Life cycle complexity, environmental change and the emerging status of salmonid proliferative kidney disease

P>1. Proliferative kidney disease (PKD) is a disease of salmonid fish caused by the endoparasitic myxozoan, Tetracapsuloides bryosalmonae, which uses freshwater bryozoans as primary hosts. Clinical PKD is characterised by a temperature-dependent proliferative and inflammatory response to parasite stages in the kidney.;2. Evidence that PKD is an emerging disease includes outbreaks in new regions, declines in Swiss brown trout populations and the adoption of expensive practices by fish farms to reduce heavy losses. Disease-related mortality in wild fish populations is almost certainly underestimated because of e.g. oversight, scavenging by wild animals, misdiagnosis and fish stocking.;3. PKD prevalences are spatially and temporally variable, range from 0 to 90-100% and are typically highest in juvenile fish.;4. Laboratory and field studies demonstrate that (i) increasing temperatures enhance disease prevalence, severity and distribution and PKD-related mortality; (ii) eutrophication may promote outbreaks. Both bryozoans and T. bryosalmonae stages in bryozoans undergo temperature- and nutrient-driven proliferation.;5. Tetracapsuloides bryosalmonae is likely to achieve persistent infection of highly clonal bryozoan hosts through vertical transmission, low virulence and host condition-dependent cycling between covert and overt infections. Exploitation of fish hosts entails massive proliferation and spore production by stages that escape the immune response. Many aspects of the parasite's life cycle remain obscure. If infectious stages are produced in all hosts then the complex life cycle includes multiple transmission routes.;6. Patterns of disease outbreaks suggest that background, subclinical infections exist under normal environmental conditions. When conditions change, outbreaks may then occur in regions where infection was hitherto unsuspected.;7. Environmental change is likely to cause PKD outbreaks in more northerly regions as warmer temperatures promote disease development, enhance bryozoan biomass and increase spore production, but may also reduce the geographical range of this unique multihost-parasite system. Coevolutionary dynamics resulting from host-parasite interactions that maximise fitness in previous environments may pose problems for sustainability, particularly in view of extensive declines in salmonid populations and degradation of many freshwater habitats.

The emergence of epitheliocystis in the upper Rhone region: evidence for Chlamydiae in wild and farm salmonid populations

We present the first study comparing epitheliocystis in a wild and farmed salmonid in Europe. Sampling three tributaries to the Lake Geneva, including one from headwaters to river mouth, revealed an unequal distribution of epitheliocystis in brown trout (Salmo trutta). When evaluated histologically and comparing sites grouped as wild versus farm, the probability of finding infected trout is higher on farms. In contrast, the infection intensities, as estimated by the number of cysts per gill arch, were higher on average and showed maximum values in the wild trout. Sequence analysis showed the most common epitheliocystis agents were Candidatus Piscichlamydia salmonis, all clustering into a single clade, whereas Candidatus Clavichlamydia salmonicola sequences cluster in two closely related sub-species, of which one was mostly found in farmed fish and the other exclusively in wild brown trout, indicating that farms are unlikely to be the source of infections in wild trout. A detailed morphological analysis of cysts using transmission electron microscopy revealed unique features illustrating the wide divergence existing between Ca. P. salmonis and Ca. C. salmonicola within the phylum Chlamydiae

No evidence for large differences in genomic methylation between wild and hatchery steelhead (Oncorhynchus mykiss)

When salmonid fish that have been raised in hatcheries spawn in the wild, they often produce fewer surviving adult offspring than wild fish. Recent data from steelhead (Oncorhynchus mykiss) in the Hood River (Oregon, USA) show that even one or two generations of hatchery culture can result in dramatic declines in fitness. Although intense domestication selection could cause such declines, it is worth considering alternative explanations. One possibility is heritable epigenetic changes induced by the hatchery environment. Here, we show, using methylation-sensitive amplified fragment length polymorphism, that hatchery and wild adult steelhead from the Hood River do not appear to differ substantially in overall levels of genomic methylation. Thus, although altered methylation of specific DNA sites or other epigenetic processes could still be important, the hatchery environment does not appear to cause a global hypo- or hypermethylation of the genome or create a large number of sites that are differentially methylated.

A natural freshwater origin for two chlamydial species, Candidatus Piscichlamydia salmonis and Candidatus Clavochlamydia salmonicola, causing mixed infections in wild brown trout (Salmo trutta)

Gill disease in salmonids is characterized by a multifactorial aetiology. Epitheliocystis of the gill lamellae caused by obligate intracellular bacteria of the order Chlamydiales is one known factor; however, their diversity has greatly complicated analyses to establish a causal relationship. In addition, tracing infections to a potential environmental source is currently impossible. In this study, we address these questions by investigating a wild brown trout (Salmo trutta) population from seven different sites within a Swiss river system. One age class of fish was followed over 18 months. Epitheliocystis occurred in a site-specific pattern, associated with peak water temperatures during summer months. No evidence of a persistent infection was found within the brown trout population, implying an as yet unknown environmental source. For the first time, we detected 'Candidatus Piscichlamydia salmonis' and 'Candidatus Clavochlamydia salmonicola' infections in the same salmonid population, including dual infections within the same fish. These organisms are strongly implicated in gill disease of caged Atlantic salmon in Norway and Ireland. The absence of aquaculture production within this river system and the distance from the sea, suggests a freshwater origin for both these bacteria and offers new possibilities to explore their ecology free from aquaculture influences.

Population transcriptomics of life-history variation in the genus Salmo

In this study, we demonstrate the power of applying complementary DNA (cDNA) microarray technology to identifying candidate loci that exhibit subtle differences in expression levels associated with a complex trait in natural populations of a nonmodel organism. Using a highly replicated experimental design involving 180 cDNA microarray experiments, we measured gene-expression levels from 1098 transcript probes in 90 individuals originating from six brown trout (Salmo trutta) and one Atlantic salmon (Salmo salar) population, which follow either a migratory or a sedentary life history. We identified several candidate genes associated with preparatory adaptations to different life histories in salmonids, including genes encoding for transaldolase 1, constitutive heat-shock protein HSC70-1 and endozepine. Some of these genes clustered into functional groups, providing insight into the physiological pathways potentially involved in the expression of life-history related phenotypic differences. Such differences included the down-regulation of genes involved in the respiratory system of future migratory individuals. In addition, we used linear discriminant analysis to identify a set of 12 genes that correctly classified immature individuals as migratory or sedentary with high accuracy. Using the expression levels of these 12 genes, 17 out of 18 individuals used for cross-validation were correctly assigned to their respective life-history phenotype. Finally, we found various candidate genes associated with physiological changes that are likely to be involved in preadaptations to seawater in anadromous populations of the genus Salmo, one of which was identified to encode for nucleophosmin 1. Our findings thus provide new molecular insights into salmonid life-history variation, opening new perspectives in the study of this complex trait.

Proliferative kidney disease in rainbow trout: time- and temperature-related renal pathology and parasite distribution

Proliferative kidney disease is a parasitic infection of salmonid fishes caused by Tetracapsuloides bryosalmonae. The main target organ of the parasite in the fish is the kidney. To investigate the influence of water temperature on the disease in fish, rainbow trout Oncorhynchus mykiss infected with T bryosalmonae were kept at 12 degrees C and 18 degrees C. The number of parasites, the type and degree of lesions in the kidney and the mortality rate was evaluated from infection until full development of disease. While mortality stayed low at 12 degrees C, it reached 77% at 18 degrees C. At 12 degrees C, pathological lesions were dominated by a multifocal proliferative and granulomatous interstitial nephritis. This was accompanied by low numbers of T. bryosalmonae, mainly located in the interstitial lesions. With progression of the disease, small numbers of parasites appeared in the excretory tubuli, and parasite DNA was detected in the urine. Parasite degeneration in the interstitium was observed at late stages of the disease. At 18 degrees C, pathological lesions in kidneys were more severe and more widely distributed, and accompanied by significantly higher parasite numbers. Distribution of parasites in the renal compartments, onset of parasite degeneration and time course of appearance of parasite DNA in urine were not clearly different from the 12 degrees C group. These findings indicate that higher mortality at 18 degrees C compared to 12 degrees C is associated with an enhanced severity of renal pathology and increased parasite numbers.

Estrogen Modulates Hepatic Gene Expression and Survival of Rainbow Trout Infected with Pathogenic Bacteria Yersinia ruckeri

In the aquatic environment, fish are exposed to various stimuli at once and have developed different response mechanisms to deal with these multiple stimuli. The current study assessed the combined impacts of estrogens and bacterial infection on the physiological status of fish. Juvenile rainbow trout were exposed to two different concentrations of 17 beta-estradiol (E2) (2 or 20 mg/kg feed) and then infected with three concentrations of Yersinia ruckeri, a bacterial pathogen causing massive losses in wild and farmed salmonid populations. Organism-level endpoints to assess the impact of the single and combined treatments included hepatic vitellogenin transcript expression to evaluate the E2 exposure efficiency and survival rate of pathogen-challenged fish. The two E2 doses increased vitellogenin levels within the physiological range. Infection with Y. ruckeri caused mortality of trout, and this effect was significantly enhanced by a simultaneous exposure to high E2 dose. The hormone reduced survival at intermediate and high (10(4) and 10(6) colony forming units, cfu) bacterial concentrations, but not for a low one (10(2) cfu). Analysis of hepatic gene expression profiles by a salmonid 2 k cDNA microarray chip revealed complex regulations of pathways involved in immune responses, stress responses, and detoxicification pathways. E2 markedly reduced the expression of several genes implicated in xenobiotic metabolism. The results suggest that the interaction between pathogen and E2 interfered with the fish's capability of clearing toxic compounds. The findings of the current study add to our understanding of multiple exposure responses in fish.

Aeromonas salmonicida subsp. salmonicida in the light of its type-three secretion system

Aeromonas salmonicida subsp. salmonicida is an important pathogen in salmonid aquaculture and is responsible for the typical furunculosis. The type-three secretion system (T3SS) is a major virulence system. In this work, we review structure and function of this highly sophisticated nanosyringe in A. salmonicida. Based on the literature as well as personal experimental observations, we document the genetic (re)organization, expression regulation, anatomy, putative functional origin and roles in the infectious process of this T3SS. We propose a model of pathogenesis where A. salmonicida induces a temporary immunosuppression state in fish in order to acquire free access to host tissues. Finally, we highlight putative important therapeutic and vaccine strategies to prevent furunculosis of salmonid fish.

Molecular crosstalk between a chemical and a biological stressor and consequences on disease manifestation in rainbow trout.

The aim of the present study was to examine the molecular and organism reaction of rainbow trout, Oncorhynchus mykiss, to the combined impact of two environmental stressors. The two stressors were the myxozoan parasite, Tetracapsuloides bryosalmonae, which is the etiological agent of proliferative kidney disease (PKD) and a natural stressor to salmonid populations, and 17β-estradiol (E2) as prototype of estrogen-active chemical stressors in the aquatic environment. Both stressors, the parasite and estrogenic contaminants, co-exist in Swiss rivers and are discussed as factors contributing to the decline of Swiss brown trout populations over the last decades. Using a microarray approach contrasting parasite-infected and non-infected rainbow trout at low or high estrogen levels, it was observed that molecular response patterns under joint exposure differed from those to the single stressors. More specifically, three major response patterns were present: (i) expression responses of gene transcripts to one stressor are weakened by the presence of the second stressor; (ii) expression responses of gene transcripts to one stressor are enhanced by the presence of the second stressor; (iii) expression responses of gene transcripts at joint treatment are dominated by one of the two stressors. Organism-level responses to concurrent E2 and parasite treatment - assessed through measuring parasite loads in the fish host and cumulative mortalities of trout - were dominated by the pathogen, with no modulating influence of E2. The findings reveal function- and level-specific responses of rainbow trout to stressor combinations, which are only partly predictable from the response to the single stressors.

Molecular epidemiology of Flavobacterium psychrophilum from Swiss fish farms

Bacterial cold water disease (BCWD) and rainbow trout fry syndrome (RTFS) caused by Flavobacterium psychrophilum are 2 of the major diseases causing high fish mortality in salmonid fish farms. The molecular epidemiology of F. psychrophilum is still largely unknown. Multilocus sequence typing (MLST) has been previously used for this pathogen and underscored a correlation between clonal complexes and host fish species. Here we used MLST to study the relationships among 112 F. psychrophilum isolates from rainbow trout Oncorhynchus mykiss and brown trout Salmo trutta fario and S. t. lacustris in Swiss fish farms between 1993 and 2012. The isolates belonged to 27 different sequence types (STs). Most of the Swiss outbreaks were associated with strains belonging to clonal complexes CC-ST2 and CC-ST90, found in both rainbow trout and brown trout and represented by several STs. Eight ST singletons could not be connected to any known clonal complex. Already reported from other parts of Europe and North America, CC-ST2 was the most frequent clonal complex observed, and it caused the majority of outbreaks in Switzerland, with CC-ST90 being the second most important type. In the tightly interconnected Swiss fish farms, no association between clonal complex and host fish was detected, but a temporal evolution of the frequency of some STs was observed. The occurrence of sporadic STs suggests high F. psychrophilum diversity and may reflect the presence of different sequence types in the environment.

First outbreak of sleeping disease in Switzerland: disease signs and virus characterization.

Sleeping disease is a contagious disease mainly of freshwater farmed rainbow trout, caused by salmonid alphavirus (SAV) Subtype 2. Here we describe the first case in Switzerland. Pathological changes ranged from acute pancreas necrosis to more chronic lesions with complete loss of exocrine pancreas and simultaneous degenerative, inflammatory and regenerative heart and muscle lesions. The partial sequencing of SAV E2 and nsp3 genes placed the Swiss SAV variant within the Subtype 2 clustering together with freshwater isolates from UK and continental Europe. Although mortality stayed low, growth rates were significantly reduced, making the disease economically relevant.

The Efficacy of a Novel Microbial 6-Phytase Expressed in Aspergillus oryzae on the Performance and Phosphorus Utilization of Cold- and Warm-Water Fish: Rainbow Trout, Oncorhynchus mykiss , and Nile Tilapia, Oreochromis niloticus

The efficacy and tolerance of a novel microbial 6-phytase were investigated in rainbow trout, Oncorhynchus mykiss, and Nile tilapia, Oreochromis niloticus. Reference diets were sufficient in available phosphorus (P). The test diet limiting in available P was supplemented with phytase at 500, 1000, or 2000 phytase units/kg feed. The enzyme was effective in increasing total P apparent digestibility coefficient in relation to increasing the dose of phytase in rainbow trout and Nile tilapia. Zinc apparent digestibility improved in relation to phytase supplementation in rainbow trout. P release due to phytase supplementation ranged from 0.06 to 0.18% P/kg feed in rainbow trout and from 0.13 to 0.26% P/kg feed in Nile tilapia. A 58-d performance trial was conducted to evaluate tolerance of fish to phytase supplementation. Dietary treatments consisted of a basal diet without phytase or supplemented with 2000 and 200,000 phytase units/kg feed. Results indicate that this novel microbial 6-phytase is well tolerated by fish. Significant improvements for growth as well as feed conversion ratio were observed when the phytase was fed at 2000 phytase units/kg feed. This phytase is proven efficient in releasing P from phytate and could be added when plants are used for fish meal replacement in diets for salmonid and omnivorous fish.

Hepeviridae: An expanding family of vertebrate viruses

The hepatitis E virus (HEV) was first identified in 1990, although hepatitis E-like diseases in humans have been recorded for a long time dating back to the 18th century. The HEV genotypes 1–4 have been subsequently detected in human hepatitis E cases with different geographical distribution and different modes of transmission. Genotypes 3 and 4 have been identified in parallel in pigs, wild boars and other animal species and their zoonotic potential has been confirmed. Until 2010, these genotypes along with avian HEV strains infecting chicken were the only known representatives of the family Hepeviridae. Thereafter, additional HEV-related viruses have been detected in wild boars, distinct HEV-like viruses were identified in rats, rabbit, ferret, mink, fox, bats and moose, and a distantly related agent was described from closely related salmonid fish. This review summarizes the characteristics of the so far known HEV-like viruses, their phylogenetic relationship, host association and proposed involvement in diseases. Based on the reviewed knowledge, a suggestion for a new taxonomic grouping scheme of the viruses within the family Hepeviridae is presented.

Proliferative kidney disease in brown trout – infection level, pathology and mortality under field conditions

Proliferative kidney disease (PKD) is an emerging disease threatening wild salmonid populations. In temperature-controlled aquaria, PKD can cause mortality rates of up to 85% in rainbow trout. So far, no data about PKD-related mortality in wild brown trout Salmo trutta fario are available. The aim of this study was to investigate mortality rates and pathology in brown trout kept in a cage within a natural river habitat known to harbor Tetracapsuloides bryosalmonae. Young-of-the-year (YOY) brown trout, free of T. bryosalmonae, were exposed in the River Wutach, in the northeast of Switzerland, during 3 summer months. Samples of wild brown trout caught by electrofishing near the cage location were examined in parallel. The incidence of PKD in cage-exposed animals (69%) was not significantly different to the disease prevalence of wild fish (82 and 80% in the upstream and downstream locations, respectively). The mortality in cageexposed animals, however, was as low as 15%. At the termination of the exposure experiment, surviving fish showed histological lesions typical for PKD regression, suggesting that many YOY brown trout survive the initial infection. Our results at the River Wutach suggest that PKD in brown trout does not always result in high mortality under natural conditions.