Biology subjects

Dong, H. T.

Publications and source records attributed to Dong, H. T..

4 recordsLinked to original sources

bioRxiv2020

Ozone nanobubble treatment effectively reduced pathogenic Gram positive and negative bacteria in freshwater and safe for tilapia

High concentrations of pathogenic bacteria in water usually results in outbreaks of bacterial diseases in farmed fish. Here, we explored the potential application of an emerging nanobubble technology in freshwater aquaculture. Specifically, we aimed to determine if this technology was effective at reducing the concentration of pathogenic bacteria in the water, and to assess whether it was safe for fish. An ozone nanobubble (NB-O3) treatment protocol was established based on examination of nanobubble size, concentration, disinfection property, and impact on fish health. A 10-min treatment with NB-O3 in 50 L water generated approximately 2-3 x 107 bubbles with majority sizes less than 130 nm and ozone level of [~]800 mV ORP. A single treatment with water contaminated with either Streptococcus agalactiae or Aeromonas veronii effectively reduced 96.11-97.92 % of the bacterial load. This same protocol was repeated 3 times with 99.93-99.99 % reduction in the bacterial concentration. In comparison, bacterial concentration the control tanks remained the same level during the experiments. In fish-cultured water with the presence of organic matter (e.g. mucus, feces, bacterial flora, feed, etc.), the disinfection property of NB-O3 was reduced i.e bacterial concentration was reduced by 42.94 %, 84.94 % and 99.27 % after the first, second and third treatments, respectively. To evaluate the safety of NB-O3 to fish, juvenile Nile tilapia were exposed to NB-O3 treatment for 10 minutes. No mortality was observed during the treatment or 48 h post treatment. Gill histology examination revealed that a single NB-O3 treatment caused no alteration morphology. However, damage in the gill filaments was noticed in the fish receiving two or three consecutive exposures within the same day. Results of all the experiments conducted in this study suggest that NB-O3 technology is promising for controlling pathogenic bacteria in aquaculture systems, and may be useful at reducing the risk of bacterial disease outbreaks in farmed fish.

microbiology↗

bioRxiv2020

Development of a SYBR Green quantitative PCR assay for detection of Lates calcarifer herpesvirus (LCHV) in farmed barramundi

Lates calcarifer herpes virus (LCHV) is a new virus of farmed barramundi in Southeast Asia. However, a rapid detection method is yet to be available for LCHV. This study, therefore, aimed to develop a rapid quantitative PCR (qPCR) detection method for LCHV and made it timely available to public for disease diagnostics and surveillance in barramundi farming countries. A newly designed primer set targeting a 93-bp fragment of the LCHV putative major envelope protein encoding gene (MEP) was used for developing and optimizing a SYBR Green based qPCR assay. The established protocol could detect as low as 10 viral copies per {micro}l of DNA template in a reaction containing spiked host DNA. No cross-amplification with genomic DNA extracted from host as well as common aquatic pathogens (12 bacteria and 3 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. We thus recommend that barramundi farming countries should promptly initiate active surveillance for LCHV in order to understand their circulation for preventing possibly negative impact to the industry. HighlightsO_LIThis study reported a new SYBR Green qPCR method for detection of LCHV C_LIO_LIThe qPCR method had detection limit of 10 copies per {micro}l plasmid DNA template when spiked with genomic DNA from the host C_LIO_LIThe aforementioned method is highly specific to LCHV C_LIO_LIValidation with clinical samples revealed that LCHV could be detected from multiple organs with fin and brain the best organs for qPCR detection C_LI

molecular biology↗

bioRxiv2019

Genetic diversity of tilapia lake virus genome segment 1 from 2011 to 2019 and a newly validated semi-nested RT-PCR method

The gene of RNA viruses, encoding RNA-directed RNA polymerase (RdRp) is relatively conserved due to its crucial function in viral genome replication and transcription making it a useful target for genetic diversity study and PCR detection. In this study, we investigated the genetic diversity of 21 tilapia lake virus (TiLV) genome segment 1 sequences predictively coding for RdRp subunit P1. Those sequences were obtained from infected fish samples collected in Ecuador, Israel, Peru, and Thailand between 2011 and 2019 (nine sequences from this study and 12 sequences from GenBank). Primers were then designed from the highly conserved regions among all 21 TiLV segment 1 sequences and used in semi-nested RT-PCR condition optimization. The result revealed that all 21 TiLV segment 1 sequences showed 95.00-99.94 and 99.00-100% nucleotide and amino acid sequence identity, respectively. These isolates were phylogenically clustered into three separate genetic clades, called i) Israeli-2011 clade (containing of TiLV isolates from Israel collected in 2011, Ecuador, and Peru isolates), ii) monophyletic Israel-2012 clade (containing only TiLV isolates collected from Israel in 2012), and iii) Thai clade (containing only sequences obtained from Thailand isolates). The newly established PCR protocol was 100 times more sensitive than our previous segment 3-based protocol when comparatively assayed with RNA extracted from infected fish. The assay was also shown to be specific when tested against negative control samples, 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 2013-2019 yielded positive test results for all samples tested, confirming that our newly designed primers and detection protocol against TiLV segment 1, have a potential application for detection of all current genetic variants of TiLV.

genetics↗

bioRxiv2019

A highly sensitive and specific SYBR Green quantitative polymerase chain reaction (qPCR) method for rapid detection of scale drop disease virus in Asian sea bass, Lates calcarifer

Scale drop disease virus (SDDV) is a novel Megalocytivirus causing scale drop disease (SDD) in Asian sea bass in Southeast Asia. In order to support disease diagnosis and surveillance, the present study developed a highly sensitive and specific SYBR Green qPCR assay for rapid detection of SDDV. Specific primers targeting a 135-bp fragment of ATPase coding gene of the SDDV genome were newly designed and subsequent gradient PCR assays were conducted to investigate their optimal annealing temperature. The optimized qPCR assay could detect as low as 2 viral copies per reaction and showed no cross amplification with DNA extracted from 12 viruses and bacteria commonly found in aquatic animals. The SDDV ATPase qPCR method was subsequently validated with field samples (n= 86). The results revealed that all clinically sick fish (n=34) from 5 affected farms gave positive results. Interestingly, 30/52 samples of apparently healthy fish from 8 unaffected farms which previously tested negative for SDDV by semi-nested PCR assay were positive by the newly developed qPCR method. This suggested that qPCR method is highly sensitive and suitable for early screening of SDDV from clinically healthy fish and for disease confirmation of sick fish. Investigation of tissue tropism and viral load of SDDV revealed systemic viral infection with relatively high viral load (8 x 102 to 6.8 x 104 copies per 200 ng of DNA template) in all 9 tested organs including eyes, brain, fin, gills, kidney, liver, kidney, spleen, and muscle. The newly developed qPCR method in this study delivered an accurate and reliable method for rapid detection of SDDV that may facilitate active surveillance and prevent widespread of the virus. HighlightsO_LIThis study developed a SYBR Green qPCR assay for rapid detection of SDDV C_LIO_LIThe developed qPCR assay is specific for SDDV with limit of detection of 2 viral copies per reaction C_LIO_LIThe assay could detect the virus from subclinically infected fish with low viral load C_LIO_LIWe recommend this qPCR assay for active surveillance and early screening of SDDV C_LI

molecular biology↗