Dynamic gill and mucus microbiomes during a gill disease episode in farmed Atlantic salmon.

Amoebic gill disease (AGD) and complex gill disease (CGD) are recurrent gill disorders in Atlantic salmon, resulting in significant aquaculture losses. The role of gill microbiomes in gill disease development is unclear. We undertook a longitudinal study to characterise the gill tissue and gill mucus microbiomes of farmed Atlantic salmon before, and during, a gill disease episode. Using a newly optimised DNA extraction protocol, we sequenced rRNA genes from microbiomes of gill samples taken from 105 individual salmon on a farm, over a summer season. The AGD aetiological agent, Neoparamoeba perurans, was PCR-quantified targeting 18S rRNA genes. Similar analyses were carried out on mucus samples. Mucus scrapings were suitable, non-lethal substitutes for characterisation of the gill prokaryotic community in this study. Gill tissue and gill mucus microbiomes changed during the campaign, correlating with N. perurans concentrations. Time explained 35% of the gill tissue and gill mucus microbiome variance, while N. perurans concentrations explained 5%. Genera including Dyadobacter, Shewanella and Pedobacter were maximally abundant in gill and mucus samples at the timepoint prior to the the detection of gill disorder signs, at T3. Shewanella was significantly more abundant before than during the gill disease episode, and we suggest this genus could be considered in future studies addressing relationships between gill disease and the gill microbiome.

Experimental infection trials with European North Atlantic ranavirus (Iridoviridae) isolated from lumpfish (Cyclopterus lumpus, L.).

European North Atlantic ranavirus (ENARV, Iridoviridae), is a ranavirus species recently isolated from lumpfish (Cyclopterus lumpus, L.), which are used as cleaner fish in Atlantic salmon (Salmo salar) farming in Northern Europe. This study aimed to investigate (1) the virulence of ENARV isolates from Ireland, Iceland and the Faroe Islands to lumpfish; (2) horizontal transmission between lumpfish; and (3) virulence to Atlantic salmon parr. Lumpfish were challenged in a cohabitation model using intraperitoneally (IP) injected shedders, and naïve cohabitants. IP challenge with isolates from Iceland (1.9 × 107 TCID50  ml-1 ) and the Faroe Islands (5.9 × 107 TCID50  ml-1 ) reduced survival in lumpfish, associated with consistent pathological changes. IP challenge with the Irish strain (8.6 × 105 TCID50  ml-1 ) did not significantly reduce survival in lumpfish, but the lower challenge titre complicated interpretation. Horizontal transmission occurred in all strains tested, but no clinical impact was demonstrated in cohabitants. Salmon parr were challenged by IP injection with the Irish isolate, no virulence or virus replication were demonstrated. A ranavirus qPCR assay, previously validated for fish ranaviruses, was first used to detect ENARV in tissues of both in lumpfish and Atlantic salmon. This study provides the first data on the assessment of virulence of ENARV isolates to lumpfish and salmon, guidelines for the diagnosis of ENARV infection, and poses a basis for further investigations into virulence markers.

Investigation of the transcriptomic response in Atlantic salmon (Salmo salar) gill exposed to Paramoeba perurans during early onset of disease.

Amoebic Gill Disease (AGD), caused by the protozoan extracellular parasite Paramoeba perurans (P. perurans) is a disease affecting Atlantic salmon (Salmo salar). This study investigated the gill transcriptomic profile of pre-clinical AGD using RNA-sequencing (RNA-seq) technology. RNA-seq libraries generated at 0, 4, 7, 14 and 16 days post infection (dpi) identified 19,251 differentially expressed genes (DEGs) of which 56.2% were up-regulated. DEGs mapped to 224 Gene Ontology (GO) terms including 140 biological processes (BP), 45 cellular components (CC), and 39 molecular functions (MF). A total of 27 reference pathways in the Kyoto Encyclopedia of Genes and Genomes (KEGG) and 15 Reactome gene sets were identified. The RNA-seq data was validated using real-time, quantitative PCR (qPCR). A host immune response though the activation of complement and the acute phase genes was evident at 7 dpi, with a concurrent immune suppression involving cytokine signalling, notably in interleukins, interferon regulatory factors and tumour necrosis factor-alpha (tnf-α) genes. Down-regulated gene expression with involvement in receptor signalling pathways (NOD-like, Toll-like and RIG-1) were also identified. The results of this study support the theory that P. perurans can evade immune surveillance during the initial stages of gill colonisation through interference of signal transduction pathways.

Differential Exoproteome and Biochemical Characterisation of Neoparamoeba perurans.

Infection with the protozoan ectoparasite Neoparamoeba perurans, the causative agent of AGD, remains a global threat to salmonid farming. This study aimed to analyse the exoproteome of both an attenuated and virulent N. perurans isolate using proteomics and cytotoxicity testing. A disproportionate presence of proteins from the co-cultured microbiota of N. perurans was revealed on searching an amalgamated database of bacterial, N. perurans and Amoebozoa proteins. LC-MS/MS identified 33 differentially expressed proteins, the majority of which were upregulated in the attenuated exoproteome. Proteins of putative interest found in both exoproteomes were maltoporin, ferrichrome-iron receptor, and putative ferric enterobactin receptor. Protease activity remained significantly elevated in the attenuated exoproteome compared with the virulent exoproteome. Similarly, the attenuated exoproteome had a significantly higher cytotoxic effect on rainbow trout gill cell line (RTgill W1) cells compared with the virulent exoproteome. The presence of a phosphatase and serine protease in the virulent exoproteome may facilitate AGD infection but do not appear to be key players in causing cytotoxicity. Altogether, this study reveals prolonged culture of N. perurans affects the exoproteome composition in favour of nutritional acquisition, and that the current culturing protocol for virulent N. perurans does not facilitate the secretion of virulence factors.

Host Response of Atlantic Salmon (Salmo salar) Re-Inoculated with Paramoeba perurans.

In aquaculture, recurrence rates of amoebic gill disease (AGD) caused by the ectoparasite Paramoeba perurans are high and no prophylactic strategies exist for disease prevention. In this study, Atlantic salmon (Salmo salar) were initially inoculated with P. perurans and following the development of amoebic gill disease were treated with freshwater immersion on day 21 and day 35 post inoculation. Fish were re-inoculated following a negative qPCR analysis for the presence of P. perurans. The gill host immune response was investigated at 7, 14, and 18 days post re-inoculation. Differential proteome expression of immune related proteins was assessed by comparison of each time point against naïve controls. In the gill, some proteins of the innate immune system were expressed in response to gill re-colonization by P. perurans, while no features of adaptive immunity were found to be differentially expressed. Many of the proteins identified are novel in the context of AGD and their expression profiles suggest that their roles in the response to disease development and progression in single or multiple infections warrant further investigation.

Investigation of the Initial Host Response of Naïve Atlantic Salmon (Salmo salar) Inoculated with Paramoeba perurans.

Amoebic Gill Disease (AGD), caused by the ectoparasite Paramoeba perurans is characterised by hyperplasia of the gill epithelium and lamellar fusion. In this study, the initial host response of naïve Atlantic salmon (Salmo salar) inoculated with P. perurans was investigated. Using gel-free proteomic techniques and mass spectrometry gill and serum samples were analysed at 7 timepoints (2, 3, 4, 7, 9, 11 and 14 days) post-inoculation with P. perurans. Differential expression of immune related proteins was assessed by comparison of protein expression from each time point against naïve controls. Few host immune molecules associated with innate immunity showed increased expression in response to gill colonisation by amoebae. Furthermore, many proteins with roles in immune signalling, phagocytosis and T-cell proliferation were found to be inhibited upon disease progression. Initially, various immune factors demonstrated the anticipated increase in expression in response to infection in the serum while some immune inhibition became apparent at the later stages of disease progression. Taken together, the pro-immune trend observed in serum, the lack of a robust early immune response in the gill and the diversity of those proteins in the gill whose altered expression negatively impact the immune response, support the concept of a pathogen-derived suppression of the host response.

Comparative proteomic profiling of newly acquired, virulent and attenuated Neoparamoeba perurans proteins associated with amoebic gill disease.

The causative agent of amoebic gill disease, Neoparamoeba perurans is reported to lose virulence during prolonged in vitro maintenance. In this study, the impact of prolonged culture on N. perurans virulence and its proteome was investigated. Two isolates, attenuated and virulent, had their virulence assessed in an experimental trial using Atlantic salmon smolts and their bacterial community composition was evaluated by 16S rRNA Illumina MiSeq sequencing. Soluble proteins were isolated from three isolates: a newly acquired, virulent and attenuated N. perurans culture. Proteins were analysed using two-dimensional electrophoresis coupled with liquid chromatography tandem mass spectrometry (LC-MS/MS). The challenge trial using naïve smolts confirmed a loss in virulence in the attenuated N. perurans culture. A greater diversity of bacterial communities was found in the microbiome of the virulent isolate in contrast to a reduction in microbial community richness in the attenuated microbiome. A collated proteome database of N. perurans, Amoebozoa and four bacterial genera resulted in 24 proteins differentially expressed between the three cultures. The present LC-MS/MS results indicate protein synthesis, oxidative stress and immunomodulation are upregulated in a newly acquired N. perurans culture and future studies may exploit these protein identifications for therapeutic purposes in infected farmed fish.

Class imbalance in gradient boosting classification algorithms: Application to experimental stroke data.

Imbalance between positive and negative outcomes, a so-called class imbalance, is a problem generally found in medical data. Imbalanced data hinder the performance of conventional classification methods which aim to improve the overall accuracy of the model without accounting for uneven distribution of the classes. To rectify this, the data can be resampled by oversampling the positive (minority) class until the classes are approximately equally represented. After that, a prediction model such as gradient boosting algorithm can be fitted with greater confidence. This classification method allows for non-linear relationships and deep interactive effects while focusing on difficult areas by iterative shifting towards problematic observations. In this study, we demonstrate application of these methods to medical data and develop a practical framework for evaluation of features contributing into the probability of stroke.

An in-vitro evaluation of the flow haemodynamic performance of Gore-Tex extracardiac conduits for univentricular circulation.

OBJECTIVE(S): The Fontan procedure is a common palliative intervention for sufferers of single ventricle congenital heart defects that results in an anastomosis of the venous return to the pulmonary arteries called the total cavopulmonary connection (TCPC). In patients with palliated single ventricular heart defects, the Fontan circulation passively directs systemic venous return to the pulmonary circulation in the absence of a functional sub-pulmonary ventricle. Therefore, the Fontan circulation is highly dependent on favourable flow and energetics, and minimal energy loss is of great importance. The majority of in vitro studies, to date, employ a rigid TCPC model. Recently, few studies have incorporated flexible TCPC models, without the inclusion of commercially available conduits used in these surgical scenarios. METHOD: The methodology set out in this study successfully utilizes patient-specific phantoms along with the corresponding flowrate waveforms to characterise the flow haemodynamic performance of extracardiac Gore-Tex conduits. This was achieved by comparing a rigid and flexible TCPC models against a flexible model with an integrated Gore-Tex conduit. RESULTS: The flexible model with the integrated Gore-Tex graft exhibited greater levels of energy losses when compared to the rigid walled model. With this, the flow fields showed greater levels of turbulence in the complaint and Gore-Tex models compared to the rigid model under ultrasound analysis. CONCLUSION: This study shows that vessel compliance along with the incorporation of Gore-Tex extracardiac conduits have significant impact on the flow haemodynamics in a patient-specific surgical scenario.

Per-Pass Analysis of Thrombus Composition in Patients With Acute Ischemic Stroke Undergoing Mechanical Thrombectomy.

Background and Purpose- Mechanical thrombectomy may involve multiple attempts to retrieve the occluding thrombus. This study examined the composition of thrombus fragments retrieved with each pass of a device during the thrombectomy procedure. Second, the per-pass composition was compared with procedural and clinical data including angiographic outcome and stroke etiology. Methods- Thrombi were retrieved from 60 patients with acute ischemic stroke, where thrombus fragments retrieved in each pass were segregated as individual samples and maintained throughout the histological analysis as independent samples. All samples were stained with hematoxylin and eosin and Martius Scarlet Blue. The relative composition of red blood cells, fibrin, and white blood cells in thrombus fragments from each pass was quantified. Results- Over the 60 cases, thrombus material was retrieved in 106 of 138 passes. The number of passes required to complete the cases ranged from 1 to 6 passes. The analysis of thrombus fragments retrieved in each pass provided a greater insight into the thrombectomy procedure progression than the overall thrombus composition; the red blood cell content of thrombus fragments retrieved in passes 1 and 2 was significantly higher than that retrieved in passes 3 to 6. The removal of thrombus material in a total of 1, 2, or 3 passes was associated with the highest percentage of final modified Thrombolysis in Cerebral Infarction score of 2c-3. There was no association between modified Thrombolysis in Cerebral Infarction score and per-pass thrombus composition. Conclusions- The differentiation achieved through the per-pass analysis of acute ischemic stroke thrombi provides a greater insight into the thrombectomy procedure progression than the combined per-case thrombus analysis. Insights gained may be a useful consideration in determining the treatment strategy as a case evolves and may be useful for the development of new devices to increase rates of 1-pass recanalization.

Gene expression analysis of Atlantic salmon gills reveals mucin 5 and interleukin 4/13 as key molecules during amoebic gill disease.

Amoebic gill disease (AGD) is one of the main diseases affecting Atlantic salmon (Salmo salar L.) mariculture. Hallmarks of AGD are hyperplasia of the lamellar epithelium and increased production of gill mucus. This study investigated the expression of genes involved in mucus secretion, cell cycle regulation, immunity and oxidative stress in gills using a targeted 21-gene PCR array. Gill samples were obtained from experimental and natural Neoparamoeba perurans infections, and sampling points included progressive infection stages and post-freshwater treatment. Up-regulation of genes related to mucin secretion and cell proliferation, and down-regulation of pro-inflammatory and pro-apoptotic genes were associated with AGD severity, while partial restoration of the gill homeostasis was detected post-treatment. Mucins and Th2 cytokines accoun ted for most of the variability observed between groups highlighting their key role in AGD. Two mucins (muc5, muc18) showed differential regulation upon disease. Substantial up-regulation of the secreted muc5 was detected in clinical AGD, and the membrane bound muc18 showed an opposite pattern. Th2 cytokines, il4/13a and il4/13b2, were significantly up-regulated from 2 days post-infection onwards, and changes were lesion-specific. Despite the differences between experimental and natural infections, both yielded comparable results that underline the importance of the studied genes in the respiratory organs of fish, and during AGD progression.

Local and systemic humoral immune response in farmed Atlantic salmon (Salmo salar L.) under a natural amoebic gill disease outbreak.

Amoebic gill disease (AGD), caused by the protozoan parasite Neoparamoeba perurans, is one of the most significant infectious diseases for Atlantic salmon (Salmo salar L.) mariculture. The present study investigated the humoral immune response (both local in gill mucus and systemic in serum) of farmed Atlantic salmon naturally infected with N. perurans in commercial sea pens, at two different stages of the disease and after freshwater treatment. Parameters analysed included activity of immune related enzymes (i.e. lysozyme, peroxidase, protease, anti-protease, esterase, alkaline phosphatase), IgM levels, and the terminal carbohydrate profile in the gill mucus. Overall, greater variations between groups were noted in the immune parameters determined in gill mucus than the equivalent in the serum. In gill mucus, IgM levels and peroxidase, lysozyme, esterase and protease activities were decreased in fish showing longer exposure time to the infection and higher disease severity, then showed a sequential increase after treatment. Results obtained highlight the capacity of gills to elicit a local response to the infection, indicate an impaired immune response at the later stages of the disease, and show partial reestablishment of the host immune status after freshwater treatment. In addition to providing data on the humoral response to AGD, this study increases knowledge on gill mucosal humoral immunity, since some of the parameters were analysed for the first time in gill mucus.

Novel methodology to replicate clot analogs with diverse composition in acute ischemic stroke.

BACKGROUND: Translational research on clot composition may be advanced by the use of clot analogs for the preclinical evaluation of mechanical thrombectomy devices. This work describes a novel set of clot analogs to represent a diverse range of fibrin and red blood cell (RBC) compositions for use in acute ischemic stroke (AIS) occlusion models. METHOD: Fresh whole blood obtained from ovine species was used to create seven different clot analog types. Five replicates were formed for each clot type. Varying amounts of whole blood constituents were mixed with thrombotic factors to create clots of varying compositions. Following histological processing, five sections from each clot were stained with H&E and Martius Scarlet Blue. Fibrin, RBC and white blood cell compositions were quantified. RESULTS: Histological examination demonstrated that the clot types had a distinct RBC and fibrin composition. No significant difference in composition was shown between replicates (p>0.05), indicating that the method of clot formation was reproducible. Percentage fibrin composition of the clot types was 1%, 8%, 31%, 38%, 64%, 79%, and 100%. A significant difference in fibrin and RBC composition between clot types was observed (p<0.05). CONCLUSIONS: Seven different clot types were developed to replicate common AIS thrombi. These clot analogs may be beneficial for the preclinical evaluation of endovascular therapies, and may be applied to interventional technique training.

Pathological and physiological effects of nicking on brown crab (Cancer pagurus) in the Irish crustacean fishery.

Nicking is used in fisheries to immobilize claws of brown crab (Cancer pagurus) in order to prevent cannibalism and fighting during storage. Nicking fractures the apodemes creating an open wound and damage to the internal claw tissues, which is the most valuable product of brown crab. In turn, this results in a reduction of quality of product and possibly compromises the host's defence mechanisms to other physiological challenges experienced throughout the post-harvest process. This study assessed the effects of nicking on the physiology and pathology of brown crab from the Irish fishery over 7 days. Results showed significantly elevated levels of muscle necrosis (P=0.005), total pathologies (P=0.022) and encirculating granulocytes in nicked crab compared to non-nicked crab. Mean glucose (212.0 µg/mL±108.4), lactate (36.52 µg/mL±38.74) and RI (11.05n±1.78) levels were higher in nicked crab indicating increased stress levels. Overall, histology results showed a significantly higher (P=0.022) occurrence of pathologies, such as melanised nodules, in nicked animals. In addition to an observed reduction in the quality of claw muscle, nicked crab also showed significantly higher (P=0.005) levels of necrosis in claw muscle. From the results of this study it is recommended that alternative retention methods are used.

Serum CRP-like protein profile in common carp Cyprinus carpio challenged with Aeromonas hydrophila and Escherichia coli lipopolysaccharide.

The potential of C-reactive protein (CRP)-like proteins to be used as a biomarker of health status in cultured carp obtained from various European fish lines has been assessed. Varying CRP-like protein levels in the serum of carp were monitored using an indirect competitive enzyme-linked immunosorbent assay. CRP-like protein basal levels in normal fish varied between carp lines, ranging on average from 2.9+/-0.15 to 12.57+/-1.19 microg ml(-1). Serum levels of CRP-like protein in carp were observed to increase several fold in fish infected with the pathogen Aeromonas hydrophila. However, carp injected with Escherichia coli lipopolysaccharide (LPS) serotype 0111:B4 did not exhibit an increase in CRP-like proteins levels.