Norwegian Isolates Research Articles

Molecular characterization of Sarcocystis species from Polish roe deer based on ssu rRNA and cox1 sequence analysis.

Sarcocysts from four Polish roe deer were collected and examined by light microscopy, small subunit ribosomal RNA (ssu rRNA), and the subunit I of cytochrome oxidase (cox1) sequence analysis. This resulted in identification of Sarcocystis gracilis, Sarcocystis oviformis, and Sarcocystis silva. However, we were unable to detect Sarcocystis capreolicanis, the fourth Sarcocystis species found previously in Norwegian roe deer. Polish sarcocysts isolated from various tissues differed in terms of their shape and size and were larger than the respective Norwegian isolates. Analysis of ssu rRNA gene revealed the lack of differences between Sarcocystis isolates belonging to one species and a very low degree of genetic diversity between Polish and Norwegian sarcocysts, ranging from 0.1 % for Sarcocystis gracilis and Sarcocystis oviformis to 0.44 % for Sarcocystis silva. Contrary to the results of the ssu rRNA analysis, small intraspecies differences in cox1 sequences were found among Polish Sarcocystis gracilis and Sarcocystis silva isolates. The comparison of Polish and Norwegian cox1 sequences representing the same Sarcocystis species revealed similar degree of sequence identity, namely 99.72 % for Sarcocystis gracilis, 98.76 % for Sarcocystis silva, and 99.85 % for Sarcocystis oviformis. Phylogenetic reconstruction and genetic population analyses showed an unexpected high degree of identity between Polish and Norwegian isolates. Moreover, cox1 gene sequences turned out to be more accurate than ssu rRNA when used to reveal phylogenetic relationships among closely related species. The results of our study revealed that the same Sarcocystis species isolated from the same hosts living in different geographic regions show a very high level of genetic similarity.Electronic supplementary materialThe online version of this article (doi:10.1007/s00436-014-3966-x) contains supplementary material, which is available to authorized users.

Same barcode, different biology: differential patterns of infectivity, specificity and pathogenicity in two almost identical parasite strains

Two Norwegian isolates of the monogenean Gyrodactylus salaris Malmberg, 1957 with identical cytochrome c oxidase subunit I barcodes from different hosts, show highly divergent biological and behavioural characteristics. The Lierelva parasite strain, typically infecting Atlantic salmon, Salmo salar L., grew exponentially on Atlantic salmon, but the Pålsbufjorden parasite strain, commonly infecting Arctic charr, Salvelinus alpinus L., grew slowly on both hosts and was non-pathogenic to Atlantic salmon. Both parasite strains reproduced successfully on Arctic charr, but the Atlantic salmon-infecting Lierelva strain grew faster on both hosts. Experiments with isolated worms revealed differences in reproductive rates which may account for the observed population differences. Atlantic salmon parasites consistently gave birth at an earlier age than the Arctic charr parasites, with the differential increasing from 1day for the first birth up to 2–4days for the third birth. Arctic charr-infecting parasites were more active on Atlantic salmon than salmon parasites on Arctic charr, a behavioural strategy leading to enhanced G. salaris mortality. Sequencing of 10kb of nuclear genomic markers revealed only four single nucleotide polymorphisms, confirming that isolates of G. salaris with differences in fitness traits influencing establishment, fecundity and behaviour may be remarkably similar at a molecular level. The framework for reporting and control of G. salaris requires re-appraisal in light of the discovery of variants with such divergent biology.

A recently introduced Dichelobacter nodosus strain caused an outbreak of footrot in Norway.

BackgroundIn 2008, an outbreak of ovine footrot occurred in Norway. Dichelobacter nodosus isolates collected between 2008 and 2011 have been characterised. Isolates defined as virulent by the gelatin gel test (GG-test) were only found in sheep in Rogaland County, where the severe cases of footrot were registered. The majority (96%) of the virulent isolates belonged to serogroup A. It is suspected that they represent a newly introduced strain, and the aim of the present study was to investigate whether they are genetically similar. Sixty-one virulent isolates from sheep and 116 benign isolates from sheep, cattle and goats were included. Four GG-test virulent isolates from Danish sheep were also included. All isolates were genotyped by pulsed-field gel electrophoresis (PFGE) and by PCR for pgr variant determination.ResultsThe Norwegian virulent isolates were assigned to 8 pulsotypes (PTs), while the benign isolates were assigned to 66 PTs. Thirty-seven (68.5%) of the 54, virulent, serogroup A isolates belonged to the same PT, and included isolates from 2008 through 2011. Isolates belonging to this PT were defined as the outbreak strain. The remaining virulent serogroup A isolates belonged to 4 PTs differing by ≤3 bands from the outbreak strain. Two virulent, Danish, serogroup A isolates differed by 2 bands from the Norwegian outbreak strain. All but 3 (95%) of the virulent isolates had the pgrA variant while 85% of the benign isolates had the pgrB variant.ConclusionThis study provides evidence that the footrot outbreak in Norway in 2008 most likely was caused by new introduction and local spread of one virulent D. nodosus strain.

The effects of photoperiod and light intensity on the sporulation of Brazilian and Norwegian isolates of Neozygites floridana

The objective of this study was to determine the effects of light intensity and duration (photoperiod) on the sporulation (discharge of primary conidia) and conidia germination (from non-infective primary conidia to infective capilliconidia) of Neozygites floridana isolates from Tetranychus urticae originating from Norway and Brazil. Two light intensities (40 and 208μmolm−2s−1), three photoperiods (24h of continuous light (24h D), 12h of darkness followed by 12h of light (12h D: 12h L) and 24h of continuous darkness (24h D)) and two temperatures (18°C and 23°C) were tested. The fungus produced similar amounts of primary conidia and capilliconidia at 12h D:12h and 24h D, indicating that the fungus discharges almost all of its conidia during the first 12h of darkness. Light had less of an effect on the production of primary conidia than on capilliconidia formation. At 24h L, capilliconidia formation was significantly lower for all tested light intensities, temperatures and isolates compared to 12h D:12h L and 24h D. At both light intensities, 24h L resulted in a significantly lower capilliconidia formation for the Norwegian isolate compared to the Brazilian isolate. Our data suggest that, even though 24h L reduced sporulation, some capilliconidia formation may occur at the low light intensities found on the underside of strawberry leaves during parts of the day as well as the top of a non-shaded strawberry leaf during the dim evening and morning hours in the tropics and during the dim, long summer days in temperate regions.

Whole-genome analysis of piscine reovirus (PRV) shows PRV represents a new genus in family Reoviridae and its genome segment S1 sequences group it into two separate sub-genotypes.

BackgroundPiscine reovirus (PRV) is a newly discovered fish reovirus of anadromous and marine fish ubiquitous among fish in Norwegian salmon farms, and likely the causative agent of heart and skeletal muscle inflammation (HSMI). HSMI is an increasingly economically significant disease in Atlantic salmon (Salmo salar) farms. The nucleotide sequence data available for PRV are limited, and there is no genetic information on this virus outside of Norway and none from wild fish.MethodsRT-PCR amplification and sequencing were used to obtain the complete viral genome of PRV (10 segments) from western Canada and Chile. The genetic diversity among the PRV strains and their relationship to Norwegian PRV isolates were determined by phylogenetic analyses and sequence identity comparisons.ResultsPRV is distantly related to members of the genera Orthoreovirus and Aquareovirus and an unambiguous new genus within the family Reoviridae. The Canadian and Norwegian PRV strains are most divergent in the segment S1 and S4 encoded proteins. Phylogenetic analysis of PRV S1 sequences, for which the largest number of complete sequences from different “isolates” is available, grouped Norwegian PRV strains into a single genotype, Genotype I, with sub-genotypes, Ia and Ib. The Canadian PRV strains matched sub-genotype Ia and Chilean PRV strains matched sub-genotype Ib.ConclusionsPRV should be considered as a member of a new genus within the family Reoviridae with two major Norwegian sub-genotypes. The Canadian PRV diverged from Norwegian sub-genotype Ia around 2007 ± 1, whereas the Chilean PRV diverged from Norwegian sub-genotype Ib around 2008 ± 1.

Molecular and epidemiological characterization of avian influenza viruses from gulls and dabbling ducks in Norway

BackgroundWild aquatic birds constitute the natural reservoir for avian influenza viruses (AIVs). Separate Eurasian and American AIV gene pools exist. Here, the prevalence and diversity of AIVs in gulls and dabbling ducks in Norway were described. The influence of host species and temporal changes on AIV prevalence was examined. Five AIVs from Norway, including three from common gull (Larus canus), were analyzed along with 10 available AIV genomes from gulls in Eurasia to search for evidence of intracontinental and intercontinental reassortment of gene segments encoding the internal viral proteins.MethodsSwabs collected from 2417 dabbling ducks and gulls in the south-west of Norway during five ordinary hunting seasons (August-December) in the period 2005–2010 were analyzed for presence of AIV. Multivariate linear regression was used to identify associations between AIV prevalence, host species and sampling time. Five AIVs from mallard (Anas platyrhynchos) (H3N8, H9N2) and common gull (H6N8, H13N2, H16N3) were full-length characterized and phylogenetically analyzed together with GenBank reference sequences.ResultsLow pathogenic AIVs were detected in 15.5% (CI: 14.1–17.0) of the samples. The overall AIV prevalence was lower in December compared to that found in August to November (p = 0.003). AIV was detected in 18.7% (CI: 16.8–20.6) of the dabbling ducks. A high AIV prevalence of 7.8% (CI; 5.9–10.0) was found in gulls. A similar temporal pattern in AIV prevalence was found in both bird groups. Thirteen hemagglutinin and eight neuraminidase subtypes were detected. No evidence of intercontinental reassortment was found. Eurasian avian (non H13 and H16) PB2 or PA genes were identified in five reference Eurasian gull (H13 and H16) AIV genomes from GenBank. The NA gene from the Norwegian H13N2 gull isolate was of Eurasian avian origin.ConclusionsThe similar temporal pattern in AIV prevalence found in dabbling ducks and gulls, the relatively high virus prevalence detected in gulls and the evidence of intracontinental reassortment in AIVs from gulls indicate that gulls that interact with dabbling ducks are likely to be mixing vessels for AIVs from waterfowl and gulls. Our results support that intercontinental reassortment is rare in AIVs from gulls in Eurasia.

Abiotic and Biotic Factors Affecting Resting Spore Formation in the Mite PathogenNeozygites floridana

Neozygites floridana is an obligate mite pathogenic fungus in the Entomophthoromycota. It has been suggested that resting spores of this fungus are produced as a strategy to survive adverse conditions. In the present study, possible mechanisms involved in the regulation of resting spore formation were investigated in the hosts Tetranychus urticae and Tetranychus evansi. Abiotic and biotic factors mimicking conditions that we, based on earlier field studies, thought might induce resting spores in temperate and tropical regions were tested with isolates from Norway and Brazil. A total of 42 combinations of conditions were tested, but only one induced the formation of a high number of resting spores in only one isolate. The Brazilian isolate ESALQ1420 produced a large number of resting spores (51.5%) in T. urticae at a temperature of 11°C, photoperiod of 10L:14D, and light intensity of 42–46 (μmol m−2 s−1) on nonsenescent plants (nondiapausing females). Resting spores of the Brazilian N. floridana isolate ESALQ1421 were found at very low levels (up to 1.0%). Small percentages of T. urticae with resting spores (0–5.0%) were found for the Norwegian isolate NCRI271/04 under the conditions tested. The percentages of resting spores found for the Norwegian isolate in our laboratory studies are similar to the prevalence reported in earlier field studies.

Characterisation of Dichelobacter nodosus isolates from Norway

An outbreak of ovine footrot in Norway in 2008, the first reported since 1948, prompted action to investigate Norwegian isolates of Dichelobacter nodosus. A total of 579 isolates from 124 different farms were characterised. These included 519 isolates from sheep, 52 isolates from cattle and 8 isolates from goats. The potential virulence of the isolates was assessed by the gelatin gel test (GG-test) and the elastin agar test, that test the heat stability and elastase activity of bacterial proteases, respectively. The isolates were also tested for the presence of intA by PCR, and allocated to serogroups by differentiation of fimA variants using multiplex PCR or sequencing. Thirty of the isolates were also serogrouped by slide agglutination. Three hundred and five isolates were defined as virulent by the GG-test. All these were from sheep from 52 farms located in the county of Rogaland in the south west of Norway. All isolates from cattle and goats were defined as benign by the GG-test. IntA was only detected in 6 (2.0%) of the virulent isolates. All serogroups except D and F were detected. Three hundred and seventy-two (64.3%) of the isolates belonged to serogroup A, and 96% of the virulent isolates belonged to this serogroup. On the grounds that virulent isolates were only found in one county, and that the majority belonged to the same serogroup (A), it is believed that a virulent D. nodosus strain was introduced to Norway relatively recently and that so far it has only spread locally.

Genetic variation in Norwegian piscine myocarditis virus in Atlantic salmon, Salmo salar L.

Cardiomyopathy syndrome (CMS) in Atlantic salmon, Salmo salar L., is a severe cardiac disease characterized by a necrotizing myocarditis involving the atrium and the spongious part of the ventricle. The disease is caused by piscine myocarditis virus (PMCV), a double-stranded RNA virus likely belonging to the family Totiviridae. The objective of this study was to evaluate the genetic variation in Norwegian PMCV isolates focusing on the putative structural proteins encoded by open reading frames (ORFs) 1 and 3. The virus isolates were sampled from a total of 36 farms along the Norwegian coastline. This study represents the first investigation of PMCV genome variation and shows that Norwegian isolates are highly similar, with the most divergent isolates sharing 98.6% nucleotide identity. Interestingly, amino acid sequence diversity within ORF3 is approximately threefold higher than for ORF1. While phylogenetic analysis based on concatenated nucleotide data covering ORF1 and ORF3 revealed four main clusters, the maximum sequence variation of 1.4% at the nucleotide level suggests that all Norwegian isolates belong to a single genogroup. Substantial sequence variation within farms was also observed, which may complicate future molecular epidemiological investigations. The genetic homogeneity among the Norwegian isolates might facilitate development of both diagnostic tools and an efficient vaccine against CMS in the future.

Characterization In Vitro and In Vivo of Pandemic (H1N1) 2009 Influenza Viruses Isolated from Patients

The first influenza pandemic of the 21st century was caused by novel H1N1 viruses that emerged in early 2009. Molecular evolutionary analyses of the 2009 pandemic influenza A H1N1 [A(H1N1)pdm09] virus revealed two major clusters, cluster I and cluster II. Although the pathogenicity of viruses belonging to cluster I, which became extinct by the end of 2009, has been examined in a nonhuman primate model, the pathogenic potential of viruses belonging to cluster II, which has spread more widely in the world, has not been studied in this animal model. Here, we characterized two Norwegian isolates belonging to cluster II, namely, A/Norway/3568/2009 (Norway3568) and A/Norway/3487-2/2009 (Norway3487), which caused distinct clinical symptoms, despite their genetic similarity. We observed more efficient replication in cultured cells and delayed virus clearance from ferret respiratory organs for Norway3487 virus, which was isolated from a severe case, compared with the efficiency of replication and time of clearance of Norway3568 virus, which was isolated from a mild case. Moreover, Norway3487 virus to some extent caused more severe lung damage in nonhuman primates than did Norway3568 virus. Our data suggest that the distinct replicative and pathogenic potentials of these two viruses may result from differences in their biological properties (e.g., the receptor-binding specificity of hemagglutinin and viral polymerase activity).

In silico evolutionary analysis of Helicobacter pylori outer membrane phospholipase A (OMPLA)

BackgroundIn the past decade, researchers have proposed that the pldA gene for outer membrane phospholipase A (OMPLA) is important for bacterial colonization of the human gastric ventricle. Several conserved Helicobacter pylori genes have distinct genotypes in different parts of the world, biogeographic patterns that can be analyzed through phylogenetic trees. The current study will shed light on the importance of the pldA gene in H. pylori. In silico sequence analysis will be used to investigate whether the bacteria are in the process of preserving, optimizing, or rejecting the pldA gene. The pldA gene will be phylogenetically compared to other housekeeping (HK) genes, and a possible origin via horizontal gene transfer (HGT) will be evaluated through both intra- and inter-species evolutionary analyses.ResultsIn this study, pldA gene sequences were phylogenetically analyzed and compared with a large reference set of concatenated HK gene sequences. A total of 246 pldA nucleotide sequences were used; 207 were from Norwegian isolates, 20 were from Korean isolates, and 19 were from the NCBI database. Best-fit evolutionary models were determined with MEGA5 ModelTest for the pldA (K80 + I + G) and HK (GTR + I + G) sequences, and maximum likelihood trees were constructed. Both HK and pldA genes showed biogeographic clustering. Horizontal gene transfer was inferred based on significantly different GC contents, the codon adaptation index, and a phylogenetic conflict between a tree of OMPLA protein sequences representing 171 species and a tree of the AtpA HK protein for 169 species. Although a vast majority of the residues in OMPLA were predicted to be under purifying selection, sites undergoing positive selection were also found.ConclusionsOur findings indicate that the pldA gene could have been more recently acquired than seven of the HK genes found in H. pylori. However, the common biogeographic patterns of both the HK and pldA sequences indicated that the transfer occurred long ago. Our results indicate that the bacterium is preserving the function of OMPLA, although some sites are still being evolutionarily optimized.

Similar Superantigen Gene Profiles and Superantigen Activity in Norwegian Isolates of Invasive and Non‐Invasive Group A Streptococci

Group A streptococcus (GAS) harbours several virulence factors, including M protein (coded by the emm gene) and superantigens (SAgs). SAgs are extracellular toxins that directly activate the immune system by cross-binding to the HLA class II molecule and T cell receptor (TCR), thereby causing activation of up to 30% of the T cells and subsequent massive secretion of cytokines. Forty-eight GAS strains isolated from patients at Norwegian hospitals between 1988 and 2004 were included in this study. Of these, 24 were invasive streptococcal toxic shock syndrome (STSS) or necrotizing fasciitis (NF) isolates and 24 were non-invasive pharyngitis isolates, matched for having the same T-type and year of isolation as the invasive isolates. The isolates were characterized by emm sequence typing, multilocus sequence typing (MLST) and SAg gene profiles. A correlation between T-type, emm type, sequence type and SAg gene profile was revealed. No difference between invasive and non-invasive isolates regarding serotype or genotype was demonstrated. Selected invasive and non-invasive isolates with identical SAg gene profiles were analysed for SAg activity in bacterial growth culture media with and without human cell culture media added. A human T cell proliferation assay was used as measurement for SAg activity and simultaneously we also measured the cytokine content in normal human peripheral blood leucocyte cell culture media. The results revealed that invasive and non-invasive isolates did not differ significantly in SAg activity as it is present in semipurified bacterial culture medium.

Molecular investigations of cytochrome c oxidase subunit I (COI) and the internal transcribed spacer (ITS) in the poultry red mite, Dermanyssus gallinae, in northern Europe and implications for its transmission between laying poultry farms

Samples of Dermanyssus gallinae (DeGeer) (Acari: Dermanyssidae) from more than 49 Norwegian and Swedish laying poultry farms, and additional samples collected from Scottish, Finnish, Danish and Dutch layer farms, were compared genetically. Analysis of partial mitochondrial gene cytochrome c oxidase subunit I (COI) sequences of mites from Norway and Sweden revealed 32 haplotypes. Only single haplotypes were found on most farms, which suggests that infections are recycled within farms and that transmission routes are few. Both Norwegian and Swedish isolates were found in the two major haplogroups, but no haplotypes were shared between Norway and Sweden, indicating little or no recent exchange of mites between these countries. There appears to be no link between haplotypes and geographical location as identical haplotypes were found in both the northern and southern Swedish locations, and haplotypes were scattered in locations between these extremes. The current data suggest that wild birds in Sweden are not a reservoir for D. gallinae infection of layer farms as their mites were genetically distinct from D. gallinae of farm layer birds. Transmission of the poultry red mite in Scandinavia is thus likely to depend on synantropic factors such as the exchange of contaminated material or infested birds between farms or facilities.

Virulence properties of Moritella viscosa extracellular products

Moritella viscosa is the causative agent of winter ulcer disease of marine fish. Knowledge of its pathogenicity is limited and there are no reports comparing the virulence properties of a collection of bacterial isolates. The in vivo and in vitro virulence of the extracellular products (ECP) of 22 M. viscosa isolates was screened. Two non-virulent Canadian isolates and a Norwegian isolate with reduced virulence produced non-lethal ECP. Correlation was obtained between cytotoxin and haemolysin production of M. viscosa. Isolates from salmon produced ECP with lower cytotoxic and haemolytic activities than ECP of isolates originating from other hosts. Correlation was not found between lethality of ECPs in salmon and cytotoxic or haemolytic activities. All isolates secreted esterases and a metallopeptidase (MvP1), degraded starch and produced siderophores. Variable levels of ECP protein concentration, different enzymatic activities and siderophore production could not explain differences in virulence. The results show that virulent M. viscosa isolates secrete a lethal toxic factor of unknown nature and that cytotoxin production may reflect host adaptation. Cell-culture models may not be optimal for determining the virulence of M. viscosa, as no association between cytotoxicity and bacterial virulence was obtained. Non-virulent strains may be useful in future research on M. viscosa virulence, as construction of mutants has not been successful.

Fusarium sibiricum sp. nov, a novel type A trichothecene-producing Fusarium from northern Asia closely related to F. sporotrichioides and F. langsethiae

Production of type A trichothecenes has been reported in the closely related species Fusarium langsethiae and F. sporotrichioides. Here, we characterized a collection of Fusarium isolates from Siberia and the Russian Far East (hereafter Asian isolates) that produce high levels of the type A trichothecene T-2 toxin and are similar in morphology to the type A trichothecene-producing F. langsethiae, and to F. poae which often produces the type B trichothecene nivalenol. The Asian isolates possess unique macroscopic and microscopic characters and have a unique TG repeat in the nuclear ribosomal intergenic spacer (IGS rDNA) region. In Asian isolates, the TRI1-TRI16 locus, which determines type A versus type B trichothecene production in other species, is more similar in organization and sequence to the TRI1-TRI16 locus in F. sporotrichioides and F. langsethiae than to that in F. poae. Phylogenetic analysis of the TRI1 and TRI16 gene coding regions indicates that the genes in the Asian isolates are more closely related to those of F. sporotrichioides than F. langsethiae. Phylogenetic analysis of the beta-tubulin, translation elongation factor, RNA polymerase II and phosphate permease gene sequences resolved the Asian isolates into a well-supported sister lineage to F. sporotrichioides, with F. langsethiae forming a sister lineage to F. sporotrichioides and the Asian isolates. The Asian isolates are conspecific with Norwegian isolate IBT 9959 based on morphological and molecular analyses. In addition, the European F. langsethiae isolates from Finland and Russia were resolved into two distinct subgroups based on analyses of translation elongation factor and IGS rDNA sequences. Nucleotide polymorphisms within the IGS rDNA were used to design PCR primers that successfully differentiated the Asian isolates from F. sporotrichioides and F. langsethiae. Based on these data, we formally propose that the Asian isolates together with Norwegian isolate IBT 9959 comprise a novel phylogenetic species, F. sibiricum, while the two subgroups of F. langsethiae only represent intraspecific groups.

Diversity, transmission and persistence of Escherichia coli in a cohort of mothers and their infants

Despite that Escherichia coli is one of the most important bacteria in early infant colonization and immune modulation, we have limited knowledge about diversity, transmission and persistence within human populations for this bacterium. Here we have utilized a novel, growth-independent, direct typing approach to describe E. coli mother-to-child transmission and persistence within infants in a well-defined cohort of 86 mothers and their infants in Norway. Seven gene multilocus sequence typing of 28 study isolates, three probiotic strains, eight Norwegian pathogenic isolates and the ECOR strain collection added a phylogenetic framework to the direct sequencing data. We found that a type B2 subpopulation of the maternal E. coli strains was the main group transmitted to the infants and that the proportion of children carrying their mothers' strain decreased as the children age. Using species richness estimates we also found a limited number of strains within the cohort compared with total E. coli diversity, constraints on infant colonization, and that infant strain diversity levels increased towards maternal diversity levels over time. This knowledge about inheritance and diversity forms a foundation for future understanding of E. coli in human health and disease.

Multiple-locus, variable number of tandem repeat analysis (MLVA) of the fish-pathogen Francisella noatunensis.

BackgroundSince Francisella noatunensis was first isolated from cultured Atlantic cod in 2004, it has emerged as a global fish pathogen causing disease in both warm and cold water species. Outbreaks of francisellosis occur in several important cultured fish species making a correct management of this disease a matter of major importance. Currently there are no vaccines or treatments available. A strain typing system for use in studies of F. noatunensis epizootics would be an important tool for disease management. However, the high genetic similarity within the Francisella spp. makes strain typing difficult, but such typing of the related human pathogen Francisella tullarensis has been performed successfully by targeting loci with higher genetic variation than the traditional signature sequences. These loci are known as Variable Numbers of Tandem Repeat (VNTR). The aim of this study is to identify possible useful VNTRs in the genome of F. noatunensis.ResultsSeven polymorphic VNTR loci were identified in the preliminary genome sequence of F. noatunensis ssp. noatunensis GM2212 isolate. These VNTR-loci were sequenced in F. noatunensis isolates collected from Atlantic cod (Gadus morhua) from Norway (n = 21), Three-line grunt (Parapristipoma trilineatum) from Japan (n = 1), Tilapia (Oreochromis spp.) from Indonesia (n = 3) and Atlantic salmon (Salmo salar) from Chile (n = 1). The Norwegian isolates presented in this study show both nine allelic profiles and clades, and that the majority of the farmed isolates belong in two clades only, while the allelic profiles from wild cod are unique.ConclusionsVNTRs can be used to separate isolates belonging to both subspecies of F. noatunensis. Low allelic diversity in F. noatunensis isolates from outbreaks in cod culture compared to isolates wild cod, indicate that transmission of these isolates may be a result of human activity. The sequence based MLVA system presented in this study should provide a good starting point for further development of a genotyping system that can be used in studies of epizootics and disease management of francisellosis.

Molecular phylodynamics and protein modeling of infectious salmon anemia virus (ISAV)

BackgroundISAV is a member of the Orthomyxoviridae family that affects salmonids with disastrous results. It was first detected in 1984 in Norway and from then on it has been reported in Canada, United States, Scotland and the Faroe Islands. Recently, an outbreak was recorded in Chile with negative consequences for the local fishing industry. However, few studies have examined available data to test hypotheses associated with the phylogeographic partitioning of the infecting viral population, the population dynamics, or the evolutionary rates and demographic history of ISAV. To explore these issues, we collected relevant sequences of genes coding for both surface proteins from Chile, Canada, and Norway. We addressed questions regarding their phylogenetic relationships, evolutionary rates, and demographic history using modern phylogenetic methods.ResultsA recombination breakpoint was consistently detected in the Hemagglutinin-Esterase (he) gene at either side of the Highly Polymorphic Region (HPR), whereas no recombination breakpoints were detected in Fusion protein (f) gene. Evolutionary relationships of ISAV revealed the 2007 Chilean outbreak group as a monophyletic clade for f that has a sister relationship to the Norwegian isolates. Their tMRCA is consistent with epidemiological data and demographic history was successfully recovered showing a profound bottleneck with further population expansion. Finally, selection analyses detected ongoing diversifying selection in f and he codons associated with protease processing and the HPR region, respectively.ConclusionsOur results are consistent with the Norwegian origin hypothesis for the Chilean outbreak clade. In particular, ISAV HPR0 genotype is not the ancestor of all ISAV strains, although SK779/06 (HPR0) shares a common ancestor with the Chilean outbreak clade. Our analyses suggest that ISAV shows hallmarks typical of RNA viruses that can be exploited in epidemiological and surveillance settings. In addition, we hypothesized that genetic diversity of the HPR region is governed by recombination, probably due to template switching and that novel fusion gene proteolytic sites confer a selective advantage for the isolates that carry them. Additionally, protein modeling allowed us to relate the results of phylogenetic studies with the predicted structures. This study demonstrates that phylogenetic methods are important tools to predict future outbreaks of ISAV and other salmon pathogens.

Bioinformatic Analysis of the Genome of Infectious Salmon Anemia Virus Associated with Outbreaks with High Mortality in Chile

The infectious salmon anemia virus (ISAV), an orthomyxovirus, is the major cause of outbreaks of high mortality rates in salmon in Chile. It has been proposed that the virulence of ISAV isolates lies mainly in hemagglutinin-esterase and fusion glycoproteins. However, based on current information, the contribution of other viral genes cannot be ruled out. To study this, we isolated and determined the complete coding sequence of two high-prevalence Chilean isolates associated with outbreaks of high mortality rates: ISAV752_09 and ISAV901_09. These isolates were compared to 15 Norwegian isolates that exhibit differences in their virulence. For this purpose, we performed bioinformatic analyses of (i) functional domains, (ii) specific mutations, (iii) Bayesian phylogenetics, and (iv) structural comparisons between ISAV and influenza virus glycoproteins by using molecular modeling. Phylogenetic analysis shows two genogroups for each protein, one of them containing the Chilean isolates. The gene sequence of the polymerase complex and nucleoprotein indicated that they are closely related to homologues from highly pathogenic Norwegian viruses. Notably, seven of the eight mutations that are present only in the Chilean isolates are on the polymerase complex and nucleoprotein. Structural modeling of hemagglutinin-esterase shows patches of variable residues on its surface. Fusion protein modeling shows that insertions are flexible regions that could affect proteolytic processing, increasing either the accessibility or the number of recognition sites for specific proteases. We found antigenic drift processes related to insertion into the isolated segment 5 of the ISAV752_09. Our results confirm the European origin of Chilean isolates to be the result of reassortments from Norwegian ancestors.

Emergence of fluoroquinolone‐resistant clonal group A: clonal analysis of Norwegian and Russian E. coli isolates

We describe a study of urinary tract and intestinal isolates of Escherichia coli from Norway and Russia using automated ribotyping, single nucleotide polymorphism analysis for clonal group A (CgA) supplemented with phylogrouping, virulence gene profiling and resistance profiling. CgA comprised 19% of the Norwegian UTI isolates from 2001. Two highly multiresistant fluoroquinolone-resistant CgA isolates were found. Ribotypes clustered into four major and six minor groups (ribogroups). Fluoroquinolone-resistant isolates and phylogroups A and B1 were associated with ribogroup (R)A. Ribogroup (R)B predominated among Russian UTI isolates and was predominantly phylogroup A and depleted in P-fimbriae. Ribogroup (R)C predominated among Norwegian UTI isolates and was rich in virulence factors (S-fimbriae, haemagglutinin and haemolysin) and predominantly phylogroup B2 and D. Ribogroup (R)G was associated with CgA and predominantly phylogroup D. Ribogroups (R)D, (R)E and (R)F had too few members for statistical analysis. The correlation between ribotype and phylogenetic group was not as strong as reported in other studies.