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Scale drop disease virus (SDDV) is one of the most important pathogens that causes scale drop disease (SDD) in Asian sea bass (Lates calcarifer). The outbreaks of this disease are one of the factors causing substantial losses in Asian sea bass aquaculture. In this study, the uracil-DNA glycosylase (UDG)-supplemented cross-priming amplification (UCPA) combined with a colorimetric detection method using the hydroxynaphthol blue (HNB) and lateral flow dipstick (LFD) for detection of SDDV was developed. The UDG was utilized to prevent carryover contamination, and the CPA reactions can be readily observed by HNB and LFD. The CPA primers and probe were designed to target the major capsid protein (MCP) gene of the SDDV. The optimized UCPA conditions were performed at the temperature of 61°C for 60 min. The UCPA assays demonstrated specificity to SDDV without cross-reaction to other tested viruses including red-spotted grouper nervous necrosis virus (RGNNV), infectious spleen and kidney necrosis virus (ISKNV) and Lates calcarifer herpes virus (LCHV), and other bacterial species commonly found in aquatic animals. The sensitivity of the UCPA-HNB and UCPA-LFD was 100 viral copies/µl and 10 pg of extracted total DNA, which was 10-fold more sensitive than that of conventional PCR. The UCPA-HNB and UCPA-LFD assays could be used to detect the SDDV infection in all 25 confirmed SDDV-infected fish samples. Therefore, the UCPA coupled with HNB and LFD was rapid, simple and effective and might be applied for diagnosis of SDDV infection., (© 2021 John Wiley & Sons Ltd.)

Asian seabass, Lates calcarifer farming in Southeast Asia, encounters serious disease challenges caused by Streptococcus agalactiae and Streptococcus iniae. However, a vaccine for disease prevention is not yet available. In this study, we investigated the mucosal and systemic antibody (IgM) response kinetics of the Asian seabass following primary immunization with oil-based formalin-killed vaccines (FKVs) prepared from S. agalactiae and S. iniae (monovalent Sa, monovalent Si, and bivalent Sa-Si) and secondary booster with the respective water-based FKVs. The efficacy of vaccines was subsequently evaluated by an experimental challenge. The results revealed similar antibody response kinetics in both systemic and mucosal systems. However, the immune response in the fish vaccinated with the monovalent vaccines was superior to those fish received the bivalent vaccine in terms of specific antibody titer. The fish that received monovalent vaccines required 1-2 weeks to raise a significant level of specific antibody titer in both systemic and mucosal systems while those vaccinated with bivalent vaccine required three weeks. Following booster at day 21, both systemic and mucosal antibody titers in all vaccinated groups had reached the peak at day 28 and gradually declined in the following weeks but remained significantly higher than control until the end of the experiment (day 63). In the challenge test, both monovalent and bivalent vaccines were found to be highly efficacious, with the relative percentage survival (RPS) ranging from 75 to 85%. In summary, this study explored the 63-days antibody response kinetics (both mucosal and systemic systems) of Asian seabass to monovalent and bivalent inactivated vaccines and confirmed that the combination of S. agalactiae and S. iniae in a single injectable vaccine is possible., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

A unique strain of Vibrio harveyi is the causative agent of scale drop and muscle necrosis disease (SDMND) in Asian sea bass (Lates calcarifer). This study investigated the protein profiles of SDMND-causing Vibrio harveyi isolates compared to the reference V. harveyi ATCC 14126 strain. A distinct protein band of 33 kDa, namely HP33, found from only V. harveyi SDMND was subjected to analysis by LC-MS/MS and the identified peptide sequences matched to an unknown hypothetical protein. Detection of HP33 coding sequence was investigated at both genomic and transcriptional levels and the results consistently supported the protein analysis. Recombinant HP33 protein was then produced using Escherichia coli system. The rHP33 protein did not cause mortality or visible clinical signs to Asian sea bass. However, the rHP33 protein was able to stimulate antibody response in Asian sea bass as evidenced by Western blotting and agglutination tests. Here, we proposed that rHP33 might be a good protein target for development of subunit vaccine and/or immunostimulant to protect Asian sea bass from SDMND., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

The 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.

A combination of eight isolates of Aeromonas hydrophila was used to produce monoclonal antibodies (MAbs). Ten different groups of MAbs specific to Aeromonas were selected. The first five groups of MAbs demonstrated high specificity and bound to only one or two isolates of A. hydrophila. The sixth and the seventh groups of MAbs were A. hydrophila specific. They recognized seven of eight A. hydrophila isolates (AH1, 2, 3, 4, 5, 6, 8); however, the MAb in the seventh group also showed cross-reactivity to one isolate of Aeromonas caviae (AC3). The eighth MAb group recognized two isolates of A. hydrophila (AH2 and AH5) and demonstrated cross-reactivity to one isolate of Aeromonas sobria (AS1) and one isolate of A. caviae (AC3). The tenth group of MAbs bound to all isolates of Aeromonas spp. tested (AH1-8, AS1-6, AC1-5, Aeromonas veronii and Aeromonas jandaei) without cross-reactivity to any of the other bacteria tested. MAbs in the ninth group showed similar specificity to those in the tenth group but did not recognize two isolates of A. sobria (AS4 and AS6) or A. jandaei. All the MAbs could be used to identify Aeromonas by dot blotting with a sensitivity ranging from 10⁵ to 10⁷ CFU mL⁻¹. However, the sensitivity of detection was increased to 10²-10³ CFU mL⁻¹ after inoculation of the sample in tryptic soy broth for 3-6 h before performing the dot blotting. The dot blot method can be used for the direct detection of A. hydrophila infection in symptomatic and asymptomatic goldfish. This study demonstrated a convenient immunological tool that can be used for the direct detection of A. hydrophila and Aeromonas infections in a complex sample without the requirement for separation of the bacteria or isolation and biochemical tests., (© 2010 Blackwell Publishing Ltd.)