Development of a molecular assay for the determination of Eimeria tenella oocyst viability

Coccidiosis is caused by apicomplexan parasites of the genus Eimeria, which infect epithelial cells of the intestinal tract causing diarrhea and negatively impacting production in the poultry industry. The self-limiting and highly immunogenic nature of infection by Eimeria spp. make live vaccination an effective means of coccidiosis control. Paramount to vaccine efficacy is the ability to administer precise numbers of viable oocysts. Unfortunately, no rapid and accurate method for determination of oocyst viability is available presently. This study presents the development of a qPCR-based assay for assessment of Eimeria tenella Tyzzer, 1929 oocyst viability. Transcriptome sequencing supported identification of three viability assay target transcripts based on significant increase in abundance with heat-stimulation. Measurement of shifts in target abundances in response to heat stimulation in oocysts, that ranged from high viability to non-infectious, was achieved via qPCR. Omission of DNase treatment supported use of background DNA in RNA samples for normalization for parasite numbers and oocyst disruption efficiency, while spike in of exogenous RNA supported normalization for variations in RNA recovery and reverse transcription efficiency. The assay demonstrated strong correlation with oocyst viability as confirmed through live infection trials, showing the highest predictive value for a transcript encoding a putative partial translationally controlled tumor protein, XM_013379639.1. This assay provides results in hours and could reduce the reliance on time-consuming and expensive live-infection trials in oocyst viability testing and could improve the accessibility and efficacy of coccidiosis vaccines. Future iterations may facilitate multivalent vaccine quality control and environmental monitoring.