An engineered Escherichia coli-ClyA-NetB outer membrane vesicle delivery system modulates jejunal microbiota in broiler chickens with necrotic enteritis

Necrotic enteritis ( NE ), caused by Clostridium perfringens ( CP ), remains one of the most economically consequential enteric diseases in broiler production, and currently available preventive strategies provide incomplete protection. Given the central role of necrotic enteritis B-like toxin ( NetB ) in NE pathogenesis, this study evaluated two NetB-targeted candidate prophylactic approaches and their effects on host immune responses and intestinal microbial dynamics. Sixty one-day-old Cobb broiler chickens were randomly assigned to four groups: an NE-positive control, an engineered Escherichia coli JC8031-treated group, a recombinant NetB-vaccinated group, and a non-challenged control. All groups except the non-challenged control were subjected to the same NE challenge conditions. NE was induced using an Eimeria -CP coinfection model combined with a high-protein diet. Challenged chickens received a 10-fold dose of COCCIVAC®-B on day 10, were fed a high-protein diet from day 15 to day 19 and were orally gavaged with the netB -positive CP54 strain from day 15 to day 18. Engineered Escherichia coli JC8031 expressing ClyA-NetB outer membrane vesicles ( OMVs ) was administered in drinking water from day 1 to day 18, whereas recombinant NetB protein was administered subcutaneously on days 1, 6, and 11. NE lesion scores, serum NetB-specific antibody responses, CP abundance, and jejunal microbiota composition were evaluated using lesion scoring, enzyme-linked immunosorbent assay, and full-length 16S rRNA gene sequencing. Both NetB-targeted approaches significantly increased serum NetB-specific antibody levels (P < 0.05), with the strongest humoral response observed after recombinant NetB immunization. However, these antibody responses did not result in significant reductions in intestinal lesion severity, NE incidence, or mortality under the present challenge conditions. Microbiota analysis showed that recombinant NetB immunization did not alter jejunal microbial composition following NE challenge. In contrast, continuous oral administration of engineered E. coli JC8031 was associated with reduced CP abundance and a shift in jejunal microbial composition toward a profile more similar to that of the non-challenged control. This pattern was supported by α-diversity indices, including Shannon diversity and Margalef richness, as well as by the relative abundance of Enterococcus faecium , which differed significantly from the NE-positive control and recombinant NetB groups but not from the non-challenged control. A relatively higher representation of Enterococcus taxa and Pediococcus acidilactici were also observed in the engineered E. coli JC8031 group, suggesting potential attenuation of CP-associated dysbiosis. Collectively, these findings indicate that induction of NetB-specific antibodies alone may be insufficient to mitigate NE outcomes under the present NE challenge conditions. Oral delivery of engineered E. coli -ClyA-NetB OMVs may provide microbiota-modulating benefits; however, further optimization is required to determine whether these effects can be translated into measurable clinical protection against NE.