Direct culture is as sensitive as bioassay to achieve in vitro isolation of Toxoplasma gondii from latently infected animal hearts

• Mouse bioassay is commonly used to isolate Toxoplasma gondii from animal tissues. • Direct culture of tissues is shown to be equally efficient to bioassay ( p = 1.0) • Isolation success trends higher in animals that have high antibody titres. • Isolation by direct culture only occurred for qPCR positive heart digest samples. • Isolation of T. gondii by direct culture improves ethics by reducing animal use. Isolation of Toxoplasma gondii from latently infected animals is most frequently achieved using bioassay in mice, but we wondered if direct cell culture could be equally efficient. Paired heart and blood samples were obtained from slaughtered ewes and the proportion of isolates acquired using direct culture of acid-pepsin digested hearts was compared with the proportion of isolates acquired by bioassay followed by culture of infected mouse tissues. Isolation was successful from 8/30 seropositive ewes by direct culture and from 9/30 via bioassay. In a second trial using lambs experimentally infected with T. gondii for 3 months, organisms were re-isolated from 4/8 hearts by direct culture and from 3/8 via bioassay. Across both trials, 12/38 isolation attempts were successful for each method ( p = 1.0). For naturally infected sheep, antibody titres ≥ 1,000 tended to favour isolation of organisms in comparison with titres between 60 and 400 ( p = 0.018, α = 0.017). Isolation by direct culture was successful from 75% of qPCR positive heart digest suspensions in comparison with 0% of qPCR negative suspensions ( p < 10 -6 ). These results indicate that the most efficient and cost-effective method to culture T. gondii from latently infected animals is to select subjects with high antibody titres and maintain direct cultures up to five weeks for those acid-pepsin heart digests that have molecular confirmation of infection. Direct isolation of T. gondii into cell culture advances lab animal welfare and is as sensitive as mouse bioassay.