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Ammocoetes of Pacific lamprey are not susceptible to common fish rhabdoviruses of the U.S. Pacific Northwest

Pacific Lampreys Entosphenus tridentatus have experienced severe population declines in recent years and efforts to develop captive rearing programs are under consideration. However, there is limited knowledge of their life history, ecology, and potential to harbor or transmit pathogens that may cause infectious disease. As a measure of the possible risks associated with introducing wild lampreys into existing fish culture facilities, larval lampreys (ammocoetes) were tested for susceptibility to infection and mortality caused by experimental exposures to the fish rhabdovirus pathogens: infectious hematopoietic necrosis virus (IHNV) and viral haemorrhagic septicaemia virus (VHSV). Two IHNV isolates, representing the U and M genogroups, and one VHSV isolate from the IVa genotype were each delivered to groups of ammocoetes by immersion at moderate and high viral doses, and by intraperitoneal injection. Ammocoetes were then held in triplicate tanks with no substrate or sediment. During 41 d of observation postchallenge there was low or no mortality in all groups, and no virus was detected in the small number of fish that died. Ammocoetes sampled for incidence of infection at 6 and 12 d after immersion challenges also had no detectable virus, and no virus was detected in surviving fish from any group. A small number of ammocoetes sampled 6 d after the injection challenge had detectable virus, but at levels below the original quantity of virus injected. Overall there was no evidence of infection, replication, or persistence of any of the viruses in any of the treatment groups. Our results suggest that Pacific Lampreys are highly unlikely to serve as hosts that maintain or transmit these viruses.

Journal of Aquatic Animal Health↗

Genetic variation in bacterial kidney disease (BKD) susceptibility in Lake Michigan Chinook Salmon and its progenitor population from the Puget Sound

Mass mortality events in wild fish due to infectious diseases are troubling, especially given the potential for long-term, population-level consequences. Evolutionary theory predicts that populations with sufficient genetic variation will adapt in response to pathogen pressure. Chinook Salmon Oncorhynchus tshawytscha were introduced into Lake Michigan in the late 1960s from a Washington State hatchery population. In the late 1980s, collapse of the forage base and nutritional stress in Lake Michigan were thought to contribute to die-offs of Chinook Salmon due to bacterial kidney disease (BKD). Previously, we demonstrated that Lake Michigan Chinook Salmon from a Wisconsin hatchery have greater survival following BKD challenge relative to their progenitor population. Here, we evaluated whether the phenotypic divergence of these populations in BKD susceptibility was due to selection rather than genetic drift. Comparison of the overall magnitude of quantitative trait to neutral marker divergence between the populations suggested selection had occurred but a direct test of quantitative trait divergence was not significant, preventing the rejection of the null hypothesis of differentiation through genetic drift. Estimates of phenotypic variation (V P ), additive genetic variation (V A ) and narrow-sense heritability (h 2 ) were consistently higher in the Wisconsin relative to the Washington population. If selection had acted on the Wisconsin population there was no evidence of a concomitant loss of genetic variation in BKD susceptibility. The Renibacterium salmoninarum exposures were conducted at both 14°C and 9°C; the warmer temperature accelerated time to death in both populations and there was no evidence of phenotypic plasticity or a genotype-by-environment (G × E) interaction. High h 2 estimates for BKD susceptibility in the Wisconsin population, combined with a lack of phenotypic plasticity, predicts that future adaptive gains in BKD resistance are still possible and that these adaptive gains would be stable under the temperature range evaluated here.

Washington;Wisconsin↗

Susceptibility of Koi and Yellow Perch to infectious hematopoietic necrosis virus by experimental exposure

Infectious hematopoietic necrosis virus (IHNV) is a novirhabdoviral pathogen that originated in western North America among anadromous Pacific salmonids. Severe disease epidemics in the late 1970s resulting from IHNV's invasion into farmed Rainbow Trout Oncorhynchus mykiss in North America, Asia, and Europe emphasized IHNV's ability to adapt to new hosts under varying rearing conditions. Yellow Perch Perca flavescens and Koi Carp Cyprinus carpio (hereafter, “Koi”) are aquaculture-reared fish that are highly valued in sport fisheries and the ornamental fish trade, respectively, but it is unknown whether these fish species are vulnerable to IHNV infection. In this study, we exposed Yellow Perch, Koi, and steelhead (anadromous Rainbow Trout) to IHNV by intraperitoneal injection (10 6 PFU/fish) and by immersion (5.7×10 5 PFU/mL) for 7 h, and monitored fish for 28 d. The extended immersion exposure and high virus concentrations used in the challenges were to determine if the tested fish had any level of susceptibility. After experimental exposure, Yellow Perch and Koi experienced low mortality (<6%) compared with steelhead (>35%). Virus was found in dead fish of all species tested and in surviving Yellow Perch by plaque assay and quantitative reverse transcription polymerase chain reaction (qPCR), with a higher prevalence in Yellow Perch than Koi. Infectious virus was also detected in Yellow Perch out to 5 d after bath challenge. These findings indicate that Yellow Perch and Koi are highly resistant to IHNV disease under the conditions tested, but Yellow Perch are susceptible to infection and may serve as possible virus carriers.

Journal of Aquatic Animal Health↗

Florfenicol residues in Rainbow Trout after oral dosing in recirculating and flow-through culture systems

Aquaflor is a feed premix for fish containing the broad spectrum antibacterial agent florfenicol (FFC) incorporated at a ratio of 50% (w/w). To enhance the effectiveness of FFC for salmonids infected with certain isolates of Flavobacterium psychrophilum causing coldwater disease, the FFC dose must be increased from the standard 10 mg·kg −1 body weight (BW)·d −1 for 10 consecutive days. A residue depletion study was conducted to determine whether FFC residues remaining in the fillet tissue after treating fish at an increased dose would be safe for human consumption. Groups of Rainbow Trout Oncorhynchus mykiss (total n = 144; weight range, 126–617 g) were treated with FFC at 20 mg·kg −1 BW·d −1 for 10 d in a flow-through system (FTS) and a recirculating aquaculture system (RAS) each with a water temperature of ∼13°C. The two-tank RAS included a nontreated tank containing 77 fish. Fish were taken from each tank (treated tank, n = 16; nontreated tank, n = 8) at 6, 12, 24, 48, 72, 120, 240, 360, and 480 h posttreatment. Florfenicol amine (FFA) concentrations (the FFC marker residue) in skin-on fillets from treated fish were greatest at 12 h posttreatment (11.58 μg/g) in the RAS and were greatest at 6 h posttreatment (11.09 μg/g) in the FTS. The half-lives for FFA in skin-on fillets from the RAS and FTS were 20.3 and 19.7 h, respectively. Assimilation of FFC residues in the fillets of nontreated fish sharing the RAS with FFC-treated fish was minimal. Florfenicol water concentrations peaked in the RAS-treated tank and nontreated tanks at 10 h (453 μg/L) and 11 h (442 μg/L) posttreatment, respectively. Monitoring of nitrite concentrations throughout the study indicated the nitrogen oxidation efficiency of the RAS biofilter was minimally impacted by the FFC treatment.

Journal of Aquatic Animal Health↗

Infecting Pacific Herring with Ichthyophonus sp. in the laboratory

The protistan parasite Ichthyophonus sp. occurs in coastal populations of Pacific Herring Clupea pallasii throughout the northeast Pacific region, but the route(s) by which these planktivorous fish become infected is unknown. Several methods for establishing Ichthyophonus infections in laboratory challenges were examined. Infections were most effectively established after intraperitoneal (IP) injections with suspended parasite isolates from culture or after repeated feedings with infected fish tissues. Among groups that were offered the infected tissues, infection prevalence was greater after multiple feedings (65%) than after a single feeding (5%). Additionally, among groups that were exposed to parasite suspensions prepared from culture isolates, infection prevalence was greater after exposure by IP injection (74%) than after exposure via gastric intubation (12%); the flushing of parasite suspensions over the gills did not lead to infections in any of the experimental fish. Although the consumption of infected fish tissues is unlikely to be the primary route of Ichthyophonus sp. transmission in wild populations of Pacific Herring, this route may contribute to abnormally high infection prevalence in areas where juveniles have access to infected offal.

Journal of Aquatic Animal Health↗

Dermocystidium sp. infection in Blue Ridge Sculpin captured in Maryland

Raised pale cysts were observed on Blue Ridge Sculpin Cottus caeruleomentum during stream fish community surveys in Catoctin Mountain Park, Maryland. When examined histologically, preserved sculpin exhibited multiple cysts containing spherical endospores with a refractile central body characteristic of Dermocystidium spp. Cysts were not observed on the gills or internally. The portion of the watershed in which affected sculpin were observed contained lower than expected numbers of sculpin, raising concerns about the population effects of this infection. A nearby stream lacked sculpin even though they are common in this region, further suggesting the possibility of regional effects. This is the first report of a Dermocystidium infecting any fish species in the eastern United States.

Maryland↗

Optimization of a Plaque Neutralization Test (PNT) to identify the exposure history of Pacific Herring to viral hemorrhagic septicemia virus (VHSV)

Methods for a plaque neutralization test (PNT) were optimized for the detection and quantification of viral hemorrhagic septicemia virus (VHSV) neutralizing activity in the plasma of Pacific Herring Clupea pallasii. The PNT was complement dependent, as neutralizing activity was attenuated by heat inactivation; further, neutralizing activity was mostly restored by the addition of exogenous complement from specific-pathogen-free Pacific Herring. Optimal methods included the overnight incubation of VHSV aliquots in serial dilutions (starting at 1:16) of whole test plasma containing endogenous complement. The resulting viral titers were then enumerated using a viral plaque assay in 96-well microplates. Serum neutralizing activity was virus-specific as plasma from viral hemorrhagic septicemia (VHS) survivors demonstrated only negligible reactivity to infectious hematopoietic necrosis virus, a closely related rhabdovirus. Among Pacific Herring that survived VHSV exposure, neutralizing activity was detected in the plasma as early as 37 d postexposure and peaked at approximately 64 d postexposure. The onset of neutralizing activity was slightly delayed in fish reared at 7.4°C relative to those in warmer temperatures (9.9°C and 13.1°C); however, neutralizing activity persisted for at least 345 d postexposure in all temperature treatments. It is anticipated that this novel ability to assess VHSV neutralizing activity in Pacific Herring will enable retrospective comparisons between prior VHS infections and year-class recruitment failures. Additionally, the optimized PNT could be employed as a forecasting tool capable of identifying the potential for future VHS epizootics in wild Pacific Herring populations.

Journal of Aquatic Animal Health↗

Influence of temperature on the efficacy of homologous and heterologous DNA vaccines against viral hemorrhagic septicemia in Pacific Herring

Homologous and heterologous (genogroup Ia) DNA vaccines against viral hemorrhagic septicemia virus (genogroup IVa) conferred partial protection in Pacific Herring Clupea pallasii . Early protection at 2 weeks postvaccination (PV) was low and occurred only at an elevated temperature (12.6°C, 189 degree days), where the relative percent survival following viral exposure was similar for the two vaccines (IVa and Ia) and higher than that of negative controls at the same temperature. Late protection at 10 weeks PV was induced by both vaccines but was higher with the homologous vaccine at both 9.0°C and 12.6°C. Virus neutralization titers were detected among 55% of all vaccinated fish at 10 weeks PV. The results suggest that the immune response profile triggered by DNA vaccination of herring was similar to that reported for Rainbow Trout Oncorhynchus mykiss by Lorenzen and LaPatra in 2005, who found interferon responses in the early days PV and the transition to adaptive response later. However, the protective effect was far less prominent in herring, possibly reflecting different physiologies or adaptations of the two fish species.

Journal of Aquatic Animal Health↗

Detection of Nanophyetus salmincola in water, snails, and fish tissues by quantitative polymerase chain reaction

We report the development and validation of two quantitative PCR (qPCR) assays to detect Nanophyetus salmincola DNA in water samples and in fish and snail tissues. Analytical and diagnostic validation demonstrated good sensitivity, specificity, and repeatability of both qPCR assays. The N. salmincola DNA copy number in kidney tissue was significantly correlated with metacercaria counts based on microscopy. Extraction methods were optimized for the sensitive qPCR detection of N. salmincola DNA in settled water samples. Artificially spiked samples suggested that the 1-cercaria/L threshold corresponded to an estimated log 10 copies per liter ≥ 6.0. Significant correlation of DNA copy number per liter and microscopic counts indicated that the estimated qPCR copy number was a good predictor of the number of waterborne cercariae. However, the detection of real-world samples below the estimated 1-cercaria/L threshold suggests that the assays may also detect other N. salmincola life stages, nonintact cercariae, or free DNA that settles with the debris. In summary, the qPCR assays reported here are suitable for identifying and quantifying all life stages of N. salmincola that occur in fish tissues, snail tissues, and water.

Journal of Aquatic Animal Health↗

Identical sequence types of Yersinia ruckeri associated with lethal disease in wild-caught invasive Blue Catfish and cultured hybrid catfish (Channel Catfish ♀ × Blue Catfish ♂) from disparate aquatic ecosystems

Objective The Blue Catfish Ictalurus furcatus is commonly raised in warmwater aquaculture in the United States to produce Channel Catfish I. punctatus × Blue Catfish hybrids. It is also a prominent aquatic invasive species of concern in the mid-Atlantic region of the United States. Here, Yersina ruckeri was isolated from moribund Blue Catfish and hybrid catfish from disparate regions of the USA. The goal of the research here was to compare these Y. ruckeri strains to each other and other known strains for which adequate sequence data was available. In addition, we sought to determine if the strain from Blue Catfish was pathogenic to Rainbow Trout Oncorhynchus mykiss . Methods Moribund hybrid catfish from culture ponds in Mississippi were processed for diagnostic evaluation in March 2016. In April 2022, a moribund Blue Catfish specimen was collected from a tributary of the Nanticoke River in Maryland. Bacterial isolates were identified and characterized using biochemical tests, antimicrobial sensitivity screening, serotyping, and complete or partial genome sequencing. Disease pathology was described via histology. The isolate from Blue Catfish was used in challenge experiments to determine if it was pathogenic to Rainbow Trout. Multilocus sequencing typing was conducted using the PubMLST database. Results Biochemical testing was consistent with Y. ruckeri . A draft genome of the Y. ruckeri isolate was assembled based on Oxford Nanopore Technology sequencing and identified a single genomic replicon (3,791,418 bp) consistent in size to other Y. ruckeri genomes and a pLT plasmid (60, 933 bp). The challenge study demonstrated no significant virulence of this isolate for Rainbow Trout ( Y. ruckeri ). This isolate was most similar to other strains isolated from ictalurids. Notably, the gyrase B gene from this isolate was identical to that of archived strains isolated from moribund Mississippi hybrid catfish aquaculture during 2016 and these isolates share identical PubMLST sequence type profiles. Similarly, they shared a pLT plasmid that differed by only 6 bp. This plasmid has never been reported from trout isolates and appears to be unique to ictalurids. Conclusions Analyses here provide preliminary genetic evidence that geographically distant (Maryland and Mississippi, USA) isolates of Y. ruckeri from ictalurids are genetically similar to each other and Y. ruckeri (strain SC09) that infects ictalurids in China. This strain is not a biothreat to Rainbow Trout at typical culture temperatures.

Journal of Aquatic Animal Health↗

Non-lethal detection of Renibacterium salmoninarum in Greenback Cutthroat Trout Oncorhynchus clarkii stomias comparing mucus, blood, and ovarian fluid samples to kidney tissues

Objective Renibacterium salmoninarum , the causative agent of bacterial kidney disease, poses a major threat to both wild and aquaculture salmonid populations. Traditional detection methods typically involve lethal sampling to collect kidney tissues but are often impractical for species of conservation concern. This study evaluates nonlethal sampling techniques for detecting R. salmoninarum in Greenback Cutthroat Trout Oncorhynchus clarkii stomias by comparing mucus, blood, and ovarian fluid samples to conventional kidney tissue. Methods During the 2019 spawning season, we collected samples from 781 adult fish and tested for R. salmoninarum via direct fluorescent antibody test (DFAT) and quantitative polymerase chain reaction (qPCR). Results A total of 25 and 256 kidney tissues were positive by DFAT and qPCR, respectively. Of the three nonlethal samples tested, mucus swabs showed the highest percent correlation for detection with positive kidney tissues (DFAT = 47.6%, qPCR = 41.7%). Blood and ovarian fluid samples showed a lower percent correlation with positive kidney tissues (blood: DFAT = 12.0%, qPCR = 1.2%; ovarian fluid: DFAT = 12.5%, qPCR = 21.4%). Conclusions Our results suggest that nonlethal mucus swabbing could serve as a practical alternative for monitoring R. salmoninarum, especially in conservation efforts where minimizing fish mortality is critical.

Colorado↗

Transcriptional changes in wild Yukon River Chinook Salmon associated with Ichthyophonus infections

Objective We compared differentially expressed genes in Chinook Salmon Oncorhynchus tshawytscha with three divergent Ichthyophonus statuses (undetected, subclinical infections, or clinical disease; n = 100) to investigate associated transcriptomic responses. Disease associated with the fish parasite Ichthyophonus sp. was first diagnosed in adult Chinook Salmon from the Yukon River in the late 1980s and has subsequently been implicated in premature host mortality. Methods Chinook Salmon tissue sample collections and Ichthyophonus infection data were leveraged from a multi-agency collaboration during summer 2022 at three locations along the main-stem Yukon River that spanned nearly 2,000 km of freshwater migration (lower, middle, and upper river). We sequenced the transcriptome and compared this to infection status based on routine diagnostic procedures. Results Among the 17,569 genes for which messenger RNA was detected, we identified a transcription signature in the skeletal muscle that was associated with Ichthyophonus infections and included 53 differentially expressed genes. The differentially expressed genes and their pathways included those known for involvement in immune functions, energy synthesis, cellular breakdown, and reproduction—all processes that are known to be influenced by senescence during spawning migrations. Conclusions Results demonstrate a clear transcriptional difference between diseased fish (clinical disease group) and those in which Ichthyophonus was undetected, including identifying candidate markers for infection in this population. These results provide a foundation for development of nonlethal biomarkers to evaluate potential Ichthyophonus infections in Chinook Salmon based on gene transcription, protein products, or gene variants (e.g., polymorphisms).

Journal of Aquatic Animal Health↗

Suppression of antibody-producing cells in rainbow trout spleen sections exposed to copper in vitro

Immunosuppression was demonstrated in sections of rainbow trout Oncorhynchus mykiss (formerly Salmo gairdneri ) spleens immunized in vitro and exposed in culture to different concentrations of copper chloride. The sections were immunized with dinitrophenyl-Ficoll and cultured in Eagle's minimum essential medium with 2% fetal calf serum; half of the medium was withdrawn and replaced every other day. The passive hemolytic plaque assay was used to determine the number of antibody-producing cells 10 d after injection. In the sections cultured with the high copper concentration (100 μg/mL), all cells died; at copper concentrations of 0.1–10 μg/mL, leukocytes remained viable, but fewer antibody-producing cells were present than in organ sections cultured in medium without copper. This in vitro method reduces the number of animals needed and the length of time required to determine toxicity and immunosuppression, and it provides information on the effects of certain environmental pollutants on fish.

Journal of Aquatic Animal Health↗

Biochemical and antigenic properties of the first isolates of infectious hematopoietic necrosis virus from salmonid fish in Europe

The first isolates of infectious hematopoietic necrosis virus (IHNV) recovered from rainbow trout Oncorhynchus mykiss (formerly Salmo gairdneri ) in France and Italy were compared to six representative strains from North America by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of virion polypeptides and neutralization by monoclonal antibodies (MAbs). All three IHNV isolates from Europe had similar polypeptide profiles when compared by SDS-PAGE. An analysis of the antigenic relatedness of the European isolates to representative strains from North America showed that they were clearly different from viruses obtained from salmonids in California. The RB/B5 MAb, which was developed against virus isolated from adult steelhead (anadromous rainbow trout) reared in central Oregon, neutralized all isolates examined. The 193–110/B4 MAb, developed against IHNV isolated from infected yearling rainbow trout in southern Idaho, neutralized all isolates tested except those from California. The SRCV/A4 MAb, developed against Sacramento River chinook virus (SRCV) isolated from adult spring chinook salmon O. tshawytscha in central California, was the least reactive, and strong neutralization was observed only with the SRCV strain of IHNV from California. However, partial reactivity of the virus isolates from France with the SRCV/A4 MAb distinguished them from the virus recovered from salmonids in Italy.

Journal of Aquatic Animal Health↗

Cytopathology and coagulopathy associated with viral erythrocytic necrosis in chum salmon

The 8-month cytopathologic progression of viral erythrocytic necrosis (VEN) disease in chum salmon Oncorhynchus keta is described. Single to multiple acidophilic, cytoplasmic viral inclusion bodies developed first in mature erythrocytes and then, within 1–2 months, all morphologically identifiable hemopoietic cell types contained VEN inclusions. Cytologic analysis indicated that multinucleate giant erythroblasts, ineffective erythropoiesis, and abnormal erythroid cell maturation occurred. A significant increase in blood coagulation time occurred concomitantly. This severe and chronic blood dyscrasia accounts for some of the pathophysiologic sequelae previously observed.

Journal of Aquatic Animal Health↗

Enhanced detection of infectious hematopoietic necrosis virus by pretreatment of cell monolayers with polyethylene glycol

To improve quantification of very low levels of infectious hematopoietic necrosis virus (IHNV) in samples of tissue, ovarian fluid, or natural water supplies, we tested the ability of polyethylene glycol (PEG) to enhance the sensitivity and speed of the plaque assay system. We compared 4, 7, and 10% solutions of PEG of molecular weight 6,000, 8,000, or 20,000 applied at selected volumes and for various durations. When cell monolayers of epithelioma papulosum cyprini (EPC), fathead minnow (FHM), chinook salmon embryo (CHSE-214), and bluegill fry (BF2) were pretreated with 7% PEG-20,000, they produced 4-17-fold increases in plaque assay titers of IHNV. The plaque assay titers of viral hemorrhagic septicemia virus, chum salmon reovirus, and chinook salmon paramyxovirus were also enhanced by exposure of CHSE-214 cells to PEG, but the titers of infectious pancreatic necrosis virus and Oncorhynchus masou virus were not substantially changed. Plaques formed by IHNV on PEG-treated EPC cells incubated at 15°C had a larger mean diameter at 6 d than those on control cells at 8 d; this suggests the assay could be shortened by use of PEG. Pretreatment of EPC cell monolayers with PEG enabled detection of IHNV in some samples that appeared negative with untreated cells. For example, when ovarian fluid samples from chinook salmon Oncorhynchus tshawytscha were inoculated onto untreated monolayers of EPC cells, IHNV was detected in only 11 of 51 samples; 17 of the samples were positive when PEG-treated EPC cells were used. PDF

Journal of Aquatic Animal Health↗

Adverse effects of heated Bouin's solution on fish preservation

Rainbow trout Oncorhynchus mykiss (formerly Salmo gairdneri ) and lake trout Salvelinus namaycush preserved by immersion in Bouin's solution at 48°C became severely deformed due to muscle contraction. This was reflected in tissue changes, indicating that this technique induces undesirable artifacts that are likely to interfere with histological examination.

Journal of Aquatic Animal Health↗

Ultrastructure of the spleen and head kidney of striped bass

The spleen and head kidney of striped bass Morone saxatilis were examined by electron microscopy, including examination of ultrastructural localization of peroxidase. The spleen is composed of a loosely organized reticulum that supports the red and white pulp. The principal hematopoietic and mature blood cells observed were erythrocytes, erythroblasts, lymphocytes, macrophages, thrombocytes, and aggregates of pigmented macrophages. In addition, limited numbers of neutrophils, neutrophilic myelocytes (immature neutrophils), plasma cells, eosinophils and monocytes were identified. Reticular cells and fibers appeared to provide a structural framework for both cells and blood sinuses in the spleen. Like the spleen, the head kidney contained erythrocytes, macrophages, monocytes, lymphocytes, and other less frequently observed cell types. A distinct difference in the cytologic composition of the two organs, however, was in the greater number of neutrophilic myelocytes found in the head kidney, where these cells were often arranged in groups. Positive peroxidase reactions were observed for neutrophils, neutrophilic myelocytes, macrophage phagolysosomes, and pigmented macrophages in both the spleen and head kidney.

Journal of Aquatic Animal Health↗