Rapid Apta-Chromogenic Detection Method for Nitrofuran Metabolite Determination.

Nitrofuran (NF) contamination in food products is a global problem resulting in the banned utilization and importation of nitrofuran contaminated products. A novel chromogenic detection method using a specific DNA aptamer with high affinity and specificity to nitrofurans was developed. Single-stranded DNA aptamers specific to nitrofuran metabolites, including 3-amino-2-oxazolidinone (AOZ), 3-amino-5-methylmorpholino-2-oxazolidinone (AMOZ), and 1-aminohydantoin (AHD), were isolated using magnetic bead-SELEX. The colorimetric detection of nitrofurans using gold nanoparticles (AuNPs) exhibited an AOZ detection range of 0.01-0.06 ppb with a limit of detection (LOD) of 0.03 ppb. At the same time, this system could detect AMOZ and AHD at a range of 0.06 ppb and 10 ppb, respectively. The fast nitrofuran extraction method was optimized for food, such as fish tissues and honey, adjusted to be completed within 3-6 h. This novel apta-chromogenic detection method could detect NF metabolites with a sensitivity below the minimum required performance limit (MPRL). This analysis will be valuable for screening, with a shortened time of detection for aquaculture products such as shrimp and fish muscle tissues.

A reusable screen-printed carbon electrode-based aptasensor for the determination of chloramphenicol in food and environment samples.

An electrochemical aptasensor was developed for the determination of chloramphenicol (CAP) in fresh foods and food products. The aptasensor was developed using Prussian blue (PB) and chitosan (CS) film. PB acts as a redox probe for detection and CS acts as a sorption material. The aptamer (Apt) was immobilized on a screen-printed carbon electrode (SPCE) modified with gold nanoparticles (AuNPs). Under optimum conditions, the linearity of the aptasensor was between 1.0 and 6.0 × 106 ng L-1 with a detection limit of 0.65 and a quantification limit of 2.15 ng L-1. The electrode could be regenerated up to 24 times without the use of chemicals. The aptasensor showed good repeatability (RSD <11.2%) and good reproducibility (RSD <7.7%). The proposed method successfully quantified CAP in milk, shrimp pond water and shrimp meat with good accuracy (recovery = 88.0 ± 0.6% to 100 ± 2%). The proposed aptasensor could be especially useful in agriculture to ensure the quality of food and the environment and could be used to determine other antibiotics.

Identification of a unique conserved region from a kinetoplastid genome of Leishmania orientalis (formerly named Leishmania siamensis) strain PCM2 in Thailand.

Mitochondrial DNAs (mtDNAs) appear in almost all eukaryotic species and are useful molecular markers for phylogenetic studies and species identification. Kinetoplast DNAs (kDNAs) are structurally complex circular mtDNA networks in kinetoplastids, divided into maxicircles and minicircles. Despite several kDNAs of many Leishmania species being examined, the kDNAs of the new species, Leishmania orientalis (formerly named Leishmania siamensis) strain PCM2, have not been explored. This study aimed to investigate the maxicircle and minicircle DNAs of L. orientalis strain PCM2 using hybrid genome sequencing technologies and bioinformatic analyses. The kDNA sequences were isolated and assembled using the SPAdes hybrid assembler from the Illumina short-read and PacBio long-read data. Circular contigs of the maxicircle and minicircle DNAs were reconstructed and confirmed by BLASTn and rKOMICs programs. The kDNA genome was annotated by BLASTn before the genome comparison and phylogenetic analysis by progressiveMauve, MAFFT, and MEGA programs. The maxicircle of L. orientalis strain PCM2 (18,215 bp) showed 99.92% similarity and gene arrangement to Leishmania enriettii strain LEM3045 maxicircle with variation in the 12s rRNA gene and divergent region. Phylogenetics of the whole sequence, coding regions, divergent regions, and 12s rRNA gene also confirmed this relationship and subgenera separation. The identified 105 classes of minicircles (402-1177 bp) were clustered monophyletically and related to the Leishmania donovani minicircles. The kinetoplast maxicircle and minicircle DNAs of L. orientalis strain PCM2 contained a unique conserved region potentially useful for specific diagnosis of L. orientalis and further exploration of this parasite population genetics in Thailand and related regions.

Cocultures of Enterococcus faecium and Aeromonas veronii Induce the Secretion of Bacteriocin-like Substances against Aeromonas.

Lactic acid bacteria (LAB) were screened from Lutjanus russellii (red sea bass), and their antimicrobial activities were evaluated against two Aeromonas species isolated from the Nile tilapia, namely, Aeromonas veronii (AV) and Aeromonas jandaei (AJ). Three LAB isolates, Enterococcus faecium MU8 (EF_8), Enterococcus faecalis MU2 (EFL_2), and E. faecalis MU9 (EFL_9), were found to inhibit both AV and AJ; however, their cell-free supernatant (CFS) did not do so. Interestingly, bacteriocin-like substances (BLS) induced by cocultures of EF_8 with AV exhibited the highest antimicrobial activity against both Aeromonas sp. The size of BLS was less than 1.0 kDa; the purified BLS were susceptible to proteinase K digestion, indicating that they are peptides. BLS contained 13 identified peptides derived from E. faecium, as determined by liquid chromatography-tandem mass spectrometry. Cocultures of Gram-positive-producing and -inducing LAB strains have been used to increase bacteriocin yields. To our knowledge, this is the first report describing inducible BLS produced by cocultures of Gram-positive-producing and Gram-negative-inducing strains.

Evaluation of Bacillus spp. as Potent Probiotics with Reduction in AHPND-Related Mortality and Facilitating Growth Performance of Pacific White Shrimp (Litopenaeus vannamei) Farms.

Acute hepatopancreatic necrosis disease (AHPND) is a serious bacterial disease affecting shrimp aquaculture worldwide. In this study, natural microbes were used in disease prevention and control. Probiotics derived from Bacillus spp. were isolated from the stomachs of AHPND-surviving Pacific white shrimp Litopenaeus vannamei (22 isolates) and mangrove forest soil near the shrimp farms (10 isolates). Bacillus spp. were genetically identified and characterized based on the availability of antimicrobial peptide (AMP)-related genes. The phenotypic characterization of all Bacillus spp. was determined based on their capability to inhibit AHPND-causing strains of Vibrio parahaemolyticus (VPAHPND). The results showed that Bacillus spp. without AMP-related genes were incapable of inhibiting VPAHPND in vitro, while other Bacillus spp. harboring at least two AMP-related genes exhibited diverse inhibition activities. Interestingly, K3 [B. subtilis (srfAA+ and bacA+)], isolated from shrimp, exerted remarkable inhibition against VPAHPND (80% survival) in Pacific white shrimp and maintained a reduction in shrimp mortality within different ranges of salinity (75-95% survival). Moreover, with different strains of VPAHPND, B. subtilis (K3) showed outstanding protection, and the survival rate of shrimp remained stable among the tested groups (80-95% survival). Thus, B. subtilis (K3) was further used to determine its efficiency in shrimp farms in different locations of Vietnam. Lower disease occurrences (2 ponds out of 30 ponds) and greater production efficiency were noticeable in the B. subtilis (K3)-treated farms. Taking the results of this study together, the heat-shock isolation and genotypic-phenotypic characterization of Bacillus spp. enable the selection of probiotics that control AHPND in Pacific white shrimp. Consequently, greater disease prevention and growth performance were affirmed to be beneficial in the use of these probiotics in shrimp cultivation, which will sustain shrimp aquaculture and be environmentally friendly.

Identification of a conserved maxicircle and unique minicircles as part of the mitochondrial genome of Leishmania martiniquensis strain PCM3 in Thailand.

BACKGROUND: The mitochondrial DNA of trypanosomatids, including Leishmania, is known as kinetoplast DNAs (kDNAs). The kDNAs form networks of hundreds of DNA circles that are evidently interlocked and require complex RNA editing. Previous studies showed that kDNA played a role in drug resistance, adaptation, and survival of Leishmania. Leishmania martiniquensis is one of the most frequently observed species in Thailand, and its kDNAs have not been illustrated. METHODS: This study aimed to extract the kDNA sequences from Illumina short-read and PacBio long-read whole-genome sequence data of L. martiniquensis strain PCM3 priorly isolated from the southern province of Thailand. A circular maxicircle DNA was reconstructed by de novo assembly using the SPAdes program, while the minicircle sequences were retrieved and assembled by the rKOMIC tool. The kDNA contigs were confirmed by blasting to the NCBI database, followed by comparative genomic and phylogenetic analysis. RESULTS: We successfully constructed the complete circular sequence of the maxicircle (19,008 bp) and 214 classes of the minicircles from L. martiniquensis strain PCM3. The genome comparison and annotation showed that the maxicircle structure of L. martiniquensis strain PCM3 was similar to those of L. enriettii strain LEM3045 (84.29%), L. arabica strain LEM1108 (82.79%), and L. tarentolae (79.2%). Phylogenetic analysis also showed unique evolution of the minicircles of L. martiniquensis strain PCM3 from other examined Leishmania species. CONCLUSIONS: This was the first report of the complete maxicircle and 214 minicircles of L. martiniquensis strain PCM3 using integrated whole-genome sequencing data. The information will be helpful for further improvement of diagnosis methods and monitoring genetic diversity changes of this parasite.

Design of a Chimeric Multi-Epitope Vaccine (CMEV) against Both Leishmania martiniquensis and Leishmania orientalis Parasites Using Immunoinformatic Approaches.

Leishmaniasis is a parasitic disease caused by protozoan flagellates of the genus Leishmania. Recently, Leishmania martiniquensis and Leishmania orientalis, emerging species of Leishmania, were isolated from patients in Thailand. Development of the vaccine is demanded; however, genetic differences between the two species make it difficult to design a vaccine that is effective for both species. In this study, we applied immuno-informatic approaches to design a chimeric multi-epitope vaccine (CMEV) against both L. martiniquensis and L. orientalis. We identified seven helper T lymphocyte (HTL) epitopes, sixteen cytotoxic T lymphocyte (CTL) epitopes, and eleven B-cell epitopes from sixteen conserved antigenic proteins found in both species. All these epitopes were joined together, and to further enhance immunogenicity, protein and peptides adjuvant were also added at the N-terminal of the molecule by using specific linkers. The candidate CMEV was subsequently analyzed from the perspectives of the antigenicity, allergenicity, and physiochemical properties. The interaction of the designed multi-epitope vaccine and immune receptor (TLR4) of the host were evaluated based on molecular dockings of the predicted 3D structures. Finally, in silico cloning was performed to construct the expression vaccine vector. Docking analysis showed that the vaccine/TLR4 complex took a stable form. Based on the predicted immunogenicity, physicochemical, and structural properties in silico, the vaccine candidate was expected to be appropriately expressed in bacterial expression systems and show the potential to induce a host immune response. This study proposes the experimental validation of the efficacy of the candidate vaccine construct against the two Leishmania.

Trial Evaluation of Protection and Immunogenicity of Piscine Bivalent Streptococcal Vaccine: From the Lab to the Farms.

Streptococcosis is one of the major diseases that causes devastation to farmed fish, leading to significant economic losses all around the world. Currently, two serotypes of Streptococcus agalactiae, serotype Ia and III, have been identified as virulent strains and major causative agents of the disease in farmed Nile tilapia (Oreochromis niloticus Linn.) in Thailand. Upon inactivated vaccine development, monovalent inactivated whole-cell vaccines demonstrated high specific antibody production against homologous serotypes and limited production with heterologous serotypes. However, for higher efficacy, a bivalent streptococcal vaccine was designed to maximize protective immunity to both serotypes. Interestingly, our bivalent vaccine could successfully induce specific antibody production against both serotypes with similar levels, and the response could extend over the 8 weeks of the experimental period. Evaluation of vaccines in the laboratory scale revealed relative percent survival (RPS) of vaccinated tilapia to serotype Ia (81.2 ± 9.4%) and serotype III (72.2 ± 4.8%), respectively. The efficacy of the bivalent vaccine showed significant RPS higher than the monovalent vaccine (p < 0.05) at 30 days, and the protection of all those vaccines was reduced thereafter. Evaluation of the vaccine in a farm trial in different locations in Thailand revealed the efficacy of the bivalent vaccine in increasing the production yield by greater than 80% in all tested farms in 2015 and 2021. Taken together, this study affirms the efficacy of the bivalent streptococcal vaccine in the prevention of streptococcus disease in Nile tilapia, which could be used in different areas. This vaccine development could be effectively applied in the tilapia culture industry.

Adeno-associated virus 9 vector-mediated cardiac-selective expression of human secretory leukocyte protease inhibitor attenuates myocardial ischemia/reperfusion injury.

Protease enzymes contribute to the initiation of cardiac remodeling and heart failure after myocardial ischemic/reperfusion (I/R) injury. Protease inhibitors attenuate protease activity and limit left ventricular dysfunction and remodeling. Previous studies showed the cardioprotective effect of secretory leukocyte protease inhibitor (SLPI) against I/R injury. However, overexpression of SLPI gene in cardiovascular diseases has only been investigated in an in vitro experiment. Here, cardiac-selective expression of the human secretory leukocyte protease inhibitor (hSLPI) gene and its effect on I/R injury were investigated. Adeno-associated virus (AAV) serotype 9 carrying hSLPI under the control of cardiac-selective expression promoter (cardiac troponin, cTn) was intravenously administered to Sprague-Dawley rats for 4 weeks prior to coronary artery ligation. The results showed that myocardial-selective expression of hSLPI significantly reduced infarct size, cardiac troponin I (cTnI), creatine kinase-MB (CK-MB), and myoglobin levels that all served to improve cardiac function. Moreover, overexpression of hSLPI showed a reduction in inflammatory cytokines, oxidatively modified protein carbonyl (PC) content, ischemia-modified albumin (IMA), and necrosis and cardiac tissue degeneration. In conclusion, this is the first study to demonstrate cardiac-selective gene delivery of hSLPI providing cardioprotection against myocardial I/R injury in an in vivo model.

Making the Most of Its Short Reads: A Bioinformatics Workflow for Analysing the Short-Read-Only Data of Leishmania orientalis (Formerly Named Leishmania siamensis) Isolate PCM2 in Thailand.

BACKGROUND: Leishmania orientalis (formerly named Leishmania siamensis) has been neglected for years in Thailand. The genomic study of L. orientalis has gained much attention recently after the release of the first high-quality reference genome of the isolate LSCM4. The integrative approach of multiple sequencing platforms for whole-genome sequencing has proven effective at the expense of considerably expensive costs. This study presents a preliminary bioinformatic workflow including the use of multi-step de novo assembly coupled with the reference-based assembly method to produce high-quality genomic drafts from the short-read Illumina sequence data of L. orientalis isolate PCM2. RESULTS: The integrating multi-step de novo assembly by MEGAHIT and SPAdes with the reference-based method using the L. enriettii genome and salvaging the unmapped reads resulted in the 30.27 Mb genomic draft of L. orientalis isolate PCM2 with 3367 contigs and 8887 predicted genes. The results from the integrated approach showed the best integrity, coverage, and contig alignment when compared to the genome of L. orientalis isolate LSCM4 collected from the northern province of Thailand. Similar patterns of gene ratios and frequency were observed from the GO biological process annotation. Fifty GO terms were assigned to the assembled genomes, and 23 of these (accounting for 61.6% of the annotated genes) showed higher gene counts and ratios when results from our workflow were compared to those of the LSCM4 isolate. CONCLUSIONS: These results indicated that our proposed bioinformatic workflow produced an acceptable-quality genome of L. orientalis strain PCM2 for functional genomic analysis, maximising the usage of the short-read data. This workflow would give extensive information required for identifying strain-specific markers and virulence-associated genes useful for drug and vaccine development before a more exhaustive and expensive investigation.

Combining segments 9 and 10 in DNA and recombinant protein vaccines conferred superior protection against tilapia lake virus in hybrid red tilapia (oreochromis sp.) compared to single segment vaccines.

Tilapia lake virus (TiLV) now affects Nile tilapia culture worldwide, with no available commercial vaccine for disease prevention. DNA and recombinant protein-based vaccines were developed and tested following viral isolation and characterization. The viral strain isolated from diseased hybrid red tilapia (Oreochromis sp.) shared high levels of morphological and genomic similarity (95.49-99.52%) with other TiLV isolates in the GenBank database. TiLV segment 9 (Tis9) and segment 10 (Tis10) DNA vaccines (pcDNA-Tis9 and pcDNA-Tis10) and recombinant protein vaccines (Tis9 and Tis10) were prepared and tested for their efficacy in juvenile hybrid red tilapia. Fish were immunized with either single vaccines (pcDNA-Tis9, pcDNA-Tis10, Tis9 and Tis10) or combined vaccines (pcDNA-Tis9 + pcDNA-Tis10 and Tis9 + Tis10) by intramuscular injection and intraperitoneal injection for DNA and protein vaccines, respectively. Negative controls were injected with PBS or a naked pcDNA3.1 vector in the same manner. An experimental challenge with TiLV was carried out at 4 weeks post-vaccination (wpv) by intraperitoneal injection with a dose of 1 × 105 TCID50 per fish. Relative percent survival (RPS) ranged from 16.67 ± 00.00 to 61.11 ± 9.62%. The Tis10 and pcDNA-Tis10 vaccines conferred better protection compared to Tis9 and pcDNA-Tis9. Highest levels of protection were observed in pcDNA-Tis9 + pcDNA-Tis10 (61.11 ± 9.62%) and Tis9 + Tis10 (55.56 ± 9.62%) groups. Specific antibody was detected in all vaccinated groups at 1-4 wpv by Dot Blot method, with the highest integrated density at 2 and 3 wpv. In silico analysis of Tis9 and Tis10 revealed a number of B-cell epitopes in their coil structure, possibly reflecting their immunogenicity. Findings suggested that the combination of Tis9 and Tis10 in DNA and recombinant protein vaccine showed high efficacy for the prevention of TiLV disease in hybrid red tilapia.

A Novel Efficient Piscine Oral Nano-Vaccine Delivery System: Modified Halloysite Nanotubes (HNTs) Preventing Streptococcosis Disease in Tilapia (Oreochromis sp.).

Generally, the injection method is recommended as the best efficient method for vaccine applications in fish. However, labor-intensive and difficult injection for certain fish sizes is always considered as a limitation to aquatic animals. To demonstrate the effectiveness of a novel oral delivery system for the piscine vaccine with nano-delivery made from nano clay, halloysite nanotubes (HNTs) and their modified forms were loaded with killed vaccines, and we determined the ability of the system in releasing vaccines in a mimic digestive system. The efficaciousness of the oral piscine vaccine nano-delivery system was evaluated for its level of antibody production and for the level of disease prevention in tilapia. Herein, unmodified HNTs (H) and modified HNTs [HNT-Chitosan (HC), HNT-APTES (HA) and HNT-APTES-Chitosan (HAC)] successfully harbored streptococcal bivalent vaccine with inactivated S. agalactiae, designated as HF, HAF, HCF and HACF. The releasing of the loading antigens in the mimic digestive tract demonstrated a diverse pattern of protein releasing depending on the types of HNTs. Remarkably, HCF could properly release loading antigens with relevance to the increasing pH buffer. The oral vaccines revealed the greatest elevation of specific antibodies to S. agalactiae serotype Ia in HCF orally administered fish and to some extent in serotype III. The efficacy of streptococcal disease protection was determined by continually feeding with HF-, HAF-, HCF- and HACF-coated feed pellets for 7 days in the 1st and 3rd week. HCF showed significant RPS (75.00 ± 10.83%) among the other tested groups. Interestingly, the HCF-treated group exhibited noticeable efficacy similar to the bivalent-vaccine-injected group (RPS 81.25 ± 0.00%). This novel nano-delivery system for the fish vaccine was successfully developed and exhibited appropriated immune stimulation and promised disease prevention through oral administration. This delivery system can greatly support animals' immune stimulation, which conquers the limitation in vaccine applications in aquaculture systems. Moreover, this delivery system can be applied to carrying diverse types of biologics, including DNA, RNA and subunit protein vaccines.

Anti-Protease Activity Deficient Secretory Leukocyte Protease Inhibitor (SLPI) Exerts Cardioprotective Effect against Myocardial Ischaemia/Reperfusion.

Inhibition of proteases shows therapeutic potential. Our previous studies demonstrated the cardioprotection by the Secretory Leukocyte Protease Inhibitor (SLPI) against myocardial ischaemia/reperfusion (I/R) injury. However, it is unclear whether the cardioprotective effect of SLPI seen in our previous works is due to the inhibition of protease enzymes. Several studies demonstrate that the anti-protease independent activity of SLPI could provide therapeutic benefits. Here, we show for the first time that recombinant protein of anti-protease deficient mutant SLPI (L72K, M73G, L74G) (mt-SLPI) could significantly reduce cell death and intracellular reactive oxygen species (ROS) production against an in vitro simulated I/R injury. Furthermore, post-ischaemic treatment of mt-SLPI is found to significantly reduce infarct size and cardiac biomarkers lactate dehydrogenase (LDH) and creatine kinase-MB (CK-MB) activity, improve cardiac functions, attenuate I/R induced-p38 MAPK phosphorylation, and reduce apoptotic regulatory protein levels, including Bax, cleaved-Caspase-3 and total Capase-8, in rats subjected to an in vivo I/R injury. Additionally, the beneficial effect of mt-SLPI was not significantly different from the wildtype (wt-SLPI). In summary, SLPI could provide cardioprotection without anti-protease activity, which could be more clinically beneficial in terms of providing cardioprotection without interfering with basal serine protease activity.

Comparative Draft Genomes of Leishmania orientalis Isolate PCM2 (Formerly Named Leishmania siamensis) and Leishmania martiniquensis Isolate PCM3 from the Southern Province of Thailand.

(1) Background: Autochthonous leishmaniasis, a sandfly-borne disease caused by the protozoan parasites Leishmania orientalis (formerly named Leishmania siamensis) and Leishmania martiniquensis, has been reported for immunocompromised and immunocompetent patients in the southern province of Thailand. Apart from the recent genomes of the northern isolates, limited information is known on the emergence and genetics of these parasites. (2) Methods: This study sequenced and compared the genomes of L. orientalis isolate PCM2 and L. martiniquensis isolate PCM3 with those of the northern isolates and other 14 Leishmania species using short-read whole-genome sequencing methods and comparative bioinformatic analyses. (3) Results: The genomes of the southern isolates of L. orientalis and L. martiniquensis were 30.01 Mbp and 32.39 Mbp, and the comparison with the genomes of the northern isolates revealed species-level similarity with a level of genome and proteome variation, suggesting the different strains. Comparative proteome analysis showed six protein groups with 53 unique proteins for the strain PCM2 and 97 for the strain PCM3. Certain proteins were related to virulence, drug resistance, and stress response. (4) Conclusion: Therefore, the findings could indicate the need for more genetic and population genomic investigation, and the close monitoring of L. orientalis and L. martiniquensis in Thailand and neighboring regions.

Challenges in Veterinary Vaccine Development.

Animals provide food and clothing in addition to other value-added products. Changes in diet and lifestyle have increased the consumption and the use of animal products. Infectious diseases in animals are a major threat to global animal health and its welfare; their effective control is crucial for agronomic health, for safeguarding food security and also alleviating rural poverty. Development of vaccines has led to increased production of healthy poultry, livestock, and fish. Animal production increases have alleviated food insecurity. In addition, development of effective vaccines has led to healthier companion animals. However, challenges remain including climate change that has led to enhancement in vectors and pathogens that may lead to emergent diseases in animals. Preventing transmission of emerging infectious diseases at the animal-human interface is critically important for protecting the world population from epizootics and pandemics. Hence, there is a need to develop new vaccines to prevent diseases in animals. This review describes the broad challenges to be considered in the development of vaccines for animals.

Novel Vaccine Development for Fish Culture Based on the Multiepitope Concept.

For the past several decades, aquaculture all around the world have been retarded by various disease outbreaks caused by many pathogens including parasites, bacteria, and viruses. Apart from being harmful to human health, the emerging diseases also dramatically affect the farm animals such as livestock and aquatic animals. To cope with this problem, one of the effective prophylactic measures is the application of vaccine. However, the traditional vaccines still have some limitations and several drawbacks; thus there is a need for the development of novel advanced vaccine such as chimeric multiepitope vaccine. Based on the current understanding of genomics and immunoproteomics together with the present bioinformatics tools, the researchers can identify the potential targeted epitopes being recognizable by the immune cells. Additionally, another critical point that should be considered for designing the chimeric multiepitope vaccine is the exposure of all those epitopes to the host organism. Thus, selecting an appropriate linker and joining each identified epitope in a suitable site can create the ideal protein structure protruding all the selected epitopes on its surface. Herein, our study would provide the fundamental platform to develop the multiepitope B-cell vaccine for the prevention and control of the aquatic animal disease starting with the epitope prediction until in vivo testing the multiepitope vaccine efficacy.

Identification and Distribution of Wedge Clams (Donacidae: Bivalvia) in Thailand by Geometric Morphometric and Molecular Analysis.

The phylogenetic relationship of living Thai Donacidae was herein studied. Two methodologies, geometric morphometrics (GM) and genetic analysis of COI sequences, were combined and applied to identify the valid taxa and explain biodiversity and the distribution pattern in this family. A total of 587 living specimens were tested to analyze the shape and size patterns by Elliptic Fourier Analysis (EFA). Shell identification and GenBank sequences were added to construct the phylogenetic relationship and haplotype network. Centroid size was used to identify the specimens to the subgenus level. Donax (Hecuba) scortum, was easily distinguished from other species by Principal Component analysis (PCA) of shell size and shape. Donax (Dentilatona) incarnatus and Donax (Deltachion) semisulcatus semisulcatus were identified using Canonical Variates Analysis (CVA). Pairwise comparison of EFA was used for species level recognition, particularly shape overlap was observed for medium and small shell size. Based on genetic distance and haplotype network of COI sequences, Donax (Latona) faba and D. (Latona) solidus could be grouped in the same clade. Intraspecific and interspecific genetic data variation of some common species in different geographical localities of Thailand was observed. Three distribution patterns of Donax species were observed along the two-marine system of Thailand.

Molecular Characterization and Cross-Allergenicity of Tropomyosin from Freshwater Crustaceans.

Tropomyosin is a major allergen responsible for cross-allergenicity in a number of shellfish species. Although extensively characterized in marine crustaceans, the information of tropomyosin is limited to a few freshwater crustacean species. As a result, more cross-reactivity evidence and information of tropomyosin at the molecular level are required for the detection of freshwater crustaceans in the food industry. In this study, we explored tropomyosin allergenicity in four freshwater crustacean species: prawn (Macrobrachium rosenbergii and Macrobrachium lanchesteri) and crayfish (Procambarus clarkii and Cherax quadricarinatus). Immunoblotting, liquid chromatography-tandem mass spectrometry, and immunoprecipitation studies indicated that tropomyosin was recognized by the sera's IgE of crustacean-allergic volunteers. Cloning and characterization of nucleotide sequences of tropomyosin cDNA from M. lanchesteri and C. quadricarinatus revealed highly conserved amino acid sequences with other crustaceans. This study emphasized the role of tropomyosin as a universal marker for the detection of both freshwater and marine crustaceans in the food industry.

Biosensors for the detection of organophosphate exposure by a new diethyl thiophosphate-specific aptamer.

OBJECTIVE: An aptamer specifically binding to diethyl thiophosphate (DETP) was constructed and incorporated in an optical sensor and electrochemical techniques to enable the specific measurement of DETP as a metabolite and a biomarker of organophosphate exposure. RESULTS: A DETP-bound aptamer was selected from the library using capillary electrophoresis-systematic evolution of ligands by exponential enrichment (CE-SELEX). A colorimetric method revealed that the aptamer had the highest affinity for DETP, with a mean Kd value (± SD) of 0.103 ± 0.014 µM. The docking results and changes in resistance showed that the selectivity of the aptamer for DETP was higher than that for the similar structures of dithiophosphate (DEDTP) and diethyl phosphate (DEP). The altered amplitude of cyclic voltammetry showed a linear range of DETP detection covering 0.0001-10 µg/ml with a limit of detection of 0.007 µg/ml. The recovery value of a real sample of pH 7 was 97.2%. CONCLUSIONS: The current method showed great promise in using the DETP-specific aptamer to detect the exposure history to organophosphates by measuring their metabolites, although degradation of organophosphate parent compounds might occur.

Refolded recombinant major capsid protein (MCP) from Infectious Spleen and Kidney Necrosis Virus (ISKNV) effectively stimulates serum specific antibody and immune related genes response in Nile tilapia (Oreochromis niloticus).

Infectious spleen and kidney necrosis virus (ISKNV) is a causative agent of high mortality in fish resulting in significant economic loss to the fish industry in many countries. The major capsid protein (MCP) (ORF006) is an important structural component that mediates virus entry into the host cell, therefore it is a good candidate antigen of ISKNV for subunit vaccine development. In this study, MCP of ISKNV was successfully produced in Escherichia coli strain Ril and was purified as the soluble form by refolding recombinant MCP using urea in combination with dialysis process. The refolded recombinant MCP protein had ability of oligomerization to become trimer like native MCP protein. Fish immunized with refolded recombinant MCP showed significantly higher serum antibody titer than fish immunized with insoluble form of the protein (p < 0.05) at 21, 28- and 35-day post-immunization (dpi). Analysis of immune-related genes response in spleen and kidney of fish immunized with refolded recombinant MCP suggested that MHC-I, MHC-II, IL-1ß and IL-4 genes were also significantly expressed relative to the group immunized with insoluble protein (p < 0.05) at 14, 21, 28- and 35-day post immunization. The highest serum antibody and immune related genes response were found at 28 day post immunization. Therefore, refolded recombinant MCP should be better than previously reported insoluble form as the candidate subunit vaccine to prevent infection of Nile tilapia from ISKNV.