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Publication Metadata only Piper betle leaf extract inhibits multiple aquatic bacterial pathogens and in vivo streptococcus agalactiae infection in nile tilapia(2018-05-01) Gabriel Arome Ataguba; Ha Thanh Dong; Triwit Rattanarojpong; Saengchan Senapin; Krishna Rugmini Salin; Mahidol University; Thailand National Center for Genetic Engineering and Biotechnology; Asian Institute of Technology Thailand; University of Agriculture, Makurdi; King Mongkut s University of Technology Thonburi© 2018, Central Fisheries Research Inst. All rights reserved. An in vitro assessment of antimicrobial properties of aqueous and ethanol extracts from solo garlic (Allium sativum), garlic chive (Allium tuberosum) and betel leaves (Piper betle) on six bacterial pathogens in aquaculture, and a challenge of Nile tilapia, Oreochromis niloticus with Streptococcus agalactiae were performed. Generally, minimum inhibitory concentrations (MIC) ranged from 26.63 to 53.25 mg mL-1 for aqueous solo garlic (G) and 14.60 to 29.20 mg mL-1 for garlic chive extracts for all pathogens tested. Ethanol extract of betel leaves (P) exhibited the strongest antibacterial activity (0.15-0.60 mg mL-1). P and G incorporated in feed at high and low doses as multiples of MIC [High; H (10X for PH and 3X for GH) and Low; L (3X for PL and 1X for GL)] were fed to tilapia followed by in vivo challenge against S. agalactiae (1 × 108 CFU mL-1). Ethanol extract of P. betle significantly improved survival (P<0.05; PH=100%, PL =77%). White blood cells (WBC), lymphocytes and monocytes differed significantly (P<0.05) among treatments and the highest WBC value (1.175 × 103) was for PH. Use of ethanol extract of Piper betle seems promising for sustainable disease management in aquaculture.Publication Metadata only A sensitive and specific SYBR Green-based qPCR assay for detecting scale drop disease virus (SDDV) in Asian sea bass(2020-05-14) Sukhontip Sriisan; Chuenchit Boonchird; Siripong Thitamadee; Molruedee Sonthi; Ha Thanh Dong; Saengchan Senapin; Mahidol Universityto target a 135 bp fragment of the SDDV ATPase gene. The optimized SDDV qPCR assay reliably detected 2 copies of a plasmid dsDNA control and did not cross-amplify DNA to any of 12 viral or bacterial pathogens commonly found in aquatic animals. When assessedPublication Open Access False rumours of disease outbreaks caused by infectious myonecrosis virus (IMNV) in the whiteleg shrimp in Asia(2011) Saengchan Senapin; Kornsunee Phiwsaiya; Warachin Gangnonngiw; Flegel, Timothy W.; Mahidol University. Faculty of Science. Center of Excellence for Shrimp Molecular Biology and Biotechnology (Centex Shrimp)since January 2007 in the Quarterly Report on Aquatic Animal Diseases (QAAD) from the Network of Aquaculture Centers in Asia Pacific (NACA). In most cases, our shrimp samples for which tissue sections were possible showed signs of muscle cramp syndromePublication Metadata only Development of a SYBR Green quantitative PCR assay for detection of Lates calcarifer herpesvirus (LCHV) in farmed barramundi(2020-01-01) Watcharachai Meemetta; Jose A. Domingos; Ha Thanh Dong; Saengchan Senapin; James Cook University, Singapore; Suan Sunandha Rajabhat University; Mahidol University; Thailand National Center for Genetic Engineering and Biotechnologyhost as well as common aquatic pathogens (12 bacteria and 4 viruses) were observed. Validation test of the method with clinical samples revealed that the virus was detected in multiple organs of the clinically sick fish but not in the healthy fish. WePublication Metadata only False rumours of disease outbreaks caused by infectious myonecrosis virus (IMNV) in the whiteleg shrimp in Asia(2011-08-05) Saengchan Senapin; Kornsunee Phiwsaiya; Warachin Gangnonngiw; Timothy W. Flegel; Thailand National Center for Genetic Engineering and Biotechnology; Mahidol University2007 in the Quarterly Report on Aquatic Animal Diseases (QAAD) from the Network of Aquaculture Centers in Asia Pacific (NACA). In most cases, our shrimp samples for which tissue sections were possible showed signs of muscle cramp syndrome that alsoPublication Metadata only Rapid genotyping of tilapia lake virus (TiLV) using Nanopore sequencing(2021-10-01) Jerome Delamare-Deboutteville; Suwimon Taengphu; Han Ming Gan; Pattanapon Kayansamruaj; Partho Pratim Debnath; Andrew Barnes; Shaun Wilkinson; Minami Kawasaki; Chadag Vishnumurthy Mohan; Saengchan Senapin; Ha Thanh Dong; Suan Sunandha Rajabhat University; The University of Queensland; WorldFish; Kasetsart University; Mahidol University; Thailand National Center for Genetic Engineering and Biotechnology; Victoria University of Wellington; Wilderlab; GeneSEQ Sdn Bhdin regional aquatic animal health diagnostic laboratories in low- and medium-income countries, for fast identification and genotyping of emerging infectious pathogens from field samples within a single day.Publication Metadata only Genetic diversity of tilapia lake virus genome segment 1 from 2011 to 2019 and a newly validated semi-nested RT-PCR method(2020-09-15) Suwimon Taengphu; Pakkakul Sangsuriya; Kornsunee Phiwsaiya; Partho Pratim Debnath; Jerome Delamare-Deboutteville; Chadag Vishnumurthy Mohan; Ha Thanh Dong; Saengchan Senapin; Suan Sunandha Rajabhat University; WorldFish; Mahidol University; Thailand National Center for Genetic Engineering and Biotechnology, i.e. RNA extracted from clinical healthy tilapia and from bacterial and viral pathogens (other than TiLV) commonly found in aquatic animals. Validation experiment with RNA extracted from naturally infected fish specimens collected in 2013–2019Publication Metadata only Production of monoclonal antibodies specific to Macrobrachium rosenbergii nodavirus using recombinant capsid protein(2012-03-20) Pradit Wangman; Saengchan Senapin; Parin Chaivisuthangkura; Siwaporn Longyant; Sombat Rukpratanporn; Paisarn Sithigorngul; Srinakharinwirot University; Mahidol University; Thailand National Center for Genetic Engineering and Biotechnology; Chulalongkorn UniversityThe gene encoding the capsid protein of Macrobrachium rosenbergii nodavirus (MrNV) was cloned into pGEX-6P-1 expression vector and then transformed into the Escherichia coli strain BL21. After induction, capsid protein-glutathione-S- transferase (GST-MrNV; 64 kDa) was produced. The recombinant protein was separated using SDS-PAGE, excised from the gel, electro-eluted and then used for immunization for monoclonal antibody (MAb) production. Four MAbs specific to the capsid protein were selected and could be used to detect natural MrNV infections in M. rosenbergii by dot blotting, Western blotting and immunohistochemistry without cross-reaction with uninfected shrimp tissues or other common shrimp viruses. The detection sensitivity of the MAbs was 10 fmol μl-1 of the GST-MrNV, as determined using dot blotting. However, the sensitivity of the MAb on dot blotting with homogenate from naturally infected M. rosenbergii was approximately 200-fold lower than that of 1-step RT-PCR. Immunohistochemical analysis using these MAbs with infected shrimp tissues demonstrated staining in the muscles, nerve cord, gill, heart, loose connective tissue and inter-tubular tissue of the hepatopancreas. Although the positive reactions occurred in small focal areas, the immunoreactivity was clearly demonstrated. The MAbs targeted different epitopes of the capsid protein and will be used to develop a simple immunoassay strip test for rapid detection of MrNV. © Inter-Research 2012.Publication Metadata only Monoclonal antibodies against extra small virus show that it co-localizes with Macrobrachium rosenbergii nodavirus(2012-07-25) Siwaporn Longyant; Saengchan Senapin; Sirijantra Sanont; Pradit Wangman; Parin Chaivisuthangkura; Sombat Rukpratanporn; Paisarn Sithigorngul; Srinakharinwirot University; Mahidol University; Thailand National Center for Genetic Engineering and Biotechnology; Chulalongkorn UniversityThe capsid protein (CP) gene of extra small virus (XSV) expressed in Escherichia coli as a 42 kDa glutathione S- ENGLtransferase (GST)-ENGLfusion protein (GST-XCP) or a 20 kDa His6-fusion protein (His6-XCP) were purified by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), combined, and used to immunize Swiss mice to produce monoclonal antibodies (MAbs). Using dot blot, Western blot, and immunohistochemistry (IHC) methods, 4 MAbs specific to the XSV CP detected XSV in the freshwater prawn Macrobrachium rosenbergii without crossreaction to host proteins or to proteins of Macrobrachium rosenbergii nodavirus (MrNV) or 5 of the most pathogenic viruses of penaeid shrimp. In dot blots, the combined MAbs could detect down to ~10 to 20 fmol μl-1 of purified GST-XCP protein, which was somewhat more sensitive compared to any single MAb. Used in conjunction with an MrNV-specific MAb, white tail disease (WTD) was diagnosed more effectively. However, the sensitivity at which the combined 4 MAbs detected XSV CP was 1000-fold lower than XSV RNA detected by RT-PCR. IHC analysis of M. rosenbergii tissue sections using the MAbs showed XSV infection to co-localize at variable loads with MrNV infection in heart and muscle cells as well as cells of connective tissues in the hepatopancreas. Since XSV histopathology remained prominent in tissues of some prawns in which MAb reactivity for MrNV was low compared to MAb reactivity for XSV, XSV might play some role in WTD severity. © Inter-Research 2012.Publication Metadata only Duplex PCR assay and in situ hybridization for detection of Francisella spp. and Francisella noatunensis subsp. orientalis in red tilapia(2016-06-15) Ha T. Dong; Warachin Gangnonngiw; Kornsunee Phiwsaiya; Walaiporn Charoensapsri; Vuong V. Nguyen; Pål Nilsen; Padmaja J. Pradeep; Boonsirm Withyachumnarnkul; Saengchan Senapin; Channarong Rodkhum; Chulalongkorn University; Thailand National Center for Genetic Engineering and Biotechnology; Mahidol University; National Broodstock Center for Mariculture Species; Zoetis Inc., USA; Prince of Songkla University; Shrimp Genetic Improvement Center© Inter-Research 2016. Conventional isolation and identification based on phenotypic characteristics is challenging with the highly fastidious, intracellular bacterium Francisella noatunensis subsp. orientalis (Fno). Here, we developed a duplex PCR method for simultaneous detection of the Francisella genus and Fno in one PCR reaction and an in situ hybridization method for paraffin section based diagnosis of Fno. The PCR results showed genus- and species-specific bands (1140 and 203 bp) from Fno but only one genus-specific band (1140 bp) from F. noatunensis subsp. Noatunensis. Sensitivity of the duplex PCR assay revealed a detection limit of 20 to 200 fg genomic DNA (∼10 to 100 genome equivalents) depending on DNA template extraction methods. The newly developed duplex PCR assay could be used to detect Fno from clinically sick fish exhibiting signs of visceral granulomas and would also be able to detect Fno infection in naturally diseased fish without symptoms of francisellosis, indicating potential application for diagnosis of field samples. The in situ hybridization assay using Fno species-specific probe revealed positive signals in multiple organs including the spleen, liver, kidney, gills and intestine of infected fish.