Growth characteristics of the intracellular pathogen,Piscirickettsia salmonis,in tissue culture and cell‐free media

Piscirickettsia salmonis is an intracellular bacterium that was first isolated and identified in fish cells. Several types of cell lines have been explored for their ability to provide the bacterium with a host cell to replicate in. Tissue culture has been used for growth and cultivation for nearly two decades, until the facultative nature of P. salmonis was confirmed upon the development of blood‐ and cysteine‐based agar. Since then, research has continued to drive the creation of novel agar and broth formulations in order to improve the efficacy of cultivation of P. salmonis. Until now, the techniques and components used for growth have not been thoroughly discussed. In this review, the methods and formulations for growth of P. salmonis in tissue culture and cell‐free media will be examined.     

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.     

Stocking density and Piscirickettsia salmonis infection effect on Patagonian blennie (Eleginops maclovinus,Cuvier 1830) skeletal muscle intermediate metabolism

The need to expand aquaculture production has led to other fish to be considered as potential species for culture, such as the sub-Antarctic notothenioid Eleginops maclovinus (Valenciennes, 1830). The aim of this study was to determine the cumulative effect of density and pathogen infection by protein extract of Piscirickettsia salmonis on skeletal muscle metabolism. In a first experiment, specimens were submitted to three different stocking densities: (1) 3.1 kg m^?3, (2) 15 kg m^?3 and (3) 60 kg m^?3, for a period of 10 days. In a second experiment, metabolic changes caused by an infection of P. salmonis protein extract (a single injection of 0.5 μL P. salmonis protein extract g body weight^?1 was inoculated in the fish) and its combined effect with stocking density was assessed during a period of 10 days. This study concludes that stress caused by high stocking density led to the reorganization of some metabolic routes to fulfill skeletal muscle energy needs. Furthermore, infection response by pathogen P. salmonis differed when stocking density increased, suggesting an increase of energy needs with density in skeletal muscle of infected fish.     

Comprehensive antibiotic susceptibility profiling of Chilean Piscirickettsia salmonis field isolates

Antibiotics have been extensively used against infections produced by Piscirickettsia salmonis, a fish pathogen and causative agent of piscirickettsiosis and one of the major concerns for the Chilean salmon industry. Therefore, the emergence of resistant phenotypes is to be expected. With the aim of obtaining a landscape of the antimicrobial resistance of P. salmonis in Chile, the susceptibility profiles for quinolones, florfenicol and oxytetracycline (OTC) of 292 field isolates derived from main rearing areas, different hosts and collected over 5 years were assessed. The results allowed for the determination of epidemiological cut‐off values that were used to characterize the pathogen population. This work represents the first large‐scale field study addressing the antimicrobial susceptibility of P. salmonis, providing evidence of the existence of resistant types with a high incidence of resistance to quinolones. Remarkably, despite the amounts and frequency of therapies, our results disclosed that the issue of resistance to florfenicol and OTC is still in the onset.     

Piscirickettsiosis and Piscirickettsia salmonis in fish: a review

The bacterium Piscirickettsia salmonis is the aetiological agent of piscirickettsiosis a severe disease that has caused major economic losses in the aquaculture industry since its appearance in 1989. Recent reports of P. salmonis or P. salmonis‐like organisms in new fish hosts and geographical regions have increased interest in the bacterium. Because this gram‐negative bacterium is still poorly understood, many relevant aspects of its life cycle, virulence and pathogenesis must be investigated before prophylactic procedures can be properly designed. The development of effective control strategies for the disease has been limited due to a lack of knowledge about the biology, intracellular growth, transmission and virulence of the organism. Piscirickettsiosis has been difficult to control; the failure of antibiotic treatment is common, and currently used vaccines show variable long‐term efficacy. This review summarizes the biology and characteristics of the bacterium, including its virulence; the infective strategy of P. salmonis for survival and evasion of the host immune response; the host immune response to invasion by this pathogen; and newly described features of the pathology, pathogenesis, epidemiology and transmission. Current approaches to the prevention of and treatment for piscirickettsiosis are discussed.     

Association of red‐mark syndrome with a Rickettsia‐like organism and its connection with strawberry disease in the USA

Red‐mark syndrome (RMS), a disease seen mostly in rainbow trout, Oncorhynchus mykiss, is of unknown aetiology. The research presented here indicates the presence of an intracellular bacterium in RMS‐affected fish. A positive reaction was observed using immunohistochemistry (IHC) with skin lesions, liver, kidney and spleen of affected fish sampled from several locations within the United Kingdom using two different polyclonal antisera raised against Piscirickettsia salmonis. The same reaction was also seen with a number of different anti‐P. salmonis monoclonal antibodies (MAbs). A disease with similar clinical signs to RMS, referred to as strawberry disease (SD), has been reported in the USA. A Rickettsia‐like organism (RLO) has recently been associated with SD based on analysis of 16S rDNA sequences. Using the same panel of anti‐P. salmonis antibodies used to screen the RMS samples, similar staining was obtained in tissue of SD‐affected fish by IHC. A polymerase chain reaction (PCR) using RLO‐specific primers was also performed on RMS‐affected fish from the United Kingdom, and the samples were positive for the RLO 16S rRNA sequence. These findings suggest that the same aetiological agent may be responsible for RMS in the United Kingdom and SD in the USA.     

The Three‐spined Stickleback,Gasterosteus aculeatus Linnaeus 1758, plays a minor role as a host of Lepeophtheirus salmonis (Krøyer 1837) in the Gulf of Maine

The sea louse, Lepeophtheirus salmonis (Krøyer 1837), is a significant parasite of farmed salmon throughout the Northern Hemisphere. Management of on‐farm louse populations can be improved by understanding the role that wild fish play in sustaining and providing refuge for the local population of sea lice. In this study, 1,064 sticklebacks were captured. Of these animals, 176 individuals were carrying a total of 238 sea lice, yielding a prevalence and intensity of 16.5% and 1.4 lice per fish, respectively. Detailed examination of the sea lice on the three‐spined sticklebacks captured in Cobscook Bay found two L. salmonis individuals using three‐spined sticklebacks as hosts. A 2012 survey of wild fish in Cobscook Bay, Maine, found multiple wild hosts for Caligus elongatus (von Nordmann 1832), including three‐spined sticklebacks (Gasterosteus aculeatus L.), but no L. salmonis were found in this earlier study.     

Fecundity of the sea louse Caligus rogercresseyi on its native host Eleginops maclovinus captured near salmon farms in southern Chile

The sea louse (Caligus rogercresseyi) is the most significant parasitic pathogen in Chilean salmon farms, and it infects farmed salmon and native host fish. Fecundity is one of the most important parameters for understanding the population dynamics of a species; however, this information is scarce for this parasite. The fecundity of C. rogercresseyi females collected from native hosts (Eleginops maclovinus) captured near salmon farms in southern Chile was measured to evaluate the reproductive output of this parasite on this host fish. From June 2008 to May 2009, 212 specimens of E. maclovinus were examined. Each fish was measured, and all its parasites were collected, sorted and counted. Seventy‐nine ovigerous C. rogercresseyi females (OFs) were measured. Total body length, egg string length and total number of eggs per string were recorded for each parasite. Ovigerous females body length varied between 3.9 and 5.0 mm. Fecundity varied between 12 and 56 eggs string^?1, and it was correlated with OF body length. Temporal variations in OF fecundity were explained by co‐variation in OF body length, but not by month. Ovigerous females on E. maclovinus were smaller and showed lower fecundity than OFs on farmed salmon. Our results suggest that native hosts play a secondary role in C. rogercresseyi egg production in Codigue bay.     

Multilocus sequence typing detects new Piscirickettsia salmonis hybrid genogroup in Chilean fish farms: Evidence for genetic diversity and population structure

Piscirickettsia salmonisis the causative bacterial pathogen of piscirickettsiosis, a salmonid disease that causes notable mortalities in the worldwide aquaculture industry. Published research describes the phenotypic traits, virulence factors, pathogenicity and antibiotic‐resistance potential for various P. salmonisstrains. However, evolutionary and genetic information is scarce for P. salmonis. The present study used multilocus sequence typing (MLST) to gain insight into the population structure and evolution of P. salmonis. Forty‐two Chilean P. salmonisisolates, as well as the type strain LF‐89^T, were recovered from diseased Salmo salar, Oncorhynchus kisutchand Oncorhynchus mykissfrom two Chilean Regions. MLST assessed the loci sequences of dnaK, efp, fumC, glyA, murG, rpoD and trpB. Bioinformatics analyses established the genetic diversity among P. salmonis isolates (H = 0.5810). A total of 23 sequence types (ST) were identified, 53.48% of which were represented by ST1, ST5 and ST2. Population structure analysis through polymorphism patterns showed few polymorphic sites (218 nucleotides from 4,010 bp), while dN/dS ratio analysis indicated purifying selection for dnaK, epf, fumC, murG, and rpoD but neutral selection for the trpB loci. The standardized index of association indicated strong linkage disequilibrium, suggesting clonal population structure. However, recombination events were detected in a group of seven isolates. Findings included genogroups homologous to the LF‐89^T and EM‐90 strains, as well as a seven‐isolate hybrid genogroup recovered from both assessed regions (three O. mykiss and four S. salar isolates). The presented MLST scheme has comparative potential, with promising applications in studying distinct P. salmonis isolates (e.g., from different hosts, farms, geographical areas) and in understanding the epidemiology of this pathogen.     

Synergistic osmoregulatory dysfunction during salmon lice (Lepeophtheirus salmonis) and infectious hematopoietic necrosis virus co‐infection in sockeye salmon (Oncorhynchus nerka) smolts

While co‐infections are common in both wild and cultured fish, knowledge of the interactive effects of multiple pathogens on host physiology, gene expression and immune response is limited. To evaluate the impact of co‐infection on host survival, physiology and gene expression, sockeye salmon Oncorhynchus nerka smolts were infected with the salmon louse Lepeophtheirus salmonis (V?/SL+), infectious hematopoietic necrosis virus (IHNV; V+/SL?), both (V+/SL+), or neither (V?/SL?). Survival in the V+/SL+ group was significantly lower than the V?/SL? and V?/SL+ groups (p = 0.024). Co‐infected salmon had elevated osmoregulatory indicators and lowered haematocrit values as compared to the uninfected control. Expression of 12 genes associated with the host immune response was analysed in anterior kidney and skin. The only evidence of L. salmonis‐induced modulation of the host antiviral response was down‐regulation of mhc I although the possibility of modulation cannot be ruled out for mx‐1 and rsad2. Co‐infection did not influence the expression of genes associated with the host response to L. salmonis. Therefore, we conclude that the reduced survival in co‐infected sockeye salmon resulted from the osmoregulatory consequences of the sea lice infections which were amplified due to infection with IHNV.     

The placement of Gyrodactylus salmonis (Yin & Sproston) in the molecular phylogeny of studied members of the Gyrodactylus wageneri‐group parasitizing salmonids

The molecular phylogeny of Gyrodactylus salmonis from brook charr, Salvelinus fontinalis, rainbow trout, Oncorhynchus mykiss, cutthroat trout, O. clarkii, and Atlantic salmon, Salmo salar, in Canada is presented using sequences from ITS‐rDNA and the mitochondrial COX 1 gene. Sequence variation among G. salmonis specimens from the different North American hosts was consistent with within‐species variation reported for other Gyrodactylus. Sequence data are compared to those from other members of the wageneri group parasitizing salmoniform fishes in northern Europe (G. derjavini, G. derjavinoides, G. lavareti, G. salaris, G. salvelini, G. teuchis and G. truttae) and Asia (G. brachymystacis). Sequence divergence between G. salmonis and the recently described G. salvelini on Arctic charr, Salvelinus alpinus, in Finland was consistent with within‐species levels of variation in Gyrodactylus; however, phylogenetic analyses and morphological comparisons provided evidence of their distinctiveness such that they appear to be sister species. They grouped with G. lavareti (a parasite of a coregonid) to form a clade separate from European and Asian species of the wageneri lineage known from salmonid fish. Further study of gyrodactylids from across salmonid, coregonid and thymallid fish in the northern hemisphere would shed more light on the phylogeography of these parasites and serves as an important backdrop to understanding the evolution of their emergent virulence.     

Effect of different sources and forms of dietary carbohydrates on growth performance,body indices and lipogenesis activity of striped catfish Pangasianodon hypophthalmus fingerlings

A 12‐week feeding trial was conducted to determine a suitable carbohydrate source and form for Pangasianodon hypophthalmus. Ten isoenergetic, isonitrogenous and isolipidic diets, including five carbohydrate sources (dextrin, potato starch, wheat starch, corn starch and sago starch) and two carbohydrate forms (raw and cooked), were formulated. Results showed that specific growth rate, weight gain percentage, whole‐body lipid and 6‐phosphogluconate dehydrogenase enzyme activity of fish fed with cooked carbohydrates were significantly lower (p < .05) than those fed with raw carbohydrates (p < .05). Similarly, the fatty acid synthase enzyme activity of fish fed with the cooked form of carbohydrates was significantly lower (p < .05) than that fed with the raw form of carbohydrates except fish fed with cooked potato starch. The blood glucose level and malic enzyme activity of fish fed with cooked carbohydrate were significantly (p < .05) higher than those fed with raw carbohydrate. In summary, this study showed that cooked starch is an unsuitable dietary carbohydrate form for Pangasianodon hypophthalmus fingerlings. The raw forms of dextrin starch and potato are suitable dietary carbohydrate sources for further scientific studies on the carbohydrate utilization of P. hypophthalmus.     

Factors associated with severity of naturally occurring piscirickettsiosis in netpen- and tank-reared juvenile Atlantic salmon at a research aquarium in western Canada

The opportunistic examination of factors associated with an outbreak of piscirickettsiosis (SRS) is described in Atlantic salmon Salmo salar post-smolts held in an open netpen or in tanks supplied with raw sea water at a research aquarium in western Canada. During the outbreak, seawater temperature was significantly higher and salinity significantly lower in the netpen compared with the tanks. Mortality in the netpen began approximately 3 weeks prior to that in the tanks, and cumulative mortality in the netpen (34%) was significantly higher than in the tanks (12%). Piscirickettsia salmonis was confirmed by qPCR in tissues from moribund and dead fish and from colonies grown on enriched blood agar medium. Neither P. salmonis nor SRS were observed in salmon held concurrently in UV-irradiated sea water. The elevated mortality was curtailed by treatment with oxytetracycline. These observations further indicate warmer, less saline and periodically hypoxic seawater are risk factors for SRS. UV irradiation of sea water is shown to be a tool for SRS management in fish-holding facilities.     

Early‐stage sea lice recruits on Atlantic salmon are freshwater sensitive

Sea lice are significant parasites of marine and brackish farmed fishes. Freshwater bathing is a potential control option against numerous sea lice species, although has been viewed as futile against those that are capable of tolerating freshwater for extended periods. By comparing freshwater survival times across host‐attached stages of Lepeophtheirus salmonis (Krøyer), a key parasite in Atlantic salmon farming, we show the first attached (copepodid) stage undergoes 96–100% mortality after 1 h in freshwater, whereas later attached stages can tolerate up to 8 days. Thus, regular freshwater bathing methods targeting the more susceptible attached copepodid stage may successfully treat against L. salmonis and potentially other sea lice on fish cultured in marine and brackish waters.     

Evaluation of carbohydrate/lipid ratios on growth and metabolic response in tropical gar (Atractosteus tropicus) juvenile

Five experimental diets with different carbohydrates/lipid ratio (CHO:L, 0.75, 1.28, 2.10, 2.52, 4.63) were formulated. Fish were randomly assigned by triplicate (10 fish per tank, 0.50 ± 0.01 g). Growth was registered every 15 days until end of the experiment (45 days). Samples were taken for analysis of chemical carcass composition, blood chemistry, glycogen and lipid liver content, digestive and metabolic enzyme activities. Results showed that survival, growth performance parameters and plasma glucose were not affected by treatments (p > 0.05). Lipids of carcass and liver, as well as triglycerides and plasma cholesterol increase significantly as CHO:L ratio decreased. While an inversely proportional tendency was observed for carcass protein and liver glycogen (p < 0.05). Digestive enzymes did not show significant differences among treatments (p > 0.05). Finally, Hexokinase (HK), glucokinase (GK), phosphoenol pyruvate carboxykinase (PEPCK) and fructose‐1,6‐biphosphase (FBPase) showed high regulation by carbohydrates up to the CHO:L ratio of 2.10 (p < 0.05), while pyruvate kinase (PK) activity was not significantly affected by the CHO:L ratio. By the other side, glucose‐6‐phosphate dehydrogenase (G6PD) tends to significantly increase as the CHO:L ratio increases. We conclude that A. tropicus fry have a high capacity to utilize carbohydrates substituting lipids as energy source in balanced diets.     

Determination of minimal concentration of Piscirickettsia salmonis in water columns to establish a fallowing period in salmon farms

A highly sensitive real‐time PCR procedure to detect and quantify the number of Pisciricketsia salmonis units in seawater samples from affected farm sites has been developed. The purpose was to determine a fallowing period that would allow safe restocking of the target farm with new fish. Bacterial load was determined in water samples by comparing the obtained amplification values against a standard curve generated by the amplification of known concentrations of the ITS‐ribosomal component of P. salmonis DNA, cloned in a suitable vector. The standard curve was linear over the range of 10¹–10¹⁰ log units. Target samples were taken every 10 days over a 40‐day period, at 5 m depth and at the surface. In a highly affected area of southern Chile, the number of bacterial units in farm water decreased to zero at day 50. Therefore, a fallowing period of 50 days post‐removal of cages of affected fish appears to be appropriate before restocking. This procedure could be adapted to control disease problems because of other pathogens in fish farm waters.     

Assessing the impacts of the introduced channel catfish Ictalurus punctatus using the comparative functional response approach

In the metric “Relative Impact Potential” (RIP), the functional response (FR) of a non‐native species can be compared with that of a native analogue and combined with the species abundance to predict its environmental impact. Here, using the River Guaraguaçu (Brazil) as a case study, this methodology was implemented to identify the impacts of the non‐native channel catfish Ictalurus punctatus (Rafinesque) compared with a native species Rhamdia quelen (Quoy & Gaimard) towards small prey fish. Both species exhibited Type II FRs, but handling times were lower for I. punctatus, resulting in a greater maximum feeding rate in this species. Consequently, an RIP > 1 was found, indicating that I. punctatus represents a superior impact to prey compared with its native analogue. These results demonstrate that I. punctatus is a potential threat to small endangered fish species; therefore, policies to avoid escapes from aquaculture should be created and the abundance of I. punctatus controlled.     

Starch and lipid in diets for dourado Salminus brasiliensis (Cuvier 1816): growth,nutrient utilization and digestive enzymes

Carbohydrates and lipids are the major and cheaper non‐protein energy sources for fish diets. This study aimed at determining ideal relationship between dietary starch and lipids and their influence on digestive enzyme profile of juvenile dourado. Trials were set up in a closed‐loop system with controlled temperature, dissolved oxygen, pH and total ammonia. Experimental extruded diets formulated to contain different starch (St) to lipids (Lip) ratio (St : Lip = 0 : 1, 0.3 : 1, 0.7 : 1, 1.0 : 1, 1.4 : 1, 2.0 : 1, 2.3 : 1 to 3.3 : 1) were fed to juvenile dourado (3.34 ± 0.16 g) and stocked into 30 polyethylene tanks (330 L, 30 fish per tank) in a completely randomized design with unbalanced repetition (n = 3 or 4), in two daily meals (1000 and 1530) until satiation, for 95 days. Dietary starch contents higher than 145.2 g kg^‐1 (St : Lip > 1.0 : 1.0) hampered the performance of fish, but, paradoxically, the inclusion of carbohydrates in the diet elicited better utilization of dietary protein and energy by the species. Fish adapted digestive enzyme profile to dietary starch contents through increasing secretion of alkaline proteases and non‐specific lipases, but secretion of amylase was not altered. Physiological responses show the limitation of species on the use of dietary carbohydrates.     

Impacts of parasites on marine survival of Atlantic salmon: a meta‐analysis

Parasites can, in theory, have large impacts on the survival of fish populations. One method to evaluate such impacts on anadromous species is to apply manipulative field experiments in which parallel groups of antiparasitically treated and non‐treated fish are simultaneously released and then subsequently recaptured as returning adults. A systematic review and meta‐analysis on all such Norwegian studies on Salmo salar provided a data set for the time period 1996 to 2011 on 118 release groups comprising 657 624 fish released and 3989 recaptured. The overall risk ratio (RR) was estimated to be 1.18 (95% CI: 1.07–1.30). The effect varied strongly between groups, (Higgins I² = 40.1%). Over 70% of this heterogeneity could be explained by the release location, time period and baseline survival. The most important predictor variable was baseline survival. In groups with low recapture in the control group (low baseline survival), the effect of treatment was high (RR = 1.7), while in groups with high recapture in the control group (high baseline survival), there was no effect of treatment (RR ~ 1.00). The most prevalent parasite in the region affected by the drugs administered was Lepeophtheirus salmonis. Hence, the meta‐analysis supports the hypothesis that L. salmonis contributes to the mortality of S. salar during outward migration. However, the effect of treatment was not consistent, but was evidently strongly modulated by other risk factors. The results suggest that the population‐level effects of parasites cannot be estimated independently of other factors affecting the marine survival of S. salar.