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Improved diagnosis of Gyrodactylus parasites infecting aquacultured species.


To improve diagnosis of Gyrodactylus parasites infecting aquacultured salmonid species in Europe, to provide information on the degree of variability in and reliability of genetic and morphological factors used to identify Gyrodactylus species and to compare pathogenicity of G. salaris from different areas of Europe.

This project aims to produce criteria for Gyrodactylus diagnosis, integrating pathogenicity, morphological and molecular investigations detailed in Section 2. Such integrated studies have been recommended by the leading expert on Gyrodactylus (Malmberg and Malmberg, 1993) and are viewed as the best approach for modern parasitology (see Bush et al., 1995). This project will carry out the first such work and will provide a sound basis for the design of sampling and identification programmes for this important fish pathogen.

Gyrodactylus salaris is a parasite which can infect several species of salmonid fish and feeds on host mucus and skin. In general, Gyrodactylus infections of wild fish are of limited pathogenicity but the effect of G. salaris on populations of wild Atlantic salmon in Norway has been a notable exception. G. salaris has resulted in the almost total depletion of juvenile salmon in affected Norwegian rivers. The original source of G. salaris in Norway is believed to be fish from an infected hatchery and apart from the Baltic region, all reports of G. salaris in Europe are from farmed rainbow trout. The parasite spread rapidly within Norway as fish were moved to stock farms and rivers. To date, 39 rivers and 37 hatcheries have been infected (Mo, 1994). Eradication of this parasite from aquaculture facilities is normally achieved by destocking and disinfection of the facility. In rivers, an expected increase in survival rate among salmon pair over time does not seem to occur, possibly because naive fish with no previous exposure to G. salaris are frequently introduced to the pathogen through reproduction of strayers from other rivers and escapees from fish farms. In some of the most badly affected rivers, the salmon population has become extinct. So far, 23 rivers have been cleared by rote none treatment to kill all fish and restocked with parasite-free fish. Rivers are intensively studied for tive years before they are declared free from G. salaris. At present, 10 rivers have achieved this declaration while 11 are in the process. In two rivers the parasite has reappeared after the treatment. G. salaris has resulted in losses of over 25% of the total wild salmon catch in Norway (Egidius et al., 1991 ) and the eradication programme currently costs 3.5 million kroner per year. Such drastic eradication
Improved diagnosis of Gyrodactylus parasites infecting aquacultured species; (Gyrodactylus diagnosis) techniques in river systems may not be economically or ecologically viable in other areas of the community and closure and drying to clear farms of infection would have severe economic impact as detailed in sections 4 and 5.

G. salaris has been demonstrated to be at least equally pathogenic, if not more so, to salmon from European origins other than Norway (Bakke and MacKenzie, 1993). Large and economically important salmon populations exist in the UK, Finland and Russia and are at risk from the importation of G. salaris with live fish, particularly rainbow trout. This strongly indicates the necessity for strict controls to prevent further spread of this parasite in Europe.
EC decision 96/490/EC allows for one year a ban on the import of live salmonids from freshwater into the UK and Ireland unless from areas free of G. salaris. It is also an expressed intention of the EC to introduce a proposal to change the status of G. salaris from a List to List II category disease under Community Directive 91/67/EEC. This is in recognition of its status as a disease which is present in some parts of the EU but exotic to others and because of its severe economic implications.
In order to implement the surveillance testing and laboratory examination required in order to achieve approved zone status under Directive 91/67/EEC, reliable and practical methods of pathogen diagnosis are required and guidelines need to be given on the criteria used for pathogen identification.

To date, there are no such guidelines on the identification and discrimination of Gyrodactylus within Europe and in particular, on the identification of pathogenic forms. Examination of the shape and size of the opisthaptoral hooks has been the peer-accepted method used in Gyrodactylus species descriptions and identifications. However, species differences in morphology can be very slight (Malmberg,1970) and structural similarities in different species can complicate identification (Ergens,1983). In the case of G. salaris, identification can be difficult as the morphological features used show wide variations within a species and may overlap with other species (Ergens, 1983; Mo, 1991 a,b,c, 1994; Prost, 1991). Such variations have prompted the search for improved methods of identification such as SEM study of the hooks (Mo and Appleby, 1990; Shinn et al., 1991,1993; 1995). However, these methods may not be suitable for routine use in a diagnostic laboratory. Implementation of routine monitoring for pathogens requires reliable and straightforward methods which can be readily applied throughout the community. Development and improvement of diagnostic methods is a dynamic process involving application of new techniques and detailed knowledge of the reliability of features used. In order to assess the probability of misidentification of gyrodactylids, it is important to know the degree to which diagnostic characteristics overlap between species (see Malmberg, 1993).

New molecular genetics techniques have been applied to the study of Gyrodactylus. Examination of the ribosomal RNA gene array (rDNA) has led to the development of DNA probes and restriction fragment length polymorphisms (RFLP) to discriminate Gyrodactylus species commonly found on salmon and trout in Europe (Cunningham et al., 1995a,b; Cunningham, in press). However, the regions of rDNA studied so far are not sufficiently variable to reveal differences between G. salaris and G. thymalli, two species that have different host specificities and pathogenicity but are difficult to separate by morphology alone (Malmberg, 1987, 1993; Mo,1994). The ribosomal spacer region has been found to be more variable between closely related species than the ribosomal genes themselves (Adlard et al., 1993; Kane and Rollinson, 1994; Luton et al., 1992). Examination of more variable regions such as the external transcribed spacer and non-transcribed spacer regions of rDNA are required to locate nucleotide differences between G. salaris and G. thymalli. The technique of random amplified polymorphic DNA (RAPD) has also been applied to the study of gyrodactylids (Cunningham and Mo, in press). This provides an alternative method of locating polymorphic DNA of potential value in diagnostic tests and can be used to examine the degree of similarity or relatedness between species or strains of parasite.

To date, no studies have been carried out to investigate the variation in genetic factors within species of Gyrodactylus infecting aquacultured fish species or fish inhabiting watercourses which may provide a source of infection for aquaculture facilities. Knowledge of such variation and of any diagnostic molecular features common to more than one species is vital in order to assess the probability of misidentification, to validate new methods of identification and to avoid false results.

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Scottish Office Agriculture, Environment and Fisheries Department
Victoria road
AB11 9DB Aberdeen
United Kingdom

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