Bull. Eur. Ass. Fish Pathol., 29(4) 2009, 123 Gyrodactylids on Danish salmonids with emphasis on wild Atlantic salmon Salmo salar T. R. Jørgensen, L.v. G. Jørgensen, R. D. Heinecke, P. W. Kania and K. Buchmann* Department of Veterinary Pathobiology, Section of Fish Diseases, University of Copenhagen, Faculty of Life Sciences, Frederiksberg, Denmark Abstract The present work reports the occurrence of gyrodactylids on salmonids (brown trout, rainbow trout, Atlantic salmon) in Danish waters. The report is supplemented with results from a new investigation on wild Atlantic salmon. Thus one hundred and sixty gyrodactylids collected from 41 Danish wild Atlantic salmon parr in River Hjortvad å (Western part of Denmark) were diagnosed by molecular techniques. The internal transcribed spacer (ITS) region of the rdna was analyzed by PCR restriction fragment length polymorphism (RFLP). A total of three different gyrodactylid species were found to infect wild salmon. Gyrodactylus derjavinoides clearly dominated in numbers, but Gyrodactylus teuchis and Gyrodactylus truttae were also found. G. salaris was not recorded in this study. Findings are analyzed with reference to previous studies on gyrodactylids infecting Danish salmonids. Confirmed occurrence of Gyrodactylus species infecting salmonids in Denmark comprises G. derjavinoides and G. teuchis (rainbow trout, brown trout and Atlantic salmon), G. truttae (brown trout and Atlantic salmon) and a non-pathogenic form of G. salaris (rainbow trout and Atlantic salmon). Introduction Parasitic infection of Atlantic salmon with the monogenean gyrodactylid Gyrodactylus salaris, is a serious problem in Norwegian rivers, where wild salmon stocks have been severely decimated due to infection with this ectoparasite (Johnsen, 1978; Johnsen & Jensen, 1988; Mo, 1994; Appleby et al., 1997; Bakke et al., 2007). Laboratory studies on wild Atlantic salmon from Danish rivers have clearly shown that these salmon strains are highly susceptible towards infection with Norwegian G. salaris (Dalgaard et al., 2004; Heinecke et al., 2007; Kania et al., 2007). This has raised the question if a G. salaris epidemic could occur in Denmark. Previous studies on gyrodactylids infecting Danish salmonids mainly focused on brown trout and rainbow trout (Buchmann & Bresciani, 1997; Buchmann et al., 2000; Nielsen and Buchmann 2001; Cunningham et al., 2001). However, a recent study on gyrodactylid infections of wild Atlantic salmon in Danish rivers has demonstrated the occurrence of five different Gyrodactylus species; G. derjavinoides G. truttae, G. teuchis, G. arcuatus and a non-pathogenic form of G. salaris (Jørgensen et al., 2008). G. derjavinoides clearly dominated in numbers on wild Danish salmon, and in general G. teuchis * Corresponding author s E-mail: kub@life.ku.dk
Bull. Eur. Ass. Fish Pathol., 29(4) 2009, 124 was more prevalent then G. truttae. Only one single specimen of a non-pathogenic form of G. salaris and one of G. arcuatus were found. G. arcuatus normally infects three-spined stickleback and was considered an accidental parasite on wild Atlantic salmon in Danish rivers. The single specimen of G. salaris (nonpathogenic to salmon) showed the same mutation in the ITS region as described in a G. salaris variant isolated from rainbow trout in Danish fish farms. Challenge studies using this particular variant have clearly showed that this G. salaris strain is non-pathogenic towards East Atlantic salmon (Jørgensen et al., 2007). Furthermore, another G. salaris like variant (Gx) previously isolated from cultured Danish rainbow trout has shown a correspondingly low pathogenicity towards east Atlantic salmon (Lindenstrøm et al., 2003). The aim of the present study was to provide additional information on gyrodactylid infections of wild Danish Atlantic salmon and provide an update on the present Gyrodactylus situation on salmonids in Denmark. Materials and methods Sampling A total of 41 specimens of 0+ and 1+ wild salmon par were sampled by electro-fishing in River Hjortvad å (Jutland, Western part of Denmark) during August 2006 (water temperature 13-15 C). Immediately upon capture, salmon were killed in an overdose of MS222. Fins were separated from the body and all parts were conserved in 96% ethanol until further analysis where these items were examined under a dissection microscope (1-40 x magnification). PCR-RFLP A total of 423 gyrodactylids were recovered and 160 of these gyrodactylids were subsampled and analyzed using PCR-RFLP analysis of the ITS (internal transcribed spacer) region of the rdna gene array (1300 bp) (Cunningham, 1997). In brief, the following procedure was used: Parasites preserved in 96% ethanol were dried and placed in 200 μl PCR tubes containing 7.5 μl lysis buffer (Tween 20 [0.45%], Proteinase K [60 μl ml 1 ], 10 mm Tris and 1 mm EDTA) at 65 C until complete digestion of soft parts (confirmed by microscopy). Inactivation of Proteinase K was done at 95 C for 10 min. PCR was performed with Taq polymerase (Bioline no. BIO21040), at an annealing temperature of 55 C (35 cycles). Specific primers used were: Forward primer 5 -TTTCCGTAGGTGAACCT-3 and reverse primer 5 -TCCTCCGCTTAGTGATA-3. The PCR products were digested with restriction enzyme BsuRI (HaeIII; Fermentas, Copenhagen, Denmark) and the band patterns were visualized on a 2% ethidium bromide stained agarose gel. Morphology Morphological identification of recovered parasites was performed by mounting isolated opisthaptors in ammonium-picrate-glycerine (Malmberg, 1970) and subsequent analysis using a Leica DMLB light microscope. Results Micrographs of the species found to infect wild Danish salmon are presented in Fig. 1. Molecular diagnostics (PCR-RFLP) of 160 Gyrodactylus specimens were conducted, resulting in identification of 3 different species (Cunningham, 1997). G. derjavinoides
Bull. Eur. Ass. Fish Pathol., 29(4) 2009, 125 Figure 1. Micrographs of Gyrodactylid species occurring on Danish salmonids. A: G. derjavinoides; B: G. salaris; C: G. teuchis; D: G. truttae. Table 1. Gyrodactylus species from 41 infected salmon parr (present investigation). Number of parasites, prevalence and mean intensity. Parasite species G. derjavinoides G. teuchis G. truttae Number of parasites 156 3 1 Prevalence 100% 7.3% 2.4% Mean intensity including all Gyrodactylus species 9.6 dominated in numbers and only few G. teuchis and G. truttae were observed (Table 1). In order to compare the present data with previous findings a list of gyrodactylids on Danish salmonids (lit. survey) is provided in Table 2. Likewise the occurrence of gyrodactylids in salmonids in countries adjacent to Denmark is given in Table 3. Discussion The finding of G. derjavinoides, G. teuchis and G. truttae on wild salmon parr from River Hjortvad å corresponds with previous investigations by Jørgensen et al. (2008) who showed that salmon parr from this, and 5 additional rivers, were infected with these three species. In addition, these authors recovered one single specimen of G. salaris and one single specimen of G. arcuatus from salmon parr. G. derjavinoides clearly
Bull. Eur. Ass. Fish Pathol., 29(4) 2009, 126 Table 2. Updated list based on literature data of Gyrodactylus species infecting Danish salmonids (Buchmann and Bresciani, 1997; Buchmann et al., 2000; Nielsen and Buchmann, 2001; Lindenstrøm et al., 2003; Cunningham et al.,. 2001; Jørgensen et al., 2008). Salmonid species Gyrodactylus species Reference Salmo salar (wild) Salmo trutta (wild) G. derjavinoides, G. truttae, G. teuchis, G. salaris*, G, arcuatus G. derjavinoides, G. truttae, G. teuchis Buchmann et al., 2000 Jørgensen et al., 2008 Buchmann et al., 2000 Cunningham et al., 2001 Thymallus thymallus (wild) G. sp. Buchmann et al., 2000 Oncorhynchus mykiss (wild) G. derjavinoides, G. truttae Buchmann et al., 2000 Oncorhynchus mykiss (farmed) G. derjavinoides, G. salaris (non-pathogenic towards Atlantic salmon), G.x (G. salaris morphotype) Buchmann & Bresciani, 1997 Buchmann et al., 2000 Nielsen & Buchmann, 2001 Lindenstrøm et al., 2003 * Showing an ITS-sequence affiliating it with the non-pathogenic G. salaris form. Table 3. Literature data on the occurrence of gyrodactylids on salmonids in countries adjacent to Denmark: Norway, Sweden, Finland, Germany and Poland. Salmonid species Country Gyrodactylus species Reference Norway G. salaris Mo, 1991a; Hansen et al., 2003; Cunningham et al., 2000 Salmo salar Sweden G. salaris Malmberg & Malmberg, 1993; Hansen et al., 2003 Finland G. salaris Kinnunen & Valtonen, 1996 Salmo trutta Norway G. salaris Johnsen & Jensen, 1988 Poland G. salaris Prost 1991; Rokicka et al., 2007 Sweden G. salaris Cunningham et al., 2003; Hansen et al., 2003 Oncorhynchus Norway G. salaris Mo, 1991b; Cunningham et al., 2003 mykiss Finland G. salaris Cunningham et al., 2003 Poland G. salaris Prost, 1991; Rokicka et al., 2007 Germany G. salaris Cunningham et al., 2003 Salvelinus alpinus Norway G. salaris Robertsen et al., 2007
Bull. Eur. Ass. Fish Pathol., 29(4) 2009, 127 dominated the parasite population on wild Danish salmon parr, and only few G. teuchis and G. truttae were recovered. The present study did not identify any G. salaris infection, which confirms that Danish salmon stocks do not face an immediate threat from this source, reflecting that G. salaris in Denmark probably is represented exclusively by the nonpathogenic form (Jørgensen et al., 2007). An update (based on previously published data) on Gyrodactylus species found to infect Danish salmonids are presented in Table 2. Thus a total of five Gyrodactylus species have been recovered from Danish salmonids. Atlantic salmon is not a good host for G. derjavinoides but may sustain low populations (Buchmann & Uldal, 1997; Olafsdottir et al., 2003). Brown trout Salmo trutta in Danish rivers are heavily infected with G. derjavinoides (Buchmann et al., 2000) and may represent an important reservoir from where parasites translocate to salmon. In addition, numerous rainbow trout farms are associated with the rivers sampled in the present study. These farms have shown relatively high G. derjavinoides intensities (Buchmann & Bresciani, 1997), which may also represent a parasite reservoir. Accordingly, it is possible that wild brown trout and rainbow trout farms act as reservoirs, continually supplying the associated river system with G. derjavinoides. In both cases a transmission of G. derjavinoides to salmon parr is possible by direct or indirect transfer from rainbow trout. G. derjavinoides may cause morbidity and mortality among brown trout (Ergens, 1980; Malmberg, 1993) and it is possible that this parasite affects brown trout populations in Danish rivers. However, the recorded infection level on Atlantic salmon in Danish rivers is of such a low level that adverse impacts are less likely. Up until now only non-pathogenic strains of G. salaris have been found in Danish rivers and trout farms, which indicates that no introduction of the pathogenic form of G. salaris from Sweden and Norway has occurred so far (Lindenstrøm et al., 2003; Jørgensen et al., 2007; Jørgensen et al., 2008). G salaris has been recovered from several salmonid species in countries close to Denmark. Thus, the parasite has been found to infect rainbow trout in German trout farms (Cunningham et al., 2003). The pathogenic status of this particular strain is unknown. However, when analyzing the IGS (intergenic spacer) region of the ribosomal RNA gene array, a close relationship between the non-pathogenic Danish rainbow trout strain and the German rainbow trout strain was found (Cunningham et al., 2003; Jørgensen et al., 2007). In Norway, Sweden and Finland G. salaris has been recorded on both rainbow trout and Atlantic salmon (Mo, 1991a; Mo, 1991b; Kinnunen & Valtonen, 1996; Cunningham et al., 2000; Cunningham et al., 2003; Hansen et al., 2003; Bakke et al., 2007). In addition the parasite has been recovered in small numbers from brown trout in Norway (Johnsen & Jensen, 1988) but do not propagate on this host species. Furthermore, recent studies have shown that a G. salaris variant infects both anadromous and resident arctic charr Salvelinus alpinus, in Norway (Bakke et al., 2007; Robertsen et al., 2007) and propagate well on this host (Bakke et al., 1996). In addition, laboratory challenge infections demonstrated that the G. salaris strain from Norwegian arctic charr propagates well on both arctic charr and rainbow trout. However, it was shown that the parasite strain was nonpathogenic towards the two East Atlantic salmon stocks that were tested (Olstad et al.,
Bull. Eur. Ass. Fish Pathol., 29(4) 2009, 128 2007). Thus this is the first record of variation in host preference among Norwegian G. salaris strains. In Poland G. salaris has been shown to infect brown trout, grayling and rainbow trout but has not been recorded from salmon (Prost 1991; Rokicka et al., 2007). The PCR-RFLP method applied by Rokicka et al., (2007) was able to discriminate between the strain reported by Lindenstrøm et al., (2003) and previous reported strains. However, the method was not able to discriminate between pathogenic strains and the non-pathogenic strain recently reported from Denmark (Jørgensen et al., 2007). Thus, at the present time we have no information about the pathogenicity of Polish and German strains of G. salaris towards Atlantic salmon. Therefore it should be a priority to apply diagnostic methods capable of discriminating between pathogenic and nonpathogenic strains when monitoring the G. salaris situation in countries where susceptible salmon populations exist. Until the present time, the UK has been successful in preventing the introduction of G. salaris. However, continuous investigation on species discrimination and screening for the parasite is being carried out, in order to prevent a severe epidemic among the highly susceptible wild British salmon (Bakke & MacKenzie, 1993; Shinn et al., 1995; Dalgaard et al., 2003). Further, due to the confirmed high susceptibility of Danish strains of Atlantic salmon (River Skjern å and River Stor å) (Dalgaard et al., 2004; Heinecke et al., 2007) it is recommended to restrict import of live fish from infected areas into Denmark, in order to prevent accidental introduction of the pathogenic form which in the worse case scenario could elicit severe epidemics. Acknowledgement This work was funded by the Danish Ministry of the Environment. References Appleby C, Mo TA and Aase IL (1997). The effect of Gyrodactylus salaris (Monogenea) on the epidermis of Atlantic salmon, Salmo salar, parr in the river Batnfjordselva, Norway. Journal of Parasitology 83, 1173-1174. Bakke TA and Mackenzie K (1993). Comparative susceptibility of native Scottish and Norwegian stocks of Atlantic salmon, Salmo salar L, to Gyrodactylus salaris Malmberg: laboratory experiments. Fisheries Research 17, 69-85. Bakke TA, Jansen PA, and Harris PD (1996). Differences in susceptibility of anadromous and resident stocks of arctic charr to infections of Gyrodactylus salaris under experimental conditions. Journal of Fish Biology 49, 341-351. Bakke TA, Cable J and Harris PD (2007). The biology of gyrodactylid monogeneans: The Russian- doll killers. Advances in Parasitology 64, 162-330. Buchmann K and Uldal A (1997). Gyrodactylus derjavini infections in four salmonids: Comparative host susceptibility and site selection of parasites. Diseases of Aquatic Organisms 28, 13-22. Buchmann K and Bresciani J (1997). Parasitic infections in pond-reared rainbow trout Oncorhynchus mykiss in Denmark. Diseases of Aquatic Organisms 28, 125-138. Buchmann K, Lindenstrøm T, Nielsen ME and Bresciani J (2000). Diagnosis and occurence of ectoparasite infections (Gyrodactylus spp.) in Danish salmonids. Dansk VeterinærTidskrift 83, 15-19. Cunningham CO (1997). Species variation within the internal transcribed spacer
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