Abstract
Feline upper respiratory tract disease (FURTD) poses a significant health threat to captive Panthera leo (African lion) populations, with Felid alphaherpesvirus 1 (FeHV-1) and Chlamydia felis acting as primary, often co-infectious, pathogens. Rapid and reliable differentiation of these agents is vital for effective clinical management, quarantine enforcement, and disease prevention in zoological collections. In this study, we developed and validated a TaqMan-based multiplex real-time quantitative polymerase chain reaction (mqPCR) assay designed for simultaneous, early detection of FeHV-1 (targeting the glycoprotein B gene) and C. felis (targeting the outer membrane protein A gene) in captive lions. Oropharyngeal, nasal, and conjunctival swab samples were collected from 142 captive African lions displaying acute, chronic, or subclinical respiratory and ocular signs, as well as asymptomatic controls, across 12 zoological institutions. Compared with singleplex qPCR reference standard assays, the mqPCR assay demonstrated diagnostic sensitivities of 96.8% (95% CI: 91.0–99.3%) for FeHV-1 and 95.2% (95% CI: 88.3–98.7%) for C. felis, alongside high diagnostic specificities of 98.7% and 99.1%, respectively. The analytical limit of detection was established at 10 genomic copies per reaction for both targets, with no cross-reactivity observed against feline calicivirus, Mycoplasma felis, or Bordetella bronchiseptica. Co-infections were identified in 22.5% of symptomatic animals. The mqPCR assay offers a highly sensitive, specific, and cost-effective tool for routine surveillance and early clinical diagnosis, facilitating rapid intervention and biosecurity protocols in managed Panthera leo herds.
