title: Detection and quantification of Histomonas meleagridis by real-time PCR targeting single copy genes.
abstract: Histomonas meleagridis, a protozoan parasite that can infect gallinaceous birds, affects mainly the liver and caeca of infected birds. As a consequence of the recent ban of chemotherapeuticals in Europe and the USA, histomonosis gained somewhat more attention due to its re-emergence and the fact that there is no effective treatment available. Therefore, special attention is now also given towards the diagnosis and the control of the disease. In the actual study we report the development of highly specific and sensitive real-time PCR methods for detection and quantification of the parasite, based on two protein coding genes, Fe-hydrogenase (FeHYD) and rpb1. Both genes seem to be in a single copy in H. meleagridis as shown by southern blotting and absolute quantification using real-time PCRs on samples containing a known amount of the parasite. The real-time PCR assays based on FeHYD and rpb1 genes were found to be an efficient method for the quantification and detection of H. meleagridis in in vitro grown cultures, tissues of infected birds and in faecal samples. Both real-time PCRs were able to detect up to a single cell in in vitro cultures of H. meleagridis and in fecal samples spiked with H. meleagridis. Finally, qPCR assays were shown to be highly specific for H. meleagridis as samples containing either of the two H. meleagridis genotypes were positive, whereas samples containing other protozoa such as Tetratrichomonas gallinarum, Trichomonas gallinae, Simplicimonas sp., Tritrichomonas sp., Parahistomonas wenrichi, Dientamoebidae sp. and Blastocystis sp. were all negative.
authors: Clinic for Poultry and Fish Medicine, Department for Farm Animals and Veterinary Public Health, University of Veterinary Medicine, Veterinaerplatz 1, 1210 Vienna, Austria.
authors: Imtiaz
authors: I
authors: Hussain
authors: Clinic for Poultry and Fish Medicine, Department for Farm Animals and Veterinary Public Health, University of Veterinary Medicine, Veterinaerplatz 1, 1210 Vienna, Austria.
authors: Barbara
authors: B
authors: Jaskulska
authors: Clinic for Poultry and Fish Medicine, Department for Farm Animals and Veterinary Public Health, University of Veterinary Medicine, Veterinaerplatz 1, 1210 Vienna, Austria; Christian Doppler Laboratory for Innovative Poultry Vaccines, Vienna, Austria.
authors: Michael
authors: M
authors: Hess
authors: Clinic for Poultry and Fish Medicine, Department for Farm Animals and Veterinary Public Health, University of Veterinary Medicine, Veterinaerplatz 1, 1210 Vienna, Austria. Electronic address: ivana.bilic@vetmeduni.ac.at.
authors: Ivana
authors: I
authors: Bilic
mesh_terms: D000818:Animals; D015139:Blotting, Southern; D016054:DNA, Protozoan / Q000235:genetics*; D056890:Eukaryota / Q000235:genetics* / Q000302:isolation & purification*; D011201:Poultry Diseases / Q000175:diagnosis / Q000469:parasitology*; D011528:Protozoan Infections / Q000175:diagnosis / Q000469:parasitology*; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D014422:Turkeys
chemical_list: D016054:DNA, Protozoan
keywords: Fe-hydrogenase; Histomonas meleagridis; Real-time PCR; Southern blotting; rpb1