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O. tshawytscha ǻȱȱǯǰȱŘŖŖŞǼDzȱ ȱȱȱĴȱȱ¢ȱ ayu Plecoglossus altivelis (Crosbie et al., 2010); (Marine Scotland Science unpublished data).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 ȱ ȱǻȱȱ 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. Both ȱȱȱ ȱĚ ȱȱȱȱȱ ȱȱ ȱȱȱȱȱ ȱȱȱȱȱǰȱȱȱ location in the North West as shown in Figure ȱȱȱĴȱȱȱĴȱȱ ŗǯȱȱȱȱȱȱȱȱȱ ǯȱ ȱ¢ȱȱȱȱȱȱȱ ǰȱ ǰȱȱ ȱȱ¢ȱȱȱ similar sampling approach in the current study. the North Sea or Western stock (pers. comm. ǰȱȱȱ ȱȱȱęȱȱ Cindy van Damme). The sampling location was sampled in this study, the same gill section ȱȱȱ¢ȱȱȱȱ ȱ ¢ȱȱȱȱȱȱǯȱȱȱ (approximately 50 km) where there is an es- study, the potential that gill swabs are more tablished aquaculture industry and AGD has ȱȱȱȱȱȱ previously been observed. This may contribute ȱP. perurans has arisen (pers. comm. Jamie to the increased prevalence at this location, Ǽȱǯǯȱ ȱȱȱȱȱȱ however P. perurans was not detected in other ȱȱȱȱȱȱȱȱȱȱ ȱȱȱȱ¢ȱȱǯȱ less competing DNA. As P. perurans ȱȱȱ¢ȱȱȱęǰȱ ȱȱȱȱȱȱȱȱȱȱȱ Median water temperature and salinity (with are the most important causative explanation. şśȱǼȱ ȱŝǯśȱǻŜǯŗȱȮȱŞǯşǼȱǚȱȱřŚǯŜȱ (33.5 – 35.3) ppt respectively. AGD outbreaks As only qPCR was used to detect P. perurans ȱȱȱȱȱȱȱ ȱȱȱȱȱȱěȱȱ- temperatures between 6.4 – 13.1 °C in Scotland ǯȱȱȱȱȱȱ- (Marine Scotland Science unpublished data) and logical processing in addition to qPCR to allow 224, Bull. Eur. Ass. Fish Pathol., 35(6) 2015
Salmo salar L., production cage in south ȱȱȱȱȱ- eastern Tasmania. Journal of Fish Diseases ¢ǰȱ ȱ ȱȱȱȱȱ ȱęȱ 31, 713-717. were either susceptible to P. peruransȱȱ Barta JR, Martin DS, Liberator PA, Dashkevicz causing AGD; or in a carrier state acting a res- M, Anderson JW, Feighner SD, Elbrecht A, ȱȱȱǯȱȱȱȱ ȱ Ȭ ȱǰȱ ȱǰȱȱ ęȱȱȱȱȱȱȱP. perurans in rela- ǰȱěȱȱȱȬȱ ȱ ȱ (1997). Phylogenetic relationships among ȱȱȱĴȱȱ¢ȱ ȱ ȱȱȱȱȱ ȱȱȱȱ¢ǯȱȱ ȱȱȱȱȱȱ sampling could include: targeting inshore areas ribosomal DNA sequences. Journal of ȱȱ¢ȱȱęȱȱȱ ȱȱěȱ Parasitology 83, 262-271. Dzȱȱȱȱěȱȱ Bustos PA, Young ND, Rozas MA, Bohle HM, ȱȱ¢ȱȱȱȱ¢ȱȱȱ ȱǰȱȱȱȱ ȱ BF (2011). Amoebic gill disease (AGD) in Ěȱȱȱȱȱȱȱ Atlantic salmon (Salmo salarǼȱȱȱ ȱȱǯȱ¢ȱȱȱȱ Chile. Aquaculture 310ǰȱŘŞŗȬŘŞŞǯ ȱȱP. peruransȱȱ ȱȱȱ Crosbie PBB, Bridle AR, Cadoret K and ęȱȱ ȱȱȱȱȱ Nowak BF (2012). In vitro cultured to that used by Garver et al. (2013). Neoparamoeba perurans causes amoebic ȱȱȱȱȱȱęȱ Koch’s postulates. International Journal for In conclusion P. perurans was detected in the Parasitology 42, 511-515. ȱęȱȱȱĴȱȱ Crosbie PBB, Ogawa K, Nakano D and waters and the apparent prevalence in the Nowak BF (2010). Amoebic gill disease in population, as a whole, was substantially less hatchery-reared ayu, Plecoglossus altivelis ȱŗƖǯȱȱȱȱęȱȱȱȱȱ ǻȱǭȱǼǰȱȱ ȱȱȱ P. perurans in horse mackerel. by Neoparamoeba perurans. Journal of Fish Diseases 33ǰȱŚśśȬŚśŞǯ
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