Whole-cell surface-display system in attenuated Salmonella Enteritidis for slide microagglutination-based antibody...

Microagglutination is a sensitive, rapid, and cost-effective serological assay widely used for disease diagnosis and surveillance in animals and humans. However, it traditionally relies on culturing live pathogenic bacteria as antigens, posing biosafety risks and limiting its application primarily to bacterial pathogens. Bacterial surface display technology offers a promising alternative by enabling the presentation of heterologous proteins or peptides on the bacterial surface, avoiding the need to culture the target pathogen, simplifying antigen preparation, and enabling potential diagnostic applications for non-bacterial pathogens. Despite these advantages, integration of this technology with slide-based microagglutination assays has not yet been evaluated. In this proof-of-concept, we evaluated a surface display platform in attenuated Salmonella Enteritidis for antibody detection by slide microagglutination, using the nucleocapsid (N) protein of Infectious Bronchitis Virus (IBV) as a model antigen.

The IBV N protein was fused to the N-terminal anchor domain of the ice nucleation protein (INP) and cloned into pET-28a. The resulting INP_N fusion protein was expressed in attenuated Salmonella Enteritidis, and its surface exposure was evaluated by immunofluorescence and flow cytometry on non-permeabilized cells, with flow cytometry indicating a surface display efficiency of 96.5%. Slide microagglutination was performed using 50 hen sera and inactivated Salmonella Enteritidis cells presenting surface-exposed N protein. This assay identified all positive sera and 20 of 25 negative sera, showing substantial agreement with the reference ELISA (κ = 0.8), with preliminary estimates of 100% sensitivity (95% CI, 87-100%) and 80% specificity (95% CI, 61-91%). No cross-reactivity was observed with commercial antisera against pathogens unrelated to IBV.

The attenuated Salmonella surface-display platform integrated into a slide microagglutination assay showed preliminary feasibility for antibody detection under the conditions evaluated. This approach does not require live pathogen cultivation, specialized equipment, antigen purification, or chemical conjugation steps. However, further optimization is needed to improve the platform's display efficiency and enhance specificity without compromising sensitivity. In addition, evaluation using a larger and more diverse sample set, including field samples, is required to further assess the performance and potential applicability of the assay.