16S rRNA targeted RT-PCR for the detection of Vibrio penaeicida, the pathogen of cultured kuruma prawn Penaeus japonicus.

Vibrio penaeicida is the causative bacterium of vibriosis in cultured kuruma prawn Penaeus japonicus in Japan. To develop a specific and sensitive method for the detection of the pathogen, a species-specific sequence in the 16S rRNA of V. penaeicida was determined and a polymerase chain reaction (PCR)-based method was devised on the basis of the sequence. Prior to sequencing, a part of the variable regions of the 16S rRNA was amplified by using primers designed from 2 conserved regions according to previously reported data on Vibrionaceae. The region of the 16S rRNA (nucleotide numbers 440 to 490 in Escherichia coli 16S rRNA) obtained by this procedure was found to be species-specific for V. penaeicida. It was confirmed that PCR and RT (reverse transcription)-PCR amplifications with a sense primer designed from the V. penaeicida-specific sequence were both able to differentiate V. penaeicida from other prawn-pathogenic vibrios. 16S rRNA-targeted RT-PCR was demonstrated to have 100 times higher sensitivity than 16S rDNA-targeted PCR and 10 fg of total nucleic acids extracted from cultured bacterial cells was sufficient to yield the visible fragment in gel electrophoresis. These results indicate that RT-PCR amplification with this primer is useful for specific and sensitive detection of V. penaeicida

Polymerase chain reaction (PCR) amplification of RNA of striped jack nervous necrosis (SJNNV)

The polymerase chain reaction (PCR) was used to amplify a portion of the coat protein gene (RNA2) of striped jack nervous necrosis vlrus (SJNNV), the causative agent of viral nervous necrosis (VNN) of larval striped jack Pseudocaranx dentex. Based on the sequence data of SJNNV RNA2, 2 forward (F1 and F2) and 3 reverse (RI. R2 and R3) PCR primers were synthesized and the 5 potential target regions were amplified with a combination of these primers. After reverse transcription of genomic RNA extracted from SJNNV and 25 cycles of PCR amplification, products of the expected size were detected separately on agarose gels stained with ethidium bromide. Southern blot hybridization confirmed that all of the amplified products were specific to cDNA of SJNNV RNA2. Two primer sets, F1-R2 and F2-R3, produced the specified 180 bp and 430 bp products. The PCR system, using the F2-R3 primer set, was able to detect 100 fg of SJNNV RNA after 25 cycles and was also able to efficiently amplify the target region of SJNNV gene in the total nucleic acids extracted from larval striped jack affected with VNN

Quantitative Detection of Viable Flavobacterium psychrophilum in Chum Salmon Oncorhynchus keta by Colony Blotting and Immunostaining

Quantitative detection of viable Flavobacterium psychrophilum, the etiological agent of bacterial coldwater disease, was evaluated by colony blotting and immunostaining. Bacterial colonies isolated from chum salmon Oncorhynchus keta ovarian fluids on a modified Cytophaga agar plate were blotted onto a nitrocellulose membrane and immunostained with antiserum against F. psychrophilum. Although the blotted colonies were strongly or weakly stained with the antiserum, blots from colonies of F. psychrophilum were distinguishable from those of other yellowish colonies by digital processing of the colony-blotted membrane photograph with an image-analyzing software. It was also confirmed that 12 strains of F. psychrophilum were all positive by the present method, while the subjected six isolates, which formed yellowish colonies but were not identified as F. psychrophilum by PCR targeting gyrB gene, and other reference six strains, F. branchiophilum, F. limicola, F. granuli, Pseudomonas flavescens, P. fluorescens, Chryseobacterium daecheongense, were all negative. From these results, the present procedure using colony blotting and immunostaining is useful for quantitative detection of viable F. psychrophilum from ovarian fluids and kidneys of chum salmon

Modification of KDM-2 with Culture-spent Medium for Isolation of Renibacterium salmoninarum

KDM-2 is a medium widely used for isolation of Renibacterium salmoninarum (R.s.), the causative agent of bacterial kidney disease (BKD). KDM-2 still has a problem for colonization of R.s. at low concentration levels. In the present study, we modified KDM by supplementation of the culture-spent medium of R.s. (SMRs) in substitution of FBS. No difference was observed in the growth rate of R.s. at ≥ 10^[3] cells/mL in KDM broth with 1% FBS regardless of SMRs supplementation. Growth rate of R.s. decreased at 10^[1] cells/mL of inoculation into KDM with 1% FBS, but it was recovered by supplementation of ≥ 1% SMRs into the medium. The activity of SMRs supporting bacterial growth was stable to treatment at 60℃ for 30 min and freezing at -20℃ for 7 days. At inoculation of ≤ 300 CFU of R.s., expected colony counts were obtained on the agar plates containing SMRs, while non or less than half of bacteria colonized on the agar plates without SMRs. It was thus considered that the modified KDM by supplementation of SMRs instead of FBS was convenient and inexpensive for isolation of R.s., especially at low concentration levels

Duration and Booster Effect of Phylactic Response against White Spot Syndrome Virus Infection in Kuruma Shrimp Orally Administrated with Recombinant Viral Proteins, rVP26 and rVP28

White spot syndrome virus (WSSV: a synonym of penaeid rod-shaped DNA virus, PRDV) is the causative agent of white spot disease (WSD: penaeid acute viremia, PAV), one of the most serious diseases affecting decapod crustaceans around the world. Recently, "quasi-immune response" was found in kuruma shrimp Penaeus japonicus, wherein individuals that naturally survived from WSD showed protection against a rechallenge with WSSV. The phylaxis against WSSV was also inducible by oral vaccination with recombinant WSSV proteins, rVP26 and rVP28. In the present study, kuruma shrimp orally vaccinated with rVPs were sequentially challenged with WSSV to evaluate onset and duration of phylactic response and booster effect. The phylactic response of shrimp against WSSV-challenge peaked at day 45 after the vaccination with rVP26 (RPS: 100%) and at day 55 with rVP28 (RPS: 93%), and decreased within 10-20 days. The phylaxis against WSSV-challenge was boosted by the secondary vaccination with homologous rVPs, but not by those with heterologous rVPs. The peaks of phylactic responses appeared at day 22 after the secondary vaccination more rapidly than those after the primary vaccination. These results demonstrated that the duration of phylaxis induced by oral vaccination with rVPs was relatively short, but could be extended by booster vaccination with homologous rVPs

An approach for genogrouping of Japanese isolates of aquabirnaviruses in a new genogroup, VII, based on the VP2/NS junction region

Aquabirnaviruses, represented by Infectious pancreatic necrosis virus (IPNV), have been isolated from epizootics in salmonids and a variety of aquatic animals in the world; six genogroups of aquabirnaviruses have been identified. In comparisons of nucleotide sequences of the VP2/NS junction region, maximum nucleotide diversities of 30·8 % were observed among 93 worldwide aquabirnavirus isolates. A phylogenetic tree revealed the existence of a new genogroup, VII, for Japanese aquabirnavirus isolates from marine fish and molluscan shellfish. Nucleotide diversities between genogroups VII and I–VI were 18·7 % or greater. At the nucleotide level, Japanese IPNV isolates from epizootics in salmonids were nearly identical to a genogroup I strain from the USA or Canada. It is suggested that Japanese IPNV isolates belonging to genogroup I were originally introduced from North American sources, whereas Japanese aquabirnavirus isolates of genogroup VII were from marine aquatic animals indigenous to Japan

Protection against white spot syndrome virus (WSSV) infection in kuruma shrimp orally vaccinated with WSSV rVP26 and rVP28

White spot syndrome virus (WSSV) is the causative agent of white spot disease (WSD), one of the most serious diseases affecting global shrimp farming. We compared WSSV infection induction in kuruma shrimp Marsupenaeus japonicus by oral, immersion, and intramuscular injection (IM) exposure methods and evaluated the oral vaccine prepared from the recombinant WSSV proteins rVP26 and rVP28. The 50% lethal doses (LD50) of WSSV by oral, immersion, and IM challenges were 10^[-0.4], 10^[-4.4], and 10^[-7.7] g shrimp^[-1], respectively, indicating that WSSV infection efficiency by oral challenge was significantly less than the other 2 challenge routes. However, in shrimp farms it is believed that WSSV infection is easily and commonly established by the oral route as a result of cannibalization of WSSV-infected shrimp. Kuruma shrimp vaccinated orally with WSSV rVP26 or rVP28 were challenged with WSSV by oral, immersion, and IM routes to compare protection efficacy. The relative percent survival values were 100% for oral challenge, 70 to 71% for immersion, and 34 to 61% for IM. Thus, the protection against WSSV-infection that was induced in kuruma shrimp by oral vaccination with rVP26 or rVP28 seemed equivalent to that obtained through IM vaccination

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 (≤10, 20, 40 and >40), and t 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 ≤10, 20 and 40. The offspring obtained from PCR-negative brood fish pairs in ea 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 ≥40, but did not occur in spawners with an antibody titer of ≤10. Therefore, it 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

Fish immunization using a synthetic double-stranded RNA Poly(I:C), an interferon inducer, offers protection against RGNNV, a fish nodavirus

Viral nervous necrosis (VNN), caused by a fish nodavirus, is one of the most serious fish diseases worldwide. Here we report a unique vaccination method in sevenband grouper Epinephelus septemfasciatus using a synthetic double-stranded RNA polyinosinic polycytidylic acid (Poly(I:C)), an interferon inducer, followed by challenge with a live fish nodavirus. Fish injected with Poly(I:C) at 200 µg fish(-1) were highly protected from artificial challenge with red-spotted grouper nervous necrosis virus (RGNNV) (relative percentage survival, RPS: 100%), and specific antibodies against RGNNV were detected in sera from survivors. Moreover, the surviving fish were protected from rechallenge with RGNNV (relative percent survival RPS: 100%). Thus, it was confirmed that specific immunity against RGNNV was established in sevenband grouper by injection with live RGNNV following Poly(I:C) administration. Antiviral state was induced in fish by injection with Poly(I:C) at ≥50 µg fish(-1), but no toxic response was observed in the fish even if Poly(I:C) was injected at a dose of 200 µg fish(-1). In fish injected with Poly(I:C) at 200 µg fish(-1), a high level of antiviral state of > 90% RPS against RGNNV challenge lasted for at least 4 d after Poly(I:C) injection. However, no curative effect by Poly(I:C) injection was observed in fish already infected with RGNNV. It is considered that the present immunization method using Poly(I:C) followed by a live virus injection could offer protection against various viral infections in a broader range of fish species