Aeromonas hydrophila associated with red spot disease in Macrobrachium nipponense and host immune-related gene expression profiles.

In September 2018, a serious disease causing high mortality with red spot syndrome occurred in a Macrobrachium nipponense aquaculture farm in Jintan County, Jiangsu Province, China. In this study, a pathogenic isolate 5-S3 was isolated from diseased M. nipponense and was identified as Aeromonas hydrophila by phenotypically and molecularly. The pathogenicity of the isolate 5-S3 to M. nipponense was determined by challenge experiments. Results of artificial challenge showed A. hydrophila was pathogenic to M. nipponense, the LD50 was 9.58 × 104 CFU/mL, and histopathological analysis revealed that the hepatopancreas of infected M. nipponense exhibited obvious inflammatory responses to A. hydrophila infection. The isolate showed significant phenotypical activities such as the lecithinase, esterase, caseinase and hemolysin which are indicative of their virulence potential. Besides, virulence genes such as aerA, act, fla, ahpß, alt, lip, eprCAI, hlyA, acg and gcaT were detected in the isolate 5-S3. Subsequently, the immune-related genes expression in M. nipponense were evaluated by quantitative real-time PCR (qRT-PCR), and the results showed that the expression levels of dorsal, relish, crustin1, crustin2, anti-lipopolysaccharide factors 1 (ALF1), anti-lipopolysaccharide factors 2 (ALF2), hemocyanin, i-lysozyme and prophenoloxidase were significantly up-regulated in hepatopancreas of M. nipponense after A. hydrophila infection, the stat, p38, crustin3, anti-lipopolysaccharide factors 3 (ALF3) genes had no significant change during the infection. The present results reveal that A. hydrophila was an etiological agent causing red spot syndrome and mass mortality of M. nipponense and the influence of A. hydrophila infection on host immune genes.

Biochemical characterization of a thermostable DNA ligase from the hyperthermophilic euryarchaeon Thermococcus barophilus Ch5.

DNA ligases are essential enzymes for DNA replication, repair, and recombination processes by catalyzing a nick-joining reaction in double-stranded DNA. The genome of the hyperthermophilic euryarchaeon Thermococcus barophilus Ch5 encodes a putative ATP-dependent DNA ligase (Tba ligase). Herein, we characterized the biochemical properties of the recombinant Tba ligase. The enzyme displays an optimal nick-joining activity at 65-70 °C and retains its DNA ligation activity even after heated at 100 °C for 2 h, suggesting the enzyme is a thermostable DNA ligase. The enzyme joins DNA over a wide pH spectrum ranging from 5.0-10.0, and its optimal pH is 6.0-9.0. Tba ligase activity is dependent on a divalent metal ion: Mn2+, Mg2+, or Ca2+ is an optimal ion for the enzyme activity. The enzyme activity is inhibited by NaCl with high concentrations. Tba ligase is ATP-dependent and can also use UTP as a weak cofactor; however, the enzyme with high concentrations could function without an additional nucleotide cofactor. Mass spectrometric result shows that the residue K250 of Tba ligase is AMPylated, suggesting that the enzyme is bound to AMP. The substitution of K250 of Tba ligase with Ala abolishes the enzyme activity. In addition, the mismatches at the first position 3' to the nick suppress Tba ligase activity more than those at the first position 5' to the nick. The enzyme also discriminates more effectively mismatches at 3' to the nick than those at 5' to the nick in a ligation cycling reaction, suggesting that the enzyme might have potential application in single nucleotide polymorphism.

Biologic and genomic characterization of a novel virulent Aeromonas hydrophila phage phiA051, with high homology to prophages.

Introduction: Aeromonas hydrophila is particularly harmful to freshwater aquaculture, and the search for phage is an effective biological control method, but reports of possible temperate phages and their mutants are rare in this field. In this study, a virulent phage highly homologous to prophage in the genomes of A. hydrophila was collected and preliminary biological characterization was carried out to understand its nature. Materials and methods: Water samples taken from eel ponds in Fujian, China were combined with the strain. Spot test method and double-layer agar plate assay was used for confirmation and purification. Phage virions were observed using transmission electron microscope. A total of 68 strains of Aeromonas spp. were used to determine the host range. MOI groups of 1,000, 100, 10, 1, 0.1, 0.01, 0.001, 0.0001, 0.00001 were prepared to detect the optimal MOI. The conditions of thermal stability assay were set as 30, 40, 50, 60, 70 and 80°C for 1 h, respectively, and conditions of acid and alkali stability assay were set as 2.0, 4.0, 6.0, 8.0, 10.0 and 12.0 of pH. MOI of 0.01 and 0.1, respectively, are set to determine the inhibitory capacity of phage. Results: A novel virulent A. hydrophila phage designated phiA051 has been isolated from aquaculture water. Electron microscopic observation showed that the phage phiA051 was composed of an icosahedral capsid. The phage phiA051 possesses an optimal multiplicity of infection (MOI) of 0.01, and its burst size was 108 PFU/cell. The phage maintained a high viability at temperatures of 30-50°C or pH 6.0-10.0 for 1 h. Phage phiA051 has certain potentials in rapidly inhibiting the spread of pathogen early in the outbreak, and it has a linear dsDNA with GC content of 60.55% and a total length of 32,212 bp, including 46 ORFs. Discussion: The phage phiA051 behaved as a virulent phage. However, the BLASTN result showed that 23 of the top 25 hits were genomes of Aeromonas strains. It was suggested that phiA051 was probably derived from some prophage in the chromosome of Aeromonas. Further investigation of the mechanism how phage phiA051 transforms from a temperate phage to a virulent phage will provide a unique perspective and idea to explore the potential of prophages.