Honors College
 

Document Type

Honors Thesis

Publication Date

Spring 2015

Abstract

Equine Strangles, an upper respiratory disease caused by Streptococcus equi subspecies equi (S. equi) causes significant losses in the horse industry. Surveillance for S. equi could be facilitated by quantifying S. equi in environmental samples. The objective of this project was to evaluate ELISA in quantifying S. equi using two monoclonal antibodies (MAB’s) to the SeM protein (MAB-212 for capture, and biotinylated MAB-211 for detection), with Streptococcal phage lysin releasing SeM from the cell surface. Initial results confirmed a fresh culture of S. equi and two S. equi lysates stored at -20 C for 2 years as positive, while confirming as negative a stored lysate of Streptococcus equi subspecies zooepidemicus. A standard curve was created using 12 serial dilutions at 1:10 of S. equi and colony counts determined for each of the 12 dilutions. Eight serial dilutions at 1:2 of these 12 dilutions then yielded one dilution for each well in a 96 well plate. Regression of absorbance against colony counts showed that ELISA was successful in quantifying between 78,125 CFU/mL and 15,000 CFU/mL of S. equi, with an R2 of 0.93. Water was then seeded with S. equi, and 3 swab samples taken, dispersed in PBS, and the 3 suspensions serially diluted 1:10 to provide 4 dilutions (12 total). Colony counts were determined by plating 100 μL of each dilution. All dilutions tested positive for S. equi, but there was no correlation between absorbance and colony counts (R2= 0.0028), possibly due to plate contamination.

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