Population Health and Pathobiology
Non Technical Summary
The Eimeria species are apicomplexan protozoa parasites that cause coccidiosis, most notably in chickens. Coccidiosis has a significant economic impact on the poultry production industry and is one of the most common diseases faced by poultry. An accurate and speedy diagnosis of Eimeria at the species level are both challenging and necessary. Presently, there are many means to distinguish between species of these protozoa, including oocyst morphology, pre-patent period, and site of infection or minimum sporulation time; but these methods are labor intensive, time consuming, and unreliable, particularly in cases of mixed Eimeria infection. Furthermore, these methods do not easily lend themselves to high-throughput applications. Numerous molecular approaches have been tried and tested with some degree of success. Quantitative Polymerase Chain Reaction (qPCR) assays, capable to speciate and enumerate Eimeria, have been validated; however, their practical utility and availability in laboratory settings are questionable due to the relatively high reagent cost. Recently, flow cytometry (FCM) has been explored for diagnostic quantification of oocysts of Cryptosporidium, another genus of apicomplexan parasitic alveolates affecting humans.
Animal Health Component
Research Effort Categories
Goals / Objectives
.Quantification and speciation of the most common species of Eimeria sp. in poultry can be done by FCM without the use of fluorescence antibodies. To prove this hypothesis, we will use FCM to (1) characterize each Eimeria species, (2) quantify and speciate mixed cultures of Eimeria sp., and (3) evaluate the effectiveness of the protocol with field samples. A key advantage of FCM is that it can be done quickly enabling decisions to be made immediately, whereas other diagnostic methods are slower, labor-intensive, and more expensive. In future studies, we will utilize the ability of FCM to differentiate vaccine and field strains of Eimeria species to detect changes in the permeability of their cell membranes.
The overall objective of the proposed work is to develop a non-antibody based flow cytometric analytical method for accurate and reliable diagnosis of Eimeria species of chickens. To achieve this objective, our experimental approach would first involve flow cytometric method optimization using purified individual Eimeria species oocyst preparations. This segment of work would allow us to determine the flow plot scatter (forward, FSC and side, SSC) coordinates for individual Eimeria species. Next, a mixture of oocysts from 3-4 different Eimeria species will be used to characterize individual Eimerian oocyst population such that their populations can be distinctly separated from each other. Here, we will also sort individual populations to verify their identity by Polymerase Chain Reaction (PCR). Lastly, we will extend our findings to apply to the field fecal samples collected from infected birds or those chickens vaccinated with coccidial vaccine containing four live Eimeria species.