Development of a new immunodiagnostic tool for poultry coccidiosis using an antigen capture sandwich assay based on monoclonal antibodies detecting an immunodominant antigen of Eimeria

Authors: LEE, YOUNGSUB - US Department Of Agriculture (USDA); Lillehoj, Hyun

Submitted to: Poultry Science
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 5/13/2023
Publication Date: 8/1/2023
Citation: Lee, Y., Lillehoj, H.S. 2023. Development of a new immunodiagnostic tool for poultry coccidiosis using an antigen capture sandwich assay based on monoclonal antibodies detecting an immunodominant antigen of Eimeria. Poultry Science. https://doi.org/10.1016/j.psj.2023.102790.
DOI: https://doi.org/10.1016/j.psj.2023.102790

Interpretive Summary: Avian coccidiosis, an intestinal disease caused by parasites of the genus Eimeria is an economically important disease for poultry industry worldwide. Current preventive measures for coccidiosis and other diseases associated with coccidiosis such as necrotic enteritis predominantly include chemotherapy and conventional vaccines. However, due to the continuous development of drug resistance and the fundamental disadvantage associated with the emergence of antimicrobial resistance, alternative and sustainable methods of coccidiosis prevention are timely needed. Various potential immunogenic antigens have been explored as recombinant and sub-unit vaccines with different degree of success to control coccidiosis, experimentally. However, there is a timely need for sensitive detection method for early diagnosis of coccidiosis in large poultry production system for more effective coccidiosis management. In this report, ARS scientists describe a new immunoassay that can directly detect coccidiosis in chickens in commercial production environment using serum and fecal samples for coccidiosis diagnosis using a set of antibodies that recognize the most immunodominant protein (antigen) of Eimeria parasites. More importantly, the daily changes in levels of this antigen in different samples from Eimeria-infected chickens were effectively monitored with this new antigen-capture ELISA.

Technical Abstract: Background: This study was conducted to develop an antigen-capture ELISA that detects an immunodominant antigen of Eimeria, 3-1E, using a set of 3-1E specific mouse monoclonal antibodies (mAbs). Results: 3-1E-specific antigen-capture ELISA was developed using compatible mAb pairs (#318 and #320) selected from six mAbs (#312, #317, #318, #319, #320, and #323) with high binding activity against recombinant 3-1E protein. A higher level of 3-1E was detected in the lysate of sporozoites than in sporocysts and the result of immunofluorescence assay (IFA) using two mAbs (#318 and #320) showed specific staining around the membrane of E. tenella sporozoites. In order to measure the changes in the 3-1E level during in coccidiosis, serum, feces, jejunal and cecal contents were individually collected daily for seven days post-infection with E. maxima and E. tenella. The new ELISA was sensitive and specific for 3-1E detection in all samples collected daily from E. maxima- and E. tenella-infected chickens for a week, and the detection sensitivity ranges were 2-5 ng/mL and 1-5 ng/mL in serum, 4-25 ng/mL and 4-30 ng/mL in feces, 1-3 ng/mL and 1-10 ng/mL in cecal contents, and 3-65 ng/mL and 4-22 ng/mL in jejunal contents. The overall 3-1E levels started to increase from 4 days post-infection (dpi), and the highest production was shown on 5 dpi. Among the samples collected from Eimeria-infected chickens, the highest overall detection level was found in the jejunal contents. Furthermore, the level of IFN-' in serum was significantly (P < 0.05) increased from 3 dpi and peaked on 5 dpi post E. maxima infection. Post E. tenella infection, the level of IFN-' in serum was gradually (P < 0.05) increased from 2 to 5 dpi and plateaued at 7 dpi. The level of TNF-a in serum was rapidly (P < 0.05) increased from 4 dpi and those levels were kept until 7 dpi post both Eimeria infections (E. maxima and E. tenella). Conclusions: This study demonstrated that the newly produced anti-3-1E mAbs specifically recognized E. tenella sporozoites. More importantly, the daily changes in the 3-1E levels in different samples from E. maxima- and E. tenella-infected chickens were effectively monitored with this new antigen-capture ELISA. Therefore, this new immunoassay is a sensitive diagnostic tool to monitor coccidiosis in a large field population in the commercial poultry farms using serum, feces, and gut samples during the entire period of infection cycle starting from 1 day after infection.