To observe changes on subsets of splenocytes in mice immunized with the transgenic alfalfa (Medicago sativa) vaccine containing Eg95-EgA31 fusion gene of Echinococcus granulosus dynamically,the leaf protein was extracted from the transgenic alfalfa by heat-coagulation method and prepared for a solution with a concentration of 20 g/ L.The 132 BALB/c mice were divided into 3 groups randomly and immunized intranasally or orally with the leaf protein solution once per 3 days for 2 months.At the same time,the group of intranasal immunization with leaf protein from the non-transgenic alfalfa was set as control group.4 mice randomized from each group were killed to get the spleens at Week 0,2,4,6,8,10,12,14,16,18 and 20 after last immunization,and the splenocytes were separated to measure CD_4~+and CD_8~+T cell subsets by FCM.In the oral group,CD_4~+subset increased significantly from Week 6 to Week 10 after the last immunization and reached the peak at Week 6.While CD+ 8 subset increased obviously from Week 4 to Week 12 and reached the highest level at Week 8.In the intranasal group,the significant increase of the CD_4~+subset was observed from Week 4 to Week 6 and also reached the peak at Week 6.The similar trend of CD_8~+ subset was observed from Week 4 to Week 10 and reached the highest level at Week 8.It was suggested that CD+ 4and CD+ 8subsets played an important role in the protection induced in mice immunized with the transgenic alfalfa vaccine.