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 A systematic review of published and unpublished data on six key pathogens (Renibacterium salmoninarum, Aeromonas salmonicida, IPNV, ISAV, SAV and VHSV) found in Scottish wild and farmed fish was undertaken.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

Transmission of some fish pathogenic viruses by the heron, Ardea cinerea

Abstract. Various possibilities of transmitting fish pathogenic VHS, SVC and IPN viruses by the heron, Ardea cinerea , were investigated. Shedding of IPN virus in the faeces could be demonstrated for a period of 7 days when about 1 g of IPN virus-infected trout fry was fed to the birds once only, whereas feeding larger quantities of infected fish on 5 consecutive days resulted in virus excretion for a further 5 days only. Infectious IPN virus could not be isolated from samples of blood and virus specific neutralizing antibodies could not be demonstrated in serum samples. A waterborne infection of trout fry was established by adding very small (0·2 g) quantities of faeces from herons fed IPN virus-contaminated fish to 20 1 water. Infectious SVC and VHS virus was also re-isolated from samples of food which were regurgitated by the herons at different times up to 120 min after feeding of contaminated fish. It was concluded that herons are able to act as mechanical vectors for IPN, VHS and SVC viruses and therefore, may be a potential source of infection and spread of the diseases.     

Improvement of a diagnostic procedure in surveillance of the listed fish diseases IHN and VHS

Infectious haematopoietic necrosis (IHN) and viral haemorrhagic septicaemia (VHS) are OIE‐listed and notifiable viral fish diseases which are controlled by eradication and surveillance programmes globally. The present study provides improved RT‐qPCR procedures based on recently described OIE protocols. Improvements comprise the design of a new TaqMan® probe, replacing a TaqMan® MGB probe that turned out to show impaired binding. Reason for this is SNPs detected in the nucleoprotein N gene sequences of IHNV strains targeted by the RT‐qPCR. Furthermore, the IHNV and VHSV RT‐qPCR assays were realized as one‐step and one‐run procedures supplemented by an endogenous control system. The IHNV and VHSV RT‐qPCR assays are characterized by a technical sensitivity of 19 and 190 gene equivalents (cRNA) and an analytical sensitivity of 2–7 and 13 TCID₅₀/ml, respectively. For verification purposes, 105 IHNV and 165 VHSV isolates and several non‐targeted viral and bacterial pathogens were included and returned adequate results. However, in field samples divergent results left 14 samples of 154 undetected for IHNV and one sample of 127 for VHSV using cell culture. The study shows that RT‐qPCR assays ensure facilitated and reliable testing on IHNV and VHSV in eradication and surveillance programmes.     

Comparative stability of two salmonid viruses and poliovirus in fresh, estuarine and marine waters

Abstract. The inactivation rates in the aquatic environment of two fish pathogens, infectious pancieatic necrosis virus (IPNV) and infectious haematopoietic necrosis virus (IHNV), were compared with that of poliovirus type 1 as a representative of the human enterovirus group. The survival studies were performed using untreated fresh, estuarine and sea water samples held at 15 and 20°C. The results indicated that the salmonid viruses survived longer than poliovirus in the saline waters, whereas in fresh water poliovirus was the most stable of the three viruses. IPN virus proved to be most stable in estuarine water at 15°C, whereas the survival of IHN virus was favoured in fresh water. We also observed that at 20°C the inactivation rate for each virus was independent of salt concentration in estuarine and sea water. Although temperature exhibited a marked effect on virus stability in fresh water, the salmonid viruses presented similar survival patterns at both temperatures in sea water. In general the period of greatest viral inactivation correlated with higher bacterial numbers in the waters.     

The first report of viral haemorrhagic septicaemia in farmed rainbow trout, Oncorhynchus mykiss (Walbaum), in the United Kingdom

Viral haemorrhagic septicaemia (VHS) was diagnosed in rainbow trout in the UK in May 2006. VHS virus (VHSV) was isolated from fingerlings showing typical histopathological lesions at a single rainbow trout farm site experiencing high mortality. The virus was confirmed as VHSV by serological and molecular biological tests. Phylogenetic analysis based on the complete glycoprotein gene sequence revealed that the isolate was closely related (99% nucleotide identity) to several Danish isolates from 1991 to 2000 and was assigned to VHSV genogroup Ia. The pathogenicity of the isolate was determined in infection experiments using rainbow trout fry. Following waterborne challenge, cumulative mortalities reached 96.67-100% by 12 days post-infection. This represents the first isolation of a pathogenic freshwater VHSV in the UK.     

Salmonid fish viruses and cell interactions at early steps of the infective cycle

A flow cytometric virus-binding assay that directly visualizes the binding and entry of infectious pancreatic necrosis virus (IPNV), infectious haematopoietic necrosis virus (IHNV) and virus haemorrhagic septicaemia virus (VHSV) to several cell lines was established. The highest efficiency of binding was shown by the BF-2 cell line and this was used to study, at the attachment level, the interactions of these cells with salmonid fish viruses in coinfections, and to further determine if the earliest stage of the viral growth cycle could explain the previously described loss of infectivity of IHNV when IPNV is present. Our results demonstrated that IPNV binds to around 88% of cells either in single or dual infections, whereas IHNV attachment always decreased in the presence of any of the other viruses. VHSV binding was not affected by IPNV, but coinfection with IHNV reduced the percentage of virus-binding cells, which suggests competition for viral receptors or co-receptors. Internalization of the adsorbed IHNV was not decreased by coinfection with IPNV, so the hypothetical competence could be restricted to the binding step. Treatment of the cells with antiviral agents, such as amantadine or chloroquine, did not affect the binding of IPNV and VHSV, but reduced IHNV binding by more than 30%. Tributylamine affected viral binding of the three viruses to different degrees and inhibited IPNV or IHNV entry in a large percentage of cells treated for 30 min. Tributylamine also inhibited IHNV cytopathic effects in a dose-dependent manner, decreasing the virus yield by 4 log of the 50% endpoint titre, at 10 mm concentration. IPNV was also inhibited, but at a lower level. The results of this study support the hypothesis that IHNV, in contrast to VHSV or IPNV, is less efficient at completing its growth cycle in cells with a simultaneous infection with IPNV. It can be affected at several stages of viral infection and is more sensitive to the action of antiviral compounds.     

A novel multiplex RT‐qPCR method based on dual‐labelled probes suitable for typing all known genotypes of viral haemorrhagic septicaemia virus

Viral haemorrhagic septicaemia (VHS) is a notifiable fish disease, whose causative agent is a rhabdovirus isolated from a wide range of fish species, not only in fresh but also in marine and brackish waters. Phylogenetic studies have identified four major genotypes, with a strong geographical relationship. In this study, we have designed and validated a new procedure – named binary multiplex RT‐qPCR (bmRT‐qPCR) – for simultaneous detection and typing of all four genotypes of VHSV by real‐time RT‐PCR based on dual‐labelled probes and composed by two multiplex systems designed for European and American/Asiatic isolates, respectively, using a combination of three different fluorophores. The specificity of the procedure was assessed by including a panel of 81 VHSV isolates covering all known genotypes and subtypes of the virus, and tissue material from experimentally infected rainbow trout, resulting in a correct detection and typing of all strains. The analytical sensitivity was evaluated in a comparative assay with titration in cell culture, observing that both methods provided similar limits of detection. The proposed method can be a powerful tool for epidemiological analysis of VHSV by genotyping unknown samples within a few hours.     

A sandwich ELISA to detect VHSV and IPNV in turbot

The recent demonstration that reared turbot (Scophthalmus maximus L) is a natural host for salmonid rhabdoviruses has made their rapid detection relevant to these fish species. A unique protocol to select and use non-competitive monoclonal antibodies (Mabs) for two high-sensitivity sandwich ELISAs has been developed to detect both infectious pancreatic necrosis virus (IPNV) and viral haemorrhagic septicaemia virus (VHSV) in turbot kidney extracts to assess the possibility of using them in field diagnosis. For maximum sensitivity, turbot kidney extracts can be two-fold diluted with high-ionic strength buffers and assayed for the presence of the major viral proteins (VMS rhabdovirus nucleoprotein N/Nx and/or IPN birnavirus protein VP3). The use of control plates coated with irrelevant mouse antibodies (IgG1 and IgG2a) in parallel ELISAs allows for a precise estimation of possible false positives. Turbot kidney extracts with low levels of virus might now be assayed directly without using cell culture, with high precision and in a short time during the acute phase of these viral diseases in reared turbot.     

Investigation of round goby viral haemorrhagic septicaemia outbreak in New York

Eleven viral haemorrhagic septicaemia virus (VHSV) genotype IVb isolates were sequenced, and their genetic variation explored to determine the source of a VHS outbreak on the eastern shore of Cayuga Lake. An active fish kill of round gobies (Neogobius melanostomus, Pallas) was intensively sampled at King Ferry, NY and nearby Long Point State Park in May 2017. Gross lesions observed on 67 moribund round gobies and two rock bass (Ambloplites rupestris, Rafinesque) included moderately haemorrhagic internal organs and erythematous areas on the head, flank, and fins. RT‐qPCR tests for VHSV were positive for all 69 fish. Viral isolation on epithelioma papulosum cyprinid cells showed cytopathic effect characteristic of VHSV for six round goby samples from King Ferry. The complete nucleotide sequence of the VHSV IVb genomes of five Cayuga Lake round goby isolates were derived on an Illumina platform along with 2017 VHSV IVb isolates from round gobies collected from the following: Lake Erie near Dunkirk, NY; the St. Lawrence River near Clayton and Cape Vincent, NY; and Lake St. Lawrence near Massena, NY. The phylogenetic tree created from these aligned sequences and four other complete VHSV IVb genomes shows Cayuga Lake isolates are closely related to the Lake Erie isolates.     

Comparative susceptibility among three stocks of yellow perch,Perca flavescens (Mitchill), to viral haemorrhagic septicaemia virus strain IVb from the Great Lakes

The Great Lakes strain of viral haemorrhagic septicaemia virus IVb (VHSV‐IVb) is capable of infecting a wide number of naive species and has been associated with large fish kills in the Midwestern United States since its discovery in 2005. The yellow perch, Perca flavescens (Mitchill), a freshwater species commonly found throughout inland waters of the United States and prized for its high value in sport and commercial fisheries, is a species documented in several fish kills affiliated with VHS. In the present study, differences in survival after infection with VHSV IVb were observed among juvenile fish from three yellow perch broodstocks that were originally derived from distinct wild populations, suggesting innate differences in susceptibility due to genetic variance. While all three stocks were susceptible upon waterborne exposure to VHS virus infection, fish derived from the Midwest (Lake Winnebago, WI) showed significantly lower cumulative % survival compared with two perch stocks derived from the East Coast (Perquimans River, NC and Choptank River, MD) of the United States. However, despite differences in apparent susceptibility, clinical signs did not vary between stocks and included moderate‐to‐severe haemorrhages at the pelvic and pectoral fin bases and exophthalmia. After the 28‐day challenge was complete, VHS virus was analysed in subsets of whole fish that had either survived or succumbed to the infection using both plaque assay and quantitative PCR methodologies. A direct correlation was identified between the two methods, suggesting the potential for both methods to be used to detect virus in a research setting.     

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.     

Strong genetic influence on IPN vaccination-and-challenge trials in Atlantic salmon, Salmo salar L

Two series of experimental challenge trials were performed for evaluation of multivalent oil‐adjuvanted vaccines with and without an infectious pancreatic necrosis virus (IPNV) antigen component. In both the trial series, Atlantic salmon were hatched, reared, vaccinated and subjected to temperature and light manipulation to induce smoltification. When ready for sea the fish were transported to the VESO Vikan experimental laboratory for bath or cohabitant challenge with IPNV. In the first series, four vaccination and bath challenge trials involving 2‐year classes of experimental fish were conducted. In the second series, three groups of eyed eggs of Atlantic salmon allegedly differing in their innate resistance to IPNV were used (Storset, Strand, Wetten, Kjøglum & Ramstad 2007). Hatching, rearing and smoltification were synchronized for each group, and fish from each genetic group were randomly allocated IPN vaccine, reference vaccine or saline before being placed into parallel tanks for bath or cohabitant challenge. In the first series of trials, IPN‐specific mortality commenced on day 10–12 after bath challenge. Replicates showed similar results. In trials 1 and 2 belonging to the same experimental fish year class, the average cumulative control mortality reached 60.6% and 79.5%, respectively, whereas in trials 3 and 4 belonging to the following year class the control mortality was consistently below 50%. In the second series of trials, the experimental fish originating from allegedly IPN susceptible parents consistently showed the highest cumulative mortality among the unvaccinated controls (>75%) whereas smolts derived from allegedly IPNV resistant parents showed only 26–35% control mortality. The IPN‐vaccinated fish experienced significantly improved survival vs. the fish immunized with reference vaccine, with RPS values above 75% in the IPN susceptible strain. In the IPN resistant strain, the protection outcomes were variable and in part non‐significant. The outcome of both the trial series suggests that control mortalities above 50% are necessary to reliably demonstrate specific protection with IPN vaccines.     

同步检测7种鱼类病毒的扩增子拯救多重PCR(Arm-PCR)方法的建立和应用

淋巴囊肿病毒(LCDV)、肿大细胞病毒属虹彩病毒(Mega)、赤点石斑鱼神经坏死病毒(RGNNV)、传染性造血器官坏死病毒(IHNV)、传染性胰脏坏死病毒(IPNV)、病毒性出血败血症病毒(VHSV)和传染性鲑鱼贫血症病毒(ISAV)是养殖鱼类主要的病毒性病原,危害巨大。为实现这7种病原的高通量、同步检测,本研究在分析这7种病毒基因序列的基础上,设计了9组扩增子拯救多重PCR(Arm-PCR)引物,并对扩增体系中的Taq酶、Mg2+、dNTP、Primer Mix浓度及退火温度等参数进行调整和优化,结合基因芯片检测技术,建立了同步检测7种鱼类病毒的Arm-PCR方法。优化后的Arm-PCR方法第一步PCR体系为：Taq酶(2.5 U/μl)1.0μl,10×PCR Buffer(含20 mmol/L的Mg2+)5μl,dNTP(各2.5 mmol/L)5μl,10×Primer Mix(各2μmol/L)9μl,模板1μl,ddH2O补足至50μl,退火温度为56℃。研究结果显示,该方法可以在1支反应管内对上述7种病毒的9个致病基因同步进行扩增和检测,检测灵敏度分别为101 copies/μl (RGNNV、VHSV、ISAV-NS、ISAV-MA)、102 copies/μl (LCDV、Mega、IHNV、IPNV)和103 copies/μl (大菱鲆红体病虹彩病毒,TRBIV)。该方法特异性强,与半滑舌鳎、石斑鱼、大菱鲆和牙鲆基因组DNA不产生交叉反应。本研究建立的可同步检测7种鱼类病毒的Arm-PCR方法具有高通量、高灵敏度、高准确性的优势,能有效提高工作效率,在鱼类病毒的筛查和流行病学调查领域有广泛的应用前景。     

Study of the interaction between a persistent infectious pancreatic necrosis virus (IPNV) infection and experimental infectious salmon anaemia (ISA) in Atlantic salmon, Salmo salar L.

Abstract. An infectious pancreatic necrosis virus (IPNV) carrier stock of Atlantic salmon parr (100 g) was divided between two tanks and inoculated experimentally with tissue homogenate containing the aetiologic agent of infectious salmon anaemia (ISA) and non-ISA tissue homogenate (control), respectively. Plasma and kidney samples from ISA-infected and control fish were taken twice weekly for 25 days. In the kidney samples, IPNV was quantified by a plaque assay. In plasma, anti-IPNV antibodies were measured using an indirect ELISA. The ISA-infection did not seem to activate the IPNV-infection. Neither the proportion of fish with IPNV or anti-IPNV antibodies, nor the IPNV titre or level of anti-IPNV antibodies showed any specific trend during the study. Independently of ISA, IPNV was detected in 54 out of 132 fish (41%), while 71 out of 195 fish (36%) had plasma antibodies against IPNV. No association was found between detection of IPNV, and presence or level of anti-IPNV antibodies in individual fish.     

Larval Pacific herring, Clupea pallasii (Valenciennes), are highly susceptible to viral haemorrhagic septicaemia and survivors are partially protected after their metamorphosis to juveniles

Pacific herring were susceptible to waterborne challenge with viral haemorrhagic septicaemia virus (VHSV) throughout their early life history stages, with significantly greater cumulative mortalities occurring among VHSV‐exposed groups of 9‐, 44‐, 54‐ and 76‐day‐old larvae than among respective control groups. Similarly, among 89‐day–1‐year‐old and 1+year old post‐metamorphosed juveniles, cumulative mortality was significantly greater in VHSV‐challenged groups than in respective control groups. Larval exposure to VHSV conferred partial protection to the survivors after their metamorphosis to juveniles as shown by significantly less cumulative mortalities among juvenile groups that survived a VHS epidemic as larvae than among groups that were previously naïve to VHSV. Magnitude of the protection, measured as relative per cent survival, was a direct function of larval age at first exposure and was probably a reflection of gradual developmental onset of immunocompetence. These results indicate the potential for easily overlooked VHS epizootics among wild larvae in regions where the virus is endemic and emphasize the importance of early life history stages of marine fish in influencing the ecological disease processes.     

Occurrence of viral haemorrhagic septicaemia in rainbow trout Salmo gairdneri Richardson reared in sea-water

Abstract. Viral haemorrhagic septicaemia (VHS) is reported for the first time in sea-water cultured rainbow trout. Heavy mortalities with typical signs and lesions A VHS virus (serotype 1) was isolated from the diseased fish. The mortalities were caused only by the VHS virus and 80 days post transfer of trout to sea-water the mortalities reached 85%, of the initial population.
The disease was experimentally transmitted to rainbow trout, both in sea-water 3·10⁴ pfu/ml of virus or by intramuscular injection of various doses of VHS₁ (7·10¹ 7·10⁴ or 7·10⁴ pfu per fish). Death occurred in all infected groups and started earlier in sea-water. Typical signs of VHS were observed in moribund fish. Viral multiplication was demonstrated to have occurred in fish organs.     

Replication of infectious pancreatic necrosis virus in trout leucocytes and detection of the carrier state

Abstract. The exact cellular site of replication of infectious pancreatic necrosis virus (IPNV) in carrier fish is unknown. In order to determine if IPNV replicates in trout leucocytes, we purified leucocytes from normal (non-carrier) trout and separated the cells into an adherent and a non-adherent population. IPNV replicated in less than 0-01 % of the adherent leucocytes with a yield of about 400 p.f.u./cell. IPNV also became associated with less than 0.07% of the non-adherent leucocytes; either IPNV did not replicate in these cells or the yield was, at best, only a few p.f.u./cell. Trout persistently infected with IPNV (carrier fish) were tested for the presence of IPNV in leucocytes by co-cultivating with a sensitive fish cell line; this same population of trout was also tested for IPNV by organ sampling using standard methods. Ninety-eight per cent of the trout were positive for IPNV by organ sampling, but only 75 % yielded IPNV from leucocytes. Thus a blood sample from a living fish can be used to detect the presence of IPNV.