Targeted mutagenesis in Anaplasma marginale to define virulence and vaccine development against bovine anaplasmosis

Author summaryThis is the first study describing targeted mutagenesis and its application in determining virulence and vaccine development for an Anaplasma species pathogen. The study is important in advancing similar research in related Anaplasma pathogens impacting multiple hosts. We demonstrated the successful development of a complete gene deletion mutation in A. marginale. Further, this in vivo infection study was performed in the natural host and established that growth of A. marginale is greatly limited when the phtcp gene is deleted. This study also demonstrated that cattle challenged with the virulent wild-type A. marginale four weeks after receiving the mutant A. marginale vaccine (MLAV) neither developed clinical disease nor did they exhibit hematologic abnormalities, such as anemia. On the contrary, non-vaccinated and WCAV vaccinated cattle challenged with virulent wild-type A. marginale developed clinical anaplasmosis with severe anemia, erythrocyte anisocytosis, and high-load infection in erythrocytes. Tick-borne Anaplasma species are obligate, intracellular, bacterial pathogens that cause important diseases globally in people, agricultural animals, and dogs. Targeted mutagenesis methods are yet to be developed to define genes essential for these pathogens. In addition, vaccines conferring protection against diseases caused by Anaplasma species are not available. Here, we describe a targeted mutagenesis method for deletion of the phage head-to-tail connector protein (phtcp) gene in Anaplasma marginale. The mutant did not cause disease and exhibited attenuated growth in its natural host (cattle). We then assessed its ability to confer protection against wild-type A. marginale infection challenge. Additionally, we compared vaccine protection with the mutant to that of whole cell A. marginale inactivated antigens as a vaccine (WCAV) candidate. Upon infection challenge, non-vaccinated control cattle developed severe disease, with an average 57% drop in packed cell volume (PCV) between days 26-31 post infection, an 11% peak in erythrocytic infection, and apparent anisocytosis. Conversely, following challenge, all animals receiving the live mutant did not develop clinical signs or anemia, or erythrocyte infection. In contrast, the WCAV vaccinees developed similar disease as the non-vaccinees following A. marginale infection, though the peak erythrocyte infection reduced to 6% and the PCV dropped 43%. This is the first study describing targeted mutagenesis and its application in determining in vivo virulence and vaccine development for an Anaplasma species pathogen. This study will pave the way for similar research in related Anaplasma pathogens impacting multiple hosts.