First comprehensive analysis of virulence-associated gene profiles and co-occurrence patterns of avian pathogenic Escherichia coli isolated from chickens in northern Vietnam.

Abstract

Background and aim: Avian pathogenic Escherichia coli (APEC), a major cause of colibacillosis in poultry, possesses diverse virulence-associated genes (VAGs) that contribute to extraintestinal pathogenicity. However, comprehensive information on VAG combinations and co-occurrence patterns among APEC isolates in Vietnam remains limited. This study aimed to characterize VAG prevalence, combination profiles, and co-occurrence patterns in APEC and non-pathogenic E. coli (NPEC) isolates from chickens in northern Vietnam.

Materials and methods: From August 2023 to August 2025, 515 spleen samples were collected from healthy and non-healthy chickens on 35 commercial poultry farms in Hanoi, Hai Phong, Phu Tho, Bac Ninh, and Thai Nguyen. A total of 200 E. coli isolates were recovered and screened by multiplex polymerase chain reaction for 19 VAGs: fimC , eaeA , papC , tsh , sfa_foc , iucD , iutA , irp2, fyuA , iucA , iroN , vat, hlyA , hlyF , astA , ompT , iss , cva /cvi, and colV. Isolates carrying ≥5 of nine selected APEC-associated VAGs were classified as APEC; the remainder were classified as NPEC. We compared gene prevalence, combination profiles, and pairwise co-occurrence using appropriate statistical tests and Jaccard similarity analysis.

Results: Among 200 isolates, 121 (60.5%) were classified as APEC and 79 (39.5%) as NPEC. APEC isolates most frequently carried hlyF (100%), fimC and iutA (96.7% each), ompT and iucD (95.9% each), iss (90.1%), irp2 (79.3%), and iroN (65.3%). APEC isolates contained up to 19 VAGs, whereas NPEC isolates contained up to 14. Twenty-three distinct VAG combination profiles were identified among APEC isolates, and 103/121 (85.1%) harbored >8 VAGs. In contrast, NPEC isolates displayed 13 less complex profiles containing 3-6 VAGs. Most VAGs were significantly more prevalent in APEC than in NPEC isolates, while papC , fyuA , and eaeA occurred exclusively in APEC. Co-occurrence analyses demonstrated greater complexity and stronger VAG associations among APEC isolates.

Conclusion: APEC isolates from northern Vietnam exhibited extensive VAG diversity and complex co-occurrence patterns distinct from those of NPEC isolates. These findings provide a molecular basis for improved APEC characterization, epidemiological surveillance, and targeted control of poultry colibacillosis.