Molecular epidemiology and PFGE-based genetic diversity of Clostridium perfringens in broiler chickens from Eastern Türkiye

Clostridium perfringens is a ubiquitous foodborne and opportunistic anaerobe and a leading cause of necrotic enteritis (NE) in broiler chickens, with implications for animal health, food safety, and public health. While the pore-forming NetB toxin has been identified as a major virulence determinant in NE, its prevalence shows geographical variability, and the molecular epidemiology of C. perfringens populations circulating in clinically healthy broilers with subclinical intestinal and hepatic lesions remains poorly characterized. A cross-sectional molecular epidemiological study was conducted using samples from broiler chickens originating from 100 commercial farms in Eastern Türkiye and sampled at slaughterhouses. A total of 1,000 samples (500 intestinal and 500 liver tissues) exhibiting hemorrhagic and lesion-associated tissues were analyzed. C. perfringens isolates were identified using phenotypic methods and PCR targeting the cpa and 16 S rRNA genes. Major toxin genes (cpa, cpb, etx, iap, cpb2, and cpe) were detected by multiplex PCR, and the netB gene was screened by singleplex PCR. Alpha-toxin production was quantified by ELISA, and clonal relatedness was assessed by pulsed-field gel electrophoresis (PFGE). C. perfringens was isolated from 66.0% (660/1,000) of the samples. Nearly all isolates (99.5%) carried the cpa gene and were classified as toxin type A, whereas none harbored the netB gene or other major toxin genes. Alpha-toxin expression was confirmed in 94.0% of tested isolates. PFGE analysis of 61 representative isolates identified 30 distinct pulsotypes distributed across 17 clusters, revealing pronounced genetic heterogeneity among farms and slaughterhouses. The C. perfringens population circulating in broiler production systems in Eastern Türkiye is dominated by genetically diverse, cpa-positive and netB-negative type A strains. From a One Health perspective, the high prevalence and diversity of these strains in food animals highlight their potential role as reservoirs at the animal–food–human interface, emphasizing the need for integrated molecular surveillance to mitigate risks to poultry health and public health. To our knowledge, no previous study from Türkiye has reported PFGE-based genetic diversity analysis of Clostridium perfringens isolates from broiler chickens within a slaughterhouse-linked, multi-farm epidemiological design, nor has the presence of the netB toxin gene been comprehensively evaluated in broiler-derived isolates from Türkiye.