Ticks were collected in April and SeptemberOctober 2009 and had been stored as described. Even more I. ricinus ticks had been available from a previous study from an area in the Saarland of altogether 150 km2 west of Saarbr?cken. That past examine was concerned principally with D. reticulatus as well as the I. ricinus have been collected but stored aside. Trapping of compact mammals Small mammals were trapped at three out of five internet sites in Leipzig with Sherman live traps baited with apple pieces. Usually twenty traps per web page were placed along purely natural structures or directly in front of holes inside the ground. The number of traps per night hardly ever exceeded 75 and so they all were checked twice day-to-day. Captured animals have been anesthetized with CO2 ex posure and killed humanely according on the German Animal Protection Act immediately after blood was drawn by heart puncture.
Just after recording trapping web site, species, gender, and health status, rodents have been dissected below BSL 2 ailments in the laboratory and stored at 80 C. Ticks had been collected from your modest mammals and stored at 80 C individually for every individual animal until finally species identification. DNA extraction Species identification selleck chemicals of ticks was carried out with stand ard taxonomic keys and DNA was extracted from your ticks as described and from tissues and entire body liquids together with the Qiagen DNA Mini Kit in accordance for the makers in struction for either animal tissue or blood. Tissue lysis was carried out above evening at 56 C in the thermomixer. Good quality and quantity of extracted DNA had been tested having a spectrophotometer. PCR strategies Detection of Babesia spp.
DNA in all tick and small mammal samples was carried out which has a conventional PCR focusing on the 18S rRNA gene followed by gel elec trophoresis as described. Detection of the. phago cytophilum was carried out in I. ricinus ticks only and during the little mammals having a actual time PCR as described. For a subsample with the A. phagocytophilum posi tive samples, a nested selleck inhibitor PCR focusing on a 497 bp area on the 16S rRNA gene was performed as previously described. Genomic DNA from A. phagocytophi lum cell culture and from B. microti from continuous cul ture in BALBc mice had been utilized as good controls and molecular grade water as damaging controls and have been included in each and every PCR run. Sequence analysis PCR solutions in the partial Babesia spp. 18S rRNA gene and of your partial 16S rRNA gene of the.
phagocytophilum had been purified with the QIAquick PCR Purification Kit and sequenced bidirectio nally at Eurofins MWG Operon. The evaluation of the obtained sequences was performed as described. Co infections and statistical evaluation of questing tick results Precise self-confidence intervals on the prevalences had been computed together with the Clopper and Pearson approach. For pooled samples the self confidence interval was calcu lated to the minimum infection, taking under consideration the nymphal pools and assuming that only one in the nymphs in one particular pool was contaminated.