Immune-enhancing effects of Astragalus polysaccharides and Ganoderma lucidum polysaccharides on Vibrio harveyi flgJ DNA vaccine in grouper.

Astragalus polysaccharides (APS) and Ganoderma lucidum polysaccharides (GLP) have been shown to possess strong immunoregulatory properties in aquatic animals. In this study, the fragment containing Vibrio harveyi flgJ gene was ligated into pcDNA3.1(+) vector and pcDNA3.1(+)-flgJ was constructed as DNA vaccine. APS and GLP were used as DNA vaccine adjuvants to evaluate the immunoregulatory effect by intramuscular injection to pearl gentian grouper (♀Epinephelus fuscoguttatus × â™‚E. lanceolatus). The results showed that pcDNA3.1(+)-flgJ combined with APS or GLP could significantly up-regulate the innate and adaptive immune response in fish, including serum-specific antibody titres, catalase and lysozyme activities. At the same time, DNA vaccine combined with APS or GLP significantly up-regulated the expression levels of CD8α, IgM, IL-1ß, MHC-Iα, MyD88 and TLR3 genes in thymus, head kidney, spleen and liver of pearl gentian grouper in comparison with those of the pFlgJ group. After 42 days post-vaccination, V. harveyi was used to challenge pearl gentian grouper by intraperitoneal injection. The relative percentage of survival (RPS) of pFlgJ, pFlgJ +APS, pFlgJ +GLP and pFlgJ+APS+GLP groups were 69%, 81%, 77% and 88%, respectively. These results suggested APS and GLP were potential adjuvants for DNA vaccine against V. harveyi infection in pearl gentian grouper.

Construction of a phosphodiesterase mutant and evaluation of its potential as an effective live attenuated vaccine in pearl gentian grouper (♀Epinephelus fuscoguttatus × â™‚Epinephelus lanceolatus).

Vibrio alginolyticus is a dominant pathogen that causes vibriosis of fish and shellfish. VAGM003125 is a specific phosphodiesterase bearing HD-GYP domain, which extensively regulates multicellular behavior and physiological processes in bacteria. In this study, an in-frame deleted ΔVAGM003125 mutant was constructed and changes of ΔVAGM003125 mutant in physiology and pathogenicity were examined. The potential application of ΔVAGM003125 mutant as a live attenuated vaccine was also assessed. The ΔVAGM003125 mutant displayed no significant differences in the growth rate and morphology in comparison to the wild type strain. However, the ΔVAGM003125 mutant significantly enhanced biofilm formation compared to the wild type strain. Also, the ΔVAGM003125 mutant was noted as being able to attenuate swarming motility, ECPase, and adherence compared to the wild type strain. Moreover, the ΔVAGM003125 mutant induced high antibody titers and provided effective immune protection, which was evidenced with a relative survival rate of 81% without histopathological abnormality. Following ΔVAGM003125 mutant vaccination, immune-related genes of pearl gentian grouper (♀Epinephelus fuscoguttatus × â™‚Epinephelus lanceolatus) including IgM, MHC-Iα, IL-16, IL-1, and TNF-α was up-regulated. Taken together, the present data suggested that the ΔVAGM003125 mutant might be applied as an attenuated live vaccination against V. alginolyticus during fish culture.

Characterization of fliR-deletion mutant ΔfliR from Vibrio alginolyticus and the evaluation as a live attenuated vaccine.

Vibrio alginolyticus is the common pathogen affecting various species of marine organisms. It has been demonstrated that fliR is a necessary virulence factor to adhere and infect their hosts for pathogenic bacteria. Frequent disease outbreaks in aquaculture have highlighted the necessity of developing effective vaccines. In the present study, in order to investigate the function of fliR in V.alginolyticus, the fliR deletion mutant ΔfliR was constructed and its biological properties were evaluated, additionally, the differences in gene expression levels between wild-type and ΔfliR were analyzed by transcriptomics. Finally, ΔfliR was used as a live attenuated vaccine to immunize grouper via the intraperitoneal route to evaluate its protective effect. Results show that fliR gene of V. alginolyticus was identified as being 783 bp in length, encoding 260 amino acids, and showing significant similarity to homologs of other Vibrio species. The fliR-deletion mutant ΔfliR of V. alginolyticus was successfully constructed, and its biological phenotype analysis showed no significant differences in growth capacity and extracellular enzyme activity compared to the wild-type. However, a substantial reduction of motility ability was detected in ΔfliR. Transcriptomic analysis revealed that the absence of fliR gene is responsible for a significantly decreased expression of flagellar genes, including flaA, flaB, fliS, flhB and fliM. The fliR-deletion mainly affects the related pathways involved in cell motility, membrane transport, signal transduction, carbohydrate metabolism, and amino acid metabolism in V. alginolyticus. The efficacy of ΔfliR as a candidate of live attenuated vaccine were evaluated by intraperitoneal injection in grouper. The ΔfliR provided the RPS (Relative protection rate) of 67.2% against V. alginolyticus in groupers. The ΔfliR efficiently stimulated antibody production with specific IgM still detected at 42 d post-vaccination, and significantly elevated the activity of antioxidant enzymes like Catalase (CAT), Superoxide dismutase (SOD), and lactate dehydrogenase (LDH) in the serum. The higher expression levels of immune-related genes were observed in the immune tissues of inoculated grouper compared to the control. In conclusion, ΔfliR effectively improved the immunity of inoculated fish. The results suggest that ΔfliR is an effective live attenuated vaccine against vibriosis in in grouper.

Foaming Behavior and Microcellular Morphologies of Incompatible SAN/CPE Blends with Supercritical Carbon Dioxide as a Physical Blowing Agent.

The foaming process and cellular morphologies of poly(styrene-co-acrylonitrile) (SAN)/chlorinated polyethylene (CPE) blends with supercritical carbon dioxide (scCO2) as a blowing agent were investigated in this study. As compared to pure SAN foam in the same batch, the foamed blends with various CPE elastomer content had smaller average pore size and larger cell density. This is probably related to the inhibition of bubble growth by elastomer, resulting in poor melt flowability and strong viscoelasticity, and the efficient bubble heterogeneous nucleation caused by numerous phase interfaces inside the incompletely compatible blend system. In addition, many tiny interconnected holes through the pore walls were formed to connect adjacent micropores in foamed blend samples. The formation mechanism of such interconnected pores is probably due to the fracture of stretched melt around the bubble from phase interfaces with weak interactions. These facts suggest an effective path to control pore size, cell density and even interconnected pores of blend foams depends on the compatibility of the blend system and difference in foamability of individual components in supercritical CO2.