Computational protocol: Characterization of Plasmodium relictum, a cosmopolitan agent of avian malaria

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Protocol publication

[…] Detailed microscopic analysis was carried out with various Olympus light microscopes equipped with Olympus digital cameras and imaging software. Preparations of blood stages of the lineages pSGS1, pGRW11, pGRW4, and pLZFUS01 were from collections of voucher specimens which have been deposited at P. B. Šivickis Laboratory of Parasitology, Nature Research Centre Vilnius. These were blood films from canaries whose were exposed experimentally to the parasite lineages pSGS1 (parasitaemia varied between 0.6 and 1.8%, preparation accession number 48979–48981 NS), pGRW11 (1.1–6%, 48982–48984 NS), pGRW4 (0.2–2.1%, 48985–48987 NS), and the lineage pLZFUS01 (0.5%, 48694–48696 NS) from the blood of a naturally infected Red-backed shrike Lanius collurio (for exposure description see []). Additionally, preparations with the Hawaiian isolate of P. relictum (pGRW4) were used. These were (1) 12 blood films from two individual canaries that were exposed experimentally by inoculation of infected blood (parasitaemia varies between 0.6 and 10%, accession nos. 48988–48999 NS) (for infection details see []), (2) 11 blood films from one naturally infected Apapane Himatione sanguinea (parasitaemia 22%, accession nos. 49000–49010 NS).Blood films from each infected bird were examined and the observed blood stages were morphologically compared by skilled parasitologists of avian malaria parasites at the P. B. Šivickis Laboratory of Parasitology. At least 100 fields were studied at high magnification (1000×) in each preparation. Intensity of parasitaemia was estimated as a percentage by actual counting of the number of parasites per 10,000 erythrocytes. The morphometric features studied (Table ) were those defined in []. The analyses were carried out using the ‘Statistica 7’ package as previously described []. [...] Total DNA was extracted from blood samples using an ammonium-acetate precipitation protocol []. Polymerase chain reaction using the primer set HaemNFI/NR3 and Haem/R2 was performed in order to amplify a 479 bp sequence of the parasite’s cytb gene [, ]. The total volume of the reaction mix for each sample was 25 µl, which included 12.5 µl of DreamTaq Master Mix (Thermo Fisher Scientific, Lithuania), 8.5 µl nuclease-free water, 1 µl of each primer and ~ 50 ng of a total genomic DNA template (2 µl). Negative controls (nuclease-free water) were used after each seven samples to detect possible false amplifications, and one positive control (extracted parasite DNA from a blood sample, which was confirmed positive during previous PCR testing) was used to evaluate the success of PCR if none of the samples would have been amplified.Temperatures for the PCR were as described in the original protocols. The success of the performed PCR was evaluated by running electrophoresis on a 2% agarose gel. Successfully amplified DNA was precipitated using 11 µl of 8 M NH 4Ac, 37 µl of 96% and 150 µl of 70% ethanol. After centrifugation, the supernatant was discarded, the samples were air-dried overnight, and then 16 µl of nuclease-free water was added on the precipitated DNA. Big Dye Terminator V3.1 Cycle Sequencing Kit and ABI PRISM™ 3100 capillary sequencing robot (Applied Biosystems, Foster City, California) were used for sequencing. Sequences were edited and aligned using BioEdit software []. Absence of double-base calling in sequence electropherograms was used as an indication of single infections []. Nucleotide BLAST (megablast algorithm) ( was used to compare our amplified sequences with sequences deposited in the GenBank.Molecular phylogenetic analysis was carried out using Bayesian and Maximum Likelihood algorithms. Sequences for the phylogenetic analysis were collected from GenBank and double-checked in MalAvi database []. Plasmodium falciparum was used as an outgroup. GenBank accession numbers and codes of the lineages are provided in the phylogenetic trees (Fig. ). Bayesian phylogenetic tree (Fig. a) was constructed using MrBayes version 3.1 [] software. The General Time Reversible Model (GTR) was used as suggested by the software MrModeltest 2.2 ( Analysis was run for a total of 10 million generations with a sampling frequency of every 100 generations. Before the construction of the consensus tree, 25% of the initial trees were discarded as ‘burn in’ period. The tree was visualized using the software FigTree v1.4.3 ( Maximum Likelihood tree (Fig. b) was constructed using the MEGA 7.0 [] software; it was performed with 1,000 bootstrap replications using the GTR model and the same dataset as during the Bayesian analysis.Fig. 1 The new sequence of lineage pPHCOL01 was deposited in GenBank (accession MG724747). Genetic differences between different lineages of P. relictum were calculated using the Jukes–Cantor model of substitution, as implemented in the programme MEGA 7.0 []. […]

Pipeline specifications

Software tools Statistica, BioEdit, BLASTN, MrBayes, MrModelTest, FigTree, MEGA
Applications Miscellaneous, Phylogenetics
Organisms Serinus canaria, Culex quinquefasciatus, Taeniopygia guttata, Melopsittacus undulatus, Toxoplasma gondii
Diseases Malaria