[Show abstract][Hide abstract] ABSTRACT: The Norwegian muskox (Ovibos moschatus) population lives on the high mountain plateau of Dovre and originates from animals introduced from Greenland. In the late summers of 2006 and 2012, severe outbreaks of pneumonia with mortality rates of 25-30% occurred. During the 2012 epidemic high quality samples from culled sick animals were obtained for microbiological and pathological examinations. High throughput sequencing (pyrosequencing) of pneumonic lung tissue revealed high concentrations of Mycoplasma ovipneumoniae in all six animals examined by this method and Pasteurella multocida subsp. multocida in four animals, whereas no virus sequences could be identified. Mycoplasma ovipneumoniae and P. multocida multocida were also isolated by culture. Using real time PCR on lung swabs, M. ovipneumoniae was detected in all of the 19 pneumonic lungs examined. Gross pathological examination revealed heavy consolidations primarily in the cranial parts of the lungs and it also identified one case of otitis media. Histologically, lung lesions were characterized as acute to subacute mixed exudative and moderately proliferative bronchoalveolar pneumonia. Immunohistochemical (IHC) examination revealed high load of M. ovipneumoniae antigens within lung lesions, with particularly intensive staining in the neutrophils. Similar IHC finding were observed in archived lung tissue blocks from animals examined during the 2006 epidemic. An M. ovipneumoniae specific ELISA was applied on bio-banked muskox sera from stray muskoxen killed in the period 2004-2013 and sick muskoxen culled, as well as sera from wild reindeer (Rangifer tarandus tarandus) on Dovre and muskoxen from Greenland. Serology and mycoplasma culturing was also carried out on sheep that had been on pasture in the muskox area during the outbreak in 2012. Our findings indicated separate introductions of M. ovipneumoniae infection in 2006 and 2012 from infected co-grazing sheep. Salt licks shared by the two species were a possible route of transmitting infection.
PLoS ONE 01/2014; 9(9):e106116. · 3.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Piscine orthoreovirus (PRV) is associated with heart- and skeletal muscle inflammation (HSMI) of farmed Atlantic salmon (Salmo salar). We have performed detailed sequence analysis of the PRV genome with focus on putative encoded proteins, compared with prototype strains from mammalian (MRV T3D)- and avian orthoreoviruses (ARV-138), and aquareovirus (GCRV-873). Amino acid identities were low for most gene segments but detailed sequence analysis showed that many protein motifs or key amino acid residues known to be central to protein function are conserved for most PRV proteins. For M-class proteins this included a proline residue in μ2 which, for MRV, has been shown to play a key role in both the formation and structural organization of virus inclusion bodies, and affect interferon-β signaling and induction of myocarditis. Predicted structural similarities in the inner core-forming proteins λ1 and σ2 suggest a conserved core structure. In contrast, low amino acid identities in the predicted PRV surface proteins μ1, σ1 and σ3 suggested differences regarding cellular interactions between the reovirus genera. However, for σ1, amino acid residues central for MRV binding to sialic acids, and cleavage- and myristoylation sites in μ1 required for endosomal membrane penetration during infection are partially or wholly conserved in the homologous PRV proteins. In PRV σ3 the only conserved element found was a zinc finger motif. We provide evidence that the S1 segment encoding σ3 also encodes a 124 aa (p13) protein, which appears to be localized to intracellular Golgi-like structures. The S2 and L2 gene segments are also potentially polycistronic, predicted to encode a 71 aa- (p8) and a 98 aa (p11) protein, respectively. It is concluded that PRV has more properties in common with orthoreoviruses than with aquareoviruses.
PLoS ONE 08/2013; 8(7):e70075. · 3.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: BACKGROUND: The genetic element s2m seems to represent one of very few examples of mobile genetic elements in viruses. The function remains obscure and a scattered taxonomical distribution has been reported by numerous groups. METHODS: We have searched GenBank in order to identify all viral accessions that have s2m(-like) sequence motifs. Rigorous phylogenetic analyses and constrained tree topology testing were also performed in order to investigate the apparently mobile nature of s2m. RESULTS: The stem-loop s2m structure can be found in four families of + ssRNA viruses; Astroviridae, Caliciviridae, Picornaviridae and Coronaviridae. In all of these virus families, with the possible exception of Caliciviridae, multiple gains and/or losses of s2m would have to be postulated in order to explain the distribution of this character. CONCLUSIONS: s2m appears to be a mobile genetic element with a unique evolutionary history in all of the four virus families where it can be found. Based on our findings and a review of the current literature on s2m, a hypothesis implying an RNAi-like function for the s2m element is also outlined.
[Show abstract][Hide abstract] ABSTRACT: Uncultivable HPR0 strains of infectious salmon anaemia viruses (ISAVs) infecting gills are non-virulent putative precursors of virulent ISAVs (vISAVs) causing systemic disease in farmed Atlantic salmon (Salmo salar). The transition to virulence involves two molecular events, a deletion in the highly polymorphic region (HPR) of the hemagglutinin-esterase (HE) gene and a Q266→L266 substitution or insertion next to the putative cleavage site (R267) in the fusion protein (F). We have performed ultra-deep pyrosequencing (UDPS) of these gene regions from healthy fish positive for HPR0 virus carrying full-length HPR sampled in a screening program, and a vISAV strain from an ISA outbreak at the same farming site three weeks later, and compared the mutant spectra. As the UDPS data shows the presence of both HE genotypes at both sampling times, and the outbreak strain was unlikely to be directly related to the HPR0 strain, this is the first report of a double infection with HPR0s and vISAVs. For F amplicon reads, mutation frequencies generating L266 codons in screening samples and Q266 codons in outbreak samples were not higher than at any random site. We suggest quasispecies heterogeneity as well as RNA structural properties are linked to transition to virulence. More specifically, a mechanism where selected single point mutations in the full-length HPR alter the RNA structure facilitating single- or sequential deletions in this region is proposed. The data provides stronger support for the deletion hypothesis, as opposed to recombination, as the responsible mechanism for generating the sequence deletions in HE.
PLoS ONE 01/2013; 8(11):e81571. · 3.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Cardiomyopathy syndrome (CMS) is a severe cardiac disease of sea-farmed Atlantic salmon Salmo salar L., but CMS-like lesions have also been found in wild Atlantic salmon. In 2010 a double-stranded RNA virus of the Totiviridae family, provisionally named piscine myocarditis virus (PMCV), was described as the causative agent of CMS. In the present paper we report the first detection of PMCV in wild Atlantic salmon. The study is based on screening of 797 wild Atlantic salmon by real-time RT-PCR. The samples were collected from 35 different rivers along the coast of Norway, and all individuals included in the study were classified as wild, based on visual appearance and scale reading. Two samples tested positive during PCR analysis, and the results were confirmed by sequencing.
Diseases of Aquatic Organisms 12/2012; 102(2):157-61. · 1.73 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The analytical procedure used for GM detection and quantification at the laboratory level is composed of different modules. Each module can qualitatively and/or quantitatively impact the accuracy of the analytical result. Within the Co-Extra project, we have investigated different aspects of reliability of GMO detection. We have also established a system for improving reliability of quantitative analysis in samples with low DNA content. Additionally, we present and discuss efforts to reduce the trade-off between reliability and analysis costs.
Genetically modified and non-genetically modified food supply chains: co-existence and traceability, http://onlinelibrary.wiley.com/book/10.1002/9781118373781 edited by Yves Bertheau, 10/2012: chapter Reliability and cost of GMO detection: pages 307-332; Wiley-Blackwell., ISBN: 978-1-4443-3778-5
[Show abstract][Hide abstract] ABSTRACT: The state-of-the-art technology for detection of genetically modified organisms (GMOs) is polymerase chain reaction. The targets are novel sequences such as genes, but the insertion locus sequences are also important for GMO identification. GMOs go through a series of developmental stages, in which field trials precede commercial releases. GMO developers only exceptionally publish details on inserted novel sequences unless such publication is, for example, required by legislation. Information on novel sequences of GMOs may therefore be sparse, and in some cases completely missing, making the development and implementation of detection methods extremely difficult. In this chapter, we present an overview of the problems and proposed possible solutions to challenges associated with detection and correct identification of GMOs, with particular emphasis on unknown GMOs.
Genetically modified and non-genetically modified food supply chains: co-existence and traceability, http://onlinelibrary.wiley.com/book/10.1002/9781118373781 edited by Yves Bertheau, 10/2012: chapter Towards detection of unknown GMOs: pages 367-382; Wiley-Blackwell., ISBN: 978-1-4443-3778-5
[Show abstract][Hide abstract] ABSTRACT: BACKGROUND: Tick-borne encephalitis (TBE) is among the most important vector borne diseases of humans in Europe and is currently identified as a major health problem in many countries. TBE endemic zones have expanded over the past two decades, as well as the number of reported cases within endemic areas. Multiple factors are ascribed for the increased incidence of TBE, including climatic change. The number of TBE cases has also increased in Norway over the past decade, and the human cases cluster along the southern coast of Norway. In Norway the distribution and prevalence of TBE virus (TBEV) in tick populations is largely unknown. The objectives of this study were to estimate the TBEV prevalence in Ixodes ricinus from seven locations and to assess the relationship between the TBEV prevalence and site-specific climatic variables. METHODS: A total of 5630 questing nymphs were collected and analyzed in pools of ten. All pools were screened with an in-house real-time RT-PCR, and the positive pools were pyrosequenced. Two methods, minimum infection rate (MIR) and a frequentist method (EPP) for pooled prevalence estimations were calculated and compared. Climatic data were descriptively compared to the corresponding EPP of each location in order to explain variations in TBEV prevalence. RESULTS: The seven foci of TBEV had an estimated overall prevalence (EPP) in pools of nymphs combined, of 0.53% with 95% CI (0.35--0.75), with point prevalence ranging between 0.11%--1.22%. The sites with the highest point prevalences were within the municipalities which had the highest numbers of registered TBE cases. The results indicate that the location with highest point prevalence had the highest relative mean humidity and lowest mean saturation deficit and vice versa for the lowest EPP. CONCLUSION: Our study confirms the existence of TBEV endemic foci in Norway. These results are of importance to increase the awareness of TBEV infections in Norway and could be used for public information and recommendations of TBE vaccination. EPP is the method of choice for pooled prevalence calculations, since it provides estimated prevalences with confidence intervals. Our findings emphasise the possible importance of microclimatic conditions regarding the TBEV prevalence in ticks.
[Show abstract][Hide abstract] ABSTRACT: The newly described piscine reovirus (PRV) appears to be associated with the development of heart and skeletal muscle inflammation (HSMI) in farmed Atlantic salmon Salmo salar L. PRV seems to be ubiquitous among fish in Norwegian salmon farms, but high viral loads and tissue distribution support a causal relationship between virus and disease. In order to improve understanding of the distribution of PRV in the salmon production line, we quantified PRV by using real-time PCR on heart samples collected at different points in the life cycle from pre-smolts to fish ready for slaughter. PRV positive pre-smolts were found in about 36% of the freshwater cohorts and a general increase in viral load was observed after their transfer to seawater. A reduction in viral loads was recorded when fish approached slaughter (18 mo in sea cages). Sequencing of positive samples did not support the hypothesis that outbreaks are caused by the spreading of a particular (virulent) strain of PRV.
Diseases of Aquatic Organisms 05/2012; 99(1):7-12. · 1.73 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
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.
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.
[Show abstract][Hide abstract] ABSTRACT: 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 real-time 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: sequence-unspecific 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.
Analytical and Bioanalytical Chemistry 03/2010; 396(6):2023-9. · 3.66 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
KeywordsDNA Sequence-Genetically Modified Organisms-Transgene Insert-Unauthorized GMO-Unknown GMO
[Show abstract][Hide abstract] ABSTRACT: 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.
PLoS ONE 01/2010; 5(7):e11487. · 3.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
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).
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.
[Show abstract][Hide abstract] ABSTRACT: 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.
in silico analysis-detection assay-web utility-primer design-probe design-MegaBLAST
Annals of Microbiology 01/2009; 59(2):391-393. · 1.55 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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 (LOD(abs)), defined as the lowest concentration yielding a false negative probability<5% was found to be approximately 5 PCR forming units (PFU<or=target template copies) equivalent to less than one A. astaci genome. The absolute limit of quantification (LOQ(abs)) was experimentally established as 10 times the LOD(abs). Assay performance was also assessed with samples of naturally infected and non-infected 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.
[Show abstract][Hide abstract] ABSTRACT: 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.
European Food Research and Technology 01/2008; 227(4):1149-1157. · 1.39 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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 H(2)S 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(T). 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(T) (=NCIMB 14265(T)=LMG 23800(T)). Francisella philomiragia ATCC 25015(T) (=DSM 735(T)) is reclassified as Francisella philomiragia subsp. philomiragia subsp. nov.
International journal of systematic and evolutionary microbiology 10/2007; 57(Pt 9):1960-5. · 2.11 Impact Factor