Study Of Immunization Of Cows With Helicobacter Pylori And The Immune Response

This paper was conducted to offer technical foundation to produce high anti-Hp immune milk product and related biological products, and give a reference to the development of colostrum and immune milk and also add a experimental method for subsequent further research. Helicobacter pylori (Hp) NCTC 11637 was used throughout the present work. The strain was cultured and preserved with different methods. Pregnant cows were immunized with whole bacterial antigen of Hp. The titre of anti-Hp antibody in immune serum and colostrum was detected by indirect ELISA, meanwhile, the mechanism of immune response and the bactericidal activity of colostrum were also studied. The results were as follows:1. Effect of different methods on Hp culture and preservationThe strain was cultured on Columbia Agar Base (CAB) supplemented with combined antibiotics containing 2500IU/1 Polymyxin B Sulfate, 5mg/l Trimethoprim, 10mg/1 Vancomycin HC1 and 10mg/l Amphotericin B and 6% defibrinated sheep blood or 6% horse serum respectively in a microaerophilic atmosphere of 5% O2, 10% CO2 and 85% N2 at 37 C for 72h.With the two methods, good effect was observed. In condition of sterilization with no supplementation with combined antibiotics, the strain can't be contaminated and grow well as well. When Brucella Broth was used an culture medium with combined antibiotics and horse serum in microaerophilic bag, Hp could grow rapidly but was easily to be spheroid without replacing the bag on time. And the liquid culture method was contaminated compared with solid method. The strain can be revived successively with cryopreservation at -60 C.2. Study of immunizing cows and immune responsePregnant cows were immunized with whole bacterial antigen of Hp. For immunization, the bacterial suspension was suspended in sterile saline to a total concentration of 2 X 109CFU/ml, and emulsified with an equal volume of Freund's complete or incompleteadjuvant. Cows were immunized with 2ml Hp vaccine via intramuscular injection to the neck from the last six weeks every 2 weeks until the last 2 weeks prior to calving, and the final injection was given in the last week. Blood and colostrum samples were obtained from all cows chosen for study. The litre of anti-Hp antibody in immune serum and colostrum detected by indirect ELISA showed the immune method can cause immune responseefficiently. The best reaction condition of indirect ELISA: 5X108 CFU/ml bacterium coated concentration; dilution serum samples tol/640; dilution whey samples to 1/1280; dilution HRP conjugated Rabbit anti-Bovine IgG to 1/20000, 1h incubation at 37 C ; then 20min substrate incubation at 37 C. The titre of anti-Hp IgG in serum increased gradually up to maximum P/N value 4.56 around parturition and declined during the lactation period. In the first milking day, the titre in colostrum whey of all immunized cows rose up to the highest P/N value 5.465. In the following milkings, the litres dropped quickly. In comparison with nulliparous cow, the immune response in parous cow is better. Immune colostrum and control colostrum both have obvious bactericidal activity which was lost at 56 掳C for 30min.