Four 1H12 E. ruminantium open reading frames cloned into the pCMViUBs mammalian expression vector and used as a recombinant DNA vaccine against heartwater repeatedly provided complete protection in sheep (using a cocktail or the individual ORFs) against a laboratory needle challenge while 1/5 of sheep were protected after a natural tick challenge. The lack of protection under natural field conditions could be attributed to the delivery strategy used and therefore there is a need to investigate other delivery methods. Polymeric microparticles based on PLGA polymers have been used extensively to target the delivery of vaccine to antigen presenting cells, play a role in the induction of cellular immunity and can be used as a single dose vaccine mimicking prime/boost vaccination. In this study, the four 1H12 pCMViUBs_ORFs and their respective recombinant proteins were either encapsulated into or adsorbed onto microparticles using a modified double emulsion solvent evaporation technique. The particles were formulated to release DNA on day zero and day 21 and recombinant proteins on day 42 thus mimicking a two times DNA prime/recombinant protein-boost immunization strategy. Encapsulation did not have any detrimental effects on the stability of the recombinant proteins as determined by gel electrophoresis and western blotting. The in vitro incubation of microparticles in either a Float-A-Lyzer® dialyzer or an eppendorf tube showed the potential of microparticles to be used as a vaccine because of their release profiles that mimics a heterologous prime/boost immunization strategy. Microparticles formulated using polymers with low glycolide ratios released 80% of the encapsulated proteins within the first week of in vitro incubation with most of the proteins released on day 1. Microparticles formulated using polymers with 50:50 monomer ratios released the recombinant proteins during week 1 and 3 of in vitro incubation. These microparticles did not release any protein in week 2 (day 7-14). Microparticles with 0.5% cetyltrimethylammonium bromide (CTAB) on their surfaces adsorbed DNA and released more than 40% of DNA on day 1 with 100% release by day 14. RG502H microparticles formed with PVA as the internal phase viscosity enhancer released intact DNA only from day 12 to day 21. A cocktail of these microparticles could therefore be used as an autobooster vaccine thus reducing the need for repeated immunizations needed to obtain protective immunity. Potential scientific publication Tshikhudo, N.P., Pretorius, A., Putterill, J., and van Kleef, M. 2009, “Biodegradable microparticles as a single dose delivery system for Ehrlichia ruminantium vaccines”, Journal of Controlled Release, (draft manuscript). Publication of results in conference proceedings / abstracts NanoAfrica 2009: Biodegradable microspheres as a single dose delivery system for Ehrlichia ruminantium vaccines: N. Tshikhudo, A. Pretorius, J. Putterill and M. van Kleef.