ABSTRACT
F4 (K88) and F18 fimbriaed enterotoxigenic Escherichia coli (ETEC) are the predominant causes of porcine postweaning diarrhea (PWD), and vaccines are considered the most effective preventive approach against PWD. Since heterologous DNA integrated into bacterial chromosomes could be effectively expressed with stable inheritance, we chose probiotic EcNc (E. coli Nissle 1917 prototype cured of cryptic plasmids) as a delivery vector to express the heterologous F4 or both F4 and F18 fimbriae and sequentially assessed their immune efficacy of anti-F4 and F18 fimbriae in both murine and piglet models. Employing the CRISPR-cas9 technology, yjcS, pcadA, lacZ, yieN/trkD, maeB, and nth/tppB sites in the chromosome of an EcNc strain were targeted as integration sites to integrate F4 or F18 fimbriae cluster genes under the Ptet promotor to construct two recombinant integration probiotic strains (RIPSs), i.e., nth integration strain (EcNcΔnth/tppB::PtetF4) and multiple integration strain (EcNc::PtetF18x4::PtetF4x2). Expression of F4, both F4 and F18 fimbriae on the surfaces of two RIPSs, was verified with combined methods of agglutination assay, Western blot, and immunofluorescence microscopy. The recombinant strains have improved adherence to porcine intestinal epithelial cell lines. Mice and piglets immunized with the nth integration strain and multiple integration strain through gavage developed anti-F4 and both anti-F4 and anti-F18 IgG immune responses. Moreover, the serum antibodies from the immunized mice and piglets significantly inhibited the adherence of F4+ or both F4+ and F18+ ETEC wild-type strains to porcine intestinal cell lines in vitro, indicating the potential of RIPSs as promising probiotic strains plus vaccine candidates against F4+/F18+ ETEC infection.
Subject(s)
CRISPR-Cas Systems/genetics , Chromosomes, Bacterial , Enterotoxigenic Escherichia coli/genetics , Adhesins, Escherichia coli/immunology , Animals , Antibodies, Bacterial/blood , Antibodies, Bacterial/immunology , Bacterial Adhesion , Cell Line , Enterotoxigenic Escherichia coli/immunology , Epithelial Cells/cytology , Epithelial Cells/metabolism , Escherichia coli Proteins/genetics , Escherichia coli Proteins/immunology , Female , Immunoglobulin G/immunology , Immunoglobulin G/metabolism , Mice , Mice, Inbred BALB C , Multigene Family , SwineABSTRACT
The work aims at illustrating the profile of DO and its mass transferring process in a biofilm reactor packed with a novel semi-suspended bio-carrier, and further revealing the main factors that influence the mass transferring coefficient of DO within the biofilm. Results showed that the biofilm was very easy to attach and grow on the semi-suspended bio-carrier, which obviously changed the DO profile inside and outside the biofilm. The semi-suspended bio-carrier caused three different mass transfer zones occurring in the bioreactor, including the zones of bulk solution, boundary layer and biofilm, in which, the boundary layer zone had an obvious higher mass transfer resistance. Increasing the aeration rate might improve the hydrodynamic conditions in the bioreactor and accelerate the mass transfer of DO, but it also detached the biofilm from the surface of bio-carrier, which reduced the consumption of DO, and accordingly, decreased the DO gradient in the bioreactor.
Subject(s)
Biofilms , Bioreactors , HydrodynamicsABSTRACT
Two new xanthone O-glycosides, polyhongkongenosides A (1) and B (2), together with four known xanthone glycosides, were isolated from the herbs of Polygala hongkongensis Hemsl. Their structures were elucidated on the basis of UV, IR, NMR, and MS spectral data. The antioxidant in vitro activities of 1-6 were determined by the scavenging activities against 1,1-diphenyl-2-picrylhydrazyl (DPPH) radicals and hydroxyl radicals and their reductive activities to Fe3+. Mangiferin, one of the four known xanthone glycosides, showed potential scavenging effect on DPPH and hydroxy radicals and reductive activity to Fe3+ with IC50 values of 4.7, 13.9, 23.7 microM, respectively.
Subject(s)
Antioxidants/chemistry , Glycosides/chemistry , Polygala/chemistry , Xanthones/chemistry , Biphenyl Compounds/chemistry , Hydrazines/chemistry , Molecular Structure , PicratesABSTRACT
A new dihydroisocoumarins, 6-methoxy-8-hydroxy-3-(4-hydroxyphenyl)isochroman-1-one, named as hongkongenin (1), and seven known flavonoids were isolated from the methanol extract whole plant of Polygala hongkongensis Hemsl. Their structures were established on the basis of UV, IR, NMR, and MS spectral data. It is the first report of the occurrence of isocoumarins in Polygala, and the known compounds 2-8 were isolated from this plant for the first time. Flavonoids showed potent antioxidant activities in vitro against 1,1-diphenyl-2-picrylhydrazyl (DPPH) radicals, hydroxyl radicals, and reductive activity to Fe3+.
