation was supported by a minor morphological difference with respect to the presence/absence of a rim around the pore plate, and was consistently reflected by different AZA profiles. Strains of Az. spinosum from ribotype A produce AZA-1, -2 and -33, whereas the new strains of ribotype B produce mainly AZA-11 and AZA-51. Significant sequence differences between both Az. spinosum ribotypes underline the need to redesign the currently used qPCR probes in order to detect all AZA producing Az. spinosum.The results generally underline the conclusion that for the Norwegian coast area it is important that amphidomatacean species are taken into account in future studies and monitoring programs.