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.

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.

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.

Development of uracil-DNA-glycosylase-supplemented loop-mediated isothermal amplification coupled with nanogold probe (UDG-LAMP-AuNP) for specific detection of Pseudomonas aeruginosa.

Pseudomonas aeruginosa (P. aeruginosa) is an important opportunistic pathogen that causes serious infections in humans, including keratitis in contact lens wearers. Therefore, establishing a rapid, specific and sensitive method for the identification of P. aeruginosa is imperative. In the present study, the uracil-DNA-glycosylase-supplemented loop-mediated isothermal amplification combined with nanogold labeled hybridization probe (UDG-LAMP-AuNP) was developed for the detection of P. aeruginosa. UDG-LAMP was performed to prevent carry over contamination and the LAMP reactions can be readily observed using the nanogold probe. A set of 4 primers and a hybridization probe were designed based on the ecfX gene. The UDG-LAMP reactions were performed at 65˚C for 60 min using the ratio of 40% deoxyuridine triphosphate to 60% deoxythymidine triphosphate. The detection of UDG-LAMP products using the nanogold labeled hybridization probe, which appeared as a red-purple color, was examined at 65˚C for 5 min with 40 mM MgSO4. The UDG-LAMP-AuNP demonstrated specificity to all tested isolates of P. aeruginosa without cross reaction to other bacteria. The sensitivity for the detection of pure culture was 1.6x103 colony-forming units (CFU) ml-1 or equivalent to 3 CFU per reaction while that of polymerase chain reaction was 30 CFU per reaction. The detection limit of spiked contact lenses was 1.1x103 CFU ml-1 or equivalent to 2 CFU per reaction. In conclusion, the UDG-LAMP-AuNP assay was rapid, simple, specific and was effective for the identification of P. aeruginosa in contaminated samples.

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.

Development of a PCR Assay Based on a Single-Base Pair Substitution for the Detection of Aeromonas caviae by Targeting the gyrB Gene.

Aeromonas caviae is a bacterial pathogen that causes various infectious diseases in both humans and animals. To facilitate its detection, we developed species-specific primer sets targeting polymorphisms in the gyrB gene for use in a PCR assay. The technique was able to detect 100% (29/29) of the A. caviae strains tested using either of two sets of primers (designated ACF1-ACR and ACF3-ACR), which produced 293-bp and 206-bp amplicons, respectively. Another set of primers (designated ACF2-ACR) yielded a 237-bp amplicon and exhibited 90% (26/29) positive results with respect to A. caviae. None of the primer sets exhibited cross-reactivity with 12 non-A. caviae isolates and 52 other non-Aeromonas bacteria. The detection limit using the ACF2-ACR and ACF3-ACR primer sets in pure culture was 1.6 × 10(3) CFU/mL, or 6 CFU per reaction, whereas that of the ACF1-ACR primer set was 1.6 × 10(4) CFU/mL, or 60 CFU per reaction. In the case of spiked Nile Tilapia Oreochromis niloticus, the sensitivity of all primer sets without enrichment was 1.8 × 10(4) CFU/g, or 30 CFU per reaction. Primer set ACF3-ACR was the best for a PCR assay targeting the gyrB gene, and the PCR technique developed was rapid, specific, and sensitive for the identification of A. caviae.

Rapid and Sensitive Detection of Vibrio alginolyticus by Loop-Mediated Isothermal Amplification Combined with a Lateral Flow Dipstick Targeted to the rpoX Gene.

Vibrio alginolyticus is a major bacterial pathogen causing disease in marine animals. The present study aimed to develop a loop-mediated isothermal amplification (LAMP) coupled with a lateral flow dipstick (LFD) for rapid and simple visual detection of V. alginolyticus-specific amplicons. The biotin-labeled LAMP amplicons from the targeted portion of a gene encoding rpoS-like sigma factor (rpoX) were generated at 60°C for 1 h and then hybridized with a fluorescein isothiocyanate-labeled probe for 5 min for visual detection with LFD. In pure cultures, the detection limit of the LAMP-LFD technique for V. alginolyticus was 1.8 × 10(2) CFU/mL while that of PCR was 1.8 × 10(3) CFU/mL. In spiked whiteleg shrimp samples Penaeus vannamei, the sensitivity for V. alginolyticus detection was 2 × 10(3) CFU/g (equivalent to 4 CFU per reaction) while PCR was 10 times less sensitive. The LAMP-LFD method for V. alginolyticus correctly identified 21 isolates of V. alginolyticus but did not recognize 23 non-V. alginolyticus Vibrio isolates and 15 non-Vibrio isolates. In summary, this LAMP-LFD method targeted to the rpoX gene is a convenient assay for specific identification of V. alginolyticus with high sensitivity.

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.

Immunological-based assays for specific detection of shrimp viruses.

Among shrimp viral pathogens, white spot syndrome virus (WSSV) and yellow head virus (YHV) are the most lethal agents, causing serious problems for both the whiteleg shrimp, Penaeus (Litopenaeus) vannamei, and the black tiger shrimp, Penaeus (Penaeus) monodon. Another important virus that infects P. vannamei is infectious myonecrosis virus (IMNV), which induces the white discoloration of affected muscle. In the cases of taura syndrome virus and Penaeus stylirostris densovirus (PstDNV; formerly known as infectious hypodermal and hematopoietic necrosis virus), their impacts were greatly diminished after the introduction of tolerant stocks of P. vannamei. Less important viruses are Penaeus monodon densovirus (PmDNV; formerly called hepatopancreatic parvovirus), and Penaeus monodon nucleopolyhedrovirus (PemoNPV; previously called monodon baculovirus). For freshwater prawn, Macrobrachium rosenbergii nodavirus and extra small virus are considered important viral pathogens. Monoclonal antibodies (MAbs) specific to the shrimp viruses described above have been generated and used as an alternative tool in various immunoassays such as enzyme-linked immunosorbent assay, dot blotting, Western blotting and immunohistochemistry. Some of these MAbs were further developed into immunochromatographic strip tests for the detection of WSSV, YHV, IMNV and PemoNPV and into a dual strip test for the simultaneous detection of WSSV/YHV. The strip test has the advantages of speed, as the result can be obtained within 15 min, and simplicity, as laboratory equipment and specialized skills are not required. Therefore, strip tests can be used by shrimp farmers for the pond-side monitoring of viral infection.

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.

Evaluation of monoclonal antibody based immunochromatographic strip test for direct detection of Vibrio cholerae O1 contamination in seafood samples.

A strip test for the detection of Vibrio cholerae O1 was developed using two monoclonal antibodies (MAbs), VC-223 and VC-1226, specific to the lipopolysaccharides of Vibrio cholerae O1 Inaba and Ogawa serovars. The sensitivity of the test was 5 × 10(5)cfu/mL which was similar to that of dot blot test. The detection limit could be improved to 1cfu/mL of the original bacterial content after pre-incubation of the bacterium in alkaline peptone water (APW) for 12h. Detection of V. cholerae O1 in various fresh seafood samples such as shrimp, blood clam, mussel and oyster could be performed directly with sensitivities ranged from 5 × 10(5) to 10(6)cfu/mL. After pre-enrichment of the shrimp sample in APW, the detection sensitivities increased to 10(2) to 10CFU/mL of the original bacterial content after incubation for 12 and 24h. However, the detection sensitivities were also depending on the content of the other bacteria that might inhibit the growth of V. cholerae during pre-enrichment step. The V. cholerae O1 strip test has advantages in speed, and simplicity in not requiring sophisticated equipment or specialized skills and the sample could be directly examined without requirement for sample processing.

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.

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.

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.