Toxinotyping of Clostridium perfringens strains isolated from broiler flocks with necrotic enteritis and evaluation of the effect of toxins on Leghorn Male Hepatoma cells

Background: Necrotic enteritis (NE) is an economically important disease, caused by Clostridium perfringens type G strains, and is one of the major targets of antibiotics used in poultry feed. Aims: This study aimed to genotypically characterize virulent strains of C. perfringens isolated from healthy and diseased birds in Iran. Methods: Eleven isolates were derived from necrotic enteritis cases, and 27 were from healthy chickens. Isolations were performed using blood agar. To assess whether zmp is generally associated with avian NE, 38 C. perfringens isolates were screened using PCR and western blotting. The involvement of these toxins as virulence factors was investigated using cytotoxicity assays. Results: All isolates carried the phospholipase c (plc) gene regardless of their origin and virulence. The zinc metallopeptidase (zmp) gene was found in the isolates collected from birds affected by necrotic enteritis. Furthermore, Necrotic enteritis like B (NetB) was only found in 36.36% of the isolates derived from necrotic enteritis-infected birds. Western blot analysis further confirmed the expression of Alpha toxin, NetB, and Zmp in different isolates. Incubation of Leghorn Male Hepatoma (LMH) cells with crude C. perfringens toxins indicated that the supernatants of all bacterial strains were toxic toward LMH cells at different dilutions. In addition, crude toxins of the Cp28 strain expressing Alpha toxin, Zmp, and NetB showed an approximately 50% cytotoxic dose (CD50) at a 1:34 dilution. Strain Cp119.2, which produces both ZMP and the Alpha toxin, and strain Cp48, which only produces the Alpha toxin, showed CD50 at 1:23 and 1:4 dilutions, respectively. Conclusion: It seems that both NetB and Zmp play major roles in the cytotoxicity and pathogenicity of this organism.