The objectives of this project are to use molecular and classical approaches to investigate pathogen-arthropod interactions of vector-borne agents causing diseases of human importance in the United States. Most of our effort has concentrated on Yersinia pestis, the causative agent of bubonic plague, and Borrelia hermsii, an agent of tick-borne relapsing fever. Previously we described the use of the polymerase chain reaction (PCR) to rapidly detect and identify Y. pestis in fleas. This will assist in field surveillance when determining the risk of transmission of Y. pestis by fleas to humans in areas where plague is occurring. During the last year considerable effort has been directed towards the development of a quantitative, competitive PCR that will determine the number of Y. pestis cells in individual infected fleas. This assay is a prerequisite to our studies examining the blocking phenomenon in fleas of both the wild-type Y. pestis and various isogenic mutants. The quantitative, competitive PCR, or QPCR, involves primers that will amplify two target sequences of DNA during the same reaction, the wild-type target and the same region that has been shortened by an internal deletion. Both targets are amplified during the same PCR reaction, resulting in two amplification products, the larger product amplified from the wild-type sequence, and a smaller product from the deleted form of the same sequence. In the QPCR, the amount of the two amplification products will vary depending on the amount of the two target sequences available. For our QPCR to estimate the number of Y. pestis cells in individual fleas, we have chosen the fur (ferric iron uptake regulation) gene as the target. The amplification product of the wild-type sequence is 328 base pairs. A 65- base pair fragment was deleted from a recombinant clone containing the fur gene (original clone provided by Robert Perry, Univ. of Kentucky) and this DNA provided the competitive target sequence added to the QPCR. Therefore both a 328-base pair (wild type) and a 263-base pair (deletion mutant) amplification product should result when both target sequences are available in the reaction using the one set of primers. With a constant amount of the competitor added to a series of different concentrations of the wild-type DNA, a standard curve was generated using scanning laser densitometry of the film negative of the agarose gel containing both amplification products. Using this assay with Oriental rat fleas that were experimentally infected with Y. pestis in the laboratory, we were able to detect down to 100 bacteria and distinguish levels of infection in single fleas. This will allow us to determine the magnitude of infection in fleas that are infected with mutant Y. pestis.
