Sarocha Jitrakorn

Additional random, single to multiple genome fragments of Penaeus stylirostris densovirus in the giant tiger shrimp genome have implications for viral disease diagnosis

Scattered reports of viral inserts in shrimp and insect genomes led to the hypothesis that random, autonomous insertion of such sequences occurs in these organisms and leads to specific, heritable immunity. To test the prediction regarding random insertion of viral sequences into the shrimp genome, we examined the giant tiger shrimp for random genomic insertions of Penaeus stylirostris densovirus (also called IHHNV). By PCR analysis using a set of 7 overlapping primer pairs to cover the whole IHHNV genome (4 kb), PCR failure with some pairs indicated sequence gaps that revealed a random pattern of putative viral inserts in the genomes of individual shrimp. Targeting a putative insert from one arbitrarily selected specimen, we used genome walking to reveal a viral insert linked to a host microsattelite-like fragment. This differed from 2 previously reported inserted fragments of IHHNV in P. monodon. In one specimen, 2 slightly different inserts were revealed, probably on paired chromosomes. By design and use of chimeric shrimp/virus primer pairs we proved that similar insertions occurred in several shrimp specimens, including those infected with IHHNV but showing no signs of disease. For the infected specimens, the inserts gave false positive PCR test results using 309F/R primers and a new IQ2000 test protocol currently recommended for detection of infectious IHHNV. This is the first experimental support for the hypothesis-based prediction that a random number and length of sequence fragments from a single virus genome may occur in the shrimp genome. Since some inserts can give false positive results for infectious IHHNV with the recommended methods above, they may have a negative effect on international seafood trade. In addition, discard of domesticated shrimp breeding stocks based on such false positive results might have negative consequences, if such inserts are related to shrimp viral disease tolerance, as also hypothesized.

Large-scale production and antiviral efficacy of multi-target double-stranded RNA for the prevention of white spot syndrome virus (WSSV) in shrimp

BackgroundRNA interference (RNAi) is a specific and effective approach for inhibiting viral replication by introducing double-stranded (ds)RNA targeting the viral gene. In this study, we employed a combinatorial approach to interfere multiple gene functions of white spot syndrome virus (WSSV), the most lethal shrimp virus, using a single-batch of dsRNA, so-called “multi-WSSV dsRNA.” A co-cultivation of RNase-deficient E. coli was developed to produce dsRNA targeting a major structural protein (VP28) and a hub protein (WSSV051) with high number of interacting protein partners.ResultsFor a co-cultivation of transformed E. coli, use of Terrific broth (TB) medium was shown to improve the growth of the E. coli and multi-WSSV dsRNA yields as compared to the use of Luria Bertani (LB) broth. Co-culture expression was conducted under glycerol feeding fed-batch fermentation. Estimated yield of multi-WSSV dsRNA (μg/mL culture) from the fed-batch process was 30 times higher than that obtained under a lab-scale culture with LB broth. Oral delivery of the resulting multi-WSSV dsRNA reduced % cumulative mortality and delayed average time to death compared to the non-treated group after WSSV challenge.ConclusionThe present study suggests a co-cultivation technique for production of antiviral dsRNA with multiple viral targets. The optimal multi-WSSV dsRNA production was achieved by the use of glycerol feeding fed-batch cultivation with controlled pH and dissolved oxygen. The cultivation technique developed herein should be feasible for industrial-scale RNAi applications in shrimp aquaculture. Interference of multiple viral protein functions by a single-batch dsRNA should also be an ideal approach for RNAi-mediated fighting against viruses, especially the large and complicated WSSV.

Detection of natural infection of infectious spleen and kidney necrosis virus in farmed tilapia by hydroxynapthol blue-loop-mediated isothermal amplification assay.

Infectious spleen and kidney necrosis virus (ISKNV) has recently been recognized as a causative agent of serious systemic disease in tilapia. Our objective was to establish a new colorimetric loop‐mediated isothermal amplification (LAMP) assay with pre‐addition of hydroxynapthol blue (blue‐LAMP) to investigate ISKNV transmission in tilapia.

Shewanella putrefaciens in cultured tilapia detected by a new calcein-loop-mediated isothermal amplification (Ca-LAMP) method

Shewanella putrefaciens is being increasingly isolated from a wide variety of sources and is pathogenic to many marine and freshwater fish. For better control of this pathogen, there is a need for the development of simple and inexpensive but highly specific, sensitive, and rapid detection methods suitable for application in field laboratories. Our colorogenic loop-mediated isothermal amplification (LAMP) assay combined with calcein (Ca-LAMP) for unaided visual confirmation of LAMP amplicons is a simple method for fish pathogen detection in cultured tilapia. Here, we describe the detection of S. putrefaciens using the same platform. As before, the method gave positive results (orange to green color change) in 45 min at 63°C with sensitivity 100 times higher than that of a conventional PCR assay, with no cross-amplification of other known fish bacterial pathogens tested. Using the assay with 389 samples of gonads, fertilized eggs, and fry of farmed Nile and red tilapia Oreochromis spp., 35% of samples were positive for S. putrefaciens. The highest prevalence was found in samples of gonads (55%) and fertilized eggs (55%) from adult breeding stocks, indicating that S. putrefaciens could be passed on easily to fry used for stocking production ponds. Tissue tropism assays revealed that the spleen showed the highest colonization by S. putrefaciens in naturally infected tilapia and that it would be the most suitable organ for screening and monitoring fish stocks for presence of the bacteria.

Infectious spleen and kidney necrosis disease (ISKND) outbreaks in farmed barramundi (Lates calcarifer) in Vietnam

Abstract Emergence of a disease with clinical signs resembling megalocytivirus infection seriously affected large‐scale barramundi farms in Vietnam in 2012–2014 with estimated losses reaching

Naturally concurrent infections of bacterial and viral pathogens in disease outbreaks in cultured Nile tilapia (Oreochromis niloticus) farms

435,810 per year. An oil‐based, inactivated vaccine against red sea bream iridovirus (RSIV) was applied in one farm for disease prevention without analysis of the causative agent, and the farmer reported inadequate protection. Here we describe histological and molecular analysis of the diseased fish. PCR targeting the major capsid protein (MCP) of megalocytiviruses yielded an amplicon with high sequence identity to infectious spleen and kidney necrosis virus (ISKNV) genotype II previously reported from other marine fish but not barramundi. Detection of the virus was confirmed by positive in situ hybridization results with fish tissue lesions of the kidney, liver, pancreas, and brain of the PCR‐positive samples. Based on the complete sequence of the MCP gene, the isolate showed 95.2% nucleotide sequence identity and 98.7% amino acid sequence identity (6 residue differences) with the MCP of RSIV. Prediction of antigenic determinants for MCP antigens indicated that the 6 residue differences would result in a significant difference in antigenicity of the two proteins. This was confirmed by automated homology modeling in which structure superimpositioning revealed several unique epitopes in the barramundi isolate. This probably accounted for the low efficiency of the RSIV vaccine when tested by the farmer. HighlightsThe first report on emergence of ISKD in farmed barramundi from Vietnam.The causative agent was identified as Megalocytivirus ISKNV genotype II.A Megalocytivirus RSIV vaccine conferred only partial protection.Structural analysis revealed significant differences between the two viruses.