Dataset Information

Molecular detection of hemogregarines and haemosporidians in Brazilian free-living testudines.

ABSTRACT: Morphological and molecular techniques were used to investigate the presence of hemogregarines and haemosporidians in biological samples of free-living Geoffroy's side-necked turtles (Phrynops geoffroanus) and Giant Amazon turtles (Podocnemis expansa) from Brazil. No evolutionary form of haemosporidians or hemogregarines were observed in the blood smears of 83 P. geoffroanus samples, and there were no meronts in the histological sections of 31 necropsied P. geoffroanus samples. All DNA samples extracted from P. geoffroanus tissues and blood aliquots were negative in haemosporidian PCR assays (based on the mitochondrial cytochrome b gene) and hemogregarine PCR assays (based on the 18S rRNA gene). In the analysis of blood smears of all seven Podocnemis expansa evaluated, gametocytes of hemogregarines were observed. The seven P. expansa were negative in the haemosporidian PCR assays. Moreover, hemogregarine DNA was detected in blood samples from all of the sampled P. expansa. The phylogenetic maximum likelihood inference and probabilistic Bayesian inference revealed five closely related genotypes that formed a monophyletic group. There was also a sister group to the lineage that consisted of Haemogregarina spp. of freshwater turtles from Canada, Italy, Mozambique, Kenya, Gabon, Vietnam, and China. The findings suggest that free-living P. expansa were parasitized by a new genotype or even a possible new species of the genus Haemogregarina. Haemosporidians and hemogregarines are not frequently found in P. geoffroanus in the studied region under the local conditions of that period.

SUBMITTER: Oliveira JP

PROVIDER: S-EPMC6058349 | biostudies-literature | 2018 Apr

REPOSITORIES: biostudies-literature

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Molecular detection of hemogregarines and haemosporidians in Brazilian free-living testudines.

Oliveira Juliana Paula de JP André Marcos Rogério MR Alves Júnior José Roberto Ferreira JRF Lustosa Ana Paula Gomes APG Werther Karin K

International journal for parasitology. Parasites and wildlife 20180203 1

Morphological and molecular techniques were used to investigate the presence of hemogregarines and haemosporidians in biological samples of free-living Geoffroy's side-necked turtles (Phrynops geoffroanus) and Giant Amazon turtles (Podocnemis expansa) from Brazil. No evolutionary form of haemosporidians or hemogregarines were observed in the blood smears of 83 P. geoffroanus samples, and there were no meronts in the histological sections of 31 necropsied P. geoffroanus</i ...[more]

PMID: 30050752

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Project description:BackgroundBirds possess the most diverse assemblage of haemosporidian parasites; including three genera, Plasmodium, Haemoproteus, and Leucocytozoon. Currently there are over 200 morphologically identified avian haemosporidian species, although true species richness is unknown due to great genetic diversity and insufficient sampling in highly diverse regions. Studies aimed at surveying haemosporidian diversity involve collecting and screening samples from hundreds to thousands of individuals. Currently, screening relies on microscopy and/or single or nested standard PCR. Although effective, these methods are time and resource consuming, and in the case of microscopy require substantial expertise. Here we report a newly developed real-time PCR protocol designed to quickly and reliably detect all three genera of avian haemosporidians in a single biochemical reaction.MethodsUsing available DNA sequences from avian haemosporidians we designed primers R330F and R480RL, which flank a 182 base pair fragment of mitochondrial conserved rDNA. These primers were initially tested using real-time PCR on samples from Malawi, Africa, previously screened for avian haemosporidians using traditional nested PCR. Our real time protocol was further tested on 94 samples from the Cerrado biome of Brazil, previously screened using a single PCR assay for haemosporidian parasites. These samples were also amplified using modified nested PCR protocols, allowing for comparisons between the three different screening methods (single PCR, nested PCR, real-time PCR).ResultsThe real-time PCR protocol successfully identified all three genera of avian haemosporidians from both single and mixed infections previously detected from Malawi. There was no significant difference between the three different screening protocols used for the 94 samples from the Brazilian Cerrado (χ(2) = 0.3429, df = 2, P = 0.842). After proving effective, the real-time protocol was used to screen 2113 Brazilian samples, identifying 693 positive samples.ConclusionsOur real-time PCR assay proved as effective as two widely used molecular screening techniques, single PCR and nested PCR. However, the real-time protocol has the distinct advantage of detecting all three genera in a single reaction, which significantly increases efficiency by greatly decreasing screening time and cost. Our real-time PCR protocol is therefore a valuable tool in the quickly expanding field of avian haemosporidian research.

Dataset Information

Molecular detection of hemogregarines and haemosporidians in Brazilian free-living testudines.

Publications

Molecular detection of hemogregarines and haemosporidians in Brazilian free-living testudines.

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