Contents lists available at ScienceDirect Veterinary Parasitology journal homepage: www.elsevier.com/locate/vetpar Research paper Diagnostic accuracy of the light microscope method to detect the eggs of Cardicola spp. in the gill filaments of the bluefin tuna. José F. Palacios-Abella a, ⁎ , Javier Rodríguez-Llanos a , María Víllora-Montero a , Salvatore Mele b , Juan A. Raga a , Francisco E. Montero a a Marine Zoology Unit, Cavanilles Institute of Biodiversity and Evolutionary Biology, University of Valencia, C/Catedrático José Beltrán 2, 46980 Paterna, Spain b Parassitologia e Malattie Parassitarie, Dipartimento di Medicina Veterinaria, Università di Sassari, 07100 Sassari, Italy ARTICLE INFO Keywords: Aquaculture Blood flukes Cardicola Mediterranean Sea Thunnus thynnus T-two test ABSTRACT Trematode blood flukes of the genus Cardicola are potentially lethal in bluefin tuna cultures. The present study proposed a new method to detect aporocotylid eggs in tuna gills. Aporocotylid eggs were detected by analysing a pair of gill filaments of five transversal areas of the eight holobranches of one hundred Atlantic bluefin tuna and observed with glycerol and a stereomicroscope with an oblique brightfield. Data were gathered according to holobranches, transversal areas and their combination. Eggs were uniformly distributed among the holo- branches, but they had the highest prevalence in the second and fifth transversal areas, which is controversial with respect to previous studies of egg distribution. An abbreviated method called the T-two test, which had the highest sensitivity (96.8%), is proposed for the detection of Cardicola spp. infections instead of the analysis all the holobranches. The T-two test limits the time and cost of the egg parasite screening analysis. The analyses of ten samples could be sufficient to detect the presence of parasites in farmed bluefin tuna; fish from the wild are expected to be less infected and more samples (45) would therefore be necessary. 1. Introduction Trematode blood flukes of the genus Cardicola (Aporocotylidae) have emerged as a serious pathogenic concern to the developing culture of bluefin tuna Thunnus spp. (Actinopterygii: Scombridae) (Colquitt et al., 2001; Nowak, 2004). Aporocotylid parasites live in the circula- tory system of fishes: adult blood flukes release eggs in the bloodstream, which eventually become trapped in the kidney, heart, and gills, the target location where eggs mature and hatch, breaking the gill tissues to release miracidia (Colquitt et al., 2001). The severity of the infection caused by these parasites, mainly due to the effect of their eggs in the gills and heart, decreases the health status of reared tunas (Nowak et al., 2006), reducing the productivity of tuna farming and producing mortality (Ogawa et al., 2010; Dennis et al., 2011; Shirakashi et al., 2012). Four species of aporocotylids (Cardicola forsteri Cribb, Daintith et Munday, 2000, C. opisthorchis Ogawa et al., 2011, C. orientalis Ogawa et al., 2010 and Cardicola sp. sensu Palacios-Abellaet al., 2015) have been collected from the circulatory system of bluefin tunas worldwide (Shirakashi et al., 2013; Palacios-Abella et al., 2015; Sugihara et al., 2015). Moreover, all of them have been reported in farms of Atlantic bluefin tuna (ABT) Thunnus thynnus (L.) from the Mediterranean Sea (Mladineo et al., 2008; Ruiz de Ybañez et al., 2011; Palacios-Abella et al., 2015; Forte-Gil et al., 2016). Blood flukes often go unnoticed as they live within the intricate circulatory system of these animals. In fact, the presence of apor- ocotylids is usually studied by the detection of parasite eggs trapped in gill filaments by the examination of the histological sections or fresh gills under a stereomicroscope (Colquitt et al., 2001; Aiken et al., 2006; Alama-Bermejo et al., 2011; McVay et al., 2011). Shirakashi et al. (2012) also digesting infected tuna gills to find and quantify eggs of Cardicola spp. Molecular analyses of the blood have also been per- formed to detect the presence of these parasites, being more sensible and less time-consuming than histology (Polinski et al., 2013) but more expensive, requiring specific equipment that is not widely available. These methods have also been used to detect Cardicola spp. in bluefin tuna species (Mladineo and Tudor, 2004; Ruiz de Ybañez et al., 2011; Norte dos Santos et al., 2012; Shirakashi et al., 2012; Forte-Gil et al., 2016). As the eight gills (holobranches) of one tuna are large and composed of thousands of filaments, the examination of all of them is extremely laborious, and the analyses of small samples not knowing the distribution of the egg could provide unreliable results. Determining the natural egg distribution in gills allows for the design of user-friendly http://dx.doi.org/10.1016/j.vetpar.2017.09.018 Received 23 June 2017; Received in revised form 12 September 2017; Accepted 23 September 2017 ⁎ Corresponding author at: Unidad de Zoología Marina, Institut Cavanilles de Biodiversitat i Biologia Evolutiva, Universitat de València, C/Catedrático J. Beltrán 2, 46980, Paterna, Spain. E-mail address: J.Francisco.Palacios@uv.es (J.F. Palacios-Abella). Veterinary Parasitology 247 (2017) 26–32 0304-4017/ © 2017 Elsevier B.V. All rights reserved. MARK