Collagen adhesin protein and necrotic enteritis B-like toxin as biomarkers for early diagnosis of necrotic enteritis in commercial broiler chickens

Authors: GOO, DOYUN - US Department Of Agriculture (USDA); PARK, INKYUNG - US Department Of Agriculture (USDA); NAM, HOYUM - US Department Of Agriculture (USDA); LEE, YOUNGSUB - US Department Of Agriculture (USDA); SAWALL, JOHN - Arm & Hammer Animal And Food Production; SMITH, XANDRA - Arm & Hammer Animal And Food Production; Li, Charles; Lillehoj, Hyun

Submitted to: Poultry Science
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 3/8/2023
Publication Date: 6/1/2023
Citation: Goo, D., Park, I., Nam, H., Lee, Y., Sawall, J., Smith, X., Li, C.Z., Lillehoj, H.S. 2023. Collagen adhesin protein and necrotic enteritis B-like toxin as biomarkers for early diagnosis of necrotic enteritis in commercial broiler chickens. Poultry Science. https://doi.org/10.1016/j.psj.2023.102647.
DOI: https://doi.org/10.1016/j.psj.2023.102647

Interpretive Summary: Necrotic enteritis (NE) is a devastating enteric disease caused by Clostridium perfringens type A/G which impacts global poultry industry by compromising performance, health and welfare of the chickens. There is no effective vaccines against NE. Availability of reliable detection strategy for NE that can be used in commercial poultry farms to identify susceptible chickens will allow early NE detection and reduce significant economic losses ($ 6 billion per year) associated with NE. In this report, ARS scientists describe a new immunoassay based on two biomarkers of C. perfringens pathogen which is a causative agent of NE. Normally NE is detectable in the field by gut sampling around 3-4 weeks post hatch, Using newly developed mouse monoclonal antibodies which specifically identify major virulence factors of C. perfringens, scientists demonstrated that this ELISA can detect NE-afflicted chickens using their serum feces and gut samples at much earlier ages than when NE is normally detectable in the poultry farms. Two major biomarkers, collagen binding adhesion (CNA) and NE B-like (NetB) toxin, are critical antigens associated with NE pathogenesis and this work showed that these two antigens are detectable very early in NE-afflicted chickens from commercial farms. In conclusion, this novel immunoassay for NE will allow early detection of field NE disease outbreak to reduce economic losses due to NE since these immunoassays will allow the accurate measurement of C. perfringens virulence in poultry samples to monitor flock health status or to make early detection of NE outbreak in NE-susceptible farms.

Technical Abstract: Mouse monoclonal antibodies (mAbs) reactive with Clostridium perfringens collagen adhesin protein (CNA) and necrotic enteritis B-like toxin (NetB) were developed. The best capture/detection mAb pairs were selected based on their affinity and specificity to develop sandwich enzyme-linked immunosorbent assays (ELISAs) to detect CNA and NetB proteins in jejunal digesta samples from commercial broiler farms in the United States. Prior to the analysis of samples from commercial broiler flocks, the specificity and sensitivity of the CNA and NetB ELISAs were validated using serum, jejunal digesta, and fecal samples from chickens co-infected with Eimeria maxima and CNA+/NetB+ C. perfringens in an animal model of necrotic enteritis (NE). Subsequently, a total of 251 field samples were collected from 74 commercial poultry farms. Among these, 18 samples were from 6 broiler farms that used organic materials as antibiotics (OA), and 233 samples were from 68 farms with non-antibiotics (NA). In the equally diluted 100 µl jejunal digesta samples, CNA levels ranged from 0.2 – 5.9 ng and NetB levels ranged from 0.9 – 19.1 ng. CNA and NetB levels showed a positive correlation with each other (Pearson correlation coefficient r = 0.772, P < 0.001). Samples from commercial farms in “Complex-6” showed significantly increased levels of both CNA and NetB proteins compared with those from “Complex-1, -2, -7, and -11” farms (P < 0.001). CNA and NetB levels in jejunal digesta were significantly decreased in OA farms compared with those from NA farms (P < 0.001). In conclusion, these new C. perfringens antigen-specific sandwich ELISAs offer a sensitive and specific means to detect C. perfringens CNA and NetB proteins as biomarkers of early NE occurrence in field samples from commercial broiler chickens.