Specific Pathogen-free Litopenaeus Vannamei Research Articles

English

Shrimp viral diseases have caused severe production and economic losses in the past two decades. A complete understanding of shrimp viruses is dependent upon the development of laboratory techniques for the maintenance and culturing of these viruses and host cells. This investigation was done to characterize the cell line culture from specific pathogen free Litopenaeus vannamei and its susceptibility to revise the cytopathic effects of white spot syndrome virus. A cell culture was successfully developed from insect cell SF9. Cytopathic changes like enlarged cells, focal lesions, shrunken and clumped cells were observed in white spot syndrome viruses (WSSV) infected SF9 cell cultures from 24 to 120 h duration. In the present study, conditions for the successful primary culture of insect SF9 cells for WSSV infection have been established. The conformation of WSSV infection in SF9 cell line was by polymerase chain reaction using target gene of WSSV419 like proteins and followed by electrophoresis. The WSSV result was positively as 550 bp in SF9 cell line and in the control sample. This is the first report on the development of primary cell culture of WSSV, host species of L. vannamei using insect cell line SF9.   Key words: White spot syndrome viruses (WSSV), SPF, L. vannamei, SF9 cell line, cytopathic effect, pathogen, economy.

Occurrence of white spot syndrome virus in shrimp culturing waters and its brunt in specific pathogen free Litopenaeus vannamei with particular allusion to molecular verdicts

ObjectiveTo detect the water samples and shrimp samples in white spot syndrome virus (WSSV) affecting and non-affecting zone. MethodsA total of 12 samples specific pathogen free Litopenaeus vannamei (L. vannamei); adult shrimp and larvae were randomly collected. Their genomic DNA was isolated and subjected to PCR. Histopathological identifications were carried out, and the hematopoietic tissues with basophilic intranuclear inclusion bodies characteristic were observed in moderate WSSV infected L. vannamei ResultsThe PCR analysis showed the appearance of a prominent band from the PCR amplified product of WSSV-DNA at internal control band 848 bp at non-infected areas. Although low infection positive bands (20 copies) were shown at 296 bp continued from initial stage of the infection region. On a moderate and ascetic level were observed as 650 bp and 910 bp (200 and 2 000 copies), during the severe out break periods. The gill epithelial cells were edematous and nuclei were hypertrophied with basophilic inclusions, but no pathological changes or hypertrophied nuclei were observed in any of L.vannamei tissues in WSSV uninfected region. The Intranuclear inclusion bodies characteristics of high level of WSSV infection presented in the gill region. ConclusionsThe present study is significant, which investigated the level of WSSV transmission from the infected tiger prawn P.monodon to SPFL. vannamei in the WSSV impact region of Tamil Nadu coastal waters.

Real time PCR quantification of WSSV infection in specific pathogen free (SPF) Litopenaeus vannamei (Boone, 1931) exposed to antiviral nucleotide

ObjectiveTo investigate the level of WSSV transmission from the infected tiger prawn Penaeus monodon (P. monodon) to specific pathogen free Litopenaeus vannamei (L. vannamei) in laboratory captivity condition in relation to PCR detection, histopathological observation and viral genome sequence. MethodsViral DNA was isolated from purified virions by treatment with proteinase K (0.2 mg/mL) and Sarkosyl (1%). The purity and concentration of the DNA were determined by agarose gel electrophoresis. Moribund and dead shrimp were removed and processed for indirect immunofluorescence (IIF) analysis. Histological observation of infected L. vannamei shrimps were revealed by the degenerated cells which were characterized by intranuclear inclusions in the tissues of WSSV infected mid-gut gland, lymphoid organ, gill lamellae and gut epithelium. Total DNA was extracted, from shrimp hemolymph and tissues, with a High Puree PCR template preparation kit. WSSV-DNA was detected using a commercial 2-step PCR detection kit. ResultsThe present study compares the real-time PCR results with SYBR Green I concentration ranging from 0.2 to 0.7×. The positive standard was used in the range of 102, 104 106, 108 and 1010 copies/ng of DNA in general. The PCR analysis showed the appearance of a prominent band from the PCR amplified product of WSSV-DNA at internal control band of 848 bp. Moderate and severe levels were observed as 650 bp and 910 bp (200 & 2 000 copies) in various transmission routes. The WSSV content in moribund shrimp of all the experimental species (L. vannamei) approximately ranged in nucleotide application by quantification method from 0.000 001 WSSV copies/μg of total DNA. In whole moribund infection animal, approximately 0.02 WSSV copies/μg of DNA was detected in nucleotide applied animal. ConclusionThese results indicate that wild brood stock and native culture shrimp P. monodon may be infected with WSSV and can get transferred into the SPF L. vannamei farming environment. Based on the studies, we made in captivity condition in different WSSV transmission route in dissimilar infection range with the use of nucleotide for antiviral drugs. There is an urgent need to address and develop antiviral drugs and molecular based viral genome technique for control measures to salt away the aquaculture environments.

Advances in Research of Necrotizing Hepatopancreatitis Bacterium (NHPB) Affecting Penaeid Shrimp Aquaculture

Necrotizing hepatopancreatitis (NHP) is a severe bacterial disease affecting penaeid shrimp aquaculture. NHP is caused by the gram-negative, pleomorphic, obligately intracellular NHP-bacterium (NHPB) that targets the hepatopancreas tissue of shrimp. NHPB is classified as an α -proteobacterium and is related to members of the Rickettsia. First reported in 1985 in a Texas shrimp farm, NHP has since affected shrimp farming in several North and South American countries, causing mortalities up to 95% and devastating economic losses to aquaculture crops. Because NHPB remains unculturable through traditional in vitro methods, the development of an in vivo cultivation system of NHPB in susceptible, specific pathogen-free Litopenaeus vannamei has allowed advancement of experimental research, including NHPB population biology and transmission dynamics. Current research on NHPB is discussed relative to management strategies of NHP disease in a shrimp pond. Compared to other important shrimp pathogens, NHPB is geographically limited, like that of yellowhead virus (YHV), but in contrast to the global distribution of Taura syndrome virus (TSV) and white spot syndrome virus (WSSV). Additionally, NHPB is less virulent than TSV and WSSV, and the transmission dynamics of TSV and YHV are much more complicated than that of NHPB and WSSV in penaeid shrimp.

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.

Preservation of necrotizing hepatopancreatitis bacterium (NHPB) by freezing tissue collected from experimentally infected Litopenaeus vannamei

Necrotizing hepatopancreatitis (NHP), a severe disease of penaeid shrimp, is caused by bacteria (NHPB) that have previously been demonstrated to reside in tubular epithelial hepatopancreatic (HP) cells of infected shrimp. There has yet to be a successful in vitro culture method to grow the intracellular organism; therefore, it must be propagated in vivo via transmission from NHPB-infected shrimp to healthy individuals. In our studies, NHPB propagation tanks containing infected shrimp were used to maintain a constant supply of organisms for experiments. In order to develop a method for storing infectious NHPB material for future challenge studies, we collected HP tissue containing NHPB by flash freezing whole, fresh HPs at -80 degrees C for up to 80 d and used it to successfully infect specific pathogen-free Litopenaeus vannamei per os in controlled experiments. HP tissue samples were collected from dead shrimp, and PCR was performed to confirm the presence of NHPB. Our results demonstrate that the infectivity of NHPB in tissue is not altered after being frozen at -80 degrees C when compared to NHPB in fresh tissue. Thus, the continual propagation of NHPB in vivo is not required to assure a source of the infectious agent.

Clinical effect of cidofovir and a diet supplemented with Spirulina platensis in white spot syndrome virus (WSSV) infected specific pathogen-free Litopenaeus vannamei juveniles

The antiviral product cidofovir and a diet supplemented with Spirulina platensis were tested for their efficacy to prevent or delay/reduce mortality due to white spot syndrome virus (WSSV) infection in specific pathogen free (SPF) shrimp Litopenaeus vannamei juveniles. Cidofovir was injected intramuscularly at 200 mg/kg shrimp mean body weight (MBW) at the moment of WSSV challenge. Spirulina was supplemented in the shrimp diet at 25% w/w and shrimp were fed for 4 days at 5% of the MBW per day before WSSV challenge. Shrimp were inoculated orally with WSSV at a dose of 30 SID 50 (SID 50 = shrimp infectious dose with 50% endpoint) and clinical signs and mortality were followed for 120 h post inoculation (hpi). WSSV infection status was determined by indirect immunoflourescence (IIF) in dead and survivor shrimp at the end of the trial. In two experiments, mortality was delayed approximately for 24 h by cidofovir treatment. The 100% mortality level was reached at 96–108 hpi in mock treated shrimp, whereas in cidofovir treated shrimp, 80–90% mortality was reached at the end of experiment (120 hpi). Significant differences ( p < 0.05) in the median lethal time (LT 50) of cidofovir-treated shrimp and mock-treated shrimp were found by probit analysis. A Spirulina supplemented diet delayed the onset of clinical signs for 12 h but had no effect on the cumulative mortality at the end of the experiment. This study opens perspectives for antiviral drugs to treat shrimp infected with WSSV.

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.