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

Authors: HOVE, PAIDASHE - Kansas State University; MADESH, SWETHA - Kansas State University; NAIR, ARATHY - Kansas State University; JAWORSKI, DEBORAH - Kansas State University; LIU, HUITAO - Kansas State University; FERM, JONATHAN - Kansas State University; KLEINHENZ, MICHAEL - Kansas State University; HIGHLAND, MARGARET - Kansas State University; CURTIS, ANDREW - Kansas State University; COETZEE, JOHANN - Kansas State University; Noh, Susan; WANG, YING - Kansas State University; GENDA, DOMINICA - Kansas State University; GANTA, ROMAN - Kansas State University

Submitted to: PLoS Pathogens
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 4/21/2022
Publication Date: 5/16/2022
Citation: Hove, P., Madesh, S., Nair, A., Jaworski, D., Liu, H., Ferm, J., Kleinhenz, M.D., Highland, M.A., Curtis, A.K., Coetzee, J.F., Noh, S.M., Wang, Y., Genda, D., Ganta, R.R. 2022. Targeted mutagenesis in Anaplasma marginale to define virulence and vaccine development against bovine anaplasmosis. PLoS Pathogens. 18(5). Article e1010540. https://doi.org/10.1371/journal.ppat.1010540.
DOI: https://doi.org/10.1371/journal.ppat.1010540

Interpretive Summary: Anaplasma marginale, the cause of bovine anaplasmosis, infects erythrocytes, and causes anemia, abortion, poor production, and in some cases, unexpected death in cattle worldwide. Despite the estimated major financial losses due to bovine anaplasmosis amounting to billions of US dollars each year globally, efficacious and safe vaccines are not available globally. An A. maginale gene likely involved in nutrient acquisition was knocked-out and used to immunize cattle. This study demonstrates the protective capacity of this modified live organism compared to inactivated whole cell vaccine and non-immunized controls. Overall, the animals immunized with the modified live organism were protected from anemia and no infected erythrocytes were detected in blood smears. In comparison the animals that received the whole-cell killed immunogen developed anemia and had a 40% reduction in packed cell volume and a maximum of 5% infected erythrocytes. In comparison, the non-immunized animals developed anemia and had a 52% drop in packed cell volume with 12% maximum of infected erythrocytes as measured in blood smears. This work demonstrates the potential for doing target gene knock-outs in A. marginale and lays the foundation for development of a modified live vaccine.

Technical Abstract: The Anaplasma spp. are obligate, intracellular, tick borne, bacterial pathogens that cause important diseases in people, agricultural animals, and dogs, globally. Targeted mutagenesis methods are yet to be developed to define genes essential for these pathogens. Similarly, vaccines offering sufficient protection against diseases caused by Anaplasma spp. are not available. Here, we describe a targeted mutagenesis method for Anaplasma marginale where we deleted the phage head-to-tail connector protein gene, as its ortholog is essential for in vivo growth and persistence in Ehrlichia chaffeensis, a closely related pathogen in the family Anaplasmataceae. The A. marginale mutant did not cause disease and had a growth defect in the natural host, cattle. We then assessed its ability to confer protection against virulent challenge. Additionally, we tested whole cell A. marginale antigen as a vaccine (WCAV) candidate. Upon challenge, non-vaccinated control cattle developed severe disease with nearly 52% drop in PCV between days 26-31 post infection, with a peak of ~12% infected erythrocytes at about 4 weeks post challenge. Conversely, following challenge, all animals receiving the live mutant developed no clinical signs or anemia. Infected erythrocytes were not detected in blood smears. In contrast, the WCAV vaccinees developed disease similar to the non-vaccinated controls, though the peak number of infected erythrocytes was 5% and there was a 40% reduction in PCV. This is the first study describing targeted mutagenesis and its application in determining in vivo virulence and vaccine development for an important Anaplasma spp. pathogen. The study will pave the way for similar research in related Anaplasma pathogens impacting multiple hosts.