How to define fish pathogen relatives from a 16S rRNA sequence library and Pearson correlation analysis between defined OTUs from the library: Supplementary data to the research article "Presence and habitats of bacterial fish pathogen relatives in a marine salmon post-smolt RAS".

This paper provides supplementary data to the research paper ''Presence and habitats of bacterial fish pathogen relatives in a marine salmon post-smolt RAS" [1]. Here, environmental samples from a marine recirculating aquaculture system (RAS) were subjected to microbiome studies. This data article adds value to the research article by providing open access to data files that increased information retrieval from the 16S rRNA sequence library. A fasta file of full-length 16S rRNA sequences from fish pathogenic microbes was deposited in the Mendeley data repository, a collection named "Fish Pathogen Database". Alignment of this database against the short sequences in the 16S rRNA library revealed the fish pathogen-relatives. Furthermore, a link to a CSV file containing Pearson correlation data was provided, an analysis based on the relative abundance information of all operational taxonomic units defined in the microbiome dataset. Included also, the methodological description of the Pearson correlation analysis, as well as a table where correlation data for the defined fish pathogen-relatives was retrieved from the large data file (Table 1).

Microbiome dataset from a marine recirculating aquaculture system (RAS) for salmon post-smolt production in Norway.

A marine aquaculture recycling system (RAS) for the production of post-smolt was monitored for microbial community structures during the first year of operation. Sample material was obtained monthly from the biofilter biofilm carriers, the production water (tank 3), the fish skin (tank 3) and the tank 3 wall biofilm. Additional samples were taken during outbreaks of fish skin wounds, washing of the plant, UV filtration of the inlet water and from various wall biofilms. Samples for depth profiles from all fish tanks were also collected. The sampling tools were a ladle for capturing biofilter biofilm carriers, toothbrushes for wall biofilm capture, filters for capture of water microbes and scalpels for skin tissue slicing. The sampling times were indicated by the production cycle number (cycle 2-5) and the week number within the cycle (W). Prior to bacterial community analysis, the stored samples were exposed to cell lysis and extraction of environmental DNA by commercial kits. All samples were subjected for PCR amplification of 16S rDNA sequences for library formations and prepared for Ion Torrent technology, which sequences 250 bp fragments. A total of 1.1 million reads were obtained from the 100 RAS samples analysed. The process from Ion Torren analysis to library involved bioinformatics steps with sorting, filtering, adjustment and taxonomic identification, and the final output was shown in a table as operational taxonomic units (OTUs) and relative abundance at different sampling sites and sampling time points. Of a total of 450 taxonomically assigned OTUs, 45% were classified at genus level. The 16S library raw data are deposited in the Mendeley data repository and cited in this Data in Brief article co-submitted with the article "Microbial colonization and stability in a marine post-smolt RAS inoculated with a commercial starter culture." [1]. So far, the raw data are referenced in four more publications in progress. These cover microbial shifts and enrichments between sampling times, sulfur cycling, "in vivo biofilm" and identification of relatives of fish pathogens in RAS. All library sequences are available in GenBank with accession numbers MN890148-MN891672.

CD8+ T cell gene expression analysis identifies differentially expressed genes between multiple sclerosis patients and healthy controls.

BACKGROUND: Genetic and clinical observations have indicated T cells are involved in MS pathology. There is little insight in how T cells are involved and whether or not these can be used as markers for MS. OBJECTIVES: Analysis of the gene expression profiles of circulating CD8+ T cells of MS patients compared to healthy controls. METHODS: RNA from purified CD8+ T cells was sequenced and analyzed for differential gene expression. Pathway analyses of genes at several p-value cutoffs were performed to identify putative pathways involved. RESULTS: We identified 36 genes with significant differential gene expression in MS patients. Four genes reached at least 2-fold differences in expression. The majority of differentially expressed genes was higher expressed in MS patients. Genes associated to MS in GWAS showed enrichment amongst the differentially expressed genes. We did not identify enrichment of specific pathways amongst the differentially expressed genes in MS patients. CONCLUSIONS: CD8+ T cells of MS patients show differential gene expression, with predominantly higher activity of genes in MS patients. We do not identify specific biological pathways in our study. More detailed analysis of CD8+ T cells and subtypes of these may increase understanding of how T cells are involved in MS.

Identification of a Novel Elastin-Degrading Enzyme from the Fish Pathogen Flavobacterium psychrophilum.

Hydrolytic extracellular enzymes degrading host tissues potentially play a role in bacterial pathogenesis. Flavobacterium psychrophilum is an important bacterial pathogen of salmonid fish reared in freshwater throughout the world. Diversity among isolates has been described at the phenotypic, serological, and genomic levels, but the links between these various traits remain poorly understood. Using a genome-wide association study, we identified a gene encoding a novel elastinolytic enzyme in F. psychrophilum To formally demonstrate enzymatic activity, this gene (FP0506 from strain JIP 02/86) was expressed in the elastinolysis-deficient strain OSU THCO2-90, resulting in proficient elastin-degrading cells. The encoded protein is predicted to be a cell-surface-exposed lipoprotein with no homology to previously reported elastases. FP0506 might belong to the zincin tribe and gluzincin clan of metalloproteases, and this new elastase-encoding gene seems to be present only in some members of the family FlavobacteriaceaeIMPORTANCE Elastin is an important proteinaceous component of vertebrate connective tissues (e.g., blood vessels, lung, and skin), to which it confers elasticity. Elastases have been identified in a number of pathogenic bacteria. They are thought to be required for tissue penetration and dissemination, acting as "spreading factors." Flavobacterium psychrophilum is a devastating bacterial pathogen of salmonid fish (salmon and trout) that is responsible for severe economic losses worldwide. This pathogen displays strong proteolytic activities. Using a variety of techniques, including genome comparisons, we identified a gene encoding a novel elastase in F. psychrophilum The encoded protein is predicted to be a cell-surface-exposed lipoprotein with no homology to previously reported elastases. In addition, this elastase likely belongs to a new family of proteases that seems to be present only in some members of this important group of bacteria.

Holter monitoring of small breed dogs with advanced myxomatous mitral valve disease with and without a history of syncope.

BACKGROUND: Syncope is a transient loss of consciousness occasionally occurring in dogs with advanced myxomatous mitral valve disease (MMVD). OBJECTIVE: (1) To study ECG changes during syncopal episodes in dogs with advanced MMVD and (2) to compare the occurrence of arrhythmias and changes in heart rate variability (HRV) between dogs with advanced MMVD with and without a history of syncope. ANIMALS: Forty-three privately owned dogs (<15 kg) with advanced MMVD: 21 with and 22 without a history of syncope. METHODS: Prospective study with dogs recruited for an evaluation including history, physical examination, echocardiography, and arrhythmia and HRV analysis performed on 24-hour Holter recordings. RESULTS: A syncopal episode was observed during Holter monitoring in 4 dogs: 3 dogs had sinus rhythm and 1 dog had sinus arrest followed by escape rhythm. An arrhythmia variable representing sinus arrhythmia was significantly lower in dogs with a history of syncope than in those without (P = .008). Eight of 26 HRV variables were significantly different between dogs with and without a history of syncope. CONCLUSIONS AND CLINICAL IMPORTANCE: Compared with dogs without a history of syncope, dogs with advanced MMVD and a history of syncope did not have a higher occurrence of arrhythmias, but had less sinus arrhythmia, and had changes in HRV variables representing decreased overall HRV, decreased parasympathetic, and increased sympathetic modulation of heart rate.

DNA damage leads to progressive replicative decline but extends the life span of long-lived mutant animals.

Human-nucleotide-excision repair (NER) deficiency leads to different developmental and segmental progeroid symptoms of which the pathogenesis is only partially understood. To understand the biological impact of accumulating spontaneous DNA damage, we studied the phenotypic consequences of DNA-repair deficiency in Caenorhabditis elegans. We find that DNA damage accumulation does not decrease the adult life span of post-mitotic tissue. Surprisingly, loss of functional ERCC-1/XPF even further extends the life span of long-lived daf-2 mutants, likely through an adaptive activation of stress signaling. Contrariwise, NER deficiency leads to a striking transgenerational decline in replicative capacity and viability of proliferating cells. DNA damage accumulation induces severe, stochastic impairment of development and growth, which is most pronounced in NER mutants that are also impaired in their response to ionizing radiation and inter-strand crosslinks. These results suggest that multiple DNA-repair pathways can protect against replicative decline and indicate that there might be a direct link between the severity of symptoms and the level of DNA-repair deficiency in patients.

Radial and longitudinal strain and strain rate assessed by speckle-tracking echocardiography in dogs with myxomatous mitral valve disease.

BACKGROUND: Assessment of left ventricular (LV) function using conventional echocardiographic methods is difficult in mitral regurgitation (MR) owing to altered hemodynamic loading conditions. Newer methods such as speckle-tracking echocardiography (STE) provide assessment of LV strain (St) and strain rates (SR). HYPOTHESES: Global St and SR are 1) decreased in dogs with clinical signs of congestive heart failure (CHF) due to myxomatous mitral valve disease (MMVD) compared with clinically healthy dogs, and are 2) associated with conventional echocardiographic indices of MMVD severity. ANIMALS: The study subjects were 93 privately owned dogs with different MMVD severities. METHODS: Prospectively recruited dogs were grouped according to MMVD severity based on echocardiographic evaluation of MR and presence of clinical signs. Global radial and longitudinal St, SR, and indices of LV dyssynchrony were assessed. RESULTS: On group-wise comparisons, dogs with CHF had increased global longitudinal St, global longitudinal and radial SR in systole (SRs), and early diastole (SRe) compared with dogs with no or minimal MR (all P < .04). On multiple regression analyses, these global STE variables increased with degree of MR, but associations with left atrial-to-aortic root ratio (LA/Ao) were best described by second-order polynomial equations. Thus, curvilinear relationships were found for LA/Ao and longitudinal St, SRs, and SRe (all P < .002) and radial St and SRe (all P < .001). CONCLUSIONS AND CLINICAL IMPORTANCE: Assessed by STE, LV function appeared to be augmented in moderate-to-severe disease. However, at CHF stages with greatly enlarged atria, a decrease to levels comparable to dogs with no or minimal MR was observed.

Heart rate, heart rate variability, and arrhythmias in dogs with myxomatous mitral valve disease.

BACKGROUND: Autonomic modulation of heart rhythm is thought to influence the pathophysiology of myxomatous mitral valve disease (MMVD). HYPOTHESES: (1) Holter-derived variables reflecting autonomic modulation of heart rhythm change with MMVD severity in Cavalier King Charles Spaniels (CKCS); (2) Holter-derived variables can identify MMVD severity in CKCS; and (3) Holter-derived variables in CKCS in congestive heart failure (CHF) secondary to MMVD differ from those in dogs of other breeds in CHF. ANIMALS: Ninety privately owned dogs: 70 CKCS with variable MMVD severity and 20 non-CKCS in CHF secondary to MMVD. METHODS: Dogs were prospectively recruited and divided into 5 MMVD severity groups based on history, breed, and physical and echocardiographic examination findings. Holter-derived variables included heart rate variability (HRV), heart rate (HR), and arrhythmia evaluated from 24-hour Holter recordings. RESULTS: In CKCS, 18 of 26 HRV (all P < .0002) and 3 of 9 arrhythmia (all P < .0004) variables decreased with increasing MMVD, whereas minimum and mean HR (all P < .0001) increased with increasing MMVD severity. An arrhythmia variable representing sinus arrhythmia ("premature normals") (P < .0001) and the HRV variable triangular index (TI) (P < .0001) could distinguish CKCS with moderate or severe mitral regurgitation from CKCS in CHF in specific intervals. Among dogs in CHF, Holter-derived variables did not differ among breeds. CONCLUSIONS AND CLINICAL IMPORTANCE: In CKCS, Holter-derived variables changed with MMVD severity. "Premature normals" and TI showed diagnostic potential. Breed differences were not seen among dogs in CHF secondary to MMVD.

Mycobacterium salmoniphilum infection in farmed Atlantic salmon, Salmo salar L.

Multiple greyish-white visceral nodules containing abundant rapidly growing and acid-fast bacteria, subsequently identified as Mycobacterium salmoniphilum, were detected in moribund and newly dead market-sized fish during a period of increased mortality in an Atlantic salmon, Salmo salar, farm in western Norway. Isolates cultured from diseased fish were phenotypically consistent with Mycobacterium sp. previously isolated from Atlantic salmon [MT 1890 (= NCIMB13533), MT1892, MT1900 and MT1901] in the Shetland Isles, Scotland. Partial sequences of 16S rDNA, ribosomal RNA internal transcribed spacer (ITS1), 65-kDa heat-shock protein (Hsp65) and ß subunit of RNA polymerase (rpoB) revealed 97-99% similarity with M. salmoniphilum type strain ATCC 13758(T) . The source of infection was not confirmed. Koch's postulates were fulfilled following experimental challenge of Atlantic salmon with field isolate NVI6598 (FJ616988). Mortality was recorded in experimentally infected fish; however, the infection remained subclinical in the majority of affected fish over the 131-day challenge period.

Tenacibaculum sp. associated with winter ulcers in sea-reared Atlantic salmon Salmo salar.

Coldwater-associated ulcers, i.e. winter ulcers, in seawater-reared Atlantic salmon Salmo salar L. have been reported in Norway since the late 1980s, and Moritella viscosa has been established as an important factor in the pathogenesis of this condition. As routine histopathological examination of winter ulcer cases in our laboratory revealed frequent presence in ulcers of long, slender rods clearly different from M. viscosa, a closer study focusing on these bacteria was conducted. Field cases of winter ulcers during 2 sampling periods, 1996 and 2004-2005, were investigated and long, slender rods were observed by histopathological examination in 70 and 62.5% of the ulcers examined, respectively, whereas cultivation on marine agar resulted in the isolation of yellow-pigmented colonies with long rods from 3 and 13% of the ulcers only. The isolates could be separated into 2 groups, both identified as belonging to the genus Tenacibaculum based on phenotypic characterization and 16S rRNA sequencing. Bath challenge for 7 h confirmed the ability of Group 1 bacterium to produce skin and cornea ulcers. In fish already suffering from M. viscosa-induced ulcers, co-infection with the Group 1 bacterium was established within 1 h. Ulcers from field cases of winter ulcers and from the transmission experiments tested positive by immunohistochemistry with polyclonal antiserum against the Group 1 bacterium but not the Group 2 bacterium. Our results strongly indicate the importance of the Group 1 bacterium in the pathogenesis of winter ulcers in Norway. The bacterium is difficult to isolate and is therefore likely to be underdiagnosed based on cultivation only.

Patients with problematic opioid use can be weaned from codeine without pain escalation.

BACKGROUND: Brief treatments for chronic non-malignant pain patients with problematic opioid use are warranted. The aims of the present study were to investigate (1) whether it is possible to withdraw codeine use in such patients with a brief cognitive-behavioural therapy (CBT), (2) whether this could be done without pain escalation and reduction in quality of life and (3) to explore the effects of codeine reduction on neurocognitive functioning. METHODS: Eleven patients using codeine daily corresponding to 40-100 mg morphine were included. Two specifically trained physicians treated the patients with six CBT sessions, tapering codeine gradually within 8 weeks. Codeine use, pain intensity, quality of life and neuropsychological functioning were assessed at pre-treatment to the 3-month follow-up. RESULTS: Codeine use was significantly reduced from mean 237 mg [standard deviation (SD) 65] pre-treatment to 45 mg (SD 66) post-treatment and to 48 mg (SD 65) at follow-up without significant pain escalation or reductions in quality of life. Moreover, neuropsychological functioning improved significantly on some tests, while others remained unchanged. CONCLUSION: The promising findings of codeine reduction in this weaning therapy programme for pain patients with problematic opioid use should be further evaluated in a larger randomized control trial comparing this brief CBT with both another brief treatment and attention placebo condition.

Characterization of a QTL region affecting clinical mastitis and protein yield on BTA6.

Quantitative trait loci affecting clinical mastitis were detected and fine mapped to a narrow region on bovine chromosome 6 in the Norwegian Red cattle population. The region includes the casein gene cluster and several candidate genes thought to influence clinical mastitis. The most significant results were found for SNPs within the Mucin 7 gene. This gene encodes an antimicrobial peptide and constitutes part of the first line of defence for the mucosal immune system. Detection of long haplotypes extending several Mb may indicate that artificial selection has influenced the haplotype structures in the region. A search for selection sweeps supports this observation and coincides with association results found both by single SNP and haplotype analyses. Our analyses identified haplotypes carrying quantitative trait loci alleles associated with high protein yield and simultaneously fewer incidences of clinical mastitis. The fact that such haplotypes are found in relative high frequencies in Norwegian Red may reflect the combined breeding goal that is characterized by selection for both milk production and disease resistance. The identification of these haplotypes raises the possibility of overcoming the unfavourable genetic correlation between these traits through haplotype-assisted selection.

Fine mapping of quantitative trait Loci on bovine chromosome 6 affecting calving difficulty.

Calving difficulty is an economically and ethically important trait for dairy cattle breeding. The aim of the present paper was to refine the position of a previously detected quantitative trait locus (QTL) affecting calving difficulty (direct effect) in Norwegian Red dairy cows. A granddaughter design consisting of 18 elite sire families and a total of 713 sons was genotyped for 154 markers spanning the QTL region, and the trait data were analyzed by using a combined linkage and linkage disequilibrium approach. A highly significant QTL was detected in a 150-kb interval between the markers LAP3_281 and BTA-114677. Additionally, there were some indications of a second QTL between the markers BTA-75776 and BTA-75780 located less than 500 kb apart. Several candidate genes may be identified close to these QTL. Of these, a cluster of genes expected to affect bone and cartilage formation may be of particular interest for follow-up studies.

Construction of a dense SNP map for bovine chromosome 6 to assist the assembly of the bovine genome sequence.

A linkage map was constructed for bovine chromosome 6 (BTA6), using 399 single nucleotide polymorphisms (SNPs) detected primarily from PCR-resequencing. The efficiency of SNP detection was highly dependent on the source of sequence information chosen for primer design (BAC-end sequences, introns or promoters). The SNPs were used to build a linkage map comprising 104 cM on BTA6. The SNP order in the linkage map corresponded very well with radiation hybrid (RH) maps available for BTA6 as well as with expected positions in the human comparative map, but diverged significantly from the current assembly of the bovine genome (Btau_3.1). When performing linkage analysis with the marker order suggested from the Btau_3.1 we observed an expansion of the genetic map from 104 cM to 137 cM, strongly suggesting a reordering of scaffolds in the current version of the bovine genome assembly. The extent of LD on BTA6 was evaluated by calculating the average r(2) for SNP pairs separated by given distances. The decline of LD was rapid with distance, such that r(2) was 0.1 at 100 kb. Our results indicate that linkage mapping will be a valuable source of information for correcting errors in the current bovine assembly. These errors were sufficiently frequent to be of concern for the accuracy of mapping QTL with panels of SNPs whose positions are based on the current assembly.

First cases of amoebic gill disease (AGD) in Norwegian seawater farmed Atlantic salmon, Salmo salar L., and phylogeny of the causative amoeba using 18S cDNA sequences.

Amoebic gill disease (AGD) was observed in seawater farmed Atlantic salmon at four geographically distant locations on the western coast of Norway. To the best of our knowledge, these are the first detected AGD outbreaks in Norway. The outbreaks lasted for 7-12 weeks in late autumn 2006 and were for the most part concurrent. The crude, cumulative mortality was in the range of 12-20% at three farms and 82% at a fourth. The histopathology showed uniform parasomal amoebae in lesions characteristic for AGD. Another gill disease, proliferative gill inflammation (PGI), was also present to a variable degree and the distinction between the two gill problems is discussed. Seawater temperatures were 3.5 degrees C higher than average before disease outbreaks, which subsided in early winter. The geographical and time pattern of these outbreaks strongly indicates simultaneous infection from the marine environment. Two contiguous 18S cDNA sequences, obtained by reverse transcriptase PCR from gill tissue with AGD-related lesions, showed highest similarity (99.2%) to a newly recognized species designated Neoparamoeba perurans and maximum likelihood analysis demonstrates that they represent Norwegian strains of this Neoparamoeba lineage.

The origin of selection signatures on bovine chromosome 6.

The extent and pattern of linkage disequilibrium (LD) between closely spaced markers contain information about population history, including past population size and selection history. Selection signatures can be identified by comparing the LD surrounding a putative selected allele at a locus to the putative non-selected allele. In livestock populations, locations of selection signatures identified in this way should be correlated with QTL affecting production traits, as the populations have been under strong artificial selection for these traits. We used a dense SNP map of bovine chromosome 6 to characterize the pattern of LD on this chromosome in Norwegian Red cattle, a breed which has been strongly selected for milk production. The pattern of LD was generally consistent with strong selection in regions containing QTL affecting milk production traits, including a strong selection signature in a region containing a mutation known to affect milk production. The results demonstrate that in livestock populations, the origin of selection signatures will often be QTL for livestock production traits, and illustrate the value of selection signatures in uncovering new mutations with potential effects on quantitative traits.

Vaccine-associated systemic Rhodococcus erythropolis infection in farmed atlantic salmon Salmo salar.

In 7 instances between 2000 and 2003, clinical investigation of populations of fresh- and seawater-reared, vaccinated, Atlantic salmon Salmo salar suffering total losses of between 0.1 and 35 % revealed infection with a Gram-positive rod-shaped bacterium. The isolations were geographically widespread, occurring in both Norway and Scotland. In all cases, a Gram-positive bacterium, subsequently identified as Rhodococcus erythropolis, was isolated in pure culture. Infections, although systemic, were focused within the peritoneal cavity. While initial attempts to reproduce the disease by intraperitoneal injection of unvaccinated Atlantic salmon failed, Koch's postulates were subsequently fulfilled in fish vaccinated with a commercially available oil-adjuvanted vaccine.

Fine mapping of milk production QTL on BTA6 by combined linkage and linkage disequilibrium analysis.

Combined linkage and linkage disequilibrium analysis were used to refine the position of a previously detected QTL affecting milk production traits on bovine chromosome 6. Through a series of single- and multitrait and single- and multipoint QTL analyses, the QTL could be positioned to a 7.5-cM interval surrounded by the markers BMS2508 and FBN12. The most significant results were found for fat percentage and protein percentage. This effect seemed to be caused by a QTL allele embedded in one specific marker haplotype that caused a reduction in fat and protein yields and a concomitant increase of milk yield, thus resulting in a marked reduction of fat and protein percentages.