Characterization of a novel immune deficiency gene of Macrobrachium rosenbergii reveals antibacterial and antiviral defenses.

OBJECTIVE: We sought to identify and characterize an immune deficiency (IMD) homolog from the giant freshwater prawn (also known as the giant river prawn) Macrobrachium rosenbergii. The IMD is a death-domain-containing protein that plays a crucial role as an adaptor protein in the IMD pathway-one of the most important response mechanisms to viral and bacterial invasion of invertebrates. METHODS: An IMD homolog gene from M. rosenbergii (MrIMD) was isolated using rapid amplification of complementary DNA ends. The tissue distribution and response to immune challenge of MrIMD were analyzed by real-time reverse transcription polymerase chain reaction to understand the regulatory mechanism of MrIMD messenger RNA (mRNA) expression in M. rosenbergii. RESULT: The open reading frame of MrIMD comprised 555 nucleotides encoding a protein consisting of 184 amino acids, with a conserved death domain at the C-terminus. The MrIMD protein demonstrated 53-74% similarity with IMDs from other crustaceans; the highest similarity was with the IMD from the oriental river prawn M. nipponense. Gene expression analysis revealed that MrIMD mRNA levels were highest in gill tissues. After Aeromonas hydrophila stimulation, MrIMD was significantly upregulated in the muscle, gills, and intestine, whereas there was no significant difference in the hemocytes and hepatopancreas. In the case of Macrobrachium rosenbergii nodavirus stimulation, MrIMD was dramatically upregulated in the muscle and hepatopancreas, whereas downregulation was observed in the gills. CONCLUSION: These results suggest that the MrIMD gene may play different roles in response to gram-negative bacteria and viral infection and plays a crucial role in innate immunity as an important key molecule in the defense against bacterial and viral infections.

A novel tumor necrosis factor receptor-associated factor 6 (TRAF6) gene from Macrobrachiumrosenbergii involved in antibacterial defense against Aeromonas hydrophila.

Tumor necrosis factor receptor-associated factor 6 (TRAF6) is an adapter protein that triggers downstream cascades mediated by both TNFR and the interleukin-1 receptor/Toll-like receptor (IL-1R/TLR) superfamily. TRAF6 is involved in various biological processes, including innate and adaptive immunity. In the present study, a homolog of TRAF6 from Macrobrachium rosenbergii (MrTRAF6) was identified and characterized. The full-length cDNA of MrTRAF6 consisted of 2,114 nucleotides with an open reading frame (ORF) of 1,695 nucleotides encoding a 564-amino acid protein that contained a conserved TRAF family motif including two RING-type zinc fingers and a C-terminal meprin and TRAF homology (MATH) domain. The putative amino sequence of MrTRAF6 shared 45.5-97.3% identity with TRAF6s from other crustacean species with the highest identity to Macrobrachium nipponense TRAF6. Phylogenetic analysis revealed that MrTRAF6 was closely related to TRAF6 of invertebrates and clustered with crustaceans. According to gene expression analysis, the MrTRAF6 transcript demonstrated broad expression in all tissues tested, with the highest expression level in gill and the lowest in muscle tissues. Upon immune challenge with Aeromonas hydrophila, significant upregulation of MrTRAF6 expression was found in the gill, hepatopancreas, hemocyte, and muscle. Furthermore, an RNA interference assay showed that silencing MrTRAF6 by dsRNA could reduce the expression of mannose-binding lectin (MBL) and crustin, but no significant change was detected in anti-lipopolysaccharide factor 5 (ALF5) levels. In addition, the cumulative mortality rate of MrTRAF6-silenced M. rosenbergii was significantly increased after A. hydrophila infection. These findings indicated that MrTRAF6 is involved in antibacterial activity and plays a critical role in the innate immune response of M. rosenbergii.

Molecular isolation and expression analysis of hemocyanin isoform 2 of Macrobrachium rosenbergii.

OBJECTIVE: Hemocyanin is a copper-bearing protein in the hemolymph of many arthropods and mollusks and functions as an oxygen transport and important nonspecific immune protein. METHODS: In this study, complementary DNA of hemocyanin isoform 2 of the prawn Macrobrachium rosenbergii (MrHc2) was isolated by rapid amplification of cDNA ends and mRNA expression was characterized to elucidate molecular basis of its function. RESULT: With a molecular mass of 77.3 kDa, MrHc2 contained three domains: hemocyanin-all-alpha, hemocyanin-copper-containing, and hemocyanin-immunoglobulin-like domains. Molecular phylogenetic analysis revealed that MrHc2 belongs to the γ-type subunit and is closely related to hemocyanin subunit 1 of the palaemonid shrimp Macrobrachium nipponense. In addition, MrHc2 resided in a different clade relative to hemocyanin (MrHc) of M. rosenbergii (α-type subunit) and in a different subclade relative to the hemocyanin proteins of penaeid shrimp. The messenger RNA transcript of MrHc2 was highly expressed in the hepatopancreas and weakly expressed in the gills, intestine, stomach, muscle, and hemocytes. Upon challenge with M. rosenbergii nodavirus (MrNV), the expression of MrHc2 was 1.96-, 2.93-, and 1.96-fold on days 3, 4, and 5, respectively, and then gradually declined to basal levels on day 7. CONCLUSION: This study suggests that MrHc2 plays an important role in the innate immune response of M. rosenbergii to MrNV.

Development of cross-priming amplification (CPA) combined with colorimetric and lateral flow dipstick visualization for scale drop disease virus (SDDV) detection.

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.

Development of a rapid immunochromatographic strip test for the detection of Vibrio parahaemolyticus toxin B that cause acute hepatopancreatic necrosis disease.

Here, two monoclonal antibodies (MAbs) specific to different epitopes on ToxB, a toxin produced by Vibrio parahaemolyticus that causes acute hepatopancreatic necrosis disease (VPAHPND ), were employed to develop a rapid strip test. One MAb was conjugated to colloidal gold to bind to ToxB at the application pad, and another MAb was used to capture colloidal gold MAb-protein complexes at the test line (T) on the nitrocellulose strip. To validate test performance, a downstream control line (C) of goat anti-mouse immunoglobulin G antibody was used to capture the free colloidal gold conjugate MAb. The sample in the application buffer could be applied directly to the application well, and the test result was obtained within 15 min. The sensitivity of the kit is approximately 6.25 µg/ml of toxin, which was equivalent to the toxin produced by approximately 107  cfu/ml of bacteria. This kit is convenient and easy to use since it can be used to identify VPAHPND directly using a single colony of bacteria grown on agar culture plates. Because of its high specificity and simplicity, as well as not being reliant on sophisticated equipment or specialized skills, this strip test could be used by farmers for surveillance for ToxB-producing bacteria.

Transcriptomic analysis of Macrobrachium rosenbergii (giant fresh water prawn) post-larvae in response to M. rosenbergii nodavirus (MrNV) infection: de novo assembly and functional annotation.

BACKGROUND: Macrobrachium rosenbergii, is one of a major freshwater prawn species cultured in Southeast Asia. White tail disease (WTD), caused by Macrobrachium rosenbergii nodavirus (MrNV), is a serious problem in farm cultivation and is responsible for up to 100% mortality in the post larvae stage. Molecular data on how M. rosenbergii post-larvae launches an immune response to an infection with MrNV is not currently available. We therefore compared the whole transcriptomic sequence of M. rosenbergii post-larvae before and after MrNV infection. RESULTS: Transcriptome for M. rosenbergii post-larvae demonstrated high completeness (BUSCO Complete: 83.4%, fragmentation: 13%, missing:3.3%, duplication:16.2%; highest ExN50 value: 94%). The assembled transcriptome consists of 96,362 unigenes with N50 of 1308 bp. The assembled transcriptome was successfully annotated against the NCBI non-redundant arthropod database (33.75%), UniProt database (26.73%), Gene Ontology (GO) (18.98%), Evolutionary Genealogy of Genes: Non-supervised Orthologous Groups (EggNOG) (20.88%), and Kyoto Encyclopedia of Genes and Genome pathway (KEGG) (20.46%). GO annotations included immune system process, signaling, response to stimulus, and antioxidant activity. Differential abundance analysis using EdgeR showed 2413 significantly up-regulated genes and 3125 significantly down-regulated genes during the infection of MrNV. CONCLUSIONS: This study reported a highly complete transcriptome from the post-larvae stage of giant river prawn, M. rosenbergii. Differential abundant transcripts during MrNV infection were identified and validated by qPCR, many of these differentially abundant transcripts as key players in antiviral immunity. These include known members of the innate immune response with the largest expression change occurring in the M. rosenbergii post-larvae after MrNV infection such as antiviral protein, C-type lectin, prophenol oxidase, caspase, ADP ribosylation factors, and dicer.

Molecular isolation and characterization of a spätzle gene from Macrobrachium rosenbergii.

Spätzle protein is an extracellular ligand of Toll receptor in Toll signaling pathway involved in the embryonic dorsoventral patterning and in the innate immunity. In this study, a spätzle gene of freshwater prawn, Macrobrachium rosenbergii (MrSpz) was isolated and characterized. The open reading frame of MrSpz consisted of 747 nucleotides encoding 248 amino acid residues containing a signal peptide and C-terminal spätzle activated domain. MrSpz shared high similarity to spätzle of Fenneropenaeus chinensis (FcSpz) at 92% identity and Marsupenaeus japonicus (MjSpz) at 83% identity. Phylogenetic analysis was performed and the results revealed that MrSpz was a member of the clade containing LvSpz3 of Litopenaeus vannamei, FcSpz and Penaeus monodon spätzle protein. The expression distribution at transcriptional level in various tissues of normal prawn revealed that the MrSpz was detected in gills, heart and hepatopancreas while no expression was observed in hemocyte, muscle and stomach. In the Aeromonas caviae challenged prawn, the expression level of MrSpz in hemocyte was increased gradually at 6, 12 and 24 h post-injection. Furthermore, in MrSpz knocked down prawn injected with Aeromonas caviae, the mortality rate were higher than that of non-related dsRNA group and control group. These results suggest that MrSpz protein may play a key role in the innate immunity of M. rosenbergii, especially in response to Gram-negative bacteria A. caviae invasion.

A Natural Vibrio parahaemolyticus ΔpirA Vp pirB Vp+ Mutant Kills Shrimp but Produces neither Pir Vp Toxins nor Acute Hepatopancreatic Necrosis Disease Lesions.

Acute hepatopancreatic necrosis disease (AHPND) of shrimp is caused by Vibrio parahaemolyticus isolates (VPAHPND isolates) that harbor a pVA plasmid encoding toxins PirA Vp and PirB Vp These are released from VPAHPND isolates that colonize the shrimp stomach and produce pathognomonic AHPND lesions (massive sloughing of hepatopancreatic tubule epithelial cells). PCR results indicated that V. parahaemolyticus isolate XN87 lacked pirA Vp but carried pirB Vp Unexpectedly, Western blot analysis of proteins from the culture broth of XN87 revealed the absence of both toxins, and the lack of PirB Vp was further confirmed by enzyme-linked immunosorbent assay. However, shrimp immersion challenge with XN87 resulted in 47% mortality without AHPND lesions. Instead, lesions consisted of collapsed hepatopancreatic tubule epithelia. In contrast, control shrimp challenged with typical VPAHPND isolate 5HP gave 90% mortality, accompanied by AHPND lesions. Sequence analysis revealed that the pVA plasmid of XN87 contained a mutated pirA Vp gene interrupted by the out-of-frame insertion of a transposon gene fragment. The upstream region and the beginning of the original pirA Vp gene remained intact, but the insertion caused a 2-base reading frameshift in the remainder of the pirA Vp gene sequence and in the downstream pirB Vp gene sequence. Reverse transcription-PCR and sequencing of 5HP revealed a bicistronic pirAB Vp mRNA transcript that was not produced by XN87, explaining the absence of both toxins in its culture broth. However, the virulence of XN87 revealed that some V. parahaemolyticus isolates carrying mutant pVA plasmids that produce no Pir Vp toxins can cause mortality in shrimp in ponds experiencing an outbreak of early mortality syndrome (EMS) but may not have been previously recognized to be AHPND related because they did not cause pathognomonic AHPND lesions.IMPORTANCE Shrimp acute hepatopancreatic necrosis disease (AHPND) is caused by Vibrio parahaemolyticus isolates (VPAHPND isolates) that harbor the pVA1 plasmid encoding toxins PirA Vp and PirB Vp The toxins are produced in the shrimp stomach but cause death by massive sloughing of hepatopancreatic tubule epithelial cells (pathognomonic AHPND lesions). V. parahaemolyticus isolate XN87 harbors a mutant pVA plasmid that produces no Pir toxins and does not cause AHPND lesions but still causes ∼50% shrimp mortality. Such isolates may cause a portion of the mortality in ponds experiencing an outbreak of EMS that is not ascribed to VPAHPND Thus, they pose to shrimp farmers an additional threat that would be missed by current testing for VPAHPND Moribund shrimp from ponds experiencing an outbreak of EMS that exhibit collapsed hepatopancreatic tubule epithelial cells can serve as indicators for the possible presence of such isolates, which can then be confirmed by additional PCR tests for the presence of a pVA plasmid.

Interaction study of a novel Macrobrachium rosenbergii effector caspase with B2 and capsid proteins of M. rosenbergii nodavirus reveals their roles in apoptosis.

Apoptosis is an essential immune response to protect invertebrates from virus infected cells. In shrimp, virus infection has been reported to induce apoptosis. Macrobrachium rosenbergii (Mr) was considered to be a disease-resistant host when compared to penaeid shrimps. Caspase-3 was classified as an executioner caspase which played a key role in virus-induced apoptosis. In this study, an effector caspase gene of M. rosenbergii (Mrcasp) was cloned and characterized. The open reading frame (ORF) of Mrcasp was 957 nucleotide encoding 318 amino acid with a deduced molecular mass of 35.87 kDa. RT-PCR analysis showed the presence of Mrcasp in all examined tissues. The phylogenetic tree indicated that Mrcasp was closely related with caspase 3 of shrimp. The functions of the Mrcasp, B2 and capsid proteins of M. rosenbergii nodavirus (MrNV) were assayed in Sf-9 cells. The results showed that Mrcasp induce apoptotic morphology cells; however, capsid protein of MrNV could inhibit apoptotic cells whereas B2 could neither induce nor inhibit apoptotic cells by DAPI staining. The protein interaction between Mrcasp and viral MrNV structure revealed that Mrcasp did not bind to B2 or capsid protein whereas B2 and capsid proteins could bind directly to each other. This study reported a novel sequence of a full-length Mrcasp and its functional studies indicated that Mrcasp could induce apoptotic cells. Our data is the first report demonstrating the direct protein-protein interaction between capsid protein and B2 protein of MrNV.

Molecular cloning and characterization of a Toll receptor gene from Macrobrachium rosenbergii.

Toll receptors are cell surface molecules acting as pattern recognition receptors (PRRs) that have been implicated in the signaling pathway of innate immune responses. In this study, the full-length cDNA of a Toll receptor gene of Macrobrachium rosenbergii, designated MrToll, was successfully isolated using designed degenerate primers and the rapid amplification of cDNA ends (RACE). The MrToll gene sequence contained an open reading frame (ORF) of 2799 nucleotides encoding a protein of 932 amino acid residues. The protein contained distinct structural motifs of the Toll-like receptor (TLR) family, including an extracellular domain containing 15 leucine-rich repeats (LRRs), a transmembrane segment of 23 amino acids, and a cytoplasmic Toll/interleukin-1R (TIR) domain of 139 residues. Phylogenetic analysis revealed that MrToll and Toll receptor of Marsupenaeus japonicus (MjToll) evolved closely. However, the MrToll ORF demonstrated only 48-49% identity with shrimp Toll1, suggesting that MrToll isolated from a palaemonid shrimp might belong to a novel class of Toll receptors in shrimp. The transcripts of the MrToll gene were constitutively expressed in various tissues, with high levels in hemocytes, the stomach and muscle. A reverse transcriptase PCR assay demonstrated that the expression patterns of MrToll were distinctly modulated after Aeromonas caviae stimulation, with significant enhancement at 3-12 h post-challenge and a decline to basal levels at 24 h post-challenge. In addition, when MrToll-silenced shrimp were challenged with A. caviae, there was a significant increase in mortality and bacterial CFU counts. These results suggest that MrToll might be involved in host innate defense, especially against the pathogen A. caviae.

Improved immunodetection of Taura syndrome virus using a monoclonal antibody specific for heterologously expressed VP1 capsid protein.

vp1, a gene encoding one of the capsid proteins of Taura syndrome virus, was cloned into the pGEX-6P-1 expression vector, and the resulting construct was then used to transform E. coli strain BL21. After induction, an N-terminally glutathione-S-transferase-tagged VP1 (GST-VP1) protein with a molecular mass of 80 kDa was obtained. This protein was purified by SDS-PAGE and used for immunization of Swiss mice for monoclonal antibody (MAb) production. Three MAbs specific for the VP1 protein were selected that were suitable for detecting natural TSV infection in Penaeus vannamei by dot blotting, western blotting and immunohistochemistry. This detection occurs without cross-reaction to other shrimp tissues or other common shrimp viruses. As determined by dot blotting, the detection sensitivity of the MAbs was approximately 2 fmole/spot of the GST-VP1. These MAbs showed detection sensitivity comparable to that of MAbs specific for VP2, but they exhibited stronger immunoreactivity than previously studied MAbs specific for VP3. Although the sensitivity of the MAbs to VP1 was 1,000 times lower than one-step RT-PCR, they could be used in various types of antibody-based assays to confirm and enhance the detection sensitivity of TSV infection in shrimp.

Simple and rapid detection of infectious myonecrosis virus using an immunochromatographic strip test.

A strip test was developed for detection of infectious myonecrosis virus (IMNV) using a pair of monoclonal antibodies (MAbs), called IMN7 and IMC6, that are specific for the N and C fragments, respectively, of the IMNV capsid protein. The test strips were placed in plastic cassettes and stored desiccated in sealed plastic bags. In detection assays using the test-strip cassettes, 100-µl samples of application buffer containing homogenates from muscles or pleopods of normal or IMNV-infected shrimp were applied to the cassette sample chamber. Subsequent flow through the glass-fiber pad and the nitrocellulose membrane strip led to the development of visible antibody-protein complexes within 15 min. In samples containing IMNV, viral capsid protein bound to gold-labeled IMN7 in the glass-fiber pad and the complex was subsequently captured by MAb IMC6 at the T line to form a reddish-purple band. Any unbound gold-labeled IMN7 migrated past the T line to be captured by the GAM antibody to form a band at the C line. Samples without IMNV or containing it below the test detection limit gave reddish-purple bands only at the C line. The sensitivity of the test was comparable to that of dot blot tests using single MAbs but was ~300-fold less sensitive than a one-step RT-PCR test for IMNV. Despite this lower sensitivity, the strip test has advantages of low cost, speed and simplicity (i.e., no sophisticated equipment or specialized skills required), and it is appropriate for use by farmers for pathogen confirmation when IMNV is suspected in diseased shrimp.

Improvement of immunodetection of white spot syndrome virus using a monoclonal antibody specific for heterologously expressed icp11.

The icp11 gene encoding the highly abundant DNA mimic protein of white spot syndrome virus (WSSV) was cloned into the pTYB1 and pGEX-6P-1 expression vectors and introduced into E. coli by transformation. After induction, C-terminally intein-tagged ICP11 (ICP11-intein) and N-terminally glutathione-S-transferase (GST)-tagged ICP11 (GST-ICP11) proteins with molecular masses of 64 and 35 kDa were obtained. These proteins were purified by SDS-PAGE and used for immunization of Swiss mice for monoclonal antibody (MAb) production. Two MAbs specific for ICP11 were selected; these MAbs can be used to detect natural WSSV infection in Penaeus vannamei by dot blotting, western blotting or immunohistochemistry without cross-reaction with other shrimp tissues or other common shrimp viruses. The detection sensitivity of the MAbs was approximately 0.7 fmole/spot of GST-ICP11 as determined by dot blotting. These MAbs showed stronger immunoreactivity than other MAbs from previous studies that are specific for VP28 and VP19. A combination of MAbs specific for ICP11, VP28 and VP19 increased the detection sensitivity of WSSV during early infection to a sensitivity 250 times lower than that of one-step PCR. Therefore, the MAbs specific for ICP11 could be used to confirm and enhance the detection sensitivity for WSSV infection in shrimp using various types of antibody-based assays.

Rapid identification and differentiation of Vibrio parahaemolyticus from Vibrio spp. in seafood samples using developed monoclonal antibodies.

Monoclonal antibodies (MAbs) specific to Vibrio parahaemolyticus were successfully generated. According to the specificity of V. parahaemolyticus, MAbs can be classified into 5 groups. The MAbs VP-2D and VP-11H were specific to the O2 and O4 groups of V. parahaemolyticus, respectively. The MAb VP-11B reacted with 11 out of 30 isolates of V. parahaemolyticus used in this study. The MAb VP-516 bound to 27 out of 30 isolates of V. parahaemolyticus and cross reacted with all 10 isolates of V. alginolyticus. The MAb VP-618 demonstrated positive reactivity to 29 out of 30 isolates of V. parahaemolyticus and demonstrated slight cross reactivity to 3 out of 30 isolates of V. harveyi. The sensitivity of the MAbs ranged from 10(8) to 10(7) c.f.u. ml(-1) for V. parahaemolyticus obtained from pure cultures and depended on the group of MAbs. However, the detection capability could be improved to be equivalent to that of the PCR technique following pre-incubation of the samples in alkaline peptone water (APW). Using these MAbs along with MAbs specific to V. alginolyticus (VA-165), V. cholerae (VC-63), V. harveyi (VH-9B and VH-20C) and Vibrio spp. (VC-201) from previous studies, V. parahaemolyticus could be identified and differentiated from Vibrio spp. in various seafood samples including shrimp, green mussels, blood clams and oysters by a simple dot blot immunoassay without the requirement for bacterial isolation or biochemical characterization.

Production of monoclonal antibodies specific to Macrobrachium rosenbergii nodavirus using recombinant capsid protein.

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.

Monoclonal antibodies against extra small virus show that it co-localizes with Macrobrachium rosenbergii nodavirus.

The capsid protein (CP) gene of extra small virus (XSV) expressed in Escherichia coli as a 42 kDa glutathione S-transferase (GST)-fusion 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 cross-reaction 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.

Rapid and sensitive detection of Vibrio vulnificus by loop-mediated isothermal amplification combined with lateral flow dipstick targeted to rpoS gene.

A novel loop-mediated isothermal amplification (LAMP) combined with amplicon detection by chromatographic lateral flow dipstick (LFD) assay was developed and evaluated for the detection of Vibrio vulnificus. Biotinylated LAMP amplicons were produced by a set of six designed primers that recognized the V. vulnificus RNA polymerase subunit sigma factor S (rpoS) gene followed by hybridization with an FITC-labeled probe and LFD detection. The optimized time and temperature conditions for the LAMP assay were 90 min at 65 °C. The LAMP-LFD method accurately identified 14 isolates of V. vulnificus but did not detect 25 non-vulnificus Vibrio isolates and 37 non-Vibrio isolates. The sensitivity of LAMP-LFD for V. vulnificus detection in pure culture was 1.5 × 10(3) CFU ml(-1) or equivalent to 2.8 CFU per reaction. In the case of spiked oyster samples without enrichment, the detection limit for V. vulnificus was 1.2 × 10(4) CFU g(-1) or equivalent to 11 CFU per reaction. The results show that this method appears to be accurate, precise and valuable tool for identification of V. vulnificus and can be used efficiently for detection of V. vulnificus in contaminated food sample.

Point-of-care rapid detection of Vibrio parahaemolyticus in seafood using loop-mediated isothermal amplification and graphene-based screen-printed electrochemical sensor.

Vibrio parahaemolyticus is one of the most important foodborne pathogens that cause various life-threatening diseases in human and animals. Here, we present a rapid detection platform for V. parahaemolyticus by combining loop-mediated isothermal amplification (LAMP) and disposable electrochemical sensors based on screen-printed graphene electrodes (SPGEs). The LAMP reactions using primers targeting V. parahaemolyticus toxR gene were optimized at an isothermal temperature of 65 °C, providing specific detection of V. parahaemolyticus within 45 min at the detection limit of 0.3 CFU per 25 g of raw seafood. The LAMP amplicons can be effectively detected using unmodified SPGEs, redox active molecules namely Hoechst-33258 and a portable potentiostat. Therefore, the proposed system is particularly suitable as a point-of-care device for on-site detection of foodborne pathogens.

Identification of Vibrio spp. in vibriosis Penaeus vannamei using developed monoclonal antibodies.

Immunohistochemical study using monoclonal antibodies specific to various shrimp viruses and Vibrio spp. was performed in shrimp samples died from unknown cause with symptoms of black stripes on lateral sides of cephalothorax or smoky body coloration. The positive results in muscular tissue were obtained with MAb VAL57 (specific to Vibrio spp.) and in hepatopancreas tissues with MAbs VVB158 (specific to V. vulnificus) and VPC701 (specific to V. parahaemolyticus). Twelve isolates of Vibrio spp. isolated from shrimp tissues were identified with various MAbs by dot blotting, biochemical tests and 16S rRNA gene. The results revealed three groups of V. vulnificus and one group of V. shilonii. All four groups of isolated Vibrio spp. were immunologically and biochemically different. None of the V. parahaemolyticus-like bacterium was isolated. The results demonstrated that the mortality in shrimp is accompanied by the presence of Vibrio spp.

Preferential suppression of yellow head virus (YHV) envelope protein gp116 in shrimp that survive challenge with YHV.

The DNA sequence that encodes the first 406 amino acid residues at the N-terminus of yellow head virus (YHV) protein gp116, namely N/2 gp116deltaTM, and the DNA sequence that encodes the next 392 amino acid residues at the C-terminus of gp116 (without the transmembrane region), namely C/2 gp116deltaTM, were cloned into pGEX-6P-1 plasmid and expressed in E. coli. Both recombinant proteins were expressed, purified by SDS-PAGE and used to immunize mice. The mouse anti-recombinant N/2 gp116 and C/2 gp116 antisera bound specifically to both the recombinant proteins and to natural gp116 protein in YHV-infected haemolymph as shown by Western blotting and in tissues as shown by immunohistochemistry. Immunohistochemical localization of YHV using anti-gp116 antiserum or monoclonal antibodies specific to gp116 (V3-2B), gp64 (Y18) and p20 (Y19) revealed similar immunoreactivity patterns for all these reagents in muscle and mandibular tissue in shrimp showing gross signs of yellow head disease. However, in gill, hepatopancreas, lymphoid organ and thoracic ganglion tissues from experimental YHV-infected shrimp (Penaeus vannamei and Palaemon serrifer) that did not show signs of disease, immunoreactivity to gp116 was reduced or absent while that for gp64 and p20 remained intense. Thus, some shrimp species were able to selectively inhibit the synthesis of gp116 in a manner that was associated with absence of gross signs of disease.