Sample collection
To determine the antibody (Ab) responses, the mice were bled a week after immunization and the serum was collected. Saliva sample was collected with pipetting method. Saliva samples harvested that PBS placed in the oral cavity at 3 minutes later, transferred to a microcentrifuge tube. For intestinal IgA, the large intestine was excised 5 cm (from the rectum), The saliva and, intestinal fluid from the entire gut was collected, mixed with cold saline (4 °C) and centrifuged for 10 min at 2000 ×g. The supernatants were collected and frozen. Peritoneal exudate cells were collected by intraperitoneal injection with 10 mL of DMEM medium. The cells were centrifuged at 2500 rpm for 10 min and the supernatants were discarded. These were resuspended in DMEM complete medium. Splenocytes were prepared from the spleens by chopping on sterile glass sides and centrifugation at 1400 rpm for 5 min. The erythrocytes were lysed with 1 mL of ammonium chloride (0.8 % w/v). After centrifugation (1400 rpm for 5 min), 10 mL of medium was added to the pelleted cells and filtered through a fine cell strainer (BD Falcon, BD Bioscience) of 40 μm pore size. The cell numbers and viability were assessed microscopically by the trypan blue dye exclusion technique (Xu et al., 2009).