Inactivation of koi-herpesvirus in water using bacteria isolated from carp intestines and carp habitats.

Since its first outbreak in Japan in 2003, koi-herpesvirus (KHV) remains a challenge to the carp Cyprinus carpio L. breeding industry. In this study, inactivation of KHV in water from carp habitats (carp habitat water) was investigated with the aim of developing a model for rapidly inactivating the pathogen in aquaculture effluent. Experiments with live fish showed that, in carp habitat water, KHV lost its infectivity within 3 days. Indications were that inactivation of KHV was caused by the antagonistic activity of bacteria (anti-KHV bacteria) in the water from carp habitats. Carp habitat water and the intestinal contents of carp were therefore screened for anti-KHV bacteria. Of 581 bacterial isolates, 23 showed anti-KHV activity. An effluent treatment model for the disinfection of KHV in aquaculture effluent water using anti-KHV bacteria was developed and evaluated. The model showed a decrease in cumulative mortality and in the number of KHV genome copies in kidney tissue of fish injected with treated effluent compared with a positive control. It is thought that anti-KHV bacteria isolated from the intestinal contents of carp and from carp habitat water can be used to control KHV outbreaks.

Molecular identification and transmission studies of X-cell parasites from Atlantic cod Gadus morhua (Gadiformes: Gadidae) and the northern black flounder Pseudopleuronectes obscurus (Pleuronectiformes: Pleuronectidae).

BACKGROUND: Epidermal pseudotumours from Hippoglossoides dubius and Acanthogobius flavimanus in Japan and gill lesions in Limanda limanda from the UK have been shown to be caused by phylogenetically related protozoan parasites, known collectively as X-cells. However, the phylogenetic position of the X-cell group is not well supported within any of the existing protozoan phyla and they are currently thought to be members of the Alveolata.Ultrastructural features of X-cells in fish pseudotumours are somewhat limited and no typical environmental stages, such as spores or flagellated cells, have been observed. The life cycles for these parasites have not been demonstrated and it remains unknown how transmission to a new host occurs. In the present study, pseudobranchial pseudotumours from Atlantic cod, Gadus morhua, in Iceland and epidermal pseudotumours from the northern black flounder, Pseudopleuronectes obscurus, in Japan were used in experimental transmission studies to establish whether direct transmission of the parasite is achievable. In addition, X-cells from Atlantic cod were sequenced to confirm whether they are phylogenetically related to other X-cells and epidermal pseudotumours from the northern black flounder were analysed to establish whether the same parasite is responsible for infecting different flatfish species in Japan. RESULTS: Phylogenetic analyses of small subunit ribosomal DNA (SSU rDNA) sequence data from Atlantic cod X-cells show that they are a related parasite that occupies a basal position to the clade containing other X-cell parasites. The X-cell parasite causing epidermal pseudotumours in P. obscurus is the same parasite that causes pseudotumours in H. dubius. Direct, fish to fish, transmission of the X-cell parasites used in this study, via oral feeding or injection, was not achieved. Non-amoeboid X-cells are contained within discrete sac-like structures that are loosely attached to epidermal pseudotumours in flatfish; these X-cells are able to tolerate exposure to seawater. A sensitive nested PCR assay was developed for the sub clinical detection of both parasites and to assist in future life cycle studies. PCR revealed that the parasite in P. obscurus was detectable in non-pseudotumourous areas of fish that had pseudotumours present in other areas of the body. CONCLUSIONS: The inability to successfully transmit both parasites in this study suggests that either host detachment combined with a period of independent development or an alternate host is required to complete the life cycle for X-cell parasites. Phylogenetic analyses of SSU rDNA confirm a monophyletic grouping for all sequenced X-cell parasites, but do not robustly support their placement within any established protist phylum. Analysis of SSU rDNA from X-cells in Japanese flatfish reveals that the same parasite can infect more than one species of fish.

Lymphocystis disease virus persists in the epidermal tissues of olive flounder, Paralichthys olivaceus (Temminch & Schlegel), at low temperatures.

Olive flounder artificially infected with lymphocystis disease virus (LCDV) were reared at 10, 20 and 30 degrees C for 60 days, to compare LCD-incidence. In the fish reared at 20 degrees C, lymphocystis cells appeared on the skin and fins at 35 days post-challenge, and the cumulative LCD-incidence was 80% at 60 days. High levels of LCDV, with a mean polymerase chain reaction (PCR) titre of 10(6) PCR-U mg(-1) tissue, were detected in the fins and skin of LCD-affected fish at 20 degrees C, but were not detected in the spleen, kidney, brain and intestinal tissues of these fish. No LCD clinical signs were observed in the fish reared at 10 degrees C and 30 degrees C; however, a low level of LCDV (10(3) PCR-U mg(-1) tissue) was detected in the fins and skin of these fish. By increasing the rearing temperature from 10 to 20 degrees C, lymphocystis clusters appeared on the skin and fins of the fish with no previous LCD clinical signs within 33 days after the temperature change. It was shown that permissive cells for LCDV infection exist in the epidermis of olive flounder. At low temperatures, small amounts of LCDV were able to persist over a period extended for a further 45 days in the fish epidermis, even though the fish showed no LCD clinical signs. The optimum growth temperature of LCDV is near 20 degrees C.

Non-specific adsorption of fish immunoglobulin M (IgM) to blocking reagents on ELISA plate wells.

Enzyme-linked immunosorbent assay (ELISA) is a popular technique for quantifiable detection of specific antibodies in warm-blooded animals, but it has not been accepted for detection of fish antibodies because of its low reproducibility, which is due in part to high background optical density (OD) measurements. In the present study, we report that the high background of a fish antibody-detection ELISA resulted from non-specific adsorption of fish immunoglobulin M (IgM) to blocking reagents on the ELISA plate wells. Four fish sera (from rainbow trout Oncorhynchus mykiss, masu salmon O. masou, Japanese flounder Paralichthys olivaceus and koi Cyprinus carpio) were poured into ELISA plate wells pre-blocked with several blocking reagents (skim milk, soybean milk, bovine serum albumin, fetal bovine serum, gelatin and Roche BlockingReagent) and then washed out in order to measure the remaining fish IgM on the ELISA plate wells. Significant amounts of fish IgMs (OD absorbance at 492 nm: 0.3 to 1.1) remained on the ELISA plate wells with no antigenic protein except blocking reagents. The amount of remaining fish IgMs on the ELISA plate wells decreased significantly following treatment of fish sera with skim milk. However, the specific immuno-reactivity of fish IgM was not reduced by such treatment. Thus, we conclude that treatment of fish sera with skim milk is useful in reducing the high background OD often observed in fish IgM detection ELISA.

Genotyping of Korean isolates of infectious hematopoietic necrosis virus (IHNV) based on the glycoprotein gene.

Glycoprotein (G) gene nucleotide sequences of four Korean isolates of infectious hematopoietic necrosis virus (IHNV) were analyzed to evaluate their genetic relatedness to worldwide isolates. All Korean isolates were closely related to Japanese isolates of genogroup JRt rather than to those of North American and European genogroups. It is believed that Korean IHNV has been most likely introduced from Japan to Korea by the movement of contaminated fish eggs. Among the Korean isolates, phylogenetically distinct virus types were obtained from sites north and south of a large mountain range, suggesting the possibility of more than one introduction of virus from Japan.

Nucleotide diversity of Japanese isolates of infectious hematopoietic necrosis virus (IHNV) based on the glycoprotein gene.

Infectious hematopoietic necrosis virus (IHNV), a member of the genus Novirhabdovirus, causes a highly lethal disease of salmonid fish. In the present study, G gene nucleotide sequences of 9 Japanese IHNV isolates obtained from 1971 to 1996 were analyzed to evaluate the genetic diversity and compared with IHNV isolates from North America and Europe. A radial phylogenetic tree revealed 5 major clusters including 3 genogroups (U, M and L) for North American isolates and 1 genogroup for European isolates. Five Japanese isolates from 1971 to 1982 appeared in the cluster for genogroup U, while the remaining Japanese isolates from 1980 to 1996 formed a new genogroup, JRt (Japanese rainbow trout). Maximum nucleotide diversity among the Japanese isolates was 4.5%, which was greater than that within the North American isolates (3.6%), and the degree of nucleotide diversity within Japanese isolates was increased by inclusion of the genogroup JRt isolates. It was concluded that Japanese isolates shared a common source with the genogroup U of the North American isolates and that there were large divergences between Japanese isolates before and after the 1980s.

A new genotype of lymphocystivirus, LCDV-RF, from lymphocystis diseased rockfish.

Lymphocystis disease virus (LCDV) is the causative agent of lymphocystis disease. In this study, nucleotide sequences of the major capsid protein (MCP) gene were analyzed among LCDV isolates from Japanese flounder and rockfish. A phylogenetic tree revealed three clusters for lymphocystiviruses. The first cluster included Japanese flounder isolates; the second cluster consisted of rockfish isolates; and the remaining one consisted of LCDV-1. Nucleotide sequence identities were > or =99.6% among Japanese flounder isolates and 100% among rockfish isolates, while between each cluster they were < or =85.2%. Experimental infections with Japanese flounder and rockfish isolates revealed that Japanese flounder and rockfish were infected by the respective homologous isolate but not by the heterologous isolate. These findings suggest that at least three genotypes exist in the genus Lymphocystivirus.

Oligonucleotide probe for detecting Enterobacteriaceae by in situ hybridization.

AIMS: To develop oligonucleotide probes for visualizing bacteria belonging to Enterobacteriaceae. METHODS AND RESULTS: 24-mer oligonucleotide probe (probe D) was designed by comparison of 16S rDNA sequences of 35 species of Enterobacteriaceae, eight species of Vibrionaceae and six species of Pasteurellaceae. The sequence of the probe corresponding to the complementary sequence of a position 1251-1274 of Escherichia coli 16S rRNA was found to be a highly conserved region of 16S rDNA sequence in Enterobacteriaceae different from that of Vibrionaceae and Pasteurellaceae. The fluorescent dye-labelled probe was tested for the specificity by in situ hybridization and epifluorescence microscopy. Seventy-six out of 78 strains belonging to Enterobacteriaceae were visualized in an optimal hybridization condition. No bacterial strains belonging to Vibrionaceae (31 strains) and Gram-positive bacteria (three strains) were visualized. CONCLUSIONS: In situ hybridization using probe D allows the detection of bacterial cells belonging to Enterobacteriaceae without false positive reaction. SIGNIFICANCE AND IMPACT OF THE STUDY: In situ hybridization techniques using the probe D are potential tools for detecting Enterobacteriaceae in food and water samples.

Selection of brood stock candidates of barfin flounder using an ELISA system with recombinant protein of barfin flounder nervous necrosis virus.

Barfin flounder nervous necrosis virus (BFNNV), the causative agent of viral nervous necrosis (VNN) of barfin flounder, is vertically transmitted from spawners to larvae. In the present study, an ELISA with a recombinant protein of BFNNV was performed for the detection of antibodies against BFNNV and applied for the selection of brood fish in order to prevent viral vertical transmissions. Brood stocks were divided into 4 groups based on ELISA antibody titers (< or = 10, 20, 40 and >40), and the BFNNV status of the brood stocks was determined by PCR. BFNNV was detected from the brood fish in the group with an antibody titer of >40 but not from those with titers < or = 10, 20 and 40. The offspring obtained from PCR-negative brood fish pairs in each group of ELISA antibody titers were subsequently reared for observation of VNN occurrence. VNN occurred in juveniles from 2 of 9 pairs of spawners with an antibody titer > or = 40, but did not occur in spawners with an antibody titer of < or = 10. Therefore, it was concluded that selection of brood fish using both the PCR test and ELISA antibody titers could help prevent vertical transmission of BFNNV in larval production of barfin flounder.

Vibrio spp., the dominant flora in shrimp hatchery against some fish pathogenic viruses.

Two strains of Vibrio spp., NICA 1030 and NICA 1031, which are the dominant flora in shrimp hatcheries, were studied for antiviral activity by exposing them to the infectious hematopoietic necrosis virus (IHNV) and Oncorhynchus masou virus (OMV) before being inoculated to the CHSE-214 cells. Antiviral activities were exhibited as plaque reduction percentages of 98 and 92% for the NICA 1030 and of 99 and 62% for the NICA 1031, respectively.

Disease problems of salmonid fish in Japan caused by international trade.

The author details the connection between disease outbreaks in salmonid fish and imports of salmonid eggs into Japan since the 1950s. The following diseases and species are involved: -infectious pancreatic necrosis in rainbow trout (Oncorhynchus mykiss) -infectious haematopoietic necrosis in sockeye ( of 'kokanee') salmon (O. nerka) and masu salmon (O. masu) -cold water disease, erythrocyte inclusion body syndrome, and bacterial kidney disease (caused by Renibacterium salmoninarum) in coho salmon (O. kisutch), ayu (Plecoglossus altivelis), rainbow trout and masu salmon. The author also discusses the strategies aimed at controlling the risk of disease spread through international trade in salmonid fish and fish products in Japan. Essential in these strategies are the following actions: -exchange of information on controlling disease problems -studies to establish standard methods to identify or detect fish pathogens -fish health certification.

Salmonid herpesvirus 2. Epizootiology and serological relationship.

Herpesvirus infections of kokanee salmon, masu salmon, coho salmon, and rainbow trout have been reported in Japan. The 11 herpesvirus strains isolated from kokanee salmon (NeVTA), masu salmon (YTV and 3 strains of OMV), coho salmon (CSTV, COTV and 2 strains of OKV), rainbow trout (RKV and RHV), and Herpesvirus salmonis were compared for their serological relatedness by serum cross-neutralization tests with polyclonal rabbit antiserum. The herpesvirus strains isolated in Japan were neutralized by antisera against these viruses and were found to be closely related to salmonid herpesvirus 2 (OMV strain 00-7812). These strains were, however, clearly distinguished from salmonid herpesvirus 1 (H salmonis).

Detection of viral DNA polymerase activity in salmon tumour tissue induced by herpes virus, Oncorhynchus masou virus.

DNA polymerase activities were surveyed in tumour tissue and normal tissue of cherry salmon (Oncorhynchus masou). High activity of DNA polymerase alpha was detected in the tumour tissue but not in the normal tissue. This indicates that the tumour cells replicate prosperously. Viral DNA polymerase activity was detected only in the tumour tissue, indicating that Oncorhynchus masou virus (OMV) DNA should replicate there. DNA polymerase beta activity was of same level in both tissues. This is the first evidence that herpesvirus DNA polymerase was detected in tumour tissue in association with herpesvirus.

Comparison of different Oncorhynchus masou virus (OMV) strains by DNA restriction endonuclease cleavage analysis.

Seven strains of Oncorhynchus masou virus (OMV) genomes were analyzed with the restriction endonucleases BamHI, EcoRI, HindIII and SmaI. The restriction patterns of OMV strain DNAs were divided into four groups. Restriction profiles of high passage strains (00-7812, 65th passage, and H-83, 60th passage) were different from those of low passage strains (00-7812, 8th passage, and H-83, 6th passage) when digested with BamHI, HindIII and SmaI. However, no difference was observed between the restriction patterns of high and low passage viral DNA with EcoRI. There was no distinct difference observed between the restriction patterns of tumor tissue-derived and coelomic fluid-derived strains. By using 32P-labelled DNA of standard OMV (strain 00-7812) as a probe, most of the fragments of other OMV strain DNAs were hybridized.

Inhibitory effect of halocyamine, an antimicrobial substance from ascidian hemocytes, on the growth of fish viruses and marine bacteria.

Halocyamine A, an antimicrobial substance isolated from hemocytes of the solitary ascidian Halocynthia roretzi, inhibited in vitro the growth of fish RNA viruses (infectious hematopoietic necrosis virus and infectious pancreatic necrosis virus). Pretreatment of RNA virus with halocyamine A reduced the infectivity of the virus toward host cells. The growth of marine bacteria, Achromobacter aquamarinus and Pseudomonas perfectomarinus, was also inhibited by halocyamine A but that of Alteromonas putrefaciens and Vibrio anguillarum was not. These results suggest that halocyamine may have a role in the defense mechanisms of H. roretzi against marine viruses and bacteria.

Inhibitory activity of (E)-5-(2-bromovinyl)-2'-deoxyuridine on the salmonid herpesviruses, Oncorhynchus masou virus (OMV) and Herpesvirus salmonis.

The highly potent and selective anti-herpesvirus agent, (E)-5-(2-bromovinyl)-2'deoxyuridine (BVdU), was examined for its inhibitory effect on the salmonid herpesviruses Oncorhynchus masou virus (OMV) and Herpesvirus salmonis (H. salmonis). Minimum inhibitory concentrations (MIC) of BVdU for OMV and H. salmonis were 1.25 and 3.0 micrograms/ml, respectively; these values were equal to or higher than those obtained for acyclovir or cytarabine. OMV DNA polymerase activity was reduced in a dose-dependent fashion by BVdU 5'-triphosphate (BVdUTP) within the concentration range of 3 to 30 microM. However, BVdUTP could also be substituted for the natural substrate, TTP, in the OMV DNA polymerase assay. It is postulated that the inhibitory action of BVdU on the salmonid herpesviruses is more or less similar to that on other herpesviruses and resides with respect to the inhibition of the virus DNA polymerase activity as well as incorporation of BVdU into the viral DNA.

Screening of bacteria with antiviral activity from fresh water salmonid hatcheries.

Bacteria isolated from two salmonid hatcheries were screened for antiviral activity against infectious hematopoietic necrosis virus (IHNV) to ascertain the presence of bacteria with anti-IHNV activity in the aquatic environment. Out of 710 bacterial isolates from the water and sediment samples, 190 strains showed anti-IHNV activities of more than 50% plaque reduction. These antiviral activities were detected predominantly in Pseudomonas, Aeromonas/Vibrio, and coryneforms. In one hatchery, the bacteria with antiviral activities were more prevalent in sediment samples than in water samples. Seventy-seven percent of the isolates with higher antiviral activities (greater than 90% plaque reduction) belonged to Pseudomonas.

In vitro antiviral effect of 9-(2-hydroxyethoxymethyl) guanine on the fish herpesvirus, Oncorhynchus masou virus (OMV).

The antiviral activity of 9-(2-hydroxyethoxymethyl) guanine (Acyclovir, ACV) on the salmon herpesvirus, Oncorhynchus masou virus (OMV), was studied in vitro. ACV showed high efficacy against the fish herpesvirus OMV, Herpesvirus salmonis and channel catfish virus (CCV). Cytopathic effect (CPE) induced by 100 TCID50/ml of OMV in rainbow trout gonad (RTG-2) cells was inhibited by 2.5 micrograms/ml of ACV. ACV was more effective than other compounds such as 9-beta-D-arabinofuranosyladenine (Ara-A), 5-iodo-2'-deoxyuridine (IUdR) and phosphonoacetate (PA). Growth of RTG-2 cells was considerably inhibited by ACV at 25 microgram/ml, but no morphological changes were observed in the cells. Replication of OMV in RTG-2 cells inoculated with 100 TCID50/ml was completely suppressed by 2.5 microgram/ml of ACV. Addition of ACV within 4 days post infection was effective in reducing OMV replication. In order to be effective, ACV had to be present continuously.