Molecular assay development: detecting pathogenic Rickettsia in the tick vector Amblyomma americanum Open Access

Dugan, Ellen Jennifer (2013)

Permanent URL: https://etd.library.emory.edu/concern/etds/zs25x9131?locale=en
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Abstract

Abstract

Molecular assay development: detecting pathogenic Rickettsia in the tick vector Amblyomma americanum

By Ellen Dugan

Purpose: Rickettsia amblyommii is a common and abundant member of the bacterial community of the tick vector Amblyomma americanum. While R. amblyommii is thought to cause only mild illness, if any, in humans, its presence makes the detection of rare and clinically more severe Rickettsia difficult to detect with available assays. A conventional PCR assay was developed to improve the detection of Rickettsia species of known or potential pathogenicity in the presence of R. amblyommii, in the tick vector Amblyomma americanum.

Methods: Homologous gene sequences from Rickettsia were aligned to detect polymorphisms unique to R. amblyommii. These polymorphisms were used to design 14 primer pairs. One primer pair was chosen for assay creation based on its ability to detect target Rickttsia DNA and to not detect R. amblyommii DNA. Further testing was conducted to validate the assay's sensitivity, specificity, and accuracy. Tests included annealing temperature optimization, determination of the LOD for Rickettsia, and testing of wild-caught tick samples with known and unknown Rickettsia infection.

Results: One hundred percent of 20 target Rickettsia species tested with the assay were detected and 0% of A. americanum DNA with known R. amblyommii infection detected R. amblyommii. Both results demonstrate assay specificity for the target Rickettsia species. Sensitivity testing indicated that R. parkeri could be detected in the presence of tick and R. amblyommii DNA in 92.8% of samples at a dilution as low as 1:6000 from the stock concentration. Rickettsia rhipicephali CWPP and Rickettsia 364D were detected in 100% of wild-caught naturally infected Dermacentor occidentalis ticks, indicating high accuracy of the assay to detect natural Rickettsia infection in ticks.

Conclusions: This simple, easily replicable, inexpensive, and rapid testing method detects Rickettsia other than R. amblyommii in ticks. It eliminates the need for sequencing to differentiate target Rickettsia from R. amblyommii, allowing for resources to be focused on less prevalent and abundant Rickettsia that cause more severe clinical disease.

Table of Contents

Table of Contents

Background......................................................................................................1
Amblyomma americanum………………………………………………….............................…..…........1
Rickettsia species…………………………………………………………….........……...............................2
Rickettsia amblyommii………………….........................…………………………….……..….…....…......2
Rickettsia amblyommii masking effect…………………..….........................…..…………...........3

Rickettsia species identification and detection….......................……..………..…….............4

Relevance to environmental public health…………………………......…................................…5
Methods………………………………....................................………….....…..………...6

Rickettsia samples…………………………………………..….…………..........................….....…....…..…6

Tick samples…………………………………………………..........................…..........………….....……..…7

Primer design and selection…………………………………..….…………….….................................…7

Conventional PCR screening and gel electrophoresis..………………................................….8

CFR performance measures applied to assay design …..……………..…….........................….9
Results……………………………………………………….............................……………12

CFR performance measures applied to assay results………..….........................………..…….12
Discussion…………………………………………….........................……..…..….………14

Discussion of the CFR performance measures applied to the assay ..........................…14
Conclusion……………………………………………………………..........................……16
References………………….........................………………………………………….……17
Tables………………………………………………..........................……….………………24

Figures…………………………………………...……………………………….....................................………25
Appendix…………………………………………………………………..............……..........................………32

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List of Tables

Table 1. Prevalence of R. amblyommii in comparison to the prevalence of

other bacterial species in A. americanum …………………..…………………................….….....….24
Table 2. Nucleotide sequences created as potential candidates for the assay...............24

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List of Figures

Figure 1. Distribution of tick samples across the United States …………….……........…..…..25
Figure 2. Assay sensitivity: Detection of R. parkeri inoculated into wild

A. americanum at the LOD concentration of 1:6000…………………...….............………….…...26

Figure 3. Assay specificity: annealing temperature optimization for

A. americanum DNA…………………………………………………….…………...............................…..…27
Figure 4a. Assay specificity: Spurious amplification of bands in A. americanum

with known R. amblyommii infection ……………………………………......................……….….…...28

Figure 4b. Assay specificity: High resolution testing of spurious bands for

the detection of R. amblyommii……………………………………….…......................…………..…....29
Figure 5. Assay accuracy: detection of 20 target Rickettsia species and the

absence of R. amblyommii and A. americanum detection……………….............………………...31

Figure 6. Assay accuracy: Detection of natural Rickettsia infection in D. occidentalis..…30

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