| IgY(Immunoglobulin of Yolk)is the major immunoglobulin of the ovipara,functionally equivalent to mammalian IgG.IgY is transmitted to the embryo through the yolk and becomes an antibody that protects the embryo against invasion by external pathogens.The collection and isolation of chicken antibody IgY is rapid and economical.After hens were immunized with certain microorganisms,high titer and lasting antibody IgY was obtained from the yolk.It had been used in antibacterial,antiviral,antitumor,and immunoassays.Based on the efficacy of immunoglobulin IgY in vitro and in vivo,specific IgY was found to improve the typical symptoms of periodontitis and halitosis in rats infected with oral anaerobic pathogenic bacteria(Porphyromonas gingivalis,Prevotella intermedius and Fusobacterium nucleatum),clarifying the role of the treatment with specific IgY on the oral diseases.1.The preparation and biochemical properties of IgY.An inactivated vaccine was made by adding formaldehyde and Freund’s adjuvant to P.gingivalis,P.intermedius and F.nucleatum resolution.Laying hens were immune three times by the inactivated vaccine.Then eggs were collected,and egg yolks were separated.Then,the egg yolks adequately stirred with sodium acetate-acetic acid buffer and stored at 4℃and 2-3 days in order to settle lipid material.After centrifugal,the supernatants were collected,then dialysis and concentrated at 100 k Da.Next,the IgY resolution was detected by SDS-PAGE,vacuum freezing dry,and ELISA kits.The results showed that the concentration was 360mmol/L,and the titer of IgY against P.gingivalis,P.intermedius and F.nucleatum was 1:25600,1:12800 and 1:25600,respectively.The result of SDS-PAGE showed that IgY was 180 kDa,composed of two heavy chains(67-70 kDa)and two light chains(25 kDa).2.The immune characteristics of IgY in experimental animals.IgY as an invasive antigen smeared the oral cavity of SD rats.ELISA kits detected histamine,IgE,IgM and IgG,that mean immune factors,and flow cytometer measured the numbers,proportion and ratio of CD4~+and CD8~+cells between control and IgY-treated groups.The purpose in this study was analyzed if IgY caused a immunology respond or a irritation and allergy of skin in SD rats,New Zealand rabbits and cavies,then evaluate and discuss the immunological characteristics of IgY.The results showed that the weights and food intake had no significant changes.The levels of histamine and IgM increased the 9~thh days,then reduced to the normal.The levels of Ig E and IgG had no significant changes.The numbers of CD4~+and CD8~+cells were 1902.60 and 974.00,the proportion in immune cells were 54.36%and 27.83%,and the ratio was 1.95.These results were significantly lower than the positive controls(p<0.05)and within the normal range.The skin irritation test was acute for 1-72 h and chronic for 1-14 days.There were 2animals with score>2 at 24 h and 48 h after the skin metamorphosis test.This was a mild allergy.These results showed that IgY didn’t cause immune respond in rats,had no skin irritation in New Zealand rabbits,and may cause slightly allergy in cavies.But when the IgY dosage was reduced,the skin allergy will be effectively controlled.These results suggest that IgY as an invasive antigen had no allergenicity in vivo.3.Effect of IgY on periodontitis in rats.The experimental periodontitis rat models were successfully infected by P.gingivalis,P.intermedius and F.nucleatum.During IgY treatment,the probe measured the scores for the plaque index(PI),gingival index(GI),probe on bleeding(POB),and ELISA kits detected the levels of IL-6 and TNF-α,and the maxillas were examined X-ray photographs and histopathological sections.Compared with control,the scores for PI,GI,and BOP were significantly(p<0.05)decreased 90.00%、81.82%and 100.00%,and the levels of IL-6 and TNF-αwere significantly(p<0.05)decreased 63.04%and 54.05%.The results showed that IgY improved gingival inflammation,bacterial colonization,and periodontal bleeding,relieved periodontal tissue lesion in rats with periodontitis and promoted the development of new periodontal fibers and new bone,reduced inflammation caused by periodontitis.Therefore,it was indicated that IgY can be used for the prevention and treatment of periodontitis.4.Effect of IgY on halitosis in rats.Based on pathogenesis of halitosis,the experimental halitosis rat models were induced by P.gingivalis,P.intermedius and F.nucleatum,and drinking cane sugar in order to promote the bacteria colonization.During IgY treatment,the scores for the PI,GI,and halitosis index(HI),the levels of IL-6 and TNF-α,and the changes of the classical and contents of oral anaerobic pathogenic bacteria metabolites were detected using SPME-GCMS.Compared with control,the scores for PI,GI,and HI were significantly(p<0.05)reduced 55.67%,57.14%and 50.00%,and the levels of IL-6 and TNF-αwere significantly(p<0.05)decreased 74.44%and 85.09%.Analyzed the metabolites of the oral anaerobic pathogenic bacteria,they were mainly composed by aldehydes,ketones,carboxylic acids,and nitrogen compounds.The greatest changes in metabolites were N-hexanal,2,5-dimethyl-3(2H)furanone,acetic acid,n-hexanoic acid,ammonia and vinyl isopropyl ether.The contents were significantly(p<0.05)decreased 8.84%,7.84%,8.11%,11.24%,8.51%and 12.00%,respectively,and the high-dose group was more than low-dose one.Therefore,it was indicated that IgY could relieved the problem of oral health and odours induced by oral anaerobic pathogenic bacteria,and had an effective role in the prevention and treatment of halitosis.5.Study on the growth inhibition of IgY on oral anaerobic pathogenic bacteria.The growth of oral anaerobic pathogenic bacteria in the artificial saliva simulation system,which was as culture medium was inhibited by the different dosages of IgY.Principal component analysis measured odours profile components in the artificial saliva simulation system by E-nose.The changes of relative contents of volatile sulfur compounds were detected by Halimeter.And the changes of the classical and contents of the metabolites produced by oral anaerobic pathogenic bacteria were detected using GCMS.This study also compared antibacterial effect between IgY and three bacteriostat,and optimized the IgY dosage of administration.P.gingivalis showed an S-type growth curve in artificial saliva,and the HI index showed a gradually increasing trend.The growth of oral anaerobic pathogenic bacteria in the artificial saliva simulation system was significantly(p<0.05)inhibited,specially the high-dose group at 6 h.The data of E-nose showed the remarkable differences between IgY-treated and control in principal component analysis with concentration dependence.The classification of VOCs in the artificial saliva simulation system included ketones,carboxylic acids,esters,aromatics and nitrogen compounds.The greatest changes in metabolites were2,4-di-tert-butylphenol,1,4-dioxoperhydropyrrolo[1,2-a]pyrazine,trans-erucamide,5-chloro-3-bromo-1-[(4-methylphenyl)sulfonyl]-1H-pyrrolo[2,3-b]pyridine and undecane,and the contents were significantly(p<0.05)decreased 1.01%、1.43%、1.13%、0.94%、1.15%、0.72%.The contents of VSCs were significantly(p<0.05)reduced 16~44%in different dosage groups compared to control.Thereby,it was indicated that the odours in the artificial saliva simulation system had an obvious changes,and the growth and the metabolites of oral anaerobic pathogenic bacteria were inhibited after IgY treatment.Moreover,IgY relieved the inflammation and odours induced by oral anaerobic pathogenic bacteria,and interdicted the developments of oral diseases.Compared to bacteriostat included protamine,lactoferrin and metronidazole,the results showed that IgY significantly(p<0.05)inhibited the growth of P.gingivalis and F.nucleatum,and the lactoferrin significantly(p<0.05)inhibited the growth of P.intermedius in high-dose.However,IgY has a better effect than the lactoferrin on the growth inhibition of P.intermedius in high and low dosages.Therefore,the effect of IgY on the growth inhibition of oral anaerobic pathogenic bacteria was more outstanding.Finally,the results showed that IgY in dosage from 165 to 181 mmol/L could excellently inhibit the growth and the metabolites of oral anaerobic pathogenic bacteria using response surface analysis.Thereby,IgY improved the inflammation and odours induced by P.gingivalis,P.intermedius and F.nucleatum,and prevented and treated the oral diseases infected by the oral anaerobic pathogenic bacteria.IgY was produced after challenging specific antigen,concentrated and aggregated in egg yolk.It was obtained by collecting eggs and extracted from egg yolk.This immunoglobulin that was high content and strong specificity can’t cause severe allergic reactions in SD rats,cavies and New Zealand rabbits.The typical symptoms were improved by IgY treatment in periodontitis and halitosis animal models.In artificial saliva simulation system,the growth and metabolites of oral anaerobic pathogenic bacteria were inhibited with IgY.In conclusion,IgY inhibited oral disease such as periodontitis and halitosis by inhibiting the proliferation of oral anaerobic pathogens. |
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