Workflow of isolation: Experimental infection with a low-passage strain causes higher honeybee mortality rates than the PRA-403 reference strain.

The impact of trypanosomatid parasites on honeybee health may represent a major threat to bee colonies worldwide. However, few axenic isolates have been generated to date and with no details on cell culture passages, a parameter that could influence parasite virulence. To address this question, a trypanosomatid isolation protocol was developed and a new strain was obtained, named C1. Using experimental infection of worker honeybees, we compared the virulence and mortality rates of the ATCC PRA-403 reference strain and C1 strain, the latter showing higher virulence from 10 days post-infection onward. This study highlights the impact of cell culture passages on the pathogenicity of in honeybees, providing new evidence of its negative effects on honeybee health.