Norihisa Oseko

Protection of rainbow trout from infectious hematopoietic necrosis (IHN) by injection of infectious pancreatic necrosis virus (IPNV) or Poly(I:C)

It was recently reported that prophylaxis against infectious hematopoietic necrosis virus (IHNV) in fish was induced by pre-exposure to the infectious pancreatic necrosis virus (IPNV). Here the establishment of IHNV immunity in rainbow trout Oncorhynchus mykiss was investigated by IHNV challenge following non-lethal pre-infection with IPNV. Also, synthetic double-stranded RNA polyinosinic polycytidylic acid, Poly(I:C), an inducer for interferon (IFN), was evaluated as a substitute for IPNV induction of the non-specific antiviral state and subsequent IHNV-specific immunity in fish. Rainbow trout pre-infected with IPNV were protected from IHNV challenge 7 d later (relative percentage survival, RPS: 68.8%), and IHNV-specific antibodies were detected in sera from the survivors. Moreover, these surviving fish showed 91.6% RPS when re-challenged with IHNV 28 d after the primary IHNV challenge. Thus, fish appear to acquire IHNV-specific immunity through the IHNV challenge following pre-injection with IPNV. Fish pre-injected with Poly(I:C) were also highly protected from IHNV challenge 2 d later (RPS: 95.2%), and IHNV-specific antibodies were also detected amongst survivors. The survivors showed a 100% survival rate following re-challenge with IHNV both 21 and 49 d after the primary IHNV challenge. Thus, IHNV immunity in rainbow trout is induced by challenge with live IHNV following pre-injection with either IPNV or Poly(I:C). The use of Poly(I:C) to induce an anti-viral state protecting rainbow trout from an otherwise lethal vaccination dose of IHNV may have application to a wider range of fish species and fish pathogenic viruses.

The efficacy of five avirulent Edwardsiella tarda strains in a live vaccine against Edwardsiellosis in Japanese flounder, Paralichthys olivaceus.

We evaluated the tissue persistence and live vaccine efficacy of five avirulent Edwardsiella tarda strains (E22, SU100, SU117, SU138, and SU244) isolated from the Japanese eel (Anguilla japonica) and from the environment. The live vaccines, containing a single strain, were injected intraperitoneally into Japanese flounder (Paralichthys olivaceus). Viable bacteria from all the strains (excluding SU100) were recovered from trunk-kidney tissue 28 d post-injection. Japanese flounder inoculated with E22 had the highest relative percentage survival (RPS = 45%) in an artificial challenge with virulent E. tarda (NUF806). The serum of E22-vaccinated fish had a significantly higher agglutination titer against NUF806. In contrast, there was little or no increase in the agglutination titer of the fish that were inoculated with the remaining avirulent strains. Injection with avirulent E. tarda increased the expression of cytokine genes, including interleukin-1beta (IL-1beta), type 1 interferon (IFN), and IFN-gamma in head-kidney of the Japanese flounder.

Mass mortality of giant abalone Haliotis gigantea caused by a Francisella sp. bacterium.

In February 2005, a mass mortality of giant abalone Haliotis (Nordotis) gigantea Gmelin, 1791 occurred on a private abalone farm in Shimane Prefecture, Japan. The cumulative mortality rate reached about 84%. In histological observations, bacteria-like spherical particles were found in affected animals, suggesting a bacterial infection. Many of the bacteria-like particles were found in the cells that were presumably host phagocytes. DNA was extracted from the hemolymph of a diseased abalone and a bacterial 16S rRNA gene was amplified by PCR. The bacterium was classified within the genus Francisella by gene sequence analysis. A bacterial isolate was obtained by spreading hemolymph of a diseased abalone on modified Eugon agar dissolved in 70% seawater containing 1% (w/v) hemoglobin. A gene fragment of the expected size was amplified from the bacterial isolate by PCR using specific primers for the 16S rRNA gene obtained from the diseased abalone. Experimental infections were carried out by intramuscular injection with the bacterial isolate or by immersion in the bacterial suspension using 2 species of abalone, the giant abalone and the Japanese black abalone Haliotis (Nordotis) discus discus Reeve, 1846. Most (98.6%) of the abalone challenged with the bacterial isolate died in experimental infections. These results suggest that the Francisella sp. isolate was the causative agent for the mass mortality of giant abalone. This is the first report of a pathogenic Francisella sp. isolate for mollusks.

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.

Rhabdovirus Olivaceus (Hirame Rhabdovirus)

Hirame rhabdovirus (HRV) is a fish rhabdovirus discovered from a moribund hirame (Japanese flounder, Paralichthys olivaceus) cultured in Hyogo Prefecture, Japan, at 1984. Characteristics of HRV have been found sufficiently distinct from other known fish rhabdoviruses; this virus has been named Rhabdovirus olivaceus, derived from the scientific name of the host species. This virus is pathogenic for hirame, black sea bream (Milio macrocephalus), red sea bream (Crysophrys major) and black rock fish (Sebastes inermis) among oceanic fishes, and also the salmonid species, especially rainbow trout. HRV has been found widely distributed in Japan.

Comparative Proteomic Analysis Between Virulent and Less Virulent Strains of Edwardsiella ictaluri Isolated from Ayu Plecoglossus altivelis

Outbreaks of disease caused by the infection of Edwardsiella ictaluri in ayu Plecoglossus altivelis have been reported in rivers in Japan since 2007. In this study, we performed comparative proteomic analysis between the virulent strain FPC1091 and the less virulent strain FPC1214 to identify virulence factors of E. ictaluri to ayu. In the experimental infection, cumulative mortalities of the ayu intraperitoneally injected with FPC1091 (1.2 × 10 CFU/fish) and FPC1214 (2.2 × 10 CFU/fish) were 100% and 26.7%, respectively. The genetic fingerprint profile of FPC1214 was identical to that of FPC1091 in the amplified-fragment length polymorphism (AFLP) analysis. The analysis of two-dimensional polyacrylamide gel electrophoresis and mass spectrometry between FPC1091 and FPC 1214 revealed 23 proteins that were either specific to or produced in greater amounts in the virulent strain FPC1091. These proteins included components of the type III and type VI secretion systems, and the proteins that seem to be involved in the resistance against oxidative stress by host phagocytic cells, suggesting that these proteins are the virulence factors of E. ictaluri to ayu.