Detection of betanodaviruses from different organs of broodstocks and wild juveniles of orange-spotted grouper, Epinephelus coioides

Aquaculture of grouper including orange-spotted grouper suffers losses from viral nervous necrosis (VNN). This disease usually occurs in larval stage resulting in a high rate of mortality. It is conceivable that vertical transmission from subclinically infected broodstock fish to the brood is the major route of infection. However, there are only a few studies regarding the fish subclinically infected with VNN and the evidence for a causal relationship between subclinically infected adult fish and vertical transmission remains tenuous. The purpose of this study is to reveal the distribution of the virus in asymptomatically infected fish. Six healthy broodstocks of orange-spotted groupers with body weights ranging from 5 to 12 kg were used and 14 different organs were collected. Cell culture and PCR methods were used to detect the virus. Additionally, 8 wild juveniles with body weights ranging from 2 to 4 g were also analyzed. In our results, the virus was detected by means of PCR but not by the cell culture method. All the broodstocks and wild juveniles were positive in the PCR test except for one juvenile. The highest detection rate of the virus among organs was the brain. However, the virus gene was also amplified from organs other than the central nervous system (CNS). We hypothesize that the virus was latently retained in the CNS of subclinically infected fish and when the fish immune system was weakened such latent virus was released from CNS to the peripheral organs including the gonad. This mechanism might provoke the vertical transmission infection

Distribution of nervous necrosis virus in orange-spotted grouper Epinephelus coioides with asymptomatic infection

Distribution of nervous necrosis virus in asymptomatically infected orange-spotted grouper Epinephelus coioides was determined by PCR assay in this study. Fourteen different tissues and organs were collected from apparently healthy groupers including seven individuals of broodstock and 17 juveniles with body weight ranging from 4 to 12 kg and from 2 to 9 g, respectively. No cytopathic effects were found in E-11 cells inoculated with filtrates of sample homogenates. However, all the broodstock and juveniles were positive by nested PCR test except for one juvenile. One hundred percent detection rate of the virus was obtained only from the brain.This study was supported by funds from Japan International Research Center for Agricultural Sciences (JIRCAS) Research Project "Studies on Sustainable Production Systems of Aquatic Animals in Brackish Mangrove Area"

Viral nervous necrosis (VNN) as a critical infectious disease of orange-spotted grouper, Epinephelus coioides, in the Philippines

Orange-spotted grouper, Epinephelus coioides, is a valuable commodity in the Philippines. In 2001, mass mortality occurred in the grouper larvae at Aquaculture Department, Southeast Asian Fisheries Development Center (SEAFDEC/AQD) and the disease was identified as viral nervous necrosis (VNN). Since then, the disease has been observed every year and the grouper hatcheries have been devastated. In this paper, recent studies of VNN which were conducted at the SEAFDEC/AQD from 2001 to 2006 are reviewed. 1) Susceptibility to the VNN virus was tested among fish species that were cultured in mangrove brackish are. Five representative cultured fish species including orange-spotted grouper, Asian sea bass (Lates calcarifer), mangrove red snapper (Lutjanus argentimaculatus), milkfish (Chanos chanos) and rabbitfish (Siganus guttatus) were used in the test where the virus was intraperitoneally injected into the juveniles. Although low or no mortality occurred in the challenge test, histopathological changes were observed in the brain and retina where the virus was re-isolated. The results were the same among the species except for rabbitfish which had no evidence for the infection. It was verified that the virus has a wide host range. 2) To estimate the possible risk of viral spread by vertical transmission, virus distribution was determined in asymptomatic groupers including 7 broodstock and 17 juveniles with body weights ranging from 4 to 12 kg and 2 to 9 respectively. The virus was detected by PCR method. The highest detection rate was in the brain, and the virus was also detectable in other organs such as the gills, heart, spleen, kidney, blood, esophagus, stomach, intestine, liver, gonad, swim bladder and/or skin. 3) As a possible VNN vaccine, a DNA p;asmid encoding the capsid protein of the virus was evaluated. After the challenge, the mortalities between the native and DNA-injected fish appeared significantly different (P<0.05).This study was supported by funds from Japan International Research Center for Agricultural Sciences (JIRCAS) Research Project "Studies on Sustainable Production Systems of Aquatic Animals in Mangrove Brackish Areas"

Comprehensive validation of T- and B-cell deficiency in rag1-null zebrafish: Implication for the robust innate defense mechanisms of teleosts

Abstractrag1−/− zebrafish have been employed in immunological research as a useful immunodeficient vertebrate model, but with only fragmentary evidence for the lack of functional adaptive immunity. rag1-null zebrafish exhibit differences from their human and murine counterparts in that they can be maintained without any specific pathogen-free conditions. To define the immunodeficient status of rag1−/− zebrafish, we obtained further functional evidence on T- and B-cell deficiency in the fish at the protein, cellular, and organism levels. Our developed microscale assays provided evidence that rag1−/− fish do not possess serum IgM protein, that they do not achieve specific protection even after vaccination, and that they cannot induce antigen-specific CTL activity. The mortality rate in non-vaccinated fish suggests that rag1−/− fish possess innate protection equivalent to that of rag1+/− fish. Furthermore, poly(I:C)-induced immune responses revealed that the organ that controls anti-viral immunity is shifted from the spleen to the hepatopancreas due to the absence of T- and B-cell function, implying that immune homeostasis may change to an underside mode in rag-null fish. These findings suggest that the teleost relies heavily on innate immunity. Thus, this model could better highlight innate immunity in animals that lack adaptive immunity than mouse models.</jats:p

Complementary approaches to diagnosing marine diseases: a union of the modern and the classic

Linking marine epizootics to a specific etiology is notoriously difficult. Recent diagnostic successes show that marine disease diagnosis requires both modern, cutting-edge technology (e.g. metagenomics, quantitative realtime PCR) and more classic methods (e.g. transect surveys, histopathology and cell culture). Here, we discuss how this combination of traditional and modern approaches is necessary for rapid and accurate identification of marine diseases, and emphasize how sole reliance on any one technology or technique may lead disease investigations astray. We present diagnostic approaches at different scales, from the macro (environment, community, population and organismal scales) to the micro (tissue, organ, cell and genomic scales). We use disease case studies from a broad range of taxa to illustrate diagnostic successes from combining traditional and modern diagnostic methods. Finally, we recognize the need for increased capacity of centralized databases, networks, data repositories and contingency plans for diagnosis and management of marine disease

Comprehensive validation of T- and B-cell deficiency in rag1-null zebrafish: Implication for the robust innate defense mechanisms of teleosts

rag1(-/-) zebrafish have been employed in immunological research as a useful immunodeficient vertebrate model, but with only fragmentary evidence for the lack of functional adaptive immunity. rag1-null zebrafish exhibit differences from their human and murine counterparts in that they can be maintained without any specific pathogen-free conditions. To define the immunodeficient status of rag1(-/-) zebrafish, we obtained further functional evidence on T- and B-cell deficiency in the fish at the protein, cellular, and organism levels. Our developed microscale assays provided evidence that rag1(-/-) fish do not possess serum IgM protein, that they do not achieve specific protection even after vaccination, and that they cannot induce antigen-specific CTL activity. The mortality rate in non-vaccinated fish suggests that rag1(-/-) fish possess innate protection equivalent to that of rag1(+/-) fish. Furthermore, poly(I:C)-induced immune responses revealed that the organ that controls anti-viral immunity is shifted from the spleen to the hepatopancreas due to the absence of T- and B-cell function, implying that immune homeostasis may change to an underside mode in rag-null fish. These findings suggest that the teleost relies heavily on innate immunity. Thus, this model could better highlight innate immunity in animals that lack adaptive immunity than mouse models