Biology subjects

Senapin, S.

Publications and source records attributed to Senapin, S..

15 recordsLinked to original sources

bioRxiv2023

Innovative approach for vaccinating Nile tilapia, Oreochromis niloticus against Streptococcus agalactiae using an ozone nanobubble pre-treatment, VAC in BAG and VAC in FEED

The treatment of Nile tilapia with ozone nanobubbles (ONb) prior to vaccination with an immersible heat-killed Streptococcus agalactiae (Sa) vaccine has been reported to modulate and enhance both innate and specific immunity. The efficacy of this novel vaccination strategy is explored further in field trials. This strategy involved a short-term treatment of ONb to activate the fishs immunity prior to immersion vaccination during their transportation in oxygenated plastic bags (VAC in BAG), followed by two oral boosters during the grow-out stage mixing vaccine in feed (VAC in FEED). The field trial was conducted over 112 days in open cages, comprising four groups: normal aeration control (AC), normal aeration + vaccine (AV), ONb control (NC), and ONb + vaccine (NV). The efficacy of the vaccine was evaluated by measuring specific antibodies for S. agalactiae, monitoring expressions of IgM and IgT transcripts in the gills and head kidney every two weeks, and a laboratory pathogen challenge. Results found that fish in the NV group had significant increases in anti-S. agalactiae antibodies after the primary dose, whereas fish in the AV group required an oral booster dose to produce significant anti-S. agalactiae antibodies. In the vaccinated groups (AV and NV), only IgM was observed to be upregulated at 14 days post-immersion (dpi), while this gene was upregulated in both gills and head kidney in the NC group. No statistically significant upregulation of IgT was recorded in any group at any time point. Despite a decline in the levels of specific antibodies among the vaccinated groups at the time of challenge (88 dpi), the NV and AV groups demonstrated a relative percent survival (RPS) of 50% and 46.7%, respectively, following a relatively high injection dose of S. agalactiae injection (0.1 mL of 108 CFU/mL). In summary, this ONb, VAC in BAG and VAC in FEED vaccination strategy represents a promising alternative to the undesirable handling and costly injection approach used within the Nile tilapia industry.

immunology↗

bioRxiv2023

Multiplex PCR detection and Nanopore-based rapid genotyping of tilapia (Oreochromis spp.) pathogens.

BackgroundTilapia aquaculture faces significant threats posed by four prominent pathogens: tilapia lake virus (TiLV), infectious spleen and kidney necrosis virus (ISKNV), Francisella orientalis, and Streptococcus agalactiae. Currently, employed molecular diagnostic methods for these pathogens rely on multiple singleplex PCR reactions, which are time-consuming and expensive. MethodsIn this study, we present an approach utilizing a multiplex PCR (mPCR) assay, coupled with rapid Nanopore sequencing, enabling the one-tube simultaneous detection and one-reaction Nanopore sequencing-based validation of four pathogens. ResultsOur one-tube multiplex assay exhibits a detection limit of 1,000 copies per reaction for TiLV, ISKNV, and S. agalactiae, while for F. orientalis, the detection limit is 10,000 copies per reaction. This sensitivity is sufficient for diagnosing infections and co-infections in clinical samples from sick fish, enabling rapid confirmation of the presence of pathogens. Integrating multiplex PCR and Nanopore sequencing provides an alternative approach platform for fast and precise diagnostics of major tilapia pathogens in clinically sick animals, adding to the available toolbox for disease diagnostics.

genomics↗

bioRxiv2023

A multiplexed RT-PCR Assay for Nanopore Whole Genome Sequencing of Tilapia lake virus (TiLV)

Tilapia lake virus (TiLV) is a highly contagious viral pathogen that affects tilapia, a globally significant and affordable source of fish protein. To prevent the introduction and spread of TiLV and its impact, there is an urgent need for increased surveillance, improved biosecurity measures, and continuous development of effective diagnostic and rapid sequencing methods. In this study, we have developed a multiplexed RT-PCR assay that can amplify all ten complete genomic segments of TiLV from various sources of isolation. The amplicons generated using this approach were immediately subjected to real-time sequencing on the Nanopore system. By using this approach, we have recovered and assembled 10 TiLV genomes from total RNA extracted from naturally TiLV-infected tilapia fish, concentrated tilapia rearing water, and cell culture. Our phylogenetic analysis, consisting of more than 36 TiLV genomes from both newly sequenced and publicly available TiLV genomes, provides new insights into the high genetic diversity of TiLV. This work is an essential steppingstone towards integrating rapid and real-time Nanopore-based amplicon sequencing into routine genomic surveillance of TiLV, as well as future vaccine development.

genomics↗

bioRxiv2022

A novel, ssDNA Bidnavirus in the giant freshwater prawn Macrobrachium rosenbergii

A purported parvovirus producing circular, eosinophilic inclusions in the nuclei of tubule epithelial cells of the hepatopancreas (HP) of the giant freshwater prawn Macrobrachium rosenbergii (Mr) was first reported from Malaysia in 1994. In 2009, similar inclusions in Mr from Thailand were confirmed to contain single-stranded DNA (ssDNA). In 2021, Mr samples showing similar HP inclusions were reported to give positive PCR test results for decapod Hepanhamaparvovirus (DHPV) using universal DHPV primers designed from many viral isolates of 3 penaeid shrimp species. However, the matching DNA probe revealed positive in situ hybridization (ISH) results with host HP nuclei only, and not with the intranuclear inclusions. At the time, the negative ISH result with the intranuclear inclusions has not been further elucidated. Here we describe metagenomic analysis and de novo assembly from 2016 samples of Mr with similar intranuclear inclusions that showed negative PCR test results using the DHPV universal primers. Genomes identified through metagenomics have revealed a novel ssDNA genome sequence of 6,723 bases that was confirmed by a single PCR amplicon. ISH using specific probes derived from this genome sequence demonstrated the ssDNA presented in both grossly normal nuclei and in the intranuclear inclusions. Tests with archived DNA from the samples used in the 2021 publication yielded positive PCR results for both DHPV and the novel sequence, suggesting that the shrimp had dual infections. Phylogenetic analysis of the novel sequence revealed no relationship to any known DHPV type. Instead, the sequence most closely matched the polymerase B gene in the V1 fragment from Bombyx mori bidensovirus 2 (BmBDV2) is characterized by 2 ssDNA genome fragments and has been classified in the family Bidnaviridae. The 2 BmBDV2 fragments V1 and V2 each produce their own independent capsid protein genes and virions. In contrast, the new Mr virus is independent with a single 6,723 base ssDNA genome, and we propose the name Macrobrachium hepatopancreatic bidnavirus (MHBV).

microbiology↗

bioRxiv2022

Usefulness of the pancreas as a prime target for histopathological diagnosis of Tilapia parvovirus (TiPV) infection in Nile tilapia, Oreochromis niloticus

Tilapia parvovirus (TiPV) is an emerging virus reportedly associated with disease and mortality in farmed tilapia. Although previous descriptions of histopathological changes are available, the lesions reported in these are not pathognomonic. Here, we report Cowdry type A inclusion bodies (CAIB) in the pancreas as a diagnostic histopathological feature found in adult Nile tilapia naturally infected with TiPV. This type of inclusion body has been well-known as a histopathological landmark for the diagnosis of other parvoviral infections in shrimp and terrestrial species. Interestingly, this lesion could be exclusively observed in pancreatic acinar cells, both in the hepatopancreas and pancreatic tissue along the intestine. In situ hybridization (ISH) using a TiPV-specific probe revealed the intranuclear presence of TiPV DNA in multiple tissues, including the liver, pancreas, kidney, spleen, gills, and the membrane of oocytes in the ovary. These findings suggest that although TiPV can replicate in several tissue types, CAIB manifest exclusively in pancreatic tissues. In addition to TiPV, most diseased fish were co-infected with Streptococcus agalactiae, and presented with multifocal granulomas secondary to this bacterial infection. Partial genome amplification of TiPV was successful and revealed high nucleotide identity (> 99%) to previously reported isolates. In summary, this study highlights the usefulness of pancreatic tissue as a prime target for histopathological diagnosis of TiPV in diseased Nile tilapia. This pattern may be critical when determining the presence of TiPV infection in new geographic areas, where ancillary testing may not be available. TiPV pathogenesis in this landmark organ warrants further investigation.

pathology↗

bioRxiv2022

Detection of nervous necrosis virus RGNNV genotype in pearl gentian grouper (Epinephelus lanceolatus male x E. fuscoguttatus female) fry imported to Thailand

Nervous necrosis virus (NNV) is a deadly virus that affects more than 120 fish species worldwide, but there is little information about it in Thailand. In August 2019, a population of pearl gentian grouper fry imported to Thailand experienced mass mortality. The diseased fish exhibited darkening, floating on the water surface, sleepy behaviour, and erratic swimming. We received a set of samples for disease diagnosis. PCR analysis revealed that these samples were positive for NNV but negative for Megalocytivirus ISKNV and Ranavirus. Sequencing the viruss genome and phylogenetic analysis revealed that it is a member of the red-spotted grouper nervous necrosis virus (RGNNV) genotype. In situ hybridization using an NNV-specific probe revealed localization of the virus in the vacuolation lesions of the central nervous system (brain and spinal cord) and the retina of infected fish. The virus was successfully isolated from tissue homogenate from diseased fish using the E-11 cell line. This study identified NNV (RGNNV genotype) as a virus associated with massive die-offs in imported pearl grouper fry in Thailand. Considering the infectious nature of the virus and its broad host range, appropriate biosecurity measures are needed to prevent any loss to the marine aquaculture industry. HighlightsO_LIThis study reports detection of NNV in grouper larvae imported to Thailand C_LIO_LIThe virus was assigned to the RGNNV genotype, which is the most extensively distributed genotype C_LIO_LIHistopathology revealed a pathognomonic lesion of NNV C_LIO_LIISH using an NNV-specific probe revealed localization of NNV in the CNS and retina C_LIO_LIThe virus was successfully propagated from diseased fish using the E-11 cell line C_LI

pathology↗

bioRxiv2021

Pre-treatment of Nile tilapia (Oreochromis niloticus) with ozone nanobubbles improve efficacy of heat-killed Streptococcus agalactiae immersion vaccine

Nanobubble technology has shown appealing technical benefits and potential applications in aquaculture. We recently found that treatment with ozone nanobubbles (NB-O3) activated expression of several immune-related genes leading to effective response to subsequent exposure to fish pathogens. In this study, we investigated whether pre-treatment of Nile tilapia (Oreochromis niloticus) with NB-O3 can enhance specific immune responses and improve efficacy of immersion vaccination against Streptococcus agalactiae. Spleen and head kidney of fish in the vaccinated groups showed a substantial upregulation in expression levels of three immunoglobulin classes(IgM, IgD, and IgT) compared with the unvaccinated control groups. At day 21 post-immunization, the relative expression was greatest (approx. 3.2 to 4.1 folds). Both systemic and mucosal IgM antibodies were elicited in vaccinated groups. As the result, the cumulative survival rate of the vaccinated groups was found to be higher than that of the unvaccinated groups, with a relative percent survival (RPS) ranging from 52.9-70.5%. However, fish in the vaccinated groups that received pre-treatment with NB-O3, bacterial antigen uptakes, expression levels of IgM, IgD, and IgT, as well as the specific-IgM antibody levels and percent survival, were all slightly or significantly higher than that of the vaccinated group without pre-treatment with NB-O3. Taken together, our findings suggest that utilizing pre-treatment with NB-O3 may improve the immune response and efficacy of immersion vaccination in Nile tilapia. HighlightsO_LIImmune response and efficacy of a heat-killed Streptococcus agalactiae immersion vaccine for Nile tilapia with and without pre-treatment with NB-O3 were accessed. C_LIO_LIBacterial antigen uptake in the NB-O3-VAC compared to the AT-VAC groups was increased 1.32 and 1.80-fold at 3 and 6 h post-vaccination, respectively. C_LIO_LIVaccinated group that received pre-treatment with NB-O3 had slightly to significantly higher levels of IgM, IgD, and IgT mRNA expression; IgM levels; and survival rate. C_LIO_LIPre-treatment with NB-O3 may be a novel strategy for improving efficacy of immersion vaccine in aquaculture C_LI

molecular biology↗

bioRxiv2021

Concentration and quantification of Tilapia tilapinevirus from water using a simple iron flocculation coupled with probe-based RT-qPCR

Tilapia tilapinevirus (also known as tilapia lake virus, TiLV) is an important virus responsible for die-off of farmed tilapia globally. Detection and quantification of the virus from environmental DNA/RNA (eDNA/eRNA) using pond water represents a potential, noninvasive routine approach for pathogen monitoring and early disease forecasting in aquaculture systems. Here, we report a simple iron flocculation method for viral concentration from water combined with a newly developed hydrolysis probe quantitative RT-qPCR method for detection and quantification of TiLV. The RT-qPCR method targeting a conserved region of TiLV genome segment 9 has a detection limit of 10 viral copies per {micro}L of template. The method had a 100% analytical specificity and sensitivity for TiLV. The optimized iron flocculation method was able to recover 16.11 {+/-} 3.3% of virus from water samples spiked with viral cultures. During disease outbreak cases from an open-caged system and a closed hatchery system, both tilapia and water samples were collected for detection and quantification of TiLV. The results revealed that TiLV was detected from both clinically sick fish and asymptomatic fish. Most importantly, the virus was successfully detected from water samples collected from different locations in the affected farms e.g. river water samples from affected cages (8.50 x 102 to 2.79 x 104 copies/L) and fish-rearing water samples, sewage, and reservoir (4.29 x 102 to 3.53 x 103 copies/L) from affected and unaffected ponds of the hatchery. In summary, this study suggests that the eRNA detection system using iron flocculation coupled with probe based-RT-qPCR is feasible for concentration and quantification of TiLV from water. This approach might be useful for noninvasive monitoring of TiLV in tilapia aquaculture systems and facilitating appropriate decisions on biosecurity interventions needed.

molecular biology↗

bioRxiv2021

Efficacy of heat-killed and formalin-killed vaccines against Tilapia tilapinevirus in juvenile Nile tilapia (Oreochromis niloticus)

Tilapia tilapinevirus (also known as tilapia lake virus, TiLV) is considered to be a new threat to the global tilapia industry. The objective of this study was to develop simple cell culture-based heat-killed (HKV) and formalin-killed (FKV) vaccines for the prevention of disease caused by TiLV. The fish were immunized with 100 {micro}L of either HKV or FKV by intraperitoneal injection with each vaccine containing 1.8 x 106 TCID50 inactivated virus. A booster vaccination was carried out at 21-day post vaccination (dpv) using the same protocol. The fish were then challenged with a lethal dose of TiLV at 28 dpv. The expression of five immune genes (IgM, IgD, IgT, CD4 and CD8) in the head kidney and spleen of experimental fish was assessed at 14 and 21 dpv and again after the booster vaccination at 28 dpv. TiLV-specific IgM responses were measured by ELISA at the same time points. The results showed that both vaccines conferred significant protection, with relative percentage survival (RPS) of 71.3% and 79.6% for HKV and FKV, respectively. Significant up-regulation of IgM and IgT was observed in the head kidney of fish vaccinated with HKV at 21 dpv, while IgM, IgD and CD4 expression increased in the head kidney of fish receiving FKV at the same time point. After booster vaccination, IgT and CD8 transcripts were significantly increased in the spleen of fish vaccinated with the HKV, but not with FKV. Both vaccines induced a specific IgM response in both serum and mucus. In summary, this study showed that both HKV and FKV are promising injectable vaccines for the prevention of disease caused by TiLV in Nile tilapia.

immunology↗

bioRxiv2021

Ozone nanobubble treatments improve survivability of Nile tilapia (Oreochromis niloticus) challenged with a pathogenic multidrug-resistant Aeromonas hydrophila

Multidrug-resistant (MDR) bacteria has rapidly increased in aquaculture, which highlights the risk of production losses due to diseases and potential public health concerns. Previously, we reported that ozone nanobubbles (NB-O3) were effective at reducing concentrations of pathogenic bacteria in water and modulating fish immunity against pathogens; however, multiple treatments with direct NB-O3 exposures caused alterations to the gills of exposed-fish. Here, we set up a modified recirculation system (MRS) assembled with an NB-O3 device (MRS-NB-O3) to investigate whether MRS-NB-O3 were 1) safe for tilapia (Oreochromis niloticus), 2) effective at reducing bacterial load in rearing water, and 3) improved survivability of Nile tilapia following an immersion challenge with a lethal dose of MDR Aeromonas hydrophila. The results indicated no behavioral abnormalities or mortality of Nile tilapia during the 14 day study using the MRS-NB-O3 system. In the immersion challenge, although high bacterial concentration (~2 x 107 CFU/mL) was used, multiple NB-O3 treatments in the first two days reduced the bacteria between 15.9% to 35.6% of bacterial load in water while bacterial concentration increased 13.1% to 27.9% in the untreated control. There was slight up-regulation of non-specific immune-related genes in the gills of the fish receiving NB-O3 treatments. Most importantly, this treatment significantly improved survivability of Nile tilapia with relative percent survival (RPS) of 64.7 - 66.7% in treated fish and surviving fish developed specific antibody against MDR A. hydrophila. In summary, the result suggests that NB-O3 is a promising alternative to antibiotics to control bacterial diseases, including MDR bacteria, and has high potential for application in recirculation aquaculture system (RAS). HighlightsO_LIMultiple treatments of NB-O3 in a modified recirculation system (MRS) were relatively safe for juvenile Nile tilapia C_LIO_LINB-O3 treatments in MRS significantly improved survivability of Nile tilapia challenged with multidrug-resistant (MDR) A. hydrophila with RPS of 64.7 - 66.7% C_LIO_LIConcentration of MDR A. hydrophila in MRS was reduced by 15.9 to 35.6% following each NB-O3 treatment, and increased by 13.1 to 27.9 % in untreated control C_LIO_LISurviving fish developed specific antibody IgM against MDR A. hydrophila C_LIO_LINB-O3 is a promising non-antibiotic approach to control diseases caused by MDR A. hydrophila C_LI

microbiology↗

bioRxiv2021

Dissecting the localization of Tilapia tilapinevirus in the brain of the experimentally infected Nile tilapia (Oreochromis niloticus)

Tilapia tilapinevirus or tilapia lake virus (TiLV) is an emerging virus that inflicts significant mortality on farmed tilapia globally. Previous studies reported detection of the virus in multiple organs of the infected fish; however, little is known about the in-depth localization of the virus in the central nervous system. Herein, we determined the distribution of TiLV in the entire brain of experimentally infected Nile tilapia. In situ hybridization (ISH) using TiLV-specific probes revealed that the virus was broadly distributed throughout the brain. The strongest positive signals were dominantly detected in the forebrain (responsible for learning, appetitive behavior, and attention) and the hindbrain (involved in controlling locomotion and basal physiology). The permissive cell zones for viral infection were observed mostly to be along the blood vessels and the ventricles. This indicates that the virus may productively enter into the brain through the circulatory system and widen broad regions, possibly through the cerebrospinal fluid along the ventricles, and subsequently induce the brain dysfunction. Understanding the pattern of viral localization in the brain may help elucidate the neurological disorders of the diseased fish. This study revealed the distribution of TiLV in the whole infected brain, providing new insights into fish-virus interactions and neuropathogenesis.

pathology↗

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↗