TREATMENT TRAINING RESEARCH DEVELOPMENT OF CHIKEN YOLK IMMUNOGLOBULIN (IGY) TO SOME BACTERIAL AND BIOTOXIN AGENTS Colonel A/Prof. Le Van Dong et al., Vietnam Military Medical University

INTRODUCTION Big size animals require large amount of antigens; Blood collection and Ab purification is complicated process; Blood can not be harvested very often, that limits the yield of Ab obtain from each immunized animal. Antiserum production

Chicken immune system  Chicken antibodies: IgM, IgA and IgY.  IgY can be transferred from hen’s blood to egg and accumulated in the yolk (egg yolk immunoglobulin: IgY)

Transfer of IgY from hen to chick IgY from hen blood → egg yolk → chick. IgG from mother blood → placenta and umbilical cord → fetus. Antigen

Advantages of IgY

IgY applications  Diagnosis: Immunological tests.  Treatment: Passive immunotherapy.

Specific IgYs have been made and used for diagnosis and treatment on animals  Diarrhea caused by viruses and enterobacteria;  Respiratory diseases: influenza virures A H5N1 and H1N1;  Hand foot mouth disease;  Candida albicans;  Snake venoms, toxins and other bio-toxins.

Clinical application of IgY Prevention of P. aeruginosa infection in patient having cystic fibrosis. Prevention of decay with mouth washing solution contain IgY to Staphyloccocus mutans. Prevention of gastric ulcer with yogurt contained IgY against urease from H. pylori. IgY production is simple method that can provide mass quantity of antibody with low cost, suitable for developing country.

TREATMENT TRAINING RESEARCH MATERIALS 1.Pseudomonas aeruginosa 2.Vibrio cholerae 3.Cholerae toxin 4.Urease from Helicobacter pylori

Reagents  Freund’s complete adjuvant.  Freund’s in-complete adjuvant.  Peroxidase conjugated anti-IgY antibody.  Bacterial culture media.  Reagents for IgY extraction and ELISA test.

Egg laying hens Imported hen Domestic hen

Antigen preparation and immunization

Bacterial/toxin antigens Egg laying hens Chicken eggs Egg yolk Purified IgY Protein Antigen preparation and immunization Detection of specific Ab in hen blood Detection of specific Ab in egg yolk Extraction of egg yolk protein Purification of IgY IgY-based biological products

RESULTS Seroconversion of P. aeruginose specific IgY in hen blood before and after immunization IgY specific to P. aeruginose Antibody titers (x1000) Immunization times Gr 1 Gr 2 Gr 3

IgY specific to P. aeruginose in egg yolk Reactivity of P. aeruginose specific IgY in egg yolks after 5 th immunization Egg yolk dilution Neg. Ctr. Gr 1 Gr 2 Gr 3

Reactivity of IgY specific to P. aeruginose against each bacterial strain Reactivity of IgY specific to P. aeruginose extratced from egg yolk against each bacterial strain Egg yolk dilution Neg. Ctr

IgY extraction and purification Ion-exchange chromatography spectrum of the extraction product. SDS-PAGE of extraction and purification products. Reactivity of specific IgY in each chromatography factions as tested with ELISA

P. aeruginose agglutination with IgY in liquid medium Strain No.6P11M27P1179P169HP131100P27P836P8 Agglutination reactivity (+++)(++) (+)

P. aeruginose inhibition with IgY in solid medium A) Strain 27P11 (27 mm)B) Strain 79P16 (28 mm) C) Strain 9HP13 (26 mm) D) Negative control, Strain 27P11 (10 mm)

Treatment of heat experimental burn wounds infected with P. aeruginose

Quantities of P. aeruginose at the wound surface before and after treatment Kinetic of bacterial quantities at wound surfaces during the treatment Day 2 Day 3 Day 6 Day 9 Day 12 Density of bacreria/cm 2 (Lg) 0.9% NaCl

Conclusion We have successfully applied IgY antibody technology in production of Ab antidotes to some bacteria and bio-toxins at laboratory scale. The obtained IgYs show specific reactions to immunized antigens as tested in vitro and in vivo.

TREATMENT TRAINING RESEARCH Thank you Acknowledgments Dr. Nguyễn Đặng Dũng Dr. Tim Sunnary Dr. Đỗ Hoàng Long A/Prof. Lê Thu Hồng Dr. Đỗ Minh Trung Dr. Đỗ Khắc Đại Dr. Hoàng Trung Kiên Dr. Nguyễn Ngọc Tuấn