A new viral epizootic of Anguilla japonica Temminck and Schlegel

Abstract. In 1969, a new kind of epizootic occurred among eels in Japan and virological investigation was initiated. A new virus designated eel virus european (EVE) was isolated and biological, cytological, serological tests and infectivity trials were carried out. In its CPE on RTG-2 cells and additionally in its biological properties and particle size, EVE was found to be similar to infectious pancreatic necrosis virus (IPNV). Serologically, EVE was similar to the ď Honnincthun, France, strain of IPNV. However, infectivity trials showed that EVE and IPNV differed; EVE killed Japanese eels Anguilla japonica but not rainbow trout fry Salmo gairdneri , while IPN virus killed rainbow trout fry but not eels. We consider EVE to be the primary agent causing the new epizootic and propose the name viral kidney disease for the resulting clinical condition.     

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.     

Comparative studies of Danish Flavobacterium psychrophilum isolates: ribotypes, plasmid profiles, serotypes and virulence

Ribotyping and plasmid profiling were carried out on 299 Danish Flavobacterium psychrophilum isolates from farmed rainbow trout, Oncorhynchus mykiss (Walbaum). The isolates had been characterized biochemically and serologically in another study. The isolates were very homogeneous, 254 isolates had the same ribotype A (restriction enzyme Eco RI) and 284 isolates harboured one 3.3 kb plasmid. Seventy-five per cent of the F. psychrophilum isolates had ribotype A, one 3.3 kb plasmid and belonged to either serotype Th or Fd. Virulence studies with representatives of the dominant groups classified such isolates as virulent, and an extra small or large plasmid did not change the virulence level. A relationship between the serotypes Fd and Th, certain ribotypes, and virulence was found. The isolates belonging to serotype FpT and to ribotype B were less virulent. Only a few isolates with other ribotypes were found and these were also less virulent. Isolates that did not have the 3.3 kb plasmid were also less virulent. The virulent isolates all harboured the 3.3 kb plasmid; however, there was no clear correlation between virulence and plasmid content as a 3.3 kb plasmid was also found in less virulent isolates.     

In vitro markers for virulence in Yersinia ruckeri

In this study, different traits that have been associated with bacterial virulence were studied in Yersinia ruckeri. Two isolates that had been shown to cause disease and mortality in experimentally infected rainbow trout were compared with five avirulent isolates. Both virulent isolates showed high adhesion to gill and intestinal mucus of rainbow trout, whereas the majority of non‐virulent strains demonstrated significantly lower adhesion. A decrease in adherence capability following bacterial treatment with sodium metaperiodate and proteolytic enzymes suggested the involvement of carbohydrates and proteins. All strains were able to adhere to and invade chinook salmon embryo cell line (CHSE‐214), fathead minnow epithelial cell line (FHM) and rainbow trout liver cell line (R1). One non‐virulent strain was highly adhesive and invasive in the three cell lines, whereas the virulent strains showed moderate adhesive and invasive capacity. The internalization of several isolates was inhibited by colchicine and cytochalasin‐D, suggesting that microtubules and microfilaments play a role. For all strains, intracellular survival assays showed a decrease of viable bacteria in the cells 6 h after inoculation, suggesting that Y. ruckeri is not able to multiply or survive inside cultured cells. Analysis of the susceptibility to the bactericidal effect of rainbow trout serum demonstrated that virulent Y. ruckeri strains were serum resistant, whereas non‐virulent strains were generally serum sensitive.     

Tissue distribution of infectious pancreatic necrosis virus serotype Sp in naturally infected cultured rainbow trout,Oncorhynchus mykiss (Walbaum): an immunohistochemical and nested‐PCR study

This study investigates the occurrence and distribution pattern of infectious pancreatic necrosis virus (IPNV) within the pancreas, liver, kidney and spleen of naturally infected cultured rainbow trout, Oncorhynchus mykiss (Walbaum), using immunohistochemistry (IHC). A nested PCR was also employed to confirm the presence of the virus in the pooled tissues of the specimens. All the examined tissues except spleen were immunohistochemically positive for IPNV, but staining intensity and distribution pattern varied. The kidney tubules had the most intense and widespread staining by IHC, indicating a specific tissue tropism at least for this particular serotype. The nucleotide sequence had the greatest identity with the Sp serotype confirming the presence of the nucleic acid of IPNV in the pooled tissues. Based on the present findings, it could be concluded that the absence of lesions consistent with infectious pancreatic necrosis (IPN) disease in the H&E‐stained sections cannot rule out the presence of the IPNV, and the use of an alternative rapid confirmatory method such as IHC with formalin‐fixed, paraffin‐embedded tissue sections is helpful for the final diagnosis of IPN in rainbow trout.     

Serotyping of Vibrio anguillarum isolated from diseased freshwater fish in Japan

Abstract. During the period from 1965 to 1980, 263 Vibrio anguillarum strains from ayu, Plecoglossiis altivelis (Temminck & Schlegel), two from rainbow trout. Salmo gairdneri Richardson, and two from eel, Anguilla japonica (Temminck & Schlegel), were collected from fish suffering from vibriosis in various parts of Japan. On the basis of cross-agglutination and cross-absorption tests with thermostable (O) antigens, six distinct serotypes (A, B, C, D, E and F) were established among 12 selected strains of V. anguillarum . 241 strains isolated from ayu and two strains from rainbow trout belonged to serotype A, six strains from ayu and one strain from eel to serotype B, 12 strains from ayu to serotype C, three strains from ayu to serotype D, one strain from ayu to serotype E, and one strain from eel to serotype F. V. anguillarum strains belonging to serotypes D, E and F have not been detected from ayu, rainbow trout and eel since 1973; these serotypes appear to be minor types. V. anguillarum strain NCMB 6 and 1669 belong to our serotype A and V. anguillarum 813 to our serotype C.     

Passive immunization of rainbow trout,Oncorhynchus mykiss (Walbaum), against Flavobacterium psychrophilum,the causative agent of bacterial coldwater disease and rainbow trout fry syndrome

Flavobacterium psychrophilum, the causative agent of bacterial coldwater disease (CWD) and rainbow trout fry syndrome (RTFS), causes high mortality in cultured salmonids. The present study was designed to determine the role antibody plays in conferring protection to rainbow trout fry, Oncorhynchus mykiss (Walbaum), by passive immunization with convalescent serum or serum from adult rainbow trout immunized with F. psychrophilum, and goat anti-F. psychrophilum serum. In each experiment, rainbow trout fry were injected intraperitoneally with antiserum and challenged by subcutaneous injection with a virulent strain (CSF-259-93) of F. psychrophilum 24-h post-immunization. Relative percentage survival (RPS) ranged from 9-42% when rainbow trout fry (mean weight 1.3 g) were injected with a 1:2 dilution of 25 microL of convalescent serum ranging in enzyme-linked immunosorbent assay antibody titres from 1600-102400. Rainbow trout fry (mean weight 1.0 g) passively immunized with 25 microL of serum from immunized adult fish exhibited RPS values of up to 57%. In each of these experiments, RPS increased with increasing antibody titres against F. psychrophilum. Passive immunization with 25 or 50 microL goat anti-F. psychrophilum serum, however, did not confer protection to fry (mean weight 1.3 g). These results suggest that trout antibody plays a role in conferring protection to F. psychrophilum, but antibody alone is unable to provide complete protection.     

Virulence of Flavobacterium branchiophilum in experimentally infected salmonids

Abstract. The ability of selected strains of Flavobacterium branchiophilum to attach to and colonize the gills of five species of teleosts (four salmonid and one cyprinid) and cause mortality was investigated. In virulence studies with F. branchiophilum strain LAB4a, cumulative mortality was dose-dependent in rainbow trout, Oncorhynchus mykiss (Walbaum), and ranged from 0 to 75%. However, regardless of dose, the relative amount of gill-associated F. branchiophilum antigen increased 1 h after challenge. The gill-associated F. branchiophilum antigen in fish which survived (moribund fish) increased by four to six times compared to that detected on the gills 1 h after challenge. The gill-associated antigen on moribund fish was not significantly different from that on the gills of dead fish. Flavobacterium branchiophilum strain LAB4a also attached to the gills of brook trout, Salvelinus fontinalis (Mitchell), rainbow trout, chinook salmon, Oncorhynchus tshawytscha (Walbaum), Arctic charr, Salvelinus alpinus (L.), and common shiners, Luxilus cornutus (Mitchell), following a 1-h bath exposure, and caused mortality in all species. The virulence of eight strains of F. branchiophilum for rainbow trout was examined. Both virulent and avirulent strains adhered to the gills following bath exposure (fimbriae were observed on all strains), but only virulent strains had the capacity to further colonize the gills and cause mortality.     

Demonstration of infectious pancreatic necrosis virus strain VR-299 in Japanese eel, Anguilla japonica Temminck & Schlegel

Abstract. Six isolates of infectious pancreatic necrosis virus (IPNV) were obtained from trout or eel farms in Taiwan in 1987. By using the electrophoretic analysis of ³⁵S-methionine-labelled early viral polypeptide patterns and silver-stained ds RNA gel patterns, a comparison of these isolates with the three archetypal IPNV serotypes (AB, SP and VR-299) was made. They were all identical with VR-299, both in the RNA and early viral polypeptide patterns. From the results obtained, it was apparent that the VR-299 serotype of IPNV was just as widespread in eel as in trout in Taiwan. This is the first case of VR-299 isolation from eel.     

Infectious pancreatic necrosis virus isolated from farmed rainbow trout and tilapia in Kenya is identical to European isolates

Infectious pancreatic necrosis virus (IPNV) is an aquabirnavirus that causes serious diseases in a variety of fish species worldwide. It has been isolated from a large number of healthy fresh and marine water fish. Prior to this study, there was no record of the presence of IPNV infection in Kenya. Here, the presence of IPNV in farmed rainbow trout and tilapia was examined in Nyeri County of central Kenya. Head kidney samples taken from five rainbow trout and three tilapia farms and stored in RNALater^® were processed by PCR followed by sequencing of a segment A fragment covering nucleotide positions 2,120–2,343 bp. IPNV was detected in all the farms sampled with infection ratios ranging from 0.3 to 0.78 although the infections were not associated with any specific clinical signs of disease. These findings were supported by immunohistochemistry staining of the virus in the kidney and exocrine pancreas of rainbow trout. Sequence alignment and phylogenetic analysis revealed that the Kenyan isolates were identical to European isolates, suggesting a common origin. These findings highlight the need for better biosecurity procedures with more stringent surveillance programmes and control for fish diseases, especially focusing on imported breeding materials to Kenya.     

Bacterial pathogens in rainbow trout, Oncorhynchus mykiss (Walbaum), reared at Danish freshwater farms

During a 2-year period, bacterial fish pathogens were monitored on five rainbow trout, Oncorhynchus mykiss (Walbaum), freshwater farms in Denmark. A total of 1206 fish were examined and 361 bacterial isolates were identified phenotypically. Enteric redmouth disease, furunculosis and rainbow trout fry syndrome/coldwater disease were recorded. Infections caused by Flavobacterium psychrophilum occurred most frequently, but only one outbreak of enteric redmouth disease caused by Yersinia ruckeri serotype O1 and one of furunculosis caused by Aeromonas salmonicida were recorded during the monitoring period. Flavobacterium psychrophilum was isolated on all farms, both during disease outbreaks and from fish without any signs of disease. Serological investigations of F. psychrophilum showed that serotype Th was the dominant serotype found. The serotypes Th and Fd were involved in disease outbreaks of fry and larger fish. All isolates of F. psychrophilum showed proteolytic activities; however, a few isolates, belonging to serotype FpT did not degrade elastin and were not associated with mortality. Increasing resistance problems to oxytetracycline were demonstrated. More than half of the F. psychrophilum isolates showed resistance to oxolinic acid and oxytetracycline. No antibiotic resistant isolates were found among Y. ruckeri and A. salmonicida .     

Preliminary report on hybridization experiments in trout — Growth and survival of F1 hybrids

Hybridization experiments were carried out by crossing females of common rainbow trout with the males of albino rainbow trout. Survival rates in egg, hatchling, fry and yearling stages, and the annual growth rate were higher than those of either parent.Reciprocal crosses with females of albino rainbow trout and males of common rainbow trout showed poor survival rate. Gonadial development of these hybrids was normal.     

Occurrence and prevalence of infectious pancreatic necrosis virus in rainbow trout (Oncorhynchus mykiss) cultured in cages in the Black Sea

Rainbow trout (Oncorhynchus mykiss) cultured in cage systems in the South Eastern Black Sea were surveyed for the type, occurrence and prevalence of infectious pancreatic necrosis virus (IPNV). Two nearby farms (designated as Farm A and Farm B) were visited monthly in 2007 and 2008. At each farm, 385 fish were selected randomly from five cages. Another farm with infected trout from a hatchery also was monitored for IPNV from the transfer to harvest. IPNV was found to be prevalent in both farms surveyed. In Farm A, IPNV was present throughout the growing period, from January to May, and all five randomly sampled cages tested positive for IPNV in March and April of 2007. In Farm B, IPNV was present only in February and March in 2007, and in 2008, IPNV was observed in January (two cages) and February (one cages) at low levels. Interestingly, IPNV was absent 2 weeks after transfer to the sea at 17.5°C. The same strain of IPNV, genotype III that was isolated from the same stock of fish at the hatchery, reoccurred when water temperatures dropped to 12°C in December in the Black Sea. Transferring fish to the sea at high water temperatures could lessen the negative impacts of IPNV on growth of rainbow trout in brackish water.     

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.     

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.     

Comparative in vitro studies on virulent and avirulent strains of Cryptobia salmositica (Sarcomastigophora: Kinetoplastida)

Abstract. The optimum temperature for in vitro multiplication of Cryptobia salmositica was 10°C. The avirulent strain multiplied more rapidly than the virulent strain. The haemolytic components, lytic component (LC) and immune complex-forming component (ICC), were secreted by the two strains into the culture medium and were detectable from one week post-inoculation. The haemolytic activity in the supernatant increased with increasing parasite numbers in both strains. Although cultures of the avirulent strain had higher parasite numbers than those of the virulent strain, the haemolytic activity was significantly lower than that of the virulent strain. Antiserum against ICC was produced in rabbit by immunization with ICC-coated rainbow trout red blood cells.     

Immunity induced by recombinant attenuated IHNV (infectious hematopoietic necrosis virus)‐GN438A expresses VP2 gene‐encoded IPNV (infectious pancreatic necrosis virus) against both pathogens in rainbow trout

Infectious hematopoietic necrosis virus (IHNV) and infectious pancreatic necrosis virus (IPNV) are important pathogens in rainbow trout farming worldwide. Their co‐infection is also common, which causes great economic loss in juvenile salmon species. Development of a universal virus vaccine providing broadly cross‐protective immunity will be of great importance. In this study, we generated two recombinant (r) virus (rIHNV‐N438A‐ΔNV‐EGFP and rIHNV‐N438A‐ΔNV‐VP2) replacing the NV gene of the backbone of rIHNV at the single point mutation at residue 438 with an efficient green fluorescent protein (EGFP) reporter gene and antigenic VP2 gene of IPNV. Meanwhile, we tested their efficacy against the wild‐type (wt) IHNV HLJ‐09 virus and IPNV serotype Sp virus challenge. The relative per cent survival rates of two recombinant viruses against (wt) IHNV HLJ‐09 virus challenge were 84.6% and 81.5%, respectively. Simultaneously, the relative per cent survival rate of rIHNV‐N438A‐ΔNV‐VP2 against IPNV serotype Sp virus challenge was 88.9%. It showed the two recombinant viruses had high protection rates and induced a high level of antibodies against IHNV or IPNV. Taken together, these results suggest the VP2 gene of IPNV can act as candidate gene for vaccine and attenuated multivalent live vaccines and molecular marker vaccines have potential application for viral vaccine.     

First evidence of infectious hematopoietic necrosis virus (IHNV) in the Netherlands

In spring 2008, infectious hematopoietic necrosis virus (IHNV) was detected for the first time in the Netherlands. The virus was isolated from rainbow trout, Oncorhynchus mykiss (Walbaum), from a put‐and‐take fishery with angling ponds. IHNV is the causative agent of a serious fish disease, infectious hematopoietic necrosis (IHN). From 2008 to 2011, we diagnosed eight IHNV infections in rainbow trout originating from six put‐and‐take fisheries (symptomatic and asymptomatic fish), and four IHNV infections from three rainbow trout farms (of which two were co‐infected by infectious pancreatic necrosis virus, IPNV), at water temperatures between 5 and 15 °C. At least one farm delivered trout to four of these eight IHNV‐positive farms. Mortalities related to IHNV were mostly <40%, but increased to nearly 100% in case of IHNV and IPNV co‐infection. Subsequent phylogenetic analysis revealed that these 12 isolates clustered into two different monophyletic groups within the European IHNV genogroup E. One of these two groups indicates a virus‐introduction event by a German trout import, whereas the second group indicates that IHNV was already (several years) in the Netherlands before its discovery in 2008.     

Recovery of yellow-pigmented bacteria from dead and moribund fish during outbreaks of rainbow trout, Oncorhynchus mykiss (Walbaum), fry syndrome in England

Abstract. During Autumn 1990, the possible presence of microbial pathogens was investigated in two discrete populations of anaemic (rainbow trout fry syndrome; RTFS) rainbow trout fry. Dense pure culture growth of three types of yellow pigmentcd bacteria were recovered from moribund and dead animals on low nutrient media. The cultures were identified as coryneforms. Cytophaga columnaris and Janthinobacterium sp. All taxa induced clinical disease (with similarities to RTFS) in rainbow trout fry.