Long-term shedding from fully convalesced individuals indicates that Pacific herring are a reservoir for viral hemorrhagic septicemia virus.

Processes that allow viral hemorrhagic septicemia (VHS) virus to persist in the marine environment remain enigmatic, owing largely to the presence of covert and cryptic infections in marine fishes during typical sub-epizootic periods. As such, marine host reservoirs for VHS virus have not been fully demonstrated, nor have the mechanism(s) by which infected hosts contribute to virus perpetuation and transmission. Here, we demonstrate that after surviving VHS, convalesced Pacific herring continue to shed virus at a low rate for extended periods. Further, exposure of previously naïve conspecific sentinels to this shed virus can result in infections for at least 6 mo after cessation of overt disease. This transmission mechanism was not necessarily dependent on the magnitude of the disease outbreak, as prolonged transmission occurred from 2 groups of donor herring that experienced cumulative mortalities of 4 and 29%. The results further suggest that the virus persists in association with the gills of fully recovered individuals, and long-term viral shedding or shedding relapses are related to cooler or decreasing water temperatures. These results provide support for a new VHS virus perpetuation paradigm in the marine environment, whereby the virus can be maintained in convalesced survivors and trafficked from these carriers to sympatric susceptible individuals.

Home used, patient self-managed, brain-computer interface for the management of central neuropathic pain post spinal cord injury: usability study.

BACKGROUND: Central Neuropathic Pain (CNP) is a frequent chronic condition in people with spinal cord injury (SCI). Previously, we showed that using laboratory brain-computer interface (BCI) technology for neurofeedback (NFB) training, it was possible to reduce CNP in people with SCI. In this study, we show results of patient self-managed treatment in their homes with a BCI-NFB using a consumer EEG device. METHODS: Users: People with chronic SCI (17 M, 3 F, 50.6 ± 14.1 years old), and CNP ≥4 on a Visual Numerical Scale. LOCATION: Laboratory training (up to 4 sessions) followed by home self-managed NFB. User Activity: Upregulating the EEG alpha band power by 10% above a threshold and at the same time downregulating the theta and upper beta (20-30 Hz) band power by 10% at electrode location C4. Technology: A consumer grade multichannel EEG headset (Epoch, Emotiv, USA), a tablet computer and custom made NFB software. EVALUATION: EEG analysis, before and after NFB assessment, interviews and questionnaires. RESULTS: Effectiveness: Out of 20 initially assessed participants, 15 took part in the study. Participants used the system for 6.9 ± 5.5 (median 4) weeks. Twelve participants regulated their brainwaves in a frequency specific manner and were most successful upregulating the alpha band power. However they typically upregulated power around their individual alpha peak (7.6 ± 0.8 Hz) that was lower than in people without CNP. The reduction in pain experienced was statistically significant in 12 and clinically significant (greater than 30%) in 8 participants. Efficiency: The donning was between 5 and 15 min, and approximately 10-20% of EEG data recorded in the home environment was noise. Participants were mildly stressed when self-administering NFB at home (2.4 on a scale 1-10). User satisfaction: Nine participants who completed the final assessment reported a high level of satisfaction (QUESQ, 4.5 ± 0.8), naming effectiveness, ease of use and comfort as main priorities. The main factors influencing frequency of NFB training were: health related issues, free time and pain intensity. CONCLUSION: Portable NFB is a feasible solution for home-based self-managed treatment of CNP. Compared to pharmacological treatments, NFB has less side effects and provides users with active control over pain. TRIAL REGISTRATION: GN15NE124 , Registered 9th June 2016.

Intra-Annual Changes in Waterborne Nanophyetus salmincola.

An analysis of daily water samples collected from an index site on Big Soos Creek, Washington indicated intra-annual differences in the concentrations of waterborne Nanophyetus salmincola. Waterborne concentrations, quantified as gene copies/L, peaked during the fall (October-November 2016), decreased to very low concentrations over the winter (January-March 2017), and then increased in the spring and throughout the summer. High waterborne concentrations of N. salmincola DNA (2 × 106 gene copies/L) corresponded with live N. salmincola cercariae (mean = 3 cercariae/L) that were detected in companion water samples. Spikes in waterborne N. salmincola concentrations in October and November typically coincided with increases in streamflow; this combination resulted in elevated infection pressures during high water events in the fall. The peak in waterborne N. salmincola concentrations corresponded with an accompanying peak in tissue parasite density (metacercariae/posterior kidney) in Coho Salmon Oncorhynchus kisutch that were reared in the untreated water.

Molecular testing of adult Pacific salmon and trout (Oncorhynchus spp.) for several RNA viruses demonstrates widespread distribution of piscine orthoreovirus in Alaska and Washington.

This research was initiated in conjunction with a systematic, multiagency surveillance effort in the United States (U.S.) in response to reported findings of infectious salmon anaemia virus (ISAV) RNA in British Columbia, Canada. In the systematic surveillance study reported in a companion paper, tissues from various salmonids taken from Washington and Alaska were surveyed for ISAV RNA using the U.S.-approved diagnostic method, and samples were released for use in this present study only after testing negative. Here, we tested a subset of these samples for ISAV RNA with three additional published molecular assays, as well as for RNA from salmonid alphavirus (SAV), piscine myocarditis virus (PMCV) and piscine orthoreovirus (PRV). All samples (n = 2,252; 121 stock cohorts) tested negative for RNA from ISAV, PMCV, and SAV. In contrast, there were 25 stock cohorts from Washington and Alaska that had one or more individuals test positive for PRV RNA; prevalence within stocks varied and ranged from 2% to 73%. The overall prevalence of PRV RNA-positive individuals across the study was 3.4% (77 of 2,252 fish tested). Findings of PRV RNA were most common in coho (Oncorhynchus kisutch Walbaum) and Chinook (O. tshawytscha Walbaum) salmon.

A systematic surveillance programme for infectious salmon anaemia virus supports its absence in the Pacific Northwest of the United States.

In response to reported findings of infectious salmon anaemia virus (ISAV) in British Columbia (BC), Canada, in 2011, U.S. national, state and tribal fisheries managers and fish health specialists developed and implemented a collaborative ISAV surveillance plan for the Pacific Northwest region of the United States. Accordingly, over a 3-1/2-year period, 4,962 salmonids were sampled and successfully tested by real-time reverse-transcription PCR. The sample set included multiple tissues from free-ranging Pacific salmonids from coastal regions of Alaska and Washington and farmed Atlantic salmon (Salmo salar L.) from Washington, all representing fish exposed to marine environments. The survey design targeted physiologically compromised or moribund animals more vulnerable to infection as well as species considered susceptible to ISAV. Samples were handled with a documented chain of custody and testing protocols, and criteria for interpretation of test results were defined in advance. All 4,962 completed tests were negative for ISAV RNA. Results of this surveillance effort provide sound evidence to support the absence of ISAV in represented populations of free-ranging and marine-farmed salmonids on the northwest coast of the United States.

Phylogenetics of Australasian gall flies (Diptera: Fergusoninidae): Evolutionary patterns of host-shifting and gall morphology.

This study investigated host-specificity and phylogenetic relationships in Australian galling flies, Fergusonina Malloch (Diptera: Fergusoninidae), in order to assess diversity and explore the evolutionary history of host plant affiliation and gall morphology. A DNA barcoding approach using COI data from 203 Fergusonina specimens from 5gall types on 56 host plant species indicated 85 presumptive fly species. These exhibited a high degree of host specificity; of the 40 species with multiple representatives, each fed only on a single host genus, 29 (72.5%) were strictly monophagous, and 11 (27.5%) were reared from multiple closely related hosts. COI variation within species was not correlated with either sample size or geographic distance. However variation was greater within oligophagous species, consistent with expectations of the initial stages of host-associated divergence during speciation. Phylogenetic analysis using both nuclear and mitochondrial genes revealed host genus-restricted clades but also clear evidence of multiple colonizations of both host plant genus and host species. With the exception of unilocular peagalls, evolution of gall type was somewhat constrained, but to a lesser degree than host plant association. Unilocular peagalls arose more often than any other gall type, were primarily located at the tips of the phylogeny, and did not form clades comprising more than a few species. For ecological reasons, species of this gall type are predicted to harbor substantially less genetic variation than others, possibly reducing evolutionary flexibility resulting in reduced diversification in unilocular gallers.

Changing demographics of spinal cord injury over a 20-year period: a longitudinal population-based study in Scotland.

STUDY DESIGN: A retrospective cohort study. OBJECTIVES: To review demographic trends in traumatic spinal cord injury (TSCI) and non-traumatic spinal cord injury (NTSCI). SETTING: The Queen Elizabeth National Spinal Injuries Unit (QENSIU), sole provider of treatment for TSCI in Scotland; a devolved region of the UK National Health Service. METHODS: A retrospective review of the QENSIU database was performed between 1994 and 2013. This database includes demographic and clinical data from all new TSCI patients in Scotland, as well as patients with severe NTSCI. RESULTS: Over this 20-year period there were 1638 new cases of TSCI in Scotland; 75.2% occurring in males. TSCI incidence increased non-significantly (13.3 per million population to 17.0), while there were significant increases in mean age at time of TSCI (44.1-52.6 years), the proportion of TSCIs caused by falls (41-60%), the proportion of TSCIs resulting in an American Spinal Injury Association Impairment Scale score of C and D on admission (19.7-28.6% and 34.5-39.5%, respectively) and the proportion of cervical TSCIs (58.4-66.3%). The increase in cervical TSCI was specifically due to an increase in C1-C4 lesions (21.7-31.2%). NTSCI patients (n=292) were 5 years older at injury, more likely to be female (68.1% male) and had a range of diagnoses. CONCLUSION: This study supports the suggestion that demographic profiles in SCI are subject to change. In this population, of particular concern is the increasing number of older patients and those with high level tetraplegia, due to their increased care needs. Prevention programmes, treatment pathways and service provision need to be adjusted for optimum impact, improved outcome and long-term care for their target population.

Effects of temperature on Renibacterium salmoninarum infection and transmission potential in Chinook salmon, Oncorhynchus tshawytscha (Walbaum).

Renibacterium salmoninarum is a significant pathogen of salmonids and the causative agent of bacterial kidney disease (BKD). Water temperature affects the replication rate of pathogens and the function of the fish immune system to influence the progression of disease. In addition, rapid shifts in temperature may serve as stressors that reduce host resistance. This study evaluated the effect of shifts in water temperature on established R. salmoninarum infections. We challenged Chinook salmon with R. salmoninarum at 12 °C for 2 weeks and then divided the fish into three temperature groups (8, 12 and 15 °C). Fish in the 8 °C group had significantly higher R. salmoninarum-specific mortality, kidney R. salmoninarum loads and bacterial shedding rates relative to the fish held at 12 or 15 °C. There was a trend towards suppressed bacterial load and shedding in the 15 °C group, but the results were not significant. Bacterial load was a significant predictor of shedding for the 8 and 12 °C groups but not for the 15 °C group. Overall, our results showed little effect of temperature stress on the progress of infection, but do support the conclusion that cooler water temperatures contribute to infection progression and increased transmission potential in Chinook salmon infected with R. salmoninarum.

Atlantic salmon, Salmo salar L. are broadly susceptible to isolates representing the North American genogroups of infectious hematopoietic necrosis virus.

Beginning in 1992, three epidemic waves of infectious hematopoietic necrosis, often with high mortality, occurred in farmed Atlantic salmon Salmo salar L. on the west coast of North America. We compared the virulence of eleven strains of infectious hematopoietic necrosis virus (IHNV), representing the U, M and L genogroups, in experimental challenges of juvenile Atlantic salmon in freshwater. All strains caused mortality and there was wide variation within genogroups: cumulative mortality for five U-group strains ranged from 20 to 100%, four M-group strains ranged 30-63% and two L-group strains varied from 41 to 81%. Thus, unlike Pacific salmonids, there was no apparent correlation of virulence in a particular host species with virus genogroup. The mortality patterns indicated two different phenotypes in terms of kinetics of disease progression and final per cent mortality, with nine strains having moderate virulence and two strains (from the U and L genogroups) having high virulence. These phenotypes were investigated by histopathology and immunohistochemistry to describe the variation in the course of IHNV disease in Atlantic salmon. The results from this study demonstrate that IHNV may become a major threat to farmed Atlantic salmon in other regions of the world where the virus has been, or may be, introduced.

Piscine reovirus, but not Jaundice Syndrome, was transmissible to Chinook Salmon, Oncorhynchus tshawytscha (Walbaum), Sockeye Salmon, Oncorhynchus nerka (Walbaum), and Atlantic Salmon, Salmo salar L.

A Jaundice Syndrome occurs sporadically among sea-pen-farmed Chinook Salmon in British Columbia, the westernmost province of Canada. Affected salmon are easily identified by a distinctive yellow discolouration of the abdominal and periorbital regions. Through traditional diagnostics, no bacterial or viral agents were cultured from tissues of jaundiced Chinook Salmon; however, piscine reovirus (PRV) was identified via RT-rPCR in all 10 affected fish sampled. By histopathology, Jaundice Syndrome is an acute to peracute systemic disease, and the time from first clinical signs to death is likely <48 h; renal tubular epithelial cell necrosis is the most consistent lesion. In an infectivity trial, Chinook Salmon, Sockeye Salmon and Atlantic Salmon, intraperitoneally inoculated with a PRV-positive organ homogenate from jaundiced Chinook Salmon, developed no gross or microscopic evidence of jaundice despite persistence of PRV for the 5-month holding period. The results from this study demonstrate that the Jaundice Syndrome was not transmissible by injection of material from infected fish and that PRV was not the sole aetiological factor for the condition. Additionally, these findings showed the Pacific coast strain of PRV, while transmissible, was of low pathogenicity for Atlantic Salmon, Chinook Salmon and Sockeye Salmon.

Concomitant colonic disease (Montreal L3) and re-resectional surgery are predictors of clinical recurrence following ileocolonic resection for Crohn's disease.

AIM: Ileocolonic resection is reserved for patients with moderate to severe Crohn's disease. Postoperative clinical recurrence can occur in up to 55% of patients within 5 years. Predicting the risk of recurrence is key in deciding upon appropriate treatment strategies. This study aims to determine the incidence of postoperative clinical recurrence and predictors of recurrence in a specialist institution. METHOD: The clinical case records of 142 patients who underwent either a one-stage or two-stage procedure for ileocolonic Crohn's disease from 1 January 2005 to 31 December 2010 were reviewed. Preoperative, perioperative and postoperative variables were extracted. Postoperative clinical recurrence was defined as an initiation or change in medical treatment for recurrent symptoms with endoscopic or radiological evidence of active disease. Time to clinical recurrence was measured in months after surgery. Univariate and multivariate analyses were performed. RESULTS: Over the 6-year period, follow-up data were obtained on 142 patients over a median of 28.5 months. Clinical recurrence was demonstrated in 59 (41.5%) patients. The proportion of patients with clinical recurrence at 5 years was 48.2%. Predictors of recurrence included a re-resection for recurrent disease [hazard ratio (HR) 1.9; 95% CI 1.1-3.3; P = 0.02] and ileocolonic disease (HR 1.7; 95% CI 1.0-2.9; P = 0.05). CONCLUSION: Identifying the predictors for postoperative clinical recurrence is important for determining the postoperative strategy. This study provides a unique perspective on the incidence of recurrence and associated predictors from the perspective of a specialist unit.

Bench-top validation testing of selected immunological and molecular Renibacterium salmoninarum diagnostic assays by comparison with quantitative bacteriological culture.

No gold standard assay exhibiting error-free classification of results has been identified for detection of Renibacterium salmoninarum, the causative agent of salmonid bacterial kidney disease. Validation of diagnostic assays for R. salmoninarum has been hindered by its unique characteristics and biology, and difficulties in locating suitable populations of reference test animals. Infection status of fish in test populations is often unknown, and it is commonly assumed that the assay yielding the most positive results has the highest diagnostic accuracy, without consideration of misclassification of results. In this research, quantification of R. salmoninarum in samples by bacteriological culture provided a standardized measure of viable bacteria to evaluate analytical performance characteristics (sensitivity, specificity and repeatability) of non-culture assays in three matrices (phosphate-buffered saline, ovarian fluid and kidney tissue). Non-culture assays included polyclonal enzyme-linked immunosorbent assay (ELISA), direct smear fluorescent antibody technique (FAT), membrane-filtration FAT, nested polymerase chain reaction (nested PCR) and three real-time quantitative PCR assays. Injection challenge of specific pathogen-free Chinook salmon, Oncorhynchus tshawytscha (Walbaum), with R. salmoninarum was used to estimate diagnostic sensitivity and specificity. Results did not identify a single assay demonstrating the highest analytical and diagnostic performance characteristics, but revealed strengths and weaknesses of each test.

Spinal cord injury as a result of Staphylococcus aureus pyogenic spinal infection complicating infected atopic eczema: two case reports.

INTRODUCTION: Pyogenic spinal infections (PSI) are a rare cause of spinal cord injury (SCI). These most often affect the lumbar spine, followed by the thoracic spine and least commonly the cervical spine, with Staphylococcus aureus being the most common causative organism. Atopic eczema is a dermatological condition which can lead to a breakdown of the skin's natural barrier function, allowing bacterial colonisation and infection. Haematological seeding of bacteria from a distant source of infection, including the skin and soft tissues, is a recognised aetiology of PSI. CASE PRESENTATION: We present two patients who sustained a SCI as a result of PSI secondary to infected atopic eczema. Methicillin-sensitive Staphylococcus aureus (MSSA) was identified as the causative organism in both patients. The two patients required prolonged courses of intravenous followed by oral antibiotics. Neurological outcomes varied between the two patients. One patient had incomplete tetraplegia (C3 AIS C), and upon discharge required hoisting from their bed to a power chair, had an indwelling urethral catheter and required bowel care. The other patient had incomplete paraplegia (L3 AIS D), and at discharge was independent with activities of daily living and was mobile with two elbow crutches. DISCUSSION: We believe that the two cases presented here represent the only examples of secondarily infected atopic eczema causing PSI and resultant SCI in the published literature. As SCI is a serious and potentially life-altering complication, medical professionals treating patients with atopic eczema should be aware of this risk.

Restricted growth of U-type infectious haematopoietic necrosis virus (IHNV) in rainbow trout cells may be linked to casein kinase II activity.

Previously, we demonstrated that a representative M genogroup type strain of infectious haematopoietic necrosis virus (IHNV) from rainbow trout grows well in rainbow trout-derived RTG-2 cells, but a U genogroup type strain from sockeye salmon has restricted growth, associated with reduced genome replication and mRNA transcription. Here, we analysed further the mechanisms for this growth restriction of U-type IHNV in RTG-2 cells, using strategies that assessed differences in viral genes, host immune regulation and phosphorylation. To determine whether the viral glycoprotein (G) or non-virion (NV) protein was responsible for the growth restriction, four recombinant IHNV viruses were generated in which the G gene of an infectious IHNV clone was replaced by the G gene of U- or M-type IHNV and the NV gene was replaced by NV of U- or M-type IHNV. There was no significant difference in the growth of these recombinants in RTG-2 cells, indicating that G and NV proteins are not major factors responsible for the differential growth of the U- and M-type strains. Poly I:C pretreatment of RTG-2 cells suppressed the growth of both U- and M-type IHNV, although the M virus continued to replicate at a reduced level. Both viruses induced type 1 interferon (IFN1) and the IFN1 stimulated gene Mx1, but the expression levels in M-infected cells were significantly higher than in U-infected cells and an inhibitor of the IFN1-inducible protein kinase PKR, 2-aminopurine (2-AP), did not affect the growth of U- or M-type IHNV in RTG-2 cells. These data did not indicate a role for the IFN1 system in the restricted growth of U-type IHNV in RTG-2 cells. Prediction of kinase-specific phosphorylation sites in the viral phosphoprotein (P) using the NetPhosK program revealed differences between U- and M-type P genes at five phosphorylation sites. Pretreatment of RTG-2 cells with a PKC inhibitor or a p38MAPK inhibitor did not affect the growth of the U- and M-type viruses. However, 100 µm of the casein kinase II (CKII) inhibitor, 5,6-dichloro-1-ß-d-ribofuranosylbenzimidazole (DRB), reduced the titre of the U type 8.3-fold at 24 h post-infection. In contrast, 100 µm of the CKII inhibitor reduced the titre of the M type only 1.3-fold at 48 h post-infection. Our data suggest that the different growth of U- and M-type IHNV in RTG-2 cells may be linked to a differential requirement for cellular protein kinases such as CKII for their growth.

Erratum: Genomes reveal genetic diversity of Piscine orthoreovirus in farmed and free-ranging salmonids from Canada and USA.

[This corrects the article DOI: 10.1093/ve/veaa054.].

Sequence analysis of the internal transcribed spacer (ITS) region reveals a novel clade of Ichthyophonus sp. from rainbow trout.

The mesomycetozoean parasite Ichthyophonus hoferi is most commonly associated with marine fish hosts but also occurs in some components of the freshwater rainbow trout Oncorhynchus mykiss aquaculture industry in Idaho, USA. It is not certain how the parasite was introduced into rainbow trout culture, but it might have been associated with the historical practice of feeding raw, ground common carp Cyprinus carpio that were caught by commercial fisherman. Here, we report a major genetic division between west coast freshwater and marine isolates of Ichthyophonus hoferi. Sequence differences were not detected in 2 regions of the highly conserved small subunit (18S) rDNA gene; however, nucleotide variation was seen in internal transcribed spacer loci (ITS1 and ITS2), both within and among the isolates. Intra-isolate variation ranged from 2.4 to 7.6 nucleotides over a region consisting of approximately 740 bp. Majority consensus sequences from marine/anadromous hosts differed in only 0 to 3 nucleotides (99.6 to 100% nucleotide identity), while those derived from freshwater rainbow trout had no nucleotide substitutions relative to each other. However, the consensus sequences between isolates from freshwater rainbow trout and those from marine/anadromous hosts differed in 13 to 16 nucleotides (97.8 to 98.2% nucleotide identity).

Differential growth of U and M type infectious haematopoietic necrosis virus in a rainbow trout-derived cell line, RTG-2.

Infectious haematopoietic necrosis virus (IHNV) is one of the most important viral pathogens of salmonids. In rainbow trout, IHNV isolates in the M genogroup are highly pathogenic, while U genogroup isolates are significantly less pathogenic. We show here that, at a multiplicity of infection (MOI) of 1, a representative U type strain yielded 42-fold less infectious virus than an M type strain in the rainbow trout-derived RTG-2 cell line at 24 h post-infection (p.i.). However, at an MOI of 10, there was only fivefold difference in the yield of infectious virus between the U and M strains. Quantification of extracellular viral genomic RNA suggested that the number of virus particles released from cells infected with the U strain at a MOI of 1 was 47-fold lower than from M-infected cells, but U and M virions were equally infectious by particle to infectivity ratios. At an MOI of 1, U strain intracellular viral genome accumulation and transcription were 37- and 12-fold lower, respectively, than those of the M strain at 24 h p.i. Viral nucleocapsid (N) protein accumulation in U strain infections was fivefold lower than in M strain infections. These results suggest that the block in U type strain growth in RTG-2 cells was because of the effects of reduced genome replication and transcription. The reduced growth of the U strain does not seem to be caused by defective genes, because the U and M strains grew equally well in the permissive epithelioma papulosum cyprini cell line at an MOI of 1. This suggests that host-specific factors in RTG-2 cells control the growth of the IHNV U and M strains differently, leading to growth restriction of the U type virus during the RNA synthesis step.

Genomes reveal genetic diversity of Piscine orthoreovirus in farmed and free-ranging salmonids from Canada and USA.

Piscine orthoreovirus (PRV-1) is a segmented RNA virus, which is commonly found in salmonids in the Atlantic and Pacific Oceans. PRV-1 causes the heart and skeletal muscle inflammation disease in Atlantic salmon and is associated with several other disease conditions. Previous phylogenetic studies of genome segment 1 (S1) identified four main genogroups of PRV-1 (S1 genogroups I-IV). The goal of the present study was to use Bayesian phylogenetic inference to expand our understanding of the spatial, temporal, and host patterns of PRV-1 from the waters of the northeast Pacific. To that end, we determined the coding genome sequences of fourteen PRV-1 samples that were selected to improve our knowledge of genetic diversity across a broader temporal, geographic, and host range, including the first reported genome sequences from the northwest Atlantic (Eastern Canada). Nucleotide and amino acid sequences of the concatenated genomes and their individual segments revealed that established sequences from the northeast Pacific were monophyletic in all analyses. Bayesian inference phylogenetic trees of S1 sequences using BEAST and MrBayes also found that sequences from the northeast Pacific grouped separately from sequences from other areas. One PRV-1 sample (WCAN_BC17_AS_2017) from an escaped Atlantic salmon, collected in British Columbia but derived from Icelandic broodstock, grouped with other S1 sequences from Iceland. Our concatenated genome and S1 analysis demonstrated that PRV-1 from the northeast Pacific is genetically distinct but descended from PRV-1 from the North Atlantic. However, the analyses were inconclusive as to the timing and exact source of introduction into the northeast Pacific, either from eastern North America or from European waters of the North Atlantic. There was no evidence that PRV-1 was evolving differently between free-ranging Pacific Salmon and farmed Atlantic Salmon. The northeast Pacific PRV-1 sequences fall within genogroup II based on the classification of Garseth, Ekrem, and Biering (Garseth, A. H., Ekrem, T., and Biering, E. (2013) 'Phylogenetic Evidence of Long Distance Dispersal and Transmission of Piscine Reovirus (PRV) between Farmed and Wild Atlantic Salmon', PLoS One, 8: e82202.), which also includes North Atlantic sequences from Eastern Canada, Iceland, and Norway. The additional full-genome sequences herein strengthen our understanding of phylogeographical patterns related to the northeast Pacific, but a more balanced representation of full PRV-1 genomes from across its range, as well additional sequencing of archived samples, is still needed to better understand global relationships including potential transmission links among regions.

Development and validation of a quantitative PCR to detect Parvicapsula minibicornis and comparison to histologically ranked infection of juvenile Chinook salmon, Oncorhynchus tshawytscha (Walbaum), from the Klamath River, USA.

Parvicapsula minibicornis is a myxosporean parasite that is associated with disease in Pacific salmon during their freshwater life history phase. This study reports the development of a quantitative (real-time) polymerase chain reaction (QPCR) to detect P. minibicornis DNA. The QPCR assay targets the 18S ribosomal subunit gene. A plasmid DNA control was developed to calibrate cycle threshold (C(T)) score to plasmid molecular equivalent (PME) units, a measure of gene copy number. Assay validation revealed that the QPCR was sensitive and able to detect 50 ag of plasmid DNA, which was equivalent to 12.5 PME. The QPCR assay could detect single P. minibicornis actinospores well above assay sensitivity, indicating a single spore contains at least 100 times the 18S DNA copies required for detection. The QPCR assay was repeatable and highly specific; no detectable amplification was observed using DNA from related myxozoan parasites. The method was validated using kidney tissues from 218 juvenile Chinook salmon sampled during the emigration period of March to July 2005 from the Klamath River. The QPCR assay was compared with histological examination. The QPCR assay detected P. minibicornis infection in 88.1% of the fish sampled, while histological examination detected infection in 71.1% of the fish sampled. Good concordance was found between the methods as 80% of the samples were in agreement. The majority of the disconcordant fish were positive by QPCR, with low levels of P. minibicornis DNA, but negative by histology. The majority of the fish rated histologically as having subclinical or clinical infections had high QPCR levels. The results of this study demonstrate that QPCR is a sensitive quantitative tool for evaluating P. minibicornis infection in fish health monitoring studies.