Abstract
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.
Highlights
Amoebic gill disease (AGD) is a serious disease affecting Atlantic salmon, Salmo salar (Linnaeus, 1758). and coho salmon, Oncorhynchus kisutch Karuk (Walbaum, 1792) farmed in the marine environment [1]
There was no amplification of the 18S rRNA gene for other species from the genus Neoparamoeba, N. pemaquidensis and N. branchiphila
The species-specific PCR tests were conducted for both clones when the clones were initially isolated, before the challenge to confirm the infectivity of the laboratory attenuated clone and before the inoculation of the cell monolayers (Supplementary S1)
Summary
Amoebic gill disease (AGD) is a serious disease affecting Atlantic salmon, Salmo salar (Linnaeus, 1758). and coho salmon, Oncorhynchus kisutch Karuk (Walbaum, 1792) farmed in the marine environment [1]. Amoebic gill disease (AGD) is a serious disease affecting Atlantic salmon, Salmo salar (Linnaeus, 1758). First reported in Tasmania Australia and Washington State and California USA in 1988 [2], AGD has since become endemic in Tasmania [3], and has subsequently impacted salmonid production in Scotland, France, Spain, Ireland, Norway, Chile, Canada, South Africa, Korea and Faroe Islands [1,4,5,6,7,8]. In addition to causing disease in salmonids, AGD has been reported in turbot Scophthalmus maximus L., ayu Plecoglossus altivelis (Temminck & Schlegel, 1846) and ballan wrasse Labrus bergytta (Ascanius, 1767) [see Refs. The causative agent of AGD is the protozoan Neoparamoeba perurans [see Refs. Cumulative mortalities can reach up to 50% if left untreated [16]
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