T A Mo and A Jørgensen*

Transcription

1 2017, 40, doi: /jfd A survey of the distribution of the PKD-parasite Tetracapsuloides bryosalmonae (Cnidaria: Myxozoa: Malacosporea) in salmonids in Norwegian rivers additional information gleaned from formerly collected fish T A Mo and A Jørgensen* Norwegian Veterinary Institute, Oslo, Norway This is an open access article under the terms of the Creative Commons Attribution- NonCommercial- NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is noncommercial and no modifications or adaptations are made. Abstract The malacosporean Tetracapsuloides bryosalmonae was detected in kidneys from Atlantic salmon parr in 64 of 91 sampled Norwegian rivers. Using realtime PCR, this parasite was found to be present in Atlantic salmon parr in rivers along the whole coast, from the northernmost and southernmost areas of the country. In addition, T. bryosalmonae was found in kidneys from brown trout parr in 17 of 19 sampled rivers in south-east Norway, and in Arctic charr sampled in the River Risfjordelva, located at the northernmost edge of the European mainland. In conclusion, T. bryosalmonae has a widespread distribution in salmonids in Norwegian watercourses. Proliferative kidney disease (PKD) caused by T. bryosalmonae and PKD-induced mortality has been observed in salmonids in several Norwegian rivers and it can be speculated that more PKD outbreaks will occur as a result of climate change. Keywords: anthropogenic translocation, climate change, fish disease, geographic distribution. Introduction The malacosporean parasite Tetracapsuloides bryosalmonae (Canning, Curry, Feist, Longshaw & Correspondence T A Mo, Norwegian Veterinary Institute, PO Box 750 Sentrum, 0106 Oslo, Norway ( tor-atle.mo@ vetinst.no) *Present address: Pronova BioPharma Norge AS, Sandefjord, Norway. 621 Okamura, 1999) (Cnidaria: Myxozoa) is the causative agent of proliferative kidney disease (PKD), a widespread and severe condition in salmonids in the Northern Hemisphere (Okamura et al. 2011). In Europe, PKD has mainly been reported from farmed fish, especially in introduced rainbow trout Oncorhynchus mykiss (Walbaum) (Hedrick, Macconnell & De Kinkelin 1993), but also from farmed native salmonids such as brown trout (Salmo trutta L.) and Atlantic salmon (S. salar L.) (Ellis, Mcvicar & Munro 1985). Historically, the occurrence of T. bryosalmonae in wild European salmonids has mainly been studied in countries with PKD diagnosed in farmed fish (Seagrave et al. 1981). However, the recent reported severe PKD outbreaks in wild brown trout in Switzerland (Wahli et al. 2002) and Atlantic salmon in Norway (Sterud et al. 2007) have promoted an increased interest for the presence and the distribution of T. bryosalmonae in European watercourses, and studies have been conducted in several countries (Feist et al. 2002; Kristmundsson, Antonsson & Arnason 2010; Skovgaard & Buchmann 2012; Dash & Vasem agi 2014; Jencic et al. 2014). In Norway, the presence of PKD and the associated, previously named PKX organism, has been known in salmonid hatcheries since the 1960s (T. Hastein, pers. comm., Laboratory Information Management System at the Norwegian Veterinary Institute). However, the number of outbreaks in these hatcheries has declined over the past 20 years due to closures and operational changes. In 1989 and 1991, severe outbreaks of PKD were observed among wild Atlantic salmon parr in the River

2 2017, 40, T A Mo & A Jørgensen PKD-parasite in Norway Figgjo in Rogaland County, western Norway. Apart from the observations in the veterinary diagnostic services, there have been no field studies on PKD or its causative organism until the severe outbreaks in the River Aelva in mid-norway in the early 2000s. In this river, the PKD-induced mortality caused a reduction of about 85% among Atlantic salmon parr compared with the normal mortality in apparently disease-free years (Sterud et al. 2007), and in the period , this additional mortality reduced the Atlantic salmon smolt production in the River Aelva by 50 75% (Forseth, Jørgensen & Mo 2007). After the documentation of PKD as the cause of increased mortality of salmon parr in the River Aelva, salmon parr from selected Norwegian watercourses with unexplained decline in the Atlantic salmon population were examined for the presence T. bryosalmonae. The PKD parasite was found at variable prevalence in Atlantic salmon parr in 15 of 16 studied watercourses (Forseth et al. 2007). As this result could indicate a frequent occurrence of T. bryosalmonae in Norwegian Atlantic salmon rivers, we have followed up the pilot study by examining Atlantic salmon parr from 91 rivers, brown trout from 19 rivers and Arctic charr (Salvelinus alpinus (L.)) from one river for the presence of T. bryosalmonae. The present paper summarizes the results from these analyses. Materials and methods Samples of salmonids In 2008, Atlantic salmon parr were sampled in 91 Norwegian rivers (Table 1) in the Norwegian surveillance program for the monogenean Gyrodactylus salaris, an introduced parasite causing extensive salmon parr mortality in affected rivers. The rivers were selected on a risk basis for the presence of G. salaris among the approximately 450 Norwegian Atlantic salmon rivers without considering the presence of other parasites. The surveillance program only focuses on the absence or presence of G. salaris, and no biological or environmental data are sampled. As these data are of importance to study the development of PKD and to compare the severity of disease between rivers, this study only focuses on the presence of T. bryosalmonae in the rivers. In most rivers, 30 Atlantic salmon parr were sampled. In some large rivers, a larger number of 622 parr were sampled and in small rivers, a number of 30 parr was difficult to obtain. This explains the variable number of Atlantic salmon parr examined for the presence of T. bryosalmonae in each river (see Table 1). The salmon parr were in their second growth season (based on fish length) and were sampled in August September by electrofishing. The fish were killed by a blow to the head and stored in 96% ethanol (initial concentration). The Atlantic salmon parr were stored in ethanol for 1 year until each fish was split open with scalpel, and a piece of the mid-kidney was individually sampled in a tube containing 96% ethanol. A new scalpel was used for each fish to avoid contamination of DNA from T. bryosalmonae. In total, 2819 Atlantic salmon parr from the 91 rivers were examined. In 2009, brown trout parr were sampled by electrofishing in 19 rivers in southern Norway in connection with the sampling of salmon parr in the surveillance programme for G. salaris. The brown trout parr were killed by a blow to the head and stored in 96% ethanol (initial concentration). Later the same year, each fish was sampled following the same process as described above. In total, 290 brown trout parr from the 19 rivers were examined (Table 2). In 2008, five Arctic charr (15 20 cm) were sampled by fly-fishing in the River Risfjordelva (N , E ), located a few km from the northernmost point on the European mainland. Each fish was sampled following the same process as described above. The samples of mid-kidneys from Atlantic salmon, brown trout and Arctic charr were examined for the presence of T. bryosalmonae by real-time PCR after DNA extraction. DNA extraction All Atlantic salmon samples were shipped (preserved in ethanol) to GenProbe (previously Tepnel) for DNA extraction. All samples were normalized to a concentration of 50 ng ll 1 DNA. The samples were further diluted to a concentration of 2.5 ng ll 1 upon arrival at the Norwegian Veterinary Institute. All brown trout and Arctic charr samples were subjected to DNA extraction using GeneMole â (an automatic DNA extraction machine). A small piece (5 10 mg) of kidney were transferred to an empty microcentrifuge tube where the ethanol was

3 2017, 40, T A Mo & A Jørgensen PKD-parasite in Norway Table 1 The presence of Tetracapsuloides bryosalmonae in 2819 Atlantic salmon parr from 91 Norwegian rivers. The river are organized by county from the Swedish border in the south-east and along the coast, and then by prevalence River name County # analysed # positive Prevalence Glomma Østfold Akerselva Akershus (Oslo) Lysakerelva Akershus Sandvikselva Akershus Askerelva Akershus Hølenelva Akershus Aroselva Buskerud Numedalslagen Vestfold Skienselva Telemark Herreelva Telemark Storelva Aust-Agder Nidelva Aust-Agder Audna Vest-Agder Otra Vest-Agder Lygna Vest-Agder Kvina Vest-Agder Mandalselva Vest-Agder Haelva Rogaland Ognaelva Rogaland Figgjo Rogaland Sokndalselva Rogaland Vikedalselva Rogaland Bjerkreimselva Rogaland Ardalselva Rogaland Storelva Hordaland Eidselva Sogn og Fjordane Nausta Sogn og Fjordane Arøyelva Sogn og Fjordane Gaula Sogn og Fjordane Ytredalselva Sogn og Fjordane Sogndalselva Sogn og Fjordane Aurlandselva Sogn og Fjordane Nærøydalselva Sogn og Fjordane Flamselva Sogn og Fjordane Henjaelva Sogn og Fjordane Høyangerelva Sogn og Fjordane Mørkridselva Sogn og Fjordane Vikja Sogn og Fjordane Vosso Sogn og Fjordane Oselva Møre og Romsdal Aheimselva Møre og Romsdal Vasskordelva Møre og Romsdal Aureelva Møre og Romsdal Ørstaelva Møre og Romsdal Tafjordelva Møre og Romsdal Eidsdalselva Møre og Romsdal Istadelva Møre og Romsdal Tressa Møre og Romsdal Eira Møre og Romsdal Surna Møre og Romsdal Bævra Møre og Romsdal Mittetelva Møre og Romsdal Norddalselva Møre og Romsdal Oppdølselva Møre og Romsdal Sagelva Møre og Romsdal Stordalselva Møre og Romsdal Todalselva Møre og Romsdal Valldalselva Møre og Romsdal Visa Møre og Romsdal Vigda Sør-Trøndelag Steindalselva Sør-Trøndelag Orkla Sør-Trøndelag

4 2017, 40, T A Mo & A Jørgensen PKD-parasite in Norway Table 1 Continued River name County # analysed # positive Prevalence Tangstadelva Nord-Trøndelag Namsen Nord-Trøndelag Mossa Nord-Trøndelag Mollelva Nord-Trøndelag Aursunda Nord-Trøndelag Bogna Nord-Trøndelag Stjørdalselva Nord-Trøndelag Lakselva, Saltfjorden Nordland Gardselva Nordland Lakselva, Senja Nordland Aelva Nordland Silselva Nordland Halsanelva Nordland Hestdalselva Nordland Lakselva, Vefsnfjorden Nordland Leirelva Nordland Beiarelva Nordland Ranelva Nordland Lyselva Troms Reisaelva Troms Malselva Troms Nordkjoselva Troms Kongfjordelva Finnmark Repparfjordelva Finnmark Tana Finnmark Børselva Finnmark Neidenelva Finnmark Altaelva Finnmark Vestre Jakobselv Finnmark Table 2 The presence of Tetracapsuloides bryosalmonae in 290 brown trout parr from 19 Norwegian rivers. The river are organized by county from the Swedish border in the south-east and along the coast, and then by prevalence River name County # analysed # positive Prevalence Enningdalselva Østfold Askerelva Akershus Lysakerelva Akershus Sandvikselva Akershus Hølenelva Akershus Aroselva Buskerud Bergselva Vestfold Merkedamselva Vestfold Numedalslagen Vestfold Selvikelva Vestfold Herreelva Telemark Skienselva Telemark Nesgrenda Aust-Agder Nidelva Aust-Agder Tovdalselva Vest-Agder Kvina Vest-Agder Otra Vest-Agder Audna Vest-Agder Mandalselva Vest-Agder evaporated prior to the addition of 100 ll of MOLE lysis buffer and 5 ll of proteinase K (Qiagen) and incubated overnight at 56 C. The 624 following day the samples were centrifuged at g for 3 min, and 100 ll of the lysate was subsequently used for automated DNA extraction using MoleStrips DNA tissue kit. All samples were normalized to a concentration of 2.5 ng ll 1. Real-time PCR Real-time PCR was conducted as described by Grabner & El-Matbouli (2009) with some minor changes. The real-time PCRs consisted of 12.5 ll of 2X Brilliant II SYBR Green QPCR Low Rox Master Mix (Stratagene), 0.4 lm of the primers PKD-real F and PKD-real R, 2.5 ng (1 ll) of DNA template and molecular-grade H 2 O to a total volume of 25 ll. All real-time PCRs were analysed on an Mx3005P instrument (Stratagene) using the thermal cycling conditions as defined by Grabner & El-Matbouli (2009) including a melting curve analysis at the end of each run to detect irregularities and non-specific amplifications. All experiments were analysed using the MxPro- Mx3005 v4.10 software (Stratagene) and manually checked according to the standard of the manufacturer. Assay threshold values were manually set

5 2017, 40, T A Mo & A Jørgensen PKD-parasite in Norway to determine the threshold cycle (C t ) of individual samples. Results Tetracapsuloides bryosalmonae was found in kidney samples from Atlantic salmon parr in 64 (70.3%) of the 91 rivers sampled (Table 1). A total of 987 (35%) of 2819 Atlantic salmon parr were found infected. The prevalence of T. bryosalmonae in Atlantic salmon parr in each river varied from 0% to 100% (Table 1). Tetracapsuloides bryosalmonae was found in brown trout parr in 17 (89.5%) of the 19 rivers sampled in south-east Norway (Table 2). A total of 204 (70.3%) of 290 brown trout parr were found infected. The prevalence of T. bryosalmonae in brown trout parr in each river varied from 0% to 100% (Table 2). Finally, T. bryosalmonae was found in all five Arctic charr examined from the River Risfjordelva. Discussion The malacosporean parasite T. bryosalmonae was found in Atlantic salmon parr in about two-thirds of 91 randomly selected rivers representing all the coastal counties of Norway. In addition, T. bryosalmonae was found in brown trout in most of the 19 rivers sampled in the south-east part of the country and in Arctic charr sampled in the River Risfjordelva, one of the northernmost rivers on the European mainland. Tetracapsuloides bryosalmonae was not found to be present in examined fish from several rivers on the west coast and in northern Norway. Many of these rivers, especially on the west coast, are fastflowing rivers, with bottom substratum consisting of pebble, commonly moving, especially in the spring flood. Under these conditions, bryozoans, the definitive hosts for T. bryosalmonae, will likely have reduced colonization success and growth. In addition, many of these rivers are oligotrophic with restricted food items needed for bryozoan growth (Hartikainen et al. 2009). Furthermore, the lower prevalence of T. bryosalmonae in the northernmost rivers compared with the southernmost rivers may be linked to the colder climate in Northern Norway with longer periods with ice cover of the rivers and lower yearly mean water temperatures. Interestingly, T. bryosalmonae was neither found in Atlantic salmon parr or in brown trout parr 625 sampled in the River Numedalslagen (Tables 1 and 2). This is a relatively large river in south-east Norway and the number of examined fish may have been too few to reveal the occurrence of the parasite. However, the 60 Atlantic salmon parr were sampled half and half at the uppermost and lowermost locations in the anadromous part of the river and the brown trout in a third locality. Thus, the apparent absence of T. bryosalmonae in the River Numedalslagen may be a correct observation. One possible explanation is that the necessary bryozoan hosts for T. bryosalmonae are not present in the River Numedalslagen. Although the geographical distribution of the 10 recorded freshwater bryozoans in Norway has been extensively studied, there are no data on the bryozoan fauna in the River Numedalslagen (Økland & Økland 2000, 2001, 2002, 2005; Økland et al. 2003). In 12 rivers, T. bryosalmonae was found in both Atlantic salmon parr and brown trout parr. In ten rivers, the prevalence of T. bryosalmonae was higher or much higher in brown trout parr compared with in Atlantic salmon parr (Tables 1 and 2). In one river, the parasite prevalence was almost equal in both fish species, and in another, the prevalence was higher in Atlantic salmon parr. As all fish was of similar size, considered to be in their second growth season, this observation could indicate that brown trout parr are more susceptible to T. bryosalmonae than Atlantic salmon parr in Norwegian rivers. Another explanation could be that the mortality among infected Atlantic salmon parr is higher than in brown trout parr, resulting in lower parasite prevalence among the Atlantic salmon parr survivors. However, as the two species were sampled in separate years, the differences in parasite prevalence could have other causes than differences in host susceptibility and mortality. In addition to the survey presented here, T. bryosalmonae has been found in bryozoans and salmonids in several other Norwegian watercourses (Forseth et al. 2007; Sterud et al. 2007; Eriksson-Kallio & Jøranlid 2008; Bendixby & Hals 2009; Mo et al. 2011; Kvamme 2013) and it can be concluded that T. bryosalmonae has a wide geographical distribution in salmonids in Norwegian watercourses. A similar wide geographical distribution of the PKD parasite has been also been documented in Switzerland (Wahli et al. 2007). In Norway and Switzerland, PKD and PKD-induced mortality have been observed in brown trout (Wahli

6 2017, 40, T A Mo & A Jørgensen PKD-parasite in Norway et al. 2002) and Atlantic salmon (Sterud et al. 2007), respectively, and these outbreaks have been linked to increased water temperatures. Thus, it can be speculated that more PKD outbreaks will occur as a result of climate change (Wahli et al. 2008; Okamura et al. 2011). The establishment and geographical spread of T. bryosalmonae between Norwegian watercourses is now an issue for discussion. T. bryosalmonae may have established in the rivers early after the last glacial period about years ago together with the recolonization of bryozoans and salmonids, but the parasite could also have been introduced in more recent times. Further studies are required to investigate whether the widespread and geographical distribution of T. bryosalmonae has been driven by migration infected salmonids or by the spread of infected bryozoans including their statoblasts, and if anthropogenic translocations of infected salmonids for stock enhancements have resulted in a further spread of T. bryosalmonae, especially upstream the salmonid migration barriers such as dams and waterfalls, in the watercourses. DNA analysis from a wide range of isolates offers the potential to address the questions, for example using known polymorphic microsatellite markers (Hartikainen et al. 2015). As a result of the surveys for T. bryosalmonae in several countries (Feist et al. 2002; Kristmundsson et al. 2010; Skovgaard & Buchmann 2012; Dash & Vasem agi 2014; Jencic et al. 2014), it can be expected that numerous isolates and DNA extracts are stored in many laboratories. This opens for a wide international cooperation, especially in Europe, in a project to compare the phylogenetic relationships between T. bryosalmonae isolates. These results can be used to understand the geographical origin of T. bryosalmonae, its routes of transmission and further local spread. In conclusion, the PKD-parasite T. bryosalmonae has a wide geographical distribution in salmonids in Norway from the southernmost to the northernmost rivers. An in-depth phylogenetic study to elucidate the introduction and spread of the parasite and the extent to which anthropogenic translocation of infected salmonids has contributed to the current range remains to be conducted. Acknowledgement We are grateful to Mathias V. Mo and Haavard S. Vive for taking the kidney samples from 626 Atlantic salmon parr and to Haakon Hansen for comments on the draft manuscript. References Bendixby L. & Hals P.I. (2009) Distribution and Abundance of Bryozoan Species in River Aelva and the Prevalence of the PKD Parasite Tetracapsuloides bryosalmonae. Master student thesis, pp. 51. Norwegian University of Life Sciences, As. Dash M. & Vasem agi A. (2014) Proliferative kidney disease (PKD) agent Tetracapsuloides bryosalmonae in brown trout populations in Estonia. Diseases of Aquatic Organisms 109, Ellis A.E., Mcvicar A.H. & Munro A.L.S. (1985) Proliferative kidney disease in brown trout, Salmo trutta L., and Atlantic salmon, Salmo salar L., parr: histopathological and epidemilogical observations. 8, Eriksson-Kallio A.M. & Jøranlid A.K. (2008) Proliferative Kidney Disease in Atlantic Salmon (Salmo salar) and Brown Trout (S. trutta) in the River Jølstra Histopathology and Occurrence of the Causative Agent Tetracapsuloides bryosalmonae. Master student thesis, pp. 66. Norwegian School of Veterinary Sciences, Oslo. Feist S.W., Peeler E.J., Gardiner R., Smith E. & Longshaw M. (2002) Proliferative kidney disease and renal myxosporidiosis in juvenile salmonids from rivers in England and Wales. 25, Forseth T., Jørgensen A. & Mo T.A. (2007) Pilotkartlegging av PKD i norske laksevassdrag. NINA Rapport 259, Grabner D.S. & El-Matbouli M. (2009) Comparison of the susceptibility of brown trout (Salmo trutta) and four rainbow trout (Oncorhynchus mykiss) strains to the myxozoan Tetracapsuloides bryosalmonae, the causative agent of proliferative kidney disease (PKD). Veterinary Parasitology 165, Hartikainen H., Johnes P., Moncrieff C. & Okamura B. (2009) Bryozoan populations reflect nutrient enrichment and productivity gradients in rivers. Freshwater Biology 54, Hartikainen H., Filippenko D., Okamura B. & Vasem agi A. (2015) First microsatellite loci of the myxozoan parasite Tetracapsuloides bryosalmonae, the causative agent of proliferative kidney disease (PKD). Diseases of Aquatic Organisms 113, Hedrick R.P., Macconnell E. & De Kinkelin P. (1993) Proliferative kidney disease of salmonid fish. Annual Review of Fish Diseases 3, Jencic V., Zajc U., Kusar D., Ocepek M. & Pate M. (2014) A survey on Tetracapsuloides bryosalmonae infections in Slovene fresh waters. 37, Kristmundsson A., Antonsson T. & Arnason F. (2010) First record of proliferative kidney disease in Iceland. Bulletin of the European Association of Fish Pathologists 30, 35. Kvamme E. (2013) Proliferative Kidney Disease (PKD) in Atlantic Salmon (Salmo salar), Sea Trout (Salmon trutta) and Arctic char (Salvelinus alpinus) Out-Migrant Smolts in the

7 2017, 40, T A Mo & A Jørgensen PKD-parasite in Norway River Halselva-(Histopathology? and) Occurrence and Temperature Related Changes of the Causative Agent Tetracapsuloides bryosalmonae in the Kidney. Master student thesis, pp. 57. Norwegian School of Veterinary Science, Oslo. Mo T.A., Kaada I., Jøranlid A.K. & Poppe T.T. (2011) Occurrence of Tetracapsuloides bryosalmonae in the kidney of smolts of Atlantic salmon (Salmo salar) and sea trout (S. trutta). Bulletin of the European Association of Fish Pathologists 31, Okamura B., Hartikainen H., Schmidt-Posthaus H. & Wahli T. (2011) Life cycle complexity, environmental change and the emerging status of salmonid proliferative kidney disease. Freshwater Biology 56, Økland K.A. & Økland J. (2000) Freshwater bryozoans (Bryozoa) of Norway: distribution and ecology of Cristatella mucedo and Paludicella articulata. Hydrobiologia 421, Økland K.A. & Økland J. (2001) Freshwater bryozoans (Bryozoa) of Norway II: distribution and ecology of two species of Fredericella. Hydrobiologia 459, Økland K.A. & Økland J. (2002) Freshwater bryozoans (Bryozoa) of Norway III: distribution and ecology of Plumatella fruticosa. Hydrobiologia 479, Økland J. & Økland K.A. (2005) Freshwater bryozoans (Bryozoa) of Norway V: review and comparative discussion of the distribution and ecology of the 10 species recorded. Hydrobiologia 534, Økland K.A., Økland J., Geimer G. & Massard J.A. (2003) Freshwater bryozoans (Bryozoa) of Norway IV: distribution and ecology of four species of Plumatella with notes on Hyalinella punctata. Hydrobiologia 501, Seagrave C.P., Bucke D., Hudson E.B. & Mcgregor D. (1981) A survey of the prevalence and distribution of proliferative kidney disease (PKD) in England and Wales. Journal of Fish Diseases 4, Skovgaard A. & Buchmann K. (2012) Tetracapsuloides bryosalmonae and PKD in juvenile wild salmonids in Denmark. Diseases of Aquatic Organisms 101, Sterud E., Forseth T., Ugedal O., Poppe T.T., Jørgensen A., Bruheim T., Fjeldstad H.P. & Mo T.A. (2007) Severe mortality in wild Atlantic salmon Salmo salar due to proliferative kidney disease (PKD) caused by Tetracapsuloides bryosalmonae (Myxozoa). Diseases of Aquatic Organisms 77, Wahli T., Knuesel R., Bernet D., Segner H., Pugovkin D., Burkhardt-Holm P., Escher M. & Schmidt-Posthaus H. (2002) Proliferative kidney disease in Switzerland: current state of knowledge. 25, Wahli T., Bernet D., Steiner P.A. & Schmidt-Posthaus H. (2007) Geographic distribution of Tetracapsuloides bryosalmonae infected fish in Swiss rivers: an update. Aquatic Sciences 69, Wahli T., Bernet D., Segner H. & Schmidt-Posthaus H. (2008) Role of altitude and water temperature as regulating factors for the geographical distribution of Tetracapsuloides bryosalmonae infected fishes in Switzerland. Journal of Fish Biology 73, Received: 7 April 2016 Revision received: 21 June 2016 Accepted: 23 June