Generation of Paramoeba perurans clonal cultures using flow cytometry and confirmation of virulence

Amoebic gill disease (AGD) in farmed Atlantic salmon is caused by the amoeba Paramoeba perurans. The recent establishment of in vitro culture techniques for P. perurans has provided a valuable tool for studying the parasite in detail. In this study, flow cytometry was used to generate clonal cultures from single‐sorted amoeba, and these were used to successfully establish AGD in experimental Atlantic salmon. The clonal cultures displayed differences in virulence, based on gill scores. The P. perurans load on gills, determined by qPCR analysis, showed a positive relationship with gill score, and with clonal virulence, indicating that the ability of amoebae to proliferate and/or remain attached on gills may play a role in virulence. Gill scores based on gross signs and histopathological analysis were in agreement. No association between level of gill score and specific gill arch was observed. It was found that for fish with lower gill scores based on histopathological examination, gross examination and qPCR analysis of gills from the same fish were less successful in detecting lesions and amoebae, respectively.     

Studies with an experimental model for pancreas disease of Atlantic salmon Salmo Salar L.

This paper describes reproducing pancreas disease in Atlantic salmon, Salmo salar L., parr with intraperitoneal inoculation of homogenized kidney and spleen taken From affected fish. Pancreatic necrosis and cardiomyopathy changes were induced consistently in all experimental groups. A time-course study demonstrated that pancreatic changes occurred as early as 4 days post inoculation and a repair process was evident in some fish on day 7 post inoculation. Heart lesions were first observed on day 5 post inoculation and increased in prevalence and severity as the experiment continued. Both types of lesion were serially passaged. and passively transmitted to fish cohabited with inoculated parr. The incidence of these changes was unaffected by passage of the infective homogenate through a 0.2 μ filter. The homogenate was inactivated after treatment with chloroform. Passive immunization with serum from recovered farmed salmon protected parr against challenge. It is suggested that pancreas disease is caused by a virus with a lipid envelope.     

Genotyping of Tenacibaculum maritimum isolates from farmed Atlantic salmon in Western Canada

Mouthrot infections (bacterial stomatitis) have a significant impact on the Atlantic salmon aquaculture industry in Western Canada due to economic losses and fish welfare. Bacteria isolated from lesions in the field have been identified as Tenacibaculum maritimum. Mouthrot is different to classical tenacibaculosis, which is most commonly associated with ulcerative lesions, frayed fins and tail rot. The marine fish pathogen T. maritimum is found worldwide; however, in Western Canada, the knowledge of the genetic profile of T. maritimum is limited. This study looked at increasing this knowledge by genotyping T. maritimum isolates collected from Atlantic salmon from farms in Western Canada. These genotypes were compared to other species of the genus Tenacibaculum, as well as other known sequence types within the species. The Western Canadian isolates belong to two new sequence types within the T. maritimum species. Phylogenetic analysis shows that the isolates form a distinct branch together with T. maritimum NCIMB 2154^T separate from other Tenacibaculum type strains, and they are most closely related to strains from Norway and Chile.     

Pathogenicity of Saprolegnia spp. to Atlantic salmon, Salmo salar L., eggs

Live and dead Atlantic salmon eyed eggs were challenged with eight different Saprolegnia isolates, selected because of their varied origins, known morphological characteristics and growth/germination pattern. Some isolates were also tested for pathogenicity to Atlantic salmon parr. Challenge of eggs was performed by exposure to spores in suspension or by co-incubation of live eggs with infected dead eggs. The phenotypic characteristics of the isolates were evaluated in relation to their observed pathogenicity from the challenge experiment, to identify possible virulence factors leading to egg-infection by Saprolegnia. The results from the experiments confirm that live eggs are refractory to infection with Saprolegnia spores in suspension and that an infection of live eggs can only occur from an infection nucleus represented by dead eggs or debris. It was observed that strains pathogenic to salmon parr were not particularly infective towards eggs, and the isolates that gave the highest infection rates to eggs were species considered to be saprotrophs.     

Saprolegnia molecular phylogeny among farmed teleosts in Nova Scotia,Canada

To identify the pathogens causing saprolegniosis among farmed fish in Nova Scotia, 172 infected tissues and 23 water samples were collected from six species of teleosts: Atlantic salmon (Salmo salar), brown trout (Salmo trutta), Arctic charr (Salvelinus alpinus), brook trout (Salvelinus fontinalis), striped bass (Morone saxatilis) and rainbow trout (Oncorhynchus mykiss) at nine facilities over a 600 km range. Following laboratory culture, 132 isolates were recovered. Six species of oomycetes were identified from analysis of the internal transcribed spacer (ITS) sequence of the nrDNA: Saprolegnia parasitica, Saprolegnia ferax, Saprolegnia diclina, Saprolegnia aenigmatica, Saprolegnia torulosa, Saprolegnia sp. and Pythiopsis cymosa. Further phylogenetic analyses of the ITS and cytochrome c oxidase subunit 1 (Cox1) regions revealed four strains of Saprolegnia parasitica (named here as S1, S2, S3 and S4), of which S1 and S2 were common (37% and 42% of the isolates), and two strains of S. ferax. Among S. parasitica, S2 and S3 are more closely related to each other than to S1 based on the phylogenetic analyses and predicted RNA secondary structure of the ITS region. Sexual structures with a similar morphology were formed by S1 and S3 in vitro, but were not formed by S2.     

A genetic tool for evaluating male‐mediated stock introgression in Atlantic salmon

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Surface properties of Streptococcus phocae strains isolated from diseased Atlantic salmon, Salmo salar L

Streptococcus phocae is an emerging pathogen for Chilean Atlantic salmon, Salmo salar, but the factors determining its virulence are not yet elucidated. In this work, cell surface–related properties such as hydrophobicity and haemagglutination, adhesion to mucus and cell lines, capsule detection, survival and biofilm formation in skin mucus and serum resistance of the isolates responsible for outbreaks in Atlantic salmon and seals were examined. Adhesion to hydrocarbons and the results of salt aggregation tests indicated most of the S. phocae were strongly hydrophobic. All isolates exhibited a similar ability to attach to the Chinook salmon embryo (CHSE) cells line, but were not able to enter CHSE cells. Haemagglutination was not detected. Our data clearly indicate that S. phocae can resist the killing activity of mucus and serum and proliferate in them, which could be associated with the presence of a capsular layer around the cells. Pathogenicity studies using seal and fish isolates demonstrated mortality or pathological signs in fish injected only with the Atlantic salmon isolate. No mortalities or histopathological alterations were observed in fish injected with extracellular products.     

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.     

Selective segregation in intraspecific competition between juvenile Atlantic salmon (Salmo salar) and brown trout (Salmo trutta)

Interactive segregation has been suggested as the ruling competition mechanism determining niche and niche segregation between juvenile Atlantic salmon (Salmo salar) and brown trout (Salmo trutta). Results from allopatry–sympatry observations of habitat use in both nature and in experiments were contrary to predictions derived from the interactive segregation hypothesis. Habitat use parameters under natural conditions such as distance to shore for Atlantic salmon parr were nearly identical in allopatric (mean ± SD; 3.2 ± 1.4 m) and sympatric (3.3 ± 1.4 m) situations. Occupied water depths largely reflected available water, but water depths <15 cm were avoided by salmon parr. Under experimental conditions, habitat use of allopatric salmon was density independent and salmon size had only minor effects, with smaller fish being more likely to occur in the shallow. Habitat use of salmon in sympatry with trout did not differ from allopatric salmon habitat use, and only salmon size had minor effects on depth choice – occurrence of trout or fish density had no effect. Allopatric trout was in general more frequent in the shallow habitat than salmon. Habitat use of sympatric trout was affected by the occurrence of salmon and trout size, resulting in a higher use of the shallow habitats for small trout. To conclude, selective segregation has a dominant role in salmon habitat use (not affected by trout occurrence), whereas a mixed situation occurs in trout habitat use with elements of interactive segregation when competing with Atlantic salmon (affected by salmon occurrence).     

Experimental induction of mouthrot in Atlantic salmon smolts using Tenacibaculum maritimum from Western Canada

Mouthrot, or bacterial stomatitis, is a disease which mainly affects farmed Atlantic salmon, (Salmo salar, L.), smolts recently transferred into salt water in both British Columbia (BC), Canada, and Washington State, USA. It is a significant fish welfare issue which results in economic losses due to mortality and antibiotic treatments. The associated pathogen is Tenacibaculum maritimum, a bacterium which causes significant losses in many species of farmed fish worldwide. This bacterium has not been proven to be the causative agent of mouthrot in BC despite being isolated from affected Atlantic salmon. In this study, challenge experiments were performed to determine whether mouthrot could be induced with T. maritimum isolates collected from outbreaks in Western Canada and to attempt to develop a bath challenge model. A secondary objective was to use this model to test inactivated whole‐cell vaccines for T. maritimum in Atlantic salmon smolts. This study shows that T. maritimum is the causative agent of mouthrot and that the bacteria can readily transfer horizontally within the population. Although the whole‐cell oil‐adjuvanted vaccines produced an antibody response that was partially cross‐reactive with several of the T. maritimum isolates, the vaccines did not protect the fish under the study's conditions.     

Road culvert restoration expands the habitat connectivity and production area of juvenile Atlantic salmon in a large subarctic river system

The effects of restoration of impassable road culverts on the distribution of juvenile Atlantic salmon, Salmo salar L., were evaluated in seven small tributaries of the subarctic River Teno system, northernmost Finland/Norway. Restoration enabled the passage of juvenile salmon through the culverts and increased the distribution area of salmon parr in the seven streams by tens or hundreds of metres, depending on the natural slope of the tributary with a total of ≈1 km new area for ascending juveniles. Areas upstream of the culverts were colonised after varying number of years, mostly 2–3, following restoration. Age‐1 and age‐2 parr were the first salmon age groups entering the new territory after removal of the migration barrier. Although the restoration measures were conducted at the downstream outlet area of the culverts only, the connectivity was improved and increased the production area accessible to juvenile salmon. Such removal of migration barriers and securing habitat connectivity by passable culverts should be taken into account in environmental management strategies of river systems safeguarding the essential habitats of salmonid fish.     

Effects of dietary vitamin C on growth and parr-smolt transformation in Atlantic salmon, Salmo salar L.

Atlantic salmon (Salmo salar) parr were fed fish meal based pelleted diets supplemented with graded levels of ascorbate-2-monophosphate (AP), equivalent to 0, 20, 60 and 1000 mg ascorbic acid (AA) kg”¹ throughout smoltification on a continuous light regime from February to June. No differences were observed in growth rate and body length distribution between the dietary regimes. The condition factor and the hepatosomatic index were somewhat elevated in fish fed no vitamin C throughout the smoltification, which can reflect changes in lipid metabolism in fish with suboptimal vitamin C nutrition. Sea water challenge tests (exposure to sea water with salinity of 34 gL^?1 and ambient temperature for 24 h) performed monthly did not reveal differences attributed to the vitamin C status, as measured by mortality, serum chloride and cortisol concentrations, haematological parameters and liver and head kidney ascorbate concentrations after 24 h. Elevated serum cortisol concentrations most probably reflected stress in the challenge tests, and some lower concentrations in fish fed high vitamin C levels may indicate a certain stress-ameliorating effect. The present results do not, however, support the anticipation of increased requirement of dietary vitamin C above the minimum requirement during smoltification in Atlantic salmon.     

Do habitat measurements in the vicinity of Atlantic salmon (Salmo salar) parr matter?

Atlantic salmon, Salmo salar L., parr habitat characterisation is usually performed by in situ measures of key environmental variables taken at the exact fish location if the fishing gear allows precise pinpointing of this location, or in large sampling sections covering a river reach or mesohabitat, often ignoring variability in the immediate vicinity around individual fish. These data may be critically important in the development and validation of habitat preference models. The influences of seven increasing distances of measures, the variation of the number of considered measures and the depth of velocity measurement (bottom or 0.6 of the depth) in the calculations of HSI (Habitat Suitability Index) from a multiple‐experts fuzzy model of Atlantic salmon parr habitat were tested. When a parr was present, six measures collected in a 50‐cm radius around the fish to provide an average measure as input data and velocity measured at 60% of the depth gave the highest HSI values. These results show some potential for the use of an intermediate study scale, between micro‐ and mesohabitat, and questions how fish habitat conditions are currently measured.     

Prevalence,geographic distribution and phenotypic differences of Piscirickettsia salmonis EM‐90‐like isolates

Early reports accounted for two main genotypes of Piscirickettsia salmonis, a fish pathogen and causative agent of piscirickettsiosis, placing the single isolate EM‐90 apart from the prototypic LF‐89 and related isolates. In this study, we provide evidence that, contrary to what has been supposed, the EM‐90‐like isolates are highly prevalent and disseminated across Chilean marine farms. Molecular analysis of 507 P. salmonis field isolates derived from main rearing areas, diverse hosts and collected over 6 years, revealed that nearly 50% of the entire collection were indeed typed as EM‐90‐like. Interestingly, these isolates showed a marked host preference, being recovered exclusively from Atlantic salmon (Salmo salar) samples. Although both strains produce undistinguishable pathological outcomes, differences regarding growth kinetics and susceptibility to the antibiotics and bactericidal action of serum could be identified. In sum, our results allow to conclude that the EM‐90‐like isolates represent an epidemiologically relevant group in the current situation of piscirickettsiosis. Based on the consistency between genotype and phenotype exhibited by this strain, we point out the need for genotypic studies that may be as important for the Chilean salmon industry as the continuous surveillance of antimicrobial susceptibility patterns.     

PCR survey for Paramoeba perurans in fauna,environmental samples and fish associated with marine farming sites for Atlantic salmon (Salmo salar L.)

Amoebic gill disease (AGD) caused by the amoeba Paramoeba perurans is an increasing problem in Atlantic salmon aquaculture. In the present PCR survey, the focus was to identify reservoir species or environmental samples where P. perurans could be present throughout the year, regardless of the infection status in farmed Atlantic salmon. A total of 1200 samples were collected at or in the proximity to farming sites with AGD, or with history of AGD, and analysed for the presence of P. perurans. No results supported biofouling organisms, salmon lice, biofilm or sediment to maintain P. perurans. However, during clinical AGD in Atlantic salmon, the amoeba were detected in several samples, including water, biofilm, plankton, several filter feeders and wild fish. It is likely that some of these samples were positive as a result of the continuous exposure through water. Positive wild fish may contribute to the spread of P. perurans. Cleaner fish tested positive for P. perurans when salmon tested negative, indicating that they may withhold the amoeba longer than salmon. The results demonstrate the high infection pressure produced from an AGD‐afflicted Atlantic salmon population and thus the importance of early intervention to reduce infection pressure and horizontal spread of P. perurans within farms.     

Evidence of exotoxin secretion of Piscirickettsia salmonis,the causative agent of piscirickettsiosis

Piscirickettsia salmonis is the aetiological agent of piscirickettsiosis, a disease which affects a variety of teleost species and that is particularly severe in salmonid fish. Bacterial‐free supernatants, obtained from cultures of three isolates of Piscirickettsia salmonis, were inoculated in Atlantic salmon, Salmo salar L., and in three continuous cell lines in an effort to determine the presence of secretion of extracellular products (ECPs) by this microorganism. Although steatosis was found in some liver samples, no mortalities or clinical signs occurred in the inoculated fish. Clear cytotoxicity was observed after inoculation in the cell lines CHSE‐214 and ASK, derived from salmonid tissues, but not in MDBK, which is of mammalian origin. The degree of cytotoxicity of the ECPs was different among the P. salmonis isolates tested. The isolate that evidenced the highest cytotoxicity in its ECPs exhibited only an intermediate virulence level after challenging fish with bacterial suspensions of the three P. salmonis isolates. Almost complete inhibition of the cytotoxic activity of ECPs was seen after proteinase K treatment, indicating their peptidic nature, and a total preclusion of the cytotoxicity was shown after their incubation at 50 °C for 30 min. Results show that P. salmonis can produce ECPs and at least some of them are thermolabile exotoxins that probably play a role in the pathogenesis of piscirickettsiosis.     

Use of a growth model to assess the suboptimal growth of Atlantic salmon parr in a subarctic river

This study tested the hypothesis that Atlantic salmon parr grew suboptimally according to the water temperature regime during the ice‐free season (from May to September) in the subarctic River Reisa (northern Norway). A laboratory‐based growth model was used to estimate the optimal growth which was then compared with the observed growth of the parr in the river. Water temperatures were recorded every second hour with a submersible data logger, and observed and predicted growth of several age classes were compared at two nearby nursery habitats. Except for parr aged 2, most age classes showed no significant change, or even a weight loss, from May to July, whereas from July to September, positive growth was found for all age classes coinciding with favourable temperatures above a critical temperature for growth. The parr growth rates were suboptimal, being lower than the predicted values, but values for observed and predicted body mass were not significantly different in five of 12 comparisons. Hence, the fish were sometimes unable to grow according to the optimal baseline set by the temperature regime. Therefore, factors other than temperature, such as limited food resources and resource competition, may also be a major determinant of Atlantic salmon parr growth. Our study demonstrates how growth predictions based on mathematical models may be employed by fish ecologists to identify growth deficiencies in wild fish populations.     

Lower prevalence of fin erosion in mature than in immature Atlantic salmon (Salmo salar) parr

Samples of hatchery-reared Atlantic salmon parr (age 0+ and 1+) from two different Norwegian river strains were investigated for connections between sexual maturity and prevalence of dorsal fin ray erosion. Mature males showed consistently lower prevalence as well as better recovery than immature males. Among the immature fish, no differences were detected between males and females. The results are discussed relative to morphological and physiological changes in maturing salmon parr.     

Biocontrol of saprolegniosis in rainbow trout (Oncorhynchus mykiss Walbaum) using two bacterial isolates (LE89 and LE141) of Pseudomonas fluorescens

The probiotic activity of 15 bacterial isolates that inhibit Saprolegnia parasitica in vitro was tested for the biocontrol of saprolegniosis in rainbow trout (Oncorhynchus mykiss Walbaum), adding the bacteria to tank water for 14 days at a concentration of 10⁶ bacteria ml^?1 water. Pseudomonas fluorescens LE89 and Pseudomonas fluorescens LE141 were effective in controlling experimental infection with S. parasitica since of the fish treated with LE89, 24.5% ± 16.27% (p < 0.05) became infected, as did 42.8% ± 8.41% (p < 0.05) of those treated with LE141. Given their protective effect when administered in water, their effect was also studied when administered in feed before and after experimental infection. Both bacterial isolates survived low pH levels and the action of bile, grew in skin and intestinal mucus, were resistant to several antibiotics and survived in feed; however, neither of the two isolates prevented S. parasitica infection when administered in feed.