Computational protocol: Epidemiology and pathology of avian malaria in penguins undergoing rehabilitation in Brazil

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Protocol publication

[…] Frozen samples of blood and tissues (lung, spleen or liver) were used for molecular analyses. DNA extraction was conducted using the DNEasy Blood and Tissue Kit (#69506, Qiagen) and was verified and quantified through UV spectrophotometry (Nanodrop 2000, Thermo Fisher Scientific). We used a nested polymerase chain reaction targeting the mitochondrial cytochrome b (cyt-b) gene of Haemoproteus and Plasmodium [] with 3 ng/μL of sample DNA, 0.6 μM of each primer, and GoTaq Green Master Mix 2x (M7122, Promega). Blood samples from chicken experimentally infected with Plasmodium gallinaceum and samples from chickens raised in arthropod-free environments were used as positive and negative controls, respectively. Gel electrophoresis was conducted to visualize amplification products, using 2% agarose gel, SYBR Safe (S33102, Invitrogen), and a high-resolution imaging system (Gel Doc EZ System 170–8270, Bio-Rad). PCR amplification products of positive samples were purified with Polyethylene Glycol 8000. Bi-directional sequencing with dye-terminator fluorescent labeling was performed through automated sequencing (ABI Prism 3100, Applied Biosystems); forward and reversed chromatograms were edited and consensus sequences were deposited in GenBank (Additional file ).Phylogenetic relationships among Plasmodium lineages identified in this study and related hemosporidian parasites were inferred by using sequences from reference lineages from the MalAvi database [], for which species was identified based on studies using morphological evidence, as well as penguin-infecting Plasmodium lineages from published studies (Additional file ). Sequences were aligned using ClustalW [] as implemented in MEGA 5.2.2 []. A Bayesian phylogenetic tree for the parasite sequences was produced using MrBayes 3.2.2 [] with the GTR + I + G model of nucleotide evolution, as recommended by ModelTest []. We ran two Markov chains simultaneously for 5 million generations that were sampled every 1000 generations. The first 1250 trees (25%) were discarded as a burn-in step and the remaining trees were used to calculate the posterior probabilities. […]

Pipeline specifications

Software tools Clustal W, MEGA, MrBayes, ModelTest-NG
Application Phylogenetics
Diseases Communicable Diseases, Malaria, Avian, Parasitic Diseases