Source: GTCALLISON, LLC submitted to
A DUAL COLOR PLATE AGGLUTINATION TEST FOR RAPID & SPECIFIC DETECTION OF ANTIBODIES AGAINST MYCOPLASMA GALLISEPTICUM & MYCOPLASMA SYNOVIAE
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
TERMINATED
Funding Source
SMALL BUSINESS GRANT
Reporting Frequency
Annual
Accession No.
0210065
Grant No.
2007-33610-17973
Project No.
NCK-2007-00056
Proposal No.
2007-00056
Multistate No.
(N/A)
Program Code
8.3
Project Start Date
Jun 1, 2007
Project End Date
Jan 31, 2012
Grant Year
2007
Project Director
Callison, S. A.
Recipient Organization
GTCALLISON, LLC
(N/A)
MOCKSVILLE,NC 27028
Performing Department
(N/A)
Non Technical Summary
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.
Animal Health Component
100%
Research Effort Categories
Basic
(N/A)
Applied
100%
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
3113210110050%
3113220110050%
Knowledge Area
311 - Animal Diseases;

Subject Of Investigation
3220 - Meat-type chicken, live animal; 3210 - Egg-type chicken, live animal;

Field Of Science
1100 - Bacteriology;
Goals / 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.
Project Methods
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.

Progress 06/01/07 to 01/31/12

Outputs
OUTPUTS: Our goal was to develop an improved screening method for the rapid detection of antibodies to Mycoplasma gallisepticum (MG) and Mycoplasma synoviae (MS) using a Dual Color Latex Agglutination test (DCA). Currently, a commercial latex agglutination test for the detection of antibodies to MG and/or MS does not exist, nor are there any published reports of such tests. The basic strategy for completing the proposed work in Phase I was to find intraspecies specific antigenic peptides that could be coupled to microspheres and used in a multiplex DCA test for the detection of antibodies to MG and MS. In order to find intraspecies specific antigenic peptides that contained relevant epitopes, a phage display method was used as published for Mycoplasma hyopneumoniae (Yang et al., 2005). The effort for Phase I of our research was similar to the Yang paper. Basically, several different random bacteriophage libraries were screened with several different anti-MG and anti-MS serum samples in order to find MG and MS specific antigenic peptides. This method allowed for screening a huge number of peptides at one time without any prior knowledge allowed for a greater chance of isolating conserved intraspecies specific antigenic peptides for each organism. During the Phase I efforts, a total of 419 clones isolated by biopanning from phage display libraries were analyzed by DNA sequencing. From this DNA sequence, the amino acid sequence was deduced and the resultant sequences analyzed. Using basic alignment/cluster analyses, BLAST searches, and an in-house developed proprietary scoring system, several clones from the libraries were identified as potential MG and MS specific sequences. A few potentially MG or MS specific clones were selected for further analysis by ELISA to determine the specificity and reactivity of each clone. Alternatively, GTCAllison also tried a different approach to complete the overall goal of the grant. Instead of binding peptides to microspheres, GTCAllison tried using whole MG and MS organisms for binding to microspheres. GTCAllison has shown proof of concept for binding whole MG and MS organisms to different colored microspheres and producing a viable DCA test. PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: During the Phase I efforts, a total of 419 clones isolated by biopanning from phage display libraries were analyzed by DNA sequencing. From this DNA sequence, the amino acid sequence was deduced and the resultant sequences analyzed. Using basic alignment/cluster analyses, BLAST searches, and an in-house developed proprietary scoring system based on physiochemical similarities of amino acids, several promising clones from the libraries were identified as potential MG and MS specific sequences. An initial round of potentially MG or MS specific clones were selected for further analysis by ELISA to determine the specificity and reactivity of each clone. This method was the simplest method for determining and quantifying the reactivity of each clone. Unfortunately, the ELISA screening method results showed that none of the candidate clones were able to generate enough specific signal to be useful as MG or MS specific. For this reason, GTCAllison decided to try a different approach to complete the overall goal of the grant. Instead of binding peptides to microspheres, GTCAllison switched to using whole MG and MS organisms for binding to microspheres. Using whole mycoplasma organisms offered several key advantages: 1) they were commercially available, 2) they were simple to work with, and 3) they provided a combination of natural epitopes for MG and/or MS specific antibodies to bind, which is critical in producing a commercial test that is not going to miss positives. GTCAllison has shown proof of concept for binding whole MG and MS organisms to different colored microspheres and producing a viable DCA test.

Impacts
GTCAllison learned during the ELISA analysis of potential MG and MS specific peptides that not enough specific signal was generated by the individual peptides for this strategy to lead to a viable DCA test. By switching to the binding of whole MG and MS organisms to microspheres, proof of concept was shown. The switching to whole organisms also has several key advantages: 1) they are commercially available, 2) they are simple to work with, and 3) they provide a combination of natural epitopes for MG and/or MS specific antibodies to bind, which is critical in producing a commercial test that is not going to miss positives. Although proof of concept has been established for the DCA, a technical hurdle was reached. Initial test comparison of the DCA test results with other serological test results using actual field samples showed that the DCA test had a high false positive rate. A certain false positive rate is expected and accepted when doing SPA tests for the detection of MG and MS antibodies. However, the current false positive rate was too high to be accepted and will be a critical problem to overcome before commercialization will be possible. Future efforts will focus on decreasing the false positive rate. With a decrease in the false positive rate, the goal of providing more consistent, reliable, and specific mycoplasma plate antigens in a simple and cost-effective dual color format remains within reach.

Publications

  • No publications reported this period


Progress 06/01/07 to 05/31/08

Outputs
OUTPUTS: The major output from the last year of this research grant has been the activity of conducting and analyzing experiments in the laboratory. Per our approach for accomplishing the overall goal of this grant, GTCAllison has used the technique of phage display to screen peptide libraries for specific Mycoplasma gallisepticum (MG) and Mycoplasma synoviae (MS) peptide sequences that can be used to detect specific Mycoplasma spp. specific antibodies in a simple dual colored plate agglutination assay. In the past year, a total of 384 (192 for each organism) peptide sequences have been isolated from phage display libraries using anti-MG and anti-MS serum samples. All of the peptide sequences have been aligned using amino acid alignment software to group similar sequences and to determine consensus sequences. A scoring matrix based on the physiochemical distances between different amino acids has been used to compare individual sequences to consensus sequences as a means of defining the best candidate peptide sequences to use in the next set of experiments. The next set of experiments will include ELISA based analysis of candidate peptides to determine reactivity/specificity with MG and MS specific antibodies. Candidate peptide sequences that are reactive and specific will then be used as peptides for the coating of colored microspheres and the development/testing of the dual colored plate agglutination assay. The other outputs during this past year were the attendance of the National Poultry Improvement Plan (NPIP) meeting in Portland, Maine and the American Veterinary Medical Association/American Association of Avian Pathologists (AVMA/AAAP) Conference in New Orleans, Louisiana. These conferences have been good for keeping abreast of the current needs, regulations, problems, and trends in the poultry industry for dealing with Mycoplasma spp. pathogens. There has been no dissemination of the work undertaken during this grant due to the fact that GTCAllison intends to patent intellectual property developed during the grant and license out the information/final product to a partner company. PARTICIPANTS: The two participants in this project have been Scott A. Callison, Ph.D., Founder and Manager of GTCAllison, LLC and Professor Stan Kleven, from The University of Georgia, who has been a paid consultant. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
The major outcomes/impact from the past year of the grant has been the change in knowledge for the participant. The general technique used for screening the phage display libraries has involved the development of a novel technique using magnetically coated beads to biopan for peptide sequences of interest. This method is simple and could be used for the biopanning of any phage display library. Future publication of the general method will be considered after steps have been taken to protect any intellectual property. Also, fundamental knowledge concerning novel antigenic MG and MS proteins has been obtained by performing BLAST alignments of isolated peptide sequences with publically available MG and MS genomic data. This knowledge can help in determining surface exposed proteins and the possible antigenic epitopes of such proteins. Furthermore, a scoring matrix based on physiochemical distances between different amino acids has been used to best determine how closely an individual peptide sequence matches a consensus sequence of all similar peptide sequences. This method allows for the selection of the best candidate peptides to move forward with in future experiments. There have been no real changes in action based on any outcomes from the past year. The strategy/method for obtaining the overall goal remains the same. However, after attending the NPIP and AVMA/AAAP conferences, GTCAllison believes that the need and future market for the final product of this grant continues to grow. Based on information learned at these conferences, GTCAllison will be better able to bring this final product to market. As of yet, no change in societal conditions has occurred based on the last year of the grant, but when the grant is completed and a final product produced, it should help to lower the cost of animal health and therefore lower the overall costs of poultry production, which can then be passed on as lower costs to the end consumer.

Publications

  • No publications reported this period