Infectious Bronchitis Virus (IBV) is a major threat to the poultry industry worldwide, causing significant economic losses. While the virus’s genetic structure is well understood, the specific strains circulating in Bolivia have remained uncharacterized until now. This study aimed to identify and characterize new IBV strains in Bolivia. Tissue samples from broilers exhibiting clinical signs of Infectious Bronchitis were screened to detect IBV using real-time RT-PCR (RT-qPCR). Positive samples with low cycle threshold (Ct) values were selected for sequencing the full S1 gene. Of the 12 samples analyzed, 10 were determined to be positive for IBV. However, only four samples yielded sufficient genetic material for sequencing and subsequent phylogenetic analysis. The results revealed the presence of GI-1 and GI-23 lineages, both belonging to genotype I (GI). The GI-1 lineage showed >99% sequence identity to the H120 and Massachusetts vaccine strains, suggesting a close relationship. In contrast, the GI-23 lineage clustered with other IBV strains, showing a distinct subclade that is genetically distant from Brazilian strains. No evidence of recombination was found. Furthermore, amino acid substitution analysis identified specific mutations in the S1 subunit, particularly in the hypervariable regions 1, 2, and 3. These mutations could potentially alter the virus’s antigenicity, leading to reduced vaccine efficacy. The findings of this study highlight the importance of continued and broad genomic surveillance of circulating IBV strains and the need to improve vaccination strategies in Bolivia.

Background: 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. Results: 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. Conclusions: 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.