Source: NORTH CAROLINA STATE UNIV submitted to
A NOVEL METHOD FOR ENUMERATION AND SPECIATION OF EIMERIA SPECIES OF COCCIDIAN PROTOZOA BY FLOW CYTOMETRY
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
TERMINATED
Funding Source
Reporting Frequency
Annual
Accession No.
1021299
Grant No.
(N/A)
Project No.
NCV-VMAH-0151
Proposal No.
(N/A)
Multistate No.
(N/A)
Program Code
(N/A)
Project Start Date
Sep 30, 2019
Project End Date
Jun 30, 2020
Grant Year
(N/A)
Project Director
Crespo, RO, .
Recipient Organization
NORTH CAROLINA STATE UNIV
(N/A)
RALEIGH,NC 27695
Performing Department
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
100%
Research Effort Categories
Basic
100%
Applied
(N/A)
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
3133260109080%
3133260111020%
Knowledge Area
313 - Internal Parasites in Animals;

Subject Of Investigation
3260 - Poultry meat;

Field Of Science
1090 - Immunology; 1110 - Parasitology;
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.
Project Methods
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.

Progress 09/30/19 to 06/30/20

Outputs
Target Audience:Poultry Industy Changes/Problems:The COVID-19 Pandemic caused delays in the validation. What opportunities for training and professional development has the project provided? This is part of a PhD project (Daniel Adams) Poultry residents (House Officers) collaborate in the project by processing samples, manual speciation and enumeration. Development of real time PCR assays for the speciation of coccidia How have the results been disseminated to communities of interest? Some local poultry producers have started to submit samples to our lab for oocyst counts. We provide results using the traditional microscopic count and an estimation of counts provided by flow cytometry (as statistical analysis is not completed yet). Several allied companies are aware of the study. They have contributed (or agreed to contribute) coccidia oocyst isolates or vaccines or field samples for validation What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Flow Cytometry Speciation and Enumeration of Eimeria Populations - The four different Eimeria populations we aimed to speciate in this study were successfully detected on a BD LSRII analyzer. Simple light-scatter data plotting (Forward Scatter [FSC] and Side Scatter [SSC]) was sufficient to resolve all the four populations as hypothesized. No special staining or antibody labeling procedures were necessary towards resolving different Eimeria populations. Validation of Each Gated Eimeria Population - The presence of each Eimeria species within each of the designated gates on LSRII was successfully validated by cell sorting. The four individual sorted populations from the MoFlo-XDP cell sorter were shown to be in alignment with the LSRII data. Sample collection in plain 1X PBS buffer was found to be appropriate for the downstream processing of the sorted populations (i.e for analysis on LSRII analyzer, and also for DNA preparation step for further PCR validation experiments).

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