Jiraporn Srisala

National Center for Genetic Engineering and Biotechnology (BIOTEC), Bang Kadi, Pathum Thani, Thailand

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Publications (8)15.07 Total impact

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    ABSTRACT: The Thai Department of Fisheries (DOF), 2013 estimated that outbreaks of acute early mortality (often called early mortality syndrome or EMS) in cultivated shrimp were responsible for a 33% drop in shrimp production during the first quarter of 2013. Similar early mortality in Vietnam was ascribed to specific isolates of Vibrio parahaemolyticus that caused acute hepatopancreatic necrosis disease (AHPND) but the status of EMS/AHPND in Thailand was unclear. Here we describe the isolation and characterization of bacteria isolated from the hepatopancreas (HP) of shrimp collected from an early mortality outbreak farm in Thailand. Four independent bacterial isolates were identified as V. parahaemolyticus by BLAST analysis and by gene-specific marker detection of a lecithin dependent hemolysin (LDH) considered to be specific for the species. Immersion challenges with 3 of these and a reference isolate, obtained from China in 2010, using a previously published laboratory infection model caused very high mortality accompanied by characteristic AHPND histopathology in the shrimp HP. Tests with one of these isolates (5HP) revealed that rate of mortality was dose dependent. Using the same challenge protocol, the 4th isolate (2HP) also caused high mortality, but it was not accompanied by AHPND histopathology. Instead, it caused a different histopathology of the HP including collapsed epithelia and unique vacuolization of embryonic cells (E-cells). These results revealed the possibility of diversity in isolates of V. parahaemolyticus that may cause early mortality in shrimp cultivation ponds. Genomic and episomic DNA of these isolates and isolates of V. parahaemolyticus that cause no disease need to be compared to better understand the molecular basis of bacterial virulence in AHPND.
    Aquaculture. 01/2014; s 428–429:297–302.
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    ABSTRACT: The microsporidian Enterocytozoon hepatopenaei was first described from Thailand in 2009 in farmed, indigenous giant tiger shrimp Penaeus (Penaeus) monodon. The natural reservoir for the parasite is still unknown. More recently, a microsporidian closely resembling it in morphology and tissue preference was found in Thai-farmed, exotic, whiteleg shrimp Penaeus (Litopenaeus) vannamei exhibiting white feces syndrome (WFS). Our objective was to compare the newly found pathogen with E. hepatopenaei and to determine its causal relationship with WFS. Generic primers used to amplify a fragment of the small subunit ribosomal RNA (ssu rRNA) gene for cloning and sequencing revealed that the new parasite from WFS ponds had 99% sequence identity to that of E. hepatopenaei, suggesting it was conspecific. Normal histological analysis using tissue sections stained with hematoxylin and eosin (H&E) revealed that relatively few tubule epithelial cells exhibited spores, suggesting that the infections were light. However, the H&E results were deceptive since nested PCR and in situ hybridization analysis based on the cloned ssu rRNA gene fragment revealed very heavy infections in tubule epithelial cells in the central region of the hepatopancreas in the absence of spores. Despite these results, high prevalence of E. hepatopenaei in shrimp from ponds not exhibiting WFS and a pond that had recovered from WFS indicated no direct causal association between these infections and WFS. This was supported by laboratory oral challenge trials that revealed direct horizontal transmission to uninfected shrimp but no signs of WFS. The microsporidian newly found in P. vannamei is conspecific with previously described E. hepatopenaei and it is not causally associated with WFS. However, the deceptive severity of infections (much greater than previously reported in P. monodon) would undoubtedly have a negative effect on whiteleg shrimp growth and production efficiency and this could be exacerbated by the possibility of horizontal transmission revealed by laboratory challenge tests. Thus, it is recommended that the PCR and in situ hybridization methods developed herein be used to identify the natural reservoir species so they can be eliminated from the shrimp rearing system.
    BMC Veterinary Research 07/2013; 9(1):139. · 1.86 Impact Factor
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    ABSTRACT: Disease outbreaks caused by viral pathogens constitute a major limitation to development of the shrimp aquaculture industry. Many research have been conducted to better understand how host shrimp respond to viral infections with the aim of using the gained knowledge to develop better strategies for disease management and control. One approach has been to study the interactions between host and viral proteins, and particularly host virus-binding proteins that might play an important role in the viral infection process. Within the past five years, increasing numbers of virus-binding proteins (VBPs) have been reported in shrimp. Characterization of these molecules has emphasized on their potential therapeutic applications by demonstrating their activities in inhibition of viral replication via in vivo neutralization assay. However, signaling to induce innate antiviral immune responses as a consequence of binding between viral proteins and VBPs remain to be fully elucidated.
    Fish &amp Shellfish Immunology 02/2013; · 2.96 Impact Factor
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    ABSTRACT: AIMS: Enterocytozoon hepatopenaei is an emerging microsporidian parasite that has been linked to recent losses caused by white feces syndrome (WFS) in cultivated giant or black tiger shrimp Penaeus (Penaeus) monodon and whiteleg shrimp Penaeus (Litopenaeus) vannamei in Asia. To more accurately assess its impact on shrimp production and to determine reservoir carriers for control measures, our objective was to establish a loop-mediated isothermal amplification (LAMP) assay combined with colorimetric nanogold (AuNP) for rapid, sensitive and inexpensive detection of this parasite. METHODS AND RESULTS: A set of six specific primers was designed to successfully detect the SSU rRNA gene of E. hepatopenaei by a LAMP reaction of 45 min at 65°C combined with visual detection of the amplification product via hybridization at 65°C for 5 min with a ssDNA-labled nanogold probe, followed by salt induced AuNP aggregation (total assay time approximately 50 minutes). This method gave similar results to LAMP followed by electrophoresis or spectrophotometric detection, and it was more sensitive (0.02 fg total DNA) than a conventional nested PCR reaction (0.2 fg total DNA). The new method gave negative results with shrimp DNA templates extracted from diseased shrimp containing other pathogens, indicating that the LAMP-AuNP assay was specific for E. hepatopenaei. CONCLUSIONS: Without sacrificing sensitivity or specificity, the new LAMP-AuNP assay significantly reduced the time, ease and cost for molecular detection of E. hepatopenaei in shrimp. © 2013 The Authors Journal of Applied Microbiology © 2013 The Society for Applied Microbiology.
    Journal of Applied Microbiology 02/2013; · 2.20 Impact Factor
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    ABSTRACT: Disease outbreaks caused by viral pathogens constitute a major limitation to development of the shrimp aquaculture industry. Many research have been conducted to better understand how host shrimp respond to viral infections with the aim of using the gained knowledge to develop better strategies for disease management and control. One approach has been to study the interactions between host and viral proteins, and particularly host virus-binding proteins that might play an important role in the viral infection process. Within the past five years, increasing numbers of virus-binding proteins (VBPs) have been reported in shrimp. Characterization of these molecules has emphasized on their potential therapeutic applications by demonstrating their activities in inhibition of viral replication via in vivo neutralization assay. However, signaling to induce innate antiviral immune responses as a consequence of binding between viral proteins and VBPs remain to be fully elucidated.
    Fish &amp Shellfish Immunology 09/2012; · 2.96 Impact Factor
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    ABSTRACT: White spot syndrome virus is currently the leading cause of production losses in the shrimp industry. Penaeus monodon Rab7 protein has been recognized as a viral-binding protein with an efficient protective effect against white spot syndrome infection. Plant-derived recombinant PmRab7 might serve as an alternative source for in-feed vaccination, considering the remarkable abilities of plant expression systems. PmRab7 was introduced into the Arabidopsis thaliana T87 genome. Arabidopsis-derived recombinant PmRab7 showed high binding activity against white spot syndrome virus and a viral envelope, VP28. The growth profile of Arabidopsis suspension culture expressing PmRab7 (ECR21# 35) resembled that of its counterpart. PmRab7 expression in ECR21# 35 reached its maximum level at 5 mg g(-1) dry weight in 12 days, which was higher than those previously reported in Escherichia coli and in Pichia. Co-injection of white spot syndrome virus and Arabidopsis crude extract containing PmRab7 in Litopenaeus vannamei showed an 87% increase in shrimp survival rate at 5 day after injection. In this study, we propose an alternative PmRab7 source with higher production yield, and cheaper culture media costs, that might serve the industry's need for an in-feed supplement against white spot syndrome infection.
    Journal of Biotechnology 05/2012; 161(1):60-7. · 3.18 Impact Factor
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    ABSTRACT: The aim of this study was to examine whether shrimp yellow head virus (YHV) from processed shrimp tissue infected at the pre-patent disease level could be transmitted to naïve shrimp in a laboratory setting. In a preliminary test, 120 YHV-free shrimp were injected intramuscularly with a virulent YHV stock to yield 5 × 105 (pre-patent disease level) and 2500 (carrier level) viral copies/g shrimp tissue (60 shrimp each dose). These are possible infection levels for grossly normal shrimp from normal harvest ponds (i.e., not shrimp from disease outbreak ponds). These yielded 1-step and 2-step positive (nested) RT-PCR reactions, respectively, in pleopods at 6 h post-injection of the viral stock. After being subjected to standard industrial processing conditions, only fresh frozen whole or peeled shrimp injected with pre-patent dose gave positive RT-PCR test results for YHV. None of the naïve shrimp exposed to the chopped processed products for 24 h and then reared on a standard diet for 14 days showed any significant mortality or gave any positive test results for YHV using nested RT-PCR assays. Based on these preliminary test results, a large-scale test was carried out using only the high, pre-patent injection dose with 1000 fresh frozen whole shrimp. The negative control consisted of 1000 buffer-injected shrimp. A random sample of 60 shrimp from the YHV-injected group after processing, revealed 57 positive for YHV by 1-step RT-PCR assay. Of the 3 remaining, 2 were positive and 1 negative by nested RT-PCR assay. All 60 shrimp from the buffer-injected, control group were negative for YHV by nested RT-PCR assay. Exposure of these whole shrimp to naïve shrimp resulted in no significant mortality and no positive RT-PCR test results for YHV by nested RT-PCR assay in the exposed naïve shrimp. Our results showed that grossly normal whole shrimp processed by chilling and freezing would present negligible YHV disease transmission risks, even if they were 1-step RT-PCR positive for YHV. Thus, shrimp subjected to any additional processing steps such as peeling or cooking should present even lower transmission risks.
    Aquaculture. 01/2010;
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    ABSTRACT: White spot syndrome virus (WSSV) PCR-detection methods that used electrophoresis or lateral flow chromatographic strips (LFCS) were to compare and visualize PCR amplicons. Real-time PCR was used to prepare a stock template solution containing 2.85 x 10 (6) copies WSSV/microl from WSSV-infected shrimp. Serial stock dilutions were used as templates for PCR amplification of a WSSV-specific DNA fragment that was detected either in ethidium bromide stained agarose electrophoresis gels or on a chromatographic strip where it interacted with antibody to markers labeled on hybridization complex. PCR amplification employed both 1-step PCR and semi-nested PCR methods. By using 1-step PCR, the LFCS method (100 copies) gave 10 times higher sensitivity than gel electrophoresis (10(3) copies). A combination of a semi-nested PCR with LFCS gave a comparable sensitivity to those with commercial kits for nested PCR (20 copies). In addition, LFCS confirmed amplicon identity, avoided handling of carcinogenic ethidium bromide and could be completed in approximately 20-30 min post-PCR compared with 1h for gel electrophoresis. The costs for the two methods were comparable. In conclusion, semi-nested PCR followed by LFCS is a safe and rapid alternative method for detection of WSSV that provides sensitivity similar to that obtained by standard nested PCR methods.
    Journal of Virological Methods 09/2008; 153(2):129-33. · 1.90 Impact Factor
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    ABSTRACT: In Thailand, several PCR-based methods are used by private and public service laboratories for the detection of white spot syndrome virus (WSSV) infection in penaeid shrimp post larvae (PL) before they are stocked in rearing ponds. Conflicting test results for similar samples sent to two service laboratories has decreased confidence in PCR testing. Thus, we compared the sensitivity of several PCR methods commonly employed in Thailand using Taqman real-time PCR as the gold standard with a purified WSSV template stock. Using this stock for assays, we found no significant inhibitory effect by WSSV-free host shrimp DNA over the range 0 to 300 ng per reaction or by added DNA from WSSV-infected shrimp. Real-time PCR could detect WSSV with certainty at dilutions of approximately 5 copies per reaction while 1000 copies were needed for a common one-step PCR method and 50 for a common single-tube nested PCR (1N-PCR) method. Of 2 two-tube nested PCR protocols tested, one required 100 and the other 1000 copies. In addition to these sensitivity tests, a triple-blind ring test was carried out employing sets of 10 WSSV-infected DNA extracts sent to 12 commercial and public laboratories in Thailand, without specifying the PCR method to be used. Returned results included no false positives and two false negatives, the latter both from light infection vials. This translated into a test sensitivity of 97.3% and a specificity of 100%. Overall, the results confirmed the validity of PCR-based methods in Thailand for detection of WSSV in shrimp DNA extracts.
    Aquaculture. 01/2006;