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.

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Available from: Jonas Waldenström, Jul 04, 2014
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    • "The genera Plasmodium, Haemoproteus and Leucocytozoon represent a group of vector-borne blood parasites causing a malaria-like disease in birds (e.g. Bensch et al. 2000; Waldenstöm et al. 2004; Bensch and A ˚ kesson 2003; Scheuerlein and Ricklefs 2004; Wood et al. 2007; Jenkins and Owens 2011; Swanson et al. 2014; Zhao et al. 2014). One of the features of such infections is that host species vary substantially in parasite prevalence. "
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    ABSTRACT: Seabirds are regarded as a group of species with relatively low levels or even complete lack of blood parasites. We used PCR to amplify a DNA fragment from the cytochrome b gene of the parasites to search for infections of the genera Plasmodium, Haemoproteus and Leucocytozoon in individuals of two sympatrically breeding gull species, the Herring Gull Larus argentatus, the Caspian Gull Larus cachinnans and their hybrids. Out of 56 analysed individuals, 53 (95 %) were identified as infected with Leucocytozoon, whereas three individuals carried double and triple infections with at least one Leu-cocytozoon and one Plasmodium lineages. No Haemopro-teus lineage was detected. The most common lineage (LARCAC02), for the first time reported here, was found in 51 (96 %) of all infected birds, and 14 gulls carried two Leucocytozoon lineages. We analysed the evolutionary relationship of Leucocytozoon lineages from the Herring and Caspian Gull and other bird species. Our results show that (1) the two identified Leucocytozoon lineages are not closely related as they belong to two distinctly different clusters. Moreover, (2) seabirds breeding inland could be highly infected with blood parasites and (3) this high prevalence is probably associated with areas where parasite vectors are abundant. Further studies should explore the importance of environmental factors affecting parasite prevalence, in particular within species comparisons under different environment conditions, including vector monitoring and sampling.
    Journal of Ornithology 09/2015; DOI:10.1007/s10336-015-1291-5 · 1.71 Impact Factor
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    • "DNA from the avian blood samples were extracted in the lab using the standard phenol/chloroform/isoamylalcohol method (Sambrook et al. 2002). Diluted genomic DNA (25 ng/μl) was used as a template in a polymerase chain reaction (PCR) assay for detection of the parasites using nested PCR protocols described by Waldenström et al. (2004). The amplification was evaluated by running 2.5 μl of the final PCR on a 2 % agarose gel. "
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    ABSTRACT: Escape behaviour is the behaviour that birds and other animals display when already caught by a predator. An individual exhibiting higher intensity of such anti-predator behaviour could have greater probabilities of escape from predators. Parasites are known to affect different aspects of host behaviour to increase their own fitness. Vector-transmitted parasites such as malaria parasites should gain by manipulating their hosts to enhance the probability of transmission. Several studies have shown that malaria parasites can manipulate their vectors leading to increased transmission success. However, little is known about whether malaria parasites can manipulate escape behaviour of their avian hosts thereby increasing the spread of the parasite. Here we used an experimental approach to explore if Plasmodium relictum can manipulate the escape behaviour of one of its most common avian hosts, the house sparrow Passer domesticus. We experimentally tested whether malaria parasites manipulate the escape behaviour of their avian host. We showed a decrease in the intensity of biting and tonic immobility after removal of infection with anti-malaria medication compared to pre-experimental behaviour. These outcomes suggest that infected sparrows performed more intense escape behaviour, which would increase the likelihood of individuals escaping from predators, but also benefit the parasite by increasing its transmission opportunities.
    Parasitology Research 09/2015; 114(12). DOI:10.1007/s00436-015-4693-7 · 2.10 Impact Factor
    • "DNA from blood samples was extracted by ammonium-acetate (Nicholls et al. 2000), after which the concentration of genomic DNA was adjusted to 25–35 ng/μL. Polymerase chain reactions (PCRs) were performed to amplify a region of cytochrome b gene in the mitochondrial DNA of Haemoproteus and Plasmodium parasites using the protocol described by Waldenström et al. (2004). In all PCRs, both negative (ddH2O) and positive controls (samples from birds which were previously confirmed to be infected) were included among the samples to control for possible contaminations and failures during PCRs, respectively. "
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    ABSTRACT: Several hypotheses predict a relationship between parasite burden and risk-taking behavior, but the underlying causal mechanisms are poorly understood due to the scarcity of experimental studies and the neglected focus on immune defense. Here, in 3 sets of field studies on the collared flycatcher, Ficedula albicollis, we investigated how among-male variation in flight initiation distance (FID, the distance at which an individual flee a potential predator) is linked to among-male variation in health status. First, we correlatively assessed the relationship between FID and the prevalence of haemosporidian blood parasites. We found no difference in risk-taking behavior between parasitized and nonparasitized individuals rejecting a hypothesis that predicts that malaria infection status affects the costs of predator avoidance. Second, we performed an immune challenge experiment, in which randomly chosen birds were injected with a novel antigen (sheep red blood cell) and their change in FID was compared with birds that received a placebo treatment. This experiment revealed no evidence for the immunological treatment affecting risk-taking behavior, thus we failed to obtain support for the hypothesis that posits that immediate health status mediates decisions about when to flee a predator. Finally, we detected a negative relationship between the number of alleles of the major histocompatibility complex and FID. This result, in concordance with the above negative results, supports the “avoidance” hypothesis that states that only individuals with efficient immune defense machinery are able to bear the costs of risk-taking that can emerge through the increased infection rates of risk-taker individuals.
    Behavioral Ecology 06/2015; 26(5). DOI:10.1093/beheco/arv077 · 3.18 Impact Factor
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