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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.

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