Cytauxzoon felis in a Post-Genomic Era: Taxonomy, Diagnosis, Treatment, and Prevention

Cytauxzoonosis is an emerging disease affecting felines throughout the Western hemisphere. Cytauxzoonosis is caused by the tick-transmitted parasite Cytauxzoon felis, an organism for which little is known. Since being discovered 40 years ago in Missouri, C. felis has spread across 1/3 of the United States, and is expected to continue spreading given the continental distribution of competent feline and tick hosts. C. felis is highly pathogenic to domestic cats. Disease progresses rapidly and even with the best treatment mortality remains high. No vaccine exists, so disease prevention relies on indoor confinement and acaricide prophylaxis. Given the rapid dispersal of this virulent parasite that lacks effective treatment or prevention options, further investigation is warranted.;However, the inability to culture the parasite in vitro, ethical concerns with in vivo studies, and lack of funding for feline diseases has limited C. felis research. To counteract this, we have sequenced the parasite's mitochondrial and chromosomal genomes. Using these resources, we have identified useful genetic targets that resolve the taxonomy of C. felis and improve the treatment, diagnosis and prevention of cytauxzoonosis.;The taxonomic placement of Cytauxzoon felis within Piroplasmida remains unsolved due to discrepancies between morphological and molecular data. We have clarified phylogeny of C. felis and other Piroplasmida using mitochondrial genome sequences and structures. Mitochondrial genome analysis supported the placement of C. felis within the Theileria clade, and indicated that T. equi, B. conradae, and B. microti organisms are genetically distinct lineages. Characterization of additional mitochondrial genomes and subsequent reclassification of Piroplasmida genera are merited.;Mortality of infected cats treated with atovaquone and azithromycin (A&A) is 40%. Atovaquone targets an electron transport protein encoded by mitochondrial cytb. Mutations in the cytb gene are associated with atovaquone resistance in related parasites. We hypothesized that C. felis cytb genotype would be associated with response to A&A treatment. After cytb-genotyping 69 samples, we identified a C. felis cytb genotype (cytb1) associated with increased survival of cats treated with A&A.;Given this association, we hypothesized that cytb1 could aid in the prognosis of cytauxzoonosis. By developing a quantitative PCR panel that identifies cytb1-specific SNPs with high resolution melt (HRM) analysis, we distinguished C. felis cytb1 from all other C. felis cytb genotypes with 100% sensitivity and 98.2% specificity. This assay is cost-effective and can be completed in less than 3 hours, which is important given the high cost of A&A and rapid disease course.;Diagnosis of C. felisis challenging during early infection when parasitemia is low and clinical signs remain vague. Mitochondrial genes are more sensitive molecular diagnostic targets for parasite detection than 18S in related parasites. We demonstrated that mitochondrial cox3 copy number is increased relative to 18S in blood and tissue samples from cats with acute cytauxzoonosis, and that cox3 is more sensitive for identifying early C. felis infection than 18S. This assay will aid in early detection of Cytauxzoon felis infection.;Sequencing the C. felis genome has allowed for identification of 33 vaccine candidates. We have manufactured two DNA vaccines: an expression library vaccine including all candidates and a vaccine consisting of the recently described cf76. We describe the immunization approach, serological response to vaccination, and subsequent clinical outcome following challenge with C. felis. All vaccinated cats became infected and developed disease, rendering both vaccines inadequate methods for prevention of cytauxzoonosis. However, the expression library vaccine showed evidence of aiding in disease control, providing a baseline for development of future subunit vaccines against cytauxzoonosis.;In conclusion, the mitochondrial and chromosomal genomes have clarified the taxonomy of Cytauxzoon felis and further advanced our understanding of the treatment, diagnosis, and prevention of cytauxzoonosis.