A New Nested Polymerase Chain Reaction Method Very Efficient in Detecting Plasmodium and Haemoproteus Infections From Avian Blood

Department of Animal Ecology, Lund University, Lund, Skåne, Sweden
Journal of Parasitology (Impact Factor: 1.23). 03/2004; 90(1):191-4. DOI: 10.1645/GE-3221RN
Source: PubMed


Recently, several polymerase chain reaction (PCR)-based methods for detection and genetic identification of haemosporidian parasites in avian blood have been developed. Most of these have considerably higher sensitivity compared with traditional microscope-based examinations of blood smears. These new methods have already had a strong impact on several aspects of research on avian blood parasites. In this study, we present a new nested PCR approach, building on a previously published PCR method, which has significantly improved performance. We compare the new method with some existing assays and show, by sequence-based data, that the higher detection rate is mainly due to superior detection of Plasmodium spp. infections, which often are of low intensity and, therefore, hard to detect with other methods.

Download full-text


Available from: Jonas Waldenström, Jul 04, 2014
    • "In order to determine the prevalence and strain of infection, genomic DNA was extracted and diluted to 25 ng μl −1 for use in a polymerase chain reaction (PCR). We used a two-step nested PCR approach as described inWaldenström et al. (2004), to amplify the cytochrome b gene of avian malaria parasites in the genera Plasmodium and Haemoproteus. For the first PCR we used the primers HAEMNF (5′- CATATATTAAGAGAATTATGGAG-3′) and HAEMNR2 (5′- AGAGGTGTAGCATATCTATCTAC-3′) of which final product 1 μl was taken as a template in a second PCR with the primers HAEMF (5′-ATGGTGCTTTCGATATATGCATG-3′) and HAEMR2 "

    No preview · Article · Jan 2016 · Journal of Avian Biology
  • Source
    • "The extracted DNA samples were then used for subsequent nested PCR reactions. For the mosquito samples, we used a nested PCR to identify Plasmodium/Haemoproteus infections (Waldenström et al. 2009). We did not test for leucocytozoon infections in mosquito samples. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Apicomplexan blood parasites Plasmodium and Haemoproteus (together termed “Avian malaria”) and Leucocytozoon are widespread, diverse vector-transmitted blood parasites of birds, and conditions associated with colonial nesting in herons (Ardeidae) and other waterbirds appear perfect for their transmission. Despite studies in other locations reporting high prevalence of parasites in juvenile herons, juvenile Little Egrets (Egretta garzetta) previously tested in the Camargue, Southern France, had a total absence of malaria parasites. This study tested the hypotheses that this absence was due to insufficient sensitivity of the tests of infection; an absence of infective vectors; or testing birds too early in their lives. Blood was sampled from juveniles of four species shortly before fledging: Little Egret (n = 40), Cattle Egret (Bubulcus ibis; n = 40), Black-crowned Night-Heron (Nycticorax nycticorax, n = 40), and Squacco Heron (Ardeola ralloides; n = 40). Sensitive nested-Polymerase Chain Reaction was used to test for the presence of parasites in both birds and host-seeking female mosquitoes captured around the colonies. No malaria infection was found of in any of the heron species. Four different lineages of Plasmodium were detected in pooled samples of female Culex pipiens mosquitoes, including two in potentially infective mosquitoes. These results confirm that the absence of malaria parasites previously demonstrated in Little Egret is not due to methodological limitations. Although the prevalence of infection in mosquitoes was low, conditions within the colonies were suitable for transmission of Plasmodium. These colonial heron species may have evolved strategies for resisting malaria infection through physiological or behavioral mechanisms.
    Full-text · Article · Dec 2015 · Waterbirds
    • "Additionally, DNA was extracted from blood samples using a standard ammonium acetate method. The PCR-based method for detection of haemosporidians (Waldenström et al. 2004) was used for confirming the absence or presence of Plasmodium and Haemoproteus infections, without identifying genetic lineages via sequencing of the obtained PCR products. All samples identified by microscopy as negative were confirmed by PCR-based diagnostics . "
    [Show abstract] [Hide abstract]
    ABSTRACT: Haemosporidians (protozoan blood parasites) are known to modify avian hosts’ behaviour in the acute phase but not much is known about the chronic phase. We identified blood parasites by microscopy and a polymerase chain reaction (PCR)-based method in a sample of 22 wild-caught nightingales (Luscinia megarhynchos) in northeast Bulgaria. We compared non-infected and infected birds in single behavioural traits under the hypothesis that infected birds should show more risk-taking behaviours and quicker exploration. Infected nightingales were more prone to risk-taking and this behavioural trait was significantly correlated with the intensity of infection. However, no differences were found in exploration speed or body condition and reactions to a stressful situation (weight change in captivity, handling), which might be due to either a lack of differences or the limited sample size. © 2015 Dipartimento di Biologia, Università di Firenze, Italia
    No preview · Article · Nov 2015 · Ethology Ecology and Evolution
Show more