Evaluation of the Infectious Potential of Neoparamoeba perurans Following Freshwater Bathing Treatments.
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ABSTRACT:
SUBMITTER: Taylor RS
PROVIDER: S-EPMC8145359 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description: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.
| S-EPMC8226569 | biostudies-literature
Project description:Neoparamoeba perurans, the aetiological agent of amoebic gill disease, remains a persistent threat to Atlantic salmon mariculture operations worldwide. Innovation in methods of AGD control is required yet constrained by a limited understanding of the mechanisms of amoebic gill disease pathogenesis. In the current study, a comparative transcriptome analysis of two N. perurans isolates of contrasting virulence phenotypes is presented using gill-associated, virulent (wild type) isolates, and in vitro cultured, avirulent (clonal) isolates. Differential gene expression analysis identified a total of 21,198 differentially expressed genes between the wild type and clonal isolates, with 5674 of these genes upregulated in wild type N. perurans. Gene set enrichment analysis predicted gene sets enriched in the wild type isolates including, although not limited to, cortical actin cytoskeleton, pseudopodia, phagocytosis, macropinocytic cup, and fatty acid beta-oxidation. Combined, the results from these analyses suggest that upregulated gene expression associated with lipid metabolism, oxidative stress response, protease activity, and cytoskeleton reorganisation is linked to pathogenicity in wild type N. perurans. These findings provide a foundation for future AGD research and the development of novel therapeutic and prophylactic AGD control measures for commercial aquaculture.
| S-EPMC8989968 | biostudies-literature
Project description: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.
| S-EPMC7994405 | biostudies-literature
Project description: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 IL1β, 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 IL1β, 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.
| S-EPMC6380893 | biostudies-literature
Project description:Gill health has become a significant global challenge for Atlantic salmon (Salmo salar) aquaculture, particularly during the marine phase of farming. The increasing prevalence of gill pathologies has been linked to rising seawater temperatures, underscoring the need to evaluate existing tools for monitoring gill health and to develop novel approaches for early detection. In this study, we investigated the gill responses of commercially farmed Atlantic salmon to natural infection with Neoparamoeba perurans during an outbreak of amoebic gill disease (AGD) in Tasmania. Our focus spanned the low AGD prevalence, high AGD prevalence, and post-freshwater treatment stages of the outbreak. Evaluations of gill tissue included assessments of the gross AGD score, histopathological score, abundance of N. perurans (measured by 18S rRNA gene expression), and expression levels of inflammation-related transcripts. We demonstrated a strong correlation between different measures of AGD-related gill pathology and significant differences between distinct stages of the N. perurans outbreak. Post-treatment, fish exhibited considerable variability in their responses to the freshwater bath, highlighting the necessity for personalized management strategies that consider genetic, environmental, and health status factors. The expression patterns of angiogenin-1 (ANG1) and complement C1q tumour necrosis factor-related protein 3-like (C1QTNF3) emphasize their potential as biomarkers for early detection of gill damage in salmon aquaculture worldwide.
| S-EPMC11350870 | biostudies-literature
Project description:Purpose: The aim of this study was to RNA-sequencing (RNA-seq) to investigate further the gill transcriptome during the early stage of infection, prior to the appearance of mucoid lesions on the gills of amoebic gill disease (ADG)-affected fish. Methods: 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 inoculation (dpi) RNA-seq data was validated using real-time, quantitative PCR (qPCR) analysis. Results: This study identified 29,357 Differentially Expressed Genes (DEGs) over the course of 16 days follwing exposure to N.perurans , the caustative agent of Amoebic gill disease. With many genes differentially regualted at more tehn one time point, the number of individual gene identified as down-regulated was 8,524 and up-regulated was 10,826. DEGs mapped to 224 Gene Ontology (GO) terms, 27 reference pathways in the Kyoto Encyclopedia of Genes and Genomes (KEGG) and 15 Reactome Gene Sets. Conclusions:Molecular diagnostics and histopathology, but not gill scores, provided an accurate record of disease progression during early stage onset of AGD, prior to evidence of clinical signs on the gill. At 7 days post infection (dpi), there was evidence of innate immune response activation and a concomitant immune suppression involving signalling pathways for cytokines, specifically interleukins.
2021-07-13 | GSE179972 | GEO
Project description: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 interest found in both exoproteomes were maltoporin, ferrichrome-iron receptor, and putative 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.
2021-09-10 | PXD025154 | Pride
Project description:Marine farmed Atlantic salmon (Salmo salar) are susceptible to recurrent amoebic gill disease (AGD) caused by the ectoparasite Neoparamoeba perurans over the growout production cycle. The parasite elicits a highly localized response within the gill epithelium resulting in multifocal mucoid patches at the site of parasite attachment. This host-parasite response drives a complex immune reaction, which remains poorly understood. To generate a model for host-parasite interaction during pathogenesis of AGD in Atlantic salmon the local (gill) and systemic transcriptomic response in the host, and the parasite during AGD pathogenesis was explored. A dual RNA-seq approach together with differential gene expression and system-wide statistical analyses of gene and transcription factor networks was employed. A multi-tissue transcriptomic data set was generated from the gill (including both lesioned and non-lesioned tissue), head kidney and spleen tissues naïve and AGD-affected Atlantic salmon sourced from an in vivo AGD challenge trial. Differential gene expression of the salmon host indicates local and systemic upregulation of defense and immune responses. Two transcription factors, znfOZF-like and znf70-like, and their associated gene networks significantly altered with disease state. The majority of genes in these networks are candidates for mediators of the immune response, cellular proliferation and invasion. These include Aurora kinase B-like, rho guanine nucleotide exchange factor 25-like and protein NDNF-like inhibited. Analysis of the N. perurans transcriptome during AGD pathology compared to in vitro cultured N. perurans trophozoites, as a proxy for wild type trophozoites, identified multiple gene candidates for virulence and indicates a potential master regulatory gene system analogous to the two-component PhoP/Q system. Candidate genes identified are associated with invasion of host tissue, evasion of host defense mechanisms and formation of the mucoid lesion. We generated a novel model for host-parasite interaction during AGD pathogenesis through integration of host and parasite functional profiles. Collectively, this dual transcriptomic study provides novel molecular insights into the pathology of AGD and provides alternative theories for future research in a step towards improved management of AGD.
| S-EPMC8202022 | biostudies-literature
Project description:Neoparamoba perurans, is the aetiological agent of amoebic gill disease (AGD), a disease that affects farmed Atlantic salmon worldwide. Multilocus sequence typing (MLST) and Random Amplified Polymorphic DNA (RAPD) are PCR-based typing methods that allow for the highly reproducible genetic analysis of population structure within microbial species. To the best of our knowledge, this study represents the first use of these typing methods applied to N. perurans with the objective of distinguishing geographical isolates. These analyses were applied to a total of 16 isolates from Australia, Canada, Ireland, Scotland, Norway, and the USA. All the samples from Australia came from farm sites on the island state of Tasmania. Genetic polymorphism among isolates was more evident from the RAPD analysis compared to the MLST that used conserved housekeeping genes. Both techniques consistently identified that isolates of N. perurans from Tasmania, Australia were more similar to each other than to the isolates from other countries. While genetic differences were identified between geographical isolates, a BURST analysis provided no evidence of a founder genotype. This suggests that emerging outbreaks of AGD are not due to rapid translocation of this important salmonid pathogen from the same area.
| S-EPMC6963586 | biostudies-literature
Project description:The parasitic amoeba, Neoparamoeba perurans is the causative agent of Amoebic Gill Disease in salmonids. The parasite has previously been 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. Three isolates of N. perurans maintained in culture for varying durations were compared. 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 a newly acquired, virulent and attenuated N. perurans culture and were analysed using two-dimensional electrophoresis (2D PAGE) coupled with liquid chromatography tandem mass spectrometry (LC-MS/MS). An experimental challenge trial using Atlantic salmon smolts confirmed a loss in virulence in an N. perurans culture that was maintained in vitro for 3 years. A greater diversity of bacterial communities was found in the microbiome of the virulent isolate harbouring predominant genera belonging to Pseudoaltermonas spp, Vibrio spp and Fluviicola spp. Microbial community richness was reduced in the attenuated microbiome, with a singular species, Thalassopira xiamenensis, representing a large proportion of its 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 the plausible occurrence of immunomodulation are ultimately upregulated in a newly acquired N. perurans culture and future studies may exploit these protein identifications for therapeutic purposes in infected farmed fish.
2021-09-09 | PXD022328 | Pride