Assessing the Efficacy of Three Hatchery Disinfectants for the Inactivation of a Lake Sturgeon Herpesvirus (Family: Alloherpesviridae).

Infectious diseases are a leading cause of losses in the aquaculture industry and conservation programs globally. Simultaneously, infectious diseases pose a substantial risk to fish being hatchery-reared and released into natural habitats for conservation purposes, including the Great Lakes lake sturgeon (Acipenser fulvescens, i.e., GL-LST). Recently, an alloherpesvirus (lake sturgeon herpesvirus 2, i.e., LSHV-2) capable of inducing disease and/or mortality in adult and juvenile GL-LSTs was detected in two adult GL-LST populations. To begin developing disease prevention and/or control methods, in vitro experiments were designed to determine the susceptibility of LSHV-2 to disinfectants commonly used in hatchery and aquaculture facilities (Virkon®-Aquatic: potassium peroxymonosulfate; Ovadine®: polyvinylpyrrolidone iodine complex; and Perox-Aid®: hydrogen peroxide). Cultured LSHV-2 was exposed to each disinfectant at two concentrations (Virkon®-Aquatic: 0.5% and 1%; Ovadine®: 50 and 100 ppm; and Perox-Aid®: 500 and 1000 ppm) in duplicate for durations of 1, 10, and 30 min. Following exposure, the disinfectant was neutralized, and after a 14-day incubation period on a white sturgeon × lake sturgeon hybrid cell line (WSxLS), percent reduction was calculated by comparing the 50% tissue culture infectious doses (TCID50/mL) of the virus with and without disinfectant exposure. When exposed to Perox-Aid®, LSHV-2 percent reduction ranged from 58.7% to 99.5%. When exposed to Ovadine®, the percent reduction ranged from 99.4% to 100%. Lastly, the percent reduction when exposed to Virkon®-Aquatic was 100% for both concentrations and all timepoints. The results herein provide evidence that both Virkon®-Aquatic and Ovadine® are virucidal to LSHV-2 and may represent a means to reduce virus transmission risk under field settings.

Diagnosis, isolation and description of a novel amnoonvirus recovered from diseased fancy guppies, Poecilia reticulata.

The guppy, Poecilia reticulata, is one of the most common cultured ornamental fish species, and a popular pet fish highly desired by hobbyists worldwide due to its availability of many brilliantly coloured fish of many varieties. The susceptibility of guppies to diseases presents a remarkable concern for both breeders and hobbyists. In this study, we report the emergence of disease in fancy guppies caused by a previously uncharacterized virus in the USA. This virus was isolated from moribund guppies in two separate outbreaks in California and Alabama, from December 2021 to June 2023. The infected guppies presented with acute morbidity and mortality shortly after shipping, displaying nonspecific clinical signs and gross changes including lethargy, anorexia, swimming at the water surface, gill pallor, mild to moderate coelomic distension and occasional skin lesions including protruding scales, skin ulcers and hyperaemia. Histological changes in affected fish were mild and nonspecific; however, liver and testes from moribund fish were positive for Tilapia lake virus (TiLV), the single described member in the family Amnoonviridae, using immunohistochemistry and in situ hybridization, although the latter was weak. A virus was successfully recovered following tissue inoculation on epithelioma papulosum cyprini and snakehead fish cell lines. Whole genome sequencing and phylogenetic analyses revealed nucleotide and amino acid homologies from 78.3%-91.2%, and 78.2%-97.7%, respectively, when comparing the guppy virus genomes to TiLV isolates. Based on the criteria outlined herein, we propose the classification of this new virus, fancy tailed guppy virus (FTGV), as a member of the family Amnoonviridae, with the name Tilapinevirus poikilos (from the Greek 'poikilos', meaning of many colours; various sorts, akin to 'poecilia').

Acipenserid herpesvirus 2 genome and partial validation of a qPCR for its detection in white sturgeon Acipenser transmontanus.

White sturgeon Acipenser transmontanus is the primary species used for caviar and sturgeon meat production in the USA. An important pathogen of white sturgeon is acipenserid herpesvirus 2 (AciHV-2). In this study, 4 archived isolates from temporally discrete natural outbreaks spanning the past 30 yr were sequenced via Illumina and Oxford Nanopore Technologies platforms. Assemblies of approximately 134 kb were obtained for each isolate, and the putative ATPase subunit of the terminase gene was selected as a potential quantitative PCR (qPCR) target based on sequence conservation among AciHV-2 isolates and low sequence homology with other important viral pathogens. The qPCR was repeatable and reproducible, with a linear dynamic range covering 5 orders of magnitude, an efficiency of approximately 96%, an R2 of 0.9872, and an analytical sensitivity of 103 copies per reaction after 35 cycles. There was no cross-reaction with other known viruses or closely related sturgeon species, and no inhibition by sturgeon DNA. Clinical accuracy was assessed from white sturgeon juveniles exposed to AciHV-2 by immersion. Viral culture (gold standard) and qPCR were in complete agreement for both cell culture negative and cell culture positive samples, indicating that this assay has 100% relative accuracy compared to cell culture during an active outbreak. The availability of a whole-genome sequence for AciHV-2 and a highly specific and sensitive qPCR assay for detection of AciHV-2 in white sturgeon lays a foundation for further studies on host-pathogen interactions while providing a specific and rapid test for AciHV-2 in captive and wild populations.

Comparative Evaluation of Booster Vaccine Efficacy by Intracoelomic Injection and Immersion with a Whole-Cell Killed Vaccine against Lactococcus petauri Infection in Rainbow Trout (Oncorhynchus mykiss).

Lactococcus petauri is an important emergent bacterial pathogen of salmonids in the USA. The purpose of this study was to evaluate the protection conferred to rainbow trout (Oncorhynchus mykiss) against L. petauri by formalin-killed vaccines in immersion and injectable forms, as well as the enhanced protection afforded by booster vaccination. In the first challenge, fish were immunized via intracoelomic injection (IC) or immersion (Imm) routes alone. Approximately 418 degree days (Temperature in degree Celsius × days post-immunization) (dd) Imm, or 622 dd IC post-vaccination, fish were challenged via IC with wild-type L. petauri. In the second experiment, initial Imm vaccination was followed by booster vaccination via Imm or IC routes 273 dd post-immunization along with appropriate PBS controls. The various vaccination protocol efficacies were evaluated by challenging fish with L. petauri by cohabitation with diseased fish 399 dd post-booster administration. A relative percent survival (RPS) of 89.5% and 28% was recorded in the IC and Imm single immunization treatments, respectively. In the second study, an RPS of 97.5%, 10.2%, 2.6% and -10.1% plus approximately 0%, 50%, 20%, and 30% bacterial persistence was recorded in the Imm immunized + IC boosted, Imm immunized + mock IC boosted, Imm immunized + Imm boosted, and Imm immunized + mock Imm boosted treatments, respectively. Only the Imm immunized + IC injection boosted treatments provided significant protection when compared to unvaccinated and challenged treatments (p < 0.05). In conclusion, although both Imm and IC vaccines appear safe for trout, the inactivated Imm vaccines seem to provide only mild and temporary protection against lactococcosis; whereas IC immunized trout develop a significantly stronger protective response in both challenges.

First Isolation of a Herpesvirus (Family Alloherpesviridae) from Great Lakes Lake Sturgeon (Acipenser fulvescens).

The lake sturgeon (Acipenser fulvescens; LST) is the only native sturgeon species in the Great Lakes (GL), but due to multiple factors, their current populations are estimated to be <1% of historical abundances. Little is known about infectious diseases affecting GL-LST in hatchery and wild settings. Therefore, a two-year disease surveillance study was undertaken, resulting in the detection and first in vitro isolation of a herpesvirus from grossly apparent cutaneous lesions in wild adult LST inhabiting two GL watersheds (Erie and Huron). Histological and ultrastructural examination of lesions revealed proliferative epidermitis associated with herpesvirus-like virions. A virus with identical ultrastructural characteristics was recovered from cells inoculated with lesion tissues. Partial DNA polymerase gene sequencing placed the virus within the Family Alloherpesviridae, with high similarity to a lake sturgeon herpesvirus (LSHV) from Wisconsin, USA. Genomic comparisons revealed ~84% Average Nucleotide Identity between the two isolates, leading to the proposed classification of LSHV-1 (Wisconsin) and LSHV-2 (Michigan) for the two viruses. When naïve juvenile LST were immersion-exposed to LSHV-2, severe disease and ~33% mortality occurred, with virus re-isolated from representative skin lesions, fulfilling Rivers' postulates. Results collectively show LSHV-2 is associated with epithelial changes in wild adult LST, disease and mortality in juvenile LST, and is a potential threat to GL-LST conservation.

Efficacy of a multidose acyclovir protocol against cyprinid herpesvirus 3 infection in koi (Cyprinus carpio).

OBJECTIVE: To evaluate the effect of a multidose acyclovir protocol on koi herpesvirus (KHV) viral load and mortality in a cohabitation challenge. ANIMALS: 180 koi fish. PROCEDURES: Forty fish (shedders) were immersed in a 0.5 KHV plaque-forming units/mL static bath for 8 hours. Mock shedders were treated similarly but exposed to cell culture media. KHV shedders were then transferred into 8 tanks (5 shedders per tank) containing 10 naïve fish (cohabitants) each. Fish in the acyclovir group (AT) received a 10 mg/kg acyclovir intracoelomic injection 1, 3, and 6 days after the first confirmed KHV mortality. Positive controls (PC) were treated similarly but received sterile saline injections. Negative controls (NC) were exposed to mock shedders. Morbidity and mortality were evaluated daily for 50 days post-challenge. Quantitative PCR was used to determine viral load in the gill biopsies of shedders and cohabitants collected at days 19 (T1), 22 (T2), 25 (T3), 34 (T4), and 50 (T5) post-challenge. RESULTS: Survival curves analyzed by the Gehan-Breslow-Wilcoxon method revealed a delayed onset of mortalities and a significantly lower KHV load at T2 and T3 detected in AT cohabitant fish (P = .042) compared to PC group. However, there were no significant differences in overall mortality or viral loads at T5. CLINICAL RELEVANCE: The acyclovir protocol used in this study did not control viral infection or mortality at the end of the 50-day trial. Shorter intervals between injections could improve outcomes, but the additional stress inflicted by handling should be considered. Exploring other therapeutic alternatives and doses is warranted.

In vitro susceptibility testing for the emerging pathogenic mould Veronaea botryosa and pharmacokinetic parameters of natamycin in white sturgeon (Acipenser transmontanus).

Systemic phaeohyphomycosis caused by the dematiaceous mould Veronaea botryosa is an important emergent disease affecting captive sturgeons (Acipenser spp.). The disease, colloquially known as "fluid belly," causes morbidity and mortality in adult animals resulting in significant economic losses to the aquaculture industry. Advancements in therapeutic and prophylactic protocols have been partially hampered by the lack of basic protocols to grow and manipulate the fungus in the laboratory. In this study, microbroth kinetic protocols were established to analyse V. botryosa growth in seven nutrient media at different temperatures. Generated area under the curve (AUC) indicates that potato flake dextrose broth (PFD-B) and Sabouraud dextrose broth (SD-B) incubated at 25°C provided the greatest growth. The generated protocol was then used to test the susceptibility of V. botryosa isolates to natamycin, a macrolide polyene antifungal agent used as a food preservative. SD-B and RPMI with l-glutamine (+RPMI-B) containing different concentrations of natamycin were inoculated with V. botryosa conidia and the generated growth curves were compared using cubic smoothing spline model. The non-inhibitory concentration and minimal inhibitory concentration (MIC; decrease of AUC by 90% compared with control) were determined to be <1 µg/mL and 16 µg/mL of natamycin in SD-B media. To gain an understanding of the tissue distribution of natamycin in white sturgeon, pharmacokinetics was tested. Based on pharmacokinetic parameters determined in this study and targeting a blood concentration >16 µg/mL for 24 h, an intravenous dose >1 g/kg would be needed, making the use of this drug unrealistic. The information presented in this study can be used to investigate susceptibility of pathogenic fungus to antimicrobials and disinfectants as well as support future therapeutic protocols against emerging fungal diseases like fluid belly.

Comparative Susceptibilities of Selected California Chinook Salmon and Steelhead Populations to Isolates of L Genogroup Infectious Hematopoietic Necrosis Virus (IHNV).

Salmonid species demonstrate varied susceptibility to the viral pathogen infectious hematopoietic necrosis virus (IHNV). In California conservation hatcheries, juvenile Chinook salmon (Oncorhynchus tshawytscha) have experienced disease outbreaks due to L genogroup IHNV since the 1940s, while indigenous steelhead (anadromous O. mykiss) appear relatively resistant. To characterize factors contributing to the losses of California salmonid fish due to IHNV, three populations of Chinook salmon and two populations of steelhead native to California watersheds were compared in controlled waterborne challenges with California L genogroup IHNV isolates at viral doses of 104-106 pfu mL-1. Chinook salmon fry were moderately to highly susceptible (CPM = 47-87%) when exposed to subgroup LI and LII IHNV. Susceptibility to mortality decreased with increasing age and also with a higher temperature. Mortality for steelhead fry exposed to two IHNV isolates was low (CPM = 1.3-33%). There was little intraspecies variation in susceptibility among populations of Chinook salmon and no differences in virulence between viruses strains. Viral persistence was demonstrated by the isolation of low levels of infectious IHNV from the skin of two juvenile Chinook salmon at 215 d post exposure. The persistence of the virus among Chinook salmon used for stocking into Lake Oroville may be an explanation for the severe epidemics of IHN at the Feather River hatchery in 1998-2002.

Expression of immune markers of white sturgeon (Acipenser transmontanus) during Veronaea botryosa infection at different temperatures.

Systemic phaeohyphomycosis caused by Veronaea botryosa is one of the most important emergent diseases to affect sturgeon aquaculture in North America. White sturgeon (Acipenser transmontanus) cultured at temperatures above 15 °C are at higher risk of severe disseminated disease and higher mortalities. Despite this, little is known regarding disease pathogenesis and the immune response to infection. The objective of this study was to investigate the acute (2 days post-challenge [dpc]) and chronic (32 dpc) response of white sturgeon at 13 °C and 18 °C challenged with V. botryosa via intramuscular injection, using gene expression analysis of a diverse array of soluble immune and inflammatory mediators. Significantly greater amounts of irf8 (p < 0.05) and tfg-ß (p < 0.05) genes were detected in gills of exposed fish at 18 °C when compared to those at 13 °C 32 dpc. Transcript levels of haptoglobin, serotransferrin, serum amyloid, cathelicidin, tnf-α, and il-17 were significantly increased in splenic tissues of challenged fish maintained at 18 °C late in infection (p < 0.05). However, only haptoglobin and serotransferrin transcript abundance were significantly greater in exposed fish when compared to controls 32dpc. Moreover, haptoglobin transcripts at this time point were significantly greater in exposed fish at 18 °C when compared to those challenged at 13 °C. Fewer differences were detected in fish kept at 13 °C. In agreement with transcript quantification, western blot assessment of haptoglobin showed increased levels in the challenged fish maintained at 18 °C.

Isolation, Identification and Characterization of a Novel Megalocytivirus from Cultured Tilapia (Oreochromis spp.) from Southern California, USA.

In spring 2019, diseased four-month-old tilapia (Oreochromis spp.) from an aquaculture farm in Southern California, USA were received for diagnostic evaluation with signs of lethargy, anorexia, abnormal swimming, and low-level mortalities. At necropsy, non-specific external lesions were noted including fin erosion, cutaneous melanosis, gill pallor, and coelomic distension. Internal changes included ascites, hepatomegaly, renomegaly, splenomegaly, and multifocal yellow-white nodules in the spleen and kidney. Cultures of spleen and kidney produced bacterial colonies identified as Francisella orientalis. Homogenized samples of gill, brain, liver, spleen, and kidney inoculated onto Mozambique tilapia brain cells (OmB) developed cytopathic effects, characterized by rounding of cells and detaching from the monolayer 6-10 days post-inoculation at 25 °C. Transmission electron microscopy revealed 115.4 ± 5.8 nm icosahedral virions with dense central cores in the cytoplasm of OmB cells. A consensus PCR, targeting the DNA polymerase gene of large double-stranded DNA viruses, performed on cell culture supernatant yielded a sequence consistent with an iridovirus. Phylogenetic analyses based on the concatenated full length major capsid protein and DNA polymerase gene sequences supported the tilapia virus as a novel species within the genus Megalocytivirus, most closely related to scale drop disease virus and European chub iridovirus. An intracoelomic injection challenge in Nile tilapia (O. niloticus) fingerlings resulted in 39% mortality after 16 days. Histopathology revealed necrosis of head kidney and splenic hematopoietic tissues.

Disruption of the Francisella noatunensis subsp. orientalis pdpA Gene Results in Virulence Attenuation and Protection in Zebrafish.

Several Francisella spp., including Francisella noatunensis, are regarded as important emerging pathogens of wild and farmed fish. However, very few studies have investigated the virulence factors that allow these bacterial species to be pathogenic in fish. The Francisella pathogenicity island (FPI) is a well-described, gene-dense region encoding major virulence factors for the genus Francisella. pdpA is a member of the pathogenicity-determining protein genes carried by the FPI that are implicated in the ability of the mammalian pathogen Francisella tularensis to escape and replicate in infected host cells. Using a sacB suicide approach, we generated pdpA knockouts to address the role of PdpA as a virulence factor for F. noatunensis. Because polarity can be an issue in gene-dense regions, we generated two different marker-based mutants in opposing polarity (the F. noatunensis subsp. orientalis ΔpdpA1 and ΔpdpA2 strains). Both mutants were attenuated (P < 0.0001) in zebrafish challenges and displayed impaired intracellular replication (P < 0.05) and cytotoxicity (P < 0.05), all of which could be restored to wild-type (WT) levels by complementation for the ΔpdpA1 mutant. Importantly, differences were found for bacterial burden and induction of acute-phase and proinflammatory genes for the F. noatunensis subsp. orientalis ΔpdpA1 and ΔpdpA2 mutants compared to the WT during acute infection. In addition, neither mutant resulted in significant histopathological changes. Finally, immunization with the F. noatunensis subsp. orientalis ΔpdpA1 mutant led to protection (P < 0.012) against an acute 40% lethal dose (LD40) challenge with WT F. noatunensis in the zebrafish model of infection. Taken together, the results from this study further demonstrate physiological similarities within the genus Francisella relative to their phylogenetic relationships and the utility of zebrafish for addressing virulence factors for the genus.

INTRAVENOUS IOPAMIDOL PHARMACOKINETICS IN COMMON CARP (CYPRINUS CARPIO).

Koi carp (Cyprinus carpio), a variety of common carp, has gained popularity as an ornamental fish worldwide. Their high monetary and sentimental value has necessitated the development of antemortem diagnostic options. Contrast-enhanced computed tomography (CT) scanning with intravenous iopamidol has been shown to be safe and diagnostically effective at a minimum dose of 480 mg iodine (I)/kg in koi. The purpose of this study was to evaluate the pharmacokinetic parameters of this dose of iopamidol, as well as excretory mechanisms specific to fish, using common carp as a model. Blood, posterior kidney, gill, and bile were collected, necessitating sacrificial sampling. Thirty-five adult fish were randomly divided into six sampling groups. Five sampling groups (n = 6/group) received 480 mg I/kg; the control group (n = 5) received an equivalent volume of saline. The iopamidol groups were sampled at the following time points postinjection: 5 min, 1 hr, 6 hr, 24 hr, and 48 hr. The control group was sampled at 48 hr. Concentrations of iopamidol were determined using liquid chromatography tandem mass spectrometry; noncompartmental analysis was used to calculate pharmacokinetic parameters. Total clearance (3.04 ml/hr per kilogram) was slower, the volume of distribution smaller (79.92 ml/ kg), and the elimination half-life (20.39 hr) prolonged compared to similar studies in mammals. The time-concentration profiles of kidney and gill were similar; these organs appear to be responsible for the majority of iopamidol excretion. However, that of bile was much different, showing slower, low-level accumulation with time, suggesting that in fish, multiple organ systems play a role in elimination beyond just the kidney. In particular, they may rely more heavily upon biliary excretion, which thus far has been noted only in mammals with renal impairment. Further research is warranted to investigate if the slower elimination allows diagnostic CT images to be acquired at different time points postinjection.

Pharmacokinetic and Efficacy Study of Acyclovir Against Cyprinid Herpesvirus 3 in Cyprinus carpio.

Cyprinid Herpesvirus 3 (CyHV-3), more commonly known as Koi Herpesvirus (KHV), is a re-emergent virus causing acute systemic infection with high mortality rates in koi fish (Cyprinus carpio). Survivors from outbreaks can become latent carriers, with viral reactivation under stressful conditions and permissible temperatures. No vaccines or treatments are currently available in the United States. Acyclovir has been shown effective in vitro against KHV. This study aimed to evaluate the cytotoxicity of acyclovir and cidofovir to koi fin (KF1) cells, the efficacy of a single antiviral intracoelomic dose in a koi fingerling cohabitation challenge, and the pharmacokinetics of the effective antiviral. Initially, a lactate dehydrogenase release-based assay revealed no significant acyclovir or cidofovir cytotoxicity to KF1 cells for 24 h at up to 1,500 µM. In laboratory-controlled challenges, KHV associated mortalities occurred 2 weeks post-infection. At this point, fish were treated with an antiviral (10 mg/kg acyclovir or 5 mg/kg cidofovir) or sterile phosphate-buffered solution. Morbidity and mortality were monitored for 30 days. A significant cumulative mortality reduction (p ≤ 0.05), and a 3-day mortality delay were detected in the acyclovir-treated group. Similar viral loads were detected in gills recovered from mortalities throughout the challenge and surviving fish at the end of the challenge regardless of treatment. For pharmacokinetic analysis, blood was collected at various timepoints after acyclovir administration. Liquid chromatography tandem mass spectrometry plasma analysis indicated a 141 µM peak plasma concentration at 0.75 h, a 14 h half-life, and a 0.05/h elimination rate constant. Histopathology of target tissues detected no evidence of acyclovir toxicity. Results suggest that a single 10 mg/kg dose of acyclovir administered intracoelomically to koi fingerlings is safe and reduces cumulative mortality during a KHV mortality event. However, multiple doses are probably required for effective treatment of pet fish.

Pathogenesis and immune response of Nile tilapia (Oreochromis niloticus) exposed to Tilapia lake virus by intragastric route.

Tilapia lake virus (TiLV) is regarded as one of the most important pathogens in tilapia aquaculture worldwide. Despite this, little is known regarding disease pathogenesis and immune responses to infection. The main objective of this study was to investigate the tissue distribution, histopathological changes, and immune response of fish exposed to TiLV. Nile tilapia (Oreochromis niloticus) maintained at 25 ± 2 °C were challenged with TiLV via intragastric-gavage. At 0.5, 1, 3, 5, 7, 10 and 15 days post-challenge (dpc), six fish per treatment were euthanized and subjected to complete necropsy. TiLV exposed fish presented 45% cumulative mortality at the end of the study. Gross lesions included cutaneous petechiae and ecchymoses, scale losses, skin ulcers, and exophthalmia. Mild multifocal hepatocellular degeneration and necrosis was observed as early as 3 dpc occasionally accompanied by syncytial formation, intracytoplasmic inclusion bodies, and inflammatory infiltrates of lymphocytes at subsequent time points. Necrosis of epithelial cells of the gastric glands and intestinal glands was also observed as early as 5 dpc. Intestinal samples showed reactive in situ hybridization signals as early as 1 dpc. No other lesions were observed in the brain or other organs. Histological changes were associated with viral dissemination and disease progression, as evidenced by increased TiLV detection in the intestine, gills, liver and spleen. Highest TiLV abundance was detected 7 dpc in gills, intestine, and liver showing an average of 6 LOG genome equivalent per ng of total RNA. Different transcript abundance was detected for the pro-inflammatory cytokine interleukin-1ß and interferon-induced myxovirus resistance protein gene in the mucosal sites (gills and intestine). Interferon regulatory transcription factor 3 transcript was more abundant in systemic organs (liver and spleen) while the expression in gills and intestine showed mixed expression at different time points. On the other hand, transforming growth factor ß expression patterns differed amongst the tissues with a trend towards downregulation of the gene in liver and gills, and a trend towards upregulation in the spleen and intestine. Overall, these results demonstrate the intestinal routes as a main port of entry for TiLV, which subsequently spreads systematically throughout the fish body.

First Isolation of a Novel Aquatic Flavivirus from Chinook Salmon (Oncorhynchus tshawytscha) and Its In Vivo Replication in a Piscine Animal Model.

The first isolation of a flavivirus from fish was made from moribund Chinook salmon (Oncorhynchus tshawytscha) from the Eel River, California, USA. Following the observation of cytopathic effect in a striped-snakehead fish cell line, 35-nm virions with flaviviral morphology were visualized using electron microcopy. Next-generation sequencing and rapid amplification of cDNA ends obtained the complete genome. Reverse transcriptase quantitative PCR (RT-qPCR) confirmed the presence of viral RNA in formalin-fixed tissues from the wild salmon. For the first time, in vivo replication of an aquatic flavivirus was demonstrated following intracoelomic injection in a Chinook salmon model of infection. RT-qPCR demonstrated viral replication in salmon brains up to 15 days postinjection. Infectious virus was then reisolated in culture, fulfilling Rivers' postulates. Only limited replication occurred in the kidneys of Chinook salmon or in tissues of rainbow trout (Oncorhynchus mykiss). The proposed salmon flavivirus (SFV) has a 10.3-kb genome that encodes a rare dual open reading frame, a feature uncharacteristic of classical flaviviruses. Phylogenetic analysis places SFV in a basal position among a new subgroup of recently recognized aquatic and bat flaviviruses distinct from the established mosquito-borne, tick-borne, insect-only, and unknown-vector flavivirus groups. While the pathogenic potential of the virus remains to be fully elucidated, its basal phylogeny and the in vivo infection model will allow SFV to serve as a prototype for aquatic flaviviruses. Ongoing field and laboratory studies will facilitate better understanding of the potential impacts of SFV infection on ecologically and economically important salmonid species.IMPORTANCE Chinook salmon are a keystone fish species of great ecological and commercial significance in their native northern Pacific range and in regions to which they have been introduced. Threats to salmon populations include habitat degradation, climate change, and infectious agents, including viruses. While the first isolation of a flavivirus from wild migrating salmon may indicate an emerging disease threat, characterization of the genome provides insights into the ecology and long evolutionary history of this important group of viruses affecting humans and other animals and into an expanding group of recently discovered aquatic flaviviruses.

Non-lethal diagnostic methods for koi herpesvirus in koi Cyprinus carpio.

Cyprinid herpesvirus 3, also known as koi herpesvirus (KHV), is a viral pathogen responsible for mass mortalities of carp worldwide. In this study, we compared the sensitivity and specificity of ELISA and quantitative PCR (qPCR) methods for the diagnosis of KHV in experimentally infected koi Cyprinus carpio over an 11 mo period. Koi were exposed to KHV at 18 ± 1°C (permissive temperatures for KHV disease) in laboratory-controlled conditions. At 21 d post challenge, the temperature in the system was decreased to <15°C (non-permissive temperature for KHV disease), and fish were monitored for the following 11 mo. At different time points throughout the study, samples of blood and gills were collected from exposed and control koi and subjected to qPCR and ELISA. Survival proportions of 53.3 and 98.8% in exposed and control treatments, respectively, were recorded at the end of the challenge. Traditional receiver-operating characteristic analysis was used to compare the sensitivity of the ELISA and blood and gill qPCR during permissive and non-permissive temperatures. ELISA was superior to qPCR of gills and whole-blood samples in detecting previous exposure to KHV. Similar results were obtained in a second experiment exposing koi to KHV and inducing persistent infection at >30°C (non-permissive temperature for KHV disease). Finally, KHV ELISA specificity was confirmed using cyprinid herpesvirus 1-exposed koi through a period of 3 mo. This study demonstrates that the combination of ELISA and gill qPCR should be recommended in the diagnosis of KHV exposure of suspected carrier-state fish.

Intragastric and intracoelomic injection challenge models of tilapia lake virus infection in Nile tilapia (Oreochromis niloticus L.) fingerlings.

To gain a better understanding of the pathogenesis of tilapia lake virus (TiLV) infections in Nile tilapia (Oreochromis niloticus), fingerlings were challenged with a single dose of 1 × 104  TCID50 /fish of TiLV utilizing intracoelomic/intraperitoneal (ICch ) or intragastric (IGch ) routes. Acute mortalities were present in both groups, reaching 70 and 40% in ICch and IGch after 10 days, respectively. Challenged fish presented erratic swimming, lethargy, anorexia, exophthalmia and cutaneous petechiae and ecchymoses. Histological changes in challenged groups included syncytial formation, intracytoplasmic inclusion bodies and multifocal hepatocellular degeneration and necrosis. In addition, multifocal areas of mild proliferation of glial cells and lymphocytic perivascular cuffing were observed in the brain of exposed challenged groups. TiLV RNA was detected in gills and faeces of challenged fish using quantitative reverse transcriptase-PCR, as well as in the tank water holding challenged fish. Moreover, TiLV RNA was detected in scrolls obtained from formalin-fixed paraffin-embedded tissue blocks from challenged fish. Results from this study suggest that IG methods represent an additional method to study the pathogenesis of the disease in this species, as it results in infection and diseases as in naturally occurring cases and does not bypass important mucosal immune responses as injectable routes do.

Effects of temperature on Veronaea botryosa infections in white sturgeon Acipenser transmontanus and fungal induced cytotoxicity of fish cell lines.

Veronaea botryosa is a melanized mold and cause of systemic fungal infections in cultured sturgeon (Acipenser spp.). Mortality in adult female sturgeon caused by this emergent pathogen results in significant economic losses for the caviar industry. Little is known regarding environmental conditions conducive to V. botryosa infection. This study evaluated the effect of temperature on V. botryosa infectivity and dissemination following intramuscular injection challenge of white sturgeon maintained at 13 or 18 °C for 40 days. Daily mortality was recorded and persistence of the fungus in the livers of moribund and surviving fish was investigated using culture and histopathological analysis. Fish maintained at 18 °C developed systemic phaeohyphomycosis and had significantly greater mortality than controls and fish maintained at 13 °C (p < 0.05). Challenged fish, regardless of temperature, exhibited lesions in multiple organs. However, muscle lesions, angioinvasion, and systemic dissemination were more severe and widespread in fish challenged at the higher temperature. In vitro cytotoxicity of V. botryosa was evaluated in white sturgeon skin (WSSK-1) and epithelioma papulosum cyprini (EPC) cell lines inoculated at spore:cell ratios of 1:10, 1:1 and 10:1, then incubated 15, 20 and 25 °C. Cytotoxicity, as indicated by quantifying the release of lactate dehydrogenase into culture supernatants, increased with increasing spore dose and incubation temperature in both fish cell lines. Findings suggest that temperature significantly influences the development of systemic V. botryosa infection in white sturgeon and that WSSK-1 and EPC cells are suitable in vitro models for the study of host-pathogen interactions between V. botryosa and fish epithelial cells.

Interaction of Francisella noatunensis subsp. orientalis with Oreochromis mossambicus bulbus arteriosus cell line.

Francisella noatunensis subsp. orientalis (Fno) (syn. F. asiatica) is an emergent warmwater fish pathogen and the causative agent of piscine francisellosis. Although Fno causes septicemia and can live extracellularly in infected tilapia (Oreochromis spp.), the early interaction of Fno with vasculature endothelium is unknown. In the present study, we examined the interaction of wild-type Fno (WT) and two Fno knockout [intracellular growth loci C (ΔiglC) and pathogenicity determinant protein A (ΔpdpA)] strains with the endothelial O. mossambicus bulbus arteriosus cell line (TmB) at 25 °C and 30 °C. Similar amounts of WT, ΔiglC, and ΔpdpA attached and were detected intracellularly after 5 h of incubation at both temperatures; however temperature affected attachment and uptake. While significantly greater amounts of Fno (WT, ΔiglC, and ΔpdpA) were detected intracellularly when TmB cells were incubated at 30 °C, bacteria attached to TmBs at greater levels at 25 °C. Only WT Fno was able to replicate intracellularly at 25 °C, which resulted in Fno mediated cytotoxicity and apoptosis at 24 and 72 h post-infection. WT Fno incubated at 30 °C as well as ΔiglC, and ΔpdpA incubated at 25 °C and 30 °C were all defective for survival, replication, and the ability to cause cytotoxicity in TmB. Taken together, these results demonstrate that temperature plays a vital role for Fno intracellular survival, persistence and cytotoxicity.

Co-infection of Acipenserid herpesvirus 2 (AciHV-2) and Streptococcus iniae in cultured white sturgeon Acipenser transmontanus.

A mortality event in cultured white sturgeon Acipenser transmontanus (Richardson, 1836) sub-adults was investigated. After transfer between farms, high mortality was observed in fish, associated with back arching, abnormal swimming, and ulcerative skin lesions. Necropsy of moribund individuals revealed hemorrhagic ascites and petechial hemorrhages in the coelomic peritoneum and serosa of internal organs. Acipenserid herpesvirus 2 (AciHV-2) was isolated from external tissue samples, then identified and genotyped by sequencing of the terminase and polymerase genes. In addition, Streptococcus iniae was recovered from internal organs of affected fish. Histologic changes were limited to interstitial hematopoietic areas of the kidney and consisted of small foci of necrosis accompanied by fibrin deposition, minimal inflammatory response, and small numbers of bacterial cocci compatible with streptococci. Identity was confirmed by partial sequencing of the 16S rRNA, rpoB, and gyrB genes. Genetic fingerprinting demonstrated a genetic profile distinct from S. iniae isolates recovered from previous outbreaks in wild and cultured fish in North America, South America, and the Caribbean. Although the isolates were resistant to white sturgeon complement in serum killing assays, in vivo challenges failed to fulfill Koch's postulates. However, the clinical presentation, coupled with consistent recovery of S. iniae and AciHV-2 from moribund fish, suggests viral and bacterial co-infection were the proximate cause of death. To our knowledge, this represents the first report of AciHV-2 and S. iniae co-infection in cultured white sturgeon.