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Globally, Atlantic salmon (Salmo salar Linnaeus) aquaculture is now routinely affected by amoebic gill disease (AGD; Neoparamoeba perurans). The disease proliferates throughout the summer and is implicated in decreasing tolerance of salmon to environmental perturbations, yet little empirical evidence exists to support these observations. Using salmon acclimated to 15 or 19 °C, our aim was to determine the effects of clinically light-moderate (industry-relevant) AGD on metabolism (ṀO 2rest and ṀO 2max ), aerobic scope (ṀO 2max - ṀO 2rest ), excess post-exercise oxygen consumption (EPOC), and hypoxia tolerance. An increase in ṀO 2rest (~8% and ~ 13% increase within the 15 and 19 °C acclimation groups, respectively) with increasing disease signs demonstrated an increase in baseline energy requirements as the disease progressed. Conversely, ṀO 2max remained stable at both temperatures (~364 mg O 2 kg -1  h -1 ), resulting in a decline in aerobic scope by 13 and 19% in the 15 and 19 °C groups, respectively. There was evidence of a decrease in hypoxia tolerance as the dissolved oxygen concentrations at loss of equilibrium increased by ~8% with more severe lesion coverage of the gills. These results suggest an increase in basal energy requirements and reduction in hypoxia tolerance as AGD proliferates, lending support to the idea that AGD reduces environmental tolerance. However, the lack of an effect of acclimation temperature indicates that the temperature-disease interaction may be more complicated than currently thought., (Copyright © 2021. Published by Elsevier Inc.)

Aeromonas hydrophila is an opportunistic bacteria with an overwhelming impact on fish farming industry especially with upraising of drug resistant mutants. This study aimed to evaluate and compare the therapeutic and side effects of levofloxacin (LEV), chitosan-nanoparticles (CNPs), and fructooligosaccharides (FOS) in control of this infection in tilapia. A total of 160 Nile-tilapia divided into 8-groups; G1: negative-control, G2: infected-control, G3: non-infected-(levofloxacin (LEV) 10 mg/kg bwt), G4: non-infected-(chitosan-nanoparticles (CNPs) 1 g/kg ration), G5: non-infected-(fructooligosaccharides (FOS) 20 g/kg ration), G6: infected-LEV, G7: infected-CNPs and G8: infected-FOS for 7 days. MICs were (0.125 μg/ml and 1.25 mg/ml) for LEV and CNPs respectively. No mortalities or significant adverse effects were recorded in non-infected treated-groups while infected were (20%) LEV, (30%) CNPs, (40%) FOS and (70%) G2. Aspartate aminotransferase (AST), alanine aminotransferase (ALT) and alkaline phosphatase (ALP) decreased by LEV and CNPs than FOS while all increased total protein (TP) and albumin than G2. Malondialdehyde (MDA) significantly decreased and superoxide dismutase (SOD) and reduced glutathione (GSH) increased in all infected-treated groups than G2 in various degrees. Urea and creatinine descending order were FOS, LEV then CNPs decreased significantly than G2. LEV musculature residues, using HPLC, decreased gradually till the 5th day; 621.00 ± 0.66, 270.00 ± 0.48 then 64.00 ± 0.40, and 471.00 ± 0.79, 175.00 ± 0.52 ppb then not detected at 1st, 3rd, and 5th days of withdrawal in non-infected and infected groups respectively. Finally, LEV and CNPs were superior as bactericidal, decreasing mortalities and enzyme activities while CNPs and FOS increased performance, non-specific immunity, and antioxidant biomarkers., (Copyright © 2021. Published by Elsevier Inc.)

Ferritin H can participate in the regulation of fish immunity. Tissue-specific analysis revealed that the highest expressions of Ferritin H in parental species were observed in spleen, while peaked level of Ferritin H mRNA in hybrid fish was observed in liver. In addition, A. hydrophila challenge could sharply enhance their Ferritin H mRNA expression in liver, kidney and spleen. To further investigate their roles in immune regulation, their Ferritin H fusion proteins were produced in vitro. Ferritin H fusion proteins could exhibit a direct binding activity to A. hydrophila and endotoxin in a dose-dependent manner, restrict dissemination of A. hydrophila to tissues and abrogate inflammatory cascades. Moreover, treatment with Ferritin H fusion proteins could reduce A. hydrophila-induced lipid peroxidation. These results indicated that Ferritin H in hybrid fish elicited a similar immune regulation of A. hydrophila-induced inflammatory signals in comparison with those of its parents., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Effect of selenium and acidification in freshwater environment was assessed solitary but no reports are available on the impacts of both factors act together. In the present study, effects of combined simultaneous exposure to selenium (Se) and low pH were assessed in Mozambique tilapia, Oreochromis mossambicus. Responses were measured based on antioxidant defenses (enzymatic SOD, CAT, GPx and non-enzymatic GSH), biotransformation enzyme (GST), metallothionein levels (MT), oxidative damage (LPO, CP), Na + /K + -ATPase (NKA) activity in gills and liver tissues and neurotoxicity (acetylcholinesterase, AChE) response in brain tissue. Fish were exposed to combined treatment at different pH levels (7.5, control (optimum pH for tilapia growth); 5.5, low pH) and Se concentrations (0, 10, and 100 μg L -1 ). Toxicity levels of Se were not significantly different under control and low pH indicating that pH did not affect Se toxicity. Levels of GSH and MT were enhanced in Se-exposed fish at both pH. Combined effects of high Se concentration and low pH decreased SOD and CAT activities and increased those of GPx and GST. However, organisms were not able to prevent cellular damage (LPO and CP), indicating a condition of oxidative stress. Furthermore, inhibition of Na + /K + -ATPase activity was showed. Additionally, neurotoxicity effect was observed by inhibition of cholinesterase activity in organisms exposed to Se at both pH conditions. As a result, the combined stress of selenium and freshwater acidification has a slight impact on antioxidant defense mechanisms while significantly inhibiting cholinesterase and Na+/K + -ATPase activity in fish. The mechanisms of freshwater acidification mediating the toxic effects of trace non-metal element on freshwater fish need to investigate further., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Chlorpyrifos (CPF) is an organophosphate insecticide and can cause cell death of animals. In the study, the common carp were exposed to CPF at 0 μg/L (the control group), 1.16 μg/L (the low dose group), 11.6 μg/L (the medium dose group), and 116 μg/L (the high dose group), respectively. The carp were euthanized at the 30th day and gills were collected immediately. The ultrastructural and histopathological observations showed obvious necrosis characteristics and inflammatory injury in the CPF-treated groups. CPF exposure activated the MAPK pathway, in which the mRNA and protein expressions of extracellular signal-regulated (ERK), p38 MAP kinase (p38), and c-Jun N-terminal kinase (JNK) were increased; the mRNAs and proteins of NF-κB and TNF-α were activated; and the mRNAs and proteins of necroptosis related genes were changed (the mRNA and protein expression of RIPK1, RIPK3, MLKL, and FADD were increased and caspase-8 was decreased) with concentration dependency. Taken together, we concluded that CPF exposure activated the MAPK/NF-κB/TNF-α pathway, promoted inflammatory injure and evoked necroptosis in common carp gills. In addition, CPF-induced inflammation and necroptosis was concentration dependency. The toxic effects of CPF on gills provided data for both aquaculture and toxicological studies., (Copyright © 2021. Published by Elsevier Inc.)

Metacercariae of trematodes from the genus Diplostomum are major helminth pathogens of freshwater fish, infecting the eye or the brain. The taxonomy of the genus Diplostomum is complicated, and has recently been based mainly on the molecular markers. In this study, we report the results of the morphological and molecular genetic analysis of diplostomid metacercaria from the brain of the minnow Phoxinus phoxinus from three populations in Fennoscandia (Northern Europe) and one population in Mongolia (East Asia). We obtained the data on the polymorphism of the partial mitochondrial cox1 gene and ribosomal ITS1-5.8S-ITS2 region of these parasites. РСА-based morphological analysis revealed that the parasites in the Asian and the European groups of Diplostomum sp. were distinctly different. Metacercariae from the brain of Mongolian minnows were much larger than those from the brain of Fennoscandian minnows but had much fewer excretory granules. Considering that the two study regions were separated by a distance of about 4500 km, we also tested the genetic homogeneity of their host, the minnow, using the mitochondrial cytb gene. It was shown that Diplostomum-infected minnows from Mongolia and Fennoscandia represented two previously unknown separate phylogenetic lineages of the genus Phoxinus. Both molecular and morphological analysis demonstrated that the parasites from Fennoscandia belonged the species Diplostomum phoxini, while the parasites from Mongolia belonged to a separate species, Diplostomum sp. MТ.Each of the two studied Diplostomum spp. was associated with a specific, and previously unknown, genealogical lineage of its second intermediate host, P. phoxinus., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Cyprinid herpesvirus 3 (CyHV-3) causes high mortality in carp. Emodin has been shown of the effects of antioxidant, anti-inflammatory and antiviral. In present study, we investigated the preventive effects and mechanism of emodin on CyHV-3 infection. The ornamental koi carp (Cyprinus carpio haematopterus) were intraperitoneally injected with emodin (10 mg/kg, 20 mg/kg, or 40 mg/kg). 72 h later, an intraperitoneal injection of CyHV-3 was administered, and collected the samples one week later to detect the antioxidant parameters, antioxidant genes, inflammatory genes and to perform histopathology assays. The results showed that emodin significantly suppressed CyHV-3 replication (P < 0.05), improved the koi survival rate and slowed the damage caused by CyHV-3. Emodin treatment increased the antioxidant activity and decreased the lipid peroxidation level of the koi. Compared to the CyHV-3 group, emodin treatment resulted in the same antioxidant parameters after CyHV-3 infection. Emodin treatment activated the Nuclear factorery throid 2-related factor 2/Kelch-like ECH-associated protein 1-antioxidatant response element (Nrf2/Keap1-ARE) pathway and upregulated the expression of heme oxygenase 1 (HO-1), superoxide dismutase (SOD), and catalase (CAT) in the hepatopancreas after CyHV-3 infection. Emodin activated the nuclear factor kappa-B (NF-κB) pathway and decreased the expression of interleukin-6 (IL-6), interleukin-8 (IL-8), and tumour necrosis factor-α (TNF-α) in the koi induced by CyHV-3. In conclusion, emodin treatment can suppress CyHV-3 replication and reduce the mortality of koi caused by CyHV-3. Emodin improves antioxidant function, relieves oxidative stress and inflammation cytokines via Nrf2/Keap1-ARE and NF-κB pathways, and protects against the adverse effects induced by CyHV-3., (Copyright © 2021. Published by Elsevier Inc.)

Tilapia lake virus (TiLV; genus: Tilapinevirus, family: Amnoonviridae) is a recently characterised enveloped virus with a linear, negative-sense single-stranded RNA genome, which causes high mortality in tilapia species. In the present study, we demonstrated that zebrafish (Danio rerio) larvae are susceptible to TiLV infection upon systemic injection. TiLV replicated in zebrafish larvae and caused their high mortality (of about 70%). Histopathological examination revealed that TiLV infection caused pathological abnormalities in zebrafish larvae that were well visible within the brain. Moreover, gene expression analysis revealed that TiLV infection induced up-regulation of the expression of the immune-related genes encoding pathogen recognition receptors involved in sensing of viral dsRNA (rig-I (ddx58), tlr3, tlr22), transcription factors (irf3, irf7), type I interferon (infϕ1), antiviral protein (mxa), and pro-inflammatory cytokine (il-1β). We also demonstrated the protective role of the recombinant zebrafish IFNϕ1 on the survival of zebrafish larvae during TiLV infection. Our results show the importance of type I IFN response during TiLV infection in zebrafish larvae and demonstrate that zebrafish is a good model organism to study interactions between TiLV - a newly emerging in aquaculture virus, and fish host., (Copyright © 2020 The Authors. Published by Elsevier Ltd.. All rights reserved.)

The sciaenid Spotted Seatrout (Cynoscion nebulosus) are infected by blood flukes (Cardicola spp.). A 2 year survey in estuaries of South Carolina, USA, showed that adult flukes and granulomas occurred throughout the year but their prevalence was highest in summer (61% and 84%, respectively), indicating an unusually high level of infection for wild fish. Granulomas remained after adult flukes could no longer be found. PCR-Restriction Fragment Length Polymorphism (RFLP) of a subsample of specimens allowed identification of Cardicola laruei as the only species infecting these seatrout during the period of study. Mean intensity of infection by flukes was higher in female seatrout, suggesting endocrine and/or immune system involvement. The prevalence of granulomas declined sharply in winter, indicating possible mortality of infected seatrout as this species is known to be cold-sensitive. Granulomas were studied using histology, immunohistochemistry, and transmission electron microscopy. Eggs were encapsulated by an inner core of dark epithelioid cells, and an outer core of large epithelioid cells undergoing epithelialization. Fibrosis was observed around granulomas and some granulomas detached from the surrounding damaged myocardium. Numerous inflammatory cells appeared mobilised around granulomas and pathology could be severe, in some cases showing grossly visible blister-like extrusions scattered in the damaged epicardium. At the gross level, some granulomas possessing eggs with live miracidia were observed at the surface of the epicardium. These findings suggest that granulomas carrying both dead and live eggs can clear the fish heart by host-mediated transport through the myocardium, as is known to occur in related human Schistosoma infections., (Copyright © 2020 Australian Society for Parasitology. Published by Elsevier Ltd. All rights reserved.)

Cyprinid herpesvirus 3 (CyHV-3) or koi herpesvirus is a global pathogen causing mass mortality in koi and common carp, against which improved vaccines are urgently needed. In this study we investigated the role of four nonessential, but immunogenic envelope glycoproteins encoded by members of the ORF25 gene family (ORF25, ORF65, ORF148 and ORF149) during CyHV-3 replication. Single deletion of ORF65 did not affect in vitro replication, and deletion of ORF148 even slightly enhanced virus growth on common carp brain (CCB) cells. Deletions of ORF25 or ORF149 led to reduced plaque sizes and virus titers, which was due to delayed entry into host cells. An ORF148/ORF149 double deletion mutant exhibited wild-type like growth indicating opposing functions of the two proteins. Electron microscopy of CCB cells infected with either mutant did not indicate any effects on virion formation and maturation in nucleus or cytoplasm, nor on release of enveloped particles. The ORF148, ORF149 and double deletion mutants were also tested in animal experiments using juvenile carp, and proved to be insufficiently attenuated for use as live virus vaccines. However, surviving fish were protected against challenge with wild-type CyHV-3, demonstrating that these antibody inducing proteins are dispensable for an efficient immune response in vivo., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Piscirickettsia salmonis is a highly aggressive facultative intracellular bacterium that challenges the sustainability of Chilean salmon production. Due to the limited knowledge of its biology, there is a need to identify key molecular markers that could help define the pathogenic potential of this bacterium. We think a model system should be implemented that efficiently evaluates the expression of putative bacterial markers by using validated, stable, and highly specific housekeeping genes to properly select target genes, which could lead to identifying those responsible for infection and disease induction in naturally infected fish. Here, we selected a set of validated reference or housekeeping genes for RT-qPCR expression analyses of P. salmonis under different growth and stress conditions, including an in vitro infection kinetic. After a thorough screening, we selected sdhA as the most reliable housekeeping gene able to represent stable and highly specific host reference genes for RT-qPCR-driven P. salmonis analysis., (Copyright © 2018 Elsevier B.V. All rights reserved.)

A batch of wild common carp and largemouth bass died in Andong, Gyeongsangbuk-do province, South Korea, in 2016. Moribund fish showed typical signs of spring viremia of carp (SVC) disease, which causes acute hemorrhage in the skin and ascites. Thus far, SVC disease has been detected in several regions of the world but never in South Korea. Suspecting the infectious agent to be the SCV virus (SVCV), the moribund fish were sampled and screened. The isolated virus developed a cytopathic effect in EPC cells. Both viral isolates from the common carp (ADC-SVC2016-1) and largemouth bass (ADC-SVC2016-3) were identical in terms of their genome sequence, which were 11,034 bp nucleotides in length. Genome comparison exhibited greater sequence similarity with the Asian SVCV sequences available at NCBI. Phylogenetic analysis revealed that the Korean SVCV isolates were clustered within the Asian clade. More specifically, evolutionary analysis by using the P gene sequences showed that the Korean isolates were sub-cladded within the Iai genogroup but diverged from Chinese strains of SH150514 and SH160901. The Korean isolates shared more than 98% sequence similarity with the two Chinese SVCV isolates, suggesting that the spread of SVCV originated from China. The isolated virus had cytopathic effects on EPC cells. Virus transmission studies showed that the virus exhibited the highest virulence at 15 °C, which was also dependent on the method used, with the injection method being better than the immersion and cohabitation methods. This is the first study to document that Korean SVCV isolates may be epizootic in wild common carp and other susceptible animal populations in South Korea., (Copyright © 2018 Elsevier B.V. All rights reserved.)

As one of nine piscine viruses recognized by the International Office of Epizootics, spring viraemia of carp virus (SVCV) is an important pathogen bringing high mortality to cyprinids. Up to now, there is no approved therapy on SVCV, making them strong public health threat in aquaculture. In this study, the anti-SVCV activities of 12 plant crude extracts were investigated by using epithelioma papulosum cyprini (EPC) cells. Among these plants, Psoralea corylifolia Linn. showed the highest inhibition on SVCV replication, with an inhibitory percentage of 67.98%. Further studies demonstrated that bavachin (BVN), one of the major constituents of Psoralea corylifolia Linn., was also highly effective to SVCV infection. The half maximal inhibitory concentrations (IC 50 ) of BVN on SVCV glycoprotein and nucleoprotein expression were 0.46 (0.29-0.73) and 0.31 (0.13-0.55) mg/L, respectively. In addition, SVCV-induced apoptosis which may be negative to SVCV replication was inhibited by BVN. The apoptotic cells were decreased 21.42% for BVN compared with SVCV group. These results indicated that the inhibition of BVN on SVCV replication was, in some extent, via blocking SVCV induced apoptosis. Furthermore, cellular morphological damage induced by SVCV was also blocked by BVN treatment. Mechanistically, BVN did not affect SVCV infectivity and cannot be used for prevention of SVCV infection. Time-of-addition and viral binding assays revealed that BVN mainly inhibited the early events of SVCV replication but did not interfere with SVCV adsorption. In conclusion, BVN was considered to develop as a promising agent to treat SVCV infection., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Koi herpesvirus (KHV) is a highly pathogenic virus of common carp and koi. KHV becomes latent in recovered koi or exposed koi without symptoms, and the latent infection can reactivate under stress conditions. KHV reactivation from latency often occurs when water temperature rapidly rises above 17°C. Dissolved O 2 is lower at ≥17°C than at non-stress temperatures ≤15°C. To determine whether reduced dissolved O 2 level has a role in KHV reactivation during temperature stress, KHV reactivation was investigated in KHV latently infected koi (KHV + koi) under stress temperatures by maintaining dissolved O 2 consistent with the O 2 level at 15°C. There was no significant difference in the amount of reactivated virus between KHV + koi maintained with and without O 2 supplementation during temperature stress. Both handling and sampling were found to be stressful to koi and can contribute to KHV reactivation from latency. There was an increase in KHV genome within white blood cells (WBC) during KHV reactivation, which is about 3-4 fold higher than the amount of KHV genome detectable in WBC during the latency stage. At day 15 post-temperature stress (PTS), inflammation and necrosis were observed in multiple tissues, especially in the gills, eye, intestine, skin and kidney. KHV DNA was also detectable in multiple tissues on days 6, 9 and 15 PTS. Following day 3 PTS, the plasma cortisol levels were higher than that observed in koi before temperature stress, suggesting that KHV reactivation is associated with physiological stress in KHV + koi., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Infectious hematopoietic necrosis virus (IHNV) is the etiological agent of a contagious disease (IHN) mainly in salmonid fish. In the present study, we isolated and identified IHNV in trout fry from Iranian trout farms with unexplained high mortality in 2016. The affected fry showed cumulative mortality of 90% with the gross pathological signs including exophthalmia and hemorrhage of the eye, skin darkening, abdominal distension, ulceration of the snout, and the visceral pallor and yellowish fluid in the intestine. Histopathological examination revealed marked necrosis in the anterior kidney, liver and spleen with the intracytoplasmic inclusion bodies in the liver sections. Also, intranuclear inclusion body and marginated chromatin were observable in the hematopoietic cells of the kidney. The homogenates tissues of infected fry induced IHNV-positive cytopathic effects (CPE) in EPC cells and confirmed by RT-PCR reactions and sequencing. Phylogenetic analysis revealed the Iranian IHNV isolates belonged to the European (E) genogroup with 100% identity to some Italian isolates. This is the first report of IHNV infection in farmed trout fry in Iran describing the viral isolation, clinical symptoms, histopathological findings, molecular confirmation, and genetic analysis suggestion of the specific country of origin., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Background: DNA vaccination has emerged as a promising tool against infectious diseases of farmed fish. Oral delivery allows stress-free administration that is ideal for mass immunization and of paramount importance for infectious pancreatic necrosis (IPN) and other viral disease that affect young salmonids and cause economic losses in aquaculture worldwide., Methods: We describe the development and in vivo assessment of an "in-feed" formulation strategy for oral immunization with liposomal DNA vaccines, by delivering a vaccine construct coding for an immunogenic region of the VP2 capsid protein. A challenge against IPNV was carried out to determine the vaccine efficacy, by comparing the mortality of pre-smolt Atlantic salmons immunized and non-immunized with the oral vaccine. The antibody response (ELISA) and hematological parameters after immunization were examined, as well as the vaccine effect on the growth and internal structures of fry salmons (histological analysis). The vaccine distribution in the experimental tank after oral administration was investigated by HPLC and PCR amplification., Results: The oral vaccine induced detectable levels of VP2-specific antibodies and conferred significant protection following IPNV challenge, with relative percent survivals (RPS) of 58.2%, for single dose (1mg pDNA /kg fish ⋅d), and 66% for double dose (2mg pDNA /kg fish ⋅d). We further provide evidence in favour of the vaccine safety to fish and demonstrated absence of pDNA in the tank water, but presence of vaccine residues in faeces and unconsumed feed sediments (solid wastes)., Conclusion: The delivery platform for liposomal DNA vaccination via feed was successfully proved against IPNV in Atlantic salmon, showing the oral vaccine to be immunogenic and safe for fish, and providing significant protection after oral administration. The "in-feed" technology for oral DNA vaccination holds potential to be applied against IPNV and other pathogens that currently threaten the aquaculture worldwide., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Enteromyxum scophthalmi, an intestinal myxozoan parasite, is the causative agent of a threatening disease for turbot (Scophthalmus maximus, L.) aquaculture. The colonisation of the digestive tract by this parasite leads to a cachectic syndrome associated with high morbidity and mortality rates. This myxosporidiosis has a long pre-patent period and the first detectable clinical and histopathological changes are subtle. The pathogenic mechanisms acting in the early stages of infection are still far from being fully understood. Further information on the host-parasite interaction is needed to assist in finding efficient preventive and therapeutic measures. Here, a RNA-seq-based transcriptome analysis of head kidney, spleen and pyloric caeca from experimentally-infected and control turbot was performed. Only infected fish with early signs of infection, determined by histopathology and immunohistochemical detection of E. scophthalmi, were selected. The RNA-seq analysis revealed, as expected, less intense transcriptomic changes than those previously found during later stages of the disease. Several genes involved in IFN-related pathways were up-regulated in the three organs, suggesting that the IFN-mediated immune response plays a main role in this phase of the disease. Interestingly, an opposite expression pattern had been found in a previous study on severely infected turbot. In addition, possible strategies for immune system evasion were suggested by the down-regulation of different genes encoding complement components and acute phase proteins. At the site of infection (pyloric caeca), modulation of genes related to different structural proteins was detected and the expression profile indicated the inhibition of cell proliferation and differentiation. These transcriptomic changes provide indications regarding the mechanisms of parasite attachment to and invasion of the host. The current results contribute to a better knowledge of the events that characterise the early stages of turbot enteromyxosis and provide valuable information to identify molecular markers for early detection and control of this important parasitosis., (Copyright © 2016 Australian Society for Parasitology. Published by Elsevier Ltd. All rights reserved.)

Amoebic Gill Disease affects farmed salmonids and is caused by Neoparamoeba perurans. Clonal cultures of this amoeba have been used for challenge experiments, however the effect of long-term culture on virulence has not been investigated. Here we show, using in vitro and in vivo methods, that a clone of N. perurans which was virulent 70 days after clonal culture lost virulence after 3 years in clonal culture. We propose that this is related either to the lack of attachment to the gills or the absence of an extracellular product, as shown by the lack of cytopathic effect on Chinook salmon embryo cells. The avirulent clonal culture of N. perurans allowed us to propose two potential virulence mechanisms/factors involved in Amoebic Gill Disease and is an invaluable tool for host-pathogen studies of Amoebic Gill Disease., (Copyright © 2015 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.)

The presence of a new microsporidium is believed to be responsible for an emaciative syndrome observed in farmed gilthead sea bream (Sparus aurata) from different facilities along the Spanish coast. Infected fish were approximately half the average weight and significant mortality was attributed to the condition in some facilities. Clinical signs included anorexia, cachexia and pale internal organs. The microsporidium was found mainly in the intestinal mucosa and occasionally in the submucosa. Morphological, histopathological, ultrastructural and molecular phylogenetic studies were conducted to characterise this organism. This microsporidium undergoes intranuclear development in rodlet cells and enterocytes, and cytoplasmic development mainly in enterocytes and macrophages. The nucleus-infecting plasmodium contains several diplokarya and displays polysporous development which occurs without an interfacial envelope. In the host cell cytoplasm, the parasite develops within a membrane-bound matrix. In both infection locations, the polar tube precursors appear as disks, first with lucent centres, then as fully dense disks as they fuse to form the polar filament, all before division of the plasmodium into sporoblasts. Up to 16 intranuclear spores result from the sporogonic development of a single plasmodium, whereas more than 40 spores result from several asynchronous reproductive cycles in the cytoplasmic infection. Fixed spores are ellipsoidal and diplokaryotic, with five to six coils of an isofilar polar filament in a single row. ssrDNA-based molecular phylogenetic inference places this parasite as a sister clade to crustacean-infecting species of the Enterocytozoonidae and closer to Enterocytozoon bieneusi than to other fish-infecting microsporidians presenting intranuclear development, i.e. Nucleospora, Paranucleospora and Desmozoon. Our studies result in the erection of a new species, Enterospora nucleophila, within the family Enterocytozoonidae, and the description of this family is amended accordingly to accommodate the features of known species assigned to it. Severe histopathological damage occurs in intense infections and this microsporidian is considered a serious emerging threat in sea bream production., (Copyright © 2013 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.)

Impulsive pile driving sound can cause injury to fishes, but no studies to date have examined whether such injuries include damage to sensory hair cells in the ear. Possible effects on hair cells were tested using a specially designed wave tube to expose two species, hybrid striped bass (white bass Morone chrysops × striped bass Morone saxatilis) and Mozambique tilapia (Oreochromis mossambicus), to pile driving sounds. Fish were exposed to 960 pile driving strikes at one of three treatment levels: 216, 213, or 210dB re 1 μPa(2)·s cumulative Sound Exposure Level. Both hybrid striped bass and tilapia exhibited barotraumas such as swim bladder ruptures, herniations, and hematomas to several organs. Hybrid striped bass exposed to the highest sound level had significant numbers of damaged hair cells, while no damage was found when fish were exposed at lower sound levels. Considerable hair cell damage was found in only one out of 11 tilapia specimens exposed at the highest sound level. Results suggest that impulsive sounds such as from pile driving may have a more significant effect on the swim bladders and surrounding organs than on the inner ears of fishes, at least at the sound exposure levels used in this study., (© 2013.)

Flavobacterium psychrophilum is a well-known pathogen causing significant problems in aquaculture worldwide. In recent years an increasing number of disease outbreaks caused by F. psychrophilum has been reported on juvenile and post smolts of rainbow trout (Oncorhynchus mykiss) in Norway. The current study was performed to assess the efficacy of two autogenous water-in-oil formulated vaccines containing whole cell antigens of F. psychrophilum to induce protective immunity against challenge. The vaccines were formulated either as multivalent (FLAVO AVM6) or divalent (FLAVO IPN) and administered by the intraperitoneal route. Intramuscular challenge with a field strain of F. psychrophilum was carried out 552 day degrees post vaccination, at a time when the FLAVO AVM6 and FLAVO IPN vaccinated groups had significantly higher antibody responses compared to the negative control. Results from the challenge study showed that the multivalent and the divalent vaccines had capacity to induce significant protection, with RPS60>87% and RPSend>77.5% for both vaccines. The high level of protection seen in the vaccinated groups was also reflected in the reduced ulceration rates observed at the injection site. Combining our results demonstrate that vaccination with FLAVO AVM6 and FLAVO IPN induces responses capable of protecting rainbow trout against infections with F. psychrophilum., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

The rapid doubling time and genetic relatedness of the fish pathogen Mycobacterium marinum to Mycobacterium tuberculosis has rendered the former an attractive model for investigating mycobacterial host-pathogen interactions. We employed the M. marinum-goldfish infection model to investigate the in vivo immune responses to this pathogen in the context of a natural host. Histological analysis revealed mycobacterial infiltrates in goldfish kidney and spleen tissues, peaking 28 days post infections (dpi). Quantitative gene expression analysis showed significant increases of mRNA levels of pro-inflammatory cytokines (IFNγ, IL-12p40, IL-1β1) and cytokine receptors (IFNGR1-1, TNFR2) at 7 dpi. Conversely, the gene expression levels of key anti-inflammatory cytokines TGFβ and IL-10 were elevated at 14 dpi. Furthermore, M. marinum infections markedly increased the cytokine-primed oxidative burst responses of isolated kidney phagocytes at 7 but not 56 dpi. We believe that the M. marinum-goldfish infection model will be invaluable in furthering the understanding of the mycobacterium host-pathogen interface., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Amoebic gill disease (AGD) in marine farmed Atlantic salmon is of growing concern worldwide and remains a significant health issue for salmon growers in Australia. Until now the aetiological agent, Neoparamoeba perurans, has not been amenable to in vitro culture and therefore Koch's postulates could not be fulfilled. The inability to culture the amoeba has been a limiting factor in the progression of research into AGD and required the maintenance of an on-going laboratory-based infection to supply infective material. Culture methods using malt yeast agar with sea water overlaid and subculturing every 3-4 days have resulted in the establishment of a clonal culture of N. perurans, designated clone 4. Identity of the amoeba was confirmed by PCR. After 70 days in culture clone 4 infected Atlantic salmon, causing AGD, and was re-isolated from the infected fish. Diagnosis was confirmed by histology and the infectious agent identified by PCR and in situ hybridisation using oligonucleotide primers and probes previously developed and specific to N. perurans. This study has fulfilled Koch's postulates for N. perurans as a causative agent of AGD and illustrates its free-living and parasitic nature., (Copyright © 2012 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.)

The annual fish Nothobranchius furzeri is the shortest-lived vertebrate which can be cultured in captivity. Here, we performed a histopathological analysis of age-related lesions in this species. Post-mortem analysis revealed lesions in liver (~90%), kidney (~75%), heart (~70%) and gonads (~40%) which are similar to those previously described in the small teleost Poecilia reticulata. In addition, a high incidence of neoplasias was observed in liver (~35%) and kidney (~25%). Different laboratory strains of N. furzeri show large genetic differences in longevity. Cross-sectional analysis revealed a clear age-dependent increase in the incidence of liver neoplasias which was accelerated in a short-lived strain. Cross-sectional analysis of gonads revealed sex-specific differences in the occurrence of lesions, with males being more severely affected than females. In conclusion, our analysis demonstrates that short life span in N. furzeri is a consequence of a typical teleost aging process which determines systemic failure of homeostasis functions rather than of a single organ or apparatus. Unlike other teleosts, however, this scenario is reinforced by high incidence of age-dependent neoplasias, making this species a promising model to analyze the molecular pathways of age-dependent spontaneous tumorigenesis., (Copyright © 2010 Elsevier Inc. All rights reserved.)

The metacercarial stage of trematodes is typically considered an encysted, developmentally quiescent, resting stage. Yet the metacercariae of some species of strigeoid trematode undergo extravagant development within specific tissues of their second intermediate host. Our understanding of patterns of migration, site selection and development of these types of metacercariae is known for only a few species. In this study, we characterize the invasion and development of Ornithodiplostomum sp. metacercariae in their second intermediate host, the fathead minnow, Pimephales promelas. Diplostomules completed their migration into the abdominal cavity between 15 min and 48 h p.i. Most diplostomules migrated along muscular and connective tissue then penetrated the peritoneal lining of the abdominal cavity en route to the liver or pancreas. Alternatively, some diplostomules migrated within the host's circulatory system, including the heart and arteries of the hepatic portal system. Metacercarial development in the liver and pancreas involved distinct growth, encystment and consolidation phases. Metacercarial volume increased 15-fold between 48 h and 4 weeks p.i., presumably due to absorptive and/or ingestive feeding activities within host tissues. By 2 weeks p.i., metacercariae were enveloped within a cyst wall and they were found loosely attached to the surfaces of internal tissues or unattached within the body cavity. These results emphasize the complex nature of metacercarial migration and growth and demonstrate that their growth and encystment phases occur within different habitats within their intermediate hosts., (Copyright © 2010 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.)

The myxozoan parasite Ceratomyxa shasta infects salmonids causing ceratomyxosis, a disease elicited by proliferation of the parasite in the intestine. This parasite is endemic to the Pacific Northwest of North America and salmon and trout strains from endemic river basins show increased resistance to the parasite. It has been suggested that these resistant fish (i) exclude the parasite at the site of invasion and/or (ii) prevent establishment in the intestine. Using parasites pre-labeled with a fluorescent stain, carboxyfluorescein succinimidyl diacetate (CFSE), the gills were identified as the site of attachment of C. shasta in a susceptible fish strain. In situ hybridization (ISH) of histological sections was then used to describe the invasion of the parasites in the gill filaments. To investigate differences in the progress of infection between resistant and susceptible fish, a C. shasta-susceptible strain of rainbow trout (Oncorhynchus mykiss) and a C. shasta-resistant strain of Chinook salmon (Oncorhynchus tshawytscha) were sampled at consecutive time points following exposure at an endemic site. Using ISH in both species, the parasite was observed to migrate from the gill epithelium into the gill blood vessels where replication and release of parasite stages occurred. Quantitative PCR verified entry of the parasite into the blood. Parasite levels in blood increased 4days p.i. and remained at a consistent level until the second week when parasite abundance increased further and coincided with host mortality. The timing of parasite replication and migration to the intestine were similar for both fish species. The field exposure dose was unexpectedly high and apparently overwhelmed the Chinook salmon's defenses, as no evidence of resistance to parasite penetration into the gills or prevention of parasite establishment in the intestine was observed., (Copyright 2010 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.)

The development of systemic autoimmunity may result as an undesired side-effect following vaccination, and this condition was recently shown to occur in farmed salmon (Salmo salar). Several of previously reported side-effects following vaccination of fish should therefore be reviewed in the light of this condition. Here, organs and pathological changes in three separate groups of fish severely affected by vaccination were investigated by different morphological methods (n=84). Granulomas or microgranulomas were observed at the injection site and in several organs. Mott cells were observed in all tissues examined. Pannus-like changes with lymphocyte infiltrates were observed in spines. In conclusion, the reactions following vaccination were of a systemic nature that may be explained by a pathogenetic mechanism caused by systemic autoimmunity., (Copyright 2010 Elsevier Ltd. All rights reserved.)

Edwardsiella tarda is a severe aquaculture pathogen that can infect many fish species and cause a systematic disease called edwardsiellosis, which can lead to high mortality under certain conditions. Currently, most vaccine candidates against edwardsiellosis are based on pathogenic E. tarda strains, which can be a concern in some cases. In this study, the vaccine potential of a natural E. tarda isolate, ATCC 15947, was examined in a Japanese flounder model. ATCC 15947 was found to be relatively avirulent to flounder but able to disseminate into and survive transiently in fish tissues following intraperitoneal (i.p.) injection. Fish vaccinated with ATCC 15947 via i.p. injection exhibited a high level of survival rate, which was increased to 100% by a booster injection. Fish vaccinated with ATCC 15947 orally in the form of alginate microspheres showed a moderate survival rate, while fish vaccinated with ATCC 15947 via the immersion route exhibited a low rate of survival. Following oral vaccination, ATCC 15947 could colonize transiently in the gut, liver, and spleen of the vaccinated fish. Both injection and oral vaccination with ATCC 15947 induced production of specific serum antibodies, the levels of which at different time points following vaccination were generally higher in fish vaccinated with ATCC 15947 via injection than in fish vaccinated with ATCC 15947 orally. Compared to control fish, fish vaccinated with ATCC 15947 showed enhanced serum bactericidal activity and significantly increased expression in genes encoding a number of immune-related factors. These results indicated that ATCC 15947 possesses good immunoprotective potential, which may be exploited in the development of E. tarda vaccines., (Copyright 2010 Elsevier Ltd. All rights reserved.)

Evidence for the presumed linkage between the enigmatic rodlet cells of fish and exposure to helminths is anecdotal and indirect. We evaluated the proliferation and development of rodlet cells in the optic lobes of fathead minnows exposed to cercariae of Ornithodiplostomum ptychocheilus. Mean rodlet cell densities (ca. 10/mm(2)) in the optic lobes were similar between unexposed controls and minnows with 1- and 2-week old infections. Rodlet cell densities increased at 4 weeks p.i., reaching maxima (ca. 200/mm(2)) at 6 weeks p.i., followed by a decline at 9 weeks. This temporal pattern of proliferation and maturation paralleled the development of metacercariae within the optic lobes. Unencysted metacercariae develop rapidly within tissues of the optic lobes for approximately 4 weeks after penetration by cercariae, then shift to the adjacent meninges to encyst. The former stage is associated with tissue damage, the latter with massive inflammation of the meninges. Thus, peak densities and maturation of rodlet cells correspond to the period when inflammation of the meninges caused by the large metacercarial cysts is at a maximum. Our results support recent contentions that rodlet cells comprise part of the host inflammatory defence response., (2009 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.)

Edwardsiella tarda is a severe aquaculture pathogen that can infect many different fish species cultured worldwide. Et49 is a major E. tarda antigen with weak immunoprotective potential. In this study, using Et49 as an example vaccine, the adjuvanticity of Freund's incomplete adjuvant (FIA), aluminum hydroxide, and aluminum phosphate adjuvant were evaluated in a Japanese flounder model. The results showed that the presence of FIA, aluminum hydroxide, and aluminum phosphate adjuvant increased the relative percent of survival of Et49-vaccinated fish by 47%, 19%, and 35%, respectively. Fish vaccinated with FIA-adjuvanted Et49 exhibited longer persistence of vaccine at the injection site and more severe intra-abdominal lesions than fish vaccinated with aluminum-adjuvanted Et49. Both aluminum adjuvants and, to a lesser degree, FIA augmented the production of specific serum antibodies, which reached the highest levels at 6 and 7 weeks post-vaccination. Passive immunization of Japanese flounder with sera from fish vaccinated with aluminum- and FIA-adjuvanted Et49 induced no protection against lethal E. tarda challenge. Examination of the transcription profile of immune-related genes showed that vaccination with aluminum-adjuvanted Et49 significantly enhanced the expression of the genes that are associated mainly with humoral immunity, whereas vaccination with FIA-adjuvanted Et49 induced the expression of a much broader spectrum of genes that are likely to be involved in humoral and innate cellular immunity. These results provide new insights to the action mechanisms of FIA and aluminum adjuvants in Japanese flounder and may be useful for the selection of adjuvant for vaccine formulations intended for Japanese flounder., (Copyright (c) 2009 Elsevier Ltd. All rights reserved.)

The objectives of this study were to examine the relationship between thiamine concentrations in unfertilized eggs and yolksac individuals of lake trout (Salvelinus namaycush), along with any associated histopathological changes in the tissues of alevins at the hatching stage. We address these questions in a lake trout population from different spawning grounds of Lake Michigan (North and South), known for compromised survival due to early mortality syndrome (EMS). However, a dichotomous forage base of lake trout spawning stocks, with a dietary thiaminase-rich alewife in the North, and dietary low-thiaminase round goby in the South, provides the basis for the assumption that different diets may lead to differences in severity of EMS between different stocks. Lake trout eggs of 18 females were collected and fertilized individually with the sperm of several males. The eggs, eyed embryos and newly-hatched alevins were sampled to examine thiamine utilization during embryogenesis. Progenies of females with low (< 0.73 nmol/g) and high (> 0.85 nmol/g) levels of thiamine were chosen for histological studies. The obtained results showed that total thiamine levels in the body and yolk of eyed embryos and alevins at hatching were influenced by thiamine levels of unfertilized eggs and it decreased during embryogenesis (to 51% in eyed embryos and 28% in newly-hatched alevins in comparison to unfertilized eggs). The survival of lake trout until hatching stage does not correlate with the thiamine level, however it was affected by collection site and was significantly higher in fish from the South site (Julian's Reef). At the hatching stage, no pathological changes were observed in the brain, olfactory lobe, retina or liver in embryos regardless of thiamine concentrations in unfertilized eggs. It has been concluded that an enhanced thiamine requirement for the fast muscle mass growth near the swim-up stage is responsible for overt and histopathological signs of EMS. Current study confirms earlier findings that lake trout suffering from EMS can be successfully treated by immersion in thiamine solution as late as at the swim-up stage.

Parasitic castration, the specific blocking of host reproductive output by an individual parasite, is a host-parasite interaction common to many invertebrates, particularly crustaceans, echinoderms and molluscs. It can reduce host density, alter host population dynamics and the evolution of host life history traits. Here we show that parasitisation by a single female cymothoid isopod, Anilocra apogonae, castrates its vertebrate host, the five-lined cardinalfish, Cheilodipterus quinquelineatus. Parasitised male fish fail to mouthbrood their young. The gonads of parasitised fish are smaller and parasitised female fish have substantially fewer and smaller ova than do the gonads of unparasitised fish. As for parasitic castrators of invertebrate hosts, A. apogonae on C. quinquelineatus are uniformly dispersed amongst infested hosts (one adult female isopod per host), are site specific, and their body size is highly correlated with that of their host. These isopods are large relative to the body size of their hosts, averaging 3.8% of the weight of the host. Parasitised fish also weigh less and are shorter than unparasitised fish of the same age. Despite the presence of other potential hosts, A. apogonae only infests C. quinquelineatus. The consistency of the ecological correlates amongst known parasitic castrators suggests that the parasitic castrator host-parasite relationship will be recognised for other parasites of vertebrates.

Mycobacterium fortuitum, as well as Mycobacterium marinum and Mycobacterium chelonae, are the etiological agents of fish Mycobacterioses. Mycobacteriosis has been reported to affect a wide range of freshwater and marine fish species, suggesting an ubiquitous distribution, and can cause zoonotic infections (known as "fish tank granuloma" or "swimming pool granuloma") in humans exposed to fish and contaminated water. Infection in human consists of nodular cutaneous lesions that can progress to tenosynovitis, arthritis, and osteomyelitis, depending on the immunological status. Authors describe some cases observed during routinary diagnostic activity in aquarium fish. Fish were sampled and histopathological, microbiological, and biomolecular exams were carried out. Histopathology showed systemic granulomatosis. Microbiological and biomolecular exams allowed us to identify the M. fortuitum as a main species. Finally, some considerations on the legal aspects of such disease are discussed.

Atlantic salmon paramyxovirus (ASPV) was isolated in 1995 from gills of farmed Atlantic salmon suffering from proliferative gill inflammation. The complete genome sequence of ASPV was determined, revealing a genome 16,968 nucleotides in length consisting of six non-overlapping genes coding for the nucleo- (N), phospho- (P), matrix- (M), fusion- (F), haemagglutinin-neuraminidase- (HN) and large polymerase (L) proteins in the order 3'-N-P-M-F-HN-L-5'. The various conserved features related to virus replication found in most paramyxoviruses were also found in ASPV. These include: conserved and complementary leader and trailer sequences, tri-nucleotide intergenic regions and highly conserved transcription start and stop signal sequences. The P gene expression strategy of ASPV was like that of the respiro-, morbilli- and henipaviruses, which express the P and C proteins from the primary transcript and edit a portion of the mRNA to encode V and W proteins. Sequence similarities among various features related to virus replication, pairwise comparisons of all deduced ASPV protein sequences with homologous regions from other members of the family Paramyxoviridae, and phylogenetic analyses of these amino acid sequences suggested that ASPV was a novel member of the sub-family Paramyxovirinae, most closely related to the respiroviruses.

The present experiment was conducted to examine if freshwater (FW) oxygen and carbon dioxide regimes cause physiological responses that lead to cataract formation in Atlantic salmon (Salmo salar L.) smolt. Duplicate groups of 50 g Atlantic salmon smolts were exposed to three freshwater oxygen saturation regimes (95, 112 or 125% saturation), with or without addition of carbon dioxide (measured 17-18 and 2-3 mg L(-1), respectively), for six weeks before transfer to seawater (SW). The FW exposure groups were followed up for another six weeks under a common SW regime. Fish were screened for cataract and sampled accordingly, at start, after 6 weeks in FW and after 6 weeks in SW. Increased growth related cataract incidences and severities were recorded in SW, mainly in the groups previously exposed to normoxic and hyperoxic conditions in FW, as compared to the respective groups added carbon dioxide. The concentration of histidine compounds (imidazoles) in muscle and lens tissue, used as quantitative risk markers of cataract, were lower than observed in earlier studies, however, neither were affected by the present water gas regimes in FW nor after follow up in SW. Independently of water oxygenation in FW, muscle free amino acid profiles in salmon groups concomitantly exposed to elevated carbon dioxide indicated use of selected free amino acids for energy purposes. Significantly lower abundance of heat shock protein 70 mRNA and trends towards stepwise reduction of antioxidant enzymes mRNA in the lens from fish exposed to increased water oxygenation were recorded, probably linked to increased growth and/or external stress during smoltification. This represents a first communication on using early molecular markers to express reduced protection of the fish lens against external stress to explain cataract development.

Amoebic gill disease (AGD) is a parasite-mediated proliferative gill disease capable of affecting a range of teleost hosts. While a moderate heritability for AGD resistance in Atlantic salmon has been reported previously, the mechanisms by which individuals resist the proliferative effects remain poorly understood. To gain more knowledge of this commercially important trait, we compared gill transcriptomes of two groups of Atlantic salmon, one designated putatively resistant, and one designated putatively susceptible to AGD. Utilising a 17k Atlantic salmon cDNA microarray we identified 196 transcripts that were differentially expressed between the two groups. Expression of 11 transcripts were further examined with real-time quantitative RT-PCR (qPCR) in the AGD-resistant and AGD-susceptible animals, as well as non-infected naïve fish. Gene expression determined by qPCR was in strong agreement with the microarray analysis. A large number of differentially expressed genes were involved in immune and cell cycle responses. Resistant individuals displayed significantly higher expression of genes involved in adaptive immunity and negative regulation of the cell cycle. In contrast, AGD-susceptible individuals showed higher expression of acute phase proteins and positive regulators of the cell cycle. Combined with the gill histopathology, our results suggest AGD resistance is acquired rather than innately present, and that this resistance is for the most part associated with the dysregulation of immune and cell cycle pathways.

Human infection by Mycobacterium tuberculosis is endemic, with approximately 2 billion infected and is the most common cause of adult death due to an infectious agent. Because of the slow growth rate of M. tuberculosis and risk to researchers, other species of Mycobacterium have been employed as alternative model systems to study human tuberculosis (TB). Mycobacterium marinum may be a good surrogate pathogen, conferring TB-like chronic infections in some fish. Medaka (Oryzias latipes) has been established for over five decades as a laboratory fish model for toxicology, genotoxicity, teratogenesis, carcinogenesis, classical genetics and embryology. We are investigating if medaka might also serve as a host for M. marinum in order to model human TB. We show that both acute and chronic infections are inducible in a dose dependent manner. Colonization of target organs and systemic granuloma formation has been demonstrated through the use of histology. M. marinum expressing green fluorescent protein (Gfp) was used to monitor bacterial colonization of these organs in fresh tissues as well as in intact animals. Moreover, we have employed the See-Through fish line, a variety of medaka devoid of major pigments, to monitor real-time disease progression, in living animals. We have also compared the susceptibility of another prominent fish model, zebrafish (Danio rerio), to our medaka-M. marinum model. We determined the course of infections in zebrafish is significantly more severe than in medaka. Together, these results indicate that the medaka-M. marinum model provides unique advantages for studying chronic mycobacteriosis.

One of the most common diseases that we have diagnosed in zebrafish is mycobacteriosis, caused by several Mycobacterium spp. The severity of the disease ranged from severe outbreaks to incidental infections. We conducted an in vivo study to evaluate the pathogenesis of six isolates of Mycobacterium from zebrafish with mycobacteriosis from four research facilities and one wholesale supplier of zebrafish in the United States: Mycobacterium abscessus, Mycobacterium peregrinum, Mycobacterium chelonae (2 isolates), and Mycobacterium marinum. We also included two isolates of M. marinum from other fishes. Fish were exposed by intraperitoneal injection at a target does of 5 x 10(4) bacteria/fish, and were held in static aquaria at 28 degrees C for 8 weeks. Fish were examined by histology and culture, and mortalities were recorded. The M. marinum isolates caused 100% infection and mortality between 30% and 100%. None of the other Mycobacterium species caused significant mortalities, but several of these fish had granulomatous lesions in visceral organs. Mycobacteria were consistently recovered in culture from fish exposed to M. marinum, and from only 9% of fish exposed to the other species. This study suggests that, of the isolates tested, only M. marinum is highly pathogenic and virulent to healthy zebrafish.

Flavobacterium psychrophilum is the causative agent of bacterial cold water disease (BCWD) and rainbow trout (Oncorhynchus mykiss) fry syndrome (RTFS). Logarithmic phase formalin-killed cells (FKC) of F. psychrophilum induced immunity to BCWD in ayu (Plecoglossus altivelis) by using an oral administration. In this study, we investigated the effective antigens of logarithmic phase cells in rainbow trout. Rainbow trout fry immunized with logarithmic phase FKC resulted in near complete protection, but the vaccine effect was low in fry immunized with stationary phase FKC. Scanning electron microscopy showed that logarithmic phase cells had many membrane vesicles (MVs) on the surface of F. psychrophilum cells. The MVs were released into medium at the stationary phase. MVs rich supernatant was collected from the stationary phase culture supernatant by using an ammonium precipitation method. Immunization with MVs rich supernatant combined with stationary phase FKC resulted in a relative percentage survival (RPS) of 94-100%, but immunization with MVs rich supernatant only resulted in no protection against F. psychrophilum infection. These data show that MVs have an adjuvant efficacy and suggest that combination of MVs and cells is necessary to obtain efficient protection.

The DNA vaccine pIHNw-G encodes the glycoprotein of the fish rhabdovirus infectious hematopoietic necrosis virus (IHNV). Vaccine performance in rainbow trout was measured 3, 6, 13, 24, and 25 months after vaccination. At three months all fish vaccinated with 0.1 microg pIHNw-G had detectable neutralizing antibody (NAb) and they were completely protected from lethal IHNV challenge with a relative percent survival (RPS) of 100% compared to control fish. Viral challenges at 6, 13, 24, and 25 months post-vaccination showed protection with RPS values of 47-69%, while NAb seroprevalence declined to undetectable levels. Passive transfer experiments with sera from fish after two years post-vaccination were inconsistent but significant protection was observed in some cases. The long-term duration of protection observed here defined a third temporal phase in the immune response to IHNV DNA vaccination, characterized by reduced but significant levels of protection, and decline or absence of detectable NAb titers. Examination of multiple tissues showed an absence of detectable long-term histopathological damage due to DNA vaccination.

The search for consistent patterns of organisation in parasite communities remains a central theme in parasite community ecology. However, to date, much evidence comes from studies without replication in both space and time; when replicate communities are examined, repeatable patterns are rarely observed. Here we determine, using nested subset analyses, whether the infracommunities of ectoparasites and endoparasites of a benthic marine fish (Sebastes capensis) show non-random structure. Then we examine the spatial repeatability of parasite community structure across the host's distribution in the southern Pacific, and the temporal repeatability of ectoparasite community structure from one locality. In total, 537 fish were captured from different latitudes (between 11 degrees S and 52 degrees S) along the Pacific coast of South America; a further 122 specimens were captured in two other years from one of the sampling localities, Valdivia (40 degrees S). In spite of variation in fish sizes among samples, fish size generally did not correlate with either ecto- or endoparasite species richness. The ecto- and endoparasite species richness of the component communities were also not correlated with fish sample size across the nine localities. Significant nested patterns were found in the ectoparasite communities of S. capensis at all eight localities, except at latitude 52 degrees S. Significant nested patterns were also found in the endoparasite infracommunities of S. capensis at seven of the nine localities, the exceptions being those from latitudes 11 degrees S and 20 degrees S. On a temporal scale, significant nestedness was observed in the ectoparasite infracommunities of S. capensis during each of the 3 years of sampling at Valdivia. In general, the same parasite species are responsible for the repeatability of nested patterns, though their importance varies among localities. The spatial and temporal predictability of the parasite community structure in S. capensis may be associated with the fish's benthic habitat and territorial behavior, suggesting that host biology may be a key determinant of the structure of parasite communities.

Zebrafish (Danio rerio) have been extensively utilized for understanding mechanisms of development. These studies have led to a wealth of resources including genetic tools, informational databases, and husbandry methods. In spite of all these resources, zebrafish have been underutilized for exploring pathophysiology of disease and the aging process. Zebrafish offer several advantages over mammalian models for these studies, including the ability to perform saturation mutagenesis and the capability to contain thousands of animals in a small space. In this review, we will discuss the use of mature zebrafish as an animal model and provide specific examples to support this novel use of zebrafish. Examples include demonstrating that clinical pathology can be performed in mature zebrafish and that age-associated changes in heat shock response can be observed in zebrafish. These highlights demonstrate the utility of zebrafish as a model for disease and aging.

Certain interspecific hybrids of the fish Xiphophorus spontaneously develop melanoma induced by the derepression of the Xmrk oncogene. Xmrk is a recent duplicate of an orthologue of the mammalian epidermal growth factor receptor gene Egfr. In addition to a specific overexpression in melanoma, amino-acid substitutions in the extracellular domain leading to ligand-independent dimerisation and constitutive autophosphorylation are responsible for the tumorigenic potential of Xmrk. The Xmrk receptor induces several signal transduction pathways mediating cell proliferation and resistance to apoptosis and initiating dedifferentiation. Moreover, Xmrk upregulates the expression of the secreted protein osteopontin, inducing an autocrine loop possibly allowing invasion and survival in the dermis as a first step in malignancy. Hence, Xmrk is able to induce pathways essential for a transformed phenotype. Some of these events are equivalent to those found downstream of the mammalian Egfr, but others have clearly evolved differently or are specific for pigment cells. Xmrk is potentially hazardous, nonessential and located in a very unstable genomic region. Nevertheless, Xmrk has been maintained under purifying selection in divergent Xiphophorus species. Hence, Xmrk has probably a beneficial function under certain conditions. The analysis of this function is a major challenge for future research in the Xiphophorus model.

Damselfish neurofibromatosis (DNF) is a transmissible disease characterized by peripheral nerve sheath and pigment cell tumors which occurs in bicolor damselfish (Stegastes partitus) on Florida reefs. The damselfish virus-like agent (DVLA) is associated with the development of DNF and contains a 2.4-kb DNA genome which was found at high levels in tumors and tumor-derived cell lines and at lower levels in non-tumor tissues of both spontaneously diseased fish (TF) and fish with experimentally induced tumors (EF). An analysis of transcription patterns revealed up to five DVLA derived RNAs ranging in size from 300 to 1400 bp in these cell types. DNA was the most commonly distributed DVLA component in TF and EF followed by RNA. Prevalence of transcripts varied by tissue type. The smallest transcripts were the most common in all cell types and the most complete patterns, which included the larger transcripts, were observed primarily in tumors. The presence of viral RNAs in addition to DNA in non-tumor tissues suggested these tissues were infected by DVLA and indicated a wide tissue tropism for this agent. The high levels of DVLA nucleic acids found in tumors suggest that replication is occurring there. However, the potential for DVLA replication in other tissues where only a limited range of transcripts were present is not known. The mechanism of tumorigenesis by this agent is unknown. However, the association of the larger transcripts with most tumor tissues and their absence in most non-tumor tissues suggests that these RNAs may be involved in tumor formation.

The Zebrafish International Resource Center was established to support the zebrafish research community, and includes a diagnostic service. One of the most common diseases that we have diagnosed is mycobacteriosis, which represented 18% of the diagnostic cases submitted from November 1999 to June 2003. We describe here the severity of the disease and associated pathological changes of 24 diagnostic cases from 14 laboratories. Identifications of the bacteria are provided for seven of these cases. For two cases in which culture of the organism was not successful, these identifications were based on ribosomal DNA (rDNA) sequence analysis obtained directly from infected tissues. Biochemical characteristics and rDNA sequence analysis from cultures are reported for the other isolates. Two severe outbreaks from different facilities on different continents were associated with an organism identified as Mycobacterium haemophilum based on rDNA sequence from tissues. Another severe outbreak was associated with an organism most closely related to Mycobacterium peregrinum. These species are recognized pathogens of humans, but this is the first report of them from fish. Bacteria identified as Mycobacterium chelonae or M. abscessus were recovered from fish in cases categorized as moderate disease or as an incidental finding. These findings indicate that species of Mycobacterium previously undescribed from fish (i.e., M. haemophilum and M. peregrinum) may pose significant health problems in zebrafish research facilities, whereas species and strains that are already recognized as common in fish usually cause limited disease on a population basis in zebrafish.

Two experiments were conducted to assess the physiological effects of freshwater exposure and amoebic gill disease (AGD) in marine Atlantic salmon (Salmo salar L.). The first experiment monitored marine salmon during a 3 h freshwater exposure, the standard treatment for AGD in Tasmania. The second experiment described the gill mucous cell histochemistry for freshwater adapted and seawater acclimated fish (AGD affected and unaffected) for possible correlations to ionoregulation. When exposed to freshwater, marine Atlantic salmon experienced a minor ionoregulatory dysfunction represented by a net efflux of Cl(-) ions at 3 h. AGD affected fish experienced the net efflux of Cl(-) ions 1 h sooner, and had a significantly greater net efflux of total ammonia. Changes to gill mucous cell populations corresponded to differing salinity and the presence of AGD. In AGD affected fish, these populations significantly differed between lesion and non-lesion associated areas of the gill filament. Our results have shown changes in the ionoregulatory capacity of Atlantic salmon due to freshwater exposure and AGD. Gill mucous cell histochemistry indicates the potential importance of the mucous layer in ionoregulation and disease. In comparison to previous studies on rainbow trout, these results suggest that Atlantic salmon have a greater short-term ionoregulatory capacity.

A review is presented of what we know and what we suspect regarding the formation of coronary arteriosclerotic lesions in salmonids. Coronary lesions are a fact of life for both Atlantic and Pacific species of migrating salmon. Severe forms of lesions, usually restricted to the main coronary artery, are typically found in the majority of a salmon population when they are spawning. Vascular injury to the coronary artery, as a result of the bulbus arteriosus being excessively distended, is proposed as an initiating mechanism for coronary lesion formation, possibly explaining why severe lesions are restricted primarily to the main coronary artery. Evidence is presented that coronary arteriosclerosis in salmonids develops in immature fish, well before maturation, and progresses with age. Growth and growth rate are implicated in lesion progression. A faster growth rate could produce a more stressful life style, which in turn initiates more coronary vascular injury. Dietary factors, especially polyunsaturated fatty acids (and their metabolites), can significantly stimulate vascular smooth muscle proliferation in the salmon coronary artery, but a possible linkage to the progression of coronary lesions has yet to be studied. Whether coronary lesions negatively impact blood flow to the salmon heart has not been properly studied. Nevertheless, the coronary blood supply to the heart has functional importance when salmon exercise and the coronary flow reserve may be reached when fish swim under mild hypoxic conditions. If coronary arterial lesions do adversely affect blood flow to the heart, the selective effects would be most prominent in years when upstream migration conditions are particularly severe.

Cryptosporidium molnari n. sp. is described from two teleost fish, the gilthead sea bream (Sparus aurata L.) and the European sea bass (Dicentrarchus labrax L.). The parasite was found mainly in the stomach epithelium and seldom in the intestine. Oocysts were almost spherical, with four naked sporozoites and a prominent residuum, and measured 3.23-5.45 x 3.02-5.04 (mean 4.72 x 4.47) microm in the type host, gilthead sea bream (shape index 1-1.17, mean 1.05). Sporulation was endogenous, as fully sporulated oocysts were found within the fish, both in the stomach epithelium and lumen, and in faeces. Oocysts and other stages of C. molnari fit most of the diagnostic features of the genus Cryptosporidium, but differ from hitherto described species, including piscine ones. All stages were located within a host contributed parasitophorous vacuole lined by a double host microvillar membrane. Merogonial and gamogonial stages appeared in the typical extracytoplasmic position, whereas oogonial and sporogonial stages were located deeply within the epithelium. Ultrastructural features, including the characteristic contact zone of the parasite with the host epithelial surface, were mostly coincident with those of other Cryptosporidium spp. Mitochondria were found in dividing meronts, merozoites, microgamonts and sporozoites. Pathological effects were more evident in gilthead sea bream, which also exhibited a clearly higher prevalence (24.4 versus 4.64% in sea bass). External clinical signs, consisting of whitish faeces, abdominal swelling and ascites, were rarely observed, in contrast with important histopathological damage. The wide zones of epithelium invaded by oogonial and sporogonial stages appeared necrotic, with abundant cell debris, and sloughing of epithelial cells, which detached to the lumen. No inflammation reaction was observed and the cellular reaction was limited to the cells involved in the engulfing of intraepithelial stages and debris, probably macrophages.

Viral encephalopathy and retinopathy, otherwise known as fish encephalitis or viral nervous necrosis (VNN), is an emerging problem in several farmed marine fish species in various geographic areas all over the world. Since summer 1995, heavy losses affecting mainly juvenile and adult sea bass (Dicentrarchus labrax) have been observed in several on-growing facilities in Italy. Dying fish show abnormal swimming behaviour and, at temperatures higher than 20-22 degrees C, mortality rates range between 15 and 50%. Neither significant external nor internal gross pathological signs, except frequent abnormal swim bladder hyperinflation, were detected. Histological investigations reveal vacuolations in the grey matter of the brain and spinal cord and in the granular layers of the retina. Serial tissue sections examined by an immunohistochemical method carried out with antisera against fish nodaviruses showed a positive reaction. Additionally, spherical virus-like particles 22-25 nm in diameter were detected by electron microscopy in negative stained preparations of brain tissues, and the same samples gave a positive reverse transcription-polymerase chain reaction (RT-PCR) for the T4 region of the fish nodavirus gene. These results indicate that both juvenile and adult sea bass subject to mass mortality in Italy since summer 1995 are infected with a fish nodavirus and strongly suggest that the identified virus is the cause of the observed mortality.

The aim of this study was to investigate the susceptibility of juvenile and adult seabass, which are generally thought to be refractory to nodavirus. Moreover, preliminary immunological studies were performed to examine the immune response of adult seabass. Successful transmission of the disease was experimentally demonstrated in juvenile and adult seabass as ascertained by the presence of the clinical signs of the disease, re-isolation of the virus in the SSN-1 cell line and subsequent confirmation by histopathology and immunohistochemistry. Bigger seabass not only developed the clinical disease but also suffered mortalities. Serum neutralisation titres were considered low in this study.