Aureispira anguillae sp. nov., isolated from Japanese eel Anguilla japonica leptocephali.

A novel filamentous eel-leptocephalus pathogenic marine bacterium, designated strain EL160426T, was isolated from Japanese eel, Anguilla japonica, leptocephali reared at a laboratory in Mie, Japan. In experimental infection studies on eel larvae, the strain EL160426T caused massive larval mortality and was reisolated from moribund leptocephali. Characteristically, observations of infected larvae found that EL160426T forms columnar colonies on the cranial surface of larvae. The novel isolate exhibited growth at 15-30 °C, pH 7-9, and seawater concentrations of 60-150% (W/V). Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain EL160426T was most closely related to Aureispira maritima 59SAT with 97.7% sequence similarity. The whole genome sequence analysis of the strain EL160426T showed that the strain maintained a circular chromosome with a size of approximately 7.58 Mbp and the DNA G + C content was 36.2%. The major respiratory quinone was MK-7 and the predominant cellular fatty acids were 16:0, 20:4 w6c (arachidonic acid), 17:0 iso and 16:0 N alcohol. DNA relatedness between the closest phylogenetic neighbor strain EL160426T and A. maritima (JCM23207T) was less than 13%. On the basis of the polyphasic taxonomic data, the strain represents a novel species of the genus Aureispira, for which the name Aureispira anguillae sp. nov. is proposed. The type strain is EL160426T (= JCM 35024 T = TSD-286 T).

Intra vitam assays for detecting fish infected with cyprinid herpesvirus 3 (CyHV-3).

Koi carp is one of the most sensitive variants of common carp Cyprinus carpio to cyprinid herpesvirus 3, commonly known as koi herpesvirus (KHV). Given that this species is traded at high prices throughout the world, intra vitam assays for detecting KHV in targeted fish with a high detection efficiency are essential. In this study, 4 intra vitam assays were compared with regard to their efficiency of detecting KHV in koi carp on each day after viral exposure via experimental infection. The results indicated that PCR from the gills and scales sampled by biopsy using dissecting scissors and forceps, respectively, can detect KHV for apparently longer periods than the other assays. This study also suggests that a PCR detection assay for environmental samples could be developed as a convenient intra vitam assay to confirm the presence of virus in environments inhabited by virus-shedding fish.

Establishing the optimal fetal bovine serum concentration to support replication of cyprinid herpesvirus 3 in CCB and KF-1 cell lines.

Koi herpesvirus (KHV) disease is a serious disease in cultured carp (Cyprinus carpio). CCB and KF-1 cell lines are commonly used for virus isolation and observation of cytopathic effects (CPE) in carp and koi samples. The purpose of this study was to determine the optimal concentration of fetal bovine-serum (FBS) to use for supporting the replication of cyprinid herpesvirus 3 CyHV-3 in CCB and KF-1 cell lines. The following concentrations were tested: 0%, 2%, 5%, and 10% FBS. At 7 days post-viral inoculation (dpi), CPE with clear vacuolation was observed in both cell lines when supplemented with 0 and 2% FBS, but not in those supplemented with 5% or 10% FBS. At 14 dpi, CPE was observed in both cell lines supplemented with FBS at any of the tested concentrations when a high virus titer was inoculated. However, CPE was indistinguishable between cell lines supplemented with 10% FBS when a low virus titer was inoculated. Results of qPCR indicated that the number copies of the viral genome tended to be larger in both cell lines supplemented with 10% FBS than the corresponding number in cell lines supplemented with 0%, 2%, or 5% FBS, at 7 dpi. In conclusion, we recommend using 2% FBS as supplement for isolation and diagnosis of CyHV-3 viral infection in carp samples.

The susceptibility of silver crucian carp (Carassius auratus langsdorfii) to infection with koi herpesvirus (KHV).

Koi herpesvirus (KHV) infections cause high mortality in carp (Cyprinus carpio). This study compared the susceptibility of silver crucian carp (Carassius auratus langsdorfii), also called ginbuna, and koi carp to KHV infection. Silver crucian carp and koi carp were challenged with KHV by both intraperitoneal injection and immersion, respectively, and kept in tanks at 22°C. All KHV-exposed koi carp died within 14 days post-infection (dpi), whereas no clinics nor mortality was observed in the KHV-exposed silver crucian carp. KHV DNA was detected in both koi and silver crucian carp shortly after infection. At 7 dpi, the copy numbers of KHV genome were increased in koi carp but decreased in silver crucian carp. Using reverse transcriptase PCR, KHV mRNA was detected in koi carp but not in silver crucian carp. Cell cultivation on common carp brain (CCB) cell samples from koi carp caused KHV-associated cytopathic effects in CCB cells. Therefore, we concluded that KHV replicated in koi carp but not in silver crucian carp and that silver crucian carp is not susceptible to infection with KHV.

Detection and application of circular (concatemeric) DNA as an indicator of koi herpesvirus infection.

Herpesviruses form a long continuous DNA molecule, or head-to-tail concatemer, as a replicating intermediate in the host. In this study, we developed a DNA-specific PCR assay for detecting the infection stage of koi herpesvirus (KHV) based on the presence of this 'endless' DNA. The 295 kbp double-stranded DNA KHV genome consists of a 251 kbp unique long region and two 22 kbp direct repeats (DRL and DRR) at each genome terminus. We designed a new primer set (DR primer set) based on the DR region spanning the presumed circular or concatemeric junction. Using the DR primer set, a PCR product was obtained from KHV-infected common carp brain (CCB) cells, but not from the virus-infected cell culture supernatant, implying that the PCR assay could detect intracellular virus in the host. The synthesis of a presumptive circular or concatemeric genome in virus-infected CCB cells was examined in a time-course experiment together with viral mRNA of the terminase gene, copy numbers of the viral genome, and infectious viral titer. The mRNA was first detected in the cells at 6 h post-inoculation (hpi), and the copy number of viral genome in the cells started to increase at 12 hpi. Subsequently, circular or concatemeric DNA was detected in the cells at 18 hpi, and progeny virus was detected in the cell culture supernatant at 24 hpi. These findings suggest that detection of the circular or concatemeric KHV genome with the developed PCR method can be used to determine the stage of KHV infection.

Differences in the susceptibility of Japanese indigenous and domesticated Eurasian common carp (Cyprinus carpio), identified by mitochondrial DNA typing, to cyprinid herpesvirus 3 (CyHV-3).

In 2004, a massive mortality of wild common carp (Cyprinus carpio) due to CyHV-3 infection occurred in Lake Biwa. Although common carp of two different mitochondrial types (Japanese indigenous and domesticated Eurasian) occur in the lake, the majority of the dead fish seemed to be the indigenous type. The apparent high mortality in the indigenous type implies a higher susceptibility of this type to CyHV-3. To test the hypothesis that the susceptibility of indigenous and Eurasian types differ, we performed experimental infections with CyHV-3 among 2 groups of the indigenous type, and for the Eurasian type 4 groups of domesticated common carp and 4 groups of koi carp. Fish were immersed in CyHV-3 isolate and kept at 24°C. Both groups of the indigenous type died more rapidly compared with the 8 groups of the Eurasian type. Cumulative mortality in both indigenous groups reached 95-100%, whereas the cumulative mortalities of domesticated common carp (30-95%) and koi carp (35-100%) were more varied. CyHV-3 genome in the organs of the indigenous type increased more rapidly after the viral exposure and reached higher peak levels than those of the domesticated strain. These findings revealed that susceptibility of the indigenous type of carp to CyHV-3 can be considered especially high.

Efficient establishment of primary fibroblast cultures from the hawksbill sea turtle (Eretmochelys imbricata).

The hawksbill sea turtle (Eretmochelys imbricata) is a critically endangered species at a risk of extinction. Preservation of the genomic and cellular information of endangered animals is important for future genetic and biological studies. Here, we report the efficient establishment of primary fibroblast cultures from skin tissue of the hawksbill sea turtle. We succeeded in establishing 19 primary cultures from 20 hawksbill sea turtle individuals (a success rate of 95%). These cells exhibited a fibroblast-like morphology and grew optimally at a temperature of 26°C, but experienced a loss of viability when cultured at 37°C. Chromosomal analysis using the primary cells derived here revealed that hawksbill sea turtles have a 2n = 56 karyotype. Furthermore, we showed that our primary cell cultures are free of several fish-related viruses, and this finding is important for preservation purposes. To our knowledge, this report is the first to describe primary cell cultures established from normal tissues of the hawksbill sea turtle. The results will contribute to the preservation of biodiversity, especially for the sea turtles that are critically endangered owing to human activities.

Development of mRNA-specific RT-PCR for the detection of koi herpesvirus (KHV) replication stage.

An mRNA-specific reverse transcription (RT)-PCR primer set spanning the exon junction of a spliced putative terminase gene in the koi herpesvirus (KHV) was developed to detect the replicating stage of the virus. The proposed RT-PCR amplified a target gene from the RNA template, but not from a DNA template extracted from common carp brain (CCB) cells infected with KHV. In addition, the RT-PCR did not amplify the target gene of templates extracted from specific cell lines infected with either CyHV-1 or CyHV-2. RT-PCR detected mRNA from the scales of koi experimentally infected with KHV at 24 h post exposure (hpe). However, unlike conventional PCR, RT-PCR could not detect KHV DNA in fish at 0 hpe. The results indicate that the RT-PCR developed in this study is mRNA-specific and that the assay can detect the replicating stage of KHV from both fish and cultured cells infected with the virus.

Identification of Edwardsiella ictaluri and E. tarda by species-specific polymerase chain reaction targeted to the upstream region of the fimbrial gene.

Phylogenetic analysis of nine strains of Edwardsiella ictaluri and eight strains of E. tarda (six typical motile strains and two atypical nonmotile strains) isolated from diseased fish was performed using the upstream region of the fimbrial gene cluster. Strains of E. ictaluri and E. tarda were significantly clustered into separate groups. Moreover, atypical E. tarda strains were clustered into a different group from the other strains. Three polymerase chain reaction (PCR) primer sets for differential detection of E. ictaluri as well as typical and atypical E. tarda were developed from the respective characteristic sequences. Strains of E. ictaluri, typical E. tarda, and atypical E. tarda were specifically detected by PCR using each primer set. No amplifications were observed after the use of these three primer sets with 25 other bacterial species, including fish pathogens. In addition, the three primer sets were able to detect the DNA of each target species from fish kidney and liver artificially infected with E. ictaluri or E. tarda.