Computational protocol: A new lizard malaria parasite Plasmodium intabazwe n. sp. (Apicomplexa: Haemospororida: Plasmodiidae) in the Afromontane Pseudocordylus melanotus (Sauria: Cordylidae) with a review of African saurian malaria parasites

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Protocol publication

[…] Ethanol-preserved blood from the nine parasitised individuals of the 77 collected of P. melanotus and both C. vittifer were used for molecular work. Genomic DNA of Plasmodium spp. was extracted from the samples following the standard protocol for the Kapa Express Extract kit (Kapa Biosystems, Cape Town, South Africa). Amplification of Plasmodium spp. DNA was initially completed using specific Plasmodium and Haemoproteus primer sets, with a nested-polymerase chain reaction (PCR) protocol targeting a fragment of the cytochrome-b (cyt-b) gene as detailed in []. The PCR method consisted of two parts. The primer set HAEMNF (5′-CAT ATA TTA AGA GAA TTA TGG AG-3′) and HAEMNR2 (5′-AGA GGT GTA GCA TAT CTA TCT AC-3′) was used first under the following conditions: initial denaturation at 94 °C for 3 min, followed by 20 cycles, entailing a 94 °C denaturation for 30 s, annealing at 50 °C for 30 s with an end extension at 72 °C for 45 s, and following the cycles a final extension of 72 °C for 10 min. This was followed by a second PCR using the primer set HAEMF (5′-ATG GTG CTT TCG ATA TAT GCA TG-3′) and HAEMR2 (5′-GCA TTA TCT GGA TGT GAT AAT GGT-3′) []. The PCR conditions were as follows: initial denaturation at 94 °C for 3 min, followed by 35 cycles, entailing a 94 °C denaturation for 30 s, annealing at 50 °C for 30 s with an end extension at 72 °C for 45 s, and following the cycles a final extension of 72 °C for 10 min.All PCR reactions were performed in a 25 μl volume microtube, using 12.5 μl Thermo Scientific DreamTaq PCR master mix (2×) (2× DreamTaq buffer, 0.4 mM of each dNTP, and 4 mM MgCl2), 1.25 μl of each primer, and at least 25 ng of genomic DNA for the first PCR, and 1 μl of the PCR product from the initial PCR for the second PCR. The remaining volume was made up of PCR-grade nuclease-free water (Thermo Scientific, Vilnius, Lithuania). The PCR reactions were undertaken in a Bio-Rad C1000 Touch™ Thermal Cycler PCR machine (Bio-Rad, Hemel Hempstead, UK). Resulting amplicons were visualised by means of a 1 % agarose gel stained with gel red (Biotium, USA) under UV light. The PCR products from each sample were sent to a commercial sequencing company (Inqaba Biotechnical Industries (Pty) Ltd. Pretoria, South Africa) for purification and sequencing in both directions. Resultant sequences were assembled using Geneious Ver. 7.1 (created by Biomatters. Available from http://www.geneious.com) and chromatogram-based contiguous sequences were generated, trimmed and manually corrected for ambiguous base calls. Sequences were identified using the Basic Local Alignment Search Tool (BLAST) [], and deposited in the NCBI GenBank database under the accession numbers KX121601−KX121609.Comparative sequences of Plasmodium species were downloaded from GenBank and aligned to the sequences generated within this study. Two species of Leucocytozoon, Leucocytozoon gentili and Leucocytozoon majoris (GenBank: DQ451435, DQ451439) were used as the outgroup, as species of Leucocytozoon were shown to be a sufficient outgroup of the focal taxa by []. All phylogenetic analyses were further undertaken in the bioinformatics software program Geneious Ver. 7.1. Sequences were aligned using the MUSCLE alignment tool []. To infer phylogenetic relationships a Bayesian inference (BI) method was used. A comprehensive model test was preformed to determine the most suitable nucleotide substitution model, according to the Akaike information criterion using jModelTest 2.1.7 [, ]. The best-fit model selected was the General Time Reversible with estimates of invariable sites and a discrete Gamma distribution (GTR + I + Γ). The dataset comprised 31 cytochrome b (cyt-b) mitochondrial sequences, with an alignment length of 497 nt. The BI analysis was implemented from within Geneious 7.1 using MrBayes 3.2.2 []. The Markov Chain Monte Carlo (MCMC) algorithm was run for 10 million generations. The Markov chain was sampled every 100 cycles, and the MCMC variant contained 4 chains with a temperature of 0.2. The log-likelihood values of the sample point were plotted against the generation time and the first 25 % of the trees were discarded as ‘burn-in’ with no ‘burn-in’ samples being retained. Results were visualised in Trace (implemented from within Geneious) to assess convergence and the ‘burn-in’ period. […]

Pipeline specifications

Software tools Geneious, BLASTN, MUSCLE, jModelTest, MrBayes
Applications Phylogenetics, Nucleotide sequence alignment
Organisms Toxoplasma gondii, Nitrosomonas sp.
Diseases Malaria