Examining the Vector–Host–Pathogen Interface With Quantitative Molecular Tools - Bacterial Pathogenesis: Methods and Protocols - page 117

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Examining the Vector-Host-Pathogen Interface
With Quantitative Molecular Tools
Jason E. Comer, Ellen A. Lorange, and B. Joseph Hinnebusch
Summary
We developed PCR assays to detect and quantitate Yersinia pestis , the bacterial agent
of plague, in flea vector and mammalian host tissues. Bacterial numbers in fleas, fleabite
sites, and infected lymph nodes were determined using real-time PCR with primers and
probes for a gene target on a multi-copy plasmid specific to Y. pestis . Tissue-matched
standard curves used to determine absolute bacterial numbers in unknown samples were
linear over at least five orders of magnitude. The methods were applied to studies of
transmission of Y. pestis by the rat flea Xenopsylla cheopis , but should be generally useful
to investigate the transmission dynamics of any arthropod-borne disease.
Key Words: Arthropod-borne disease; quantitative real-time polymerase chain
reaction; vector-borne transmission; vector competence.
1. Introduction
Pathogens transmitted by blood-feeding arthropods cause serious public health
problems worldwide. Despite their importance, many basic epidemiological
parameters of arthropod-borne diseases remain poorly defined because trans-
mission cycle dynamics have been difficult to study. For example, quantitative
estimates of vector competence, such as susceptibility to infection, permissiveness
of pathogen reproduction, extent of dissemination in the arthropod, and compar-
ative transmission efficiencies of different vector species of a particular pathogen
are lacking for many arthropod-borne diseases. Epidemiological modeling of
vector-borne diseases therefore often relies on indirect estimates of these param-
eters (1) . Much also remains to be learned about how specific microbial factors