A real-time, quantitative PCR protocol for assessing the relative parasitemia of Leucocytozoon in waterfowl

Journal of Microbiological Methods
By: , and 

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Abstract

Microscopic examination of blood smears can be effective at diagnosing and quantifying hematozoa infections. However, this method requires highly trained observers, is time consuming, and may be inaccurate for detection of infections at low levels of parasitemia. To develop a molecular methodology for identifying and quantifying Leucocytozoon parasite infection in wild waterfowl (Anseriformes), we designed a real-time, quantitative PCR protocol to amplify Leucocytozoon mitochondrial DNA using TaqMan fluorogenic probes and validated our methodology using blood samples collected from waterfowl in interior Alaska during late summer and autumn (n = 105). By comparing our qPCR results to those derived from a widely used nested PCR protocol, we determined that our assay showed high levels of sensitivity (91%) and specificity (100%) in detecting Leucocytozoon DNA from host blood samples. Additionally, results of a linear regression revealed significant correlation between the raw measure of parasitemia produced by our qPCR assay (Ct values) and numbers of parasites observed on blood smears (R2 = 0.694, P = 0.003), indicating that our assay can reliably determine the relative parasitemia levels among samples. This methodology provides a powerful new tool for studies assessing effects of haemosporidian infection in wild avian species.

Additional publication details

Publication type Article
Publication Subtype Journal Article
Title A real-time, quantitative PCR protocol for assessing the relative parasitemia of Leucocytozoon in waterfowl
Series title Journal of Microbiological Methods
DOI 10.1016/j.mimet.2015.01.027
Volume 111
Year Published 2015
Language English
Publisher Elsevier
Publisher location New York, NY
Contributing office(s) Alaska Science Center Biology WTEB
Description 6 p.
First page 72
Last page 77
Online Only (Y/N) N
Additional Online Files (Y/N) N
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