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.
Ehrlichia chaffeensis (ECH) is an obligate intracellular gram-negative bacterium and a frequent cause of severe and fatal tick-borne infection in people in North America. The canine is a common incidental host and a useful model for vaccine development and assessing immune responses to ECH infection. We recently established mutagenesis methods for ECH and demonstrated that immunization of dogs with an ECH mutant with a functional disruption in the ECH_0660 gene protected the vertebrate host from both tick-transmitted and intravenous wildtype ECH challenge when given 4 weeks after immunization. In the current study, we evaluated the duration of immunity offered by the ECH_0660 gene mutant live attenuated vaccine (MLAV) against wildtype infection challenge. Groups of 12 dogs were immunized with the ECH MLAV and then challenged 2, 4, 8 and 12 months later via tick-transmitted or blood-transmitted infection with wildtype ECH. Animals were monitored for 4 weeks after challenge and then sacrificed for evaluation of tissue pathology and bacterial burden. Immunization with the MLAV induced ECH-specific T cell responses, detectable by ELISPOT for IFNγ; and differentiation of IFNγ- and/or TNFα-producing ECH-specific T effector memory CD4 T cells (CD45RAneg CD45RO+ CD62Lneg). Antigen-specific recall responses were detectable in the peripheral blood of most (27/32) vaccinated dogs for 4 months after immunization, but only in 5/24 of the dogs after 8 months. This result suggests that a single MLAV vaccination induces cellular immune responses and differentiation of memory helper T cells. Future work will investigate the correlations between cellular immunity, humoral responses and bacterial load. Supported by grants from NIH (R01 AI152418)
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