Dr. Torstein Tengs

Prevalence of piscine myocarditis virus (PMCV) in marine fish species

Torstein Tengs Dr. — Wed, 23 Nov 2011 06:24:31 PST

No abstract.

A novel totivirus and piscine reovirus (PRV) in Atlantic salmon (Salmo salar)

Torstein Tengs — Thu, 02 Dec 2010 01:17:17 PST

Background

Cardiomyopathy syndrome (CMS) is a severe disease affecting large farmed Atlantic salmon. Mortality often appears without prior clinical signs, typically shortly prior to slaughter. We recently reported the finding and the complete genomic sequence of a novel piscine reovirus (PRV), which is associated with another cardiac disease in Atlantic salmon; heart and skeletal muscle inflammation (HSMI). In the present work we have studied whether PRV or other infectious agents may be involved in the etiology of CMS.

Results

Using high throughput sequencing on heart samples from natural outbreaks of CMS and from fish experimentally challenged with material from fish diagnosed with CMS a high number of sequence reads identical to the PRV genome were identified. In addition, a sequence contig from a novel totivirus could also be constructed. Using RT-qPCR, levels of PRV in tissue samples were quantified and the totivirus was detected in all samples tested from CMS fish but not in controls. In situ hybridization supported this pattern indicating a possible association between CMS and the novel piscine totivirus.

Conclusions

Although causality for CMS in Atlantic salmon could not be proven for either of the two viruses, our results are compatible with a hypothesis where, in the experimental challenge studied, PRV behaves as an opportunist whereas the totivirus might be more directly linked with the development of CMS.

Heart and skeletal muscle inflammation of farmed salmon is associated with infection with a novel reovirus

Torstein Tengs — Tue, 03 Aug 2010 02:49:42 PDT

Atlantic salmon (Salmo salar L.) mariculture has been associated with epidemics of infectious diseases that threaten not only local production, but also wild fish coming into close proximity to marine pens and fish escaping from them. Heart and skeletal muscle inflammation (HSMI) is a frequently fatal disease of farmed Atlantic salmon. First recognized in one farm in Norway in 1999, HSMI was subsequently implicated in outbreaks in other farms in Norway and the United Kingdom. Although pathology and disease transmission studies indicated an infectious basis, efforts to identify an agent were unsuccessful. Here we provide evidence that HSMI is associated with infection with piscine reovirus (PRV). PRV is a novel reovirus identified by unbiased high throughput DNA sequencing and a bioinformatics program focused on nucleotide frequency as well as sequence alignment and motif analyses. Formal implication of PRV in HSMI will require isolation in cell culture and fulfillment of Koch's postulates, or prevention or modification of disease through use of specific drugs or vaccines. Nonetheless, as our data indicate that a causal relationship is plausible, measures must be taken to control PRV not only because it threatens domestic salmon production but also due to the potential for transmission to wild salmon populations.

Comparison of nine different real-time PCR chemistries for qualitative and quantitative applications in GMO detection

Torstein Tengs — Mon, 01 Feb 2010 01:39:27 PST

Several techniques have been developed for detection and quantification of genetically modified organisms, but quantitative real-time PCR is by far the most popular approach. Among the most commonly used realtime PCR chemistries are TaqMan probes and SYBR green, but many other detection chemistries have also been developed. Because their performance has never been compared systematically, here we present an extensive evaluation of some promising chemistries: sequenceunspecific DNA labeling dyes (SYBR green), primer-based technologies (AmpliFluor, Plexor, Lux primers), and techniques involving double-labeled probes, comprising hybridization (molecular beacon) and hydrolysis (TaqMan, CPT, LNA, and MGB) probes, based on recently published experimental data. For each of the detection chemistries assays were included targeting selected loci. Real-time PCR chemistries were subsequently compared for their efficiency in PCR amplification and limits of detection and quantification. The overall applicability of the chemistries was evaluated, adding practicability and cost issues to the performance characteristics. None of the chemistries seemed to be significantly better than any other, but certain features favor LNA and MGB technology as good alternatives to TaqMan in quantification assays. SYBR green and molecular beacon assays can perform equally well but may need more optimization prior to use.

Characterization of unknown genetic modifications using high throughput sequencing and computational subtraction

Torstein Tengs — Wed, 14 Oct 2009 01:05:03 PDT

Background

When generating a genetically modified organism (GMO), the primary goal is to give a target organism one or several novel traits by using biotechnology techniques. A GMO will differ from its parental strain in that its pool of transcripts will be altered. Currently, there are no methods that are reliably able to determine if an organism has been genetically altered if the nature of the modification is unknown.

Results

We show that the concept of computational subtraction can be used to identify transgenic cDNA sequences from genetically modified plants. Our datasets include 454-type sequences from a transgenic line of Arabidopsis thaliana and published EST datasets from commercially relevant species (rice and papaya).

Conclusion

We believe that computational subtraction represents a powerful new strategy for determining if an organism has been genetically modified as well as to define the nature of the modification. Fewer assumptions have to be made compared to methods currently in use and this is an advantage particularly when working with unknown GMOs.

Non-prejudiced Detection and Characterization of Genetic Modifications

Torstein Tengs — Wed, 14 Oct 2009 00:58:38 PDT

The application of gene technology is becoming widespread much thanks to the rapid increase in technology, resource, and knowledge availability. Consequently, the diversity and number of genetically modified organisms (GMOs) that may find their way into the food chain or the environment, intended or unintended, is rapidly growing. From a safety point of view the ability to detect and characterize in detail any GMO, independent of publicly available information, is fundamental. Pre-release risk assessments of GMOs are required in most jurisdictions and are usually based on application of technologies with limited ability to detect unexpected rearrangements and insertions. We present an array-based approach to address these problems and show with three examples (GTS 40-3-2 Roundup Ready and event A5547-127 soybean as well as T25 Liberty Link Maize) that the method can detect and characterize GMOs with high accuracy while making very few prior assumptions about the actual genetic modifications or constructs in question. Based on the array results, a simple polymerase chain reaction-scheme is also described that will enable the user to characterize the inserted sequences to DNA sequence level. The method may provide the biotechnology developers and risk regulators with a useful tool to improve pre-market risk assessments as well as seed producers and other food chain and environmental stakeholders with a platform to improve their ability to detect and characterize GMOs.

UniquePrimer - a web utility for design of specific PCR primers and probes

Torstein Tengs — Wed, 14 Oct 2009 00:43:51 PDT

We have developed a web-based tool for design of specific PCR primers and probes. The program allows you to enter primer sequence information as well as an optional probe, and sequence similarity searches (MegaBLAST) will be performed to see if the sequences match the same sequence entry in the specified database. If primers (and probe) match, this will be reported. The program can handle overlapping amplicons, amplification from a single primer, ambiguous bases and other problematic cases.

A quantitative TaqMan MGB real-time polymerase chain reaction based assay for detection of the causative agent of crayfish plague Aphanomyces astaci

Torstein Tengs — Tue, 10 Feb 2009 02:25:53 PST

Here we present the development and first validation of a TaqMan minor groove binder (MGB) real-time polymerase chain reaction (RT-PCR) method for quantitative and highly specific detection of Aphanomyces astaci, the causative agent of crayfish plague. The assay specificity was experimentally assessed by testing against DNA representative of closely related oomycetes, and theoretically assessed by additional sequence similarity analyses comparing the primers and probe sequences to available sequences in EMBL/GenBank. The target of the assay is a 59 bp unique sequence motif of A. astaci found in the internal transcribed spacer 1 of the nuclear ribosomal gene cluster. A standard curve for quantification was established by setting up a four-fold dilution series with genomic A. astaci DNA. The absolute limit of detection (LODabs), defined as the lowest concentration yielding a false negative probability < 5% was found to be approximately 5 PCR forming units (PFU; target template copies) equivalent to less than one A. astaci genome. The absolute limit of quantification (LOQabs) was experimentally established as 10 times the LODabs. Assay performance was also assessed with samples of naturally infected and noninfected susceptible crayfish (Astacus astacus) and carrier crayfish (Pacifastacus leniusculus). The benefits and limitations of the method are discussed, and guidance to practical application and interpretation of analytical results is provided.

A statistical approach for evaluation of PCR results to improve the practical limit of quantification (LOQ) of GMO analyses (SIMQUANT)

Torstein Tengs — Fri, 08 Feb 2008 01:28:03 PST

The predominant approach for quantification of genetically modified organisms (GMO) is the application of quantitative real-time PCR. However, for a large number of processed food and feed products, this approach is unsuitable, because they contain low amounts (mass) of amplifiable DNA. Here we present a novel approach, ‘‘Single molecule quantification’’ (SIMQUANT) for GMO quantification of samples with extremely low amounts of DNA. The approach is based on statistics and application of multiple qualitative parallel PCRs. Here the qualitative PCRs were done using real-time PCR setup, but this is not a requirement. The difference is that the quantitative real-time PCR requires that the target copy number exceeds the absolute limit of quantification (LOQabs) and provides quantity estimates by extrapolation from a linear regressional relationship between an observed cycle threshold (Ct) value and copy numbers, while with SIMQUANT the template DNA typically contains very few, e.g., one target copy per PCR volume and the quantity is estimated on the basis of observed ratio between positive and negative individual PCRs. The components of this analysis are the numbers of test samples, the size of each sample and the outcome in number and relative ratio of positive and negative test results. The approach results in a statistical estimate of the relative GM concentration based on the probability that one or more amplifiable GM template copies are present in discrete volumes. Thus, the approach is based on the ratio of discrete volumes without or with one or more PCR-amplifiable GM target copies. The approach described here can be used reliably with more than a 100-fold improvement of the practical LOQ (LOQpract) compared to real-time quantitative PCR based on standard curves.

Microarray-based method for detection of unknown genetic modifications

Torstein Tengs — Mon, 04 Feb 2008 00:11:54 PST

Background

Due to the increased use of genetic modifications in crop improvement, there is a need to develop effective methods for the detection of both known and unknown transgene constructs in plants. We have developed a strategy for detection and characterization of unknown genetic modifications and we present a proof of concept for this method using Arabidopsis thaliana and Oryza sativa (rice). The approach relies on direct hybridization of total genomic DNA to high density microarrays designed to have probes tiled throughout a set of reference sequences.

Results

We show that by using arrays with 25 basepair probes covering both strands of a set of 235 vectors (2 million basepairs) we can detect transgene sequences in transformed lines of A. thaliana and rice without prior knowledge about the transformation vectors or the T-DNA constructs used to generate the studied plants.

Conclusions

The approach should allow the user to detect the presence of transgene sequences and get sufficient information for further characterization of unknown genetic constructs in plants. The only requirements are access to a small amount of pure transgene plant material, that the genetic construct in question is above a certain size (here [greater than or equal to] 140 basepairs) and that parts of the construct shows some degree of sequence similarity with published genetic elements.

Francisella philomiragia subsp. noatunensis subsp. nov., isolated from farmed Atlantic cod (Gadus morhua L.)

Torstein Tengs — Mon, 03 Sep 2007 06:31:05 PDT

Seven bacterial isolates from farmed Atlantic cod displaying chronic granulomatous disease were characterized by phenotypic and molecular taxonomic methods. The isolates were Gram-negative, facultatively intracellular, non-motile, strictly aerobic coccobacilli which produced H2S from cysteine-supplemented media and are therefore phenotypically consistent with members of the genus Francisella. Comparison of 16S rRNA gene sequences and six partial housekeeping gene sequences (groEL, shdA, rpoB, rpoA, pgm and atpA) confirmed the organism as a member of the genus Francisella, with Francisella philomiragia as its closest relative (99.3% 16S rRNA gene sequence similarity, 92.2–99.0% housekeeping gene sequence similarity). Despite the close relationship with F. philomiragia, isolates from Atlantic cod could be readily distinguished phenotypically and genetically from F. philomiragia ATCC 25015. DNA–DNA hybridization studies revealed a mean reassociation value of 68 %. Thus, on the basis of phenotypic and molecular genetic evidence, we propose that the strains isolated from Atlantic cod should be recognized as Francisella philomiragia subsp. noatunensis subsp. nov. with the type strain 2005/ 50/F292-6C (5NCIMB 14265T5LMG 23800). Francisella philomiragia ATCC 25015 (5DSM 735) is reclassified as Francisella philomiragia subsp. philomiragia subsp. nov.

Phenotypically different microalgal morphospecies with identical ribosomal RNA: a case of rapid adaptive evolution?

Torstein Tengs — Fri, 25 May 2007 00:02:56 PDT

The agents driving the divergence and speciation of freeliving microbial populations are still largely unknown. We investigated the dinoflagellate morphospecies Scrippsiella hangoei and Peridinium aciculiferum, which abound in the Baltic Sea and in northern temperate lakes, respectively. Electron microscopy analyses showed significant interspecific differences in the external cellular morphology, but a similar plate pattern in the characteristic dinoflagellate armor. Experimentally, S. hangoei grew in a wide range of salinities (0–30), whereas P. aciculiferum only grew in low salinities (0–3). Despite these phenotypic differences and the habitat segregation, molecular analyses showed identical ribosomal DNA sequences (ITS1, ITS2, 5.8S, SSU, and partial LSU) for both morphospecies. Yet, a strong interspecific genetic isolation was indicated by amplified fragment length polymorphism (FST = 0.76) and cytochrome b (cob) sequence divergence (õ1.90%). Phylogenetic reconstructions based on ribosomal (SSU, LSU) and mitochondrial (cob) DNA indicated a recent marine ancestor for P. aciculiferum. In conclusion, we suggest that the lacustrine P. aciculiferum and the marine-brackish S. hangoei diverged very recently, after a marine–freshwater transition that exposed the ancestral populations to different selective pressures. This hypothetical scenario agrees with mounting data indicating a significant role of natural selection in the divergence of free-living microbes, despite their virtually unrestricted dispersal capabilities. Finally, our results indicate that identical ITS rDNA sequences do not necessarily imply the same microbial species, as commonly assumed.

Equal performance of TaqMan, MGB, molecular beacon, and SYBR green-based detection assays in detection and quantification of Roundup Ready soybean

Torstein Tengs — Tue, 02 Jan 2007 00:16:56 PST

We have tested and compared the performance of 12 different assays representing four different real-time polymerase chain reaction (PCR) chemistries in the context of genetically modified organism detection. Several different molecular beacon, SYBR Green, TaqMan, and MGB assays were designed for the event specific detection and quantification of the 3' integration junction of GTS 40-3-2 (Roundup Ready) soybean. Sensitivity as well as robustness in the presence of background DNA were tested. None of the PCR-based approaches appeared to be significantly better than any of the other, but the molecular beacon assays had the lowest efficiency and also seemed more sensitive to changes in experimental setup.

Real–time PCR monitoring of estuarine water samples for Pfiesteria piscicida, a dinoflagellate associated with fish kills and human illness

Torstein Tengs — Tue, 19 Dec 2006 05:19:44 PST

No abstract.

Development of real–time PCR assays for the detection of Chattonella species in culture and environmental samples

Torstein Tengs — Tue, 19 Dec 2006 05:16:57 PST

Raphidophytes have been associated with fish kill events in Japanese, European and U.S. coastal waters, and many produce toxins that can pose a threat to human health. The recognition of raphidophytes as HAB species in mid-Atlantic estuarine waters led us to initiate molecular phylogenetic analyses of these taxa and to develop real-time PCR assays for rapid detection of important species. The molecular studies and PCR detection methods will enhance ongoing taxonomic, toxicologic and ecological of these organisms and will be a useful tool in HAB monitoring programs.

Heteroduplex mobility assay-guided sequence discovery: elucidation of the small subunit (18S) rDNA sequences of Pfiesteria piscicida and related dinoflagellates from complex algal culture and environmental sample DNA pools

Torstein Tengs — Tue, 19 Dec 2006 04:56:27 PST

The newly described heterotrophic estuarine dinoflagellate Pfiesteria piscicida has been linked with fish kills in field and laboratory settings, and with a novel clinical syndrome of impaired cognition and memory disturbance among humans after presumptive toxin exposure. As a result, there is a pressing need to better characterize the organism and these associations. Advances in Pfiesteria research have been hampered, however, by the absence of genomic sequence data. We employed a sequencing strategy directed by heteroduplex mobility assay to detect Pfiesteria piscicida 18S rDNA ‘‘signature’’ sequences in complex pools of DNA and used those data as the basis for determination of the complete P. piscicida 18S rDNA sequence. Specific PCR assays for P. piscicida and other estuarine heterotrophic dinoflagellates were developed, permitting their detection in algal cultures and in estuarine water samples collected during fish kill and fish lesion events. These tools should enhance efforts to characterize these organisms and their ecological relationships. Heteroduplex mobility assay-directed sequence discovery is broadly applicable, and may be adapted for the detection of genomic sequence data of other novel or nonculturable organisms in complex assemblages.

Phylogenetic analyses indicate that the 19'Hexanoyloxy-fucoxanthin-containing dinoflagellates have tertiary plastids of haptophyte origin

Torstein Tengs — Tue, 19 Dec 2006 04:54:25 PST

The three anomalously pigmented dinoflagellates Gymnodinium galatheanum, Gyrodinium aureolum, and Gymnodinium breve have plastids possessing 199-hexanoyloxy-fucoxanthin as the major carotenoid rather than peridinin, which is characteristic of the majority of the dinoflagellates. Analyses of SSU rDNA from the plastid and the nuclear genome of these dinoflagellate species indicate that they have acquired their plastids via endosymbiosis of a haptophyte. The dinoflagellate plastid sequences appear to have undergone rapid sequence evolution, and there is considerable divergence between the three species. However, distance, parsimony, and maximum-likelihood phylogenetic analyses of plastid SSU rRNA gene sequences place the three species within the haptophyte clade. Pavlova gyrans is the most basal branching haptophyte and is the outgroup to a clade comprising the dinoflagellate sequences and those of other haptophytes. The haptophytes themselves are thought to have plastids of a secondary origin; hence, these dinoflagellates appear to have tertiary plastids. Both molecular and morphological data divide the plastids into two groups, where G. aureolum and G. breve have similar plastid morphology and G. galatheanum has plastids with distinctive features.

Development of real-time PCR assays for rapid detection of Pfiesteria piscicida and related dinoflagellates

Torstein Tengs — Tue, 19 Dec 2006 04:52:20 PST

Pfiesteria complex species are heterotrophic and mixotrophic dinoflagellates that have been recognized as harmful algal bloom species associated with adverse fish and human health effects along the East Coast of North America, particularly in its largest (Chesapeake Bay in Maryland) and second largest (Albermarle- Pamlico Sound in North Carolina) estuaries. In response to impacts on human health and the economy, monitoring programs to detect the organism have been implemented in affected areas. However, until recently, specific identification of the two toxic species known thus far, Pfiesteria piscicida and P. shumwayae (sp. nov.), required scanning electron microscopy (SEM). SEM is a labor-intensive process in which a small number of cells can be analyzed, posing limitations when the method is applied to environmental estuarine water samples. To overcome these problems, we developed a real-time PCR-based assay that permits rapid and specific identification of these organisms in culture and heterogeneous environmental water samples. Various factors likely to be encountered when assessing environmental samples were addressed, and assay specificity was validated through screening of a comprehensive panel of cultures, including the two recognized Pfiesteria species, morphologically similar species, and a wide range of other estuarine dinoflagellates. Assay sensitivity and sample stability were established for both unpreserved and fixative (acidic Lugol’s solution)-preserved samples. The effects of background DNA on organism detection and enumeration were also explored, and based on these results, we conclude that the assay may be utilized to derive quantitative data. This real-time PCR-based method will be useful for many other applications, including adaptation for field-based technology.

Genetic polymorphism in Gymnodinium galatheanum chloroplast DNA sequences and development of a molecular detection assay

Torstein Tengs — Tue, 19 Dec 2006 04:49:29 PST

Nuclear and chloroplast-encoded small subunit ribosomal DNA sequences were obtained from several strains of the toxic dinoflagellate Gymnodinium galatheanum . Phylogenetic analyses and comparison of sequences indicate that the chloroplast sequences show a higher degree of sequence divergence than the nuclear homologue. The chloroplast sequences were chosen as targets for the development of a 5'–3' exonuclease assay for detection of the organism. The assay has a very high degree of specificity and has been used to screen environmental water samples from a fish farm where the presence of this dinoflagellate species has previously been associated with fish kills. Various hypotheses for the derived nature of the chloroplast sequences are discussed, as well as what is known about the toxicity of the species.

Classification and identification of Pfiesteria and Pfiesteria-like species

Torstein Tengs — Tue, 19 Dec 2006 04:46:46 PST

Dinoflagellates can be classified both botanically and zoologically; however, they are typically put in the botanical division Pyrrhophyta. As a group they appear most related to the protistan ciliates and apicomplexans at the ultrastructure level. Within the Pyrrhophyta are both unarmored and armored forms of the dominant, motile flagellated stage. Unarmored dinoflagellates do not have thecal or wall plates arranged in specific series, whereas armored species have plates that vary in thickness but are specific in number and arrangement. In armored dinoflagellates, the plate pattern and tabulation is a diagnostic character at the family, subfamily, and even genus levels. In most cases, the molecular characterization of dinoflagellates confirms the taxonomy on the basis of external morphology; this has been demonstrated for several groups. Together, both genetic and morphological criteria are becoming increasingly important for the characterization, separation, and identification of dinoflagellates species. Pfiesteria and Pfiesteria-like species are thinly armored forms with motile dinospore stages characterized by their distinct plate formulae. Pfiesteria piscicida is the best-known member of the genus; however, there is at least one other species. Other genetically and morphologically related genera, now grouped under the common names of “Lucy,” “Shepherd’s crook,” and cryptoperidiniopsoid, are being studied and described in separate works. All these other heterotrophic dinoflagellate groups, many of which are thought to be benign, co-occur in estuarine waters where Pfiesteria has been found. Key words: Pfiesteria, cryptoperidiniopsoid,”Lucy,” dinoflagellates, Pfiesteria-like, Kofoidian, plate patterns, morphology.