Preparation And Efficacy Evaluation Of Egg Yolk Immunoglobulin (IgY) Against Dairy Cow Mastitis

Dairy cow mastitis is one of the main causes of economic losses in dairy cow industry. Antibiotics are used widely in prevention and treatment of mastitis.With the present awareness of the problems associated with antibiotics-resistant and residue of antibiotics in milk,it is urgent to find an alternative to antibiotics for mastitis treatment.Chicken egg yolk immunoglobulin(IgY) provides an inexpensive and effective source of antibodies for the passive immunization of animals.It is a promising alternative for the treatment and prevention of bacteria infections,and has shown to be effective against a number of pathogens.In this study,specific IgY against the primary mastitis-causing pathgoens, Staphylococcus aureus and Escherichia coli,were prepared.The in vitro and in vivo activity of these specific IgY and their opsonization to phagocytes were estimated.Specific IgY were produced by hens immunized with formaldehyde killed S.aureus or E. coli O111 in long-standing immunization response(titer≥6400 for 100 days) with the highest titer of 25600.Several factors affect IgY titer during imunization including concentrations of antigens,days of hens and quaility of adjuvants.The contents of specific IgY in yolk varied at the same trend to that of IgY titer,and the highest contents of specific IgY against S.aureus and E.coli O111 were 1.86 mg/mL and 1.88 mg/mL,the average contents of total IgY were 9.82 mg/mL and 9.95 mg/mL,respectively.IgY was isolated from yolks with a purity of 94.52%and a yield of 27.68 mg/egg by water dilution,salt precipitations,ultrafiltration and gel filtration.The purity decreased to 87.09%,but the yield increased to 100.6 mg/egg without gel filtration.Specific IgY inhibited the growth of bacteria in liquid culture medium in a dose-dependent manner.The growth of S.aureus and E.coli were completely inhibited by 10 mg/mL of S.aureus-IgY and 20 mg/mL of E.coli-IgY,respectively.These specific IgY could also inhibit the growth of five strains of S.aureus and five strains of E.coli isolated from mastitic cows.Immunofluorescence and immunoelectron indicated specific IgY bound with bacteria resulting in structural alteration on cell surface.The opsonization of IgY to phagocytes in cows was detected by flow cytometry.Specific IgY enhanced the phagocytosis of polymorphonuclear neutrophil leukocytes(PMN) isolated from cow blood and milk with an optimal concentration of 1 mg/mL.The phagocytic percents of PMN in blood to S.aureus increased from 60.2%to 90.5%with IgY opsonizing,that of PMN in milk increased from 52.95%to 82.45%.The phagocytosis of PMN and macrophages(Mφ) in milk to S.aureus and E.coli were both enhanced by specific IgY.The increment of phagocytic percents for PMN and Mφto S.aureus were 29.5%and 2.7.66%, those to E.coli were 26.5%and 26.36%,respectively.The opsonization of specific IgY to PMN and Mφwas similar(p＞0.05 ).Treatments for clinical mastitis and experimental mastitis were performed by a 6-day course of intramammary infusion with IgY(200 mg) and penicillin(1000 mg).The milk color and clot recovered to normal form during the therapy course,the milk somatic cell counts and bacterial counts decreased.The cure rates for experimental mastitis by IgY and penicillin were 83.3%and 66.7%,and those for clinical mastitis were 50%and 33.3%,respectively.In conclusion,this study indicated specific IgY has potential to use as an alternative of antibiotics for mastitis therapy.