Nervous necrosis virus titration and antigen quantitation by indirect sandwich enzyme linked immunosorbent assay.

Viral nervous necrosis (VNN) is a serious disease of marine and brackishwater fishes caused by nervous necrosis virus (NNV) resulting in up to 100% mortality in early larval and juvenile stages. Adult fish when infected are asymptomatic and spread the virus vertically to the offspring through milt and eggs. Prevention of vertical transmission of the disease is by using disease free broodstock and vaccinating the brooders. Estimation of antigen content and virus titre is essential to determine antigen/virus concentration in VNN vaccine. A monoclonal antibody based indirect sandwich ELISA was developed to quantify the NNV antigen and to estimate the virus titre by TCID50 coupled ELISA. Mouse hybridoma clones secreting monoclonal antibodies (MAb) against the capsid protein of NNV was developed and characterised. The antibodies reacted specifically with the recombinant capsid protein and purified virus in western blot. Polyclonal antibodies against NNV were used as capture antibodies and MAbs were used as detection antibodies to optimise an indirect sandwich ELISA to detect and quantify capsid protein of NNV. The developed assay had a sensitivity of 390 ng/ml and could detect the virus in clinical samples. The assay coupled with TCID50 could be used to estimate the virus titre rather than by observing the CPE which is laborious and subjective.

The effect of acclimation temperature and optimal temperature gradient for egg and larvae of silver moony (Monodactylus argenteus) during the early ontogenesis.

The early life history of a fish species is regulated by temperature, the most critical environmental cue. Thus, identifying the gradient of temperature that optimises the early development of a species is a prerequisite for standardising hatchery technology. Silver moony, Monodactylus argenteus is a tropical brackishwater ornamental fish that holds scope for the Indian and Global ornamental fish industry. This study unravels the effect of water temperature increments (26, 28, 30, 32, and 34 °C) on embryonic development, hatching, and survival rate, as well as the growth profile and survival rate of larvae at 5 days post-hatch (5 dph). Experiments were conducted to find out the optimal temperature gradient for egg incubation and early larval rearing. The experiment results revealed that the embryogenesis was accelerated at increasing temperatures, especially after the gastrula stage, and apparent differences were evident in each stage. However, the morphological development profile at each embryonic stage was similar throughout the temperature range. The incubation period differed significantly (P < 0.05) between the temperature gradients. The highest rates of hatching (90-100%) and survival after 12 hph were observed at 28 ºC and 30 ºC. Hatched-out larvae demonstrated the highest total length (1.92 ± 0.02 µm) at 34 °C, and the total length decreased at lower temperature levels. The yolk sac volume of larvae was shrunken with an increase in temperature, and a significant difference was observed between the studied temperatures. However, the oil globule diameter did not differ between the different temperatures. The total length and growth rate of 5 dph larvae were significantly different among the temperature treatments and increased with increasing temperature. In contrast, the survival rate of 5 dph larvae was highest at the range of 26 ºC and 30 ºC. The results indicated that the change in temperature from the spawning temperature (29 ± 1 °C) negatively influenced embryogenesis and the early development of M. argenteus. Based on the experimental results, the growth and survival of embryo and larvae were found to be optimum at 28 to 30 ºC. This prediction is of great importance for the effective management in the hatchery production phase, and especially the temperature could be considered the critical environmental cue.

Gonadal recrudescence and annual reproductive hormone pattern of captive female Spotted Scats (Scatophagus argus).

The Spotted Scat (Scatophagus argus), an important euryhaline fish inhabiting mangrove and coastal regions of Indo-Pacific waters, is both an ornamental and food fish in India. Detailed insight into maturation of Spotted Scat when maintained in aquaculture systems, therefore, needs to be elucidated. Lack of information on annual maturation dynamics of female scat collected from their natural habitat and reared in earthen ponds is the basis of this study. Oocytes were classified into five developmental stages: pre-vitellogenic, vitellogenic, late-vitellogenic, ripe, and follicular atresia. Ovarian maturity stages were subsequently categorized as immature (Stage 1), vitellogenesis (Stage 2), maturing (Stage 3), mature (Stage 4), and spent (Stage 5). In oocytes in primary, secondary and tertiary yolk stages, there are greater concentrations of E2 in vitellogenic females between March and June. Significant increases of E2, T, and 17-OHP paralleled the increase of diameter of late-vitellogenic oocytes in maturing females during July. The completion of vitellogenesis and initiation of germinal vesicle migration in the cytoplasm were evident in mature females (Stage 4) with a decreasing trend of sex steroids in and subsequent to the month of August. There were 50 % of oocytes in the final oocyte maturation stage (FOM) (490-620 µm) until completion of Stage 4 in September. The results of this study indicate there is complete ovarian maturation in female scats captured in their natural habitat and maintained in an earthen pond, which may be important information for hatchery management for induction of spawning of Spotted Scat in aquaculture systems.

Resistance of pearlspot larvae, Etroplus suratensis, to redspotted grouper nervous necrosis virus by immersion challenge.

Viral nervous necrosis (VNN) affects more than 120 species mostly belonging to the order Perciformes. However, none of the brackishwater species belonging to the family Cichlidae under the order Perciformes are reported to be susceptible. Hence, the present experiment was undertaken to study the susceptibility of the brackishwater cichlid, pearlspot, Etroplus suratensis to NNV. Thirty-day-old pearlspot larvae were infected with NNV by immersion. Mortality was recorded till 14 days post-infection, and the infected larvae were subjected to nested RT-PCR and histology. The virus was isolated from infected larvae using SSN-1 cells. To study the replication of the virus in vitro, primary cultured brain cells of E. suratensis and IEK cells were infected with NNV. No mortality was observed in any of the control or experimentally infected larvae. However, the experimentally infected larvae were positive for NNV by nested RT-PCR and the virus was isolated using SSN-1 cells. Further, the infected pearlspot brain cells and IEK cells showed cytopathic effect at second and third passage of the virus and they were positive for NNV by nested RT-PCR. Pearlspot is relatively resistant to VNN although the virus could replicate in the larvae and in cell culture.