title: A real-time, quantitative PCR protocol for assessing the relative parasitemia of Leucocytozoon in waterfowl.
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 (R(2)=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.
authors: US Geological Survey, Alaska Science Center, 4210 University Drive, Anchorage, AK 99508, United States; Institute of Arctic Biology, Biology and Wildlife Department, University of Alaska Fairbanks, 211 Irving I, Fairbanks, AK 99775, United States. Electronic address: mmsmith@usgs.gov.
authors: Matthew M
authors: MM
authors: Smith
authors: US Geological Survey, Alaska Science Center, 4210 University Drive, Anchorage, AK 99508, United States. Electronic address: jschmutz@usgs.gov.
authors: Joel
authors: J
authors: Schmutz
authors: Hawaii Cooperative Studies Unit, University of Hawaii at Hilo, 200W. Kãwili St., Hilo, HI 96720, United States. Electronic address: chloeapelgren@gmail.com.
authors: Chloe
authors: C
authors: Apelgren
authors: US Geological Survey, Alaska Science Center, 4210 University Drive, Anchorage, AK 99508, United States. Electronic address: aramey@usgs.gov.
authors: Andrew M
authors: AM
authors: Ramey
mesh_terms: D000413:Alaska; D000818:Animals; D046090:Anseriformes / Q000469:parasitology*; D001717:Birds / Q000469:parasitology*; D004272:DNA, Mitochondrial; D016054:DNA, Protozoan; D016787:Haemosporida / Q000302:isolation & purification*; D008853:Microscopy; D018512:Parasitemia / Q000175:diagnosis / Q000662:veterinary*; D016133:Polymerase Chain Reaction; D011529:Protozoan Infections, Animal / Q000175:diagnosis* / Q000469:parasitology; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*
chemical_list: D004272:DNA, Mitochondrial; D016054:DNA, Protozoan
keywords: Leucocytozoon; Parasitemia; Waterfowl; qPCR