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

• Published in: Fish & Shellfish Immunology
• Main Authors: Cano, I, Taylor, NGH, Bayley, A, Gunning, S, McCullogh, R, Bateman, K, Nowak, BF, Paley, RK
• Format: Article in Journal/Newspaper
• Language: English
• Published: Academic Press Ltd Elsevier Science Ltd 2019
• Subjects: amoebic gill disease; AGD; Neoparamoeba perurans; RTgill-W1; Transwell®; permeable supports; AG-2; Th2; phagocytosis; Atlantic salmon
• Online Access: https://eprints.utas.edu.au/29500/; https://eprints.utas.edu.au/29500/1/129996%20-%20In%20vitro%20gill%20cell%20monolayer%20successfully%20reproduces%20in%20vivo%20Atlantic%20salmon%20host%20responses%20to%20Neoparamoeba%20perurans%20infection.pdf

An in vitro model to study the host response to Neoparamoeba perurans, the causative agent of amoebic gilldisease (AGD), was evaluated. The rainbow trout gill derived cell line, RTgill-W1, was seeded onto permeablecell culture supports and maintained asymmetrically with apical seawater. Cells were inoculated with either apassage 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 cytopathiceffect appeared and at 72 h cytolysis was observed, with total disruption of the cell monolayer at 96 h pi. Due tocell monolayer disruption, the platform could not support proliferation of amoebae, which showed a 3-logreduction in parasite 18S rRNA mRNA after 72 h (106 copies at 1 h to 103 at 72 h pi). SEM observations showedamoebae-like cells with either short pseudopodia and a malleiform shape, or, long pseudopodia embeddedwithin the gill cells and erosion of the cell monolayer. To study the host immune response, inoculated gill cellswere harvested from triplicate inserts at 0, 1, 3, 6, 24 and 48 h pi, and expression of 12 genes involved in theAtlantic salmon response to AGD was compared between infected and uninfected cells and between amoebicclones. Both clones induced similar host inmate immune responses, with the up-regulation of proinflammatorycytokine IL1β, complement C3 and cell receptor MHC-1. The Th2 pathway was up-regulated, with increasedgene expression of the transcription factor GATA3, and Th2 cytokines IL10, IL6 and IL4/13A. PCNA and AG-2were also up-regulated. The wild clone induced significantly higher up-regulation of IL1β, MHC-1, PCNA, lysozymeand IL10 than the attenuated clone for at least some exposure times, but AG-2 gene expression washigher in cells inoculated with the attenuated one. A principal component analysis showed that AG-2 and IL10were key genes in the in vitro host response to N. perurans. This in vitro model has proved to be a promising tool tostudy host responses to amoebae and may therefore reduce the requirement for in vivo studies when evaluatingalternative therapeutants to AGD control.