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.

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.

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.

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.

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.

Microarray analyses of gene expression in Japanese flounder Paralichthys olivaceus leucocytes during monogenean parasite Neoheterobothrium hirame infection.

In this study, the gene expression patterns of peripheral blood leucocytes (PBL) from Japanese flounder Paralichthys olivaceus were analyzed during the course of monogenean parasite Neoheterobothrium hirame infection in order to select candidates for molecular biomarkers of infection. cDNA microarray analysis was performed to compare the gene expression patterns of PBL between infected and non-infected fishes. Among the 797 genes analyzed, 45 genes (5.6%) changed their expression levels. These genes included specific and non-specific immune-related genes (matrix metalloproteinase[MMP]-9, MMP-13, leukotriene B4 receptor, CD20 receptor, MHC [major histocompatibility complex] Class I, MHC Class II beta-chain, immunoglobulin light chain and immunoglobulin heavy chain). Significant up- and down-regulation of some unknown genes was also observed. Several candidates for infection-marker genes were selected for further study. These genes included MMP-9, MMP-13, leukotriene b4 receptor, CD20 receptor, immunoglobulin heavy chain, immunoglobulin light chain and unknown genes coded as B613, E25, LB3(8), WE2(3), WE8-18R and WF12-18R.

Gene expression of leucocytes in vaccinated Japanese flounder (Paralichthys olivaceus) during the course of experimental infection with Edwardsiella tarda.

In this paper, we focused on the detection of differentially expressed genes in peripheral blood leucocytes (PBL) during the course of Edwardsiella tarda infection in vaccinated and non-vaccinated Japanese flounder (Paralichthys olivaceus). cDNA microarray analysis was performed to compare the gene expression patterns of the PBL between the vaccinated and non-vaccinated fish in response to E. tarda inoculation. Fish were vaccinated twice, at a two-week interval and experimentally challenged with E. tarda two weeks after the second vaccination. Among the 1187 analyzed genes, 42 genes were up-regulated during the course of infection either in vaccinated or non-vaccinated fish. These genes included immune-related genes, such as MMP-9, MMP-13, CXC chemokine, CD20 receptor and hepcidin. Some immune-related genes were down-regulated after the E. tarda challenge, i.e. interferon inducible Mx protein, MHC class II-associated invariant chain, MHC class II alpha and MHC class II beta encoding genes, immunoglobulin light chain precursor, immunoglobulin light chain and IgM. These responses are thought to be a common reaction of Japanese flounder PBL in the course of edwardsiellosis, irrespective of immunized condition. Ten genes were significantly up-regulated only in vaccinated fish, and 11 genes were significantly up-regulated only in non-vaccinated fish. These genes may have a correlation with the efficacy of vaccination, although we have no evidence to link the different gene expression patterns and the efficacy of vaccination at present.

Virus-like particles associated with mass mortalities of the pen shell Atrina pectinata in Japan.

Mass mortalities of the pen shell Atrina pectinata occurred in the fishing grounds of Ariake Bay, in southwestern Japan, during late spring and summer in 2003 and 2004. Histological examination revealed extensive necrosis in the epithelial cells of the kidney and gill, and impairment of the endothelial cells of the mantle arteria. Although cestode larvae belonging to the genus Tylocephalum were found in the mantle, adductor muscle, kidney, and digestive gland, their prevalence and the intensity of infection were low. Examinations of moribund pen shells for Haplosporidium spp. infection using PCR analysis and for Perkinsus spp. infection using Ray's fluid thioglycollate medium were negative. Unenveloped virus-like particles were detected by transmission electron microscopy in the cytoplasm of affected kidney and gill cells of moribund pen shells. They were icosahedral spherical and 50 to 55 nm in diameter. These virus-like particles found in moribund pen shells are different from those described in other marine mollusks, and may be the causative agent of the mass mortalities of pen shells.

Role of cellular response in elimination of the monogenean Neoheterobothrium hirame in Japanese flounder Paralichthys olivaceus.

Adult worms of the blood-feeding monogenean parasite Neoheterobothrium hirame, which cause anemia in the Japanese flounder Paralichthys olivaceus, attach to the host fish by embedding their posterior part deeply into the host tissue. To investigate the possibility that cellular responses of the host fish can eliminate N. hirame, flounder were experimentally infected with N. hirame larvae and reared in either fed or starved conditions. Mature parasites were identified on the buccal cavity wall of the fish 33 d post-infection (Day 33). Monocytes/macrophages and granulocytes increased rapidly in the blood and infected sites after the appearance of mature parasites. These cells adhered to the tegument of the parasites. In addition, a few cells with large electron-dense granules (DGCs) were observed in the inflammatory foci. On Day 47, the tegument of some parasites collapsed partially and were phagocytosed by the infiltrated host cells. Some infiltrated cells adhered directly to the inner tissues of the parasites. On Day 54, in the fed fish group, the loss of the tegument led to damage of the parasites' inner tissue by a large number of infiltrated cells. In this group, the elimination of the parasites was noted from Day 47 to 54. These observations probably suggest that the cellular response of the host fish destructed the parasite's posterior part embedded in the tissue, thereby eliminating the parasites. On the other hand, a high mortality was observed in the starved group. The starved fish developed much more severe anemia than the fed fish, and the elimination of the parasites was not observed in this group. The results of the present study suggest that flounder can eliminate N. hirame if they are fed sufficiently.

Detection of white spot syndrome virus from stomach tissue homogenate of the kuruma shrimp (Penaeus japonicus) by reverse passive latex agglutination.

A reversed passive latex agglutination (RPLA) assay was developed for detecting the white spot syndrome virus (WSSV), which was formally named as penaeid rod-shaped DNA virus (PRDV) in Japan, from stomach tissue homogenate of the kuruma shrimp (Penaeus japonicus). Using high-density latex particles and specific polyclonal antibody, WSSV was detectable after 4h incubation. The hemolymph, the stomach, and the gills were extracted from a shrimp that had been infected experimentally with WSSV, the virus contained in each sample was tested by the PRLA and PCR assay. It was possible to detect the WSSV only from stomach tissue homogenates by the RPLA assay. And there was an agreement between RPLA and PCR assays for WSSV detection. Considering that the RPLA assay does not require biochemical expertise and latex reagents and all apparatus can be provided as a kit, this assay can be used for virus detection in the culture pond of shrimps or in the field as a convenient method.