Improved detection of antibodies to Mycoplasma synoviae vaccine MS-H using an autologous recombinant MSPB enzyme-linked immunosorbent assay

Abstract

Mycoplasma synoviae is a poultry pathogen causing respiratory disease and synovitis. An indirect enzyme-linked immunosorbent assay (ELISA) has previously been devised in our laboratory using the major membrane antigen MSPB of M. synoviae strain WVU 1853 as antigen. However, sera from chickens inoculated with the M. synoviae vaccine strain MS-H showed lower optical densities in the assay than chickens infected with wild-type strains. In the present study, we investigate whether a low level of antibodies detected in MS-H-vaccinated birds is due to the limited ability of the vaccine to elicit antibodies, or to the reduced capacity of the antigen to specifically detect antibodies to this strain. Preliminary immunostaining experiments using native MSPBs from M. synoviae MS-H and WVU 1853 suggested that they were antigenically related but differed in at least some epitopes. Using a combination of polymerase chain reaction (PCR) and cloning, the gene encoding MSPB ( vlhA ) was cloned from strain MS-H, and its nucleotide sequence was partially determined. Analysis of the partial nucleotide sequence of the cloned vlhA gene revealed that it had a high identity (86%) with the previously published vlhA sequence from strain WVU 1853, but differed from it in several regions. Also, several nucleotide substitutions/deletions were detected in the conserved region (nucleotides 1 to 700) of the MS-H vlhA gene. A polypeptide, containing amino acids 27 to 299 of the MS-H MSPB, was expressed as a fusion protein in Escherichia coli and purified by affinity chromatography. An indirect ELISA was developed using the MS-H MSPB as coating antigen and compared with that of WVU 1853 MSPB and the commercial rapid serum agglutination test using a panel of sera from MS-vaccinated and/or challenged or unvaccinated specific pathogen free and commercial field chickens. Analysis of the absorbance values from specific pathogen free and field chicken sera showed that MS-H MSPB was species specific and more sensitive than the WVU-MSPB ELISA or the rapid serum agglutination test in detecting antibodies to the MS-H vaccine strain. These results emphasize the importance of using appropriate diagnostic antigens for sensitive detection of antibodies following vaccination or challenge with a M. synoviae strain.