Graduation Year

2023

Document Type

Thesis

Degree

M.S.P.H.

Degree Name

MS in Public Health (M.S.P.H.)

Degree Granting Department

Public Health

Major Professor

Lynn B. Martin, Ph.D.

Committee Member

Mark Marg, Ph.D.

Committee Member

Ryan McMinds, Ph.D.

Keywords

Peromyscus leucopus, Resistance, Tolerance, Zoonotic disease

Abstract

Zoonotic diseases account for a significant proportion of emerging infectious diseases (EIDs), with their dynamics being heavily influenced by the environment. While the effects of the environment on population-level disease dynamics have been extensively studied, there is limited research investigating the impact of the environment on individual-level disease susceptibility and transmission. This study aims to fill this gap by creating a tool to examine the variation in immune response to Borrelia burgdorferi, the causative agent of Lyme Disease, in two competent host species, Peromyscus leucopus and Peromyscus maniculatus. Two triplex droplet digital polymerase chain reaction (ddPCR) assays were developed to simultaneously measure six immune genes: interferon gamma (IFNγ), interleukin 10 (IL-10), interleukin 6 (IL-06), Toll-like receptor 2 (TLR2), transforming growth factor beta (TGF-β) and GATA Binding Protein 3 (GATA3). Each assay used two types of probe fluorophores (FAM and HEX) which allowed for clear, simultaneous detection. For optimization, I utilized synthetic DNA oligos as a positive control for each target and found that each triplex shows adequate specificity. I found that the amount of cDNA would properly scale with the amount of expression for each gene (corr >0.90). Next, I tested for the optimal RNA input that minimized intra- and interpersonal variation and found that 150 ng/μL produced the smallest coefficients of variability. Altogether, these results suggest that the two triplexes are a suitable method for testing the variation of immune gene expression.

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