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

Abstract

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.

Recent advances in genetic systems in obligate intracellular human-pathogenic bacteria

“…In both cases, the bacteria were washed in 300 mM sucrose. Selection of transformants has predominantly been via the use of spectinomycin and streptomycin resistance conferred by the aadA gene, and more recently gentamicin resistance has also been used (Hove et al, 2022). Interestingly, in a step toward nonantibiotic selection in A. phagocytophilum, Oliva Chavez et al (2019) tested the use of the herbicide resistance bar gene, which provides resistance to phosphinothricin (PPT), for selection of transformed bacteria.…”

“…Targeted mutagenesis in Anaplasma marginale has recently been achieved (Hove et al, 2022). In this study, the vector AM581-KO-tuf-mCherry-Gent, containing the tuf promoter-mCherry-gentamicin cassette flanked by 1.1 kb of 5′ and 3′ DNA that flank the AM581 gene, was used to a create linear PCR product that was used to delete the AM581 gene (encoding the phage headto-tail connector protein) by allelic exchange.…”

Bovine Anaplasmosis: Diagnosis, Treatment and Control Strategies

“…As mentioned before, live organisms would seem to induce complete immunity against homologous and heterologous challenge. Based on this premise, targeted mutagenesis was used for deletion of the phage head-to-tail connector protein (phtcp) gene in A. marginale (that is cultivable on tick cells) (Hove et al 2022). The resulting mutant did not cause disease and exhibited attenuated growth in its natural host (cattle).…”

“…When its ability to confer protection against wild-type A. marginale was challenged, all animals receiving the live mutant did not develop clinical signs or anemia, or erythrocyte infection. In contrast, animals immunized with whole cell inactivated vaccine developed similar disease as the nonvaccines following A. marginale infection (Hove et al 2022). This is very innovative approach as the organism is cultivable on tick cell lines, thus it is not exposed to cattle pathogens and cannot transmit them and it can be reproduced on demand.…”

“…This is very innovative approach as the organism is cultivable on tick cell lines, thus it is not exposed to cattle pathogens and cannot transmit them and it can be reproduced on demand. The mutation seems to be stable, thus it will not reverse the virulence in the host and finally it may protect a wide array of heterologous strains upon testing (Hove et al 2022).…”