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

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 (LSUrRNA) 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. 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 ParasitWirts-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 Communicated by M. Wink. Electronic supplementary material The online version of this article (

Estimating prevalence of avian haemosporidians in natural populations: a comparative study on screening protocols

“…Our comparisons to evaluate the accuracy and sensitivity across three detection methods (restriction enzyme-based PCR, nested PCR and qPCR) with varying intensity for avian haemosporidians detected in samples from India and Sweden revealed qPCR as the most accurate and sensitive method especially in the case of submicroscopic (low intensity) infections, which is concordant with previous studies conducted on avian haemosporidians [35, 51] as well as human Plasmodium [31, 32]. However, in the Swedish data set, the sensitivity of the nested PCR did not differ from the qPCR method.…”

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

“…Several studies that have produced similar real-time, quantitative PCR protocols targeting Haemoproteus and Plasmodium species in avian hosts have extrapolated their resulting Ct values into estimated parasite gene copy number, percent parasitemia, or even the estimated number of parasites per 100 erythrocytes (Bentz et al, 2006;Cellier-Holzem et al, 2010;Friedl and Groscurth, 2011). Since the goal for this study was to provide an optimized protocol for assessing the relative parasitemia for a given sample set, we chose to report our results as raw Ct values rather than transform them into other derived estimates.…”

“…Efficiency above or below this range could indicate problematic amplification of target regions (Reynisson et al, 2006); 2) R 2 values for the slope of the standard curve were N0.99; a lower value could indicate inefficient amplification of the standards (Friedl and Groscurth, 2011); 3) the standard deviation among all three replicates of each sample was less than 1.0. If samples showed a standard deviation greater than 1.0, they were re-run on separate reaction plates to determine if the original high variation was due to laboratory error (Friedl and Groscurth, 2011). If the second run of the samples also produced standard deviations greater than 1, those samples were removed from analysis.…”

“…These quantitative methods are based on the ability to monitor the amplification of a specific target sequence in real time, through the use of fluorescent probes or DNA-binding dyes (Bell and RanfordCartwright, 2002). Several studies have recently developed qPCR protocols for determining levels of infection by Haemoproteus and Plasmodium species in passerine birds (Cellier-Holzem et al, 2010;Knowles et al, 2010a;Friedl and Groscurth, 2011), but an optimized and validated qPCR methodology specific to Leucocytozoon parasites has not yet been reported.…”

A new real-time PCR protocol for detection of avian haemosporidians

“…Although real-time PCR has been used for avian haemosporidians, it has generally been used to determine level of parasitemia [ 47 – 50 ] or for detecting specific lineages [ 22 , 51 – 53 ]. The usefulness of real-time PCR as a large scale screening tool for haemosporidian DNA in avian blood samples has been only minimally explored [ 54 ] and never done for all three genera with a single reaction. Here we report the development of a real-time PCR protocol that can identify infections of any of three haemosporidian genera in a single screening reaction using a 182 bp fragment of the conserved RNA region of the mitochondrial genome.…”