Honors Theses and Capstones

Date of Award

Spring 2018

Project Type

Senior Honors Thesis

College or School

COLSA

Department

Molecular, Cellular and Biomedical Sciences

Program or Major

Biomedical Science: Medical Microbiology

Degree Name

Bachelor of Science

First Advisor

Brian Stevens

Second Advisor

Christopher Benton

Abstract

The cases of Lyme disease in New Hampshire have increased over time. There are speculations that increasing number of Lyme disease cases in New Hampshire are due to environmental factors, such as warmer climate, white-footed mouse population, white-tailed deer population, opossum population, and forestation coverage. In this study, we processed whole tick samples from 2000, 2001, and 2003 for Borrelia burgdorferi by Real-Time TaqMan PCR. In addition, we also processed homogenized tick samples from 2010, that previously tested positive for B. burgdorferi and had been stored at -80°C since 2010, for repeat B. burgdorferi testing by Real-Time TaqMan PCR. Then, the number of reported positive tick samples from the years 2000, 2001, 2003, 2009, 2010, and 2011 were correlated with white-tailed deer population and Lyme disease cases. Based on our analysis, there was an indirect relationship noted between white-tailed deer population, which is highly suggestive of the relationship between host diversity and Lyme disease cases. On the other hand, the rate of positive tick samples exhibited similar trend as Lyme disease cases. Due to poor staffing and funding issues, the NH Department of Human and Health Services were not able to obtain any tick samples in 2002, from 2004 to 2008, and from 2012 to 2017. We were unable to correlate Lyme disease cases to neither white-footed mouse population nor opossum population, since the NH Fish and Game do not keep track of these two populations. Of the 141 ticks collected in 2000, 2001, and 2003, 44 ticks tested positive for B. burgdorferi. These sample became the oldest, documented tick samples in the state of New Hampshire, which tested positive for B. burgdorferi. In addition, there were insignificant deviations noted between Ct values of the 2010 samples, which were processed in 2010 and 2018. Therefore, the unremarkable difference in Ct values suggest that cryopreservation seems to be the most optimal method of preserving DNA. It was also noted in this study, historic samples had significantly lower DNA concentration than the 2010 samples. We attributed the significant difference to time of storage and method of DNA preservation. We attempted to sequence tick samples for Next Generation Sequencing. DNA of tick samples from 2000, 2001, 2003, and 2010 were quantified in Qubit Fluorometer. However, DNA concentration of individual tick samples were insufficient for prokaryotic enrichment, thus the DNA from positive tick samples in 2010 were pooled together. The pooled DNA was reprecipitated and quantified, but the DNA concentration was still insufficient to proceed with enrichment and sequencing.

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