Immune gene expression and protective effects in goldfish (Carassius auratus L.) immunized with formalin-inactivated cyprinid herpesvirus-2 (CyHV-2) vaccine.

The goldfish hematopoietic necrosis viral disease (GHNVD) has led to worldwide economic losses in goldfish aquaculture. The present study has focused on the development of an inactivated vaccine for the cyprinid herpesvirus (CyHV-2) and to check the immunogenicity of the vaccine in the host. The fantail goldfish fin (FtGF) cell line was used in the propagation of the CyHV-2 and the viral titer obtained were of 107.8 TCID50/ml. Followed by the virus was inactivated using 0.1% formalin for 2 days. Various concentrations of formalin-inactivated CyHV-2 (1%, 0.7%, 0.5%, 0.3% and 0.1%) were studied in the FtGF cell line. Morphological changes were observed in the FtGF cell line in all other concentrations of formalin except 0.1% formalin-inactivated CyHV-2 vaccine. The goldfishes were intraperitoneally injected with 300 µl of vaccine and various immune gene responses were studied for a period of 30 days. The gene expression of the adaptive markers CD8, CD4, IFN-ϒ, the cytokines (IL-10, IL-12) was studied in kidney and spleen tissues. Formalin-inactivated CyHV-2 vaccine showed a significant up-regulation of the genes CD8 and IFN-ϒ by the 6th hr post-vaccination onwards. The experimental fish were challenged intraperitoneally with CyHV-2 virus of concentration 107.8 TCID50/ml after 30 days of post-vaccination. A significant difference in cumulative mortality rate was observed for the vaccinated fishes from the unvaccinated fishes. The relative percent survival for formalin immunized fish was 74.03%. Our results have proven that the formalin-inactivated vaccines were efficient and it resulted in triggering the immune gene expression in goldfish. The development and further enhanced studies for this vaccine will lead to a promising low-cost commercial vaccine for CyHV-2 viral infection.

Quick hassle-free detection of cyprinid herpesvirus 2 (CyHV-2) in goldfish using recombinase polymerase amplification-lateral flow dipstick (RPA-LFD) assay.

Cyprinid herpesvirus 2 (CyHV-2) is the etiological agent of herpesviral hematopoietic necrosis disease (HVHND), which causes severe mortality in ornamental goldfish (Carassius auratus), crucian carp (Carassius auratus), and gibel/prussian carp (Carassius gibelio). Quick and hassle-free point-of-care detection of CyHV-2 is vital for the maintenance of ornamental fish health. In this manuscript, we describe the development of a rapid and sensitive RPA (recombinase polymerase amplification) assay, coupled with lateral flow dipsticks (LFD), that can achieve sensitive diagnosis of CyHV-2 in goldfish within 20 min at 36 °C with the satisfactory detection limit of 102 gene copies per reaction. This is the first report wherein major capsid protein (MCP) of CyHV-2 was targeted for RPA-LFD assay development. The assay did not show any cross-reactivity with other viral pathogens like cyprinid herpesvirus 3 (CyHV-3), spring viremia of carp virus (SVCV), infectious spleen and kidney necrosis virus (ISKNV), and viral nervous necrosis virus (VNNV). Furthermore, screening of CyHV-2 infection in CyHV-2-infected goldfish did not yield any false positive/negative results. In short, the RPA-LFD assay developed in this study presents a simple, rapid, and sensitive method for point-of-care diagnosis of CyHV-2, especially under resource-limited conditions.

A peek into mass mortality caused by antimicrobial resistant Edwardsiella tarda in goldfish, Carassius auratus in Kerala.

Edwardsiella tarda is one of the serious threats affecting the worldwide aquaculture. In the present study, four isolates were recovered from diseased goldfish, showing hemorrhages, reported with 60% mass mortality in an ornamental fish farm, Ernakulam, Kerala. Based on the phenotypic and genotypic analysis, the bacteria were identified as Edwardsiella tarda, Citrobacter freundii, Acinetobacter junii and Comammonas testosteronii. Experimental challenge studies using healthy goldfish revealed that among the four isolates, E. tarda alone leads to 100% mortality of experimental fish within 175 degree days and the pathogen could be successfully re-isolated from the moribund fish. The LD50 value of E. tarda was calculated as 9.9 × 105 CFU/fish. The histopathology of the infected tissues of goldfish had shown the typical features of E .tarda infection. The pathogen was found positive for the virulence genes viz., hly, etfA, etfD and eseD as detected using PCR. Thus E. tarda was confirmed as the real causative agent of the disease outbreak. Multiple antimicrobial resistance (AMR) exhibited by the pathogen towards 19 tested antibiotics with the MAR index of 0.46 highlighted the exposure of antibiotics to the fish in the farm. The existence of antibiotic resistant genes within the plasmid as revealed through plasmid curing studies pointed out the possibility of rapid dissemination of AMR in aquaculture. Hence proper surveillance and appropriate diagnostic methods need to be implemented at regular intervals to mitigate the menace.

Infectious spleen and kidney necrosis virus-associated large-scale mortality in farmed giant gourami, Osphronemus goramy, in India.

Megalocytivirus cause diseases that have serious economic impacts on aquaculture, mainly in East and South-East Asia. Five primary genotypes are known: infectious spleen and kidney necrosis virus (ISKNV), red sea bream iridovirus (RSIV), turbot reddish body iridovirus (TRBIV), threespine stickleback iridovirus (TSIV) and scale drop disease virus (SDDV). ISKNV-mediated infectious spleen and kidney necrosis disease (ISKND) is a major viral disease in both freshwater and marine fish species. In this study, we report the isolation of ISKNV from diseased giant gourami, Osphronemus goramy, in India. Transmission electron microscopy of ultrathin sections of kidney and spleen revealed the presence of numerous polygonal naked viral particles having an outer nucleocapsid layer within the cytoplasm of enlarged cells (115-125 nm). Molecular and phylogenetic analyses confirmed the presence of ISKNV and the major capsid protein (MCP) (1,362 bp) gene in the infected fish had a high similarity to the other ISKNV-I isolates. Moreover, ISKNV was propagated in the Astronotus ocellatus fin (AOF) cell line and further confirmed genotypically. A high mortality rate (60%) was observed in gourami fish injected with ISKNV-positive tissue homogenate through challenge studies. Considering the lethal nature of ISKNV, the present study spotlights the implementation of stringent biosecurity practices for the proper control of the disease in the country.