Understanding the host response of farmed fish to blood flukes (Trematoda: Aporocotylidae) for developing new treatment strategies.

Aporocotylids (Trematoda: Digenea), also known as fish blood flukes infect the circulatory system of fish leading to serious health problems and mortality. Aporocotylids are a particular concern for farmed fish as infection intensity can increase within the farming environment and lead to mortalities. In the context of managing these infections, one of the most crucial aspects to consider is the host response of the infected fish against these blood flukes. Understanding the response is essential to improving current treatment strategies that are largely based on the use of anthelmintic praziquantel to manage infections in aquaculture. This review focuses on the current knowledge of farmed fish host responses against the different life stages of aporocotylids. New treatment strategies that are able to provide protection against reinfections should be a long-term goal and is not possible without understanding the fish response to infection and the interactions between host and parasite.

Molecular evidence for stress, inflammation and structural changes in non-specific ulcers in skin of farmed Chinook salmon (Oncorhynchus tshawytscha).

Fish skin is critical to physical defence against pathogens and there is a need to understand the physiological processes impacting ulcers and their healing. Ulcers have been reported in farmed Chinook salmon in New Zealand. This study investigated stress, immune and structural gene expression in farmed Chinook salmon skin with and without ulcers from two sites in New Zealand sampled from February (higher temperature, late summer) to May (lower temperature, late autumn). Skin samples taken adjacent to non-specific ulcers in May and control fish in February demonstrated upregulation of heat shock protein 70 relative to control fish in May. Anterior gradient 2 expression was upregulated in fish with ulcers relative to control fish (both February and May), suggesting increased mucous cell activity. Based on the results of this study, fish with non-specific ulcers showed evidence of stress, inflammation, re-epithelisation, and delayed healing near the ulcer site, elucidating the importance of these processes in the pathogenesis of non-specific ulcers in farmed chinook salmon.

Association between melanin deposits in gill tissue and microbiome across different hatchery reared Atlantic salmon.

AIMS: To investigate the relationship between microbial community profiles and gill pathology during a production cycle of Atlantic salmon in two commercial hatcheries. METHODS AND RESULTS: Relationships between gill histology, environmental conditions, and microbiome were determined using high-throughput data, including 16S rDNA amplicon sequencing data, histopathology data, and water quality parameters. Hatchery A used riverine water and operated a mixed system of recirculation aquaculture system (RAS) and flowthrough. Hatchery B was used bore water and operated a RAS. Melanin deposits, hyperplastic, and inflammatory lesions were observed histologically in the gills. A higher prevalence of melanin deposits was detected and correlated to a change in beta diversity of bacterial communities in early time points (fingerling and parr stages). High abundance of Sphaerotilus sp.,Pseudomonas sp.,Nitrospira sp.,Exiguobacterium sp.,Deinococcus sp.,and Comamonas sp. was correlated with a high prevalence of melanin in filaments. Bacterial diversity increased as the fish cohort transitioned from RAS to flowthrough in hatchery A. CONCLUSIONS: Under commercial conditions, the commensal community of gill bacteria was related to melanin prevalence.

Size-dependent resistance to amoebic gill disease in naïve Atlantic salmon (Salmo salar).

Amoebic gill disease, caused by the protozoan ectoparasite Neoparamoeba perurans, remains a significant threat to commercial Atlantic salmon aquaculture operations worldwide, despite partial control afforded by selective breeding and therapeutic intervention. Anecdotal reports from commercial producers suggest that historically, smaller Atlantic salmon smolts are more susceptible to AGD than larger smolts. Here, large (>350 g) and small (<200 g) commercially sourced, AGD-naïve Atlantic salmon cohorts were experimentally exposed to 50 N. perurans trophozoites L-1 without intervention. Progression and severity of AGD in challenged cohorts was evaluated through gill pathology, using gill score and histological examination, and quantification of gill-associated amoebae burden using qPCR. To determine the potential basis for differences in AGD susceptibility between cohorts, transcriptome analysis was conducted using RNA extracted from whole gill arches. Overall, the large Atlantic salmon cohort had significantly lower gill parasite burdens and reduced AGD-related gross pathology compared to the small cohort. Relative gill load of N. perurans appeared to be proportional to gill score in both size classes, with larger smolts typically observed to have comparatively reduced parasite burdens at a given gill score. Moreover, comparison between gene expression profiles of large and small smolts highlighted upregulation of genes consistent with elevated immune activity in large smolts. Combined, the results presented here provide strong evidence of size-dependent resistance to AGD in AGD-naïve Atlantic salmon.

Transcriptomic characterisation of a common skin lesion in farmed chinook salmon.

Little is known about host responses of farmed Chinook salmon with skin lesions, despite the lesions being associated with increased water temperatures and elevated mortality rates. To address this shortfall, a transcriptomic approach was used to characterise the molecular landscape of spot lesions, the most commonly reported lesion type in New Zealand Chinook salmon, versus healthy appearing skin in fish with and without spot lesions. Many biological (gene ontology) pathways were enriched in lesion adjacent tissue, relative to control skin tissue, including proteolysis, fin regeneration, calcium ion binding, mitochondrial transport, actin cytoskeleton organisation, epithelium development, and tissue development. In terms of specific transcripts of interest, pro-inflammatory cytokines (interleukin 1ß and tumour necrosis factor), annexin A1, mucin 2, and calreticulin were upregulated, while cathepsin H, mucin 5AC, and perforin 1 were downregulated in lesion tissue. In some instances, changes in gene expression were consistent between lesion and healthy appearing skin from the same fish relative to lesion free fish, suggesting that host responses weren't limited to the site of the lesion. Goblet cell density in skin histological sections was not different between skin sample types. Collectively, these results provide insights into the physiological changes associated with common spot lesions in farmed Chinook salmon.

Urinary calculi in larvae of striped trumpeter, Latris lineata.

Urinary calculi are observed in some cultured marine fish larvae and may negatively impact larval health and survival. This study assessed urinary calculi in striped trumpeter Latris lineata larvae from hatching to 28 days post-hatching (dph). The prevalence of urinary calculi was variable over time and ranged from 15% to 50%, whereas the average size of calculi increased with larval age. Urinary calculi were semi-translucent, light cream to white colour with irregular morphology and a uniform internal structure. The calculi resulted in pressure atrophy causing a distended epithelium of the urinary bladder of fish with calculi. The calculi were predominantly formed of calcium hydrogen phosphate (CaHPO4 ). Routine assessment of the prevalence of urinary calculi in marine fish larvae is recommended, along with other parameters, to monitor larval quality and inform hatchery management actions.

Prevalence of epitheliocystis in freshwater Atlantic salmon reared in flow-through and recirculation aquaculture systems.

Epitheliocystis, an intracellular bacterial infection in the gills and skin epithelium, has been frequently reported in Atlantic salmon (Salmo salar) during freshwater production in a number of countries. This study describes the prevalence and intensity of a natural epitheliocystis infection present in the gills of two strains of Atlantic salmon reared in either a flow-through (FT) or a recirculation aquaculture system (RAS) in Ireland. Repeated sampling of gills prior to and throughout seawater transfer, histology and quantitative real-time PCR were used to determine infection prevalence and intensity. Despite no clinical gill disease, and minor histopathological changes, epitheliocystis lesions were identified in histology at all time points. Specific PCR confirmed the presence of Candidatus Clavichlamydia salmonicola in both strains and its number of copies was correlated with intensity of epitheliocystis lesions. A significant interaction between hatchery system and fish strain on the prevalence and intensity of gill epitheliocystis was found both using histological and molecular methods. Specifically, fish from FT had higher prevalence and intensity than RAS reared fish and within FT, the Irish cohort were more affected than Icelandic.

Experimental exposure to low concentrations of Neoparamoeba perurans induces amoebic gill disease in Atlantic salmon.

Amoebic gill disease (AGD) is a significant issue in Atlantic salmon mariculture. Research on the development of treatments or vaccines uses experimental challenges where salmon is exposed to amoebae concentrations ranging from 500 to 5,000/L. However, the water concentrations of N. perurans on affected salmon farms are much lower. The lowest concentration of N. perurans previously reported to cause AGD was 10/L. Here, we report that concentrations as low as 0.1/L of N. perurans can cause AGD. We propose that concentrations of N. perurans that reflect those measured on salmon farms should be used for future experimental challenges.

Relationship between Southern Bluefin Tuna, Thunnus maccoyii, melanomacrophage centres and Cardicola spp. (Trematoda: Aporocotylidae) infection.

Southern Bluefin Tuna (SBT), Thunnus maccoyii, is ranched off Port Lincoln, South Australia and is Australia's second largest economic finfish aquaculture industry. The biggest threats to SBT health identified by the industry are the blood flukes Cardicola forsteri and C. orientalis (Trematoda: Aporocotylidae). Melanomacrophage centres (MMCs) are aggregations of pigmented macrophage like cells present in spleen, kidney and liver of teleost fish. The aim of this study was to quantify MMCs in SBT anterior kidney, liver and spleen to investigate changes in relation to Cardicola spp. Infection. Samples were collected at the end of ranching from pontoons where SBT were treated with PZQ and pontoons with untreated SBT. SBT MMC percentage of surface area cover was highest in SBT spleen and lowest in the liver. Significant positive correlations were identified between SBT MMC area and SBT size in all three organs (p < 0.05). MMC area and parasite infection showed significant positive correlations in the kidney and spleen for Cardicola spp. gill egg counts, and in the kidney for C. forsteri DNA from SBT hearts and gills (p < 0.05). MMCs area increased with increased intensity of Cardicola spp. Infection and MMCs have the potential to be used as an indicator to assess health effects that Cardicola spp. have on SBT.

Transcriptomic Profiling in Fins of Atlantic Salmon Parasitized with Sea Lice: Evidence for an Early Imbalance Between Chalimus-Induced Immunomodulation and the Host's Defense Response.

Parasitic sea lice (e.g., Lepeophtheirus salmonis) cause costly outbreaks in salmon farming. Molecular insights into parasite-induced host responses will provide the basis for improved management strategies. We investigated the early transcriptomic responses in pelvic fins of Atlantic salmon parasitized with chalimus I stage sea lice. Fin samples collected from non-infected (i.e. pre-infected) control (PRE) and at chalimus-attachment sites (ATT) and adjacent to chalimus-attachment sites (ADJ) from infected fish were used in profiling global gene expression using 44 K microarrays. We identified 6568 differentially expressed probes (DEPs, FDR < 5%) that included 1928 shared DEPs between ATT and ADJ compared to PRE. The ATT versus ADJ comparison revealed 90 DEPs, all of which were upregulated in ATT samples. Gene ontology/pathway term network analyses revealed profound changes in physiological processes, including extracellular matrix (ECM) degradation, tissue repair/remodeling and wound healing, immunity and defense, chemotaxis and signaling, antiviral response, and redox homeostasis in infected fins. The QPCR analysis of 37 microarray-identified transcripts representing these functional themes served to confirm the microarray results with a significant positive correlation (p < 0.0001). Most immune/defense-relevant transcripts were downregulated in both ATT and ADJ sites compared to PRE, suggesting that chalimus exerts immunosuppressive effects in the salmon's fins. The comparison between ATT and ADJ sites demonstrated the upregulation of a suite of immune-relevant transcripts, evidencing the salmon's attempt to mount an anti-lice response. We hypothesize that an imbalance between immunomodulation caused by chalimus during the early phase of infection and weak defense response manifested by Atlantic salmon makes it a susceptible host for L. salmonis.

In vitro gill cell monolayer successfully reproduces in vivo Atlantic salmon host responses to Neoparamoeba perurans infection.

An in vitro model to study the host response to Neoparamoeba perurans, the causative agent of amoebic gill disease (AGD), was evaluated. The rainbow trout gill derived cell line, RTgill-W1, was seeded onto permeable cell culture supports and maintained asymmetrically with apical seawater. Cells were inoculated with either a passage attenuated or a recent wild clone of N. perurans. Amoebae, loaded with phagocytosed fluorescent beads, were observed associated with host cells within 20 min post inoculation (pi). By 6 h small foci of cytopathic effect appeared and at 72 h cytolysis was observed, with total disruption of the cell monolayer at 96 h pi. Due to cell monolayer disruption, the platform could not support proliferation of amoebae, which showed a 3-log reduction in parasite 18S rRNA mRNA after 72 h (106 copies at 1 h to 103 at 72 h pi). SEM observations showed amoebae-like cells with either short pseudopodia and a malleiform shape, or, long pseudopodia embedded within the gill cells and erosion of the cell monolayer. To study the host immune response, inoculated gill cells were harvested from triplicate inserts at 0, 1, 3, 6, 24 and 48 h pi, and expression of 12 genes involved in the Atlantic salmon response to AGD was compared between infected and uninfected cells and between amoebic clones. Both clones induced similar host inmate immune responses, with the up-regulation of proinflammatory cytokine IL1ß, complement C3 and cell receptor MHC-1. The Th2 pathway was up-regulated, with increased gene expression of the transcription factor GATA3, and Th2 cytokines IL10, IL6 and IL4/13A. PCNA and AG-2 were also up-regulated. The wild clone induced significantly higher up-regulation of IL1ß, MHC-1, PCNA, lysozyme and IL10 than the attenuated clone for at least some exposure times, but AG-2 gene expression was higher in cells inoculated with the attenuated one. A principal component analysis showed that AG-2 and IL10 were key genes in the in vitro host response to N. perurans. This in vitro model has proved to be a promising tool to study host responses to amoebae and may therefore reduce the requirement for in vivo studies when evaluating alternative therapeutants to AGD control.

A highly sensitive, non-invasive qPCR-based strategy for direct quantification of Yersinia ruckeri in fish faeces.

Finfish with asymptomatic Yersinia ruckeri infections pose a major risk as they can transmit the pathogen and cause clinical outbreaks in stock populations. Current tools have insufficient quantitative ability for accurately detecting the trace levels of Y. ruckeri typically associated with asymptomatic infection, necessitate invasive or lethal sampling, or require long processing times. This study presents a highly sensitive qPCR-based method, targeting part of the Y. ruckeri 16S rRNA sequence, that is capable of detecting extremely low levels of Y. ruckeri in noninvasively collected faecal samples. Quantitative precision and accuracy of faecal sample analysis was consistent, despite the complexity of the faecal matrix. The assay demonstrated linearity over a six log-wide dynamic range. Its limit of detection (LOD) and limit of quantification (LOQ) were 4 and 10 copies of the target sequence, respectively. Sensitivity of the assay was comparable to other qPCR-based methods without requiring invasive or lethal sampling. Applicability as a screening strategy was tested using passively collected faecal samples. Asymptomatic Y. ruckeri infection was detected in all samples, although none of the fish exhibited overt infection. This method will be beneficial for finfish disease management if developed further as a noninvasive, screening tool against asymptomatic Y. ruckeri infection.

Functional Annotation of All Salmonid Genomes (FAASG): an international initiative supporting future salmonid research, conservation and aquaculture.

We describe an emerging initiative - the 'Functional Annotation of All Salmonid Genomes' (FAASG), which will leverage the extensive trait diversity that has evolved since a whole genome duplication event in the salmonid ancestor, to develop an integrative understanding of the functional genomic basis of phenotypic variation. The outcomes of FAASG will have diverse applications, ranging from improved understanding of genome evolution, to improving the efficiency and sustainability of aquaculture production, supporting the future of fundamental and applied research in an iconic fish lineage of major societal importance.

Preliminary characterization of Tasmanian aquareovirus (TSRV) isolates.

In an attempt to determine whether or not genetic variants of the Tasmanian strain of Atlantic salmon aquareovirus (TSRV) exist, 14 isolates of TSRV, originating from various locations in Tasmania, covering a 20-year period (1990-2010), obtained from various host species and tissues, and isolated on different cell lines, were selected for this study. Two categories, termed "typical" and "atypical", of variants of TSRV were identified based on preliminary genotypic and phenotypic characterization carried out on these 14 different isolates. In addition, electron microscopic examination indicated the existence of at least three variants based on viral particle size. Finally, this study demonstrated the existence of at least one new variant of TSRV isolates, other than the more commonly isolated typical TSRV isolates, in farmed Tasmanian Atlantic salmon.

Immune reactivity in early life stages of sea-cage cultured Pacific bluefin tuna naturally infected with blood flukes from genus Cardicola (Trematoda: Aporocotylidae).

Pacific bluefin tuna (PBT), Thunnus orientalis, due to its high average price on the market is an economically valuable fish species. Infections by blood flukes from the genus Cardicola (Trematoda: Aporocotylidae) represent a growing concern for the cage culture of bluefin tuna in Japan, Australia and Southern Europe. The accumulation of numerous Cardicola eggs in the fish gills causes severe pathology that has been linked to mortality in PBT juveniles up to one year old. The only effective treatment used to mitigate the infection is the oral administration of the antihelminthic drug praziquantel (PZQ) to the affected fish. However, with the need to minimise therapeutic drug use in aquaculture it is hoped that immunoprophylaxis can provide a future alternative to protect the PBT juveniles against Cardicola infection. Currently, little is known of the host immune response to these parasites and of their infection dynamics. In this study, using real-time qPCR we aimed to quantitatively detect C. orientalis and C. opisthorchis DNA within the gills and heart of cultured PBT juveniles and to investigate the host immune response at the transcriptional level in the gills. The research focused mainly during early stages of infection soon after young PBT were transferred to culture cages (from 14 to 77 days post-transfer). An increase (up to 11-fold) of immune-related genes, namely IgM, MHC-I, TCR-ß and IL-1ß was observed in the PBT gills infected with Cardicola spp. (28-77 days post-transfer). Furthermore, IgM (19-fold increase) and MHC-I (11.5-fold increase) transcription was strongly up-regulated in gill samples of PBT infected with C. orientalis relative to uninfected fish but not in fish infected with C. opisthorchis. Cardicola-specific DNA was first detected in the host 14 days post-transfer (DPT) to sea-cages which was 55 days earlier than the first detection of parasite eggs and adults by microscopy. Oral administration of PZQ did not have an immediate effect on parasite DNA presence in the host and the DNA presence started to reduce after 24 days only in the host heart. The results provide evidence of an immune response in early age sea-cage cultured juveniles of PBT naturally infected with C. orientalis and C. opisthorchis. This response, whilst not protective against primary infection, provides evidence that immunisation at an early age may have potential as a health strategy.

Effects of single and repeated infections with Neoparamoeba perurans on antibody levels and immune gene expression in Atlantic salmon (Salmo salar).

Amoebic gill disease (AGD) is the main health problem for the salmon industry in Tasmania, Australia and is now reported in most salmon producing countries. Antibody and gene expression responses to the pathogen, Neoparamoeba perurans, have been studied independently following primary exposure; however, the effects of sequential reinfection, which can often occur during net-pen culture of salmon, remain unclear. The association between the transcription of immunoglobulin (Ig) and their systemic and mucosal antibody levels in regards to AGD is unknown. Herein, we assessed the antibody responses as well as Ig transcription in the gills of Atlantic salmon infected only once and also sequentially with N. perurans. After four successive AGD challenges, no significant differences in plasma or skin mucus levels of IgM were observed between AGD-naïve and challenged fish. However, IgM gene expression in gill lesions of AGD-affected fish increased up to 31 d after infection, while no changes in IgT, TCR and CD8 transcription were observed. Changes at IgM transcription level did not match the lack of antibody response in mucus, which is possibly explained by weak correlations existing between protein and mRNA abundances in cells and tissues. In the second experiment, which investigated Ig responses to AGD at the transcriptional as well as antibody production level in salmon after a single infection, the levels of serum or skin mucus IgM antibody were not affected and no changes in the IgM or IgT transcription were induced.

Vaccination with recombinant protein (r22C03), a putative attachment factor of Neoparamoeba perurans, against AGD in Atlantic salmon (Salmo salar) and implications of a co-infection with Yersinia ruckeri.

Amoebic gill disease (AGD) affects salmonids during the marine grow-out phase in the Tasmanian industry and in other major salmonid producing countries. During the period post-transfer to seawater, the bacterial condition yersiniosis can also cause high levels of mortality in Atlantic salmon grown in Tasmania, in addition to the hatchery outbreaks. The recombinant protein r22C03, a mannose-binding protein-like (MBP-like) similar to attachment factors of other amoebae, was tested as a vaccine candidate against AGD in a large scale challenge trial. Fish were immunised with r22C03 combined with FCA via intraperitoneal (i.p.) injection, and given a booster five weeks later by either i.p. injection (RP group) or by a dip-immersion (mRP). Fish were then challenged twice with Neoparamoeba perurans: the initial challenge 16 weeks after primary immunisation was terminated due to presence of ulcerative lesions in the skin of salmon; the second challenge was carried out after five weeks of treatment with oxytetracycline. These skin lesions might have been associated with a concurrent infection with Yersinia ruckeri, which was detected by real-time qPCR in serum of a large proportion of moribund and survivor fish after the AGD challenge. Before and during the N. perurans infection, levels of antibodies against r22C03 were measured by ELISA in serum, skin mucus and supernatant from skin and gill explants. For the second challenge, the average size of AGD lesions was recorded from histology sections and survival curves were obtained. Before AGD challenge, r22C03 induced antibody responses in serum and explants with both vaccination strategies. At the end of the challenge, levels of antibodies were lower than before challenge irrespective of treatment. Both vaccinated groups presented increased serum antibody responses, while only mRP presented antibody responses in skin mucus, and no significant antibody responses were measured in the explants. Antibodies did not confer protection to N. perurans infection, as no difference was observed in the survival curves of the vaccinated and control groups, and there was no effect on the gill lesion size. The concurrent yersiniosis infection probably represented more closely infection patterns observed in commercial settings. However, it could have interfered with the survival results and with the ability of the fish to respond to the amoebae infection.

Review of the buccal-attaching fish parasite genus Glossobius Schioedte & Meinert, 1883 (Crustacea: Isopoda: Cymothoidae).

Two species of Glossobius Schioedte & Meinert, 1883 are known from Australia: Glossobius anctus Bruce & Bowman, 1989 and Glossobius impressus (Say, 1818), the latter recorded here for the first time from Australia and southern Africa. Glossobius ogasawarensis Nunomura, 1994 is here placed in synonymy with Glossobius auritus Bovallius, 1885; whereas Glossobius crassa (Dana, 1853) is removed from synonymy with G. auritus and placed into nomen dubium. Glossobius arimae Nunomura, 2001 is transferred to the genus Ceratothoa Dana, 1852. A key to the species of Glossobius is presented.

Differentially expressed proteins in gill and skin mucus of Atlantic salmon (Salmo salar) affected by amoebic gill disease.

The external surfaces of fish, such as gill and skin, are covered by mucus, which forms a thin interface between the organism and water. Amoebic gill disease (AGD) is a parasitic condition caused by Neoparamoeba perurans that affects salmonids worldwide. This disease induces excessive mucus production in the gills. The host immune response to AGD is not fully understood, and research tools such as genomics and proteomics could be useful in providing further insight. Gill and skin mucus samples were obtained from Atlantic salmon (Salmo salar) which were infected with N. perurans on four successive occasions. NanoLC tandem mass spectrometry (MS/MS) was used to identify proteins in gill and skin mucus of Atlantic salmon affected by AGD. A total of 186 and 322 non-redundant proteins were identified in gill and skin mucus respectively, based on stringent filtration criteria, and statistics demonstrated that 52 gill and 42 skin mucus proteins were differentially expressed in mucus samples from AGD-affected fish. By generating protein-protein interaction networks, some of these proteins formed part of cell to cell signalling and inflammation pathways, such as C-reactive protein, apolipoprotein 1, granulin, cathepsin, angiogenin-1. In addition to proteins that were entirely novel in the context in the host response to N. perurans, our results have confirmed the presence of protein markers in mucus that have been previously predicted on the basis of modified mRNA expression, such as anterior gradient-2 protein, annexin A-1 and complement C3 factor. This first proteomic analysis of AGD-affected salmon provides new information on the effect of AGD on protein composition of gill and skin mucus. Future research should focus on better understanding of the role these components play in the response against infection with N. perurans.