Standardized white spot syndrome virus (WSSV) inoculation procedures for intramuscular or oral routes

In the past, strategies to control white spot syndrome virus (WSSV) were mostly tested by infectivity trials in vivo using immersion or per os inoculation of undefined WSSV infectious doses, which complicated comparisons between experiments. In this study, the reproducibility of 3 defined doses (10, 30 and 90 shrimp infectious doses 50% endpoint [SID50] of WSSV was determined in 3 experiments using intramuscular (i.m.) or oral inoculation in specific pathogen-free (SPF) Litopenaeus vannamei. Reproducibility was determined by the time of onset of disease, cumulative mortality, and median lethal time (LT50). By i.m. route, the 3 doses induced disease between 24 and 36 h post inoculation (hpi). Cumulative mortality was 100% at 84 hpi with doses of 30 and 90 SID50 and 108 hpi with a dose of 10 SID50. The LT50 of the doses 10, 30 and 90 SID50 were 52, 51 and 49 hpi and were not significantly different (p > 0.05). Shrimp orally inoculated with 10, 30 or 90 SID50 developed disease between 24 and 36 hpi. Cumulative mortality was 100% at 108 hpi with doses of 30 and 90 SID50 and 120 hpi with a dose of 10 SID50. The LT50 of 10, 30 and 90 SID50 were 65, 57 and 50 hpi; these were significantly different from each other (p 50 was selected as the standard for further WSSV challenges by i.m. or oral routes. These standardized inoculation procedures may be applied to other crustacea and WSSV strains in order to achieve comparable results among experiment

Tracing sulfate recycling in the hypersaline Pétrola lake (SE Spain): A combined isotopic and microbiological approach

Sulfur (S) plays a significant role in saline environments, and sulfate (SO4 2−) is an important component of the biogeochemical S-cycle since it acts as the main electron acceptor in anoxic sediments. The purpose of this paper is to evaluate the fate of S, its origin, and processes affecting sulfate outcome in the hypersaline Pétrola Lake in the Castilla-La Mancha region (High Segura Basin, SE Spain). The lake is the terminal discharge zone of an endorheic basin with considerable anthropogenic pressures. Anthropogenic activities (mainly agricultural inputs and wastewater discharge), together with bedrock leaching of sulfate and sulfide-rich sediments, increase dissolved SO4 2− in surface and groundwater up to 123,000 mg/L. The source and fate of sulfate in this environment was investigated coupling hydrochemistry, including hydrogen sulfide (H2S) microprofiles, isotopic analyses (δ34S, δ18OSO4, δ2HH2O, δ18OH2O, and tritium), mineralogical determinations, and molecular biology tools (16S rDNA amplification and sequencing). The origin of dissolved SO4 2− in water is related to pyrite oxidation from Lower Cretaceous sediments, and secondary gypsum dissolution. Under the lake, dissolved SO4 2− decreases with depth, controlled by three main processes: (1) seasonal evaporation cycles, (2) hydrodynamic instability caused by the different density-driven groundwater flow, and (3) sulfate-reduction processes, i.e. dissimilatory bacterial sulfate reduction (BSR). These processes control the continuous recycling of sulfur in the system. Lake water and groundwater are in hydraulic connection, and a density-driven flow (DDF) is able to transport reactive organic matter and dissolved SO4 2− towards the underlying aquifer. Hydrochemical evolution in depth, H2S production (up to 0.024 nmol/cm3·s) and the presence of sulfate-reducing bacteria suggest the existence of BSR processes. However, isotope techniques are insufficient to elucidate BSR processes since their isotopic effect is masked by low isotope fractionation and high SO4 2− concentrations. The pattern here described may be found in other saline basins worldwide

The effect of raising water temperature to 33°C in <i>Penaeus vannamei</i> juveniles at different stages of infection with white spot syndrome virus (WSSV)

This study investigated the effect of high water temperature (33°C) at different stages of infection with a highly virulent and low virulent white spot syndrome virus strain (WSSV Thai-1 and WSSV Viet) in Penaeus vannamei juveniles. Shrimp were inoculated intramuscularly with either a high dose (HD) or low dose (LD). Water temperature was kept either at continuously 27°C or switched from 27°C to 33°C at 0, 12 or 24 h post inoculation (hpi) for both strains and in addition at 48 or 96 hpi for WSSV Viet. The increased temperature 33°C was maintained till the end of the experiments (120–144 hpi with WSSV Thai-1 and 240 hpi with WSSV Viet). To determine the infection status at the moment of temperature increase, five shrimp that were kept continuously at 27 °C were euthanized at 0, 12, 24, 48 and 96 hpi with each dose of two strains. WSSV infections (viral antigen VP28) in dead and euthanized shrimp were demonstrated by indirect immunofluorescence.Shrimp inoculated with HD or LD of WSSV Thai-1 and kept continuously at 27°C till euthanasia were 100% viral antigen positive from 12 (HD) or 24 hpi (LD). Shrimp inoculated with WSSV Viet were 100% positive from 24 (HD) and 48 hpi (LD). Shrimp kept at 27°C, showed clinical signs from 24 (HD) or 24–36 hpi (LD) with both strains. Cumulative mortalities reached 100% with WSSV Thai-1 at 60 (HD) or 84–144 hpi (LD) and with WSSV Viet 100% at 216 hpi (HD) or 90% at 240 hpi (LD). Switch of temperature to 33°C from 0, 12 or 24 hpi was effective in reducing mortality of shrimp inoculated with the LD of both strains and with the HD of WSSV Viet. The switch to 33°C from 24 hpi with the Thai strain (HD) and from 48 and 96 hpi with the Viet strain (LD or HD) had no effect or even accelerated the mortality rate (80–100%). All shrimp were viral antigen positive at death and euthanasia (one shrimp LD WSSV Viet) when kept continuously at 27°C. All dead and euthanized shrimp kept at 33°C from 0 or 12 hpi were viral antigen negative. With 33°C from 24, 48 or 96 hpi, all dead shrimp were viral antigen positive and euthanized shrimp were negative.This study showed that 33°C is effective to prevent disease, reduce mortality and block WSSV replication, but only in the early stages of infection

Rahman M.M., Escobedo-Bonilla C.M., Corteel M., Wille M., Alday-Sanz V., Pensaert M.B., Sorgeloos P. and Nauwynck H.J. Evaluation of virulence of three white spot shrimp Litopenaeus vannamei. Aqua 2006, Joint International Conference & Exhibition of European Aquaculture Society & World Aquaculture Society. Florence, Italy

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