| Objective To prepare the chicken IgY against Clostridium difficile,and study its therapeutic effect on Clostridium difficile-related diarrhea(CDAD)mice,as well as explore the mechanism.For the purpose of exploring a new non-antibiotic therapy of Clostridium difficile infection(CDI),meanwhile providing theoretical basis for diagnostic reagents and vaccine development of CDI.Methods(1)Preparation and identification the chicken IgY against Clostridium difficile : Cultured Clostridium difficile VPI 10463 in Anaerobic culture,collected bacteriophage through centrifuge,and detoxified by 0.4% formaldehyde,then adjusted the concentration of phage to 1×10~9CFU/ml.Eight healthy hens of 16 weeks old were selected for basic immunization after 3 weeks of adaptive feeding and immunize again after 2 weeks.Then strengthened immunization 2 times,once two weeks.Afterwards immunized 4 times with Clostridium difficile vaccine,once four weeks.One week after the initial immunization,collected eggs once every 2 days.Preparated the chicken IgY against Clostridium difficile via water dilution,ammonium sulfate precipitation and molecular sieve chromatography.Measuremented the concentration by Coomassie brilliant blue G-250 and identified the chicken IgY against Clostridium difficile by SDS-PAGE electrophoresis.Indirect ELISA method was used to assay the antibody titer and physicochemical properties of specific IgY.(2)Effect of the chicken IgY against Clostridium difficile on Clostridium difficile in vitro : Cell adhesion was used to study the effect of specific IgY on the adhesion of Clostridium difficile.Growth inhibition experiments was used to study the effect of specific IgY on the growth of Clostridium difficile.Cell cytotoxicity and neutralization experiment was used to study the effect of specific IgY on TcdA and TcdB.(3)The therapeutic effect of the chicken IgY against Clostridium difficile on CDAD mice: Establish the model of CDAD,30 CDAD mice were randomly divided into 5 groups,6 rats per group.Mice were given 11.86 mg /ml,1.19 mg/ml,0.12 mg/ml specific IgY by gavage,the CDAD model group received normal saline,the negative control group received negative IgY.Each group of animals were intragastrically administered daily with 0.5ml/kg for three days.Sacrificed the mice and separated cecum for making pathological section after 24 hours at the last treatment.And measured the content of DAO,D-lactic acid and TNF-α,while measure the content of water in ileal tissue.Result(1)The specific IgY protein concentration was 11.86mg/ml.The results of SDS-PAGE showed the specific IgY was composed of H chain and L chain,and the molecular weight were 68 kDa and 38 kDa,respectively.Indirect ELISA showed specific IgY antibody titer was 1:8192.The valence of antibody remained stable when the specific IgY was treated under 63.5℃,and the antibody would inactivated be treated more than 80℃.The titer of specific IgY did not change in acid conditions.(2)The results of adhesion experiments: In middle and high IgY dose group,the adhesion number of Clostridium difficile in Lovo cells was significantly lower than low dose IgY group,blank control group and negative control group(P<0.01).In low dose IgY group,the adhesion number of Clostridium difficile was significantly lower than the negative control group and the blank control group(P<0.05).Growth inhibition test showed that there was no significant difference in the growth curve of Clostridium difficile between different dose groups(P>0.05).Cell cytotoxicity and neutralization experiment showed that the titer of specific IgY to TcdA and TcdB were 2.2×2~7 CU/mg and 2.0×2~7 CU/mg,respectively.(3)The results of cecal histopathology: In the negative control group and the CDAD model group,the cecal mucosa exfoliated with a large amount of inflammatory cells infiltration.The cecal mucosa was intact and no inflammatory cell infiltration in the high IgY group.The cecal mucosa exfoliated with a few inflammatory cells infiltration in the low IgY group.(4)The results of instestinal barrier and inflammatory factors: In high IgY group,the activation of DAO in cecal tissue was significantly higher compared to the CDAD model group and the negative control group(P<0.01).In middle dosage IgY group,the activation of DAO was higher compared to the negative control(P<0.05).The content of TNF-α was significantly lower in middle dasage and high dosage IgY group than low dosage IgY group,CDAD model group and negative control group(P<0.05).The content of D-lactic acid and the moisture content were not statistically significant in different groups(P>0.05).Conclusion(1)We successfully prepared the specific IgY which has high concentration,purity,titer and stable physico-chemical properties for the first time by immunizated Hailan chicken with Clostridium difficile vaccine.(2)The specific IgY could inhibit the adhesion of Clostridium difficile to intestinal epithelial cells and neutralize Clostridium difficile toxin.(3)Oral administration of specific IgY could improve the damage of intestinal mucosal epithelial cells induced by Clostridium difficile infection effectively,as wellas reduced the inflammatory reaction of intestinal mucosa in mice.Thus,the specific IgY had a good therapeutic effect on CDAD. |
PDF Full Text Request