Date of Award

2011

Degree Type

Thesis

Degree Name

Master of Science in Biomedical Sciences

First Advisor

C Scott Little, PhD

Second Advisor

Denah M Appelt, PhD

Third Advisor

Brian J Balin, PhD

Fourth Advisor

Marina D'Angelo, PhD

Fifth Advisor

Marcus Bell, PhD, Program Director, Graduate Program in Biomedical Sciences

Abstract

Exogenous bacteria, such as Chlamydia pneumoniae, may be a cause of inflammation that contributes to the progression of Alzheimer's disease (AD). Inflammation has been previously established as a contributor to AD progression. Balin et al. first reported C. pneumoniae in the brains of post-mortem late onset AD patients. PCR analysis showed that 17/19 AD patients were positive for the organism in areas of the brain with typical AD-related neuropathology while 18/19 control patients were PCR-negative. Gerard et al. determined, using real-time PCR, that C. pneumoniae-infected cells colocalize with both neuritic senile plaques and neurofibrillary tangles, the defining pathologies of AD.

Several research groups have independently replicated detection of C. pneumoniae in AD brains, whereas a number of research groups did not detect the organism. The variability in detection results demonstrates the need for a standardized technique to produce accurate and dependable results amongst research groups.

The goal of this work is to optimize a sensitive and specific technique to detect C. pneumoniae in experimentally infected olfactory bulb (OB) and frontal cortex (FC) tissue from mice after 3 days and 14 days following direct intracranial infection with 1Xl03 infectious forming units (IFU) using a PrimerDesign™ genesig 16S ribosomal RNA sequence RT-PCR advanced Chlamydia detection kit.

Chlamydia pneumoniae was successfully detected in 2X1 06 monocytic cell line THP-1 infected with C. pneumoniae for 48 hours at a multiplicity of infection (MOl) of 0.75. The lowest amount detected was at a dilution of 1:500,000 (0.04ng total DNA), which was approximately 3 infected cells of the 2X 1 06 total. Experimental samples were considered positive if a sample amplified a Chlamydia-specific 16S RNA sequence in at least 1 reaction, crossing the threshold (CT) at less than 40 cycles, which was based on a dilution series of positive control infected THP-1 cells.

C. pneumoniae was also detected in CNS tissue following inoculation at both day 3 and day 14 samples using RT -PCR. Total DNA content of 400 and 800ng/reaction were not sufficient amounts to accurately and consistently detect C. pneumoniae. However, when using a total DNA content of 1500ng/reaction, C. pneumoniae was detectable when present in both day 3 and day 14 samples.

Trends in the data show that by increasing the total DNA content, samples that were previously inconsistently positive, or even negative, now showed presence of Chlamydia-specific sequence amplification. In 24 samples tested at 400ng/reaction, 9 were detected and only 3 of those 9 were considered positive. Conversely, at 1500ng/reaction, 34 of 44 samples amplified Chlamydia-specific 16S RNA, 9 of which were considered positive.

A total of 16 experimental tissue samples were analyzed, 8 at day 3 and 8 at day 14-post infection. Samples 005 and 053 were injected with vehicle only and served as negative controls. Day 3 samples considered positive were FC-006, 007, and 008 as well as OB-007 and 008. Positive day 14 samples were FC-056 and OB-055 and 056.

Assigning undetected samples a CT value of 50, the last cycle for the reaction, the average CT value at 400ng/reaction is 47.4 and at 800ng/reaction it is 47.6. Raising the content to 1500ng/reaction gives a mean CT value of 44.4. Furthermore, 30 of the 48 samples were undetected at both 400 and 800ng/reaction while at 1500ng/reaction only 10 of the 44 reactions were undetected.

These results suggest that a PrimerDesign™ genesig 16s ribosomal RNA sequence R T-PCR advanced Chlamydia detection kit is suitable for detection of C. pneumoniae in experimental samples. We were also able to determine that 1500ng/reaction is a suitable minimum for total DNA content as we were able to produce consistent results using 1500ng/reaction. One future consideration would be to increase the total DNA content to 2000-2500ng/reaction. Doing so may result in increased sensitivity and lower, more consistent, CT values that parallel the infeceted THP-1 positive control.