Development of a Quantitative Polymerase Chain Reaction Procedure for Analysis of Cytokine Responses to Strongylus Vulgaris in Ponies.
Date of Award
Doctor of Philosophy (PhD)
Biomedical and Veterinary Medical Sciences - Comparative Biomedical Sciences
David W. Horohov
The effect of oral vaccination with radiation-attenuated Strongylus vulgaris larvae on the cytokine responses of ponies to oral challenge with S. vulgaris was examined. Message was quantified using RT-PCR by interpolation against a plasmid-derived standard curve. PCR product was detected using the Perkin-Elmer QPCR System 5000. Standard curves were analyzed with a two parameter exponential rise to maximum fit of the data. By altering the cycling parameters to maintain $\beta$-actin amplification within its exponential phase, $\beta$-actin was used as a normalization factor. The assay resolves 2 fold changes in message, has a linear dynamic range on the order of 3 logs, and is precise with errors in the range of 10% for replicate amplifications from the same sample. The error between replicate RNA samples from separate extractions of the same cell population, following $\beta$-actin normalization, was 13-27%. Equine Interleukin (IL)6, IL10, and $\beta$-actin were cloned and sequenced for use in this assay. 10 ponies were reared parasite-free. Four received 500 attenuated S. vulgaris L$\sb3$ 9 and 6 weeks prior to challenge with 1000 nonirradiated L$\sb3.$ Four nonvaccinated ponies were also challenged. Control ponies were neither vaccinated nor challenged. Four days prior to challenge, and on days 4, 9, and 14 post-challenge, peripheral blood mononuclear cells (PBMC) and colonic lymph nodes (CLN) were collected. Prior to necropsy on D14, CLN were obtained surgically. PBMC and CLN cells (CLNC) were assayed for IL2, IL4, IL5, IL10, and IFN$\gamma.$ Vaccinates and nonvaccinates demonstrated TH2-like cytokine patterns with significant elevations in IL4 and IL5 in response to S. vulgaris challenge. Vaccinates produced more IL4 and IL5 than nonvaccinates prior to challenge (not statistically significant), and significantly more IL4 in response to challenge, first in CLNC at D4 and then PBMC at D9. IL4 in CLNC of nonvaccinates increased significantly by D9. Thus, ponies appear to generate TH2-like responses to gastrointestinal nematodes and anamnestic IL4 production may be one component of the protective immune response generated by vaccination. Strongylus vulgaris-specific antibody isotypes and lymphoproliferative responses were also examined.
Swiderski, Cyprianna Ellen, "Development of a Quantitative Polymerase Chain Reaction Procedure for Analysis of Cytokine Responses to Strongylus Vulgaris in Ponies." (1998). LSU Historical Dissertations and Theses. 6640.