Preliminary Studies On The Preparation And Application Of Chicken Escherichia Coli O₁and O₇₈ Ghosts

The Escherichia coli infection is caused by avian pathogenic Escherichia coli (Avain pathogenic Escherichia coli, APEC) and is a local or systemic infection disease called avian Escherichia coli disease, the main clinical symptoms include embryonic death, Escherichia coli septicemia, swollen head syndromeomphalitis yolk, sac infection, E. coli granuloma, the main pathological changes of the disease are airbag diseases, omphalitis, cellulitis, peritonitis, salpingitis, synovitis, the whole eye inflammatory and so on.In this study, the two serotypes O1and O78clinical isolates of Escherichia coli, was transformed to Escherichia coli recombinant bacteria. Through the lysis expressing experiments, we prepared Escherichia coli ghosts of two serotypes, and this laid the foundation for further studying the Escherichia coli bacteria ghosts vaccine. The results achieved by the present study are as follows:1. The preparation of different serotypes O1、O78of chicken Escherichia coli ghostsThe lysis gene E was obtained by PCR amplification from PhiX174using specific primers, cloned into pMD-18T vector and sequenced to find one mutation. After gene-directed mutagenesis the276bp DNA fragment containing modified gene E cut off from pMD18T-Em was inserted into the corresponding sites of pBV220which was designated as pBV220-Em. The plasmid pBV220-Em was eletroporated into the chicken Escherichia coli to prepare recombinant Escherichia coli O1-E, O78-E. The chicken Escherichia coli carrying pBV220-Em were generated successfully increasing the incubation temperature from29℃up to42℃to induce the expression of the gene E. The lysis was observed to begain at about1h, and competed about2h-2.5h, At the end of the lysis inactivation efficiency of the Escherichia coli ghosts was determined as95%by plating and CFU counting. However, no living cell was detected by the freeze thawing inactivated, or adding chloramphenicol inactivated.2. Chickens were immunized with Escherichia coli ghosts to study the cross-protection8-day-old chickens were injected subcutaneously different serotypes O1, O78, respectively, and the LD50was determined. LD50of E. coli O78strain was8.98×106CFU/mL, while E. coli O1strain was non-pathogenic.7-day-old chickens were immunized with Escherichia coli O1-E, O78-E ghosts and formalin killed Escherichia coli O1or O78with adjuvant respectively. Two weeks after vaccination, chickens were subcutaneously challenged with two fold of LD50Escherichia coli O78. The results of immunoprotection experiment showed that the immunoprotection effect of bacterial ghosts immunized groups was obviously better than formalin inactivated bacteria groups, Escherichia coli O78-E ghost showed the protection rate was80%, and Escherichia coli O1-E ghost showed the protection rate was70%. Formalin inactivated O1, O78vaccines showed the protection rate was50%,60%, respectively. The immunoprotection of the bacteria ghost O1-E was higher than the ghost O78-E, the ghost vaccines had good cross-protective immunity, which was to provide a reliable theoretical basis for further study of avian Escherichia coli vaccines.