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 A systematic review of published and unpublished data on six key pathogens (Renibacterium salmoninarum, Aeromonas salmonicida, IPNV, ISAV, SAV and VHSV) found in Scottish wild and farmed fish was undertaken.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.     

Infectious pancreatic necrosis virus in Atlantic salmon, Salmo salar L., post-smolts in the Shetland Isles, Scotland: virus identification, histopathology, immunohistochemistry and genetic comparison with Scottish mainland isolates

During mid-June 1999 peak mortalities of 11% of the total stock per week were seen at a sea cage site of Atlantic salmon, Salmo salar L., post-smolts in the Shetland Isles, Scotland. Virus was isolated on chinook salmon embryo (CHSE) cells in a standard diagnostic test and infectious pancreatic necrosis virus (IPNV) identified by enzyme-linked immunosorbent assay. IPNV was confirmed as serogroup A by a cell immunofluorescent antibody test using the cross-reactive monoclonal antibody AS-1. Four weeks after the main outbreak, virus titres in surviving moribund fish were assayed at >10(10) TCID50 g(-1) kidney. Histopathology of moribund fish was characterized by pancreatic acinar cell necrosis and a marked catarrhal enteritis of the intestinal mucosa. In the liver, necrosis, leucocytic infiltration and a generalized cell vacuolation were noted. IPNV-specific immunostaining was demonstrated in pancreas, liver, heart, gill and kidney tissue. The nucleotide sequence of the coding region of segment A was determined from the Shetland isolate. A 1180 bp fragment of the VP2 gene of this isolate was compared with a 1979 reference isolate from mainland Scottish Atlantic salmon, La/79 and another more recent mainland isolate, 432/00. Both A2 isolates were derived from carrier fish without signs of IPN and serotyped by a plaque neutralization test. The Shetland isolate shows a different nucleotide and amino acid sequence compared with the two isolates from carrier fish. These latter isolates showed identical amino acid sequences in the fragment examined, despite the 21 years separating the isolations. Sequence comparisons with other A2 (Sp) isolates on the database confirm all three Scottish isolates are A2 (Sp).     

Distribution of infectious pancreatic necrosis virus (IPNV) in wild marine fish from Scottish waters with respect to clinically infected aquaculture sites producing Atlantic salmon, Salmo salar L

This study represents the first large-scale investigation of IPNV in Scottish wild marine fish. Kidney samples were taken from 30 627 fish comprising 37 species and 45 isolations were made from nine different species, illustrating these as reservoirs of IPNV in Scottish waters. The estimated prevalence of IPNV in the Scottish marine environment was low at 0.15% (90% confidence intervals, (CI) of 0.11-0.19%). This was significantly greater in fish caught less than 5.0 km from IPN-positive fish farms in Shetland, at 0.58% (90% CI of 0.45-0.77%). This prevalence persisted and did not significantly decrease over the 16-month period of study. The estimated prevalence of IPNV for each positive species was less than 1% with the statistically non-significant exceptions of flounder, Platichthys flesus (L.), at 12.5% (90% CI of 0.64-47.06%) and saithe, Pollachius virens (L.), at 1.11% (90% CI of 0.49-2.19%). The 45 isolates were titrated and all but two were below the detection limit of the test (<55 PFU g(-1)). Titres of 3.8 x 10(2) PFU g(-1) and 2.8 x 10(1) PFU g(-1) were calculated from common dab, Limanda limanda (L.), and saithe, respectively. This study provides evidence that clinical outbreaks of IPN in farmed Atlantic salmon may cause a localized small increase in the prevalence of IPNV in wild marine fish.     

Health status of two salmonid aquaculture facilities in North Portugal: characterization of the bacterial and viral pathogens causing notifiable diseases

A comparative bacteriological and virological survey was conducted in two fish farms in the North of Portugal. The fish species examined included cultured rainbow trout, Oncorhynchus mykiss (Walbaum), and brown trout, Salmo trutta L., as well as wild fish captured near both facilities. The microbial load in the internal organs of apparently healthy fish was nonitored over a year, an all the disease problems occurring during this period were investigated. Although both farms presented intermediate levels of infection(30–40% infected fish), farm B showed the poorest microbiological quality since constant but low mortalities were observed throughout the year. Flavobacterium and Psedomonas-Xanthomonas were the predominant bacterial groups, comprising around 40–50% of the isolates from each farm. In farm B, members of the Enterobacteriaceae and mortile Aeromonas also showed significant prevalence (about 20%). The only outbreak of a notifiable disease was an occurrence of furunculosis, caused by Aeromonas salmonicida subsp. salmoncida, in farm A. However, Yersinia ruckeri was isolated not only from diseased fish, but also from asymptomatic fish, usually in mixed infections with motile Aeromonas or infections with motile Aeromonas or infectious pancreatic necrosis virus (IPNV). While Y. ruckeri isolates associated with mortalities belonged to the serotype O1 (subgroup a), those isolated from asymtomatic fish corresponded to serotype O3. Two strains of IPNV (serotype Ab) were isolated in farm B, which represents the first viral agent detected in Portuguese aquaculture. Qualitative and quantitative differences in microbial load were observed between cultured and wild fish. No notifiable bacterial or viral pathogens were detected in any of the feral species studied.     

传染性胰腺坏死病毒VP3蛋白的原核表达及抗原性分析

应用RT-PCR方法扩增了IPNV编码内衣壳VP3蛋白的基因615bp,将VP3基因克隆至原核表达载体pET30b,并在大肠杆菌BL21中得到了表达。通过SDS-PAGE分析表明,重组菌诱导后得到了预期大小约30ku的VP3蛋白,与理论值相符,经薄层扫描分析表明目的蛋白表达量可占菌体总蛋白的30%。用镍离子亲和层析柱纯化可溶性的VP3蛋白,并制备抗血清。Western-blotting结果显示,VP3蛋白可被兔抗IPNV阳性血清识别;间接ELISA结果显示,IPNV细胞培养物作为抗原,兔抗VP3蛋白高免血清稀释度为1∶25600时,P/N>2,抗血清可与IPNV全病毒发生反应,以上两项结果说明,表达的VP3蛋白与天然的IPNVVP3蛋白一样具有相同的抗原性。试验利用原核表达系统成功地高效表达了IPNVVP3蛋白,融合蛋白以可溶性形式存在,并制备了高效价的抗血清。     

Study of virulence in field isolates of infectious pancreatic necrosis virus obtained from the northern part of Norway

In order to study the variety of infectious pancreatic necrosis virus (IPNV) strains involved in outbreaks of infectious pancreatic necrosis (IPN) in Atlantic salmon fish farms, samples were collected from 19 different outbreaks of IPN in the northern part of Norway. The main objective of this study was to examine whether IPNV isolates of different virulence were involved in the outbreaks and could explain the variable IPN protection observed in vaccinated post‐smolts in the field. Both the molecular basis of virulence of all field isolates and virulence expressed by mortality after bath challenge of unvaccinated post‐smolts with eight of the isolates were studied. Very little variation among the field isolates was detected when the 578‐bp variable region encoding the VP2 protein known to be involved in virulence was sequenced. The cumulative mortality after experimental challenge with field isolates genetically characterized as highly virulent was always high (40–56%), while the cumulative mortality of the same strains in vaccinated post‐smolts during the field outbreaks varied from 1 to 50%. Although the tested samples came from fish vaccinated with the same vaccine product, the protection against IPN varied. These results demonstrate that differences in virulence of the isolates were not the main reason for the variation in mortality in the field outbreaks. Most of the field isolates were of high virulence, which is shown in experimental challenges to be important for mortality, but clearly other factors that might affect the susceptibility of IPN also play an important role in the outcome of an IPNV infection.     

传染性胰腺坏死病病毒分离株VP2基因抗原表位区融合表达及免疫特性的分析

利用RT-PCR方法扩增出IPNV-ZYX分离株主要结构蛋白VP2的抗原表位区基因(616 bp), 命名为IPNV VP2 COE, 将其克隆到pCold TF表达载体中构建重组质粒pCold TF-VP2 COE, 在大肠杆菌BL21(DH5α)感受态表达, 经SDS-PAGE电泳分析, 表达蛋白约78 ku, 用镍离子亲和层析柱纯化该蛋白, 制备抗血清, 间接ELISA结果显示, IPNV (ATCC VR-1318)细胞培养物与鼠抗VP2 COE蛋白血清发生特异性反应, 效价为1∶12 800; 间接免疫荧光结果显示, 鼠抗VP2 COE血清可与黑龙江某渔场已知感染IPNV虹鳟肝组织产生特异性的荧光, 以上两项结果表明, 表达IPNV VP2 COE蛋白具有良好的免疫原性和免疫反应性, 为IPNV检测方法的建立及疫苗的制备提供理论依据     

A comparative histopathological, immunohistochemical and nested-PCR study for diagnosis of infectious pancreatic necrosis in the naturally infected cultured rainbow trout (Oncorhynchus mykiss)

To confirm the diagnosis of the infectious pancreatic necrosis, the pancreas, liver, kidney and spleen specimens from 140 rainbow trout (Oncorhynchus mykiss) of weight ranges from 15 to 250?g were processed for routine histopathological and immunohistochemical (IHC) studies together with a nested-PCR using primers that amplify a 164-bp product. Of the 140 fish samples, 37 (26.4%) had a final diagnosis of IPN on the basis of hematoxylin and eosin staining (H&E), while 39/45 (86.6%) were positive by IHC method. With the nested-PCR, 59/140 (42.1%) fish samples were positive. The IHC and nested-PCR showed higher prevalence than histopathology (P?<?0.05). Based on the nested-PCR, as the fish weight increased, IPN positive results decreased (P?<?0.05). However, IHC method detected IPNV constantly in these weight ranges. Only IPNV serotype Sp was identified by nucleotide sequencing and immunohistochemistry, and antiserum to IPNV serotype Ab and IHNV showed negative results in IHC. This is the first comparative diagnostic study of IPN at different weight ranges in cultured salmonids.     

Dispersal of large farmed Atlantic salmon, Salmo salar, from simulated escapes at fish farms in Norway and Scotland

Abstract  Individually tagged farmed Atlantic salmon, Salmo salar L., were released from fish farms in simulated escapes in Scotland ( n  =   678) and Norway ( n  =   597) to compare migratory behaviour and dispersal. Large fish (510–870 mm fork length) were released to simulate the escape of aquaculture growers. Fish were released in spring and all recaptures of tagged fish were reported during summer and autumn of the year of release. Recapture rates were respectively 7.0 and 0.6% of the salmon released in Norway and Scotland, indicating a higher mortality or a lower exploitation or reporting rate for Scottish fish. Recaptures of Norwegian fish were all from Norway and mostly within 150 km of the release site; 64% were taken by anglers in fresh water. By contrast, the three salmon recaptured from the release in Scotland were reported from Norway (Hardangerfjord and Lofoten) and western Sweden (River Göta); two detached tags were found on beaches in Scotland north of the release site. These findings establish the capacity for long distance dispersal among escapees from aquaculture facilities and suggest a net easterly bias in long distance dispersal of salmon escaping from Scottish fish farms.     

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.     

Isolation of infectious pancreatic necrosis (IPN) virus from non-salmonid fish

Abstract. The isolation and characterization of infectious pancreatic necrosis (IPN) virus from a goldfish, discus fish and bream is described. The fish from which the isolates were recovered showed no pathological signs of IPN. All three virus isolates were neutralized by antiserum to IPN, strain Ab, but not by antiserum to the Sp or VR-299 strains. They were morphologically identical to IPN virus in negative stain electron microscopy, grew in the cytoplasm of BF-2 cells, as shown by immuno-fluorescence and, like IPNV, were stable to heating, lipid solvents and acid pH.     

Evaluation of a rapid coagglutination (COA) test for the detection of infectious pancreatic necrosis virus (IPNV) in tissue samples of Atlantic salmon, Salmo salar L.

The field use of a staphylococcal coagglutination (COA) test for the detection of infectious pancreatic necrosis virus (IPNV) in tissue samples from Atlantic salmon, Salmo salar L., was evaluated. The COA test was compared with an immunohistochemical (IHC) method for the detection of clinical outbreaks of infectious pancreatic necrosis (IPN). The present paper describes the evaluation of 320 COA test results performed at local fish health laboratories in Norway from 1994 to 1996, and COA test results from two infection trials with IPNV. The agreement between the COA test and the IHC was very good. The agreement beyond chance, measured as kappa values, was 0.74 in individuals and 0.90 in pooled samples. Thus, the COA test was suited for the detection of outbreaks of IPN. Covert infections with IPNV remained undetected by the COA test. The minimum IPNV titre needed to obtain a positive COA test was ≈ 10⁵ TCID₅₀ mL^–1.     

Viral haemorrhagic septicaemia virus in marine fish and its implications for fish farming--a review

Viral haemorrhagic septicaemia virus (VHSV) has, in recent decades, been isolated from an increasing number of free-living marine fish species. So far, it has been isolated from at least 48 fish species from the northern hemisphere, including North America, Asia and Europe, and fifteen different species including herring, sprat, cod, Norway pout and flatfish from northern European waters. The high number of VHSV isolations from the Baltic Sea, Kattegat, Skagerrak, the North Sea and waters around Scotland indicate that the virus is endemic in these waters. The VHSV isolates originating from wild marine fish show no to low pathogenicity to rainbow trout and Atlantic salmon, although several are pathogenic for turbot. Marine VHSV isolates are so far serologically indistinguishable from freshwater isolates. Genotyping based on VHSV G- and N-genes reveals four groups indicating the geographical origin of the isolates, with one group representing traditional European freshwater isolates and isolates of north European marine origin, a second group of marine isolates from the Baltic Sea, a third group of isolates from the North Sea, and a group representing North American isolates. Examples of possible transfer of virus from free-living marine fish to farmed fish are discussed, as are measures to prevent introduction of VHSV from the marine environment to aquaculture.     

Infectious pancreatic necrosis virus (IPNV) serotype Sp is prevalent in Turkish rainbow trout farms

Infectious pancreatic necrosis virus (IPNV) is a common pathogen of rainbow trout (Oncorhynchus mykiss) in Turkey. We found that 455 of 1,676 sample pools tested were IPNV positive. Positive samples were found in all geographical regions where sampling was conducted. Sequence and phylogenetic analyses of VP2 from 30 isolates representing all regions showed that the viruses were highly similar in sequence and grouped within Genogroup 5 (serotype Sp‐A2). No correlations between sequences, sampling sites or geographical origins were identified. Although clinical disease was evident in several farms, analyses of the amino acid sequence of VP2 showed that all virus strains harboured the P₂₁₇T₂₂₁ motif, assumed to be associated with low virulence. We conclude that IPNV is prevalent in Turkish rainbow trout farms and that the viruses are very homogenous and likely to be of European origin. Frequent exchange of eggs and live fish within the farming industry may explain the homogeneity of the IPNV.     

The prevalence of escaped farmed salmon, Salmo salar L., in the River Ewe, western Scotland, with notes on their ages, weights and spawning distribution

Abstract  The prevalence of escaped farmed Atlantic salmon, Salmo salar L., in the River Ewe, western Scotland, was assessed. After the establishment of smolt cages in the catchment and marine cages near the river mouth during 1986–1987, approximately 425 000 parr and smolts, and 122 000 growers have escaped. Between 1987 and 2001, farmed salmon occurred in the rod fishery in 13 of the 15 years, contributing at least 5.8% of the total catch, with a maximum annual frequency of 27.1%. It was estimated that <1% of fish escaping from the marine cages entered the river, but contributed at least 27% of potential anadromous spawners in 1997. Radiotagged, farmed fish in 2001 probably spawned in three subcatchments also used by tagged wild fish. Despite the likelihood of hybridisation there was no change in the median weight or marine age of wild fish, but smolt age decreased significantly ( P  < 0.02). The Ewe has a depleted wild salmon population (≤900 anadromous adults), and further genetic introgression by escapees should be prevented.     

Comparison of mortality and viral load in rainbow trout (Oncorhynchus mykiss) infected with infectious pancreatic necrosis virus (IPNV) genogroups 1 and 5

Infectious pancreatic necrosis virus (IPNV) is the aetiological agent of a highly contagious disease that affects farmed salmonids. IPNV isolates have been phylogenetically classified into eight genogroups, of which two are present in Chile, genogroups 1 and 5. Here, we compare the mortality rate caused by isolates from both genogroups in rainbow trout (Oncorhynchus mykiss) fry to determine if there is an association between host susceptibility and phylogenetic characterization of IPNV. Fish were challenged by immersion with one of four isolates (two for each genogroup), and mortality curves were assessed after 30 days. Viral load was measured in all mortalities and in live fish sampled at 1, 7 and 20 days post-infection. Although mortality was low throughout the challenge, differences were found between fish infected with different isolates. Both isolates from genogroup 1 caused greater cumulative mortalities than either of the isolates from genogroup 5. When combined, the overall mortality rate of fish challenged with genogroup 1 isolates was significantly higher than those infected with genogroup 5. However, viral load was lower on trout infected with genogroup 1 isolates. These results suggest that rainbow trout are more susceptible to IPNV isolates from genogroup 1 than genogroup 5.     

Provisional epidemiological cutoff values for standard broth microdilution susceptibility testing of Flavobacterium columnare

The gliding aquatic bacterium Flavobacterium columnare causes columnaris disease, a common problem for wild and farmed freshwater fish worldwide. Recently, a broth microdilution method was standardized to test the susceptibility of F. columnare against antimicrobials commonly used in aquaculture. We used this new method to measure the minimal inhibitory concentrations (MICs) of ten antimicrobials against 120 F. columnare isolates. The resulting MIC frequency distributions for each antimicrobial (1 MIC/isolate) were used to estimate epidemiological cut‐off values (ECVs) which separate isolates with typical wild‐type (WT) susceptibility from isolates with decreased non‐wild‐type (NWT) susceptibility. We identified 22 NWT isolates with elevated MICs relative to the ECV that covered 99.9% of the MIC distribution against one or more of the antimicrobials: ampicillin, enrofloxacin, erythromycin, florfenicol, flumequine, oxolinic acid or oxytetracycline. Ten of the NWT isolates had decreased susceptibility to a single antimicrobial class, six isolates to two antimicrobial classes and six isolates to three or more antimicrobial classes. The MIC frequency distributions and provisional cut‐off values provide data needed to set epidemiological cut‐off values to monitor for the development of antimicrobial resistance among F. columnare.     

An understated danger: Antimicrobial resistance in aquaculture and pet fish in Switzerland,a retrospective study from 2000 to 2017

Aquaculture is a rapidly growing field of food production. However, morbidity and mortality are higher in aquaculture species than in domestic animals. Bacterial diseases are a leading cause of farmed fish morbidity and are often treated with antimicrobials. Since most Swiss fish farms release effluents directly into surface water without treatment and since aquaculture fish are consumed by humans, antimicrobial resistance (AMR) and multi-resistance in aquaculture fish are important for environmental and public health. In this study, AMR tests for 14 antimicrobials were performed on 1,448 isolates from 1,134 diagnostic laboratory submissions from farmed and ornamental fish submissions for the period from 2000 to 2017. Amoxicillin, gentamycin and norfloxacin had the lowest proportion of resistant samples. However, AMR was highly variable over time. Resistance proportions were higher in: (a) ornamental fish compared with farmed fish, (b) fish from recirculation systems compared with those from other farming systems and (c) isolates originating from skin compared with those originating from inner organs. Multiple resistances were common. The results of this study provide useful data for Swiss fish veterinarians and some interesting hypotheses about risk factors for AMR in aquaculture and pet fish in Switzerland. However, further research is needed to define risk factors.     

Antigenic analysis of reference strains and Norwegian field strains of aquatic birnaviruses by the use of six monoclonal antibodies produced against the infectious pancreatic necrosis virus N1 strain

Abstract. Six monoclonal antibodies (MAbs) produced against the infectious pancreatic necrosis virus (IPNV) N1 strain were used in a dot-blot assay to examine reference strains of the nine proposed serotypes and a representative selection of 81 Norwegian aquatic birnavirus isolates. These isolates had earlier been serotyped by use of a panel of 11 MAbs produced against other strains of IPNV. Correlations between the reaction patterns of the two panels of MAbs were found. All reference strains and field isolates shared two epitopes, one on VP2 and one on VP3. Seventy-seven of the field isolates reacted identically with the N1 strain (positive with all six MAbs). The Sp type strain was positive with five of the MAbs and was different from all the field strains. The other reference strains (WB, VR299, Ab, Ja, Te, He, C1, C2 and C3) were positive with two to four of the MAbs. Together with previously published data, these findings indicate that most Norwegian isolates are closely related to, or identical with, the N1 strain and belong to the Sp serotype. No correlation between the health status of Atlantic salmon and antigenicity of the isolates was found. Testing of the reference strains in ELISA revealed some discrepancies with the dot-blot results.