title: PCR amplification of a multi-copy mitochondrial gene (cox3) improves detection of Cytauxzoon felis infection as compared to a ribosomal gene (18S).
abstract: Cytauxzoon felis is a tick-transmitted protozoan parasite that infects felids. Clinical disease caused by acute C. felis infection rapidly progresses in domestic cats, leading to high morbidity and mortality. Accurately diagnosing cytauxzoonosis as soon as possible during acute infection would allow for earlier initiation of antiprotozoal therapy which could lead to higher survival rates. Molecular detection of parasite rRNA genes (18S) by PCR has previously been shown to be a sensitive method of diagnosing C. felis infections. Based on evidence from related apicomplexan species, we hypothesized that C. felis mitochondrial genes would exist at higher copy numbers than 18S and would be a more sensitive diagnostic target. In this study we have designed a PCR assay targeting the C. felis mitochondrial gene cytochrome c oxidase subunit III (cox3). Herein we demonstrate that (1) the cox3 PCR can detect as low as 1 copy of DNA target and can detect C. felis in samples with known mitochondrial sequence heterogeneity, (2) cox3 copy number is increased relative to 18S in blood and tissue samples from acutely infected cats, and (3) the cox3 PCR is more sensitive than 18S PCR for detection of C. felis during early infections.
authors.affiliation: North Carolina State University, College of Veterinary Medicine, 1060 William Moore Dr., Raleigh, NC 27607, United States.
authors.forename: Megan E
authors.initials: ME
authors.lastname: Schreeg
authors.affiliation: North Carolina State University, College of Veterinary Medicine, 1060 William Moore Dr., Raleigh, NC 27607, United States.
authors.forename: Henry S
authors.initials: HS
authors.lastname: Marr
authors.affiliation: North Carolina State University, College of Sciences, Campus Box 8201/4216, Broughton Hall, Raleigh, NC 27695, United States.
authors.forename: Emily H
authors.initials: EH
authors.lastname: Griffith
authors.affiliation: North Carolina State University, College of Veterinary Medicine, 1060 William Moore Dr., Raleigh, NC 27607, United States.
authors.forename: Jaime L
authors.initials: JL
authors.lastname: Tarigo
authors.affiliation: North Carolina State University, College of Agriculture and Life Sciences, 317A Ricks Hall 1, Lampe Dr., Raleigh, NC 27695, United States.
authors.forename: David M
authors.initials: DM
authors.lastname: Bird
authors.affiliation: Oklahoma State University, Center for Veterinary Health Sciences, 205 McElroy Hall, Stillwater, OK 74078, United States.
authors.forename: Mason V
authors.initials: MV
authors.lastname: Reichard
authors.affiliation: University of Missouri, College of Veterinary Medicine, 900 E. Campus Dr., Columbia, MO, 65211, United States.
authors.forename: Leah A
authors.initials: LA
authors.lastname: Cohn
authors.affiliation: North Carolina State University, College of Veterinary Medicine, 1060 William Moore Dr., Raleigh, NC 27607, United States.
authors.forename: Michael G
authors.initials: MG
authors.lastname: Levy
authors.affiliation: North Carolina State University, College of Veterinary Medicine, 1060 William Moore Dr., Raleigh, NC 27607, United States. Electronic address: ajbirken@ncsu.edu.
authors.forename: Adam J
authors.initials: AJ
authors.lastname: Birkenheuer
mesh_terms: D000818:Animals; D002371:Cat Diseases / Q000175:diagnosis*; D002415:Cats; D016054:DNA, Protozoan / Q000097:blood / Q000235:genetics; D003576:Electron Transport Complex IV / Q000235:genetics*; D018628:Gene Dosage; D016787:Haemosporida / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000592:standards / Q000662:veterinary*; D011529:Protozoan Infections, Animal / Q000175:diagnosis*; D012337:RNA, Ribosomal, 18S / Q000235:genetics; D012680:Sensitivity and Specificity
chemical_list: D016054:DNA, Protozoan; D012337:RNA, Ribosomal, 18S; D003576:Electron Transport Complex IV
keywords: Cytauxzoonosis; Cytochrome c oxidase; Feline; Mitochondria; Molecular diagnostic assays; Piroplasmosis