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Journal of Ornithology

, Volume 153, Issue 1, pp 239–247 | Cite as

A real-time PCR protocol for simple and fast quantification of blood parasite infections in evolutionary and ecological studies and some data on intensities of blood parasite infections in a subtropical weaverbird

  • Thomas W. P. FriedlEmail author
  • Elisabeth Groscurth
Technical Notes

Abstract

In several fields of research, such as immunoecology, evolutionary ecology, sexual selection, parasitology or host-parasite coevolution, a reliable quantitative assessment of blood parasite infections is necessary for testing specific predictions regarding relationships between the degree of infections and various parameters of interest. Here, we present a relatively simple, fast and reliable protocol based on quantitative real-time PCR to determine the intensity of infections with blood parasites of the genus Plasmodium and/or Haemoproteus in blood samples of birds, using male Red Bishops (Euplectes orix; Ploceidae, Passeriformes) as example. The intensity of infections is assessed by amplification of a specific 85-bp fragment within the plastid-like large subunit ribosomal-RNA (LSU-rRNA) gene, which is conservative across a range of Plasmodium and Haemoproteus species. By measuring the accumulation of the product during the PCR (in real-time) using a fluorescent labelled oligonucleotide probe, a threshold can be determined at which the fluorescence of the product raises above background level. The starting quantity of blood parasites in the investigated blood samples is then calculated by comparison with thresholds determined for standards of known quantity (clones of a 594-bp fragment within the LSU-rRNA gene from Plasmodium falciparum including the target sequence) in the same PCR reaction. With this method, blood parasites were detected in 123 out of 127 samples from male Red Bishops, with a median of 0.059 blood parasites per 100 blood cells (range 0–19.2 blood parasites per 100 blood cells). The method described here produces consistent and reproducible data, can easily be modified and extended to detect and quantify blood parasites at different systematic levels, and thus has broad application to many researchers in the field of evolutionary and behavioral ecology.

Keywords

Avian haemosporidians Haemoproteus Intensity of infections Plasmodium Quantitative real-time PCR 

Zusammenfassung

Ein Real-Time PCR Protokoll zur einfachen und schnellen Quantifizierung von Blutparasitenbefall in evolutionsbiologischen und ökologischen Studien, und einige Daten zu Intensitäten von Blutparasitenbefall bei subtropischen Webervögeln

In vielen Forschungsfeldern wie Immunoökologie, Evolutionsökologie, sexuelle Selektion, Parasitologie und Parasit-Wirts-Koevolution ist eine verlässliche Quantifizierung von Blutparasitenbefall notwendig, um spezifische Vorhersagen zum Zusammenhang zwischen der Intensität von Blutparasitenbefall und verschiedenen Parametern von Interesse zu testen. Hier stellen wir am Beispiel von Männchen des Oryxwebers (Euplectes orix; Ploceidae, Passeriformes) ein relativ einfaches, schnelles und verlässliches, auf quantitativer Real-Time PCR beruhendes Protokoll vor, mit dem die Intensität des Befalls mit Blutparasiten der Gattungen Plasmodium und/oder Haemoproteus bestimmt werden kann. Die Intensität des Befalls wird mittels Amplifikation eines 85 bp Fragments des Gens der ribosomalen RNA aus der großen Untereinheit des Ribosoms (LSU-rRNA) ermittelt, welches spezifisch für die beiden Gattungen Plasmodium und Haemoproteus ist. Durch die Messung der Zunahme des PCR-Produkts mittels einer mit einem Fluoreszenzfarbstoff markierten Oligonukleotidsonde in Echtzeit kann eine Schwelle bestimmt werden, bei der die durch die Produktamplifikation bedingte Fluoreszenz über die Hintergrundfluoreszenz ansteigt. Die Menge an Blutparasiten in den untersuchten Blutproben wird dann durch den Vergleich mit in derselben PCR-Reaktion ermittelten Schwellenwerten von Standards mit bekannter Menge an Blutparasiten-DNA (Klone eines 594 bp Fragments des LSU-rRNA Gens von Plasmodium falciparum, welches die Zielsequenz beinhaltet) bestimmt. Mit dieser Methode wurden Blutparasiten in 123 von 127 Blutproben von Oryxwebermännchen mit einem Median von 0.059 Blutparasiten pro 100 Blutzellen (Bereich 0 bis 19.2 Blutparasiten pro 100 Blutzellen) detektiert. Die hier beschriebene Methode ergibt konsistente und reproduzierbare Daten, kann leicht für die Detektion und Quantifizierung von Blutparasiten verschiedener taxonomischer Ebenen modifiziert und erweitert werden, und ist demzufolge von großem Interesse für viele Forscher auf den Gebieten Evolutionsbiologie und Verhaltensökologie.

Notes

Acknowledgments

We are grateful to the National Parks Board of South Africa and the Chief Directorate Environmental Affairs of the Department of Economic Affairs, Environment and Tourism of the Eastern Cape Province for permission to conduct this study in the Addo Elephant National Park. We would like to thank the whole park staff of the Addo Elephant National Park, in particular John and Melanie Adendorff, for continuous support, Nicole Geberzahn and Lars Holst Hansen for their help in collecting Red Bishop blood samples in the field, Armin Michel for advice regarding primer and probe design, and Thomas Jacobs from the Bernhard-Nocht-Institut für Tropenmedizin for providing DNA samples of Plasmodium falciparum. An anonymous reviewer provided useful comments on an earlier version of this manuscript. This study was funded by the Deutsche Forschungsgemeinschaft (DFG Kl 13-1,2 and FR 2096/2-2). The experiments and procedures conducted here comply with German and South African law.

Supplementary material

10336_2011_735_MOESM1_ESM.doc (104 kb)
Supplementary material 1 (DOC 139 kb)

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Copyright information

© Dt. Ornithologen-Gesellschaft e.V. 2011

Authors and Affiliations

  1. 1.AG Zoophysiologie und Verhalten, Institut für Biologie und UmweltwissenschaftenCarl von Ossietzky Universität OldenburgOldenburgGermany

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