Development of molecular diagnostics for the characterization of Neoparamoeba pemaquidensis Page, 1987

Neoparamoeba pemaquidensis Page, 1987 is an ongoing pathogen for commercial finfish aquaculture and has also sporadically been associated with mass mortality outbreaks of invertebrates. Despite the ubiquity and importance of this amphizoic amoeba, our understanding of its biology as applied to host range, pathogenicity, tissue tropism and geographic distribution, is severely lacking. This confusion may stem from the inability of current diagnostic tests based on morphology, immunology and molecular biology to differentiate strains at the subspecies level. This study focused on the identification of a subspecies marker able to characterize Neoparamoeba pemaquidensis strains. The inter-strain and intra-strain variability of the amoeba Internal Transcribed Spacer (ITS) region was estimated. This hypervariable region showed discriminative inter-strain variability among individual amoeba isolates. However, high levels of intra-genomic microheterogeneity were found among sequenced ITS regions. Further investigations on the ITS region of the Neoparamoeba eukaryotic endosymbiont, renamed Ichthyobodo necator Related Organism, revealed pertinent inter-strain variability and significantly lower levels of microheterogeneity. Phylogenetic and ParaFit coevolution analyses involving Neoparamoeba pemaquidensis isolates and their respective endosymbionts confirmed a significant coevolutionary relationship between the two protists. The combination of non-shared microheterogeneity and coevolution, presents the endosymbiont marker as a complementary or alternative target to differentiate Neoparamoeba strains. Polymerase Chain Reaction Restriction Fragment Length Polymorphism (PCR-RFLP) diagnostic tests based on both ITS regions were developed. The investigations centred on the complications of the amoeba ITS microheterogeneity in the development of a subspecies marker and the use of the endosymbiont ITS region as an internal marker. Both amoeba and endosymbiont ITS PCR-RFLP analyses were successfully used to detect and characterize a N. pemaquidensis isolate from an episode of Amoebic Gill Disease in Atlantic salmon, Salmo salar, from the west coast of North America (Washington State, USA).