Targeted mutagenesis in <i>Ehrlichia canis</i> deleting the phage head-to-tail connector protein gene and its assessment as a vaccine candidate preventing canine ehrlichiosis

<i>Ehrlichia canis</i> is primarily a <i>Rhipicephalus sanguineus</i> tick-borne rickettsial pathogen initially identified as causing canine monocytic ehrlichiosis, and infections in people have also been reported in Venezuela, Mexico, and parts of Europe. It is of high importance to have a vaccine suitable in protecting the canine host, which will aid in lessening <i>E. canis</i> infections also in people. Gene inactivation mutations in the phage head-to-tail connector protein genes ( <i>phtcp</i> ) from <i>E. chaffeensis</i> and <i>A. marginale</i> caused attenuated growth, and prior infection with the mutated bacteria induced protective immunity against wild-type bacterial infections in natural hosts, independent of blood-borne infection or tick-transmission infection. In the current study, we describe the development of targeted mutagenesis for the first time in <i>E. canis</i> genome and with a novel modification to avoid introducing antibiotic resistance cassettes to delete the <i>phtcp</i> ortholog from <i>E. canis</i> . The mutated <i>E. canis</i> was then assessed for its <i>in vivo</i> growth and the induction of host immunity exerted following the mutant infection aiding to protect against wild-type infection challenge in the canine host. We assessed systemic pathogen loads, hematological parameters, IgG immune responses, and plasma cytokines following the mutant infection relative to uninfected dogs. Similarly, the assessments were carried out following wild-type pathogen infections in dogs with or without prior mutant infection challenges. The study demonstrates that prior infection of dogs with the mutant induces immunity to prevent infection establishment by wild-type <i>E. canis.</i> Similarly, the mutant infection resulted in clear biological differences compared to the wild-type infection. This study establishes that the molecular genetic methods are broadly applicable to pathogens belonging to the family Anaplasmataceae and that the modified live vaccines with <i>phtcp</i> gene orthologs are valuable in reducing the diseases caused by the tick-borne rickettsial pathogens belong to Anaplasmataceae, including <i>E. canis</i> .