Development and evaluation of lateral-flow test for rapid detection of Mycoplasma gallisepticum and Mycoplasma synoviae in poultry clinical samples

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Background: The UK chicken poultry industry loses millions of eggs and hundreds of tons of meat due to Mycoplasma gallisepticum (MG) and Mycoplasma synoviae (MS). These pathogens cause respiratory, reproductive and musculoskeletal infections, resulting in illnesses, increased medication costs and drops in egg production. To date, for MG and MS, there are no pen-side rapid diagnostic lateral flow tests available, for either antibody or antigen detection.Aims: Development and evaluation of lateral flow (LF) test for detection of MG and MS antibodies or antigens in poultry clinical samples. Hypotheses and objectives (48 months PhD):Months 1-12: Development of MG/MS lateral flow (LF) antibody (Ab) test. Synthesis, purification and quantification of recombinant protein against cytadhesin proteins of MG (mgc2, GapA and PlpA)(1, 2) and MS (vlhA) (3). This will be achieved by plasmid recombination and expression in E. coli (4). Sonicated MG/MS (5) will also be included. LF Ab test development will include preparation of colloidal gold (CG) nanoparticles, conjugation of the CG with the antisera, nitrocellulose membrane, sample and conjugation pads (6). Month 6-18: Development of MG/MS LF antigen (At) test to identify surface proteins of MG/MS in clinical samples. A panel of MG/MS surface protein monoclonal antibodies (mAb) (above) will be prepared and purified. Subsequently, the mAb biotinylated will use a biotin-spacer and colloidal-gold conjugated (7). LF test strip, as above, will be used and go through optimisation of components involved. Sensitivity and specificity of Ab/At detection will be established.Month 12-24: Optimisation for detection of both MG/MS in single LF strip test. Following successful detection and optimisation of MG and MS in single test (above), the procedures will be modified to test MG/MS antibodies or antigens in a single strip. Month 25-30: In vivo experimental infection of MG/MS: SPF birds will be inoculated via oculonasal (8). Blood and oropharyngeal-choanal cleft swabs will be collected weekly up to 6 weeks post challenge. Month 31-36: Internal validation of LF using samples (above) from experimental chickens. The blood/sera and swabs (above) will be used in the LF Ab and At test respectively. Test devices will be optimised. Month 37-42: Validation of the LF using clinical samples. Samples from Aviagen flocks tested in the farms and in their laboratory. Results cross-compared against commercial MG/MS ELISAs and PCRs. Month 42-48: Completion of work and thesis writing. Complete laboratory work and data analysis, also writing thesis. Last 3 months: thesis submission, viva and publications.