Anaplasmosis, a hemolytic disease of cattle caused by the tick-borne pathogen Anaplasma marginale (Rickettsiales: Anaplasmataceae) has been controlled using killed vaccines made with antigen harvested from infected bovine erythrocytes. We recently developed a cell culture system for propagation of A. marginale in a continuous tick cell line. In this study, we performed a cattle trial to compare the bovine response to vaccination with A. marginale harvested from tick cell culture or bovine erythrocytes. All immunized and control cattle were then challenge-exposed by allowing male Dermacentor variabilis infected with A. marginale to feed and transmit the pathogen. Nine yearling cattle (three per group) were used for this study and were immunized with cell culture-derived A. marginale, erythrocyte-derived A. marginale or received adjuvant only to serve as controls. Each vaccine dose contained approximately 2 x 10(10) A. marginale and three immunizations were administered at weeks 1, 4 and 6. At week 8, cattle were challenge-exposed by allowing 60 D. variabilis male that were infected with A. marginale as adults to feed on the cattle. Antibody responses of cattle against major surface proteins (MSP) 1a, 1b and 5, as determined by ELISAs, peaked 2 weeks after the last immunization. Cattle immunized with infected IDE8 cell-derived antigens had a preferential recognition for MSP1b while cattle immunized with erythrocyte-derived antigens had a preferential recognition for MSP1a. Protection efficacy was evaluated using the percent infected erythrocytes (PPE), the packed cell volume (PCV), and the prepatent period. A. marginale-immunized cattle showed lower PPE and higher PCV values when compared to control animals and did not display clinical anaplasmosis. The cell culture-derived A. marginale shows promise for use as antigen in development of a new killed vaccine for anaplasmosis.