David B. Groman

First detection, isolation and molecular characterization of infectious salmon anaemia virus associated with clinical disease in farmed Atlantic salmon (Salmo salar) in Chile

BackgroundInfectious salmon anaemia (ISA) is a viral disease of marine-farmed Atlantic salmon (Salmo salar) caused by ISA virus (ISAV), which belongs to the genus Isavirus, family Orthomyxoviridae. The virus is considered to be carried by marine wild fish and for over 25 years has caused major disease outbreaks in marine-farmed Atlantic salmon in the Northern hemisphere. In the Southern hemisphere, ISAV was first detected in Chile in 1999 in marine-farmed Coho salmon (Oncorhynchus kisutch). In contrast to the classical presentation of ISA in Atlantic salmon, the presence of ISAV in Chile until now has only been associated with a clinical condition called Icterus Syndrome in Coho salmon and virus isolation has not always been possible. During the winter of 2007, unexplained mortalities were registered in market-size Atlantic salmon in a grow-out site located in Chiloé in Region X of Chile. We report here the diagnostic findings of the first significant clinical outbreak of ISA in marine-farmed Atlantic salmon in Chile and the first characterization of the ISAV isolated from the affected fish.ResultsIn mid-June 2007, an Atlantic salmon marine farm site located in central Chiloé Island in Region X of Chile registered a sudden increase in mortality following recovery from an outbreak of Pisciricketsiosis, which rose to a cumulative mortality of 13.6% by harvest time. Based on the clinical signs and lesions in the affected fish, and laboratory tests performed on the fish tissues, a confirmatory diagnosis of ISA was made; the first time ISA in its classical presentation and for the first time affecting farmed Atlantic salmon in Chile. Rapid sequencing of the virus-specific RT-PCR products amplified from the fish tissues identified the virus to belong to the European genotype (Genotype I) of the highly polymorphic region (HPR) group HPR 7b, but with an 11-amino acid insert in the fusion glycoprotein, and ability to cause cytopathic effects (CPE) in CHSE-214 cell line, characteristics which make it distinct from common European Genotype ISAV isolates from Europe and North America.ConclusionIn conclusion, the present work constitutes the first report of a case of ISA in farmed Atlantic salmon in Chile. The clinical signs and lesions are consistent with the classical descriptions of the disease in marine-farmed Atlantic salmon in the Northern hemisphere. The outbreak was caused by ISAV of European genotype (or Genotype I) of HPR 7b but distinct from common European Genotype ISAV isolates.

Identification and Characterization of a Piscine Neuropathy and Nodavirus from Juvenile Atlantic Cod from the Atlantic Coast of North America

Abstract 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...

Nonlesions, misdiagnoses, missed diagnoses, and other interpretive challenges in fish histopathology studies: a guide for investigators, authors, reviewers, and readers.

Differentiating salient histopathologic changes from normal anatomic features or tissue artifacts can be decidedly challenging, especially for the novice fish pathologist. As a consequence, findings of questionable accuracy may be reported inadvertently, and the potential negative impacts of publishing inaccurate histopathologic interpretations are not always fully appreciated. The objectives of this article are to illustrate a number of specific morphologic findings in commonly examined fish tissues (e.g., gills, liver, kidney, and gonads) that are frequently either misdiagnosed or underdiagnosed, and to address related issues involving the interpretation of histopathologic data. To enhance the utility of this article as a guide, photomicrographs of normal and abnormal specimens are presented. General recommendations for generating and publishing results from histopathology studies are additionally provided. It is hoped that the furnished information will be a useful resource for manuscript generation, by helping authors, reviewers, and readers to critically assess fish histopathologic data.

Infectious salmon anaemia virus replication and induction of alpha interferon in Atlantic salmon erythrocytes

BackgroundInfectious salmon anaemia (ISA) virus (ISAV), which causes ISA in marine-farmed Atlantic salmon, is an orthomyxovirus belonging to the genus Isavirus, family Orthomyxoviridae. ISAV agglutinates erythrocytes of several fish species and it is generally accepted that the ISAV receptor destroying enzyme dissolves this haemagglutination except for Atlantic salmon erythrocytes. Recent work indicates that ISAV isolates that are able to elute from Atlantic salmon erythrocytes cause low mortality in challenge experiments using Atlantic salmon. Previous work on ISAV-induced haemagglutination using the highly pathogenic ISAV strain NBISA01 and the low pathogenic ISAV strain RPC/NB-04-0851, showed endocytosis of NBISA01 but not RPC/NB-04-0851. Real-time RT-PCR was used to assess the viral RNA levels in the ISAV-induced haemagglutination reaction samples, and we observed a slight increase in viral RNA transcripts by 36 hours in the haemagglutination reaction with NBISA01 virus when the experiment was terminated. However, a longer sampling interval was considered necessary to confirm ISAV replication in fish erythrocytes and to determine if the infected cells mounted any innate immune response. This study examined the possible ISAV replication and Type I interferon (IFN) system gene induction in Atlantic salmon erythrocytes following ISAV haemagglutination.ResultsHaemagglutination assays were performed using Atlantic salmon erythrocytes and one haemagglutination unit of the two ISAV strains, NBISA01 and RPC/NB-04-0851, of differing genotypes and pathogenicities. Haemagglutination induced by the highly pathogenic NBISA01 but not the low pathogenic RPC/NB-04-0851 resulted in productive infection as evidenced by increased ISAV segment 8 transcripts and increase in the median tissue culture infectious dose (TCID50) by 5 days of incubation. Moreover, reverse transcription (RT) quantitative PCR used to compare mRNA levels of key Type I IFN system genes in erythrocyte lysates of haemagglutination reactions with the two ISAV strains showed a higher relative fold increase of IFN-α in NBISA01 haemagglutinations compared to RPC/NB-04-085-1 haemagglutinations (33.0 – 44.26 relative fold increase compared to 11.29). Erythrocytes exposed to heat-inactivated virus or to polyinosinic:polycytidylic acid (polyI:C) or to L-15 medium alone (negative control assays) had minimal late induction (<3.5 relative fold increase) of STAT1 and/or ISG15 and Mx genes, whereas erythrocytes exposed to UV-inactivated virus lacked any cytokine induction.ConclusionISAV-induced haemagglutination by a highly pathogenic virus strain results in virus uptake and productive infection of Atlantic salmon erythrocytes accompanied by significant induction of IFN-α. This study also highlights the critical role of ISAV strain variation in the initial stages of the virus-cell interaction during haemagglutination, and possibly in the pathogenesis of ISA. Moreover, the study shows for the first time that fish erythrocytes immunologically respond to ISAV infection.

Cohabitation Transmission of Infectious Salmon Anemia Virus among Freshwater-Reared Atlantic Salmon

Abstract The present study examined factors affecting horizontal transmission of infectious salmon anemia virus (ISAV) among naive Atlantic salmon Salmo salar maintained in freshwater. The ratio of injected to naive cohabitants was varied and mortality was monitored. Whether ISAV transmission in freshwater required contact among salmon was also examined. Pathognomonic histological lesions in fish infected by injection or cohabitation were compared. In trial 1, duplicate tanks containing 0 + 25, 2 + 23, 4 + 21, 6 + 19, or 8 + 17 fish (injected + naive, respectively) were established. Mean days to onset of mortality and mean days to death (mdd) were significantly less among injected fish (16.9 and 18.8 d, respectively) than in fish infected by cohabitation (33.4 and 43.8 d). Cumulative mortality in naive cohabitants in trial 1 was significantly greater in groups containing six injected fish compared with those containing either two or eight injected fish. In trial 2, triplicate tanks of 0 + 25, 4 + 21, and ...

Virulence and Pathogenicity of Infectious Salmon Anemia Virus Isolated from Farmed Salmon in Atlantic Canada

Abstract Infectious salmon anemia virus (ISAV) was isolated from farmed Atlantic salmon Salmo salar associated with an outbreak of hemorrhagic kidney syndrome in the Bay of Fundy, Canada. The virus induced cytopathic effects in salmon head kidney cell line SHK-1 from Atlantic salmon and was positively confirmed as ISAV by an indirect fluorescent antibody test and by reverse transcriptase polymerase chain reaction. Atlantic salmon parr injected with ISAV from the SHK-1 line experienced significant reductions in hematocrits as early as 5 d postinfection (DPI). Mortality began 17 DPI and reached 76% by 24 DPI at a water temperature of 11°C. In a second trial, similarly high mortality occurred in salmon parr injected with 10-fold dilutions of supernatant from ISAV-infected SHK-1 cultures. The ISAV was reisolated from eight randomly selected salmon that died after experimental infection. Microscopic pathological changes among infected fish included congestion and necrosis, seen in the livers from 7 of 19 sampl...

Case Report: Viral Infectious Pancreatic Necrosis in Farmed Rainbow Trout from Mexico.

This case report provides pathologic and confirmatory diagnostic documentation of the first reported clinical epizootic of infectious pancreatic necrosis (IPN) in farmed rainbow trout Oncorhynchus mykiss from central Mexico. Both the gross and microscopic pathology were consistent with IPN. A virus was isolated in cell culture with the cytopathic effect typical of the IPN virus (IPNV). Positive identification as IPNV was achieved by means of an IPNV-specific indirect fluorescent antibody test and reverse transcription-polymerase chain reaction. Further genotyping identified this isolate as the Buhl strain of IPNV, which is a member of the West Buxton (A1) serotype of aquatic birnavirus serogroup A.

Massive Mortality of Common Carp (Cyprinus carpio carpio) in the St. Lawrence River in 2001: Diagnostic Investigation and Experimental Induction of Lymphocytic Encephalitis

A massive fish kill affecting exclusively common carp (Cyprinus carpio carpio) in the St. Lawrence River, Québec, Canada, during the summer of 2001 was investigated by use of laboratory diagnostic methods and by an attempt to experimentally induce the disease. The ultimate causes of mortality were opportunistic bacterial infections with Aeromonas hydrophila and Flavobacterium sp. secondary to immunosuppression induced by physiologic (i.e., spawning) and environmental (i.e., high temperatures and low water levels) stressors, and possibly enhanced by an infection causing lymphocytic encephalitis observed in 9 of 18 (50%) fish examined. Experimental induction of disease was attempted in captured wild carp by administration of crude and filtered (particulate <0.22 μm) inocula prepared from a homogenate of tissues from carp affected by the natural outbreak. Although significant clinical disease or mortality was not induced by experimental challenge, lymphocytic encephalitis similar to the one observed in naturally affected carp was induced in four of seven (57%) fish administered crude inoculum and four of seven (57%) fish administered filtered inoculum. None of the control fish inoculated with sterile phosphate-buffered saline (n = 6) were affected by encephalitis. The cause of the encephalitis observed in carp from the natural outbreak and in experimentally inoculated fish could not be determined by use of virus isolation and transmission electron microscopy.

Correlation of virus replication in tissues with histologic lesions in Atlantic salmon experimentally infected with infectious salmon anemia virus.

We have studied the replication of virus in tissues and development of lesions associated with infectious salmon anemia virus (ISAV) infection in Atlantic salmon using in situ hybridization (ISH) with a riboprobe targeting ISAV RNA segment 7 messenger RNA. Fish were infected with three ISAV isolates (U55751, RPC-01-0593-1, Norway 810/9/99) and then euthanatized sequentially at 3, 6, 10, and 13 days postinoculation (dpi) and thereafter once a week for 8 weeks. Severe histopathologic lesions were observed in tissues from all groups beginning at the onset of mortality. The severe histopathologic lesions correlated with maximum intensity and frequency of ISH signals (P < 0.001). There was a strong association between the hybridization signals and severity of lesions in the liver, kidney, and heart (R = 0.81, 0.70, and 0.78, respectively; P < 0.001). The distribution of ISH signals indicated the presence of a viremia because signals were observed predominantly in individual blood cells and endothelial cells, and possibly hematopoietic cells of head kidney, but not in the necrotic hepatocytes and renal epithelium. Of the organs sampled, the heart was the first and last to show ISH signals, possibly because of increased activity of the endocardial endothelial cells and the underlining macrophages, which continuously trap and remove circulating virus, and therefore represents the best tissue sample for screening of suspected infected fish. On the basis of mortality, severity of lesions, and intensity and frequency of ISH signals, ISAV isolate Norway 810/9/99 was the most virulent and U5575-1 the least virulent isolate studied.

Case Report: Outbreak of Bumper Car Disease Caused by Anophryoides haemophila in a Lobster Holding Facility in Nova Scotia, Canada

Abstract This case report provides pathologic and confirmatory molecular characterization of an outbreak of bumper car disease caused by the scuticociliate Anophryoides haemophila in American lobster Homarus americanus, from a commercial holding facility in Nova Scotia, Canada. Although sporadically and anecdotally reported to be present in Atlantic Canada, this is the first report detailing observations from a natural outbreak of bumper car disease by gross and microscopic pathology and its positive identification by small subunit rRNA gene sequencing. Randomly amplified polymorphic DNA analysis confirmed that the genotype of the present outbreak isolate is identical to the original isolate of A. haemophila reported from Maine lobsters in 1993.