Competitive, quantitative PCR analysis of Haemobartonella felis in the blood of experimentally infected cats

The DNA from the organism, Haemobartonella felis, was extracted from the leukocyte-poor peripheral blood of foul experimentally infected cats. Since these organisms are currently unculturable, a competitive, quantitative PCR method developed by Zachar et al. [Nucleic Acid Res. 21 (1993) 2017-2018] was used to estimate the numbers of H. felis organisms in the blood of these cats. This estimation was based on the assumption that there is only one copy of the 16S rRNA gene in the genome of H. Sells. It was also based on the efficiency of the DNA extraction, lysing efficiency, as well as the difference in amplification rates between a cloned version of the 16S rRNA gene and genomic DNA from the organism. The number of organisms in the peripheral blood of the cats at peak bacteremia was estimated to be between 3.0 10(5) and 1.1 . 10(8) per microliter of blood. Using this method, the sensitivity of the PCR was determined by estimating the lowest limits of detection. It was determined that as few as 52 organisms were detectable by PCR. (C) 1999 Elsevier Science B.V. All rights reserved.