Preliminary Study On Out Membrane Structure And Immunogenicity Changes Of Drug-resistant Aeromonas Hydrophila

In this study, identification and pathogenicity of six strains Aeromonas hydrophila sensitivite to tetracyclines and fluoroquinolones, with sub-inhibitory concentration subculture method, artificial induction of resistant to tetracycline, doxycycline, norfloxacin and levofloxacin resistance A. hydrophila were obtained. The differences about the major protective antigen lipopolysaccharide and outer membrane protein of outer membrane of sensitivite and resistant A. hydrophila were compared. Channel catfish(Ictalurus punctatus Rafinsque) were immunized with outer membrane proteins, lipopolysaccharide and formalin killed sensitive and resistant A. hydrophila to analysis the immunogenicity changes of drug-resistant A. hydrophila. The results were summarized as follows:1. The morphological, physiological and biochemical characterization of isolated strains were in line with the national standard of GB/T18652-2002 "Pathogenic A.hydrophila testing methods", so it was identified as A. hydrophila. Hemolytic experiments and artificial infection experiments confirmed that six isolated strains were strong pathogenicity. In addition, the minimum inhibitory concentration (MIC) to tetracycline hydrochloride were that Ah563, Ah638, Ah28 and Ah15 of as an intermediary strains,6 strains to doxycycline hydrochloride, norfloxacin and levofloxacin were sensitive.2. A. hydrophila was grown at 28℃for 72 h and used it to test for the development of resistance after 9 sequential subcultures in sub-inhibitory concentrations of two tetracyclines(doxycycline and tetracycline) and two fluoroquinolones (norfloxacin and levofloxacin). After 9 subcultures the MIC to tetracyclines was 8～32 times greater than initial values, MIC values to fluoroquinolones was 125～7997 times greater than initial values, and drug-resistance were stability. The doxycycline-resistant isolate was resistant to tetracycline and fluoroquinolones, and the levofloxacin-resistant isolate was resistant to tetracyclines and norfloxacin. The drug-resistance stability test indicated that long-term storage of the bacteria at 4℃could reduce their resistance to antimicrobials.3. Changes in OMP and LPS in cells of strains of sensitive and resistant A. hydrophlia had been examined through SDS-PAGE gel electrophoresis. All the resistant strains had altered OMP and LPS profiles, but the changes varied from strain to strain. All resistant strains had alterations in OMP profiles, including the increased expression of one protein band and the decreased expression of three protein bands. Less dense bands of the fastest-migrating bands were observed in the LPS from Nor-resistant strains, but loss of the fastest-migrating bands were showed in the LPS from Lev-resistant strains, and proteinase K digestion group showed a lower band density.4. Channel catfish(Ictalurus punctatus Rafinsque) were immunized with LPS, OMP and formalin killed sensitive and resistant A. hydrophila. The immune response against six vaccines and protective immunity by challenging with live sensitive and resistant A. hydrophila of I. punetaus were studied by determining agglutination antibody titers, phagocytic activity of phagocyies in blood, lysozyme activity and the relative percentage survival. The result showed that six vaccines had significantly higher immunogenicity and antigenicity than the control group. The determining agglutination antibody titers, phagocytic activity of phagocyies in blood, lysozyme activity of fish immunized by the three vaccines of resistant A. hydrophila were a little higher than sensitive A. hydrophila vaccines. The relative percentage survival of immunized goups were 33.3%～83.3%, which was in the sequence of LPS group> OMP group> FAH group>> control group. The relative percentage survival of resistant A. hydrophila vaccines were a little higher than sensitive A. hydrophila vaccines.