Isolation And Identification Of Edwardsiella Ictaluri And Its Ghost Vaccine Development

Edwardsiella ictaluri is a fish pathogen with a wide range of hosts,characterized by long incubation period,rapid transmission and high lethality rate,which has caused huge economic losses to the related aquaculture industry.As a new type of vaccine,the bacterial ghost has advantages that traditional vaccines do not possess.In this paper,we investigate it from the following three aspects.(1)Using all-male yellow catfish(Pelteobagrus fulvidraco)with red head disease as the research object,the pathogen was isolated and purified from the lesion site,and identified by morphology,physiological and biochemical characteristics,16S rRNA gene sequence and specific PCR amplification detection.The drug sensitivity characteristics and pathogenicity of it were analyzed by drug sensitivity assay and zebrafish(Danio rerio)artificial infection test respectively.The results showed that the dominant strain isolated from the lesions of the diseased all-male yellow catfish was Edwardsiella ictaluri,which was highly pathogenic with an LD50 of 2.3×105 CFU.The symptoms of zebrafish infected with this strain were similar to those of natural disease.The drug sensitivity assay results showed that the strain was highly sensitive to 28 antibiotics such as penicillin,chloramphenicol,and tetracycline,moderately sensitive to erythromycin,and resistant to 6 drugs such as benzimidazole,cloramphenicol,and vancomycin.(2)To overcome the technical barrier that the temperature-controlled expression system is not suitable for the preparation of E.ictaluri ghosts(EIG),the lysis plasmids pFGH06-BAD-E and pFGH06-BAD-EN containing lysis gene E and its fusion with Staphylococcal nuclease A(SNA)were constructed based on the arabinose induction expression system,while the control plasmids pFGH06-BAD-MCS were constructed using multi cloning site(MCS)substitution.The above plasmids were respectively transformed into E.ictaluri YCH and then induced for expression.The lysis kinetics,nuclease activity,genomic electrophoresis and electron microscope observation were carried out for each recombinant bacteria.The results showed that although the OD600 value of recombinant YCH(pFGH06-BAD-E)decreased more rapidly,the OD600 value of recombinant YCH(pFGH06-BAD-EN)decreased more persistently,reaching a minimum value at 9 h after induction and remaining stable,with a smaller minimum value and a higher lysis rate of 99.991%;The nuclease activity was detected in the supernatant of the culture medium of recombinant YCH(pFGH06-BAD-EN)and intracellularly at 0.5 h after induction,and the intracellular nuclease activity started to decline rapidly after reaching the maximum value 1 h after induction,while the nuclease activity in the supernatant continued to rise slowly until reaching the maximum value 10 h after induction,and no nuclease activity was detected in the culture supernatant or intracellular of recombinant bacteria YCH(pFGH06-BAD-E)and YCH(pFGH06-BAD-MCS);The genome concentration of recombinant bacterium YCH(pFGH06-BAD-EN)tended to decrease more significantly than that of YCH(pFGH06-BAD-E),and the genome was completely degraded 6 h after induction,whereas the genomes of YCH(pFGH06-BAD-E)and YCH(pFGH06-BAD-MCS)were always detectable throughout the experimental period,and the latter genome was increasing in amount.It was observed by scanning electron microscopy that E.ictaluri carrying the lysis plasmid was successfully induced to produce ghosts.The external morphology of the ghosts was similar to that of the non-lysed bacteria,but was more crumpled,and transmembrane tunnel could be seen at both ends or in the center of the ghosts.The above results showed that the combined effect of lysis gene E and SNA and the replacement of the inducible expression system could achieve the preparation of efficient and safe E.ictaluri ghosts.This study may provide new ideas and references for the control of E.ictaluri and the diseases caused by them.(3)Using zebrafish as the model animal,the immunization effect of E.ictaluri ghosts vaccine was evaluated in terms of leukocyte phagocytic activity and immune protection rate The results showed that the phagocytic index and percentage of phagocytosis in the EIG group were significantly higher than those in the formalin killed cells(FKC)vaccine group and the PBS(phosphate buffered saline)control group(p<0.05),and the relative immune protection rate in the EIG group(78%)was significantly higher than that in the FKC group(59%)(p<0.05).This indicates that E.ictaluri ghosts vaccine has better immune protection than traditional vaccines and is expected to have a broad application prospect in the future prevention and control of E.ictaluri and its caused diseases.