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Proper sample size must be considered when designing infectious-agent prevalence studies for mixed-stock fisheries, because bias and uncertainty complicate interpretation of apparent (test)-prevalence estimates. Sample size varies between stocks and are often smaller than expected during wild-salmonid surveys. Our case example of 2010–2016 survey of sockeye salmon (Oncorhynchus nerka) from different stocks of origin in British Columbia, Canada, illustrated the effect of sample size on apparent-prevalence interpretation. Molecular testing (viral RNA RT-qPCR) for infectious hematopoietic necrosis virus (IHNv) revealed large differences in apparent prevalence across wild salmon stocks (much higher from Chilko Lake) and sampling location (fresh water or marine), indicating differences in both stock and host life-stage effects. Ten of the 13 marine non-Chilko stock–years with IHNv-positive results had small sample sizes (

Wild Pacific salmonids (WPS) are economically and culturally important to the Pacific North region. Most recently, some populations of WPS have been in decline. Of hypothesized factors contributing to the decline, infectious agents have been postulated to increase the risk of mortality in Pacific salmon. We present a literature review of both published journal and unpublished data to describe the distribution of infectious agents reported in wild Pacific salmonid populations in British Columbia (BC), Canada. We targeted 10 infectious agents, considered to potentially cause severe economic losses in Atlantic salmon or be of conservation concern for wild salmon in BC. The findings indicated a low frequency of infectious hematopoietic necrosis virus, piscine orthoreovirus, viral haemorrhagic septicaemia virus, Aeromonas salmonicida, Renibacterium salmoninarum, Piscirickettsia salmonis and other Rickettsia-like organisms, Yersinia ruckeri, Tenacibaculum maritimum and Moritella viscosa. No positive results were reported for infestations with Paramoeba perurans in peer-reviewed papers and the DFO Fish Pathology Program database. This review synthesizes existing information, as well as gaps therein, that can support the design and implementation of a long-term surveillance programme of infectious agents in wild salmonids in BC.

The avermectins, emamectin benzoate (EMB) and ivermectin (IVM) have been commonly used in North America over the last two decades to control the salmon louse, Lepeophtheirus salmonis, infections in farmed Atlantic salmon. Emamectin benzoate, trade name SLICE ™, was used heavily in the Eastern Canadian industry between the years 2000–2008, due to its long lasting protection and efficacy against all parasitic life stages. However, over reliance on this drug soon resulted in reduced sensitivity in many L. salmonis populations, resulting more recently in uses of higher treatment dosages and switching to the use of IVM. For these reasons, we investigated the effects of different dosages of EMB and multiple IVM treatments on baseline immunophysiological indicators, anti-viral responses and protection against subsequent salmon lice exposure in salmon smolts. Different doses of EMB or repeated treatment with IVM did not affect feeding behaviour in salmon, however by the end of the second IVM treatment, some neurotoxicity was observed. A single (1×) EMB dose (50 μg/Kg) administered for 7 consecutive days had no significant effect on L. salmonis abundance and development, whereas triple the dosage (150 μg/Kg) significantly reduced lice development, thereby eliminating subsequent stress responses in salmon associated with lice development to pre-adult stages. Emamectin benzoate and IVM treatment did not significantly impact expression of resting antigen presentation molecules in salmon (MH class I or II), however they did inhibit short-term (6 h) induced responses to the ISA virus. The impact of gender, as previously shown, had the greatest effect on louse transcriptomic regulation, but avermectin treatment also caused perturbations in gene expression. Transcriptome differences between lice on control and 1× EMB treated fish were larger than those observed for IVM or 3× EMB. Nearly half of the transcripts differentially expressed by IVM were also affected by one of the EMB treatments. Transcriptomic results from the louse suggest a high degree of similarity and concordance within and across studies in avermectin treatment, with gender of louse and dosage of drug significantly impacting the outcomes.

Background Aquatic Rhabdoviruses are globally significant pathogens associated with disease in both wild and cultured fish. Infectious hematopoietic necrosis virus (IHNV) is a Rhabdovirus that causes the internationally regulated disease infectious hematopoietic necrosis (IHN) in most species of salmon. Yet not all naïve salmon exposed to IHNV become diseased, and the mechanisms by which some individuals evade or rapidly clear infection following exposure are poorly understood. Here we used RNA-sequencing to evaluate transcriptomic changes in Sockeye salmon, a keystone species in the North Pacific and natural host for IHNV, to evaluate the consequences of IHNV exposure and/or infection on host cell transcriptional pathways. Results Immersion challenge of Sockeye salmon smolts with IHNV resulted in approximately 33% infection prevalence, where both prevalence and viral kidney load peaked at 7 days post challenge (dpc). De novo assembly of kidney transcriptomes at 7 dpc revealed that both infected and exposed but noninfected individuals experienced substantial transcriptomic modification; however, stark variation in gene expression patterns were observed between exposed but noninfected, infected, and unexposed populations. GO and KEGG pathway enrichment in concert with differential expression analysis identified that kidney responses in exposed but noninfected fish emphasized a global pattern of transcriptional down-regulation, particularly for pathways involved in DNA transcription, protein biosynthesis and macromolecule metabolism. In contrast, transcriptomes of infected fish demonstrated a global emphasis of transcriptional up-regulation highlighting pathways involved in antiviral response, inflammation, apoptosis, and RNA processing. Quantitative PCR was subsequently used to highlight differential and time-specific regulation of acute phase, antiviral, inflammatory, cell boundary, and metabolic responsive transcripts in both infected and exposed but noninfected groups. Conclusions Waterborne exposure with IHNV has a dramatic effect on the Sockeye salmon kidney transcriptome that is discrete between resistant and acutely susceptible individuals. Here we identify that metabolic, acute phase and cell boundary pathways are transcriptionally affected by IHNV and tissue responses to local infection are highly divergent from those generated as part of a disseminated response. These data suggest that primary resistance of naïve fish to IHNV may involve global responses that encourage reduced cellular signaling rather than promoting classical innate antiviral responses.

This chapter summarises our understanding of the direct effects that parasitic crustaceans have on their invertebrate and vertebrate hosts. At the individual host level, the effects of infection with parasitic Crustacea with respect to host pathological changes and the development of disease states are reasonably well understood. However, we have a much poorer understanding of how infection affects the physiological, immunological and reproductive status of hosts, with much of what is known arising from studies of sea lice (caligid copepods) infections of salmonids. Quantifying sublethal impacts of parasitic Crustacea infection on the biology and ecology of hosts is especially challenging even under controlled laboratory conditions. This is due to the complex and poorly understood interactions between parasite, host and environmental determinants, which ultimately influence the outcome and magnitude of the effect. There is very limited information on the effects that parasitic Crustacea have on their hosts at a population level, as well as on the indirect effects that they may have on species that interact with their hosts (community level effects). Our relatively good understanding of effects of sea lice on salmonids has been brought about due to the large economic impact that these parasites have on farmed salmonids and the necessity to develop new methods for their control. Unfortunately, the progress for other parasites and hosts is much slower which is in part due to the lack of experimental systems, research tools and funding.

14 páginas, 1 figura, 3 tablas.-- Simone Sühnel ... et al., The protozoans Perkinsus marinus, Perkinsus olseni, Bonamia ostreae, Bonamia exitiosa, and Marteilia refringens are responsible for some of the most detrimental diseases in the production of cultivated shellfish worldwide and are classified as notifiable diseases by the World Organization for Animal Health (OIE). This study examined the general health status of wild and cultured bivalves from southern Brazil and included diagnostic tests for the presence of Perkinsus sp., Bonamia sp., andMarteilia sp. Cultured bivalves included the mangrove oyster Crassostrea gasar (syn. Crassostrea brasiliana), the brown mussel Perna perna, the lion's paw scallop Nodipecten nodosus, andwing pearl oyster Pteria hirundo. Themangrove oyster Crassostrea rhizophorae and the carib pointed venus clam Anomalocardia brasiliana (syn. Anomalocardia flexuosa) were collected from wild populations. A variety of parasitic or commensal organisms were detected by histology including Ancistrocoma-like and Spenophrya-like organisms; Bucephalus genus, Nematopsis sp., Steinhausia sp., and Tylocephalum sp.; unidentified trematode; unknown protozoan and metazoans; and an amoeba parasite. Hemocytic infiltration was most commonly associated with parasitized animals. Histological, culture and molecular diagnostic tests did not find any evidence of the presence of OIE-listed pathogens or related species in this study. Although the current survey did not identify any pathogens or diseases of concern, it provides baseline health assessment data for these species against which any future disease developments or significant changes in population health can be compared. These data are also valuable with respect to the development and implementation of public policies related to aquatic animal health, We would like to thank to the Ministry of Fisheries and Aquaculture, Brazil, for the research support provided to the Fundaçao de Amparo à Pesquisa do Estado de Santa Catarina (FAPESC), Brazil; the National Council of Technological and ScientificDevelopment, Brazil (CNPq-558222/2008-0), for supporting themacro- andmicroscopical analysis and for providing a grant (CNPq-301072/2007-8); the Coordenaçao de Aperfeiçoamento de Pessoal de Nivel Superior (CAPES), Brazil, for post-doctoral scholarship support for SS; the Pacific Biologic Station and Vancouver Island University (VIU) in Canada for supporting the molecular studies

A method to estimate the mean standard length ( L ¯ ) of juvenile salmon in aggregations using multi-frequency (67 and 125 kHz) acoustic analysis was developed. An autonomous, single beam echosounder (Acoustic Zooplankton and Fish Profiler (AZFP), ASL Environmental Sciences) was deployed on the seafloor in Okisollo Channel, British Columbia during peak juvenile salmon migration season in 2015 and 2016. A positive correlation was found between the difference in the mean volume backscattering strength at 67 and 125 kHz (ΔMVBS67-125kHz) of juvenile salmon aggregations and the mean standard length of juvenile salmon collected with a purse seiner. A relationship of the form Δ M V B S 67 - 125 k H z = A l o g 10 L ¯ + B was established. High variability was observed due to differences in salmon species composition and lower sampling sizes at the tail end of the migration periods, but a consistent relationship with comparable parameters was found. We suggest that this method is valuable particularly in the case of long-term monitoring of species that are known to aggregate in schools of individuals with similar size, as it can be used to estimate mean size without the use of invasive capture techniques.

The phenology of long-distance migrations can influence individual fitness, moderate population dynamics and regulate the availability of ecosystem services to other trophic levels. Phenology varies within and among populations, and can be influenced by conditions individuals experience both prior to departure and encounter en route. Assessing how intrinsic and extrinsic factors (e.g., individual physical condition vs. environmental conditions) interact to influence variation in migratory phenologies across ecological scales is often limited due to logistical constraints associated with tracking large numbers of individuals from multiple populations simultaneously. We used two natural tags, DNA and otolith microstructure analysis, to estimate the relative influence of individual traits (life-history strategy, body size at departure and growth during migration), population-specific behaviours and interannual variability on the phenology of marine migrations in juvenile sockeye salmon Oncorhynchus nerka. We show that the timing and duration of juvenile sockeye salmon migrations were correlated with both life-history strategy and body size, while migration duration was also correlated with departure timing and growth rates during migration. Even after accounting for the effect of individual traits, several populations exhibited distinct migration phenologies. Finally, we observed substantial interannual and residual variation, suggesting stochastic environmental conditions moderate the influence of carry-over effects that develop prior to departure, as well as population-specific strategies. Migratory phenologies are shaped by complex interactions between drivers acting at multiple ecological and temporal scales. Given evidence that intraspecific diversity can stabilize ecological systems, conservation efforts should seek to maintain migratory variation among populations and preserve locally adapted phenotypes; however, variation within populations, which may buffer systems from environmental stochasticity, should also be regularly assessed and preserved.

Background Piscine reovirus (PRV) has been associated with the serious disease known as Heart and Skeletal Muscle Inflammation (HSMI) in cultured Atlantic salmon Salmo salar in Norway. PRV is also prevalent in wild and farmed salmon without overt disease manifestations, suggesting multifactorial triggers or PRV variant-specific factors are required to initiate disease. In this study, we explore the head kidney transcriptome of Sockeye salmon Oncorhynchus nerka during early PRV infection to identify host responses in the absence of disease in hopes of elucidating mechanisms by which PRV may directly alter host functions and contribute to the development of a disease state. We further investigate the role of PRV as a coinfecting agent following superinfection with infectious hematopoietic necrosis virus (IHNV) – a highly pathogenic rhabdovirus endemic to the west coast of North America. Results Challenge of Sockeye salmon with PRV resulted in high quantities of viral transcripts to become present in the blood and kidney of infected fish without manifestations of disease. De novo transcriptome assembly of over 2.3 billion paired RNA-seq reads from the head kidneys of 36 fish identified more than 320,000 putative unigenes, of which less than 20 were suggested to be differentially expressed in response to PRV at either 2 or 3 weeks post challenge by DESeq2 and edgeR analysis. Of these, only one, Ependymin, was confirmed to be differentially expressed by qPCR in an expanded sample set. In contrast, IHNV induced substantial transcriptional changes (differential expression of > 20,000 unigenes) which included transcripts involved in antiviral and inflammatory response pathways. Prior infection with PRV had no significant effect on host responses to superinfecting IHNV, nor did host responses initiated by IHNV exposure influence increasing PRV loads. Conclusions PRV does not substantially alter the head kidney transcriptome of Sockeye salmon during early (2 to 3 week) infection and dissemination in a period of significant increasing viral load, nor does the presence of PRV change the host transcriptional response to an IHNV superinfection. Further, concurrent infections of PRV and IHNV do not appear to significantly influence the infectivity or severity of IHNV associated disease, or conversely, PRV load. Electronic supplementary material The online version of this article (doi:10.1186/s12864-016-3196-y) contains supplementary material, which is available to authorized users.

Background Anti-Müllerian hormone (amh) or Müllerian-inhibiting substance (mis) is a member of the transforming growth factor-β family of hormones. This gene plays a key role in vertebrate male sex-determination by inhibiting the development of the Müllerian ducts, and has been shown to be the master sex-determinant in the Patagonian pejerrey. Results In the lingcod, Ophiodon elongatus, both males and females share one copy of amh, however we have identified a second duplicate copy that appears solely in the male individuals. We have developed a PCR-based assay targeting the TGF-β domain of amh that provides a simple method with which to sex lingcod from a small amount of tissue. An analysis across 57 individuals gave a 100 % success rate in identifying the phenotypic sex. Conclusions We present a simple method to sex lingcod through non-lethal tissue sampling. A third, independent, male-specific duplication of amh in a teleost fish has been identified in the lingcod. Electronic supplementary material The online version of this article (doi:10.1186/s13104-016-2030-6) contains supplementary material, which is available to authorized users.

Cortisol is released into the bloodstream of fish in response to exogenous stimuli referred to as stressors, and long-term or repetitive exposure to stressors can lead to poor growth and disease susceptibility in aquaculture-reared fish. Thus, as has been shown for other fish species (e.g. rainbow trout), the identification of fish with an attenuated cortisol response to stress may be beneficial to the emerging cod aquaculture industry. In this study, we: 1) examined if differences in the magnitude of the cortisol response to a standardized stressor (30 s handling) could be consistently detected within a multi-family population of cod; 2) determined if these differences were related to growth, and independent of stressor type; and 3) assessed whether single nucleotide polymorphisms (SNPs) identified by the Atlantic Cod Genomics and Broodstock Development Project (CGP) were correlated significantly with the magnitude of cortisol response. In this study, we were able to consistently separate fish with a high (HR) cortisol response from those with a low cortisol response (LR) to handling and heat stress. We showed that the percentage of HR and LR responders differed dramatically among the 10 cod families (with some families having no LR or HR individuals), and that significant negative correlations existed between cortisol responsiveness (total Z score) and wet mass, specific growth rate and condition factor; LR fish were approx. 55% heavier at the end of the year long study. Finally, we identified four polymorphic SNPs (QTLs) that were significantly associated with the magnitude of cortisol response to handling stress. This research suggests that there is a genetic component to this phenotype (HR vs. LR) in Atlantic cod, and that responsiveness to stress at the juvenile stage may be an important trait to consider in broodstock selection.

Myxobolus arcticus Pugachev and Khokhlov, 1979 is a freshwater myxosporean par- asite infecting the nerve tissues of salmonid fishes throughout the Pacific region of Far East Asia and North America. The principal fish host is sockeye salmon Oncorhynchus nerka in North America and masu salmon O. masou in Japan. Actinospores of M. arcticus were isolated from the lumbriculid oligochaetes Lumbriculus variegatus and Stylodrilus heringianus in Japan and Canada, respectively. Morphological comparisons indicated that Japanese actinospores from L. variegatus have significantly shorter caudal projections than Canadian isolates from S. hering - ianus, whereas the corresponding myxospores are indistinguishable. Transmission experiments showed that sockeye salmon were rarely susceptible to the Japanese actinospores, while masu salmon are highly susceptible to this parasite. Sequences of 4560 base pairs of the ribosomal RNA (rRNA) gene, including small subunit (SSU) and internal transcribed spacer (ITS) regions, from Japanese and Canadian isolates had a high similarity over 99.9%, suggesting that they may be conspecific. However, the biological data indicate that they are at least distinct strains. M. arcticus may be geographically isolated due to the specific homing migration of the anadromous fish hosts and has specialized its morphology and host selection for its local environment in the ongoing pro- cess of differentiation, potentially leading to speciation.

Chemokines are a large, diverse group of small cytokines that can be classified into several families, including the CC chemokines that are characterized by two adjacent cysteines near their amino terminus. CC chemokines play a pivotal role in host defense mechanisms by inducing leukocyte chemotaxis under physiological and inflammatory conditions. Analysis of CC chemokines from teleost fishes indicates that the number of CC chemokine genes and their tissue expression patterns vary largely in this group of vertebrates. Here we describe 32 distinct CC chemokine sequences from Atlantic cod (Gadus morhua) identified by analysis of approximately 206,000 ESTs. Phylogenetic analysis of Atlantic cod CC chemokines placed these sequences in seven clusters, most likely resulting from species-specific gene duplications, and two unique sequences; 12 of these CC chemokines, including at least one member of each cluster, were analyzed by QPCR using four immune-related tissues (head kidney, liver, spleen and blood) obtained from unstimulated, polyriboinosinic polyribocytidylic acid (pIC)-stimulated and formalin-killed atypical Aeromonas salmonicida-stimulated individuals. EST abundance and QPCR analysis indicate that the expression of closely related CC chemokines GmSCYA101 and GmSCYA102, GmSCYA108 and GmSCYA109 or GmSCYA122 and GmSCYA124 can be highly tissue-specific despite substantial sequence identity. Stimulation with the viral mimic pIC or formalin-killed atypical A. salmonicida resulted in increased expression of most of the CC chemokines, indicating that they can be regarded as either inducible (inflammatory) or dual-function rather than constitutive (homeostatic). Tissue specificity, and the level of induction, varied broadly; for example, GmSCYA123 was at least 4-fold up-regulated by both inducers in all tissues analyzed, whereas pIC increased the expression of GmSCYA124 in liver over 1500 times.

Nodaviruses and other RNA viruses have a profoundly negative impact on the global aquaculture industry. Nodaviruses target nervous tissue causing viral nervous necrosis, a disease characterized by neurological damage, swimming abnormalities, and morbidity. This study used functional genomic techniques to study the Atlantic cod ( Gadus morhua ) brain transcript expression responses to asymptomatic high nodavirus carrier state and intraperitoneal injection of polyriboinosinic polyribocytidylic acid (pIC). Reciprocal suppression subtractive hybridization (SSH) cDNA libraries enriched for virus-responsive brain transcripts were constructed and characterized. We generated 1,938 expressed sequence tags (ESTs) from a forward brain SSH library (enriched for transcripts upregulated by nodavirus and/or pIC) and 1,980 ESTs from a reverse brain SSH library (enriched for transcripts downregulated by nodavirus and/or pIC). To examine the effect of nodavirus carrier state on individual brain gene expression in asymptomatic cod, 27 transcripts of interest were selected for quantitative reverse transcription-polymerase chain reaction (QPCR) studies. Transcripts found to be >10-fold upregulated in individuals with a high nodavirus carrier state relative to those in a no/low nodavirus carrier state were identified as ISG15, IL8, DHX58 (alias LGP2), ZNFX1, RSAD2 (alias viperin), and SACS (sacsin, alias spastic ataxia of Charlevoix-Saguenay). These and other SSH-identified transcripts were also found by QPCR to be significantly ( P < 0.05) upregulated by pIC compared with saline-injected controls within 72 h of injection. Several transcripts identified in the reverse SSH library, including two putative ubiquitination pathway members (HERC4 and SUMO2), were found to be significantly ( P < 0.05) downregulated in individuals with a high nodavirus carrier state. Our data shows that Atlantic cod brains have a strong interferon pathway response to asymptomatic high nodavirus carrier state and that many interferon pathway and other immune relevant transcripts are significantly induced in brain by both nodavirus and pIC.

The Olympia oyster (Ostrea lurida) is the only oyster species native to British Columbia, Canada. Despite management regulations designed to protect this species, populations continue to decline in many locations. In an effort to determine whether parasites or disease are contributing to the population decline, oysters were collected from 5 locations and examined for the presence of pathogens and disease using histology and polymerase chain reaction (PCR) assays. Five parasite/symbiotic organisms were detected by histology, including Rickettsia-like prokaryotes, Mytilicola sp., Rhynchodida-like ciliates, encapsulated copepods, and an unknown protist. Six pathologies of unknown etiology were also detected, including hemocytic neoplasia, basophilic inclusions in the digestive gland epithelium, metaplasia of digestive gland tubules, diapedesis, and diffuse and focal hemocyte infiltration. Despite the variety of histological findings, most were detected at low prevalence and intensity, and are not be...

Physiological changes, elicited in animal immune tissues by exposure to pathogens, may be studied using functional genomics approaches. We created and characterized reciprocal suppression subtractive hybridization (SSH) cDNA libraries to identify differentially expressed genes in spleen and head kidney tissues of Atlantic cod ( Gadus morhua) challenged with intraperitoneal injections of formalin-killed, atypical Aeromonas salmonicida. Of 4,154 ESTs from four cDNA libraries, 10 genes with immune-relevant functional annotations were selected for QPCR studies using individual fish templates to assess biological variability. Genes confirmed by QPCR as upregulated by A. salmonicida included interleukin-1β, interleukin-8, a small inducible cytokine, interferon regulatory factor 1 (IRF1), ferritin heavy subunit, cathelicidin, and hepcidin. This study is the first large-scale discovery of bacteria-responsive genes in cod and the first to demonstrate upregulation of IRF1 in fish immune tissues as a result of bacterial antigen stimulation. Given the importance of IRF1 in vertebrate immune responses to viral and bacterial pathogens, the full-length cDNA sequence of Atlantic cod IRF1 was obtained and compared with putative orthologous sequences from other organisms. Functional annotations of assembled SSH library ESTs showed that bacterial antigen stimulation caused changes in many biological processes including chemotaxis, regulation of apoptosis, antimicrobial peptide production, and iron homeostasis. Moreover, differences in spleen and head kidney gene expression responses to the bacterial antigens pointed to a potential role for the cod spleen in blood-borne pathogen clearance. Our data show that Atlantic cod immune tissue responses to bacterial antigens are similar to those seen in other fish species and higher vertebrates.

18 páginas, 3 tablas, To investigate the immunological responses of turbot to nodavirus infection or pIC stimulation, we constructed cDNA libraries from liver, kidney and gill tissues of nodavirusinfected fish and examined the differential gene expression within turbot kidney in response to nodavirus infection or pIC stimulation using a turbot cDNA microarray. Turbot were experimentally infected with nodavirus and samples of each tissue were collected at selected time points post-infection. Using equal amount of total RNA at each sampling time, we made three tissue-specific cDNA libraries. After sequencing 3230 clones we obtained 3173 (98.2%) high quality sequences from our liver, kidney and gill libraries. Of these 2568 (80.9%) were identified as known genes and 605 (19.1%) as unknown genes. A total of 768 unique genes were identified. The two largest groups resulting from the classification of ESTs according to function were the cell/organism defense genes (71 uni-genes) and apoptosis-related process (23 uni-genes). Using these clones, a 1920 element cDNA microarray was constructed and used to investigate the differential gene expression within turbot in response to experimental nodavirus infection, This work was supported by the NRC-CSIC Canadian-Spanish Cooperation Program. A. Montes gratefully acknowledges the Ministerio de Educación y Ciencia, the CSIC (I3P program) for a research fellowship.

O-acetylation is one of the major modifications of sialic acids that significantly alters biological properties of the parent molecule. These O-acetylated forms are components of the cellular membrane and can affect physiological and pathological responses. Understanding the role of N-glycans in physiology is of increasing relevance to cellular biologists in various disciplines who study glycoproteomics yet lack information regarding the function of the attached glycans. It is well known that stress may decrease immune function in fish; however, there are only few suitable biomarkers available to monitor the physiological responses under the stress conditions. This study is the first report on the effect of stress on the profile of O-acetylation of sialic acids in fish serum. In order to preserve the relevant structural characteristics as much as possible, native N-glycans were directly analyzed using CE-MS. We have characterized the N-glycans in serum of salmon (Salmo salar) exposed to long-term handling stress (15 s out of the water, daily for 4 wk) and compared with the results obtained from sera of control fish. The results indicated that major N-glycans in salmon serum contained mono-acetylated sialic acids (83%), and that the O-acetylation pattern of sialic acids could be altered by long-term stress.

The effect of reduced feed ration on infestation levels with the sea louse Lepeophtheirus salmonis and gene expression in juvenile pink salmon Oncorhynchus gorbuscha was tested in three laboratory trials. Body weight was significantly lower among fish on the reduced ration for 27, 34, or 65 d than fish on the full ration. Neither the prevalence nor the abundance of L. salmonis differed between fish on full and reduced rations at any time in any trial. In trial 2, sea louse rejection was delayed among fish on reduced rations; however, the parasite was ultimately rejected from all fish in this trial regardless of ration. Proinflammatory gene expression in salmon exposed to L. salmonis was modulated by reduced rations. There was a reduction in the expression of interleukin-8 in pink salmon on reduced rations 7 d after exposure but not 14 d after exposure. In contrast, the 7-d expression of interleukin-1 beta (IL-1beta) was reduced in exposed pink salmon regardless of ration. By day 14, however, expression of IL-1beta was increased in association with reduced rations among exposed salmon. Similarly, the expression of tumor necrosis factor alpha (TNFalpha) was increased 14 d after exposure among salmon on a reduced ration. There was no evidence that short-duration exposure of otherwise healthy juvenile pink salmon to a reduced ration affected susceptibility to L. salmonis. The expression data do not suggest an obvious mechanism of louse rejection; rather, they indicate that a more comprehensive suite of inflammatory pathways should be surveyed to better understand the early pink salmon response to L. salmonis.

In this study we measured plasma cortisol, plasma glucose, plasma sodium and potassium, and liver and gill hsp70 levels in juvenile matrinxã (Brycon amazonicus) subjected to a 96 h exposure to phenol (0, 0.2, and 2.0 ppm), and the effect of this exposure on their ability to respond to a subsequent handling stress. Fish were sampled prior to initiation of exposure and 96 h, and at 1, 6, 12, and 24 h post-handling stress. During the 96 h exposure, plasma cortisol and glucose levels remained unchanged in all treatments. While plasma sodium levels were significantly reduced in all groups, plasma potassium levels only decreased in fish exposed to 0 and 0.2 ppm of phenol. Liver hsp70 levels decreased significantly at 96 h in fish exposed to 2.0 ppm of phenol. All groups, except fish exposed to 0.2 ppm of phenol, were able to increase plasma cortisol and glucose levels after handling stress. Fish exposed to 2.0 ppm of phenol showed decreased gill and liver hsp70 levels after the handling stress. Our data suggest that exposure to phenol may compromise the ability of matrinxã to elicit physiological responses to a subsequent stressor.

Sea-caged cod are limited in their movements in the water column, and thus can be exposed to large seasonal ( approximately 0-20 degrees C) temperature fluctuations. To investigate the physiological response of Atlantic cod to summer-like increases in temperature, we exposed 10 degrees C acclimated juvenile cod to a graded thermal challenge (1 degrees C increase every 5 days) and measured: (1) plasma cortisol and glucose levels; (2) the respiratory burst activity of blood leukocytes; and (3) the expression of specific immune-related genes [MHC Class I, Interleukin-1beta (IL-1beta), beta2-microglobulin (beta2-M), Immunoglobulin M (IgM)-light (L) and -heavy (H) chains] in the blood using quantitative reverse transcription-polymerase chain reaction (QRT-PCR). The experiment was stopped at 19.1 degrees C, with 26.7% of the fish surviving to this point. Plasma glucose levels increased slightly at 16 and 18 degrees C (by 1.39- and 1.74-fold, respectively), in contrast, cortisol levels were elevated significantly (by 2.9-fold) at 16 degrees C but returned to control levels thereafter. The effect of increasing temperature on the expression of immune related genes in blood cells (leukocytes) was variable and depended on the gene of interest. The expression of IgM-H remained stable for the duration of the experiment. In contrast, IL-1beta expression was increased significantly (by approximately 25-fold) at 19 degrees C as compared to time-matched control fish, and changes in the expression of beta2-M, MHC Class I and IgM-L followed a pattern similar to that seen for cortisol: increasing at 16 degrees C (by 4.2-, 5.3- and 17-fold, respectively), but returning to pre-stress levels by 19 degrees C. Interestingly, increasing temperatures had no effect on respiratory burst activity. This study is the first to examine the effects of a chronic regimen of increasing temperature on the stress physiology and immunology of a marine teleost, and suggests that immune function is influenced by complex interactions between thermal effects and temperature-induced stress (elevated circulating cortisol levels).

Laboratory-reared pink and chum salmon juveniles (approximately 2g) received an intraperitoneal injection with a commercial, unadjuvanted Aeromonas salmonicida bacterin or sterile saline. Relative to elongation factor-1A, expression levels of genes encoding the proinflammatory cytokines interleukin-1beta-1 (IL-1beta), tumour necrosis factor-alpha-1 (TNFalpha) and interleukin-8 (IL-8) in pools of kidney and liver were examined 6- and 24-h after injection. Expression of IL-1beta was significantly elevated in pink and chum salmon by 6-h, and declined in pink salmon but not in chum salmon by 24-h. Similarly, expression of TNFalpha was significantly elevated in both species at 6h and only in chum salmon after 24-h. Expression of IL-8 was significantly elevated in both species at 6- and 24-h after injection. Expression of the three proinflammatory cytokine genes differed between salmon species both in the timing and magnitude of their expression. The significance of these differences with respect to immune function in these fish requires further research.

Piscine reovirus (PRV) is a double stranded non-enveloped RNA virus detected in farmed and wild salmonids. This study examined the phylogenetic relationships among different PRV sequence types present in samples from salmonids in Western Canada and the US, including Alaska (US), British Columbia (Canada) and Washington State (US). Tissues testing positive for PRV were partially sequenced for segment S1, producing 71 sequences that grouped into 10 unique sequence types. Sequence analysis revealed no identifiable geographical or temporal variation among the sequence types. Identical sequence types were found in fish sampled in 2001, 2005 and 2014. In addition, PRV positive samples from fish derived from Alaska, British Columbia and Washington State share identical sequence types. Comparative analysis of the phylogenetic tree indicated that Canada/US Pacific Northwest sequences formed a subgroup with some Norwegian sequence types (group II), distinct from other Norwegian and Chilean sequences (groups I, III and IV). Representative PRV positive samples from farmed and wild fish in British Columbia and Washington State were subjected to genome sequencing using next generation sequencing methods. Individual analysis of each of the 10 partial segments indicated that the Canadian and US PRV sequence types clustered separately from available whole genome sequences of some Norwegian and Chilean sequences for all segments except the segment S4. In summary, PRV was genetically homogenous over a large geographic distance (Alaska to Washington State), and the sequence types were relatively stable over a 13 year period.

The recently described type III secretion system (TTSS) of Aeromonas salmonicida subsp. salmonicida has been linked to virulence in salmonids. In this study, three TTSS effector genes, aexT, aopH or aopO, were inactivated by deletion, as was ascC, the gene encoding the outer-membrane pore of the secretion apparatus. Effects on virulence were assayed by live challenge of Atlantic salmon (Salmo salar). The ΔascC mutant strain was avirulent by both intraperitoneal (i.p.) injection and immersion, did not appear to establish a clinically inapparent infection and did not confer protection from subsequent rechallenge with the parental strain. 1H NMR spectroscopy-based metabolite profiling of plasma from all fish showed significant differences in the metabolite profiles between the animals exposed to the parental strain or ΔascC. The experimental infection by immersion with ΔaopO was indistinguishable from that of the parental strain, that of ΔaexT was delayed, whilst the virulence of ΔaopH was reduced significantly but not abolished. By i.p. injection, ΔaexT, ΔaopH and ΔaopO caused an experimental disease indistinguishable from that of the parental strain. These data demonstrate that while the TTSS is absolutely essential for virulence of A. salmonicida subsp. salmonicida in Atlantic salmon, removal of individual effectors has little influence on virulence but has a significant effect on colonization. The ΔascC i.p. injection data also suggest that in addition to host invasion there is a second step in A. salmonicida pathogenesis that requires an active TTSS.

The objective of the present study was to describe the histological and physiological development of the gastrointestinal system in Atlantic halibut from the time of first-feeding until metamorphosis. At first-feeding, the gastrointestinal (GI) tract is divided into anterior, mid and hindgut regions. The liver is present, as is the pancreas. During development the pancreas changes from a compact organ to a diffuse tissue interspersed through much of the mesentery surrounding the GI tract. Functional gastric glands are not present until approximately 66 days post-hatch (dph). Using primers based upon winter flounder digestive enzyme gene sequences, we were able to amplify partial sequences for bile salt-activated lipase (BAL), trypsinogen (TRP), and pepsinogen (PEP) from RNA extracted from whole larvae and juveniles using Reverse Transcription-PCR (RT-PCR). PCR products were sequenced and the sequences used to design halibut gene-specific primers for BAL and PEP. RT-PCR analysis revealed that BAL and TRP gene expression was evident at least from the time of first-feeding but PEP gene expression was not detectable until 80 dph. In situ hybridization using molecular probes from winter flounder sequences localized expression of BAL and TRP to the exocrine pancreas. PEP expression was only localized to the glandular regions of the stomach. These data provide a first step toward understanding the molecular events governing the ontogeny of digestive capacity in Atlantic halibut.

Toll-like receptors (TLRs) are involved in the innate immune response against microbial pathogens in vertebrates and insects. The extracellular region of a TLR recognizes pathogen-associated molecules, while the intracellular region initiates the signaling pathway leading to immune response. Membrane-bound TLRs have been found in most vertebrates, but few soluble forms have been reported. A novel transcript corresponding to a portion of a soluble TLR was identified in liver of infected Atlantic salmon. The complete coding sequence of this TLR was obtained and BLASTN analysis showed the highest sequence identity to a recently described full-length cDNA sequence of a soluble TLR5 from rainbow trout (GenBank Accession No.: AB062504). The deduced protein is 40% identical to the mammalian counterpart of the leucine-rich repeat (LRR)/LRR-like motifs of TLR5. Based on the structure of human TLRs, it contains 21 LRRs with conserved LxxLxLxxNx*xx*xxxxFxxL pattern. Since TLR5 is essential for the recognition of bacterial flagellins, we hypothesize that flagellin and perhaps some other pathogen-derived factors from Aeromonas salmonicida bind to this soluble TLR through an unknown binding domain within the LRR.

Recently, less toxic and more effective anaesthetics are essential for marine fishes. Lidocaine belongs to a group of anaesthetics which are used as local anaesthetic in human medicine. This chemical was tested fer winter flounder, Pleunnectes americanus. Anaesthetic effect of lidocaine hydrochloride-sodium bicarbonate mixture (lidocaine ) was tested for the winter flounder at five different temperature regimes: Anaesthetic dose and temperature-dependent relationship in exposure and recovery time were observed for the winter flounder of mean total length. Based on the results, anaesthetic lidocaine showed rapid exposure time and rapid recovery time for winter flounder. The results indicate that lidocaine can be used as suitable anaesthetic for this species.

In the present work, we report the characterization of the partial cDNA sequence of a bile salt-activated lipase (BAL) cDNA from haddock. The predicted polypeptide encoded by the cDNA sequence contains the bile salt-binding site characteristic of all BALs at amino acid positions 36–45, and the lipid-binding site thus far only reported in fish BALs starting at position 345. Other features of BAL are also present including: the active site serine motif at positions 111–117, the catalytic triad formed by the residues S114, D239 and H358, and an N-glycosylation site at position 107. The relative levels of BAL gene expression were determined in haddock larvae during ontogeny by reverse transcription–polymerase chain reaction (RT–PCR) with the earliest detectable transcript levels identified at hatch. Using in situ hybridization, the BAL transcripts were localized consistently in the pancreas of haddock larvae from mouth opening until 401 degree days (DD). Using biochemical techniques, the specific activity of BAL was found to decline significantly over time. Our results also suggest that haddock larvae are capable of digesting lipids at the time of mouth opening.

Piscine reovirus (PRV) has been associated with the serious disease known as Heart and Skeletal Muscle Inflammation (HSMI) in cultured Atlantic salmon Salmo salar in Norway. Recent studies have also found PRV to be prevalent in wild (Oncorhynchus spp.) and farmed Chinook (O. tshawytscha) and Atlantic salmon from the west coast of North America. Interestingly, HSMI or other disease associated with PRV infection has not been seen, suggesting factors beyond the simple presence of PRV are required to initiate disease. Here, we explore whether the presence of PRV could influence how Atlantic and Pacific salmon respond to additional infectious agents or have other impacts on host physiology and/or ecological performance. To this end we have studied the transcriptional responses of Sockeye (O. nerka) and Atlantic salmon head kidney and blood to infection with PRV, Sockeye head kidney to co-infection of PRV and Infectious Hematopoietic Necrosis Virus (IHNV), and Atlantic salmon erythrocytes to infection with PRV under in vivo and ex vivo conditions. We have used laboratory challenges and transcript profiling by RNA Sequencing (RNA-seq) and RT-qPCR to study these responses at high PRV loads. We found that infection with PRV alone caused a very limited immune response at the transcript level in blood and head kidney of Sockeye and Atlantic salmon. Infection with PRV also had little to no effect on the subsequent immune response at the transcript level to IHNV in Sockeye head kidney. Interestingly, the well develop immune response to IHNV did not have a measurable effect on PRV loads or infectivity. In contrast, ex vivo Atlantic salmon erythrocytes demonstrate substantial innate antiviral responsiveness to exposure with PRV. The potential mechanisms for evasion of host immune responses as well as the possible role of PRV in HSMI or other disease manifestations are discussed.

Aeromonas salmonicida subsp. salmonicida is a facultatively intracellular gram-negative bacterium that is the etiological agent of furunculosis, a bacterial septicemia of salmonids that causes significant economic loss to the salmon farming industry. The mechanisms by which A . salmonicida evades intracellular killing may be relevant in understanding virulence and the eventual design of appropriate treatment strategies for furunculosis. We have identified two open reading frames (ORFs) and related upstream sequences that code for two putative superoxide dismutases (SODs), sodA and sodB . The sodA gene encoded a protein of 204 amino acids with a molecular mass of approximately 23.0 kDa (SodA) that had high similarity to other prokaryotic Mn-SODs. The sodB gene encoded a protein of 194 amino acids with a molecular mass of approximately 22.3 kDa that had high similarity to other prokaryotic Fe-SODs. Two enzymes with activities consistent with both these ORFs were identified by inhibition of O 2 − -catalyzed tetrazolium salt reduction in both gels and microtiter plate assays. The two enzymes differed in their expression patterns in in vivo- and in vitro-cultured bacteria. The regulatory sequences upstream of putative sodA were consistent with these differences. We could not identify other SOD isozymes such as sodC either functionally or through data mining. Levels of SOD were significantly higher in virulent than in avirulent strains of A . salmonicida subsp. salmonicida strain A449 when cultured in vitro and in vivo.

The partial sequencing of two lipases from winter flounder Pseudopleuronectes americanus, one most closely related to gastric, lingual and lysosomal acid lipase from other vertebrates and one most closely related to bile salt-activated lipase, is reported. Biochemical analyses of enzymatic activity demonstrated the greater contribution made by bile salt-activated lipase relative to neutral bile salt-independent lipase. Using molecular techniques, the tissue-specific expression of bile salt-activated lipase in pancreatic tissue and acid triacylglycerol lipase in a wide variety of organs was demonstrated. Furthermore, the developmental expression of these types of lipase in larval fish was established.

In order to establish effective procedures for chromosome manipulations in the winter flounder Pseudopleuronectes americanus, temperature-dependent measures of mitotic intervals (τ0) were determined. Mitotic intervals (τ0) were determined by averaging the duration of the first and third embryonic divisions over a range of temperatures from 0 to 20 °C. At higher temperatures, eggs developed faster and underwent more identical development. Mitotic intervals for winter flounder were 98.5±1.3 min at 0 °C, 79.5±1.7 min at 5 °C, 59.5±1.7 min at 10 °C, 38.7±1.8 min at 15 °C and 23.9±2.2 min at 20 °C. There were strong, negative correlations between mitotic interval and water temperature for all five temperatures application (Y=−19.00X+117.40, R2=0.9969, where Y is mitotic interval and X is temperature).

In 1999, disease outbreaks in juvenile Atlantic cod Gadus morhua that showed the classic signs of viral encephalopathy and retinopathy (VER) were reported in Nova Scotia. Brain and retinal tissues from moribund cod showed diffuse degenerative vacuolative encephalopathy and degenerative histiocytic retinitis. The affected brain and retinal tissues were observed to be positive for nodaviral antigens by means of immunohistochemical techniques. We partially characterized a nodavirus-like agent from brain and eye tissues and cell culture using reverse-transcriptase polymerase chain reaction and primer sets originally designed for amplification of white trevally Caranx dentex (also known as striped jack Pseudocaranx dentex) and Atlantic halibut Hippoglossus hippoglossus nervous necrosis virus coat (capsid) proteins. Sequencing of the T2 region of the coat protein revealed high similarities (>85% nucleotide identity) to the coat protein genes of other fish nodavirus strains, especially those of Atlantic...

Mikrocytos mackini is a microcell parasite that usually infects Crassostrea gigas distributed along the Pacific Northwest coast of North America. For many years, M. mackini was the only known species in the genus, but there have been multiple recent findings of genetically divergent forms of Mikrocytos in different hosts and in distantly located geographic locations. This note describes M. boweri sp. nov. found in Olympia oysters Ostrea lurida collected from and native to British Columbia, Canada, primarily using a molecular taxonomic approach.

The objective of the present study was to understand how and when the frontal filament (FF) in the salmon louse Lepeophtheirus salmonis is produced by examining the sequence of morphological changes leading to FF production in the copepodid and early chalimus stages. Atlantic salmon (Salmo salar) were heavily infested with newly molted copepodids. Sea lice were sampled prior to infestation and at 1, 2, 3, 4, 5, 6, 7, 8, and 9 days postinfestation. FF morphogenesis from newly molted copepodid to chalimus II, i.e., through 2 molts, was studied using high-resolution light microscopy of serial transverse and sagittal resin sections. Three groups of cells, identified as A, B, and C, are thought to be involved in the production of the secretions (S1 and S2) that form the filament material. The amount and shape of S1 and S2 and their association with B- and C-group cells, respectively, changed with the molt cycle. The following scenario for FF development is proposed: the first secretion to form after the molt for both copepodid and chalimus stages is S1, and it is formed by B-group cells and becomes the basal plate of the external FE C-group cells produce S2 during mid-intermolt to premolt stage. The S2 becomes the stem of the external FE In premolt larvae, S1 and S2 were contained within a cuticle-lined invagination that had a form similar to that of the extruded filament. The axial duct present in both copepodid and chalimus originates from the A-group cells and probably carries a secretion used to attach the filament to the host. This study provides strong evidence that L. salmonis produces a new filament with each molt, creating the possibility of using a sea lice control method based on interference with filament production more feasible.

As part of an investigation of the biochemical interactions between the salmon louse Lepeophtheirus salmonis and Atlantic salmon Salmo salar, we characterized protease activity in the skin mucus of noninfected Atlantic salmon and Atlantic salmon infected with L. salmonis and in an L. salmonis whole-body homogenate. Zymography revealed that mucus from infected salmon contained a series of low-molecular-mass (17-22 kDa) serine proteases that were not present in the mucus of noninfected salmon. Based on molecular mass, inhibition studies, and affinity chromatography, the series of proteases was identified as being trypsin-like. Similar proteases were observed in the L. salmonis homogenate and in mucus from noninfected Atlantic salmon following a 1-hr incubation with live L. salmonis. An antibody raised against Atlantic salmon trypsin failed to recognize any proteases in the mucus of noninfected salmon or infected salmon or in the L. salmonis homogenate. Collectively, these findings suggest that the trypsin-like proteases present in the mucus of infected Atlantic salmon were produced by L. salmonis, possibly to aid in feeding and evasion of host immune responses.

Haddock (Melanogrammus aeglefinus) is a promising new species for aquaculture in Eastern Canada. Because haddock aquaculture could involve overwintering of fish in sea cages in icy near-shore areas, haddock freeze resistance was studied. Measurements were done on the blood of wild haddock collected during the winter from Georges and St. Pierre Banks as well as from cultured haddock originating from Northeast Bank in the Bay of Fundy. Freezing points were within the normal range for teleost blood, suggesting no freeze resistance adaptations. Glycerol in blood samples from Georges Bank and from most cultured haddock was not above normal physiological levels whereas St. Pierre Bank and Sandy Cove fish showed elevated glycerol levels. However, glycerol in the latter group was far lower than amounts required to depress the blood freezing point to a safe level in icy seawater. Thermal hysteresis measurements revealed no antifreeze protein (AFP) activity in any of the samples. However, ice crystal morphology revealed small amounts of AFP in a haddock sample collected in sub-zero seawater. The haddock antifreeze differed from the antifreeze glycoproteins (AFGPs) of other gadids in that it was active in the presence of peanut lectin and inactivated by EDTA. Neither the glycerol levels nor the trace level of AFP shown here would be sufficient to protect this species from freezing in icy seawater during winter.

We investigated the effects of a calcium-dependent mannose-binding lectin isolated from the serum of Atlantic salmon on Aeromonas salmonicida viability and the anti-A. salmonicida activity of Atlantic salmon macrophages. In the absence of other factors, binding of this lectin at concentrations of 0.8, 4.0 and 20.0 ng ml(-1) to virulent A. salmonicida failed to significantly reduce (P0.05) cell viability. However, binding of the lectin to A. salmonicida did result in significant (Por = 0.05) dose-dependent increases in phagocytosis, and bactericidal activity. Significant increases (Por = 0.05) were also observed in phagocyte respiratory burst activity within the lectin concentration range of 4.0-20.0 ng ml(-1) but the stimulation was not dose dependent at these lectin concentrations. At the lowest lectin concentration tested (0.32 ng ml(-1)), a significant decrease (Por = 0.05) in respiratory burst was observed. The structure and activity of this lectin are similar to that of mammalian mannose-binding lectins, which are known to play a pivotal role in innate immunity. The presence of this lectin may be an important defense mechanism against Gram-negative bacteria such as A. salmonicida.

Perkinsus qugwadi, a pathogenic protozoan parasite of Yesso scallops Patinopecten yessoensis, is found only in cultured populations in British Columbia, Canada. This pathogen was first identified in 1988 and caused significant mortalities at some locations during the early 1990s. Prevalence of infection decreased dramatically following 1995, and the disease was last reported in 1997, leading to speculation that the Yesso scallop stocks in Canada had developed resistance to the disease, or that P. qugwadi had disappeared. However, the present study revealed that infection with P. qugwadi and associated mortality is still occurring in scallops from at least one location in British Columbia. One of the PCR tests developed for P. qugwadi detected the parasite in a 105-fold dilution of DNA extracted from a heavily infected sample and detected 52% more positive scallops than histology; however, the assay also cross-reacted with P. honshuensis and P. olseni. The other PCR test was less sensitive and detected 34% more positives, but did not react to any of the other Perkinsus species tested, suggesting that these PCR tests are powerful tools for screening for the presence of P. qugwadi. Phylogenetic analysis of 1796 bp of SSU rRNA gene sequence clearly indicated that P. qugwadi is positioned basally to other Perkinsus species.

Heart and skeletal muscle inflammation (HSMI) is a significant and often fatal disease of cultured Atlantic salmon in Norway. The consistent presence of Piscine orthoreovirus (PRV) in HSMI diseased fish along with the correlation of viral load and antigen with development of lesions has supported the supposition that PRV is the etiologic agent of this condition; yet the absence of an in vitro culture system to demonstrate disease causation and the widespread prevalence of this virus in the absence of disease continues to obfuscate the etiological role of PRV with regard to HSMI. In this study, we explore the infectivity and disease causing potential of PRV from western North America—a region now considered endemic for PRV but without manifestation of HSMI—in challenge experiments modeled upon previous reports associating PRV with HSMI. We identified that western North American PRV is highly infective by intraperitoneal injection in Atlantic salmon as well as through cohabitation of both Atlantic and Sockeye salmon. High prevalence of viral RNA in peripheral blood of infected fish persisted for as long as 59 weeks post-challenge. Nevertheless, no microscopic lesions, disease, or mortality could be attributed to the presence of PRV, and only a minor transcriptional induction of the antiviral Mx gene occurred in blood and kidney samples during log-linear replication of viral RNA. Comparative analysis of the S1 segment of PRV identified high similarity between this North American sequence and previous sequences associated with HSMI, suggesting that factors such as viral co-infection, alternate PRV strains, host condition, or specific environmental circumstances may be required to cause this disease.