Michael Snow

Functional Characterization of a Nonmammalian IL-21: Rainbow Trout Oncorhynchus mykiss IL-21 Upregulates the Expression of the Th Cell Signature Cytokines IFN-γ, IL-10, and IL-22

In mammals, IL-21 is a common γ chain cytokine produced by activated CD4+ T cells and NKT cells that acts on multiple lineages of cells. Although IL-21 has also been discovered in birds, amphibians, and fish, to date, no functional studies have been reported for any nonmammalian IL-21 molecule. We have sequenced an IL-21 gene (tIL-21) in rainbow trout, which has a six-exon/five-intron structure, is expressed in immune tissues, and is induced by bacterial and viral infection and the T cell stimulant PHA. In contrast to mammals, calcium ionophore and PMA act synergistically to induce tIL-21. Recombinant tIL-21 (rtIL-21) induced a rapid and long-lasting (4–72 h) induction of expression of IFN-γ, IL-10, and IL-22, signature cytokines for Th1-, Th2-, and Th17-type responses, respectively, in head kidney leukocytes. However, rtIL-21 had little effects on the expression of other cytokines studied. rtIL-21 maintained the expression of CD8α, CD8β, and IgM at a late stage of stimulation when their expression was significantly decreased in controls and increased the expression of the Th cell markers CD4, T-bet, and GATA3. Intraperitoneal injection of rtIL-21 confirmed the in vitro bioactivity and increased the expression of IFN-γ, IL-10, IL-21, IL-22, CD8, and IgM. Inhibition experiments revealed that the activation of JAK/STAT3, Akt1/2, and PI3K pathways were responsible for rtIL-21 action. This study helps to clarify the role of IL-21 in lower vertebrates for the first time, to our knowledge, and suggests IL-21 is a likely key regulator of T and B cell function in fish.

A low-pathogenic variant of infectious salmon anemia virus (ISAV-HPR0) is highly prevalent and causes a non-clinical transient infection in farmed Atlantic salmon (Salmo salar L.) in the Faroe Islands

Infectious salmon anemia virus (ISAV) is an orthomyxovirus responsible for a significant disease of farmed Atlantic salmon. Fallowing and re-establishment of the Atlantic salmon farming industry in the Faroes following a recent devastating infectious salmon anaemia (ISA) disease epidemic provided a unique opportunity to study the risk of re-emergence of disease. Over 53 months, 2787 of 34 573 (8.1%) apparently healthy Atlantic salmon analysed tested positive for ISAV by RT-PCR. Sequence analysis revealed the putative low-pathogenic ISAV-HPR0 subtype in all cases. Results demonstrated that ISAV-HPR0 appeared as a seasonal and transient infection without detectable ISA mortality or pathology. This finding, coupled to an apparent gill tropism of ISAV-HPR0, suggests ISAV-HPR0 causes a subclinical respiratory infection more like seasonal influenza, as opposed to the systemic infection and serious disease caused by highly pathogenic ISAV. The mean time before marine sites became infected was 7.7 months after transfer to seawater of the fish, suggesting a potentially unknown marine reservoir of infection. Sequence analysis identified two main subtypes of ISAV-HPR0 sequences, one of which showed close genetic association with ISAV isolates responsible for the disease outbreak in the Faroes. Thus ISAV-HPR0 might represent an ancestor of pathogenic variants and thus be a potential risk factor in the emergence of new strains of disease-causing ISAV. Our data, however, suggest that the risk of emergence of pathogenic ISAV variants from a reservoir of ISAV-HPR0 is low. This risk is probably being further reduced by practical management strategies adopted in the Faroes and aimed at reducing the potential for maintenance and adaptation of ISAV-HPR0.

Development and validation of a reverse transcription quantitative PCR for universal detection of viral hemorrhagic septicemia virus.

Viral hemorrhagic septicemia virus (VHSV) infects over 70 fish species inhabiting marine, brackish or freshwater environments throughout the Northern Hemisphere. Over its geographic range, 4 VHSV genotypes and multiple subtypes exist. Here, we describe the development and validation of a rapid, sensitive and specific real-time reverse transcription quantitative PCR assay (RT-qPCR) that amplifies sequence from representative isolates of all VHSV genotypes (I, II, III and IV). The pan-specific VHSV RT-qPCR assay reliably detects 100 copies of VHSV nucleoprotein RNA without cross-reacting with infectious hematopoietic necrosis virus, spring viremia of carp virus or aquatic birnavirus. Test performance characteristics evaluated on experimentally infected Atlantic salmon Salmo salar L. revealed a diagnostic sensitivity (DSe) > or = 93% and specificity (DSp) = 100%. The repeatability and reproducibility of the procedure was exceptionally high, with 93% agreement among test results within and between 2 laboratories. Furthermore, proficiency testing demonstrated the VHSV RT-qPCR assay to be easily transferred to and performed by a total of 9 technicians representing 4 laboratories in 2 countries. The assay performed equivalent to the traditional detection method of virus isolation via cell culture with the advantage of faster turnaround times and high throughput capacity, further suggesting the suitability of the use of this VHSV RT-qPCR in a diagnostic setting.

Surveillance for infectious salmon anaemia virus HPR0 in marine Atlantic salmon farms across Scotland

Infectious salmon anaemia virus (ISAV) is a serious and commercially important pathogen of Atlantic salmon. Multiple viruses have been defined based on a highly polymorphic region (HPR) of the haemagglutinin-esterase (HE) protein encoded by genomic segment 6. The viruses causing disease outbreaks in farms to date all have deletions in this region with respect to a putative ancestral variant with a longer HPR (HPR0). The presence of HPR0 nucleic acid has been detected in many countries including Scotland, where it has mostly been associated with healthy wild and farmed fish. Pathogenic ISAVs appear to have been derived from HPR0 ancestors on multiple independent occasions, which suggests that the presence of HPR0 could represent a risk factor in the re-emergence of infectious salmon anaemia (ISA) disease. In order to better understand this potential risk factor, anonymous samples of gill and heart tissues from marine Atlantic salmon farms throughout Scotland were collected and screened for the presence of ISAV RNA. Since it has not been possible to isolate HPR0 in conventional ISA-permissive cell cultures, a sensitive real-time RT-PCR method was employed for the detection of viral RNA. DNA sequencing was carried out on the positive samples to determine their HPR sequence. ISAV RNA was detected in 6 samples originating from 4 different locations and sequence analysis indicated the viruses were of the HPR0 type. Full length segment 6 sequence analysis of 1 positive sample indicated that it was most similar to a European genotype sequence previously obtained from North America.

Development of a sensitive and controlled real-time RT-PCR assay for viral haemorrhagic septicaemia virus (VHSV) in marine salmonid aquaculture

A survey was undertaken to determine the potential distribution of viral haemorrhagic septicaemia virus (VHSV) in marine cage-based salmonid farms in Scotland. A rapid, accurate and sensitive quantitative real-time RT-PCR (qRT-PCR) assay was developed, targeting a conserved region of the nucleoprotein (N) gene of the virus. The qRT-PCR assay was shown to be more sensitive than the conventional VHSV RT-PCR. A validation protocol included several different virus isolates as the target and confirmed that the assay could detect all European VHSV genotypes (I, II and III). Both endogenous and exogenous controls were designed to control for integrity of template and distinguish between true VHSV positives and contamination with the positive control material. In total, the universal European VHSV qRT-PCR assay with exogenous positive control was applied to screen 2040 individual Atlantic salmon Salmo salar and 150 individual rainbow trout Oncorhynchus mykiss. No evidence of the presence of VHSV in association with either salmonid species in Scottish marine farms was detected. However, both marine Atlantic salmon and rainbow trout are still considered possible carriers of VHSV, which remains a potential threat to freshwater farming. Therefore, a continued surveillance of these species in marine environment is recommended.

Selection for upper thermal tolerance in rainbow trout ( Oncorhynchus mykiss Walbaum)

ABSTRACT Rainbow trout (Oncorhynchus mykiss Walbaum) in southern Western Australia have undergone passive selection for over 19 generations to survive high water temperatures. Based on the conceptual model of ‘oxygen- and capacity-limited thermal tolerance’, we measured critical thermal maximum (CTmax), maximum heart rate (fH,max) and aerobic scope to test the hypothesis that these rainbow trout can maintain aerobic scope at high temperatures through a robust cardiac performance supporting oxygen delivery. Across five family groups CTmax averaged 29.0±0.02°C. Aerobic scope was maximized at 15.8±0.3°C (Topt), while the upper pejus temperature (Tpej, set at 90% of maximum aerobic scope) was 19.9±0.3°C. Although aerobic scope decreased at temperatures above Topt, the value at 25°C remained well over 40% of the maximum. Furthermore, pharmacologically stimulated fH,max increased with temperature, reaching a peak value between 23.5±0.4 and 24.0±0.4°C (Tmax) for three family groups. The Arrhenius breakpoint temperature (TAB) for fH,max was 20.3±0.3 to 20.7±0.4°C, while the average Q10 breakpoint temperature (TQB, when the incremental Q10<1.6) for fH,max was 21.6±0.2 to 22.0±0.4°C. Collectively, fH,max progressively became less temperature dependent beyond 20°C (TAB and TQB), which coincides with the upper Tpej for aerobic scope. Although upper thermal performance indices for both aerobic scope and fH,max were compared among family groups in this population, appreciable differences were not evident. Compared with other populations of rainbow trout, the present assessment is consistent with the prediction that this strain has undergone selection and shows the ability to tolerate higher water temperatures. Summary: Rainbow trout in Pemberton Freshwater Research Centre, Western Australia, demonstrated their impressive thermal performance in terms of cardiorespiratory function.

Histology, immunocytochemistry and qRT-PCR analysis of Atlantic salmon, Salmo salar L., post-smolts following infection with infectious pancreatic necrosis virus (IPNV)

Infectious pancreatic necrosis (IPN) is a very serious viral disease in terms of its impact on production of Atlantic salmon, Salmo salar L., fry and post-smolts. Post-smolts of Atlantic salmon were injected with infectious pancreatic necrosis virus (IPNV) and cohabited with naive fish to produce natural infection. Cohabitant fish were sampled every 2 days, up to day 36 post-infection (p.i.). From 90 cohabitant fish, 11 (12.2%) were positive by immunohistochemistry (IHC). The first detection of IPNV by IHC occurred on day 16 p.i. which coincided with the onset of mortality in this group. Besides the pancreas, the liver was found to be a key target organ for IPNV. For the first time, the virus was observed in the islets of Langerhans and in the kidney corpuscles of Stannius which suggests that the virus could affect the fishs metabolism. The liver of two fish, which showed the most widespread presence of IPNV by IHC, had a pathology including focal necrosis and widespread presence of apoptotic hepatocytes, many of which did not stain for virus by IHC. Up-regulation of cytokine gene expression was found only in the IHC-positive (IHC+ve) fish and reflected the level of infection as determined by IHC positivity of the liver. In most fish, interferon (IFN), Mx, γIFN and γIP were up-regulated in liver and kidney, while only IFN and Mx were up-regulated in gill. IL1β and TNFα were not induced in any tissue. The gill showed variable levels of constitutive expression of IL1β and γIFN. The two fish with liver pathology had the highest level of IFN expression, especially relative to the level of Mx expression, in the liver compared with the other IHC+ve fish which did not have a liver pathology. The results suggest that following widespread infection of hepatocytes, the cells may over-produce IFN, resulting in apoptosis of neighbouring cells with subsequent death from liver failure.

Development of a widely applicable positive control strategy to support detection of infectious salmon anaemia virus (ISAV) using Taqman real-time PCR

Real-time PCR assays are being increasingly applied to the detection of fish pathogens due to their sensitivity, specificity and potential for high throughput sample processing. Such assays allow for the ready and efficient inclusion of appropriate quality controls which are fundamental to scientific integrity and to satisfying the demands of diagnostic test accreditation. In this article, we report development of a universal positive control strategy for real-time PCR assays, which has been used to support and improve a previously published method for detection of infectious salmon anaemia virus (ISAV). The strategy employed uses an RNA mimic template, which is based on the ISAV segment 8 target sequence but includes an artificial universal positive control sequence. Inclusion of this sequence, which is targeted by a second specific probe carrying a different fluorophore to the primary assay, allows for convenient screening of all real-time PCR reactions for the presence of contaminating positive control material. The development of readily distinguishable artificial positive control material offers distinct advantages to real-time PCR assays over using control material derived from clinical material.

The contribution of molecular epidemiology to the understanding and control of viral diseases of salmonid aquaculture.

Molecular epidemiology is a science which utilizes molecular biology to define the distribution of disease in a population (descriptive epidemiology) and relies heavily on integration of traditional (or analytical) epidemiological approaches to identify the etiological determinants of this distribution. The study of viral pathogens of aquaculture has provided many exciting opportunities to apply such tools. This review considers the extent to which molecular epidemiological studies have contributed to better understanding and control of disease in aquaculture, drawing on examples of viral diseases of salmonid fish of commercial significance including viral haemorrhagic septicaemia virus (VHSV), salmonid alphavirus (SAV) and infectious salmon anaemia virus (ISAV). Significant outcomes of molecular epidemiological studies include:Improved taxonomic classification of virusesA better understanding of the natural distribution of virusesAn improved understanding of the origins of viral pathogens in aquacultureAn improved understanding of the risks of translocation of pathogens outwith their natural host rangeAn increased ability to trace the source of new disease outbreaksDevelopment of a basis for ensuring development of appropriate diagnostic toolsAn ability to classify isolates and thus target future research aimed at better understanding biological functionWhile molecular epidemiological studies have no doubt already made a significant contribution in these areas, the advent of new technologies such as pyrosequencing heralds a quantum leap in the ability to generate descriptive molecular sequence data. The ability of molecular epidemiology to fulfil its potential to translate complex disease pathways into relevant fish health policy is thus unlikely to be limited by the generation of descriptive molecular markers. More likely, full realisation of the potential to better explain viral transmission pathways will be dependent on the ability to assimilate and analyse knowledge from a range of more traditional information sources. The development of methods to systematically record and share such epidemiologically important information thus represents a major challenge for fish health professionals in making the best future use of molecular data in supporting fish health policy and disease control.

Application of a sensitive, specific and controlled real‐time PCR assay to surveillance indicates a low prevalence of viral haemorrhagic septicaemia virus (VHSV) in wild herring, Clupea harengus L., in Scottish waters

Surveillance data on the distribution of viral haemorrhagic septicaemia virus (VHSV) in the North Sea (UK), targeting Atlantic herring in areas with previous virus detection, were obtained from research cruises conducted during 2005. The sensitive molecular approach of real-time RT-PCR (qRT-PCR) was applied alongside a newly developed endogenous positive control assay specific for herring (elongation factor 1α) to ensure integrity of template. Three hundred and five pools from 1937 individual herring were tested, and no evidence of VHSV in association with wild Atlantic herring was detected. Samples were obtained from Scottish waters where marine aquaculture is conducted. The results confirm that previous tissue culture studies have most likely not significantly underestimated the prevalence of carrier herring in this area. The significance of migratory species such as herring as a reservoir species for VHSV, with the potential to translocate virus genotypes between geographical areas, is discussed.