Correlation Between Avian Pathogenic Clostridium perfringens Spore Load and Occurrence of Necrotic Enteritis on Broiler Chicken Farms.

Abstract

Necrotic enteritis (NE) is a significant intestinal disease in poultry caused by the spore-forming, obligate anaerobe Clostridium perfringens. Historically, antimicrobials have controlled NE but growing concerns over antibiotic resistance have led to reduced use, resulting in disease resurgence. Clostridium perfringens spores persist in poultry environments, and their accumulation is believed to contribute to NE outbreaks. However, tools to assess spore load and predict outbreak risk are limited. To address this, a selective egg yolk agar medium was developed using potassium chlorate to inhibit aerobic bacteria, combined with heat treatment to enumerate C. perfringens spores in poultry litter. Matrix-assisted laser desorption-ionization time of flight mass spectrometry confirmed that 80% of lecithinase-positive colonies on chlorate-supplemented agar were C. perfringens. When comparing reinforced Clostridium agar and Clostridium perfringens agar (CPA) as bases, chlorate did not significantly reduce lecithinase-positive aerobes. Ninety-nine percent of lecithinase-positive black colonies on CPA were strict anaerobes, and 85% were confirmed as C. perfringens by PCR. A significant correlation (χ² test; P < 0.05) was observed between spore abundance in litter and a farm's NE history. However, spore abundance did not correlate with increased sporulation efficiency in C. perfringens isolates (P = 0.86). Notably, the presence of netB-positive C. perfringens was strongly associated with prior NE outbreaks (P < 0.01). This study demonstrated CPA-based egg yolk agar as an effective diagnostic medium for detecting C. perfringens in poultry litter.