Differing Resistance of Atlantic Salmon Strains and Rainbow Trout to Gyrodactylus salaris Infection

Abstract

Laboratory studies were conducted on the susceptibility of different strains of Atlantic salmon Salmo salar and rainbow trout Oncorhynchus mykiss to infection with the monogenean Gyrodactylus salaris. This parasite, probably originating from the Baltic Sea region, is known to minimally affect Neva River (Baltic Sea) Atlantic salmon. However, following its introduction into Norway, G. salaris has caused severe mortality and morbidity among Norwegian Atlantic salmon, which are considered a highly susceptible strain. The cohabitation experiment included one stock of rainbow trout and four different strains of Atlantic salmon from the Baltic Sea region (Mörrum River, Sweden), Europe (Skjern River, Denmark; Conon River, Scotland), and North America (Bristol Cove River, Canada). Fish were exposed to a Norwegian strain of G. salaris, and parasite population development and distribution were monitored for 7 weeks. Rainbow trout exhibited low susceptibility to G. salaris infection, whereas Conon River and Skjern River Atlantic salmon were highly susceptible and exhibited high mortality rates. Mörrum River Atlantic salmon exhibited intermediate susceptibility and low mortality. Bristol Cove River Atlantic salmon harbored relatively low parasite numbers, but fish mortality was high. Our experiment showed that the Danish Skjern River strain of Atlantic salmon is highly susceptible to G. salaris infection, further supporting the hypothesis that Atlantic Ocean strains are more susceptible to G. salaris infection than are Baltic strains.     

The First Isolation in North America of Infectious,Hematopoietic Necrosis Virus (IHNV) and Viral Hemorrhagic Septicemia Virus (VHSV) in Coho Salmon from the Same Watershed

Abstract

In November 1989, infectious hematopoietic necrosis virus (IHNV) was found for the first time in the Soleduck River at the Washington Department of Fisheries Soleduck Hatchery. The virus was isolated from ovarian fluid and kidney-spleen tissue pools from chinook salmon Oncorhynchus tshawytscha and ovarian fluid pools from coho salmon O. kisutch returning to the Soleduck Hatchery. The virus was identified as IHNV by neutralization assays. In December 1989, the virus causing viral hemorrhagic septicemia (VHSV) was found in ovarian fluid and milt pools from wild coho salmon obtained from the Soleduck and Bogachiel rivers and held at the Soleduck Hatchery. The virus was identified as VHSV by neutralization and immunoblot assays. These findings and their implications for routine broodstock sampling are discussed.     

Detection of infectious pancreatic necrosis virus (IPNV) RNA by hybridization with an oligonucleotide DNA probe

Marine farming of Atlantic salmon (Salmo salar) is growing rapidly in Norway, and fish diseases have now become one of the biggest obstacles for this new industry. Infectious pancreatic necrosis virus (IPNV) is commonly found in fish from Norwegian sea farms. Diagnosis of IPNV infection is, at present, mainly based on virus isolation in cell cultures and identification by neutralization tests (NT). A DNA-RNA hybridization assay was developed using a 24 base DNA oligonucleotide probe. This is homologous to a part of the nucleotide sequence of the IPNV genome coding for a protease. RNA extracted from IPNV and harvest from infected cell cultures were fixed to nylon filters and hybridized with the 32P end-labelled probe. The results showed that the probe specifically identified IPNV from these two materials, both for the three different virus strains (Ab, Sp and VR-299) used, and for several different field isolates. It did not hybridize with reoviruses or non-infected cell cultures used as controls. These results indicate that the probe is not serotype specific, and furthermore that RNA extraction is not required before hybridization. This method may be a useful alternative to NT for routine identification of IPNV, particularly when non-radioactive labelling of the probe is introduced.     

The persistence of infectious pancreatic necrosis virus in Atlantic salmon

Atlantic salmon leucocytes from Infectious Pancreatic Necrosis Virus (IPNV) carriers showed a suppressed response to phytohemagglutinin stimulation compared with uninfected controls. A significant degree of inhibition of DNA synthesis was observed using 3H-thymidine incorporation. IPNV was isolated from 41% of the stimulated leucocyte cultures supernatants, while only 6% of the unstimulated cultures were found to be positive.     

Inoculation of infectious pancreatic necrosis virus serotype Sp did not cause pancreas disease in Atlantic salmon (Salmo salar L.).

Atlantic salmon were selected from a fish farm with no previous record of pancreas disease (PD) or infectious pancreatic necrosis virus (IPNV) infection. Groups of fish were inoculated with either IPNV (strain Sp) from cell culture, organ material from fish with PD or control material as phosphate-buffered saline (PBS). Virological, histological and immunohistochemical examinations were carried out throughout the experiment. None of the fish died or showed clinical symptoms of PD. Histological examination revealed no pathological changes, and immunohistochemical studies were negative. Virus was isolated only sporadically from the group inoculated with organ material, whereas it was isolated consistently from the group inoculated with virus propagated in cell culture, as well as from in-contact control fish after the first week. In a latent carrier test, changes were entirely lacking in the first mentioned group, and were only slight in the last mentioned group. The data suggest that PD is not a transmissible disease, and that IPNV isolated from a PD outbreak does not play any part in the etiology of this disease.     

Quantitation of immune response gene expression and cellular localisation of interleukin-1beta mRNA in Atlantic salmon, Salmo salar L., affected by amoebic gill disease (AGD)

The characterisation of selected immune response genes during amoebic gill disease (AGD) in Atlantic salmon, Salmo salar L., was performed using semi-quantitative RT-PCR, quantitative real-time RT-PCR (qRT-PCR), and in situ hybridisation (ISH). The immune response genes of interest were interleukin-1beta (IL-1beta), inducible nitric oxide synthase (iNOS), serum amyloid A (SAA), and serum amyloid P-like pentraxin (SAP). Atlantic salmon were inoculated with the ectoparasite Neoparamoeba sp., the causative agent of AGD, and gill, liver and anterior kidney tissue sampled at 0, 7 and 14 d post-inoculation (p.i.). Semi-quantitative RT-PCR was performed on the tissue samples to identify up/down-regulated mRNA expression relative to uninfected control fish and normalised to the housekeeping gene, beta-actin. Interleukin-1beta (IL-1beta) was the only immune response gene of those investigated whose mRNA was differentially regulated in any of the tissues and was found to be up-regulated in the gills by semi-quantitative RT-PCR. Increased gill IL-1beta mRNA expression was then accurately quantitated and confirmed using probe-based qRT-PCR. The cellular localisation of the IL-1beta mRNA expression in the gills of uninfected and infected fish was then determined by ISH using an IL-1beta-specific biotinylated cRNA probe. Expression of IL-1beta mRNA was localised to filament and lamellar epithelium pavement cells in gills of uninfected and infected Atlantic salmon. These data implicate the involvement of IL-1beta at the site of infection, the gills, of Atlantic salmon during AGD. This work supports previous studies that suggest IL-1beta is important in the regulation of the fish immune response to parasitic infection but additionally shows the cellular localisation of fish IL-1beta mRNA expression during infection.     

Infectious Hematopoietic Necrosis Virus (IHNV) in Coho Salmon

Abstract

Infectious hematopoietic necrosis virus (IHNV) causes important losses of chinook salmon Oncorhynchus tshawytscha, sockeye salmon Oncorhynchus nerka, and rainbow trout and steelhead Oncorhynchus mykiss on the west coast of North America. Although coho salmon Oncorhynchus kisutch are considered resistant to IHNV infection, the virus was detected in numerous adult coho salmon returning to Trinity River Hatchery, California, in 1985 and 1986. The virus was isolated from internal organs and ovarian fluids of these fish. Antigenic and structural polypeptides of the viruses were identical in adult coho and chinook salmon collected at the same location. Chinook salmon and rainbow trout alevins exhibited high degrees of susceptibility to IHNV obtained from adult coho and chinook salmon. Coho salmon alevins were resistant to both virus isolants.     

Antigen dose and humoral immune response correspond with protection for inactivated infectious pancreatic necrosis virus vaccines in Atlantic salmon (Salmo salar L)

An enduring challenge in the vaccinology of infectious pancreatic necrosis virus (IPNV) is the lack of correlation between neutralizing antibodies and protection against mortality. To better understand the immunological basis of vaccine protection, an efficacy trial including Atlantic salmon (Salmo salar L.) vaccinated with a high antigen (HiAg) or low antigen (LoAg) dose vaccine was carried out in a cohabitation challenge model using the highly virulent Norwegian Sp strain NVI015. To pinpoint the immunological basis of vaccine protection, pathogenic mechanisms of IPNV were unraveled in control fish while obtaining feedback on mechanisms of protection in the vaccinated fish. During the incubation period, infection rates were highest in control fish, followed by the LoAg group with the lowest infections being in the HiAg group. Although both the liver and pancreas are target organs prone to tissue damage, infection in the liver was delayed until acute infection in most fish. A correlate of pathology determined as the cutoff threshold of viral copy numbers linked to tissue damage in target organs was estimated at ≥ 10^7.0, which corresponded with an increase in mortality. The kinetics of IFNα and Mx expression suggests that these genes can be used as biomarkers of IPNV infection progression. Mechanisms of vaccine protection involved reducing infection rates, preventing infection of the liver and reducing virus replication in target organs to levels below the correlate of pathology. Overall, the study shows that antigen dose corresponds with vaccine efficacy and that antibody levels can be used as a signature of protective immunity against pathological disease and mortality.     

Gene expression studies of host response to Salmonid alphavirus subtype 3 experimental infections in Atlantic salmon

Salmonid alphavirus subtype-3 (SAV-3) infection in Atlantic salmon is exclusively found in Norway. The salmonid alphaviruses have been well characterized at the genome level but there is limited information about the host-pathogen interaction phenomena. This study was undertaken to characterize the replication and spread of SAV-3 in internal organs of experimentally infected Atlantic salmon and the subsequent innate and adaptive immune responses. In addition, suitability of a cohabitation challenge model for this virus was also examined. Groups of fish were infected by intramuscular injection (IM), cohabited (CO) or kept uninfected in a separate tank. Samples of pancreas, kidney, spleen, heart and skeletal muscles were collected at 2, 4 and 8 weeks post infection (wpi). Pathological changes were assessed by histology concurrently with viral loads and mRNA expression of immune genes by real time RT-PCR. Pathological changes were only observed in the pancreas and heart (target organs) of both IM and CO groups, with changes appearing first in the pancreas (2 wpi) in the former. Lesions with increasing severity over time coincided with high viral loads despite significant induction of IFN-α, Mx and ISG15. IFN-γ and MHC-I were expressed in all tissues examined and their induction appeared in parallel with that of IL-10. Inflammatory genes TNF-α, IL-12 and IL-8 were only induced in the heart during pathology while T cell-related genes CD3ε, CD4, CD8, TCR-α and MHC-II were expressed in target organs at 8 wpi. These findings suggest that the onset of innate responses came too late to limit virus replication. Furthermore, SAV-3 infections in Atlantic salmon induce Th1/cytotoxic responses in common with other alphaviruses infecting higher vertebrates. Our findings demonstrate that SAV-3 can be transmitted via the water making it suitable for a cohabitation challenge model.     

Primary Isolation and Characterization of Tenacibaculum maritimum from Chilean Atlantic Salmon Mortalities Associated with a Pseudochattonella spp. Algal Bloom

Abstract

This study presents the first isolation of Tenacibaculum maritimum from farmed Atlantic Salmon Salmo salar in Chile. The isolate, designated T. maritimum Ch-2402, was isolated from gills of Atlantic Salmon at a farm located in region X, Los Lagos, Chile, during the harmful algal bloom caused by Pseudochattonella spp. in February 2016. The algal bloom is reported to have caused 40,000 metric tons of mortality in this salmon farming area. The bacterium T. maritimum, which causes tenacibaculosis, is recognized as an important pathogen of marine fish worldwide. Genetic, phylogenetic, and phenotypic characterizations were used to describe the T. maritimum Ch-2402 isolate. The isolate was similar to the type strain of T. maritimum but was genetically unique. Tenacibaculum dicentrarchi isolates were also recovered during sampling from the same farm. Based on the fact that T. maritimum has been shown to cause disease in Atlantic Salmon in other regions, the presence of this bacterium poses a potential risk of disease to fish in the Chilean aquaculture industry.

Received November 6, 2016; accepted May 29, 2017 Published online July 26, 2017     

Persistence of infectious pancreatic necrosis virus (IPNV) in Scottish salmon (Salmo salar L.) farms

Infectious pancreatic necrosis is a disease that is causing increasing loses to Scottish Atlantic-salmon farms. I asked the question: is infection of individual salmon farms persistent or transient? Using Fisheries Research Services' Fish Health Inspectors' data, conditional probabilities that a farm would (time 0) be infected were estimated for farms that had been infected [P(I(0)|I(-T))] or free [P(I(0)|F(-T))] from infectious pancreatic necrosis virus (IPNV) when a sample was taken at some earlier time (-T). A logistic-regression model was used to estimate these conditional probabilities with T; this model was multilevel to account for regional and inter-annual level differences in prevalence of IPNV. In freshwater, conditional probabilities remained substantially different for periods of at least 4 years, so, although many farms did change infection status, IPNV either persisted or recurred at specific freshwater farms. Marine farms showed similar conditional probabilities after about 2 years following a positive or a negative sample, indicating that infection was transient. Management of larger areas and exchanges between farms might be more effective than farm-level management at controlling marine IPNV.     

Prevalence of Infectious Pancreatic Necrosis Virus on Salmonid Fish Farms in Spain

Abstract

In Spain, salmonid fish farming was commercially developed in the 1960s, and now there are 140 private farms that depend heavily on imported embryonate eggs. Infectious pancreatic necrosis was first clinically diagnosed in Spain in 1970, but the virus (IPNV) was not isolated and identified until 1980. Since that time, researchers have isolated IPNV from other samples in Spain. A diagnostic survey was conducted to determine how prevalent IPNV is on fish farms in Spain and whether the virus has been responsible for some of the major financial losses occurring every year on these farms. In total, 236 samplings of rainbow trout Oncorhynchus mykiss from 31 farms in eight hydrographic areas were done over a 3-year period. Infectious pancreatic necrosis virus was isolated in 94 cases, and serotyping of the viral strains revealed that 81% of these isolates were strain Sp and 19% were strain Ab. Neither IPNV strain VR-299 nor rhabdovirus (as infectious hematopoietic necrosis virus or viral hemorrhagic septicemia virus) was detected in any of the samples.     

Ichthyophoniasis: An Emerging Disease of Chinook Salmon in the Yukon River

Abstract

Before 1985, Ichthyophonus was unreported among Pacific salmon Oncorhynchus spp. from the Yukon River; now it infects more than 40% of returning adult Chinook salmon O. tshawytscha. Overall infection prevalence reached about 45% in the Yukon River and about 30% in the Tanana River between 1999 and 2003. Mean infection prevalence was greater in females than males in the main-stem Yukon River during each of the 5 years of the study, but the infection prevalence in males increased each year until the difference was no longer significant. Clinical signs of ichthyophoniasis (presence of visible punctate white lesions in internal organs) were least at the mouth of the Yukon River (～10%) but increased to 29% when fish reached the middle Yukon River and was 22% at the upper Tanana River. However, clinical signs increased each year from 7% in 1999 to 27% in 2003 at the mouth of the river. As fish approached the upper reaches of the Yukon River (Canada) and the spawning areas of the Chena and Salcha rivers (Alaska), infection prevalence dropped significantly to less than 15% in females on the Yukon River and less than 10% for both sexes in the Chena and Salcha rivers, presumably because of mortality among infected prespawn fish. Age was not a factor in infection prevalence, nor was the position of fish within the run. The source of infection was not determined, but Ichthyophonus was not found in 400 Pacific herring Clupea pallasi from the Bering Sea or in 120 outmigrating juvenile Chinook salmon from two drainages in Alaska and Canada. Freshwater burbot Lota lota from the middle Yukon River were subclinically infected with Ichthyophonus, but the origin and relationship of this agent to the Chinook salmon isolate is unknown.     

Molecular characterization of the VP1 gene of a Mexican isolate of infectious pancreatic necrosis virus

The presence of infectious pancreatic necrosis virus (IPNV) in salmonids predominantly produces a high mortality rate in first-feeding fry. Genomic analysis of the vp2 gene sequence is most commonly used to determine the genetic diversity of IPNV isolates. Recently, information obtained from the vp1 gene allowed for efficient analysis of the genetic diversity of IPNV. In this study, the vp1 gene from a Mexican IPNV isolate was characterized and compared with IPNV isolates from Europe, North America, and Asia. The results indicate that the Mexican isolate is most closely related genetically to the 2310 strain from Spain.     

Assessment of the Risk of White Sturgeon to become Infected and Potential Carriers of Infectious Pancreatic Necrosis Virus

Abstract

Little scientific information is available to assess whether White Sturgeon Acipenser transmontanus can become infected and potential carriers of infectious pancreatic necrosis virus (IPNV). To assess this risk, monitoring results of adult and progeny White Sturgeon were examined from waters historically stocked with salmonid fish known to be IPNV carriers. From 1999 through 2004 White Sturgeon from a total of 30 separate families whose parentage came from waters historically stocked with IPNV carrier fish were tested. Duplicate groups of 25 juvenile Snake River White Sturgeon were waterborne exposed to 1.0×10⁴ 50% tissue culture infective dose (TCID50)/mL of water for 1 h and an additional group was injected intraperitoneally with 1.0×10⁵ TCID50/fish. A negative control group was handled similarly but was not exposed to the virus. No morbidity was detected in any of the treatment groups or the negative control. At 34, 40, 47, and 54 d postexposure to IPNV, virus reisolation was attempted on five fish from each group, and an additional five fish from each group were examined for histological changes consistent with an IPNV infection. At 34 and 40 d postinjection with IPNV, 20% (one of five) of the fish tested positive for the virus per sample interval; however, fish from groups that were waterborne-exposed to IPNV were all negative. At 47 and 54 d after exposure or injection with IPNV an additional five fish from each group were tested at each sample interval and all results were negative. Histological analysis of target tissue obtained from five fish per group at 34 and 54 d postinfection also failed to detect any consistent change associated with an IPNV infection. These results suggest that the risk of White Sturgeon to become infected and develop into potential carriers of IPNV is negligible.

Received May 21, 2013; accepted July 8, 2013     

The role of fish movements and the spread of infectious salmon anemia virus (ISAV) in Chile, 2007–2009

Infectious salmon anemia virus (ISAV) infection is a constant major threat to farmed and wild Atlantic salmon worldwide. Many epidemics have recently been reported in the most important salmon farming regions of the world, including Chile (2007–2009), where ISAV generated the most important disease and economic crisis in history of the salmon industry of the country. The spread of ISAV within a region is most likely by local or neighborhood spread from an infected farm; however, there is evidence that anthropogenic activities, such as movement of live or harvested fish or their byproduct, may have played a more important role than environmental or passive transmission in the 2007–2009 outbreak. Atlantic salmon farms (n = 421) were retrospectively followed from stocking to harvesting in southern Chile at the time of the ISAV epidemic (2007–2009). The effect of husbandry and spatial risk factors, in addition to contact-network risk factors, which were obtained from the social network analyses, on time to first ISAV infection was estimated using a multivariable Cox proportional hazards model. Five variables were retained in the final fitted model: co-existing multiple generations on a farm (hazard ratio [HR] = 2.585), mean smolt weight at stocking greater than 120 g (HR = 1.165), farm area (per km²) (HR = 1.005), and increased number of shipments entering a farm, i.e. the farm input degree (HR = 1.876) were associated with reduced time to infection; whereas time-to-infection was longer for farms located farther from an ongoing ISAV outbreak (HR = 0.943). It was demonstrated that movements of latently infected fish resulted in approximately 7 outbreaks, and potentially explain about 6% of the total number of cases during the epidemic. Results from this study provide new information about the mechanisms of spread of ISAV in one the largest documented ISAV epidemics in the world. Findings may be used to support the design and implementation of risk-based surveillance and control programs that may help to prevent, detect and control future ISAV outbreaks.     

Survey of pathogens in hatchery Chinook salmon with different out-migration histories through the Snake and Columbia rivers

The operation of the Federal Columbia River Power System (FCRPS) has negatively affected threatened and endangered salmonid populations in the Pacific Northwest. Barging Snake River spring Chinook salmon Oncorhynchus tshawytscha through the FCRPS is one effort to mitigate the effect of the hydrosystem on juvenile salmon out-migration. However, little is known about the occurrence and transmission of infectious agents in barged juvenile salmon relative to juvenile salmon that remain in-river to navigate to the ocean. We conducted a survey of hatchery-reared spring Chinook salmon at various points along their out-migration path as they left their natal hatcheries and either migrated in-river or were barged through the FCRPS. Salmon kidneys were screened by polymerase chain reaction for nine pathogens and one family of water molds. Eight pathogens were detected; the most prevalent were Renibacterium salmoninarum and infectious hematopoietic necrosis virus. Species in the family Saprolegniaceae were also commonly detected. Pathogen prevalence was significantly greater in fish that were barged through the FCRPS than in fish left to out-migrate in-river. These results suggest that the transmission of infectious agents to susceptible juvenile salmon occurs during the barging process. Therefore, management activities that reduce pathogen exposure during barging may increase the survival of juvenile Chinook salmon after they are released.