First report on detection of necrotic enteritis (NE) B-like toxin in biological samples from NE-afflicted chickens by capture enzyme-linked immunosorbent assay

Authors: LEE, KYUNG-WOO - US Department Of Agriculture (USDA); Lillehoj, Hyun; KIM, WOOHYUN - US Department Of Agriculture (USDA); PARK, INKYUNG - US Department Of Agriculture (USDA); Li, Charles; LU, MINGMIN - US Department Of Agriculture (USDA); HOFACRE, CHARLES - University Of Georgia

Submitted to: Poultry Science
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 4/5/2021
Publication Date: 4/20/2021
Citation: Lee, K., Lillehoj, H.S., Kim, W., Park, I., Li, C.Z., Lu, M., Hofacre, C.L. 2021. First report on detection of necrotic enteritis (NE) B-like toxin in biological samples from NE-afflicted chickens by capture enzyme-linked immunosorbent assay. Poultry Science. https://doi.org/10.1016/j.psj.2021.101190.
DOI: https://doi.org/10.1016/j.psj.2021.101190

Interpretive Summary: Necrotic enteritis (NE) is an enteric bacterial disease caused by Clostridium perfringens in chickens and has gained much attention recently in the post antibiotic era. NetB toxin secreted by C. perfringens type G has been identified as the major toxin involved in pathogenesis of NE in broiler chickens. Currently, there is a need to develop a sensitive ELISA which can identify susceptible chickens in poultry farms. In this paper, ARS scientists and a colleague in South Korea developed a monoclonal-based NetB-specific capture ELISA and showed that this in vitro assay detects native NetB toxin present in the biological samples of NE-afflicted chickens. In this study we showed that this new assay can detect NetB toxin in the gut content and fecal droppings collected from poultry farms enabling identification of poultry farms with potential NE disease outbreak. Availability of this assay will facilitate early monitoring of the presence of NetB toxin in fecal droppings from NE-afflicted broilers in commercial farms.

Technical Abstract: Necrotic enteritis (NE) is a devastating enteric disease caused by Clostridium perfringens type G. NE B-like (NetB) toxin, one of pore-forming toxins secreted by pathogenic C. perfringens type G, has been proposed to be the main virulent factor in NE pathogenesis. The present study investigated to detect the presence of NetB toxin in biological samples of NE-afflicted chickens using NetB-specific monoclonal-based enzyme-linked immunosorbent assay. Biological samples including serum, digesta and fecal droppings were obtained from previous three NE studies (thus, designated as Trials 1 to 3). In Trials 1 and 2, broiler chicks were infected with Eimeria maxima 41A on day 14, and followed by netB-positive C. perfringens Del-1 strain on day 18. Serum samples were obtained on 20 days post-hatch (i.e., 2 days post C. perfringens infection). In addition, various samples including serum, gut digesta and fecal droppings were obtained that had been collected on 0, 6, 24, and 30 h post C. perfringens infection. In Trial 3, broiler chicks were indirectly infected with litter-contaminated E. maxima on day 14, and followed by netB-positive C. perfringens via drinking water on days 18, 19, and 20. Serum samples and fecal droppings were obtained on 21 days post-hatch (i.e., 1 day post last C. perfringens infection). The results showed that NetB toxin was not detected in serum samples on Trials 1 and 3. No NetB toxins were detected in all samples obtained before C. perfringens infection in Trial 2. Low but detectable NetB toxins were found in serum samples obtained on 6 hours post C. perfringens infection in Trial 2. While NetB toxin in digesta and fecal droppings was detected on 6 hours post C. perfringens infection, its level was plateaued on 24 and 30 hours post C. perfringens infection. In Trial 3, NetB toxins were detected in fecal droppings from NE group and their concentration ranged from 2.9 to 3.1 ng per g of wet feces. In Trial 2, NE-specific lesions were not seen on 0 and 6 h post C. perfringens infection, but chickens exhibited moderate to severe following 24 h post infection leading to moderate association (r = +0.527) between NE lesion and NetB toxin in gut digesta. Here, we first report the presence of native NetB toxins in biological samples from NE-induced chickens using NetB-specific monoclonal-based capture ELISA.