Development and Validation of an In-House Indirect ELISA for Serological Detection of Gallibacterium anatis in Laying Hens Using Local Field Isolates.

Gallibacterium anatis is an emerging opportunistic pathogen associated with respiratory and reproductive tract infections in poultry, causing reduced egg production and economic losses in laying hens. Serological detection is important for surveillance; however, no validated in-house enzyme-linked immunosorbent assay (ELISA) based on local Indonesian isolates is available. This study aimed to develop and validate an indirect ELISA for detecting antibodies against G. anatis in laying hens. Five archived isolates were characterized by PCR targeting gyrB, gtxA, and flfA virulence-associated genes, followed by growth rate and biofilm formation analyses. Isolate B116, which showed the highest growth rate and strongest biofilm-forming ability, was selected for antigen preparation. Four antigen extraction methods, including heat treatment at 56°C and 96°C, capsular extraction, and sonication, were evaluated to determine the best coating antigen. Checkerboard titration was performed to optimize antigen concentration, serum dilution, and HRP-conjugated rabbit anti-chicken IgG dilution. Cross-reactivity was assessed using positive sera against Pasteurella multocida, Avibacterium paragallinarum, Escherichia coli, Salmonella sp., and Clostridium perfringens. The optimal ELISA conditions were 7.11 μg/mL coating antigen concentration, 1:200 serum dilution, and 1:8000 conjugate dilution. The cutoff values were 0.156 and 0.189 using mean ± 2SD and mean ± 3SD. Compared with PCR, the in-house ELISA showed 85.0% sensitivity and 94.7% specificity. Field serum testing revealed the highest seropositivity in laying hens aged 29-48 weeks. The developed in-house ELISA demonstrated good diagnostic performance and may serve as a practical tool for large-scale serological surveillance and epidemiological monitoring of G. anatis infection in poultry farms.