Pathogenic avian mycoplasmas continue to be potential problems for the commercial poultry industry worldwide. The economic impact of avian mycoplasma diseases stems from increased condemnation and downgrading of carcasses at processing, reduced efficiency of feed and egg production, and increased costs due to prevention and control programs that require surveillance, medication, and vaccination. Mycoplasma gallisepticum (MG) and Mycoplasma synoviae (MS) are the two most economically important avian mycoplasmas. Serological surveillance has been one of the most common and effective methods employed in the mycoplasma prevention and control programs. Due to the cost, sensitivity, and rapidity, the serum plate agglutination (SPA) test has become the most common screening method of choice for the detection of antibodies to MG and MS. However, the problem is that the SPA test lacks specificity and always generates a low percentage of false positives. Furthermore, in recent years, the SPA assay has become more problematic due to the inconsistent availability and quality of plate antigen, in particular for MS. The purpose of this project is to develop an improved screening method for the rapid detection of antibodies to pathogenic avian mycoplasmas using a dual color plate agglutination assay that employs colored microspheres coupled with Mycoplasma spp. specific antigenic peptides as an indicator system. The assay will provide more consistent, reliable, specific, and sensitive mycoplasma plate antigens in a simple and cost-effective dual color format. OBJECTIVES: Our goal is to develop an improved screening method for the rapid detection of antibodies to pathogenic avian mycoplasmas. We will demonstrate detection of Mycoplasma gallisepticum (MG) and Mycoplasma synoviae (MS) specific antibodies in serum samples using the agglutination of colored microspheres coupled with Mycoplasma spp. specific antigenic peptides as an indicator system. This should provide more consistent, reliable, and specific mycoplasma plate antigens in a simple and cost-effective dual color format. To complete the overall goal of the grant, the research plan is broken up into four separate objectives to be accomplished over an 8 month period. Objective 1 is to find intraspecies specific antigenic peptides for MG and MS by screening random phage display peptide libraries. A large number of clones from the phage display libraries will be sequenced and analyzed to find species specific antigenic peptides for MG and MS. Three months are allotted for the completion of this objective. Objective 2 is to provide proof of concept for the dual color plate agglutination assay. Antigenic peptides will be coupled to colored microspheres. Each candidate peptide will be tested with a panel of known MG and MS positive and negative serum samples to evaluate reactivity, specificity, and sensitivity. All peptides specific for each organism that show a high degree of specificity and sensitivity will be combined and coated onto different colored microspheres for each organism and mixed. The mixture will be reevaluated with the panel of known MG and MS positive and negative serum samples to provide proof of concept for the dual color plate agglutination assay. Two months are allotted for the completion of this objective. Objective 3 is the experimental and clinical evaluation of the dual color plate antigen assay. The evaluation will include 200 to 500 serum samples. Direct comparisons of the dual color assay and the current serum plate agglutination (SPA) assay, hemagglutination inhibition (HI) assay, and ELISA assay will be performed. Two months are allotted for the completion of this objective. Objective 4 is the generation of a final technical report that details the research activities of the work. The report will be sent to the granting agency and the data contained within used for the prosecution of patents to protect intellectual property and for publication of appropriate scientific manuscripts. APPROACH: The basic strategy for completing the proposed work is to find intraspecies specific antigenic peptides that can be coupled to microspheres and used in a multiplex serum plate agglutination (SPA) assay for the specific detection of antibodies to Mycoplasma gallisepticum (MG) and Mycoplasma synoviae (MS). In order to find intraspecies specific antigenic peptides that contain relevant epitopes, a phage display method will be used. Basically, several different random bacteriophage libraries will be screened with several different anti-MG and anti-MS serum samples in order to find MG and MS specific antigenic peptides. This method is preferred over the traditional PEPSCAN method because over 2 billion random peptide sequences can be screened with a serum sample for only a fraction of the cost that would be required for synthesizing overlapping peptides for just one protein. Furthermore, due to the antigenic relatedness of MG and MS, the approximately 200-300 coding genes of unknown function within each genome, and the well-known phase variation exhibited by mycoplasmas, the choice of candidate proteins to screen by PEPSCAN is unclear. A method like phage display that allows for screening a large number of peptides at one time without any prior knowledge should allow for a greater chance of isolating conserved intraspecies specific antigenic peptides for each organism. After determining species specific antigenic peptides for MG and MS, corresponding antigenic peptides will be coupled to different colored microspheres for each organism and each used as SPA antigen. This method should be preferred over the current use of whole organism for several reasons. Mycoplasmas are difficult to grow in culture and generally the yield of organisms per batch is low. Also, swine serum is a required component in the mycoplasma growth media and has been implicated as a factor in non-specific SPA reactions. Growth medias using liposomes to replace serum have been studied, but are not commonly used. The antigenic relatedness of MG and MS has also been implicated in non-specific SPA reactions due to the use of whole organism as antigen. Therefore, using peptides bound to microspheres should help to eliminate possible sources of non-specific SPA reactions. Furthermore, it should make the antigen production process simpler, more efficient, and more standardized
