Bull. Eur. Ass. Fish Pathol., 35(6) 2015, 217 NOTE ȱȱParamoeba perurans in Ĵȱȱ ȱęȱ H. E. B. Stagg*, M. Hall, I. S. Wallace, C. C. Pert, S. Garcia Perez and C. Collins Marine Scotland Science, Marine Laboratory, Aberdeen, AB11 9DB Abstract ȱȱParamoeba perurans, ȱȱȱȱȱȱ ȱȱ¢ȱȱ ȱ ȱęȱȱĴȱȱ ȱǻȱƽȱŘǰřŚŞǼǯȱOverall, the apparent prevalence was low. A ȱęǰȱȱȱȱTrachurus trachurus, ȱǯȱȱȱȱęȱȱȱȱ ȱP. perurans in horse mackerel. Paramoeba perurans is an amoeba parasite and the Salmo salar and rainbow trout Oncorhynchus ȱȱȱȱȱȱǻ Ǽȱ mykiss (Munday et al., 1990); coho salmon O. (Young et al., 2007, Crosbie et al., 2012). The kisutchȱǻ ȱȱǯǰȱŗşŞŞǼDzȱ Scophthalmus ȱ ȱęȱȱȱȱȱŘŖŖŜȱ maximus ǻ¢ȱȱǯǰȱŗşşŞǼDzȱȱȱDicen- with additional outbreaks occurring since 2011 trarchus labrax (Dykova et al., 2000); chinook ȱȱȱ¢ȱ ȱȱȱęȱ salmon O. tshawytscha ǻȱȱǯǰȱŘŖŖŞǼDzȱ ȱȱȱĴȱȱ¢ȱ ayu Plecoglossus altivelis (Crosbie et al., 2010); (Marine Scotland Science unpublished data). ballan wrasse Labrus bergylta (Karlsbakk et al., ȱȱȱȱęȱȱȱ 2013); blue warehou Seriolella brama (Adams (Shinn et al., 2014) especially in the Australian ȱǯǰȱŘŖŖŞǼDzȱȱȱȱDiplodus puntazzo ȱȱ¢ȱȱȱ (Dykova and Novoa, 2001). ȱȱȱȱȱęȱȱȱ ŗşŞŚȱǻ¢ǰȱŗşŞŜǼǯȱȱȱȱȱȱ ȱȱȱ ȱęȱȱȱȱȱȱ reported in the USA (Kent et al., ŗşŞŞǼǰȱ ȱ P. peruransȱȱȱȱȱȱȱȱ (Rodger and McArdle, 1996), the Mediterranean ȱ¢ȱȱȱȱȱȱȱ ǻ¢ȱȱǯǰȱŗşşŞǼǰȱ ȱȱǻȱȱ ȱȱȱ ȱȱȱȱ ǯǰȱŘŖŖŞǼǰȱ ¢ȱǻȱȱǯǰȱŘŖŖŞǼǰȱ ȱ ȱȱęǯȱȱǰȱP. perurans has only (Crosbie et al., 2010), Chile (Bustos et al., 2011) ȱȱȱęȱȱȱ- ȱȱȱȱǻȱȱǯǰȱŘŖŗŚǼǯȱ- ǯȱȱȱȱȱȱ¢ȱȱ ceptible species to AGD include: Atlantic salmon ȱȱ ȱParamoeba ǯȱȱ ȱęȱ * Corresponding author’s e-mail: [email protected] ŘŗŞǰȱǯȱǯȱǯȱȱǯǰȱřśǻŜǼȱŘŖŗś ǻȱȱǯǰȱŘŖŖŞǼȱ ȱȱȱ ȱ ȱȱȱȱȱ¢ȱȱȱ ȱȱȱȱ¢ȱȱȱȱ ȱ ȱȱęȱȱȱ¢ȱǻ ȱ ȱȱ in Tasmania and tested ȱǯǰȱŘŖŖŗǼǯȱ¢ȱęȱ ȱȱ ȱ using histological and immunohistochemical each haul based on the approximate proportion techniques however, the amoeba species was ȱȱȱȱȱȱǰȱȱ ȱȱȱȱȱȱȱȱȱ ȱ ȱȱȱȱȱ ȱȱP. perurans here ȱȱęǯȱ ȱȱęǯȱȱȱ¢ȱ ȱ ȱȱ¢ȱȱȱȱȱ ȱ ȱȱȱȱ ȱȱȱȱȱ ęȱȱȱȱęȱȱȱP. perurans ȱȱȱȱP. peruransȱȱ ȱȱ ǻȬ ȱȱǯǰȱŘŖŖŘǼǰȱęȱȱ- ęȱȱȱ ǯȱȱȱȱȱ ȱȱȱȱȱȱȱ ȱȱřŘśȱ species sampled is shown in Table 1. A section ȱęȱȱȱ¡ȱȱȱ ȱȱȱȱęȱȱǰȱȱȱȱ ǯȱȱęȱ ȱȱȱȱ¢ȱ ȱȱȱęȱȱȱȱȱȱĚ- ȱȱȱȱȱ ȱ ęǰȱ ȱ¢ȱȱȱŗŖŖƖȱȱ AGD was present. This study was conducted (Sigma) and stored at 4 °C prior to processing. when Neoparamoeba pemaquidensis was thought ȱȱȱȱȱȱ ǰȱȱȱ ȱȱ ȱȱȱȱěȱ ȱȱȱP. perurans was not determined, (Qiagen) with 7 mm stainless steel beads using no paramoeba, or AGD lesions were observed the Qiagen TissueLyser system (Qiagen) at a ȱ¢ȱȱȱȱǯ ¢ȱȱŘśȱ £ȱȱȱȱȱŚȱǯȱ ȱȱ ȱ¡ȱȱśȱȱȱ ȱ¢ȱȱȱȱȱP. perurans was ȱȱ¢¢ȱȱȱ ȱȱ ȱȱȱ ȱȱęȱȱȱ QIAsymphony DNA DSP kit (Qiagen) using ȱȱȱǰȱȱȱȱȱ the Tissue LC 200 DSP protocol with an elution ȱȱȱȱ ȱęȱȱȱȱ ȱȱŘŖŖȱΐǯȱ reservoir species. Determining this background ȱ ȱȱȱȱȱȱ- Real-time polymerase chain reaction (qPCR) ȱ¢ȱȱȱǰȱǰȱ ȱȱȱȱȱȱ- ȱȱȱ¢ȱȱȱǰȱ ¢ȱŚŞŖȱ ȱȱȱȱȱ ȱȱȱȱȱP. perurans Toughmix (Quanta BioScience) with primers may be expected during an AGD outbreak in and probes as described by Fringuellli et al. ȱȱǰȱ ȱȱę¢ȱ (2012) and detailed in Table 2. A single universal targeted in this survey. ęȱȱȱǰȱȱȱ ŗŞȱǰȱ ȱȱȱȱȱȱȱȱ ȱȱ¢ǰȱęȱ ȱȱ¢ȱȱ DNA quality and quantity. Samples were con- ȱǻȱȱŚŜȱȮȱŗşŘȱǼȱȱ- ȱȱȱȱȱȱȱȱȱ ȱȱȱĴȱȱ£ȱǻȱ ȱȱęȱ ȱǯȱȱ ŗǼȱȱȱȱ ȱȱ¢ȱȱȱ positive by qPCR, and samples generating am- ęȱ ȱȱȱŘŖŗřǯȱȱ ȱ biguous results, where only one replicate had methods have previously been reported as a a late Cp value, were subjected to nested PCR Bull. Eur. Ass. Fish Pathol., 35(6) 2015, 219 Figure 1.ȱęȱ ȱȱȱĴȱȱ ǯȱȱȱǻS) indicates sampling location ȱȱęȱ ȱȱȱParamoeba perurans. White circle ({) indicates sampling location where one ęȱ ȱȱȱP. perurans. 220, Bull. Eur. Ass. Fish Pathol., 35(6) 2015 Table 1.ȱȱȱȱęȱȱȱȱȱȱȱǰȱȱ¢ȱǯ Fish Species Number Number Common Name Scientięc Name Sampled Positive ȱǻęǼ Lophius piscatorius 30 Blue whiting Micromesistius poutassou 163 0 Common dab Limanda limanda 95 0 Cod Gadus morhua 45 0 ȱ Phycis blennoides 50 Grey gurnard Eutrigla gurnardus 45 0 Goldsinny wrasse Ctenolabrus rupestris 10 Haddock ȱę ŘşŞ 0 Hake Merluccius merluccius 57 0 Herring Clupea harengus 259 0 Horse mackerel (scad) Trachurus trachurus 21 Lesser argentine Argentina sphyraena 16 0 Ling Molva molva 20 Long rough dab Hippoglossoides platessoides ŘŞ 0 Lemon sole ȱĴ 12 0 Lumpsucker Cyclopterus lumpus 10 Common dragonet Callionymus lyra 70 Mackerel Scomber scombrus 12 0 Megrim ȱ Ĝ 40 Norway pout Trisopterus esmarkii 597 0 Poor cod Trisopterus minutus 147 0 Plaice Pleuronectes platessa 62 0 Saithe Pollachius virens 54 0 Red gurnard Aspitrigla cuculus 22 0 Silvery pout Gadiculus argenteus 10 Sprat ĴȱĴ 134 0 Sea trout ȱĴ 10 Whiting Merlangius merlangus 275 0 Bull. Eur. Ass. Fish Pathol., 35(6) 2015, 221 Table 2. Primer and probe sequences used in this study. The Paramoeba perurans qPCR assay has been ȱ¢ȱȱȱǯȱǻŘŖŗŘǼǰȱȱȱȱ¢ȱȱȱȱȱ¢ȱȱȱŗŞȱ ȱȱȱȱȱȱȱȱȱęȱǯȱȱęȱȱȱȱ ȱȱȱ ȱȱŗȱǻȱȱǯǰȱŗşşŝǼȱȱȱȱŗŖŚśȱǻȱȱǯǰȱŘŖŖŞǼǰȱȱȱȱ ȱȱȱȱȱȱȱȱ¢ȱȱȱǯȱǻŘŖŖŞǼǯ Assay Primer/probe name Primer/probe sequence Peru For 5’-GTTCTTTCGGGAGCTGGGAG-3’ Paramoeba perurans Peru Rev 5’-GAACTATCGCCGGCACAAAAG-3’ QPCR Peru probe 6FAM CAATGCCATTCTTTTCGGA-MGB UNIFISHFOR 5’-CCTGCGGCTTAATTTGACTCA-3’ Endogenous control UNIFISHREV 5’-AAAGAGCTATCAATCTGTCAATCCTTT-3’ QPCR UNIFISHPROBE 6FAM-CTCACCCGGCCCGGACACG-MGB ȱȱęȱ ERB1 5’-ACCTGGTTGATCCTGCCAG-3’ round Np1045r 5’-CTGTCCCTTTTAATCATTACACTTC-3’ Nested PCR second YOUNG F 5’-ATCTTGACYGGTTCTTTCGRGA-3’ round YOUNG R 5’-ATAGGTCTGCTTATCACTYATTCT-3’ ȱȱȱȱǰȱȱȱęȱ ȱȱȱȱȱȱ negative results by an alternative method, re- water were included in extraction, qPCR and ¢ǯȱ ȱ¢ȱ¢ȱȱȱ ȱǯȱȱȱȱȱ ȱ¢ǰȱȱȱȱ¢ȱ ȱȱ ȱȱ¡ȱȱȱȱP. perurans ȱȱȱ¢ȱȱȱȱȱȱ ȱǻȱęȱ¢ȱǰȱȱ assay (data not shown). et al., 2012) were ran in both qPCR and PCR. ȱȱ ȱȱȱȱȱȱ ȱȱȱ ȱȱ¢ȱȱ ȱȱǰȱȱȱȱȱ ȱǭȱȱǻǰȱȱȱ ambiguous result samples, in a 25 μl reaction ȱǰȱ¢ȱȱǰȱǰȱ as previously described (Snow et al., 2004) gen- www.dnaseq.co.uk) using the second round erating a 636 bp product using primer sets as nested PCR primers. The sequences were ana- ȱȱȱŘǯȱȱęȱȱȱ ¢ȱȱȱřǯŖȱ ȱǻ ȱ ȱ ȱȱȱ ȱȱ Codes Corporation, Ann Arbor, MI, USA) and ERB1 (Barta et al., 1997) and reverse primer BLASTn searches in GenBank. Np1045r (Steinum et al., 2ŖŖŞǼȱ ȱȱ- ȱȱȱŚŝȱǚǯȱȱȱȱȱ ȱȱȱȱęȱȱȱ the nested PCR used primers and conditions ȱP. perurans and the apparent prevalence, as described by Young et al.ȱǻŘŖŖŞǼǯȱȱ ȱȱȱȱȱȱęȱ- round PCR products were visualised by gel ȱȱȱȱȱȱęȱȱ ȱȱęȱȱ ȱ ȱȱǰȱ ȱǯȱęȱ ȱęȱȱǻǼǯȱ intervals (CI) were estimated by logistic regres- 222, Bull. Eur. Ass. Fish Pathol., 35(6) 2015 sion as Wald-type intervals on the log-odds ity (R) representing guanine or adenine, which scale using Taylor linearised standard errors ȱ¢ȱȱȱǰȱȱȱ ȱ ȱȱȱȱȱ¢ȱǯȱ ȱȱȱȱȱȱȱȱ Calculations were carried out within the R sta- the salmon louse. The horse mackerel isolate tistical environment 3.1.2 (R Core Team, 2014) ȱ ȱşşǯŞƖȱ¢ǰȱǻȱȱ utilising the supplementary R package survey ěǼȱȱP. perurans (EU326494.1) isolated 3.30-3 (Lumley, 2004). ȱȱȱȱȱ ¢ȱǻ¢ȱȱ ǯǰȱŘŖŖŞǼǰȱ ȱȱȱȱěȱ ȱȱȱŘǰřŚŞȱęȱȱȱȱȱ ȱȱȱěȱȱȮȱŚřŞȱȱȱȱ ǻȱȱȱŗǼȱ ȱȱȱP. perurans ȱǯȱȱȱ ȱĴȱ ¢ȱǯȱȱȱǰȱȱȱȱ- generic P. perurans ȱǻ şŞşŞŞŗǼǰȱşşǯŜƖȱ erel Trachurus trachurus,ȱȱȱȱP. ¢ȱǻŘȱȱěǼȱ ȱǰȱ perurans. ¢ȱ¡ȱȱȱȱ ȱȱěȱȱȱŚŚşȱȱȱ qPCR results where only one replicate tested previously discussed and a second nucleotide ǰȱ ȱȱȱȱ ȱȱȱ ěȱȱȱŚŚŖȱȱȱȱȱ P. perurans ¢ȱȱȱȱȱȱ isolate. At both positions there were ambiguity reported as negative. All other samples were in the culture isolate so again, this may not be ȱȱP. perurans by qPCR. These results ȱȱěǯȱȱȱ¡ȱȱȱ are summarised in Table 1. All negative controls ȱȱ ȱȱęǰȱ- had acceptable results. All endogenous controls cating that there was no contamination, and the ȱȱȱȱȱȱŘŖǯ ȱȱ ȱęȱ¢ȱȱȱ can be assumed that this is a true positive result. A partial sequence (495 nts, GenBank acces- ȱȱ şŞşŞŞŖǼȱȱȱŗŞȱȱȱP. ȱȱȱP. perurans positive sampled peruransȱ ȱȱȱȱȱȱ ęȱ ȱŖǯŖŚřƖȱ ȱȱşśƖȱ ȱȱŖǯŖŖŜȮŖǯŘşřƖǯȱ ȱǯȱȱȱęȱP. ȱȱȱȱȱȱŖǯŖśŖƖȱ perurans and the sequence was most similar ǻŖǯŖŖŝȬŖǯřŜŚƖǼDzȱȱȱȱȱȱȱ to P. peruransȱȱȱȱȱȱȱ ȱȱ¢ȱȱę¢ȱȱȱ wrasse ȱ ¢ǯȱę¢ǰȱȱȱ ȱ assay or gill sampling technique but does rep- ŗŖŖƖȱȱȱ ŗŝşśŘŖǯŗȱǻǼȱȱ ȱȱȱȱȱȱȱȱ ȱşşǯŞƖȱ¢ȱǻȱȱěǼȱ ȱǯ to KF146711.1, KF146712.1, KF146713.1 (Karls- bakk et al., 2013). The horse mackerel isolate A previous study (Douglas-Helders et al., 2002) ȱ ȱşşǯŞƖȱ¢ȱǻȱȱ ȱȱ ȱęȱȱȱȱęȱ ěǼȱȱP. perurans (GU574794.1) isolated ȱȱP. peruransȱȱȱřŘśȱ ȱȱȱǰȱLepeophtheirus salmonis, on ȱęȱȱȱȱȱȱȱ Atlantic salmon in USA (Nowak et al., 2010). ǯȱ ȱȱȱȱȱȱ ȱȱȱěȱȱȱȱ ȱȱȱ ȱȱ ȱȱȱ ȱ ȱȱȱȱȱŚŚşȱȱȱȱ Ĵȱ ȱȱP. perurans was unlikely to mackerel partial sequence, a nucleotide ambigu- ȱȱȱȱȱȱ£ȱȱřŘśǯ Bull. Eur. Ass. Fish Pathol., 35(6) 2015, 223 ȱȱȱȱȱȱȱȱ- as high as 20 °C in other countries (Munday et ȱȱ ȱǰȱȱȱȱȱȱȱ al., 1990). It has been reported that salinity is ¢ȱȱȱ¢ǰȱȱ a more relevant contributing environmental ȱęȱȱȱ ȱȱȱȱ¡ȱ ȱ ȱ ȱȱȱȱ- ȱȬęȱȱǯȱȱȱ ȱǃřŘȱȱǻ¢ȱȱǯǰȱŘŖŖŗǼǯȱȱȱ current samples were screened using molecular ěȱȱ ȱȱȱȱȱȱ techniques and tissue integrity is not critical, the Scotland with outbreaks typically occurring ęȱȱȱ ȱȱȱȱ¡- ȱȱȱ ǰȱȱȱȱȱ sure to external environment might increase the (Marine Scotland Science unpublished data), ȱȱP. perurans detection. However, ȱȱȱȱ¢ȱȱȱ such an assumption is not valid as intake and ȱǰȱȱȱȱȱP. per- ¡ȱȱ ȱȱȱȱ¢ȱȱȱ urans might be expected. other gill arches and regions within those gills being more disposed to capturing and retaining There were two horse mackerel sampled in amoebae. Adams and Nowak (2001) analysed this survey - it is curious that the one positive ȱȱȱȱȱȱȱȱ ȱ ȱȱȱȱȱȱȱ ȱȱ ǯȱ¢ȱȱę¢ȱȱȱ ȱ ȱȱȱȱȱęȱ in the dorsal section compared to median and ȱȱȱȱȱȱȱȱ ventral sections and suggested that this might ȱȱęȱȱȱP. perurans.