A unique lectin composing of fibrinogen-like domain from Fenneropenaeus merguiensis contributed in shrimp immune defense and firstly found to mediate encapsulation.

In invertebrates, both fibrinogen-related proteins (FREPs) and C-type lectins are acknowledged to act as pattern recognition receptors (PRRs) to participate particularly in an innate immunity. Hereby, a unique C-type lectin designated as FmLFd was isolated from the hemocytes of Fenneropenaeus merguiensis. FmLFd contained one open reading frame which encoding a peptide of 312 amino acid residues and a signal peptide of 18 amino acids. The primary sequence of FmLFd was composed of a fibrinogen-like domain (Fd) with a Ca2+-binding site and possessing specificity to bind N-acetyl glucosamine (GlcNAc). The FmLFd transcripts were detected mainly in hemocytes of healthy shrimp. The expression of FmLFd was significantly up-regulated upon challenge shrimp with Vibrio parahaemolyticus and Vibrio harveyi which more potent than by white spot syndrome virus (WSSV). The knocking down shrimp with FmLFd double-stranded RNA caused dramatical gene down-regulation. The gene silencing with co-injection of pathogens resulted in reduction of the shrimp survival rate. Recombinant protein of FmLFd (rFmLFd) could agglutinate and bind directly to both Gram-negative and Gram-positive bacteria in a Ca2+-dependent manner and showed the sugar specificity to GlcNAc and bacterial saccharides; peptidoglycan (PGN), lipopolysaccharide (LPS) and lipoteichoic acid (LTA). Recombinant protein of Fd domain (rFd) displayed the lower activity and specificity only to PGN. The binding between recombinant proteins of FmLFd and its domain confirming by ELISA demonstrated that both rFmLFd and rFd could bind to PGN, LPS and LTA with the highest affinity respected to PGN including a less extent of rFd. Besides, rFmLFd but not rFd could bind to WSSV proteins with the highest binding affinity to capsid VP15 and decreasing in order to envelope VP28 and tegument VP39A, respectively. It was presumed that entire molecule of FmLFd exhibited the antimicrobial ability by inhibiting the growth of pathogenic V. parahaemolyticus and this action was not affected by GlcNAc. Otherwise, FmLFd, a lectin containing fibrinogen-like domain, was firstly reported to be capable of promoting encapsulation by hemocytes. Altogether, we concluded that FmLFd belonged to a FREP family indentified by the existence of a conserved fibrinogen-like domain with possessing an ability to bind GlcNAc. It was a new C-type lectin existed in F. merguiensis and might presumably act as a kind of PRRs to participate in the shrimp immune defense towards bacterial and viral pathogens.

FmLC5, a putative galactose-binding C-type lectin with two QPD motifs from the hemocytes of Fenneropenaeus merguiensis participates in shrimp immune defense.

Crustaceans are deficient in adaptive immune system. They depend completely on an innate immunity to protect themselves from invading microorganisms. One kind of pattern recognition receptors that contribute roles in the innate immunity is lectin. A new C-type lectin gene designated as FmLC5 was isolated from Fenneropenaeus merguiensis. Its full-length cDNA is composed of 1526bp and one open reading frame of 852bp encoding a peptide of 284 amino acids. The deduced amino acid sequence of FmLC5 comprises a signal peptide of 20 contiguous amino acids with a molecular mass of 31.47kDa and an isoelectric point of 4.35. The primary structure of FmLC5 consists of two similar carbohydrate recognition domains (CRDs), each CRD contains a Ca2+ binding site-2 and a QPD motif specific for galactose-binding. The FmLC5 transcripts were detected only in the hemocytes analyzed by RT-PCR and in situ hybridization. The FmLC5 expression was significantly up-regulated after challenge with Vibrio harveyi, white spot syndrome virus (WSSV) or lipopolysaccharide. RNAi-based silencing with co-injection by V. harveyi or WSSV resulted in critical suppression of the FmLC5 expression, increasing in mortality and reduction of the median lethal time. These results conclude that FmLC5 is unique putative galactose-binding C-type lectin in F. merguiensis that may contribute as receptor molecule in the immune response to defend the shrimp from pathogenic bacteria and viruses.

A systematic review and meta-analysis of the global prevalence and relationships among Burkholderia pseudomallei sequence types isolated from humans, animals, and the environment.

Background and Aim: Burkholderia pseudomallei, a highly pathogenic bacterium responsible for melioidosis, exhibits ecological ubiquity and thrives within soil and water reservoirs, posing significant infection risks to humans and animals through direct contact. The aim of this study was to elucidate the genetic diversity and prevalence patterns of B. pseudomallei sequence types (STs) across a global spectrum and to understand the relationships between strains isolated from different sources. Materials and Methods: We performed a systematic review and meta-analysis in this study. Extensive research was carried out across three comprehensive databases, including PubMed, Scopus, and ScienceDirect with data collected from 1924 to 2023. Results: A total of 40 carefully selected articles contributed 2737 B. pseudomallei isolates attributed to 729 distinct STs and were incorporated into the systematic review. Among these, ST46 emerged as the most prominent, featuring in 35% of the articles and demonstrating a dominant prevalence, particularly within Southeast Asia. Moreover, ST51 consistently appeared across human, animal, and environmental studies. Subsequently, we performed a meta-analysis, focusing on nine specific STs: ST46, ST51, ST54, ST70, ST84, ST109, ST289, ST325, and ST376. Surprisingly, no statistically significant differences in their pooled prevalence proportions were observed across these compartments for ST46, ST70, ST289, ST325, and ST376 (all p > 0.69). Conversely, the remaining STs, including ST51, ST54, ST84, and ST109, displayed notable variations in their prevalence among the three domains (all p < 0.04). Notably, the pooled prevalence of ST51 in animals and environmental samples surpassed that found in human isolates (p < 0.01). Conclusion: To the best of our knowledge, this study is the first systematic review and meta-analysis to investigate the intricate relationships between STs and their sources and contributes significantly to our understanding of B. pseudomallei diversity within the One Health framework.

Diagnostic accuracy of automation and non-automation techniques for identifying Burkholderia pseudomallei: A systematic review and meta-analysis.

BACKGROUND: Burkholderia pseudomallei, a Gram-negative pathogen, causes melioidosis. Although various clinical laboratory identification methods exist, culture-based techniques lack comprehensive evaluation. Thus, this systematic review and meta-analysis aimed to assess the diagnostic accuracy of culture-based automation and non-automation methods. METHODS: Data were collected via PubMed/MEDLINE, EMBASE, and Scopus using specific search strategies. Selected studies underwent bias assessment using QUADAS-2. Sensitivity and specificity were computed, generating pooled estimates. Heterogeneity was assessed using I2 statistics. RESULTS: The review encompassed 20 studies with 2988 B. pseudomallei samples and 753 non-B. pseudomallei samples. Automation-based methods, particularly with updating databases, exhibited high pooled sensitivity (82.79%; 95% CI 64.44-95.85%) and specificity (99.94%; 95% CI 98.93-100.00%). Subgroup analysis highlighted superior sensitivity for updating-database automation (96.42%, 95% CI 90.01-99.87%) compared to non-updating (3.31%, 95% CI 0.00-10.28%), while specificity remained high at 99.94% (95% CI 98.93-100%). Non-automation methods displayed varying sensitivity and specificity. In-house latex agglutination demonstrated the highest sensitivity (100%; 95% CI 98.49-100%), followed by commercial latex agglutination (99.24%; 95% CI 96.64-100%). However, API 20E had the lowest sensitivity (19.42%; 95% CI 12.94-28.10%). Overall, non-automation tools showed sensitivity of 88.34% (95% CI 77.30-96.25%) and specificity of 90.76% (95% CI 78.45-98.57%). CONCLUSION: The study underscores automation's crucial role in accurately identifying B. pseudomallei, supporting evidence-based melioidosis management decisions. Automation technologies, especially those with updating databases, provide reliable and efficient identification.

Determination of the efficacy of using a serine protease gene as a DNA vaccine to protect against Vibrio parahaemolyticus infection in Litopenaeus vannamei.

Serine proteases are proteolytic enzymes that exhibit biological roles in many biological systems. Previously, a Vibrio parahaemolyticus serine protease was reported to be a virulence factor. Here, the serine protease gene of V. parahaemolyticus was investigated as a DNA vaccine against V. parahaemolyticus in Litopenaeus vannamei. The serine protease gene was mutated to replace the conserved residues His82, Asp131 and Ser231 with Gly, Asp and Pro, respectively. Then, a pcDNA3.1 vector to express mutVpSP (mutant serine protease) was constructed for in vitro and in vivo DNA vaccine investigation. In vivo mutVpSP transcriptional analysis revealed expression in various immunized white shrimp tissues, such as hemocytes, hepatopancreas, stomach, intestine, gills, and muscle. The efficiency of prevention of V. parahaemolyticus infection was investigated in vaccinated shrimp, and the lowest cumulative mortality percentage was 30%, while the control shrimp had a 60% cumulative mortality rate. The immune system was stimulated in shrimp vaccinated with the DNA vaccine. The mRNA expression of the shrimp immune-responsive genes phenoloxidase, peroxinectin and C-type lectin was significantly upregulated. Additionally, the humoral and cellular immune responses, including the PO, phagocytic, and encapsulation activities and nodule formation, were elevated. These results suggested that the serine protease could be a V. parahaemolyticus virulence determinant and that this DNA vaccine could be applied as an effective vaccine candidate for control of acute hepatopancreatic necrosis disease syndrome (AHPND) in shrimp.

Antioxidants, inhibits the growth of foodborne pathogens and reduces nitric oxide activity in LPS-stimulated RAW 264.7 cells of nipa palm vinegar.

Nipa palm vinegar (NPV) made from the sap of nipa palm (Nypa fruticans Wurmb.) has long been used as a local food seasoning and folk medicine. This study compared the bioactive compounds, antioxidant, in vitro anti-inflammatory and antimicrobial activities of three NPVs obtained from different plantations based on varied soil and water salinity levels, including fresh water NPV, brackish water NPV and saline water NPV. The analysis results revealed that total phenolic content of saline water NPV had statistically significantly lower than both fresh water and brackish water NPV (p < 0.0001). Furthermore percentage of acetic acid in brackish water NPV had statistically significantly lower than both fresh water and saline water. NPV (p = 0.002). Nevertheless, total flavonoid and pH, were not significantly different (p = 0.144 and 0.066, respectively). The antioxidant activities using three ABTS, DPPH and FRAP methods displayed similar patterns, in which saline water NPV showed the highest antioxidant activities, followed by brackish water and fresh water NPV, respectively. Antimicrobial activity was examined for seven enteropathogenic bacteria. The tested NPVs were found inhibitive against all test cultures with a minimum inhibitory concentration (MIC) of ≤ 7.8 µL/mL. The cytotoxicity of the NPV obtained from different plantations by MTT assay revealed low cytotoxicity. Anti-inflammatory activity was also carried out through the inhibition of nitric oxide production. The fresh water NPV exhibited the highest anti-inflammatory activity with IC50 17.59 ± 0.17 µL/mL, followed by saline and brackish water NPV with IC50 18.12 ± 0.49 and 28.29 ± 2.64 µL/mL, respectively. The findings indicated that NPV from different soil salinities could potentially be natural functional food and developed to antimicrobial and anti-inflammatory medicinal agents with safety.

Use of Recombinant Escherichia coli Strains in Immunofluorescence Assays for Melioidosis Diagnosis.

Burkholderia pseudomallei is a Gram-negative bacterium and the causative agent of melioidosis in humans and animals in the tropics. The clinical manifestations of melioidosis are diverse, ranging from localized infections to whole-body sepsis. The effective serological method is crucial for the point-of-care diagnosis of melioidosis. The aim of this study was to develop indirect immunofluorescence assay (IFA)-based methods for detecting immunoglobulin G (IgG) antibodies in melioidosis patients. These methods use whole-cell antigens made from recombinant E. coli strains that express major B. pseudomallei antigens, including TssM, OmpH, AhpC, BimA, and Hcp1. A total of 271 serum samples from culture-confirmed melioidosis patients (n = 81), patients with other known infections (n = 70), and healthy donors (n = 120) were tested. Our study showed that the recombinant TssM strain had the highest performance, with 92.6% sensitivity, 100% specificity, 100% positive predictive value, 96.9% negative predictive value, 97.8% efficiency, 97.0% accuracy, and no cross-reactivity. The method agreement analysis based on k efficiency calculations showed that all five IFA methods perfectly agreed with the standard culturing method, while the traditional indirect hemagglutination (IHA) method moderately agreed with the culture. In summary, our investigations showed that the TssM-IFA method could be used for melioidosis diagnosis.