Detection of Tilapia Lake Virus in Clinical Samples by Culturing and Nested Reverse Transcription-PCR.

Tilapia are an important group of farmed fish that serve as a significant protein source worldwide. In recent years, substantial mortality of wild tilapia has been observed in the Sea of Galilee and in commercial ponds in Israel and Ecuador. We have identified the etiological agent of these mass die-offs as a novel orthomyxo-like virus and named it tilapia lake virus (TiLV). Here, we provide the conditions for efficient isolation, culturing, and quantification of the virus, including the use of susceptible fish cell lines. Moreover, we describe a sensitive nested reverse transcription-PCR (RT-PCR) assay allowing the rapid detection of TiLV in fish organs. This assay revealed, for the first time to our knowledge, the presence of TiLV in diseased Colombian tilapia, indicating a wider distribution of this emerging pathogen and stressing the risk that TiLV poses for the global tilapia industry. Overall, the described procedures should provide the tilapia aquaculture industry with important tools for the detection and containment of this pathogen.

Pathogenesis of experimental salmonid alphavirus infection in vivo: an ultrastructural insight.

Salmonid alphavirus (SAV) is an enveloped, single-stranded, positive sense RNA virus belonging to the family Togaviridae. It causes economically devastating disease in cultured salmonids. The characteristic features of SAV infection include severe histopathological changes in the heart, pancreas and skeletal muscles of diseased fish. Although the presence of virus has been reported in a wider range of tissues, the mechanisms responsible for viral tissue tropism and for lesion development during the disease are not clearly described or understood. Previously, we have described membrane-dependent morphogenesis of SAV and associated apoptosis-mediated cell death in vitro. The aims of the present study were to explore ultrastructural changes associated with SAV infection in vivo. Cytolytic changes were observed in heart, but not in gill and head-kidney of virus-infected fish, although they still exhibited signs of SAV morphogenesis. Ultrastructural changes associated with virus replication were also noted in leukocytes in the head kidney of virus-infected fish. These results further describe the presence of degenerative lesions in the heart as expected, but not in the gills and in the kidney.

Infectious agents and their physiological correlates in early marine Chinook salmon (Oncorhynchus tshawytscha).

The early marine life of Pacific salmon is believed to be a critical period limiting population-level survival. Recent evidence suggests that some infectious agents are associated with survival but linkages with underlying physiological mechanisms are lacking. While challenge studies can demonstrate cause and effect relationships between infection and pathological change or mortality, in some cases pathological change may only manifest in the presence of environmental stressors; thus, it is important to gain context from field observations. Herein, we examined physiological correlates with infectious agent loads in Chinook salmon during their first ocean year. We measured physiology at the molecular (gene expression), metabolic (plasma chemistry) and cellular (histopathology) levels. Of 46 assayed infectious agents, 27 were detected, including viruses, bacteria and parasites. This exploratory study identified.a strong molecular response to viral disease and pathological change consistent with jaundice/anemia associated with Piscine orthoreovirus,strong molecular signals of gill inflammation and immune response associated with gill agents `Candidatus Branchiomonas cysticola' and Parvicapsula pseudobranchicola,a general downregulation of gill immune response associated with Parvicapsula minibicornis complementary to that of P. pseudobranchicola.Importantly, our study provides the first evidence that the molecular activation of viral disease response and the lesions observed during the development of the PRV-related disease jaundice/anemia in farmed Chinook salmon are also observed in wild juvenile Chinook salmon.

Draft genome sequence of a nonhemolytic fish-pathogenic Streptococcus agalactiae strain.

Streptococcus agalactiae is a significant Gram-positive bacterial pathogen of terrestrial and aquatic animals. A subpopulation of nonhemolytic strains which appear to be pathogenic only for poikilotherms exists. We report here the first draft genome sequence of a nonhemolytic S. agalactiae isolate recovered from a diseased fish.

Discovery and surveillance of viruses from salmon in British Columbia using viral immune-response biomarkers, metatranscriptomics, and high-throughput RT-PCR.

The emergence of infectious agents poses a continual economic and environmental challenge to aquaculture production, yet the diversity, abundance, and epidemiology of aquatic viruses are poorly characterised. In this study, we applied salmon host transcriptional biomarkers to identify and select fish in a viral disease state, but only those that were negative for known viruses based on RT-PCR screening. These fish were selected for metatranscriptomic sequencing to discover potential viral pathogens of dead and dying farmed Atlantic (Salmo salar) and Chinook (Oncorhynchus tshawytscha) salmon in British Columbia (BC). We found that the application of the biomarker panel increased the probability of discovering viruses in aquaculture populations. We discovered two viruses that have not previously been characterised in Atlantic salmon farms in BC (Atlantic salmon calicivirus and Cutthroat trout virus-2), as well as partially sequenced three putative novel viruses. To determine the epidemiology of the newly discovered or emerging viruses, we conducted high-throughput reverse transcription polymerase chain reaction (RT-PCR) and screened over 9,000 farmed and wild salmon sampled over one decade. Atlantic salmon calicivirus and Cutthroat trout virus-2 were in more than half of the farmed Atlantic salmon we tested. Importantly we detected some of the viruses we first discovered in farmed Atlantic salmon in Chinook salmon, suggesting a broad host range. Finally, we applied in situ hybridisation to determine infection and found differing cell tropism for each virus tested. Our study demonstrates that continual discovery and surveillance of emerging viruses in these ecologically important salmon will be vital for management of both aquaculture and wild resources in the future.

Jellyfish as vectors of bacterial disease for farmed salmon (Salmo salar).

Swarms or blooms of jellyfish are increasingly problematic and can result in high mortality rates of farmed fish. Small species of jellyfish, such as Phialella quadrata (13 mm in diameter), are capable of passing through the mesh of sea cages and being sucked into the mouth of fish during respiration. Results of the current study show that the initial damage to gills of farmed Atlantic salmon, likely produced by nematocyst-derived toxins from the jellyfish, was compounded by secondary bacterial infection with Tenacibaculum maritimum. Results also demonstrate that these filamentous bacteria were present on the mouth of the jellyfish and that their DNA sequences were almost identical to those of bacteria present on the salmon gills. This suggests that the bacterial lesions were not the result of an opportunistic infection of damaged tissue, as previously thought. Instead, P. quadrata is probably acting as a vector for this particular bacterial pathogen, and it is the first time that evidence to support such a link has been presented. No prior literature describing the presence of bacteria associated with jellyfish, except studies about their decay, could be found. It is not known if all jellyfish of this and other species carry similar bacteria or the relationship to each other. Their source, the role they play under other circumstances, and indeed whether the jellyfish were themselves diseased are also not known. The high proteolytic capabilities of T. maritimum mean that partially digested gill tissues were readily available to the jellyfish, which rely heavily on intracellular digestion for their nutrition.

Genome Sequence of Tilapia Lake Virus Associated with Syncytial Hepatitis of Tilapia in an Ecuadorian Aquaculture Facility.

Here, we report the complete coding sequences of tilapia lake virus (TiLV) associated with syncytial hepatitis of tilapia (SHT). The TiLV strain was sequenced from the liver RNA extract of a moribund Nile tilapia (Oreochromis niloticus) fingerling from an Ecuadorian aquaculture facility in 2012.

Endangered wild salmon infected by newly discovered viruses.

The collapse of iconic, keystone populations of sockeye (Oncorhynchus nerka) and Chinook (Oncorhynchus tshawytscha) salmon in the Northeast Pacific is of great concern. It is thought that infectious disease may contribute to declines, but little is known about viruses endemic to Pacific salmon. Metatranscriptomic sequencing and surveillance of dead and moribund cultured Chinook salmon revealed a novel arenavirus, reovirus and nidovirus. Sequencing revealed two different arenavirus variants which each infect wild Chinook and sockeye salmon. In situ hybridisation localised arenavirus mostly to blood cells. Population surveys of >6000 wild juvenile Chinook and sockeye salmon showed divergent distributions of viruses, implying different epidemiological processes. The discovery in dead and dying farmed salmon of previously unrecognised viruses that are also widely distributed in wild salmon, emphasizes the potential role that viral disease may play in the population dynamics of wild fish stocks, and the threat that these viruses may pose to aquaculture.

STREPTOCOCCUS PHOCAE IN MARINE MAMMALS OF NORTHEASTERN PACIFIC AND ARCTIC CANADA: A RETROSPECTIVE ANALYSIS OF 85 POSTMORTEM INVESTIGATIONS.

: Streptococcus phocae is a pathogen of marine mammals, although its pathogenicity remains poorly understood. Recovery of this bacterium from asymptomatic carriers suggests that it is an opportunistic pathogen. We investigated the role of S. phocae in naturally occurring disease and its significance as a pathogen based on postmortem investigations. Between 2007 and 2012, 1,696 whole carcasses, tissue samples, or both were submitted from the northeastern Pacific and Arctic Canada for diagnostic testing. Streptococcus phocae was cultured from phocids ( n=66), otariids ( n=12), harbor porpoises ( Phocoena phocoena; n=5), and sea otters ( Enhydra lutris; n=2). Pathologic manifestations of S. phocae-associated disease included localized, as well as systemic, inflammatory lesions with common findings of suppurative bronchopneumonia ( n=17) and bacteremia ( n=27). Lung lesions were frequently culture-positive for S. phocae, suggesting commensal colonization of the oropharynx with subsequent opportunistic infection of the respiratory tract during tissue injury, coinfection, immunosuppression, or other debilitating conditions. The presence of a positive spleen culture, and interpretations at necropsy and histopathology, were used to determine the presence of S. phocae bacteremia. Less frequent lesions that were culture positive for S. phocae included abscesses ( n=9), meningitis ( n=7), and cellulitis ( n=1). The majority of cases with S. phocae lesions featured pre-existing conditions that presumably contributed to some degree of debilitation or immunosuppression, including emaciation ( n=29), liver mercury accumulation ( n=29), trauma ( n=22), severe pulmonary or cardiovascular nematodiasis ( n=9), concurrent bacterial or viral infections ( n=8), or sarcocystosis ( n=6). These findings suggest that S. phocae could be characterized as an opportunistic pathogen, associated with debilitating conditions in stranded and rehabilitating marine mammals. Wildlife investigators can use these results to draw more definitive conclusions regarding positive S. phocae cultures during postmortem studies in marine mammals.

Heart and skeletal muscle inflammation (HSMI) disease diagnosed on a British Columbia salmon farm through a longitudinal farm study.

Heart and skeletal muscle inflammation (HSMI) is an emerging disease of marine-farmed Atlantic Salmon (Salmo salar), first recognized in 1999 in Norway, and later also reported in Scotland and Chile. We undertook a longitudinal study involving health evaluation over an entire marine production cycle on one salmon farm in British Columbia (Canada). In previous production cycles at this farm site and others in the vicinity, cardiac lesions not linked to a specific infectious agent or disease were identified. Histologic assessments of both live and moribund fish samples collected at the farm during the longitudinal study documented at the population level the development, peak, and recovery phases of HSMI. The fish underwent histopathological evaluation of all tissues, Twort's Gram staining, immunohistochemistry, and molecular quantification in heart tissue of 44 agents known or suspected to cause disease in salmon. Our analysis showed evidence of HSMI histopathological lesions over an 11-month timespan, with the prevalence of lesions peaking at 80-100% in sampled fish, despite mild clinical signs with no associated elevation in mortalities reported at the farm level. Diffuse mononuclear inflammation and myodegeneration, consistent with HSMI, was the predominant histologic observation in affected heart and skeletal muscle. Infective agent monitoring identified three agents at high prevalence in salmon heart tissue, including Piscine orthoreovirus (PRV), and parasites Paranucleospora theridion and Kudoa thyrsites. However, PRV alone was statistically correlated with the occurrence and severity of histopathological lesions in the heart. Immunohistochemical staining further localized PRV throughout HSMI development, with the virus found mainly within red blood cells in early cases, moving into the cardiomyocytes within or, more often, on the periphery of the inflammatory reaction during the peak disease, and reducing to low or undetectable levels later in the production cycle. This study represents the first longitudinal assessment of HSMI in a salmon farm in British Columbia, providing new insights on the pathogenesis of the disease.

Impact of Salmonid alphavirus infection in diploid and triploid Atlantic salmon (Salmo salar L.) fry.

With increasing interest in the use of triploid salmon in commercial aquaculture, gaining an understanding of how economically important pathogens affect triploid stocks is important. To compare the susceptibility of diploid and triploid Atlantic salmon (Salmo salar L.) to viral pathogens, fry were experimentally infected with Salmonid alphavirus sub-type 1 (SAV1), the aetiological agent of pancreas disease (PD) affecting Atlantic salmon aquaculture in Europe. Three groups of fry were exposed to the virus via different routes of infection: intraperitoneal injection (IP), bath immersion, or cohabitation (co-hab) and untreated fry were used as a control group. Mortalities commenced in the co-hab challenged diploid and triploid fish from 11 days post infection (dpi), and the experiment was terminated at 17 dpi. Both diploid and triploid IP challenged groups had similar levels of cumulative mortality at the end of the experimental period (41.1% and 38.9% respectively), and these were significantly higher (p < 0.01) than for the other challenge routes. A TaqMan-based quantitative PCR was used to assess SAV load in the heart, a main target organ of the virus, and also liver, which does not normally display any pathological changes during clinical infections, but exhibited severe degenerative lesions in the present study. The median viral RNA copy number was higher in diploid fish compared to triploid fish in both the heart and the liver of all three challenged groups. However, a significant statistical difference (p < 0.05) was only apparent in the liver of the co-hab groups. Diploid fry also displayed significantly higher levels of pancreatic and myocardial degeneration than triploids. This study showed that both diploid and triploid fry are susceptible to experimental SAV1 infection. The lower virus load seen in the triploids compared to the diploids may possibly be related to differences in cell metabolism between the two groups, however, further investigation is necessary to confirm this and also to assess the outcome of PD outbreaks in other developmental stages of the fish when maintained in commercial production systems.

Case Report: Strawberry Disease in Farmed Chilean Rainbow Trout.

Strawberry disease is a chronic, nonlethal skin condition that affects Rainbow Trout Oncorhynchus mykiss in the United States and several European countries, where it is also known as red-mark syndrome. We provide the first identification and characterization of three strawberry disease outbreaks occurring at two aquaculture farms in southern Chile. Clinically affected fish weighing an average of 400 g presented multiple bright-red, usually raised, skin lesions on the flank, ventral surface, and dorsal surface. A PCR using Rickettsia-like-organism (RLO)-specific primers was performed on nine affected fish, and all skin samples were positive for the RLO 16S ribosomal RNA sequence. All PCR results for Flavobacterium psychrophilum and other bacterial and viral pathogens were negative. Histopathological examination of the skin lesions revealed extensive dermatitis, with severe lymphocytic infiltration in advanced cases. This report is the first to describe strawberry disease in farmed Chilean Rainbow Trout. Additional studies are needed to evaluate the risk for Rainbow Trout culture; fish challenge experiments should be performed to fulfill Koch's postulates and to demonstrate that RLO is the cause of this disease. Received December 27, 2014; accepted October 23, 2015.

Characterization of a Novel Orthomyxo-like Virus Causing Mass Die-Offs of Tilapia.

UNLABELLED: Tilapia are an important global food source due to their omnivorous diet, tolerance for high-density aquaculture, and relative disease resistance. Since 2009, tilapia aquaculture has been threatened by mass die-offs in farmed fish in Israel and Ecuador. Here we report evidence implicating a novel orthomyxo-like virus in these outbreaks. The tilapia lake virus (TiLV) has a 10-segment, negative-sense RNA genome. The largest segment, segment 1, contains an open reading frame with weak sequence homology to the influenza C virus PB1 subunit. The other nine segments showed no homology to other viruses but have conserved, complementary sequences at their 5' and 3' termini, consistent with the genome organization found in other orthomyxoviruses. In situ hybridization indicates TiLV replication and transcription at sites of pathology in the liver and central nervous system of tilapia with disease. IMPORTANCE: The economic impact of worldwide trade in tilapia is estimated at $7.5 billion U.S. dollars (USD) annually. The infectious agent implicated in mass tilapia die-offs in two continents poses a threat to the global tilapia industry, which not only provides inexpensive dietary protein but also is a major employer in the developing world. Here we report characterization of the causative agent as a novel orthomyxo-like virus, tilapia lake virus (TiLV). We also describe complete genomic and protein sequences that will facilitate TiLV detection and containment and enable vaccine development.

Ultrastructure of Spironucleus salmonis n. comb. (formerly Octomitus salmonis sensu Moore 1922, Davis 1926, and Hexamita salmonis sensu Ferguson 1979), with a guide to spironucleus species.

Diplomonad flagellates can be associated with significant morbidity and mortality in fishes, particularly in farmed salmonids. Diagnosis using only light microscopy is limited, and species cannot be confirmed. We therefore undertook a comprehensive transmission electron microscopy study of 20 trophozoites from the intestine of farmed juvenile rainbow trout Oncorhynchus mykiss from Northern Ireland (reported as Hexamita salmonis by Ferguson in 1979). Re-assignment to the genus Spironucleus was determined based on the anteriorly tapering and intertwined elongate nuclei, anterior-medial kinetosomes, and the presence of a flagellar pocket. At the species level we observed a tri-radiate pattern of microtubules in the opening of the asymetrical striated lamina, comprising 3 to 5 microtubules following the edge of the flagellar pocket, 3 radiating away from the opening of the striated lamina, and 4 to 5 curving over the striated lamina. We observed electron-dense plaques adjacent to the kinetosomes, electron-dense bodies, numerous free ribosomes, aggregations of glycogen, bowl-shaped membranous structures, rough endoplasmic reticulum, and a novel distinctive pyriform sac of densely packed free ribosomes at the posterior of the cell (8-shaped in transverse section, and delineated by furrowed endoplasmic reticulum). We now propose to rename H. salmonis sensu Ferguson (1979) Spironucleus salmonis. Our review of the morphology of Octomitus salmonis illustrated by Moore (1922a,b) and Davis (1926), has shown that this organism also belongs to the genus Spironucleus. We synonymise H. salmonis sensu Ferguson (1979) with O. salmonis sensu Moore (1922) and Davis (1926), and rename them S. salmonis. An expanded diagnostic guide, including new cytoplasmic elements, is presented for the 4 species of Spironucleus from fishes (S. barkhanus, S. salmonis, S. torosa and S. vortens). We recommend that type descriptions of diplomonads be based on organisms taken directly from the host rather than from in vitro culture in order to reliably include the cytoplasmic organelles.

Phenotypic and genotypic heterogeneity among Streptococcus iniae isolates recovered from cultured and wild fish in North America, Central America and the Caribbean islands.

Streptococcus iniae, the etiological agent of streptococcosis in fish, is an important pathogen of cultured and wild fish worldwide. During the last decade outbreaks of streptococcosis have occurred in a wide range of cultured and wild fish in the Americas and Caribbean islands. To gain a better understanding of the epizootiology of S. iniae in the western hemisphere, over 30 S. iniae isolates recovered from different fish species and geographic locations were characterized phenotypically and genetically. Species identities were determined biochemically and confirmed by amplification and sequencing of the 16S rRNA gene. Repetitive-element palindromic PCR fingerprinting as well as biochemical and antimicrobial susceptibility profiles suggest that a single strain of S. iniae was responsible for two different disease outbreaks among reef fishes in the Caribbean, one in 1999 and another in 2008. Interestingly, a majority of the isolates recovered from cultured fish in the Americas were genetically distinct from the Caribbean isolates and exhibited a trend toward higher minimal inhibitory concentration with respect to several antibiotics as well as greater genetic variability. The biological significance of this genetic variability is unclear, but it could have implications for future vaccine development and treatment.

Novel brain lesions caused by Edwardsiella tarda in a red tilapia (Oreochromis spp.).

The histological lesions caused by Edwardsiella tarda in a variety of fish species, including tilapia, have been well characterized. There are apparent differences in the type of inflammatory response manifested by these different species, which may be due to the fish species itself, the phase of infection, or the virulence factors produced by different strains of E. tarda. In catfish, systemic abscesses involving muscles of the flank or caudal peduncle are the most common lesions. By contrast, infection in tilapia and red sea bream is more likely to be associated with granulomatous inflammation. Necrotic meningitis, encephalitis, and vasculitis with fibrinoid necrosis of the blood vessels walls, as well as the formation of a plaque-like structure in the brain, are described in the current study. The presence of E. tarda was confirmed by microbiological isolation and a positive nested polymerase chain reaction in paraffin wax-embedded tilapia tissues.

Characterization of a novel alloherpesvirus from Atlantic cod (Gadus morhua).

Alloherpesviruses affect freshwater and marine fish species. The aim of the current study was to characterize a novel alloherpesvirus in Atlantic cod (Gadus morhua). Samples were processed for histopathology, transmission electron microscopy (TEM), virus isolation, molecular characterization, and in situ hybridization (ISH). Histopathology revealed that the infection was restricted to the gills and that it induced cytomegaly in infected cells. By TEM, numerous viral particles with morphology compatible with a herpesvirus were observed inside the cytomegalic cells. To characterize this new agent, polymerase chain reaction amplified regions of the ATPase subunit of the terminase, and DNA polymerase genes were sequenced. Phylogenetic analysis revealed strongest similarity with alloherpesviruses belonging to the genus Ictalurivirus and Salmonivirus. The ISH showed specific labeling of nuclear inclusions in the cytomegalic cells. While virus isolation was unsuccessful, the results obtained through different diagnostic tests in the present study confirm the discovery of a new alloherpesvirus affecting Atlantic cod. The authors propose the formal species designation Gadid herpesvirus 1 (GaHV-1) to be considered for approval by the International Committee on the Taxonomy of Viruses.

A prospective longitudinal study of "Candidatus arthromitus"-associated rainbow trout gastroenteritis in the UK.

Rainbow trout gastroenteritis (RTGE) is an emerging disease of farmed rainbow trout (Oncorhynchus mykiss) reported in Croatia, France, Italy, Spain and the UK. The impact of RTGE at the site-level varies, and daily mortalities of 0.5-1% are common. Gross lesions include severe enteritis with congestion and oedema, and the segmented filamentous bacterium "Candidatus arthromitus" has been suggested as a possible aetiological agent. This paper presents findings from a prospective longitudinal epidemiology study that took place from June to November 2006 in 12 RTGE-positive UK sites, examining the impact and presentation of the disease, spread pattern, risk factors for disease presence and severity and the efficacy of current control strategies. RTGE was present in a total of 164 production units across the 12 sites, representing 39% of all stocked units. Fish losses due to RTGE totalled 61.4tonnes. The plotting of epidemic curves revealed a propagating epidemic pattern in 51% affected units, suggesting RTGE is infectious. This was supported by risk analysis of fish transfers and site layouts, which revealed an increased risk of becoming RTGE positive after contiguity or fish transfer to and from a previously affected unit. Conditional multivariate logistic regression analysis identified eight variables significantly associated with the presence of RTGE. Seven variables were risk factors, including higher stocking densities, mortalities due to predation or handling, higher water temperature, higher daily feed input and contiguity or fish movement to and from a previous case. One variable was protective: the absence of an aeration system in a unit. General linear modelling identified a significant association of mean feed input per fish during an outbreak with RTGE cumulative mortality. Finally, great variability was observed with regards to the control strategies targeted to RTGE in these sites, suggesting an absence of a common strategy. In feed NaCl treatments apparently presented a palliative effect if given to affected fish during the clinical outbreaks, but they did not prevent recurrence.