Are brown trout Salmo trutta fario and rainbow trout Oncorhynchus mykiss two of a kind? A comparative study of salmonids to temperature‐influenced Tetracapsuloides bryosalmonae infection

Proliferative kidney disease (PKD) of salmonids caused by Tetracapsuloides bryosalmonae causes high mortalities of wild brown trout (Salmo trutta fario) and farmed rainbow trout (Oncorhynchus mykiss) at elevated water temperatures. Here the aim was to compare the temperature‐dependent modulation of T. bryosalmonae in the two salmonid host species, which display different temperature optima. We used a novel experimental set‐up in which we exposed brown trout and rainbow trout to an identical quantified low concentration of T. bryosalmonae for a short time period (1 hr). We followed the development of the parasite in the fish hosts for 70 days. PKD prevalence and parasite kinetics were assessed using qPCR. Exposures were performed at temperatures (12°C and 15°C) that reflect an environmental scenario that may occur in the natural habitat of salmonids. T. bryosalmonae infection was confirmed earliest in brown trout kept at 15°C (day 7 post‐exposure) while, in all other groups, T. bryosalmonae was not confirmed until day 15 post‐exposure. Moreover, significantly greater infection prevalence and a faster increase of parasite intensity were observed in brown trout kept at 15°C than in all other groups. These results indicate that PKD is differentially modulated by water temperature in related host species.     

A survey on Tetracapsuloides bryosalmonae infections in Slovene fresh waters

Slovenia has no history of health problems related to proliferative kidney disease (PKD) either in farmed or in wild fish. However, due to the past molecular evidence for the presence of Tetracapsuloides bryosalmonae DNA in tissues of some fish from open waters, a survey was conducted on wild salmonids that were primarily sampled for other purposes. In winter 2010–2011, specimens from a total of 244 rainbow trout, Oncorhynchus mykiss (Walbaum), and brown trout, Salmo trutta L., from 30 bodies of fresh water were examined for T. bryosalmonae using a PCR method. The sampled fish showed no clinical signs or gross pathological lesions characteristic of PKD. Nineteen (7.8%) fish from seven (23.3%) fresh waters were positive for T. bryosalmonae. The identity of PCR amplicons was confirmed by sequencing. With one exception, all the positive fish were found in waters from the regions where the average yearly temperatures and the environmental pollution are higher. This preliminary countrywide survey provided the first insight into the situation regarding T. bryosalmonae infection of wild salmonids in Slovenia.     

Patterns of Tetracapsuloides bryosalmonae infection of three salmonid species in large,deep Norwegian lakes

Proliferative kidney disease (PKD), caused by the myxozoan endoparasite Tetracapsuloides bryosalmonae, is of serious ecological and economical concern to wild and farmed salmonids. Wild salmonid populations have declined due to PKD, primarily in rivers, in Europe and North America. Deep lakes are also important habitats for salmonids, and this work aimed to investigate parasite presence in five deep Norwegian lakes. Kidney samples from three salmonid species from deep lakes were collected and tested using real-time PCR to detect PKD parasite presence. We present the first detection of T. bryosalmonae in European whitefish in Norway for the first time, as well as the first published documentation of the parasite in kidneys of Arctic charr, brown trout and whitefish in four lakes. The observed prevalence of the parasite was higher in populations of brown trout than of Arctic charr and whitefish. The parasite was detected in farmed, but not in wild, charr in one lake. This suggests a possible link with a depth of fish habitat and fewer T. bryosalmonae-infected and PKD-affected fish. Towards a warmer climate, cold hypolimnion in deep lakes may act as a refuge for wild salmonids, while cold deep water may be used to control PKD in farmed salmonids.     

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.     

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

The malacosporean Tetracapsuloides bryosalmonae was detected in kidneys from Atlantic salmon parr in 64 of 91 sampled Norwegian rivers. Using real‐time 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.     

Distribution and prevalence of T. bryosalmonae in Austria: A first survey of trout from rivers with a shrinking population

The first evidence of proliferative kidney disease (PKD) in an Austrian river (the River Kamp) was documented in 2016, and no information on the PKD infection status of trout in other rivers was available. Since then, brown trout (Salmo trutta fario) and rainbow trout (Oncorhynchus mykiss) have been collected from rivers in Upper and Lower Austria for different diagnostic purposes. In this study, we summarize the recent findings of a first survey concerning the distribution of Tetracapsuloides bryosalmonae, the causative agent of proliferative kidney disease (PKD), from these samples. Between September 2015 and October 2017, a total of 280 brown trout and 39 rainbow trout were collected from 21 rivers in the provinces of Upper and Lower Austria. T. bryosalmonae was detected by PCR of kidney tissue in 17 of 21 sampled rivers and in 138 of 280 brown trout as well as in 11 of 39 rainbow trout. Pathological signs of PKD (e.g., hypertrophy of the kidney) were observed in 33 analysed brown trout and six rainbow trout samples. No correlations between fish infected by T. bryosalmonae and the parameters size and age class, condition factor, geological origin of the streams and distribution within the river course were found, while positively tested fish are significantly increased at sampling sites exceeding water temperatures of 15°C for median periods of 115 days. The prevalence within the affected streams or stream sections is highly variable, and in single rivers, infection rates of up to 90% are confirmed.     

Development of improved PCR to prevent false positives and false negatives in the detection of Tetracapsula bryosalmonae, the causative agent of proliferative kidney disease

Proliferative kidney disease (PKD) is an economically significant disease caused by the myxozoan parasite Tetracapsula bryosalmonae. Polymerase chain reaction (PCR) protocols using primers specific for the small subunit ribosomal RNA (18S rDNA) gene of the parasite enable detection, however, false positive and negative results can render detection inconclusive. In this study a decontamination protocol was developed, using hydroxylamine hydrochloride (H), to prevent false positives by blocking re‐amplification of carry‐over contaminants. A mimic molecule was also developed and used as a competitive internal standard coamplified with target DNA in PCRs, revealing both true and false negatives. The sensitivity of one new and two existing primer sets was assessed with all primers detecting DNA equivalent to at least eight parasite cells per gram of tissue. This improved PCR protocol canprovide more reliable testing for T. bryosalmonae.     

Establishment of medium for laboratory cultivation and maintenance of Fredericella sultana for in vivo experiments with Tetracapsuloides bryosalmonae (Myxozoa)

The freshwater bryozoan Fredericella sultana (Blumenbach) is the most common invertebrate host of the myxozoan parasite Tetracapsuloides bryosalmonae, the causative agent of proliferative kidney disease in salmonid fish. Culture media play an important role in hatching of statoblasts and maintaining clean bryozoan colonies for Malacosporea research. We developed a novel culture medium, Bryozoan Medium C (BMC), for the cultivation and maintenance of F. sultana under laboratory conditions. Statoblasts of F. sultana were successfully hatched to produce transparent‐walled, specific pathogen‐free (SPF) colonies that were maintained >12 months in BMC at pH 6.65. Tetracapsuloides bryosalmonae was successfully transmitted from infected brown trout, Salmo trutta L., to newly hatched F. sultana colonies in BMC, then from the infected bryozoan to SPF brown trout. This study demonstrated the utility of BMC (pH 6.65) for hatching statoblasts, long‐term cultivation of clean and transparent bryozoan colonies and maintenance of the Tetracapsuloides bryosalmonae life cycle in the laboratory for molecular genetic research and other studies such as host–parasiteinteraction.     

The occurrence of proliferative kidney disease (PKD) in cultured and wild fish: further investigations

Abstract. In an investigation of the occurrence of proliferative kidney disease (PKD) in freshwater fish other than rainbow trout, 18 species of wild fish and seven species of fish raised in cultivation wore sampled from waters where the disease occurred annually in rainbow trout, Oncorhynchus mykiss (Richardson). Results revealed that certain wild stocks of brown trout. Salmo trutta L., grayling, Thymallus thymallus L., and pike, Esox lucius L., were infected with PKD, as were cultivated Atlantic salmon, Salmo salar L., parr, brown trout and char, Salvelinus alpinus (L.). Microscopical examination revealed the presence of the PKX cell in these species and also intraluminal protozoa possibly related to the PKX cell, which were not found in the rainbow trout. Other species of freshwater fish had myxosporidan infections but, unlike PKD infection, there was little host/parasite tissue response. The PKX cell as a myxosporidan stage is discussed and the presence of the disease in wild fish is reported.     

Piscine orthoreovirus‐3 is prevalent in wild seatrout (Salmo trutta L.) in Norway

In 2017, a PCR‐based survey for Piscine orthoreovirus‐3 (PRV‐3) was conducted in wild anadromous and non‐anadromous salmonids in Norway. In seatrout (anadromous Salmo trutta L.), the virus was present in 16.6% of the fish and in 15 of 21 investigated rivers. Four of 221 (1.8%) Atlantic salmon (Salmo salar L.) from three of 15 rivers were also PCR‐positive, with Ct‐values indicating low amounts of viral RNA. All anadromous Arctic char (Salvelinus alpinus L.) were PCR‐negative. Neither non‐anadromous trout (brown trout) nor landlocked salmon were PRV‐3 positive. Altogether, these findings suggest that in Norway PRV‐3 is more prevalent in the marine environment. In contrast, PRV‐3 is present in areas with intensive inland farming in continental Europe. PRV‐3 genome sequences from Norwegian seatrout grouped together with sequences from rainbow trout (Oncorhynchus mykiss Walbaum) in Norway and Coho salmon (Oncorhynchus kisutch Walbaum) in Chile. At present, the origin of the virus remains unknown. Nevertheless, the study highlights the value of safeguarding native fish by upholding natural and artificial barriers that hinder introduction and spread, on a local or national scale, of alien fish species and their pathogens. Accordingly, further investigations of freshwater reservoirs and interactions with farmed salmonids are warranted.     

Demonstrating the practical impact of studies on biotic interactions and adaptation of a threatened unionoid mussel (Margaritifera margaritifera) to its host fish (Salmo trutta)

1. To understand the ecological factors behind the decline of functionally important threatened species with complex life cycles, many different life‐cycle stages need to be investigated. The highly threatened unionoid freshwater mussels, with their complex life cycle, including a parasitic stage on host fish, often have a large influence on biodiversity and ecosystem functioning.
2. The overall aim of the present article is to summarize and discuss the impact of two articles published in Aquatic Conservation: Marine and Freshwater Ecosystems (AQC) on biotic interactions and adaptation of a threatened unionoid mussel (Margaritifera margaritifera) to its host fish (Salmo trutta).
3. The two AQC publications described research on the influence of population size and density of mussels and host fish, and host–parasite interactions between mussels and their host fish, on the recruitment of juvenile mussels.
4. The results from these publications filled gaps in knowledge and resulted in recommendations and incentives for conservation. The results and method development have been used in practical conservation work with threatened mussel species and have been implemented and cited in management handbooks. The outcome of the publications has been implemented in large conservation and restoration projects, and in several recent scientific publications.
5. Specifically, the results from one publication showed that ecological parameters such as mussel and host fish density and population size influenced recruitment of the threatened freshwater pearl mussel. The results from the second publication showed that understanding host–parasite interactions is important for comparing the suitability of host fish strains, and that host fish strains differ in their suitability for mussel infestations. In combination, the articles show that integrating ecological parameters of threatened mussels and their host fish with host–parasite interaction experiments can be an important influence on conservation recommendations, adaptive management and national management programmes for threatened species.

     

Atlantic salmon infected with salmon lice are more susceptible to new lice infections

Aggregation is commonly observed for macroparasites, but its adaptive value remains unclear. Heavy infestations intensities may lead to a decrease in some fitness‐related traits of parasites (e.g. parasite fecundity or survival). However, to a dioecious parasite, increased aggregation could also increase the chance of finding individuals of the opposite sex. In a laboratory experiment, we tested if previous experience with salmon lice (Lepeophtheirus salmonis) affected susceptibility of Atlantic salmon (Salmo salar) to later exposure to the same parasite species. We found that currently infected fish got higher intensities of new lice than naive fish. This suggests that hosts already carrying parasites are more susceptible to new lice infections. For this dioecious parasite, such positive density dependence might be adaptive, ensuring successful reproduction under conditions of low lice densities by increasing the probability of both sexes infecting the same host.     

A survey of salmon gill poxvirus (SGPV) in wild salmonids in Norway

In 2016, the Norwegian health monitoring programme for wild salmonids conducted a real‐time PCR‐based screening for salmon gill poxvirus (SGPV) in anadromous Arctic char (Salvelinus alpinus L.), anadromous and non‐anadromous Atlantic salmon (Salmo salar L.) and trout (Salmo trutta L.). SGPV was widely distributed in wild Atlantic salmon returning from marine migration. In addition, characteristic gill lesions, including apoptosis, were detected in this species. A low amount of SGPV DNA, as indicated by high Ct‐values, was detected in anadromous trout, but only in fish cohabiting with SGPV‐positive salmon. SGPV was not detected in trout and salmon from non‐anadromous water courses, and thus seems to be primarily linked to the marine environment. This could indicate that trout are not a natural host for the virus. SGPV was not detected in Arctic char but, due to a low sample size, these results are inconclusive. The use of freshwater from anadromous water sources may constitute a risk of introducing SGPV to aquaculture facilities. Moreover, SGPV‐infected Atlantic salmon farms will hold considerable potential for virus propagation and spillback to wild populations. This interaction should therefore be further investigated.     

Suitability of different salmonid strains as hosts for the endangered freshwater pearl mussel (Margaritifera margaritifera L.)

• 1. The complex life cycle of endangered European freshwater pearl mussel Margaritifera margaritifera L. involves an obligatory parasitic phase on a host fish. Knowledge on the host–parasite interaction and on the suitability of different host fish species and strains is required both for the management of wild fish and mussel populations as well as for improving the efficiency of captive breeding methods.
• 2. In this study, the suitability of different salmonid strains for hosting glochidia was tested, including Danube salmon (Hucho hucho L.) and three brown trout (Salmo trutta L.) strains from inside and outside the freshwater pearl mussel distribution range. All brown trout strains as well as Danube salmon were successfully infected with freshwater pearl mussel glochidia and encystment of mussel larvae was detected.
• 3. One brown trout strain originating from the natural pearl mussel distribution range was identified as the most suitable host, revealing the highest fish‐weight‐normalized infection rates and highest glochidial growth rates, whereas endemic Danube salmon was least suitable. Under natural conditions, the role of Danube salmon may be attributed to the long‐distance dispersal of glochidia in the Danube system, whereas sedentary brown trout appear to be the most important hosts at a local scale.
• 4. Successful infection of suitable hosts and the maintenance of these host–parasite systems in calcareous water were demonstrated in this study. These results indicate that neither the infection process nor the encystment phase of freshwater pearl mussels is dependent on low lime concentrations.
• 5. The results of this study suggest that careful selection and management of appropriate host fish strains is mandatory for sustainable conservation management of freshwater pearl mussel populations. Copyright © 2010 John Wiley & Sons, Ltd.

     

Development of a novel PCR assay based on the 16S-23S rRNA internal transcribed spacer region for the detection of Lactococcus garvieae

Lactococcus garvieae is recognized as an emerging pathogen in fish. Several PCR‐based methods have been developed for the detection and identification of L. garvieae; however, the sensitivity of these methods is still in question regarding the discrimination of this organism from other closely related species. Two primers, ITSLg30F and ITSLg319R, were designed from the sequence in the 16S–23S internal transcribed spacer (ITS) region and used for the specific detection of L. garvieae. L. garvieae strains including fish isolates were positive by this method. In contrast, previously developed PCR methods showed false‐positive results with non‐L. garvieae species. Our results indicate that a PCR method using the newly designed ITS primer set provides a sensitive and efficient tool for the detection of L. garvieae in fish and aquaculture environments.     

Microhabitat selection of Gyrodactylus salaris Malmberg on different salmonids

The microhabitat selection of the ectoparasite Gyrodactylus salaris (Lærdalselva strain, Norway) was investigated concurrently with studies on the parasite population growth on five strains of Atlantic salmon, Salmo salar L., and a strain of Danish rainbow trout, Oncorhynchus mykiss (Walbaum). The salmon used were hatchery‐reared parr of East Atlantic strains [River Conon (Scotland), River Storå (western Denmark) and River Ätran (western Sweden)] and Baltic strains [Lule and Ume (eastern Sweden)]. The location and numbers of parasites were recorded on anaesthetized fish once a week from week 0 to week 8. The mean abundance of G. salaris steadily increased to high levels on the River Conon, Storå and Ätran strains until the end of the experiment. The mean abundance of G. salaris on the two Baltic strains (River Lule älv and River Ume älv) initially increased but after 4–7 weeks the growth of the parasite infrapopulations decreased markedly. The Danish rainbow trout strain showed the lowest abundances of all the fish species and strains. Gyrodactylus salaris preferentially selected the fins and head region when colonising the hosts (all species and strains). Increasing percentages of G. salaris on the tail fins of the East Atlantic strains and rainbow trout were found during the course of infection, whereas the two Baltic salmon strains experienced a decreasing percentage of parasites in this microhabitat.     

First isolation of hirame rhabdovirus from freshwater fish in Europe

A rhabdovirus was isolated in cell culture inoculated with tissue material from diseased grayling, Thymallus thymallus (L.), originating from a fish farm affected by a mortality episode in Poland. Diagnostics tests showed that the virus was not related to novirhabdoviruses known in Europe, nor to vesiculovirus‐like species, except perch rhabdovirus (PRhV) with which it shared moderate serological relations. However, RT‐PCR with PRhV probes gave negative results. To identify the virus, a random‐priming sequence‐independent single primer amplification was adopted. Surprisingly, two of the obtained sequences exhibited a high identity (>99%) with hirame rhabdovirus (HIRRV), a novirhabdovirus usually found in fish in marine Asiatic countries, for instance Japan, China and Korea. The full‐length sequence of the phosphoprotein gene (P) demonstrated a higher identity of the present isolate with HIRRV from China compared with the Korean isolate. An identical viral sequence was also found in brown trout, Salmo trutta trutta L., affected by mortalities in a second farm in the same region, after a likely contamination from the grayling farm. To our knowledge, this is the first report of HIRRV in Europe, and in two hosts from fresh water that have not been described before as susceptible species.