A flow cytometric method for enumeration and speciation of coccidia affecting broiler chickens

Production losses, mortality, and control measures associated with coccidiosis, caused by Eimera species, cost the broiler industry over $14 billion a year. Current means to distinguish Eimeria species such as oocyst morphology, pre-patent period and site of infection are subjective, labor intensive or unsuitable for high-throughput applications. Although Polymerase Chain Reaction (PCR) techniques have been validated, the target gene cannot differentiate relative abundance of each species in mixed infections. In this study, we developed a non-antibody-based flow cytometry high throughput method to simultaneously enumerate and speciate four Eimeria species, E. acervulina, E. mitis, E. maxima, and E. tenella, using commercial coccidia vaccine as well as field fecal samples. Our findings showed that the four Eimeria oocyst populations could be distinctly speciated based on their size and granularity (shape) via scatter plotting. These distinct populations were sorted and confirmed by quantitative real-time PCR assay. Finally, the flow cytometry findings were applied to enumerate and speciate oocysts from fecal samples collected from commercial broiler flocks vaccinated for coccidiosis at day of hatch and the results were validated against the conventional manual method of floatation and microscopic examination. Collectively, the findings of this study suggested that non-antibody based Flow Cytometry technique can be successful in the simultaneous enumeration and speciation of coccidia. Further development and validation is needed to make this diagnostic tool useful for field applications at a much larger scale as well as to speciate other Eimeria species.