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[…] Determined 18S rRNA gene nucleotide sequences were used as query in a BLASTn search in order to identify and download most closely related 18S rRNA gene sequences of well-defined piroplasmid species from GenBank. In addition, 18S rRNA gene sequences of representative piroplasmid species were downloaded in order to allow species delineation in the phylogenetic analysis. A multiple alignment of the hypervariable region of 95 18S rRNA gene sequences comprising selected and analysed sequences including the 18S rRNA gene of Cardiosporidium cionae was done using MUSCLE []. Positions containing gaps and missing data were eliminated from the 514 nucleotide-alignment to finally result in 385 positions in the final dataset. After estimation of shape parameter, the K2 + G + I parameter model was applied to generate a maximum likelihood tree []. Phylogenetic analysis was carried out using the MEGA6 software []. [...] Differences in the prevalence of infection with Babesia spp. in questing ticks, ticks attached to rodents, and in rodents were analysed between habitats, years, and rodent species and genders applying Fisher’s exact test, supplemented with Mantel-Haenszel common odds ratio estimate and its 95 % confidence interval in cases when two prevalences were compared. Rodents positive for spleen, blood and/or lungs were considered Babesia-positive. The 95 % confidence intervals of the prevalences in questing ticks, rodent-attached ticks and rodents were computed using a bootstrap technique. Logistic regression was used to estimate the effect of habitat type and year on the probability of tick infection and the effect of habitat type, rodent species and gender on the probability of rodent infection. Backward stepwise method was used to find the set of variables significantly affecting the probability of tick and rodent infection. Tests for the significance of the effects in the model were performed via the Wald statistic. Results on the presence of A. phagocytophilum and Candidatus N. mikurensis (CNM) in the same questing ticks, and rodents available from previous studies [, ], were used to calculate the probability of co-infections with Babesia spp. and analyse the dependence of the microorganisms on the habitat type using Fisher’s exact test. Differences were considered significant at P < 0.05 in all tests. Statistical analyses were performed with IBM SPSS Statistics, version 22 [] and Statistica software, version 12 []. […]

Pipeline specifications

Software tools BLASTN, MUSCLE, MEGA, SPSS, Statistica
Organisms Ixodes ricinus, Anaplasma phagocytophilum, Brucella microti
Diseases Babesiosis, Disease, Infection, Sprains and Strains, Wiskott-Aldrich Syndrome, Zoonoses, Tick-Borne Diseases, Precursor Cell Lymphoblastic Leukemia-Lymphoma, Coinfection