An old unknown: 40 years of crayfish plague monitoring in Switzerland, the water tower of Europe.

The oomycete Aphanomyces astaci is the causative agent of crayfish plague, a disease threatening susceptible freshwater crayfish species in Europe. To detect its spatiotemporal occurrence in Switzerland, we reviewed (1) the literature regarding occurrence of crayfish plague and North American crayfish carrier species and (2) the necropsy report archive of the Institute for Fish and Wildlife Health (FIWI) from 1968 to 2020. In the past, crayfish plague was diagnosed through several methods: conventional PCR, culture, and histology. When available, we re-evaluated archived Bouin's or formalin-fixed, paraffin-embedded samples collected during necropsies (1991-2020) with a recently published quantitative PCR. Literature research revealed putative reports of crayfish plague in Switzerland between the 1870s and 1910s and the first occurrence of three North American crayfish species between the late 1970s and 1990s. Finally, 54 (28.1%) cases were classified as positive and 9 (4.7%) cases as suspicious. The total number of positive cases increased by 14 (14.7%) after re-evaluation of samples. The earliest diagnosis of crayfish plague was performed in 1980 and the earliest biomolecular confirmation of A. astaci DNA dated 1991. Between 1980-1990, 1991-2000 and 2001-2010 crayfish plague spread from one to two and finally three catchment basins, respectively. Similar to other European countries, crayfish plague has occurred in Switzerland in two waves: the first at the end of the 19th and the second at the end of the 20th century in association with the first occurrence of North American crayfish species. The spread from one catchment basin to another suggests a human-mediated pathogen dispersal.

Detection of novel RNA viruses in wild noble crayfish (Astacus astacus): A virome analysis in Swiss water bodies.

European native crayfish populations are undergoing a strong decline due to environmental factors and the introduction of highly competitive non-native species. Pathogens are an additional threat to native crayfish. However, aside from the crayfish plague, other infectious diseases are still widely unknown. This study aimed to investigate viruses present in seven populations of wild noble crayfish (Astacus astacus) in Switzerland, through high-throughput sequencing. Sequence analysis revealed the presence of 11 novel RNA viruses (one bunya-like, four hepe-like, two dicistro-like, three picorna-like, and one permutotetra-like) in the samples. The discovery of a novel bunya-like virus in noble crayfish without associated mortality or macroscopical alterations is of particular interest since it is closely related to the bunya-like brown spot virus, a virus described in 2019 from diseased native white-clawed crayfish (Austropotamobius pallipes) during a mass mortality event in France. It seems that these two closely related viruses have very different impacts on their respective hosts, raising the need for further investigations on virulence factors and host susceptibility towards these viruses. This study provides a basis for future investigations, permitting to gradually fill the knowledge gap in crayfish viral diseases.

Application of UV-C irradiation prevented a severe outbreak of proliferative kidney disease in rainbow trout aquaculture.

There is an urgent need to establish protocols on how to protect salmonids in aquaculture from outbreaks of proliferative kidney disease (PKD). For this purpose, systems for a continuous application of peracetic acid (PAA, 0.1 mg l-1) and of ultraviolet C light (UV-C, 323.5-158.6 mW s cm-2) were installed in the inlet of raceway-channels within a sub-unit of a commercial rainbow trout Oncorhynchus mykiss farm. After 127 d of rearing, a fish health examination was conducted. Fish in the control and PAA treatment groups showed signs of PKD. In contrast, fish in the UV-C treatment group showed almost no signs of disease based on clinical examinations and necropsy. This observation indicates that UV-C irradiation could be a promising tool to protect fish from PKD in the future.

Amoeba species colonizing the gills of rainbow trout (Oncorhynchus mykiss) in Swiss aquaculture.

Nodular gill disease (NGD) is an infectious condition characterized by proliferative gill lesions leading to respiratory problems, oxygen deficiency and mortality in fish. Globally, NGD primarily impacts freshwater salmonids in intensive aquaculture systems. In recent years, numerous outbreaks of severe gill disease have affected more than half of the larger rainbow trout (Oncorhynchus mykiss) farms in Switzerland, mainly during spring and early summer. Mortality has reached up to 50% in cases where no treatment was administered. Freshwater amoeba are the presumed aetiologic agent of NGD. The gross gill score (GS) categorising severity of gill pathology is a valuable first-line diagnostic tool aiding fish farmers in identifying and quantifying amoebic gill disease (AGD) in farmed marine salmonids. In this study, the GS was adapted to the NGD outbreak in farmed trout in Switzerland. In addition to scoring disease severity, gill swabs from NGD-affected rainbow trout were sampled and amoeba were cultured from these swabs. Morphologic and molecular methods identified six amoeba strains: Cochliopodium sp., Naegleria sp., Vannella sp., Ripella sp., Saccamoeba sp. and Mycamoeba sp. However, the importance of the different amoeba species for the onset and progression of NGD still has to be evaluated. This paper presents the first description of NGD with associated amoeba infection in farmed rainbow trout in Switzerland.

Fatal Diplostomum phoxini infection in captive Atlantic puffin Fratercula arctica chicks following ingestion of infected European minnows Phoxinus phoxinus.

Conservation of endangered animal species is a major task of zoos. Husbandry and breeding of Atlantic puffins Fratercula arctica in captivity is challenging. In 2019, the entire chick population (n = 4 chicks) in Berne Animal Park's Atlantic puffin colony (Bern, Switzerland) died within 7 d. Due to supply constraints, the chicks had been fed with wild-caught European minnows Phoxinus phoxinus. At necropsy, the main pathological finding in all deceased puffin chicks was a multifocal, moderate to severe subacute heterophilic and granulomatous enteritis with intralesional adult trematodes and eggs. Metacercariae surrounded by few necrotic cells and scattered macrophages were found in the brain and spinal cord of the food fish. Additional microbiological analyses of both the puffin chicks and fish were unremarkable. Diplostomum phoxini DNA could be identified in formalin-fixed paraffin-embedded tissue from the small intestine of all puffin chicks and European minnows following PCR and sequencing of the 18S ribosomal RNA gene and the internal transcribed spacer (ITS1) region. This report illustrates the importance of intensive health checks of food fish for animal species kept in captivity.

Mitigating human impacts including climate change on proliferative kidney disease in salmonids of running waters.

Over the last two decades, an increasing number of reports have identified a decline in salmonid populations, possibly linked to infection with the parasite Tetracapsuloides bryosalmonae and the corresponding disease, that is, proliferative kidney disease (PKD). The life cycle of this myxozoan parasite includes sessile bryozoan species as invertebrate host, which facilitates the distribution of the parasite in running waters. As the disease outcome is temperature dependent, the impact of the disease on salmonid populations is increasing with global warming due to climate change. The goal of this review is to provide a detailed overview of measures to mitigate the effects of PKD on salmonid populations. It first summarizes the parasite life cycle, temperature-driven disease dynamics and new immunological and molecular research into disease resistance and, based on this, discusses management possibilities. Sophisticated management actions focusing on local adaptation of salmonid populations, restoration of the riverine ecosystem and keeping water temperatures cool are necessary to reduce the negative effects of PKD. Such actions include temporary stocking with PKD-resistant salmonids, as this may assist in conserving current populations that fail to reproduce.

Further spread of perch rhabdovirus on European percid farms.

Variants of perch rhabdovirus (PRV) circulate across European percid farms via the fish trade. To trace their circulation, they are usually isolated by cell culture and subsequently identified genetically by sequencing partial or complete genes. Here, a newly developed nested PCR-based method was used to amplify and sequence the complete N and P genes directly from clinical samples obtained during an outbreak on a farm as well as from four batches of fish sampled from two other farms in another country. In an attempt to trace the origin of the five detected viruses, their N and P sequences were concatenated and compared with related viruses. One virus found in pike-perch was highly related to a virus isolated in 2016 in Belgium. Two other viruses detected on a single farm were distinct from one another, with one being almost identical to another virus isolated in 2016 in Belgium and the other being more closely related to a subgroup with different origins, France and Belgium. Two other viruses found in perch from a third farm were identical and were more related to a subgroup of viruses isolated in France. Identifying variants by a direct PCR approach will help to prevent further dissemination in farms.

Immunohistochemical investigation of epithelial, mesenchymal, neuroectodermal, immune and endocrine markers in sterlet (Acipenser ruthenus), shortnose sturgeon (Acipenser brevirostrum) and common carp (Cyprinus carpio).

Immunohistochemistry (IHC) is a laboratory method widely used to characterize tissue and cell origin, both in human and veterinary medicine. In fish, however, little is known about staining characteristics of most tissue types, and especially for less studied chondrostean fish. The aim of this study was to examine the specificity of various immunohistochemical markers in tissues of chondrostean and teleostean fish and to validate diagnostic tests. Sterlet (Acipenser ruthenus L.), shortnose sturgeon (Acipenser brevirostrum) and common carp (Cyprinus carpio L.) were examined. Markers were chosen as representatives of epithelial (cytokeratin AE1/AE3), mesenchymal (vimentin), neuroectodermal (S-100 protein), lymphoid (leukocyte common antigen, LCA) and endocrine (thyroglobulin, thyroxin) tissues and organs. Applied antibodies were of monoclonal or polyclonal mammalian origin and primarily intended for human medicine research or diagnostic application. No species differences were obvious while examining sterlet, shortnose sturgeon and carp. Cytokeratin AE1/AE3, vimentin, S-100 protein and thyroxin were positive on targeted tissues and structures. Leukocyte common antigen (LCA) and thyroglobulin were negative on targeted structures, however, and with clear cross-reactivity on non-targeted tissues (vascular wall, granulocytes). Conclusive results were obtained when using polyclonal antibodies with dilution adjusted to laboratory practice, while application of ready-to-use (RTU) kits with pre-diluted antibodies or monoclonal antibodies often showed conflicting or inconclusive results.

Novel Filoviruses, Hantavirus, and Rhabdovirus in Freshwater Fish, Switzerland, 2017.

European perch (Perca fluviatilis) are increasingly farmed as a human food source. Viral infections of European perch remain largely unexplored, thereby putting farm populations at incalculable risk for devastating fish epizootics and presenting a potential hazard to consumers. To address these concerns, we applied metatranscriptomics to identify disease-associated viruses in European perch farmed in Switzerland. Unexpectedly, in clinically diseased fish we detected novel freshwater fish filoviruses, a novel freshwater fish hantavirus, and a previously unknown rhabdovirus. Hantavirus titers were high, and we demonstrated virus in macrophages and gill endothelial cells by using in situ hybridization. Rhabdovirus titers in organ samples were low, but virus could be isolated on cell culture. Our data add to the hypothesis that filoviruses, hantaviruses, and rhabdoviruses are globally distributed common fish commensals, pathogens, or both. Our findings shed new light on negative-sense RNA virus diversity and evolution.

It's a hard knock life for some: Heterogeneity in infection life history of salmonids influences parasite disease outcomes.

Heterogeneity in immunity occurs across numerous disease systems with individuals from the same population having diverse disease outcomes. Proliferative kidney disease (PKD) caused by Tetracapsuloides bryosalmonae, is a persistent parasitic disease negatively impacting both wild and farmed salmonids. Little is known of how PKD is spread or maintained within wild susceptible populations. We investigated an aspect of fish disease that has been largely overlooked, that is, the role of the host phenotypic heterogeneity in disease outcome. We examined how host susceptibility to T. bryosalmonae infection, and the disease PKD, varied across different infection life-history stages and how it differs between naïve, re-infected and persistently infected hosts. We investigated the response to parasite exposure in host phenotypes with (a) different ages and (b) heterogeneous infection life histories. Among (a) the age phenotypes were young-of-the-year (YOY) fish and juvenile 1+ fish (fish older than one) and, for (b) juvenile 1+ infection survivors were either re-exposed or not re- exposed to the parasite and response phenotypes were assigned post-hoc dependant on infection status. In fish not re-exposed this included fish that cleared infection (CI) or had a persistent infection (PI). In fish re-exposed these included fish that were re-infected (RI), or re-exposed and uninfected (RCI). We assessed both parasite-centric (infection prevalence, parasite burden, malacospore transmission) and host-centric parameters (growth rates, disease severity, infection tolerance and the immune response). In (a), YOY fish, parasite success and disease severity were greater and differences in the immune response occurred, demonstrating an ontogenetic decline of susceptibility in older fish. In (b), in PI and RI fish, parasite success and disease severity were comparable. However, expression of several adaptive immunity markers was greater in RI fish, indicating concomitant immunity, as re-exposure did not intensify infection. We demonstrate the relevance of heterogeneity in infection life history on disease outcome and describe several distinctive features of immune ontogeny and protective immunity in this model not previously reported. The relevance of such themes on a population level requires greater research in many aquatic disease systems to generate clearer framework for understanding the spread and maintenance of aquatic pathogens.

Scuticociliate (Philasterides dicentrarchi) infection cluster in a multispecies marine aquarium system.

Scuticociliatosis, caused by ciliated protozoa of the subclass Scuticociliatia, has been associated with high mortalities in marine fish. Environmental factors such as an increase in water temperature can enhance this disease. The aim of the present report is to describe the occurrence of a cluster of cases of scuticociliatosis in a multispecies marine cold-water system in a public aquarium. Philasterides dicentrarchi was identified by PCR in formalin-fixed tissues of some of the fish showing meningitis or meningoencephalitis, dermatitis and myositis with intralesional protozoa. An increase in water temperature of approximately 2°C was identified as a potential contributing factor for this cluster of infections. Higher temperature may have enhanced the propagation or pathogenicity of scuticociliates or increased host susceptibility of some species of fish, especially wolf-eel Anarrhichthys ocellatus and spotted ratfish Hydrolagus colliei. This report also highlights the complexity of dealing with mixed species systems housing fish from different natural ecozones.

Sudden outbreak of metastatic intestinal adenocarcinoma in rainbow trout Oncorhynchus mykiss.

Intestinal adenocarcinomas are uncommon in fishes. To date, they have been reported in zebrafish Danio rerio, blue gularis Fundulopanchax sjostedti, koi carp Cyprinus carpio koi, Atlantic salmon Salmo salar and rainbow trout Oncorhynchus mykiss. Metastases are even rarer and have been observed so far at very low prevalence, only in feed-induced adenocarcinoma in Atlantic salmon and rainbow trout. Intestinal adenocarcinoma with liver and heart metastases and mesenteric invasion was found in approximately 33% of 4 yr old rainbow trout from a Slovene hatchery with 2000 breeding trout. During stripping, lumps in the abdominal cavity were palpated in one-third of the breeding fish; some of the fish were anorectic and lethargic, and mortality was slightly increased. Affected trout were euthanized and 4 were submitted for necropsy and histopathology. Necropsy revealed firm, whitish, irregularly lobular masses originating from the intestine. Histologically, the intestinal masses showed a prominent proliferation of tall columnar neoplastic epithelial cells arranged in dense irregular islands or solid areas and papillotubular protuberances. Solid areas of neoplastic cells were also observed in the mesentery of all trout and in the liver of one trout, whereas minute groups of neoplastic cells were seen in the vessels of the intestinal mucosa in all trout and in the myocardium and the liver of one trout. Epithelial origin of neoplastic cells was confirmed by expression of the cytokeratin marker AE1/AE3. The intestinal masses were diagnosed as intestinal adenocarcinoma with mesenteric invasion and metastases to the liver and heart. The cause of intestinal adenocarcinoma was not determined.

Comparison of diagnostic methods for Tetracapsuloides bryosalmonae detection in salmonid fish.

Diagnostic accuracy of pathogen detection depends upon the selection of suitable tests. Problems can arise when the selected diagnostic test gives false-positive or false-negative results, which can affect control measures, with consequences for the population health. The aim of this study was to compare sensitivity of different diagnostic methods IHC, PCR and qPCR detecting Tetracapsuloides bryosalmonae, the causative agent of proliferative kidney disease in salmonid fish and as a consequence differences in disease prevalence. We analysed tissue from 388 salmonid specimens sampled from a recirculating system and rivers in the Czech Republic. Overall prevalence of T. bryosalmonae was extremely high at 92.0%, based on positive results of at least one of the above-mentioned screening methods. IHC resulted in a much lower detection rate (30.2%) than both PCR methods (qPCR32: 65.4%, PCR: 81.9%). While qPCR32 produced a good match with IHC (60.8%), all other methods differed significantly (p < .001) in the proportion of samples determined positive. Both PCR methods showed similar sensitivity, though specificity (i.e., the proportion of non-diseased fish classified correctly) differed significantly (p < .05). Sample preservation method significantly (p < .05) influenced the results of PCR, with a much lower DNA yield extracted from paraffin-embedded samples. Use of different methods that differ in diagnostic sensitivity and specificity resulted in random and systematic diagnosis errors, illustrating the importance of interpreting the results of each method carefully.

Preliminary investigation of an idiopathic muscle disease in farmed burbot Lota lota.

The rearing of less established fish species in recirculating aquaculture systems (RASs) is increasing, but may require adaptations of the rearing facilities if health impairments occur. We observed several health issues in burbot Lota lota reared for up to 2 yr in a RAS and used microbiological, histological and molecular-biological methods to identify the causative agents. Minor skin trauma led to the development of ulcers. In addition, several fillets of burbot showed pronounced granulomatous inflammation and calcification with signs of muscle fiber degeneration which resembled a condition called 'sandy flesh disease' in North American walleye. Several infectious agents were able to be excluded as a cause for the disease. Carnobacterium maltaromaticum was isolated in high numbers in some of the affected muscle tissue. However, the role of this bacterium or other causative agents or husbandry conditions remains to be elucidated.

Integrated field, laboratory, and theoretical study of PKD spread in a Swiss prealpine river.

Proliferative kidney disease (PKD) is a major threat to wild and farmed salmonid populations because of its lethal effect at high water temperatures. Its causative agent, the myxozoan Tetracapsuloides bryosalmonae, has a complex lifecycle exploiting freshwater bryozoans as primary hosts and salmonids as secondary hosts. We carried out an integrated study of PKD in a prealpine Swiss river (the Wigger). During a 3-year period, data on fish abundance, disease prevalence, concentration of primary hosts' DNA in environmental samples [environmental DNA (eDNA)], hydrological variables, and water temperatures gathered at various locations within the catchment were integrated into a newly developed metacommunity model, which includes ecological and epidemiological dynamics of fish and bryozoans, connectivity effects, and hydrothermal drivers. Infection dynamics were captured well by the epidemiological model, especially with regard to the spatial prevalence patterns. PKD prevalence in the sampled sites for both young-of-the-year (YOY) and adult brown trout attained 100% at the end of summer, while seasonal population decay was higher in YOY than in adults. We introduce a method based on decay distance of eDNA signal predicting local species' density, accounting for variation in environmental drivers (such as morphology and geology). The model provides a whole-network overview of the disease prevalence. In this study, we show how spatial and environmental characteristics of river networks can be used to study epidemiology and disease dynamics of waterborne diseases.

An understated danger: Antimicrobial resistance in aquaculture and pet fish in Switzerland, a retrospective study from 2000 to 2017.

Aquaculture is a rapidly growing field of food production. However, morbidity and mortality are higher in aquaculture species than in domestic animals. Bacterial diseases are a leading cause of farmed fish morbidity and are often treated with antimicrobials. Since most Swiss fish farms release effluents directly into surface water without treatment and since aquaculture fish are consumed by humans, antimicrobial resistance (AMR) and multi-resistance in aquaculture fish are important for environmental and public health. In this study, AMR tests for 14 antimicrobials were performed on 1,448 isolates from 1,134 diagnostic laboratory submissions from farmed and ornamental fish submissions for the period from 2000 to 2017. Amoxicillin, gentamycin and norfloxacin had the lowest proportion of resistant samples. However, AMR was highly variable over time. Resistance proportions were higher in: (a) ornamental fish compared with farmed fish, (b) fish from recirculation systems compared with those from other farming systems and (c) isolates originating from skin compared with those originating from inner organs. Multiple resistances were common. The results of this study provide useful data for Swiss fish veterinarians and some interesting hypotheses about risk factors for AMR in aquaculture and pet fish in Switzerland. However, further research is needed to define risk factors.

Mysterious syndrome causing high mortality in wild brown trout in Eastern Switzerland, pathology and search for a possible cause.

Since 2016, annually occurring species-specific die-offs of brown trout (Salmo trutta fario) occurred in the Thur River, situated in the Eastern part of Switzerland. These events lead to drastically reduced population densities in the impacted river regions. Clinical signs in brown trout and mortality were restricted to few weeks in August/September. To characterize the syndrome and to find possible causes, from end of March to November 2018, one-year-old brown trout (Salmo trutta fario) and rainbow trout (Oncorhynchus mykiss) were exposed to water from Thur River, fish were sampled regularly and screened for infectious agents, including viral metagenomics, and pathology was described. Starting approximately four months post-exposure, brown trout showed severe lymphohistiocytic pancarditis and necrotizing and haemorrhagic hepatitis. These lesions were recorded until the end of the experiment in November. Rainbow trout were not affected at any point in time. No infectious agents could be identified so far as cause of disease, especially no viral aetiology. Even if pathogenesis and pathology point in the direction of an infectious agent, a causative relationship could not be confirmed and aetiology remains unclear.

Field study indicating susceptibility differences between salmonid species and their lineages to proliferative kidney disease.

Tetracapsuloides bryosalmonae (Myxozoa: Malacosporea) is the causative agent of proliferative kidney disease (PKD), which affects both wild and farmed salmonid fish. The objective of this study was to outline differences in susceptibility to PKD in different salmonid species, hybrids and breeding lineages. Susceptibility to T. bryosalmonae infection was established based on cumulative mortality, pathological findings and detection of T. bryosalmonae in the kidney using immunohistochemistry and molecular methods. Determination of pure and hybrid individuals of different species in the genus Salvelinus, and dissimilarity of rainbow trout lineages, was performed using traditional polymerase chain reaction (PCR) and microsatellite analyses. Rainbow trout displayed higher disease severity compared with brook trout and Alsatian charr. Moreover, the results indicated differences in infection susceptibility, not only among different salmonid species but also among different lineages of charr and rainbow trout. Our study indicated that some salmonid species and even different lineages of the same species are more suitable for farming under PKD pressure.

Systemic infection in European perch with thermoadapted virulent Aeromonas salmonicida (Perca fluviatilis).

In non-salmonid fish, Aeromonas salmonicidacan cause local infections with severe skin ulcerations, known as atypical furunculosis. In this study, we present a systemic infection by a virulent A. salmonicidain European perch (Perca fluviatilis).This infection was diagnosed in a Swiss warm water recirculation aquaculture system. The isolate of A.  salmonicida encodes a type three secretion system (TTSS) most likely located on a plasmid similar to pAsa5/pASvirA, which is known to specify one of the main virulence attributes of the species A. salmonicida. However, the genes specifying the TTSS of the perch isolate show a higher temperature tolerance than strains isolated from cold-water fish. The function of the TTSS in virulence was verified in a cytotoxicity test using bluegill fry and epithelioma papulosum cyprinid cells.