Simultaneous detection of multiple bacterial and viral aquatic pathogens using a fluorogenic loop-mediated isothermal amplification-based dual-sample microfluidic chip.

Rapid and user-friendly diagnostic tests are necessary for early diagnosis and immediate detection of diseases, particularly for on-site screening of pathogenic microorganisms in aquaculture. In this study, we developed a dual-sample microfluidic chip integrated with a real-time fluorogenic loop-mediated isothermal amplification assay (dual-sample on-chip LAMP) to simultaneously detect 10 pathogenic microorganisms, that is Aeromonas hydrophila, Edwardsiella tarda, Vibrio harveyi, V. alginolyticus, V. anguillarum, V. parahaemolyticus, V. vulnificus, infectious hypodermal and haematopoietic necrosis virus, infectious spleen and kidney necrosis virus, and white spot syndrome virus. This on-chip LAMP provided a nearly automated protocol that can analyse two samples simultaneously, and the tests achieved limits of detection (LOD) ranging from 100 to 10-1  pg/µl for genomic DNA of tested bacteria and 10-4 to 10-5  pg/µl for recombinant plasmid DNA of tested viruses, with run times averaging less than 30 min. The coefficient of variation for the time-to-positive value was less than 10%, reflecting a robust reproducibility. The clinical sensitivity and specificity were 93.52% and 85.53%, respectively, compared to conventional microbiological or clinical methods. The on-chip LAMP assay provides an effective dual-sample and multiple pathogen analysis, and thus would be applicable to on-site detection and routine monitoring of multiple pathogens in aquaculture.

Molecular characterization of a cDNA encoding Na+/K+/2Cl- cotransporter in the gill of mud crab (Scylla paramamosain) during the molt cycle: Implication of its function in osmoregulation.

Although iono-regulatory processes are critical for survival of crustaceans during the molt cycle, the mechanisms involved are still not clear. The Na+/K+/2Cl- cotransporter (NKCC), a SLC12A family protein that transports Na+, K+ and 2Cl- into cells, is essential for cell ionic and osmotic regulation. To better understand the role of NKCC in the molt osmoregulation, we cloned and characterized a NKCC gene from the mud crab, Scylla paramamosain (designated as SpNKCC). The predicted SpNKCC protein is well conserved, and phylogenetic analysis revealed that this protein was clustered with crustacean NKCC. Expression of SpNKCC was detected in all the tissues examined but was highest in the posterior gills. Transmission electron microscopy revealed that posterior gills had a thick type of epithelium for ion regulation while the anterior gills possessed a thin phenotype related to gas exchange. During the molting cycle, hemolymph osmolality and ion concentrations (Na+ and Cl-) increased significantly over the postmolt period, remained stable in the intermolt and premolt stages and then decreased at ecdysis. Meanwhile, the expression of SpNKCC mRNA was significantly elevated (26.7 to 338.8-fold) at the ion re-establishing stages (postmolt) as compared with baseline molt level. This pattern was consistent with the coordinated regulation of Na+/K+-ATPase α-subunit (NKA α), carbonic anhydrase cytoplasmic (CAc) isoform and Na+/H+ exchanger (NHE) genes in the posterior gills. These data suggest that SpNKCC may be important in mediating branchial ion uptake during the molt cycle, especially at the postmolt stages.

Two colistin resistance-producing Aeromonas strains, isolated from coastal waters in Zhejiang, China: characteristics, multi-drug resistance and pathogenicity.

Introduction: Aeromonas spp. are ubiquitous inhabitants of ecosystems, and many species are opportunistically pathogenic to humans and animals. Multidrug-resistant (MDR) Aeromonas species have been widely detected in hospitals, urban rivers, livestock, and aquatic animals. Results: In this study, we identified two Aeromonas isolates, namely Aeromonas veronii 0728Q8Av and Aeromonas caviae 1029Y16Ac, from coastal waters in Zhejiang, China. Both isolates exhibited typical biochemical characteristics and conferred MDR to 11 kinds of antibiotics, remaining susceptible to ceftazidime. Whole-genome sequencing revealed that both isolates harbored multiple antibiotic resistance genes (ARGs) and several mobile genetic elements (MGEs) on the chromosomes, each containing a resistance genomic island (GI), a typical class 1 integron, a transposon, and various insertion sequences (ISs). Most ARGs were situated within the multiple resistance GI, which contained a class 1 integron and a transposon in both Aeromonas isolates. Furthermore, a chromosomal mcr-3.16 gene was identified in A. veronii 0728Q8Av, while a chromosomal mcr-3.3 was found in A. caviae 1029Y16Ac. Both mcr-3 variants were not located within but were distanced from the multidrug resistance GI on the chromosome, flanking by multiple ISs. In addition, a mcr-3-like was found adjacent to mcr-3.16 to form a tandem mcr-3.16-mcr-3-like-dgkA structure; yet, Escherichia coli carrying the recombinants of mcr-3-like did not exhibit resistance to colistin. And an incomplete mcr-3-like was found adjacent to mcr-3.3 in A. caviae 1029Y16Ac, suggesting the possibility that mcr-3 variants originated from Aeromonas species. In vivo bacterial pathogenicity test indicated that A. veronii 0728Q8Av exhibited moderate pathogenicity towards infected ayu, while A. caviae 1029Y16Ac was non-virulent. Discussion: Thus, both Aeromonas species deserve further attention regarding their antimicrobial resistance and pathogenicity.

A novel antiviral coumarin derivative as a potential agent against WSSV infection in shrimp seedling culture.

White spot syndrome virus (WSSV), a double-stranded DNA virus that infects crustaceans, is the most serious viral pathogen affecting shrimp farming worldwide. To reduce the economic losses caused by WSSV, we screened a novel coumarin derivative from a small molecule drug library, N-(4-((4-(((2-oxo-2H-chromen-7-yl)oxy)methyl)-1H-1,2,3-triazol-1-yl)sulfonyl)phenyl)acetamide (N2905), to evaluate its anti-WSSV effects in vivo. We determined that compound N2905, up to a concentration of 20 mg/L, significantly decreased the number of WSSV copies in Litopenaeus vannamei post-larvae, with a maximum inhibitory rate of > 90 %, and increased the survival rate of WSSV-infected post-larvae. Pre-treatment and post-treatment assays indicated that N2905 could treat, but not prevent, WSSV infections. When WSSV was preincubated with N2905 for 1-4 h, the incidence of viral infections was significantly reduced and survival time of post-larvae extended to 120 h. A stability study of N2905 provided a reference for its practical use. Considering the antiviral stability of N2905 in culture water within 2 d, continuous N2905 exchange was performed, showing a significant decrease in viral load at 120 h post-infection (hpi) and a 55 % increase in survival of WSSV-infected post-larvae. Overall, our study demonstrated the potential of N2905 as an antiviral agent.

[Effects of three antigens extracted from Vibrio vulnificus on the immunological protection of Nibea albiflora].

Nibea albiflora was immunized by intraperitoneal injection with either Lipopolysaccharide (LPS) or outer membrane protein (OMP) extracted from Vibrio vulnificus or formalin killed Vibrio vulnificus (FKC). The influence of the three antigens on the immunological function of Nibea albiflora was determined at different time points following the injection by testing the agglutinating antibody titers of the serum, lysozyme activity of the serum, phagocytic activity of the blood and the relative survival percentage. The results showed that the three antigens have higher immunogenicity and antigenicity than the control group(injection with sterile saline). The agglutinating antibody titers of the immune challenged groups increased quickly, and were highest on the day 28. The lysozyme activity and phagocytic activity were raised significantly (P<0.01), reaching their top value on day 21, and then the index gradually reduced. The immunological indexes of three immune groups were higher than the control group (P<0.05). The agglutinating antibody titers of the LPS group or the OMP group were lower than the control group, but the relative survival percentage was adverse when challenged with a Vibrio vulnificus infection. The order of relative survival percentage was group LPS>OMP>FKC>Control.