Susceptibility of corkwing wrasse Symphodus melops, goldsinny wrasse Ctenolabrus rupestis, and Atlantic salmon Salmo salar smolt, to experimental challenge with Vibrio tapetis and Vibrio splendidus isolated from corkwing wrasse

The virulence of two Vibrio strains, previously isolated from diseased corkwing wrasse Symphodus melops and identified as V. tapetis and V. splendidus, to corkwing and goldsinny wrasse Ctenolabrus rupestris and to Atlantic salmon Salmo salar, was studied under laboratory conditions. Both bacteria were shown to be opportunistically pathogenic to corkwing wrasse, causing significantly higher mortality in the challenged groups than in the controls. Bacterial cultivation of kidney samples and re-isolation of V. tapetis and V. splendidus from most mortalities confirmed the two strains as the probable cause of mortality in the challenged groups. The control group also suffered relatively high mortality, but no specific pathogens that were suspected to be the main cause of death were isolated, other than a mixture of Vibrio spp. and, in the case of one individual, atypical Aeromonas salmonicida. Following injection challenge with both bacterial strains, no mortality was recorded in Atlantic salmon. In bath challenge trials with goldsinny wrasse, only slight mortality was observed in the challenged groups and the unchallenged control group. Bacterial examination showed that atypical Aeromonas salmonicida was the probable cause of death in both bath challenged and control groups of goldsinny wrasse, and no indication of infection by any Vibrio sp. was found.     

A historical review of the key bacterial and viral pathogens of Scottish wild fish

Thousands of Scottish wild fish were screened for pathogens by Marine Scotland Science. A systematic review of published and unpublished data on six key pathogens (Renibacterium salmoninarum, Aeromonas salmonicida, IPNV, ISAV, SAV and VHSV) found in Scottish wild and farmed fish was undertaken. Despite many reported cases in farmed fish, there was a limited number of positive samples from Scottish wild fish, however, there was evidence for interactions between wild and farmed fish. A slightly elevated IPNV prevalence was reported in wild marine fish caught close to Atlantic salmon, Salmo salar L., farms that had undergone clinical IPN. Salmonid alphavirus was isolated from wild marine fish caught near Atlantic salmon farms with a SAV infection history. Isolations of VHSV were made from cleaner wrasse (Labridae) used on Scottish Atlantic salmon farms and VHSV was detected in local wild marine fish. However, these pathogens have been detected in wild marine fish caught remotely from aquaculture sites. These data suggest that despite the large number of samples taken, there is limited evidence for clinical disease in wild fish due to these pathogens (although BKD and furunculosis historically occurred) and they are likely to have had a minimal impact on Scottish wild fish.     

Aeromonas salmonicida infects Atlantic salmon (Salmo salar) erythrocytes

Aeromonas salmonicida subsp. salmonicida (hereafter A. salmonicida) is the aetiological agent of furunculosis in marine and freshwater fish. Once A. salmonicida invade the fish host through skin, gut or gills, it spreads and colonizes the head kidney, liver, spleen and brain. A. salmonicida infects leucocytes and exhibits an extracellular phase in the blood of the host; however, it is unknown whether A. salmonicida have an intraerythrocytic phase. Here, we evaluate whether A. salmonicida infects Atlantic salmon (Salmo salar) erythrocytes in vitro and in vivo. A. salmonicida did not kill primary S. salar erythrocytes, even in the presence of high bacterial loads, but A. salmonicida invaded the S. salar erythrocytes in the absence of evident haemolysis. Naïve Atlantic salmon smolts intraperitoneally infected with A. salmonicida showed bacteraemia 5 days post‐infection and the presence of intraerythrocytic A. salmonicida. Our results reveal a novel intraerythrocytic phase during A. salmonicida infection.     

Development and Application of a Quantitative Real‐time Polymerase Chain Reaction Assay for the Detection of Aeromonas salmonicida

A rapid, economical, specific, and sensitive quantitative real‐time polymerase chain reaction (qPCR) assay coupled with SYBR Green I chemistry was developed for the quantitative detection of Aeromonas salmonicida from farmed Atlantic salmon, Salmo salar, with the symptoms of furunculosis. The set of primers designed from the virulence array protein (vapA) gene was specific to A. salmonicida. Compared with the conventional PCR, qPCR had a lower detection limit of 5.6 copies of the positive plasmids. The standard curve, which showed the relationship between the copies of A. salmonicida and its quantification cycle (C_q) value, could be described as follows: log (copies of A. salmonicida) = ?0.3213 C_q + 10.721. The quantitative detection of copies of A. salmonicida in different tissues of the moribund Atlantic salmon showed that A. salmonicida could be detected in all tissues; the spleen contained the largest number of A. salmonicida and then the kidney. These results suggest that the qPCR assay reported here is a specific, sensitive, and quantitative method for detecting A. salmonicida. It can be used for the routine tests of A. salmonicida in local aquaculture enterprise and for the research of infection routes of A. salmonicida to Atlantic salmon.     

Infectious pancreatic necrosis virus: experimental infection of goldsinny wrasse, Ctenolabrus rupestris L. (Labridae)

The use of wrasse as cleaner fish in farming of Atlantic salmon, Salmo salar L., is now widespread in Scotland, Ireland and Norway, but little is known about the susceptibility of these fish to the common pathogens of cage-cultured salmon. The susceptibility of goldsinny wrasse, Ctenolabrus rupestris L., to infectious pancreatic necrosis virus (IPNV) was investigated. Captive-bred C. rupestris were infected with IPNV-Sp (Shetland strain) using a bath challenge method. Two infection doses were used, low (4.1 × 10⁵ pfu mL⁻⁻¹) and high (2.4 × 10⁶ pfu mL⁻⁻¹), with two replicates for each experiment. Bathing times were 1 h for the low dose and 5 h for the high dose. A maximum prevalence of infection (30%) was seen 2 weeks post-infection in replicate 1 of the low dose experiment and was accompanied by low tissue titres. The tissue titres dropped to an undetectable level by 4 weeks post-infection. In the high dose experiment, a high prevalence of infection was seen along with moderate titres in tissues as well as a marked pathological response. Both prevalence and intensity declined rapidly and the fish recovered. In the high dose experiment, faecal titres followed the same pattern as those in the tissues. The implications for the use of wrasse in the farming of Atlantic salmon are discussed.     

Aeromonas salmonicida infection levels in pre‐ and post‐stocked cleaner fish assessed by culture and an amended qPCR assay

Due to increasing resistance to chemical therapeutants, the use of ‘cleaner fish’ (primarily wrasse, Labridae, species) has become popular in European salmon farming for biocontrol of the salmon louse, Lepeophtheirus salmonis (Krøyer). While being efficient de‐licers, cleaner fish mortality levels in salmon cages are commonly high, and systemic bacterial infections constitute a major problem. Atypical furunculosis, caused by Aeromonas salmonicida A‐layer types V and VI, is among the most common diagnoses reached in clinical investigations. A previously described real‐time PCR (qPCR), targeting the A. salmonicida A‐layer gene (vapA), was modified and validated for specific and sensitive detection of all presently recognized A‐layer types of this bacterium. Before stocking and during episodes of increased mortality in salmon cages, cleaner fish (primarily wild‐caught wrasse) were sampled and screened for A. salmonicida by qPCR and culture. Culture indicated that systemic bacterial infections are mainly contracted after salmon farm stocking, and qPCR revealed A. salmonicida prevalences of approximately 4% and 68% in pre‐ and post‐stocked cleaner fish, respectively. This underpins A. salmonicida's relevance as a contributing factor to cleaner fish mortality and emphasizes the need for implementation of preventive measures (e.g. vaccination) if current levels of cleaner fish use are to be continued or expanded.     

Polyphasic characterization of Aeromonas salmonicida isolates recovered from salmonid and non‐salmonid fish

Michigan's fisheries rely primarily upon the hatchery propagation of salmonid fish for release in public waters. One limitation on the success of these efforts is the presence of bacterial pathogens, including Aeromonas salmonicida, the causative agent of furunculosis. This study was undertaken to determine the prevalence of A. salmonicida in Michigan fish, as well as to determine whether biochemical or gene sequence variability exists among Michigan isolates. A total of 2202 wild, feral and hatchery‐propagated fish from Michigan were examined for the presence of A. salmonicida. The examined fish included Chinook salmon, Oncorhynchus tshawytscha (Walbaum), coho salmon, O. kisutcha (Walbaum), steelhead trout, O. mykiss (Walbaum), Atlantic salmon, Salmo salar L., brook trout, Salvelinus fontinalis (Mitchill), and yellow perch, Perca flavescens (Mitchill). Among these, 234 fish yielded a brown pigment‐producing bacterium that was presumptively identified as A. salmonicida. Further phenotypic and phylogenetic analyses identified representative isolates as Aeromonas salmonicida subsp. salmonicida and revealed some genetic and biochemical variability. Logistic regression analyses showed that infection prevalence varied according to fish species/strain, year and gender, whereby Chinook salmon and females had the highest infection prevalence. Moreover, this pathogen was found in six fish species from eight sites, demonstrating its widespread nature within Michigan.     

Use of mesocosms in larval rearing of saithe [Pollachius virens (L.)], goldsinny [Ctenolabrus rupestris (L.)], and corkwing [Crenilabrus melops (L.)]

Pilot experiments were carried out in mesocosms (5.3 m³ plastic bag enclosures) for larval rearing of the commercially important saithe [Pollachius virens (L.)], and two wrasse species, goldsinny [Ctenolabrus rupestris (L.)] and corkwing [Crenilabrus melops (L.)] which are both used as cleaner-fishes for control of sea-lice infestations in Atlantic salmon farming. Fertilized saithe eggs were collected from natural spawning in a simple and inexpensive tank system. Goldsinny and corkwing eggs were obtained by stripping mature individuals collected from the sea. Egg mortality during incubation was low in all species. Larvae were released in the rearing enclosures a few days after hatching and fed natural plankton (mainly copepod nauplii). Survival through metamorphosis was low for saithe (3%), which may be attributed to specific environmental requirements, in terms of water quality, in this species. Survival in the wrasse was good (20–40%), indicating that the use of mesocosms may have potential for mass-production of these species.     

A mortality event in wrasse species (Labridae) associated with the presence of viral haemorrhagic septicaemia virus

Viral haemorrhagic septicaemia (VHS) is an infectious disease of farmed and wild fish and has an extensive host range in both freshwater and marine environments. In December 2012, a wrasse population consisting of ballan, Labrus bergylta (Ascanius), corkwing, Symphodus melops (L.), cuckoo, Labrus mixtus L., goldsinny, Ctenolabrus rupestris (L.), and rock cook, Centrolabrus exoletus (L.), held at a marine hatchery in the Shetland Isles, Scotland, experienced a mortality event. Approximately 10 000 wrasse were being held at the facility on behalf of an Atlantic salmon, Salmo salar L., aquaculture company prior to being deployed for the biological control of parasites on marine pen Atlantic salmon, aquaculture sites. Fish Health Inspectors from Marine Scotland Science initiated a diagnostic investigation, and subsequent diagnostic testing confirmed the site to be VHSV positive by qRT-PCR and virus isolation followed by ELISA. A VHSV genotype-specific qRT-PCR assay revealed that the isolates belonged to genotype III, the European marine strain of the virus. The virus genotype was further confirmed by nucleic acid sequencing of the partial nucleoprotein (N) and glycoprotein (G) genes followed by BLAST nucleotide searches. This study reports for the first time the detection of VHSV within multiple wrasse species and highlights the need for a comprehensive risk-based approach to the use of wrasse and other finfish species as biological controls within the aquaculture industry.     

Antibiotic protection against recrudescence of latent Aeromonas salmonicida during furunculosis vaccination

Problems of temporary immunosuppression following vaccination against Aeromonas salmonicida infection had to be overcome in the development of a furunculosis vaccine. Empirical observations have indicated that immunosuppression persists for some time after vaccination, rendering fish, especially subclinical carriers of A. salmonicida, highly vulnerable to bacterial invasion. The efficacy of simultaneous application of furunculosis vaccine and a long-lasting amoxycillin preparation to a population of Atlantic salmon smolts was evaluated. Control groups were treated with either vaccine alone, amoxycillin alone or were untreated. Moderate stress, simulating smolt transfer with a 5°C temperature rise, resulted in a rapid outbreak in mortalities reaching 100% in the vaccinates. Losses among the untreated controls were more gradual and rose to about 50%. Both amoxycillin-treated groups survived well. Further severe stress resulted in total mortalities among the untreated fish but no further losses in the amoxycillin groups. Four months after vaccination, evidence of a specific immune response was confirmed by ELISA, demonstrating circulating antibodies in the blood of vaccinates. In a severe and in a moderate challenge with A. salmonicida., the relative specific protection was 63 and 86%, respectively. Thus, effective protection against furunculosis was achieved without jeopardizing the stock during the vaccination process and with elimination of the carrier state.     

Genetic population structure of goldsinny wrasse, Ctenolabrus rupestris (L.), in Norway: implications for future management of parasite cleaners in the salmon farming industry

The application of goldsinny wrasse, Ctenolabrus rupestris (L.), as parasite cleaners in the salmon farming industry has led to transport of live wrasse between regions in Norway. Owing to the operation of salmon farms, some of the transferred wrasse escape from the salmon net pens and can therefore mix and potentially interbreed with the local populations. The genetic effects of such interbreeding are unknown and information about the genetic population structure of this species in Norwegian inshore waters is needed to evaluate the potential effects. A genetic study based on polymorphic tissue enzymes was therefore carried out. Four of the 35 protein coding loci screened were polymorphic at the 99% level, and only two at the 95% level. The allele frequencies of the IDDH-1*; PGM-1*; GPI-1* and GPI-2* loci were compared in wrasse samples (1442 specimens) collected at 12 localities. Significant differences were found for PGM-1* among the samples collected at two inner fjord localities. Significant differences at the IDDH-1*, PGM-1* and GPI-2* loci were found when data from a sample of transferred wrasse caught in southern Norway were compared with local wrasse in the recipient areas.     

Investigations on the role of the salmon louse,Lepeophtheirus salmonis (Caligidae), as a vector in the transmission of Aeromonas salmonicida subsp. salmonicida

A bacteria–parasite challenge model was used to study the role of sea lice, Lepeophtheirus salmonis (Copepoda), as a vector of Aeromonas salmonicida subsp. salmonicida. Three hypotheses were tested: (i) L. salmonis can acquire A. salmonicida subsp. salmonicida via water bath exposure; (ii) L. salmonis can acquire the bacteria via parasitizing infected Atlantic salmon, Salmo salar; and (iii) L. salmonis can transmit the bacteria to naïve Atlantic salmon via parasitism. Adult L. salmonis exposed to varying A. salmonicida subsp. salmonicida suspensions (10¹–10⁷ cells mL^?1) for 1.0, 3.0 or 6.0 h acquired the bacteria externally (12.5–100%) and internally (10.0–100%), with higher prevalences associated with the highest concentrations and exposures. After exposure to 10⁷ cells mL^?1, viable A. salmonicida subsp. salmonicida could be isolated from the external carapace of L. salmonis for 120 h. Lepeophtheirus salmonis also acquired the bacteria externally and internally from parasitizing infected fish. Bacterial transmission was observed only when L. salmonis had acquired the pathogen internally via feeding on ‘donor fish’ and then by parasitizing smaller (<50 g) ‘naive’ fish. Under specific experimental conditions, L. salmonis can transfer A. salmonicida subsp. salmonicida via parasitism; however, its role as a mechanical or biological vector was not defined.     

Genetic variation in susceptibility of Atlantic salmon, Salmo salar L., to furunculosis, BKD and cold water vibriosis

Genetic variation in susceptibility of Atlantic salmon, Salmo salar L., to furunculosis, bacterial kidney disease (BKD) and cold water vibriosis was studied by challenge testing one-year-old fingerlings. Fish from 81 full-sib families within 32 sire progeny groups were infected with Aeromonas salmonicida, Renibacterium salmoninarum and Vibrio salmonicida. Estimated heritabilities were relatively low, being highest for BKD (h²= 0.23) and lowest for cold water vibriosis (h²= 0.13). Genetic correlations between the ability to survive the diseases were all positive, but the magnitude of the genetic correlation between furunculosis and BKD may be biased upwards because some of the dead BKD fish were also infected with furunculosis. The application of selection to develop resistant populations of Atlantic salmon is advocated. Challenge testing seems to be a feasible method, with relatively low costs and easy management. The future response to selection will depend on the relationships between results from a challenge test and mortalities under farming conditions and between disease resistance and other traits in the breeding goal.     

Influence of dietary vitamin B6 on tissue vitamin B6 contents and immunity in Atlantic salmon, Salmo salar L.

Atlantic salmon, Salmo salar L., (14 g) were fed a practical fish-meal-based diet supplemented with 0. 10, 20. 40, 80 and 160 mg pyridoxine (PN) per kg feed for 20 weeks. Tissue vitamin B₆ contents were significantly reduced in fish fed the non-supplemented diet compared with fish fed PN-supplemented diets. Serum haemolytic complement activity and head kidney lysozyme activity, and the specific antibody response following immunization with Vibrio salmonicida. were not influenced by the dietary regimes. Challenge with Aeromonas salmonicida showed that increasing the dietary levels of vitamin B₆ did not improve the resistance to furunculosis. Growth, mortality and haematology were not affected by supplementing a practical diet with vitamin B₆ In conclusion, feed levels of vitamin B₆ higher than the minimum dietary requirement did not enhance immune functions and disease resistance in Atlantic salmon.     

Diseases of north European wrasse (Labridae) and possible interactions with cohabited farmed salmon, Salmo salar L

There have been several reported studies of wrasse health but none of these has shown transmission of wrasse diseases when stocked with farmed Atlantic salmon. Most of the studies have focussed on bacterial and parasite issues, including treatment of bacterial diseases with antibiotics and vaccination of wrasse. Classical and atypical furunculosis have been reported in wrasse following stress, and wrasse have been susceptible to vibrio infection. Further study is required on the vaccination of wrasse for furunculosis with latent carrier status to maximize survival. There are studies on viral diseases such as infectious pancreatic necrosis, infectious salmon anaemia and pancreas disease and although these did not give any undue concern for salmon health, there is also scope for further study in this area. Resident parasite communities of wrasse are largely host-specific and do not appear to be a threat to salmon. Given that wrasse have not, to date, been a vector of disease in salmon, attention should be placed on maintaining best practice in cohabiting wrasse with salmon. Other issues that should be addressed are good welfare of wrasse in pens and identifying measures of this, the identification of losses of wrasse in pens, being alert to potential emerging diseases through health screening of mortalities and assessing the risks associated with carrying forward wrasse from one salmon production cycle to the next. Issues of exploitation by fishing on wild wrasse stocks and improved biosecurity may be addressed by the increased movement by the industry to the stocking of farmed wrasse.     

Cold‐water vibriosis. The current status of knowledge

The current review for the first time summarizes the findings of the 30 years of research on cold‐water vibriosis (CWV). The diseased caused by Aliivibrio salmonicida (earlier known as Vibrio salmonicida) was for the first time described in 1986 and became one of the most important bacterial diseases in salmon aquaculture. The lack of appropriate vaccine hampered development of Atlantic salmon aquaculture until the late 1980s when a novel vaccine allowed dramatic increase in the Atlantic salmon farming. In December 2007, the genus Vibrio was split into two genera and several bacterial species including V. salmonicida were transferred to genus Aliivibrio. The change of the names create significant difficulties with the designation of the CWV disease agent since its abbreviation A. salmonicida became similar to another well‐known salmon pathogen Aeromonas salmonicida (A. salmonicida). The disease was considered as controlled by vaccination, but reappeared at Atlantic salmon farms in 2011, this time affecting vaccinated Atlantic salmon. The current review summarizes the knowledge on pathogenesis, vaccination and treatment of CWV and proposes further directions for studying the disease.     

The Physical Condition and Welfare of Five Species of Wild‐caught Wrasse Stocked under Aquaculture Conditions and when Stocked in Atlantic Salmon,Salmo salar,Production Cages

Several indices were examined to assess the physical condition of wrasse stocked on Atlantic salmon, Salmo salar, farms as cleaner fish, and included examination of eye condition, snout erosion, skin hemorrhaging, and erosion and splitting of dorsal, pectoral, anal and caudal fins. Baseline values were determined for five wrasse species: goldsinny, Ctenolabrus rupestris; rock cook, Centrolabrus exoletus; corkwing, Crenilabrus melops; cuckoo, Labrus mixtus; and ballan, Labrus bergylta, held in a farm environment for 3 mo prior to transfer to salmon farms. The caudal fin was most affected by injury. The fin erosion index (FEI) was low in all species and below 0.6. The fin splitting index (FSI) was the most prominent index and was significantly higher in the caudal fin (FSI > 2) compared with other fins (FSI < 0.5), and also significantly higher in corkwing and rock cook compared with the other wrasse species. The FEI and FSI were also calculated for a group of ballan wrasse before stocking on a seawater farm, during the first winter and upon harvesting. There were no significant differences in the scores of fin erosion and fin splitting in any of the samples, although the indices were marginally poorer in winter.     

Evaluation of florfenicol in Atlantic salmon,Salmo salar L.: efficacy against furunculosis due to Aeromonas salmonicida and cold water vibriosis due to Vibrio salmonicida

Two replicated controlled trials were conducted to determine the efficacy of florfenicol against Aeromonas salmonicida and Vibrio salmonicida infections in Atlantic salmon, Salmo salar L., smolts kept in 25‰ salt water. Infection with A. salmonicida was treated with florfenicol, oxolinic acid, oxytetracycline, trimethoprim/sulphadiazine or flumequine, whereas the V. salmonicida infection was treated with florfenicol or oxolinic acid only. A. salmonicida infection was induced by the introduction of cohabitant fish previously inoculated intraperitoneally. Medication started simultaneously in all test tanks on the first day of specific mortality among test fish. V. salmonicida infection was induced by intraperitoneal inoculation of all test fish. Medication started 1 day after infection. Medicated feeds were produced by coating the antibacterials on standard feed pellets, and administered twice daily for 10 consecutive days. With the dose used in the present trials, florfenicol was highly effective in reducing specific mortalities due to both infections. It was slightly more effective than oxolinic acid and trimethoprim/sulphadiazine against A. salmonicida infection. There was no significant difference between florfenicol and oxolinic acid in reducing specific mortalities due to V. salmonicida.     

The efficacy of a single intraperitoneal injection of either flumequine or oxytetracycline hydrochloride in prevention of outbreaks of atypical Aeromonas salmonicida infection in goldsinny wrasse, Ctenolabrus rupestris L., following stress.

In this investigation, the efficacy of a single intraperitonealinjection of either flumequine or oxytetracycline hydrochloride to preventoutbreak of atypical Aeromonas salmonicida infection ingoldsinny wrasse, Ctenolabrus rupestris, following stresswas studied. Six groups of goldsinny wrasse, each of 50 individuals, weretreated with an intraperitoneal injection of either propylene glycol : saline(50 : 50), 200 mg kg^–1 of oxytetracycline or 50mg kg^–1 of flumequine dissolved in propyleneglycol : saline (50 : 50). Three days following medication the fish in allgroups were stressed by an intraperitoneal injection of prednisolone acetate(0.05 ml) and a rise in seawater temperature from 9 to 11°C. Mortality was observed daily for 21 days. Flumequine wasthe more effective with a mean cumulative mortality of 5% compared tooxytetracycline with 54%. The mean cumulative mortality in the unmedicatedcontrol groups was 84%. Bacterial examination of kidneys from dead fishconfirmed the presence of atypical A. salmonicida as theprobable cause of death. Minimum inhibitory concentration (MIC) values forflumequine and oxytetracycline against the isolated A.salmonicida were determined to 0.13 gml^–1, and 2.0 g ml^–1,respectively.     

The impact of Aeromonas salmonicida infection on behaviour and physiology of Atlantic salmon (Salmo salar L.)

This study examined the effect of Aeromonas salmonicida infection on the swimming behaviour and physiology of Atlantic salmon (Salmo salar L.). Fish were injected in the dorsal muscle with either 100 μL bacterium solution (3.05 × 10⁷CFU mL^?1) Aeromonas salmonicida, or 100 μL 0.9% NaCl (as control group). Compared with the control group, the pathogen injected group significantly impaired the critical swimming speed (U_crit) and exhausting time (P < 0.05), which were reduced by 37% and 39% at severe time (day 6, when most of the fish challenged by Aeromonas salmonicida died) respectively. Furthermore, the blood parameters related to their swimming behaviour were also influenced significantly by pathogen injection (P < 0.05). The results showed that the high density lipoprotein (HDL), haemoglobin and total protein decreased by about 63%, 49% and 74% at the end, respectively, while lactate increased by about 29% on day 6. The results suggested that the swimming performance of Atlantic salmon might be a useful indicator of disease, and it was feasible to warn the outbreaks of acute disease by fish behaviour.