Development of PCR tests for the detection and quantification of Philasterides dicentrarchi in infected fish and in seawater samples

Abstract

Philasterides dicentrarchi is a free-living ciliate that can behave as an endoparasite and cause scuticociliatosis, an important disease that affects farmed and aquarium fish worldwide. Scuticociliatosis can also be caused by other ciliates belonging to several genera of the subclass Scuticociliatia, some of which have not always been correctly identified in the past. Although the abundance of ciliates varies greatly, seawater may contain 1-150 ciliates mL-1 belonging to many taxa. The identification and quantification of opportunistic pathogens such as P. dicentrarchi is therefore not an easy task. The aim of this study was to develop PCR and qPCR methods for the specific detection and quantification of Philasterides dicentrarchi in infected fish and in seawater samples. We began by designing primers to amplify β-tubulin and the ITS region or rDNA genes and then tested these against cultures of P. dicentrarchi, Miamiensis avidus and several other fresh and seawater ciliate species. For each gene, we optimized the PCR conditions for the specific detection of P. dicentrarchi. We also determined the number of gene copies that can be detected per reaction as well as the detection limit of P. dicentrarchi in water samples. Finally, we tested the method in seawater samples obtained from fish farms, after concentrating the ciliates by filtration. This method represents a promising tool for detecting P. dicentrarchi in infected fish, monitoring the concentrations of ciliates and determining the relationship between the numbers of P. dicentrarchi in seawater and the risk of scuticociliatosis.