Crassostrea gigas oysters as a shrimp farm bioindicator of white spot syndrome virus.

This study explored whether Crassostrea gigas oysters can be used as a bioindicator of white spot syndrome virus (WSSV) in shrimp farm water canals. Bioassays showed that C. gigas can accumulate WSSV in their gills and digestive glands but do not become infected, either by exposure to seawater containing WSSV or by cohabitation with infected shrimp. The use of a WSSV nested PCR to screen oysters placed in water canals at the entry of a shrimp farm allowed WSSV to be detected 16 d prior to the disease occurring. The finding that C. gigas can concentrate small amounts of WSSV present in seawater without being harmed makes it an ideal sentinel species at shrimp farms.

Virulence of white spot syndrome virus (WSSV) isolates may be correlated with the degree of replication in gills of Penaeus vannamei juveniles.

A standardized inoculation model was used in 2 separate experiments to gauge the virulence of 3 white spot syndrome virus (WSSV) isolates from Thailand and Vietnam (WSSV Thai-1, WSSV Thai-2, and WSSV Viet) in Penaeus vannamei juveniles. Mortality patterns (Expt 1) were compared and WSSV-positive cells quantified (Expt 2) in tissues following intramuscular inoculation of shrimp with the most (WSSV Thai-1) and least (WSSV Viet) virulent isolates as determined by Expt 1. The results of Expt 1 demonstrated that mortalities began at 36 h post inoculation (hpi) for both Thai isolate groups and at 36 to 60 hpi for the Viet isolate group. Cumulative mortality reached 100% 96 to 240 h later in shrimp challenged with the WSSV Viet isolate compared to shrimp challenged with the Thai isolates. WSSV infection was verified in all groups by indirect immunofluorescence. In Expt 2, WSSV-infected cells were quantified by immunohistochemical analysis of both dead and time-course sampled shrimp. WSSV-positive cells were detected in tissues of Thai-1 inoculated dead and euthanized shrimp from 24 hpi onwards and from 36 hpi onwards in shrimp injected with the Viet isolate. Significantly more infected cells were found in tissues of dead shrimp inoculated with the Thai-1 than in Viet isolate-inoculated shrimp. In these experiments, substantial differences in virulence were demonstrated between the WSSV isolates. The Vietnamese isolate induced a more chronic disease and mortality pattern than was found for the Thai isolates, possibly because it infected fewer cells. This difference was most pronounced in gills.

Pathogenesis of a Thai strain of white spot syndrome virus (WSSV) in juvenile, specific pathogen-free Litopenaeus vannamei.

White spot syndrome virus (WSSV) causes disease and mortality in cultured and wild shrimp. A standardized WSSV oral inoculation procedure was used in specific pathogen-free (SPF) Litopenaeus vannamei (also called Penaeus vannamei) to determine the primary sites of replication (portal of entry), to analyze the viral spread and to propose the cause of death. Shrimp were inoculated orally with a low (10(1.5) shrimp infectious dose 50% endpoint [SID50]) or a high (10(4) SID50) dose. Per dose, 6 shrimp were collected at 0, 6, 12, 18, 24, 36, 48 and 60 h post inoculation (hpi). WSSV-infected cells were located in tissues by immunohistochemistry and in hemolymph by indirect immunofluorescence. Cell-free hemolymph was examined for WSSV DNA using 1-step PCR. Tissues and cell-free hemolymph were first positive at 18 hpi (low dose) or at 12 hpi (high dose). With the 2 doses, primary replication was found in cells of the foregut and gills. The antennal gland was an additional primary replication site at the high dose. WSSV-infected cells were found in the hemolymph starting from 36 hpi. At 60 hpi, the percentage of WSSV-infected cells was 36 for the epithelial cells of the foregut and 27 for the epithelial cells of the integument; the number of WSSV-infected cells per mm2 was 98 for the gills, 26 for the antennal gland, 78 for the hematopoietic tissue and 49 for the lymphoid organ. Areas of necrosis were observed in infected tissues starting from 48 hpi (low dose) or 36 hpi (high dose). Since the foregut, gills, antennal gland and integument are essential for the maintenance of shrimp homeostasis, it is likely that WSSV infection leads to death due to their dysfunction.

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 < 0.05). A dose of 30 SID50 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 experiments.

In vivo titration of white spot syndrome virus (WSSV) in specific pathogen-free Litopenaeus vannamei by intramuscular and oral routes.

White spot syndrome virus (WSSV) is a devastating pathogen in shrimp aquaculture. Standardized challenge procedures using a known amount of infectious virus would assist in evaluating strategies to reduce its impact. In this study, the shrimp infectious dose 50% endpoint (SID50 ml(-1)) of a Thai isolate of WSSV was determined by intramuscular inoculation (i.m.) in 60 and 135 d old specific pathogen-free (SPF) Litopenaeus vannamei using indirect immunofluorescence (IIF) and 1-step polymerase chain reaction (PCR). Also, the lethal dose 50% endpoint (LD50 ml(-1)) was determined from the proportion of dead shrimp. The median virus infection titers in 60 and 135 d old juveniles were 10(6.8) and 10(6.5) SID50 ml(-1), respectively. These titers were not significantly different (p > or = 0.05). The titration of the WSSV stock by oral intubation in 80 d old juveniles resulted in approximately 10-fold reduction in virus titer compared to i.m. inoculation. This lower titer is probably the result of physical and chemical barriers in the digestive tract of shrimp that hinder WSSV infectivity. The titers determined by infection were identical to the titers determined by mortality in all experiments using both i.m. and oral routes at 120 h post inoculation (hpi), indicating that every infected shrimp died. The determination of WSSV titers for dilutions administered by i.m. and oral routes constitutes the first step towards the standardization of challenge procedures to evaluate strategies to reduce WSSV infection.

A review on the morphology, molecular characterization, morphogenesis and pathogenesis of white spot syndrome virus.

Since it first appeared in 1992, white spot syndrome virus (WSSV) has become the most threatening infectious agent in shrimp aquaculture. Within a decade, this pathogen has spread to all the main shrimp farming areas and has caused enormous economic losses amounting to more than seven billion US dollars. At present, biosecurity methods used to exclude pathogens in shrimp farms include disinfecting ponds and water, preventing the entrance of animals that may carry infectious agents and stocking ponds with specific pathogen-free post-larvae. The combination of these practices increases biosecurity in shrimp farming facilities and may contribute to reduce the risk of a WSSV outbreak. Although several control methods have shown some efficacy against WSSV under experimental conditions, no therapeutic products or strategies are available to effectively control WSSV in the field. Furthermore, differences in virulence and clinical outcome of WSSV infections have been reported. The sequencing and characterization of different strains of WSSV has begun to determine aspects of its biology, virulence and pathogenesis. Knowledge on these aspects is critical for developing effective control methods. The aim of this review is to present an update of the knowledge generated so far on different aspects of WSSV organization, morphogenesis, pathology and pathogenesis.