Development of a Prototype Immuno-Lateral Flow Assay for Antigen Detection of Avibacterium paragallinarum Isolates
Open Access
- Author:
- Anil, Gayatri
- Area of Honors:
- Veterinary and Biomedical Sciences
- Degree:
- Bachelor of Science
- Document Type:
- Thesis
- Thesis Supervisors:
- Bhushan M Jayarao, Thesis Supervisor
Robert John Vansaun, Thesis Honors Advisor - Keywords:
- Infectious coryza
Lateral flow assay
Avibacterium paragallinarum - Abstract:
- Infectious coryza (IC) is a highly contagious respiratory disease in chickens caused by the bacterium Avibacterium paragallinarum (formerly known as Haemophilus paragallinarum). Rapid, accurate diagnostic tools are needed to contain infections within and between poultry operations. Immuno-lateral flow assays (ILFA) can yield results in less than 30 min and are suitable diagnostic tools to detect IC. In this study, we identified immunodominant epitopes of A. paragallinarum and tested their suitability to be used in a lateral flow assay. Ninety-nine B-cell epitopes were predicted from sixteen antigens of the bacteria using bioinformatics tools. Out of the ninety-nine epitopes, two epitopes were chosen based on their peptide length, hydrophilicity, membrane location, and conserved nature among Avibacterium paragallinarum genome sequences. These two epitopes, which are located on the filamentous hemagglutinin outer membrane protein gene ibpA (HA) and TonB transporter (TonB) proteins of the bacterium, were synthesized as peptides. Hyperimmune polyclonal serum was generated against these peptides, confirming their immunogenic properties. The antibody concentration and pH conditions were optimized to conjugate the generated hyperimmune serum with colloidal gold. When tested in a prototype ILFA, the conjugated polyclonal antibodies were shown to be reactive against their respective peptides. In addition, the preliminary sandwich ILFA design using TonB conjugate and HA capture antibodies was able to successfully identify A. paragallinarum positive samples directly from chicken nasal swabs. These results suggest that the epitopes chosen on the HA and TonB antigens are suitable targets for detecting A. paragallinarum and can be used to develop an antigen-detection ILFA for on-farm diagnosis of IC.