The Preparation And Evaluation Of Immune Efficacy Of Enterotoxigenic Escherichia Coli Ghosts

Calf diarrhea refers to an acute diarrhea in cattle 7 hours to 12 days after born,usually caused by infectious factors such as intestinal bacteria,virus,parasites or non-infectious factors such as malnutrition,bad feeding environment.Severe calf diarrhea could lead to dehydration,causing mass dead in calves.Calf diarrhea is one of the most common issues in cow or beef breeding worldwide,it costs large economic loss and restricts the development of cattle industry.In various factors leading to the calf diarrhea,Enterotoxigenic Escherichia coli(ETEC)is the main pathogen,meanwhile is one of the pathogens of cattle enterotoksemia.Currently,vaccination is commonly used to prevent the calf diarrhea.However,commercial vaccines hardly can offer a robust immunoprotection since Escherichia coli serotype is various and the epidemiological characteristic is different in different regions.Therefore,the development of new-type vaccines is important to prevent calf diarrhea.Bacterial ghost is a new-type vaccine in recent years.Bacteria are transformed into non-cytoplasma,non-activity empty bacteria by the way of gene inactivation while the antigenic structures on the outer membrane of the bacteria and the thallus are remained intact.Bacterial ghost as a vaccine strategy has been the focus of domestic and foreign researches because of its many advantages.For example,it has high immunogenicity,the simple preparation process,easiness of storage,convenient transportation,and also is in the nature of immune adjuvant which can enhance the immune response.Based on the clinical samples collected in some dairy farms in Heilongjiang province of dead calves due to diarrhea,this research isolated the pathogenic E.coli strains and detected the virulence factors of these strains,and classified the pathogenic E.coli.The results indicated that ETEC was the main pathogen of calves diarrhea.On the basis,we were also able to select one vaccine candidate strain and prepared the ETEC ghost vaccine.The immune effect of ETEC ghost vaccine was evaluated by animal experiment.30 mice were randomly divided into three groups,10 mice each group: one group for ghost vaccine,one group for formaldehyde inactivated vaccine,one group for control.All vaccines were vaccinated by the way of abdominal injection with the concentration of 1×108 CFU,once every two weeks for three times.Blood samples of immunized mice were collected one week after each vaccination.Indirect ELISA assay was conducted to detect the level of serum specific antibody.After the last vaccination,challenging immunized mice with ETEC.Observed and recorded the deaths of the mice within one week after challenging.Evaluated the immune effect of ETEC ghost vaccine by the protection of vaccine in mice and the level of serum specific antibody.The results showed that ETEC ghost vaccine could have high protection rate on infected mice,and also could induce high antibody levels.Comprehensive results of this study indicated that ETEC ghost vaccine was safe and had the satisfied immunogenicity,which could induce stimulate the animal bodies to produce a better immune response.Meanwhile,ETEC ghost vaccine could be used as the new-type vaccine candidates for the prevention of calf diarrhea caused by ETEC.Besides,this study provided the basis for the preparation of ETEC vaccines.