Genetic Analysis of Betanodaviruses in Subclinically Infected Aquarium Fish and Invertebrates

Betanodaviruses causing viral nervous necrosis (VNN) have been detected and isolated from several species of cultured marine fish worldwide. In Korea, VNN was identified in several species of cultured marine fish. This study presents data on the amplified nested PCR product (420 bp) of 11 nodavirus strains from different species of apparently healthy aquarium fish and invertebrates collected in one private commercial aquarium in Korea. Phylogenetic analyses based on the partial nucleotide sequence (177 bases) of the RNA2 coat protein gene were identical to the redspotted grouper nervous necrosis virus (RGNNV) genotype (96%–100%). The presence of the RGNNV type of betanodaviruses in these subclinically infected aquarium fish and invertebrates imported from different countries probably indicates that the samples were contaminated inside the aquarium and represents a serious challenge for its management of viral nervous necrosis. These positive samples can be an inoculum source of betanodavirus infection to other susceptible fish species inside the aquarium.     

Phylogenetic analysis of the major capsid protein gene of iridovirus isolates from cultured flounders Paralichthys olivaceus in Korea

In 2003, 13 isolates of iridovirus were obtained from cultured flounders Paralichthys olivaceus during epizootics in Korea. The full open reading frames (ORFs) encoding the major capsid protein (MCP) (1362 bp) from the 13 flounder iridoviruses (FLIVs) were sequenced and the deduced amino acid sequences were phylogenetically analyzed. Phylogenetic analysis of the MCP revealed that all 13 FLIVs were the same species as rock bream iridovirus (RBIV), red sea bream iridovirus (RSIV), and infectious spleen and kidney necrosis virus (ISKNV), and were grouped into an unknown genus which was different from the 2 genera known to infect fish, Ranavirus and Lymphocystivirus. This is the first report on the isolation and phylogenetic analysis of the iridovirus of unknown genus from flounders during epizootics.     

Enhanced propagation of fish nodaviruses in BF-2 cells persistently infected with snakehead retrovirus (SnRV)

Fish nodaviruses are causative agents of viral nervous necrosis causing high mortality in cultured marine fishes around the world. The first successful isolation of fish nodavirus was made with SSN-1 cells, which are persistently infected with snakehead retrovirus (SnRV). In the present study, a BF-2 cell line persistently infected with SnRV (PI-BF-2) was established to evaluate the influence of SnRV on the production of fish nodavirus. The PI-BF-2 cells were slightly more slender than BF-2 cells, but no difference was observed in propagation rate between both cell lines. No difference was observed in production of SnRV between PI-BF-2 and SSN-1 cell lines. Although both PI-BF-2 and BF-2 cell lines showed no cytopathic effect (CPE) after inoculation of striped jack nervous necrosis virus (SJNNV) and redspotted grouper nervous necrosis virus (RGNNV), these fish nodaviruses could be amplified in BF-2 cells, and moreover, production of fish nodaviruses in the PI-BF-2 cell line was more than 40 times higher than in BF-2 cells. Thus, it was concluded that BF-2 cell permissiveness to fish nodaviruses was enhanced by persistent infection with SnRV. Furthermore, homologous cDNA to genomic RNA of SJNNV was detected from both PI-BF-2 and SSN-1 cell lines persistently infected with SnRV. The amount of nodavirus cDNA in SJNNV-inoculated PI-BF-2 cells was clearly lower than that in SJNNV-inoculated SSN-1 cells.     

Survey of Zoonotic Trematode Metacercariae in Fish from Water systems of Geum-gang (River) in Republic of Korea

The infection status of zoonotic trematode metacercariae (ZTM) was surveyed in freshwater fishes from the water systems of Geum-gang (River) in the Republic of Korea (Korea). A total of 1,161 freshwater fishes from 6 local sites of Geum-gang were examined with the artificial digestion method for 4 years (2012–2015). Clonorchis sinensis metacercariae were detected in 122 (37.2%) out of 328 fishes in the positive fish species from 4 surveyed areas, and their mean intensity was 43 per fish infected. Metagonimus spp. metacercariae were found in 432 (51.7%) out of 835 fishes in the positive fish species from all 6 surveyed areas, and their mean intensity was 30 per fish infected. Centrocestus armatus metacercariae were detected in 285 (75.0%) out of 380 fishes in the positive fish species from 6 surveyed areas, and their mean intensity was 2,100 per fish infected. Echinostoma spp. metacercariae were found in 56 (19.7%) out of 284 fishes in the positive fish species from 5 surveyed areas, and their mean intensity was 10 per fish infected. Clinostomum complanatum metacercariae were detected in 98 (57.3%) out of 171 fishes in the positive fish species from only 2 surveyed areas, and their mean intensity was 11 per fish infected. Conclusively, the endemicity of ZTM is not so high in fishes from water systems of Geum-gang in Korea although it is more or less different by fish species, surveyed areas and ZTM species.     

Outbreaks and risks of infectious spleen and kidney necrosis virus disease in freshwater ornamental fishes

We examined the distribution of iridoviruses in 10 freshwater ornamental fish species hatched in Korea and imported from other Asian countries using both 1-step and 2-step polymerase chain reation (PCR). None of the 10 fish species analyzed were free of iridovirus as shown by 2-step PCR positive results, and 3 species yielded 1-step PCR positive results with associated mortality. Cloned PCR amplicons of the adenosine triphosphatase (ATPase) and major capsid protein (MCP) genes in genomic DNA of iridovirus showed the same nucleotide sequences as that of infectious spleen and kidney necrosis virus (ISKNV) isolated from the mandarinfish Siniperca chuatsi. These results indicate the presence of ISKNV disease in various ornamental fish as new host species and that the disease is widespread throughout different Asian countries including Korea, Singapore and China. Such infections were either clinical with associated mortality (and 1-step PCR positive) or asymptomatic in fish that were externally healthy (and only positive in 2-step PCR). Molecular analyses of the K2 region performed on iridovirus samples isolated from freshwater ornamental fishes revealed deletion/insertion of repetitive sequences of various lengths (42 to 339 bp), depending on the ISKNV isolates, without substitutions. Experimental infection of pearl gourami Trichogaster leeri and silver gourami T. microlepis with a tissue homogenate of pearl gourami infected by ISKNV induced 70 and 20% cumulative mortalities in the pearl and silver gourami, respectively.     

Isolation of Aeromonas salmonicida from masu salmon in the Republic of Korea

The isolation and identification of the Gram-negative fish pathogen, Aeromonas salmonicida salmonicida, from masu salmon (Oncorhynchus masou) at the Yang Yang Salmon Hatchery in the Republic of Korea is described. The bacterium possessed certain characteristics in common with isolates from fish in Japan. It is an important cause of mortality among cultured salmonids in the Republic of Korea. This is the first report of this fish pathogen from the Asian mainland.     

Molecular detection and characterization of human enteroviruses in Korean surface water

In this study, the genetic epidemiology of enteroviruses (EVs) in Korean surface water was evaluated by conducting phylogenetic analyses of the nucleotide sequences of the 5' non-coding region (5' NCR), which was determined by RT-PCR analysis of total culturable virus assay-positive samples. The results showed that the nucleotide sequences of the EVs could be classified into 4 genetic clusters, and that the predominant presence of Korea EVs were very similar to echoviruses type 30. Interestingly, two nucleotide sequences were very similar to those of coxsackievirus type B1 isolated from aseptic meningitis patients in Seoul, Korea, implying the possibility of a common source for the viruses circulated in water systems and humans. In addition, 3 nucleotide sequences clustered strongly with the nucleotide sequences from China or Japan, and one fell into the same cluster as echovirus type 11 from Taiwan, which suggests that EVs in Asia may have evolved in a region-specific manner. Taken together, the results of this study revealed that EVs from Korea surface waters could be genetically classified as coxsackieviruses or echoviruses, and that they evolved in Asia in a region-specific manner.     

Anisakis pegreffii Larvae in Sea Eels (Astroconger myriaster) from the South Sea,Republic of Korea

Anisakis simplex sensu stricto (s.s.), Anisakis pegreffii, Anisakis berlandi (=A. simplex sp. C), and Anisakis typica are the 4 major species of Anisakis type I larvae. In the Republic of Korea (Korea), A. pegreffii, A. berlandi, and A. typica larvae in fish hosts has seldom been documented. In this study, molecular analysis was performed on Anisakis larvae from the sea eels (Astroconger myriaster), the major source of human anisakiasis in Korea, collected from Tongyeong City, a southern coastal area of Korea. All 20 sea eels examined were infected with Anisakis type I larvae (160 larvae; 8 per fish). Their species were analyzed using PCR-RFLP patterns and nucleotide sequences of internal transcribed spacers (ITS1, 5.8 subunit gene, and ITS2) and mitochondrial cytochrome c oxidase 2 (cox2). Most (86.8%; 112/129) of the Anisakis type I larvae were A. pegreffii, and 7.8% (10/129) were A. typica. The remaining 5.4% (7/129) was not identified. Thus, A. pegreffii is the major species of anisakid larvae in sea eels of the southern coast of Korea.     

Low resolution of mitochondrial COI barcodes for identifying species of the genus Larus (Charadriiformes: Laridae)

We evaluated whether cytochrome c oxidase subunit I (COI) barcodes that have been previously suggested for birds are useful for identifying species of the genus Larus, which are resident or migratory birds in Korea. We found 31 intra- or interspecific COI haplotypes from 12 of 13 Larus species in Korea. Haplotype analyses showed that the COI barcodes could not distinguish some interspecific haplotypes from 6 of 12 Larus species because there were no nucleotide substitutions among their COI haplotypes. The neighbor-joining tree formed shallow branches in the clades expected for L. saundersi, L. crassirostris, L. canus, L relictus, and L. ridbundus. In the nine Larus species, COI haplotypes were not grouped as distinct entities that were correctly assigned to their corresponding species, resulting in polytypic clades. These results indicate that the COI sequences need to be cautiously selected as a DNA barcode for identifying species of Korean Larus birds.     

Strain Variation in Mycobacteriummarinum Fish Isolates

A molecular characterization of two Mycobacterium marinum genes, 16S rRNA and hsp65, was carried out with a total of 21 isolates from various species of fish from both marine and freshwater environments of Israel, Europe, and the Far East. The nucleotide sequences of both genes revealed that all M. marinum isolates from fish in Israel belonged to two different strains, one infecting marine (cultured and wild) fish and the other infecting freshwater (cultured) fish. A restriction enzyme map based on the nucleotide sequences of both genes confirmed the divergence of the Israeli marine isolates from the freshwater isolates and differentiated the Israeli isolates from the foreign isolates, with the exception of one of three Greek isolates from marine fish which was identical to the Israeli marine isolates. The second isolate from Greece exhibited a single base alteration in the 16S rRNA sequence, whereas the third isolate was most likely a new Mycobacterium species. Isolates from Denmark and Thailand shared high sequence homology to complete identity with reference strain ATCC 927. Combined analysis of the two gene sequences increased the detection of intraspecific variations and was thus of importance in studying the taxonomy and epidemiology of this aquatic pathogen. Whether the Israeli M. marinum strain infecting marine fish is endemic to the Red Sea and found extremely susceptible hosts in the exotic species imported for aquaculture or rather was accidentally introduced with occasional imports of fingerlings from the Mediterranean Sea could not be determined.     

Strain variation in Mycobacterium marinum fish isolates

A molecular characterization of two Mycobacterium marinum genes, 16S rRNA and hsp65, was carried out with a total of 21 isolates from various species of fish from both marine and freshwater environments of Israel, Europe, and the Far East. The nucleotide sequences of both genes revealed that all M. marinum isolates from fish in Israel belonged to two different strains, one infecting marine (cultured and wild) fish and the other infecting freshwater (cultured) fish. A restriction enzyme map based on the nucleotide sequences of both genes confirmed the divergence of the Israeli marine isolates from the freshwater isolates and differentiated the Israeli isolates from the foreign isolates, with the exception of one of three Greek isolates from marine fish which was identical to the Israeli marine isolates. The second isolate from Greece exhibited a single base alteration in the 16S rRNA sequence, whereas the third isolate was most likely a new Mycobacterium species. Isolates from Denmark and Thailand shared high sequence homology to complete identity with reference strain ATCC 927. Combined analysis of the two gene sequences increased the detection of intraspecific variations and was thus of importance in studying the taxonomy and epidemiology of this aquatic pathogen. Whether the Israeli M. marinum strain infecting marine fish is endemic to the Red Sea and found extremely susceptible hosts in the exotic species imported for aquaculture or rather was accidentally introduced with occasional imports of fingerlings from the Mediterranean Sea could not be determined.     

Spread of Neoheterobothrium hirame (Monogenea), a serious pest of olive flounder Paralichthys olivaceus, to Korea

Neoheterobothrium hirame is a large, blood-feeding gill-worm infecting the highly prized olive flounder Paralichthys olivaceus in Japan. There is strong evidence that this worm is the primary cause of anaemia, a common and serious condition causing losses among both wild and cultured olive flounders. N. hirame was first detected in Japanese waters less than a decade ago, and its population then proliferated and spread throughout most of Japan, except Hokkaido. In neighbouring Korea, olive flounder is the most important species of cultured marine fish, and production currently exceeds that in Japan. However, until now, there have been no reports of any monogeneans or anaemia among olive flounders in Korea. Our survey conducted in 2000 of 100 cultured individuals from 4 provinces revealed 2 immature specimens of N. hirame: 1 from a land-based pond-culture system in southern Cheju Island (off the SW coast of Korea) and the other from a floating net cage near Yosu (in the mid-S part of the peninsula). The geographic range of this pathogen may have been enlarged as a result of introduction(s) of infected broodstock from Japan, but this seems unlikely. (The raising of this species in hatcheries developed in Korea in 1985, 7 years before the earliest detection of the worm in Japan.) Low numbers of flounders were also clearly anaemic. This, and the current rarity of N. hirame in Korean farms, appears to favour the hypothesis of a more recent, natural dispersal of the worm, during migrations of infected flounder across the narrow and shallow Tsushima and Korea Straits. Regardless of route of entry, we expect this pathogen will have an impact on Korean flounder fisheries equally serious to that being experienced in Japan.     

Genetic variation and geographic distribution of megalocytiviruses

Viruses belonging to the genus Megalocytivirus in the family Iridoviridae have caused mass mortalities in marine and freshwater fish in Asian countries. In this study, partial major capsid protein (MCP) gene of seven Japanese and six Korean megalocytiviruses was sequenced and compared with the known megalocytiviruses to evaluate genetic variation and geographic distribution of the viruses. Comparison of MCP gene nucleotide sequences revealed sequence identity of 92.8% or greater among these 48 isolates. A phylogenetic tree clearly revealed three clusters: genotype I including nine Japanese isolates, thirteen Korean isolates, one Chinese isolates, one Thailand isolate and one South China Sea isolate; genotype II including five freshwater fish isolates in Southeast Asian countries and Australia; and the remaining genotype III mainly consisted of flatfish isolate in Korea and China. This suggests that viruses belonging to the genotype I widely distribute among various fish species in many Asian countries. Conversely, the epidemic viruses belonged to genotype II and III are may be still locally spreading and constrained in their prevalence to the limited host fish species, i.e., genotype II viruses mainly distribute in Southeast Asian countries, whereas genotype III viruses distribute in flatfish species in Korea and China.     

Taxonomic reappraisal on<Emphasis Type="Italic">Suaeda australis</Emphasis> (Chenopodiaceae) in korea based on the morphological and molecular characteristics

We used morphological and molecular characteristics to perform a taxonomic reappraisal ofSuaeda australis (Brown) Moquin-Tandon from Korea. Populations of this species are dispersed at the bottoms of sand zones, and usually exhibit a depressed habit. Except for their total heights and leaf lengths, the morphology of these plants does not differ from that ofS. maritima. Molecular traits were examined based on ITS and psbB-psbH spacer region sequences. The former region included ITS-1, 5.8S, and ITS-2, which were 629 nucleotide bases long. Pair-wise distances (p-distance) among KoreanSuaeda species ranged from 1.12 to 17.84. The psbB-psbH spacer region sequences were 618 nucleotide bases long, and were conserved in the alignment of KoreanSuaeda species. In our ML and MrBayesian analysis of ITS sequences aligned with other sequences from GenBank, the plants of KoreanSuaeda made three clades: 1)S. japonica;S. australis, andS. maritima;2)S. malacosperma; and 3) S.glauca. However, the psbB-psbH region sequences could not be resolved amongS. japonica, S. maritima, andS. australis from Korea. Molecular and vegetative characteristics indicated that the plants now classified asS. australis from Korea should instead be named as S.maritima (L.) Dumont.     

Molecular detection and characterization of nodavirus in several marine fish species from the northeastern Atlantic

Nodaviruses (NNV) are responsible for causing disease outbreaks mainly in hatchery-reared larvae and juveniles of a wide variety of fishes throughout the world. This disease has seriously limited the culture of marine fishes over the last decade. In the Atlantic provinces of Canada, disease caused by a nodavirus was first reported in juvenile Atlantic cod being reared in Nova Scotia, in 1999. More recently, disease outbreaks caused by nodavirus have been identified in hatchery-reared Atlantic cod and haddock in Newfoundland and New Brunswick, respectively, and along the east coast of the USA. The presence of NNV in wild Atlantic cod adults and wild winter flounder has also been reported. Nodaviruses were isolated from cultured Atlantic cod and haddock, as well as from wild winter flounder from a variety of geographical localities, and their virus coat (capsid) protein genes were partially sequenced. An analysis of the data indicates that all of the nodaviruses isolated from eastern North America were closely related to one another, but that they were distinct from the European isolates already sequenced. Regardless of host species, isolates from close geographical localities were more similar than those from distant geographical areas. At the protein level, differences in coat protein sequences were seen only for strains isolated from Atlantic cod originating from Newfoundland. Our results suggest that NNV may have been present in the Atlantic off Canada and on the east coast of the USA for some time, and has evolved to form a monophyletic group, distinct from other isolates found in cold-water species. Non-lethal methods for detection of NNV are necessary to develop management strategies for this disease, and would be an asset to diagnosticians and producers. Based on the results of this study, new primers were designed and developed for an improved RT-PCR assay able to detect North Atlantic nodaviruses in ovarian fluids, eggs and other tissues. The application of this test to field samples is discussed.     

Characterization of Cryptocaryon irritans,a parasite isolated from marine fishes in Taiwan

The ciliated protozoan parasite Cryptocaryon irritans infecting marine fishes in Taiwan is described. Developmental characteristics and sequences of the ribosomal DNA regions such as part of 18 S, the entire first internal transcribed spacer, and part of 5.8 S of various Taiwan isolates of C. irritans were investigated. A total of 5 isolates was obtained from different fish-host species and localities, the majority from cultured fish species. C. irritans from Taiwan is able to shift its developmental characteristics, i.e. from non-adherent to adherent tomonts, from individualistic to aggregate-forming tomonts, from infection of the gills only to infection of the gills and body. Thus, it is not possible to classify strains of C. irritans on the basis of these parameters. Premature tomonts that developed from dead fishes were able to produce theronts that could infect fish host. Isolates from Pingtung and the USA had identical nucleotide sequences while an isolate from Malaysia was identical to an Israel isolate. Percentage variation among pairs of Taiwan isolates showed a higher degree of variation than isolate sequences listed in GenBank. Sequence analysis revealed highly aberrant isolates in Taiwan, and a phylogenetic tree distinguished a marine and a low-salinity variant. C. irritans from marine fishes in Taiwan, therefore, display some characteristics not previously reported. Since manipulation of salinity in brackishwater ponds and marine cage sites is not feasible, there is a need to develop new strategies for the control and prevention of cryptocaryoniasis.