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Development of a non-lethal hydrogen peroxide treatment for surveillance of Gyrodactylus salaris on trout farms and its application to testing wild salmon populations.

ABSTRACT: This study documents the development of a non-lethal sampling method to recover gyrodactylid parasites from large numbers of fish that will underpin an improved surveillance strategy for Gyrodactylus salaris. A review of published literature identified over 80 compounds that have previously been tested against gyrodactylids or closely related parasite species. Five safe and relatively fast-acting compounds were selected for testing to determine their efficiency in removing gyrodactylids from host fish in small-scale aquaria trials using three-spined stickleback infected with Gyrodactylus gasterostei as a model host-parasite system. The most effective compound was hydrogen peroxide; short-duration exposure (3 min) achieved a parasite detection sensitivity of 80%-89%. The practicality of exposing farmed salmonids to hydrogen peroxide for G. salaris surveillance was tested in the field by conducting a parasite recovery trial using a brown trout stock endemically infected with G. truttae and G. derjavinoides and comparing this to the whole-body examination procedure currently conducted by UK authorities. Significantly more parasites were recovered after exposing fish to hydrogen peroxide and filtering the treatment solution than by direct whole-body examination of killed fish (mean: 225 vs. 138 parasites per fish). The gyrodactylid recovery rate of the two methods was 84.6% and 51.9%, respectively. A comparison of timings for the two methods indicated scope for significant time savings in adopting the chemical screening method. The study demonstrated that hydrogen peroxide bath treatment may be successfully applied to the surveillance of gyrodactylid parasites and established as a non-lethal method for sampling farmed and wild fish. This approach has the potential to reduce resources required to collect and isolate parasites for diagnostic testing and improve the sensitivity and confidence of surveillance programmes designed to demonstrate freedom from disease, thus underpinning a robust and defensible surveillance strategy for G. salaris for the UK aquatic animal disease contingency plan.

SUBMITTER: Thrush MA

PROVIDER: S-EPMC6851698 | biostudies-literature | 2019 Sep

REPOSITORIES: biostudies-literature

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Development of a non-lethal hydrogen peroxide treatment for surveillance of Gyrodactylus salaris on trout farms and its application to testing wild salmon populations.

Thrush Mark A MA Hill Tom T Taylor Nick G H NGH

Transboundary and emerging diseases 20190618 5

This study documents the development of a non-lethal sampling method to recover gyrodactylid parasites from large numbers of fish that will underpin an improved surveillance strategy for Gyrodactylus salaris. A review of published literature identified over 80 compounds that have previously been tested against gyrodactylids or closely related parasite species. Five safe and relatively fast-acting compounds were selected for testing to determine their efficiency in removing gyrodactylids from hos ...[more]

PMID: 31155828

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Project description:The salmon parasite Gyrodactylus salaris Malmberg, 1957 has caused high mortalities in many Atlantic salmon, Salmo salar, populations, mainly in Norway. The parasite is also present in several countries across mainland Europe, principally on rainbow trout, Oncorhynchus mykiss, where infections do not seem to result in mortalities. There are still European countries where there are potential salmonid hosts for G. salaris but where the occurrence of G. salaris is unknown, mainly due to lack of investigations and surveillance. Gyrodactylus salaris is frequently present on rainbow trout in low numbers and pose a risk of infection to local salmonid populations if these fish are subsequently translocated to new localities.Farmed rainbow trout Oncorhynchus mykiss (n?=?340), brook trout, Salvelinus fontinalis (n?=?186), and brown trout, Salmo trutta (n?=?7), and wild brown trout (n?=?10) from one river in Romania were sampled in 2008 and examined for the presence of Gyrodactylus spp. Alltogether 187 specimens of Gyrodactylus spp. were recovered from the fish. A subsample of 76 specimens representing the different fish species and localities were subjected to species identification and genetic characterization through sequencing of the ribosomal internal transcribed spacer 2 (ITS2) and mitochondrial cytochrome c oxidase subunit 1 (cox1).Two species of Gyrodactylus were found, G. salaris and G. truttae Gläser, 1974. This is the first time G. salaris is diagnosed in Romania. Gyrodactylus salaris was found to infect rainbow trout, brown trout and brook trout in eight of the 12 farms examined. The prevalence and intensity of infections were generally low in all farms. Gyrodactylus truttae was present on brook trout in one farm and on wild brown trout in the river studied. This also represents the first record of this parasite in Romania. Analyses of sequences of the cox1 gene of G. salaris from Romania revealed four haplotypes, all previously undescribed. While it is not unlikely that the infections in Romanian fish farms originate directly from imported rainbow trout, the current data is not sufficient to conclude on this and does not exclude that the infections can originate from hosts in the local water systems. The study shows that there are still unknown populations and variants (haplotypes) of G. salaris present in European rainbow trout aquaculture, all or many of them with unknown biological characteristics such as host specificity and virulence. As some strains might be pathogenic to Atlantic salmon, the importance of carrying out surveillance and keeping a high focus on control with import and export of live fish for aquaculture purposes is important.Gyrodactylus salaris and G. truttae are for the first time found on salmonids in Romania. All mitochondrial haplotypes recovered were previously undescribed and this indicates that there is still an unknown diversity of this parasite present in localities not previously examined. The virulence of the haplotypes found in Romania is unknown and requires establishing.

Project description:BackgroundGyrodactylus salaris Malmberg, 1957 is an OIE (Office International des Epizooties)-listed parasitic pathogen and had until the current study been reported from 19 countries across Europe, although many of these records require confirmation. The last comprehensive evaluation regarding the distribution of G. salaris, however, was made in 2007, although some of the states identified as being G. salaris-positive were ascribed this status based on misidentifications, on partial data resulting from either morphological or molecular tests, or from records that have not been revisited since their early reporting. It is thus important to go through the reports on G. salaris to obtain a status for each country.MethodsTo provide a revised update of the G. salaris distribution, a literature review was necessary. This literature, however, was not always readily accessible and, in certain cases, the article only made superficial reference to the parasite without providing details or data to support the identification. In most cases, the original specimens were not deposited in a national collection. Additional Gyrodactylus material for the current study was obtained from selected salmonid populations with the aim to contribute to current understanding regarding the distribution of G. salaris. Additional parasite material collected for this study was processed following standard procedures for species identification in Gyrodactylus [1].ResultsFrom the work conducted in the current study, G. salaris is reported from a further three regions in Italy, alongside three other species, and appears to occur extensively throughout central Italy without causing significant mortalities to its rainbow trout, Oncorhynchus mykiss (Walbaum), host. The analysis of archive material from G. salaris-positive farms would suggest that G. salaris has been in this country since at least 2000. Material obtained from rainbow trout from Finland and Germany are confirmed as G. salaris, supporting existing data for these countries. No specimens of G. salaris, however, were found in the additional Gyrodactylus material obtained from rainbow trout reared in Portugal and Spain. A morphologically similar species, Gyrodactylus teuchis Lautraite, Blanc, Thiery, Daniel et Vigneulle, 1999, however, was found.ConclusionsFollowing the present review, Gyrodactylus salaris is reported from 23 out of 50 recognised states throughout Europe; only records from 14 of these states have been confirmed by either morphology and/or by an appropriate molecular test and are considered valid, while only nine of these records have been confirmed by a combination of both methods.

Project description:BACKGROUND:Environmental DNA (eDNA) monitoring is growing increasingly popular in aquatic systems as a valuable complementary method to conventional monitoring. However, such tools have not yet been extensively applied for metazoan fish parasite monitoring. The fish ectoparasite Gyrodactylus salaris, introduced into Norway in 1975, has caused severe damage to Atlantic salmon populations and fisheries. Successful eradication of the parasite has been carried out in several river systems in Norway, and Atlantic salmon remain infected in only seven rivers, including three in the Drammen region. In this particular infection region, a prerequisite for treatment is to establish whether G. salaris is also present on rainbow trout upstream of the salmon migration barrier. Here, we developed and tested eDNA approaches to complement conventional surveillance methods. METHODS:Water samples (2 × 5 l) were filtered on-site through glass fibre filters from nine locations in the Drammen watercourse, and DNA was extracted with a CTAB protocol. We developed a qPCR assay for G. salaris targeting the nuclear ribosomal ITS1 region, and we implemented published assays targeting the mitochondrial cytochrome-b and NADH-regions for Atlantic salmon and rainbow trout, respectively. All assays were transferred successfully to droplet digital PCR (ddPCR). RESULTS:All qPCR/ddPCR assays performed well both on tissue samples and on field samples, demonstrating the applicability of eDNA detection for G. salaris, rainbow trout and Atlantic salmon in natural water systems. With ddPCR we eliminated a low cross-amplification of Gyrodactylus derjavinoides observed using qPCR, thus increasing specificity and sensitivity substantially. Duplex ddPCR for G. salaris and Atlantic salmon was successfully implemented and can be used as a method in future surveillance programs. The presence of G. salaris eDNA in the infected River Lierelva was documented, while not elsewhere. Rainbow trout eDNA was only detected at localities where the positives could be attributed to eDNA release from upstream land-based rainbow trout farms. Electrofishing supported the absence of rainbow trout in all of the localities. CONCLUSIONS:We provide a reliable field and laboratory protocol for eDNA detection of G. salaris, Atlantic salmon and rainbow trout, that can complement conventional surveillance programs and substantially reduce the sacrifice of live fish. We also show that ddPCR outperforms qPCR with respect to the specific detection of G. salaris.

Project description:Gyrodactylus salaris (Monogenea, Platyhelminthes) is a notifiable freshwater pathogen responsible for causing catastrophic damage to wild Atlantic salmon stocks, most notably in Norway. In some strains of Baltic salmon (e.g., from the river Neva) however, the impact is greatly reduced due to some form of innate resistance that regulates parasite numbers, resulting in fewer host mortalities. Gyrodactylus salaris is known from 17 European states; its status in a further 35 states remains unknown; the UK, the Republic of Ireland and certain watersheds in Finland are free of the parasite. Thus, the parasite poses a serious threat if it emerges in Atlantic salmon rearing regions throughout Europe. At present, infections are generally controlled via extreme measures such as the treatment of entire river catchments with the biocide rotenone, in order to remove all hosts, before restocking with the original genetic stock. The use of rotenone in this way in EU countries is unlikely as it would be in contravention of the Water Framework Directive. Not only are such treatments economically and environmentally costly, they also eradicate the potential for any host/parasite evolutionary process to occur. Based on previous studies, UK salmon stocks have been shown to be highly susceptible to infection, analogous to Norwegian stocks. The present study investigates the impact of a G. salaris outbreak within a naïve salmon population in order to determine long-term consequences of infection and the likelihood of coexistence. Simulation of the salmon/ G. salaris system was carried out via a deterministic mathematical modelling approach to examine the dynamics of host-pathogen interactions. Results indicated that in order for highly susceptible Atlantic strains to evolve a resistance, both a moderate-strong deceleratingly costly trade-off on birth rate and a lower overall cost of the immune response are required. The present study provides insights into the potential long term impact of G. salaris if introduced into G. salaris-free territories and suggests that in the absence of external controls salmon populations are likely to recover to high densities nearing 90% of that observed pre-infection.

Project description:BACKGROUND: Describing and evaluating miRNA inventories with Next Generation Sequencing is a goal of scientists from a wide range of fields. It requires high purity, high quality, and high yield RNA extractions that do not only contain abundant ribosomal RNAs but are also enriched in miRNAs. Here we compare 6 disparate and commercially available totalRNA extraction kits for their suitability for miRNA-preparations from Gyrodactylus salaris, an important but small (500 ?m in length) monogenean pathogen of Norwegian Atlantic salmon (Salmo salar). FINDINGS: We evaluated 1 salt precipitation method (MasterPure™ Complete RNA Purification Kit, Epicentre), 2 Phenol based extraction methods (mirVana Kit, Ambion, and Trizol Plus Kit, Invitrogen), 1 paramagnetic bead extraction method (RNA Tissue kit, GeneMole) and 2 purification methods based on spin column chromatography using a proprietary resin as separation matrix (Phenol-free Total RNA Purification Kit, Amresco, and ZR MicroPrep Kit, Zymo Research). The quality of the extractions from 1, 10 and 100 individuals, respectively, was assessed in terms of totalRNA yield, RNA integrity, and smallRNA and miRNA yield. The 6 RNA extraction methods yielded considerably different total RNA extracts, with striking differences in low molecular weight RNA yield. The Phenol-free Total RNA Purification Kit (Amresco) showed the highest totalRNA yield, but the best miRNA/totalRNA ratio was obtained with the ZR MicroPrep Kit (Zymo Research). It was not possible to extract electrophoretically detectable miRNAs from Gyrodactylus salaris with the RNA Tissue Kit (GeneMole) or the Trizol Plus Kit (Invitrogen). CONCLUSIONS: We present an optimized extraction protocol for single and small numbers of Gyrodactylus salaris from infected Atlantic salmon that delivers a totalRNA yield suitable for downstream next generation sequencing analyses of miRNA. Two of the six tested totalRNA kits/methods were not suitable for the extraction of miRNAs from Gyrodactylus salaris.

Dataset Information

Development of a non-lethal hydrogen peroxide treatment for surveillance of Gyrodactylus salaris on trout farms and its application to testing wild salmon populations.

Publications

Development of a non-lethal hydrogen peroxide treatment for surveillance of Gyrodactylus salaris on trout farms and its application to testing wild salmon populations.

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