High-throughput OpenArray™: novel, swift TaqMan® Real-Time PCR chip for diagnostic biomarkers in Oncorhynchus tshawytscha

Abstract

In 2014, aquaculture replaced capture fisheries as the primary source of fish supply for human consumption for the first time in history, but to date, fish domestication has not developed enough to cope with the disease-related challenges. Members of the Salmonidae family are among the most popular commercialized fish species and salmon aquaculture has been the fastest growing food production system worldwide. An OpenArray™ device for Chinook Salmon (Oncorhynchus tshawytscha) health surveillance was validated in this thesis. On each microscope slide–sized OpenArray™ plate, 28 gene transcripts from 48 samples can be quantified using TaqMan® RT-qPCR. To evaluate the function of the chip, fish culture water, mucus, head kidney, spleen, and gill tissues were collected from Chinook salmon that were intraperitoneally injected with live Vibrio anguillarum within the course of 96 h. Based on the on-chip primer efficiency results, primers for 27 of the genes were considered acceptable for future validation. Based on the transcript expression patterns revealed by the OpenArray™ TaqMan® RT-qPCR chip, 6 immune genes (calm, mhc-i, mhc- ii, il-1β, il-8, tapbp) were selected and validated by SYBR RT-qPCR. Pearson’s correlation analysis revealed that a moderate to strong correlation existed between the results generated by both RT-qPCR methods for the 6 genes analysed. Both OpenArray™ TaqMan® and SYBR RT-qPCR detected significantly increased il- 1β and il-8 expression levels in spleen tissue at 72 h and 96 h, although the peaks of the expression were not recorded within the time point analysed. In head kidney tissue, il-1β expression level was also increased at 72 h, but there was no difference observed for il-8 between the control and infected groups within any time points. A longer response initiation time was observed in the Chinook salmon model in this trial. Comparing to previous studies in related salmon species, the pro-inflammatory cytokine il-1β was observed to increase as early as 6 h post infection in vivo. Nevertheless, mucus samples showed great potential as a minimally invasive sampling method if the transcripts were quantified by SYBR RT-qPCR. By comparison, in mucus the OpenArray™ TaqMan® RT-qPCR chip did not report as many quantification data as in spleen and head kidney tissue. In general, both RT-qPCR methods revealed an early systematic immune response induced by Vibrio anguillarum in spleen and head kidney, but the biological significance of the delayed immune response initiation will require further analysis to understand. The OpenArray™ TaqMan® RT-qPCR chip validated in this thesis was designed to be specific to salmon species under the order Salmoniformes. By validating this health monitoring tool in other salmon species, it is possible that the interspecies physiological differences can be horizontally compared. This will enable the development of species- specific, more effective prophylactic and therapeutic approaches against the salmon diseases.