Development of different diagnostic techniques for Endolimax piscium (archamoebae) and their applicability in Solea senegalensis clinical samples

Systemic amoebiasis of sole is caused by Endolimax piscium, a cryptic parasitic archamoeba whose epidemiology and pathogeny are yet unknown. To establish reliable detection methods for this parasite, a battery of molecular diagnostic tools (ISH, PCR and qPCR) were developed and evaluated with a panel of clinical samples from symptomatic diseased fish and from apparently normal animals of different stocks. As there is neither enough background information on the epidemiology of the disease nor a validated reference method, comparison of tests used a composite reference method approach. The ISH technique was the most specific and sensitive in intestine samples and particularly useful as a reference confirmatory method, while the best method in muscle samples was qPCR. Application of the tests to asymptomatic fish demonstrated presence of parasites in a large proportion (>25%) of their intestines, suggesting that this is the point of entry of the amoebae and the initial stage in the development of the disease. The triggering factors that facilitate the breaching of the intestinal barrier by E. piscium, causing granulomatous lesions in other organs and systemic spreading, are not completely understood but our results point to the connective tissue as a preferential target for parasite development and migration.This work was funded through CSIC Internal Research Programmes (Intramural project 201330E025). Additional funding was granted by the regional Government (Generalitat Valenciana PROMETEO 2010/006 and ISIC 2012/003). M. Constenla was supported by a Ph.D. student grant of the Universitat Autònoma de Barcelona (‘P.I.F. Programme’).Peer reviewe