Rapid Diagnostic Assay for Streptococcus equi

Abstract Research Plan In Scientific Terms: Scientific Importance: . Laboratory diagnosis of equine strangles requires culture and PCR of specimens from the nasal cavity and sometimes from the guttural pouch. These procedures are time consuming (2-3 days or longer) and entail delays in decisions concerning isolation and transportation. A rapid test to presumptively identifY infected horses would be very helpful in strangles prevention and management. S. zooepidemicus shares many antigens with S. equi - its clonal descendent. A major hurdle therefore in the design of a test specific for S. equi is the identification of an immunogenic surface exposed component not present on S. zooepidemicus that is expressed abundantly enough to be readily detectable. A secreted product is less attractive because it is likely to be diluted in secretions and/or degraded. The hypothesis that drives this proposal is that S. equi expresses a surface exposed antigen in an amount detectable by specific antibody in a capture assay. Recent studies in our laboratory have identified surface exposed proteins (Se44.2, Se45.5, Se46.8, Se75.3 and Se18.9) in addition to the antiphagocytic SeM that have extensive domains unique to S. equi and which might be useful in its detection. The proposed research will evaluate protein specific rabbit antibodies that have been covalently linked to magnetic beads in a capture assay for S. equi in nasal washes or swabs. Beads with attached S. equi will be separated using a magnet, washed, and mixed with an enzymically labeled (eg. Alkaline phosphatase) affinity purified antibodies to specific proteins or the group C carbohydrate antigen of S. equi followed by a substrate that generates a distinctive color. The specific aims are to: 1. subclone fragments of se42.2, se46.8, se45.5, se75.3, se18.9 and seM encoding peptides specific for S. equi; express and purifY recombinant polypeptides. 2. prepare antibodies to each polypeptide in rabbits; test antibodies for reactivity with a set of S. zooepidemicus and S. equi in absorption experiments 3. covalently link antibodies to magnetic beads and determine relative capture ability of each antibody in dilute suspensions of S. equi and S. zooepidemicus in saline. 4. biotinylate rabbit antibodies to each fusion peptide and to Lancefield group C carbohydrate and test each by ELISA for ability to detect small numbers of S. equi captured on sensitized magnetic beads. 5. evaluate the sensitivity and specificity of the capture assay on a series of nasal washes of known bacteriologic and PCR status from experimental infections and from outbreaks of strangles in Kentucky and elsewhere in the eastern U.S. Although the proposed assay will be based on magnetic beads the results should provide a basis for adaptation to a proprietary lateral flow ELISA format as currently widely used for equine influenza and for a variety of pathogens in dogs and cats and S. pyogenes in humans. Importance to the Equine Industry: The goal of this research is development and preliminary evaluation of a diagnostic test for Streptococcus equi that can be performed in the field or in the veterinarian's office and yield results in less than 2 hours. Tests currently in use require 2 to 3 days before results are available. A rapid diagnostic test would for example allow speedy decisions about shipping, isolation, cessation of shedding and documentation of infection status on US Health Certificates that accompany horses to competitions and/or race tracks. Although strangles was estimated to account for 20% of cases of upper respiratory disease (IURD) in 1998-1999 (Gross et al 2000; AAEP Proceedings Vol. 46) many of these cases were not confirmed by isolation of S. equi. A rapid test performed by the veterinarian would allow more accurate documentation of strangles incidence. Documentation of vaccine efficacy would also be improved. Rapid tests are widely used by physicians for diagnosis of S. pyogenes and by small animal veterinarians for heartworm, Lyme disease, parvovirus, feline FIV and LV etc. These tests are available in lateral flow ELISA formats in disposable plastic holders that incorporate proprietary technologies readily adaptable to nasal washes or swabs from horses. Abstract of Research Plan in Lay Language: Scientific Importance: A rapid test for S. equi will require use of an antibody that does not react with S. zooepidemicus, a close relative of S. equi, but normally present in the equine nasopharynx. Our laboratory has identified surface exposed proteins on S. equi that differ from those on S. zooepidemicus. Antibodies prepared against parts of these proteins unique to S. equi will be attached to magnetic beads and tested for binding of small numbers of S. equi in equine nasopharyngeal washes. Bound S. equi will be detected using an enzymically labeled antibody to one of the proteins or to the carbohydrate cell wall component of S. equi. An enzyme substrate that changes color will serve as an indicator of a positive or negative reaction. The assay will be validated on nasopharyngeal washes and swabs from strangles outbreaks of known S. equi culture and PCR status. Importance to the Equine Industry: Strangles caused by S. equi continues to be highly prevalent in N. America and elsewhere and caused serious disruption to training and movement of horses at racing stables in Kentucky, Florida and the N. Eastern U.S. in spring of2005. Laboratory diagnosis of infection requires culture and PCR of specimens from the nasal cavity and sometimes from the guttural pouch. These procedures are expensive, time-consuming and result in delays in decisions concerning movement and transportation. A test for S. equi, which could rapidly be applied by the practicing veterinarian, would greatly facilitate control and management of strangles. Positive animals could be quickly isolated before their infection was transmitted to other horses. Cessation of shedding could also be determined and discussions regarding shipping and training quickly made.