First report of viable Toxoplasma gondii isolation from African lions (Panthera leo): Evidence for their status as intermediate hosts.

Toxoplasma gondii can cause lifelong infection in most definitive and intermediate hosts. Serological evidence of T. gondii infection has been documented in both captive and free-ranging lions. We collected samples from 40 captive African lions (Panthera leo) in China from 2017 to 2025 and investigated T. gondii in these animals via serological, bioassay, and molecular methods. The overall prevalence of T. gondii in the 40 lions was 45.0% (95% CI: 30.70-60.18%). Sex, age, phenotype, and sampling date were not risk factors for susceptibility to T. gondii infection. A viable T. gondii isolate (designated as TgLionCHn1) was obtained from the tissues of one lion via mouse bioassay and amplified by in vitro cultivation. Parasite DNA was extracted from the cell culture, and the genotype was determined by polymerase chain reaction - restriction fragment length polymorphism analysis using 10 genetic markers (SAG1, SAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1, and Apico) and two virulence genes (ROP18/ROP5). The genotype of the T. gondii isolate TgLionCHn1 (designated as ToxoDB #348) exhibited a previously unreported ROP18/ROP5 allelic combination (2/1). Mice infected with ≥1 tachyzoite died from acute toxoplasmosis at 9.2 ± 0.6 days post infection, indicating that TgLionCHn1 is virulent in mice. Our assessment of this viable isolate from the tissues of a dead lion provides the first direct evidence that a lion can serve as an intermediate host for T. gondii. Seropositive lions may shed T. gondii oocysts, highlighting zoonotic risks in zoo environments and the need for improved biosecurity measures.