Qiamp tissue kit

Genomic DNA was extracted from ethanol-stored foot tissues using silica columns (QIAmp Tissue kit, QIAGEN) for the host genetics component of the study [27 (link)].
Molecular detection of parasites was performed on genomic DNA extracts prepared from the different tissue samples using the QiAmp® DNeasy Blood & Tissue Kit (Qiagen, Courtaboeuf, France), according to the manufacturer’s instructions.

Prior to myxospore exposures, oligochaetes (N = 20/lineage group) were randomly collected from the bulk cultures. Total genomic DNA was extracted from individual oligochaetes using the QIAmp tissue kit (Qiagen), initially screened with the Tt specific primers (Beauchamp et al. 2001 (link)) and verified with the mt 16S rDNA lineage-specific PCR (Beauchamp et al. 2002 (link)). Oligochaetes that were negative for these tests were considered non-Tt. Although Tt has evolved into extended species complex as found in Europe (Marotta et al. 2014 ), the mt 16S rDNA lineage-specific PCR (Beauchamp et al. 2001 (link)) was considered the most appropriate marker for specific identification of Tt strains, thus only four Tt lineages could be distinguished in the current study using the assay. The proportion of Tt lineage type was calculated as the number of oligochaetes positive for the lineage over the number of oligochaetes examined in each group. Lineage typing was also conducted at 80 days post exposure (pe) and at termination of the study at 6 months as described below in the non-shifted and shifted studies.

Genomic DNA from Coxiella burnetii was isolated using the Qiamp Tissue kit (Qiagen, Hilden, Germany) according to the manufacturer's specifications. Primers were designed based on the genome sequence of C. burnetii RSA 493/Nine Mile phase I (NCBI: NC_002971). Four full-length genes (CBU_1718, CBU_0092, CBU_0937 and CBU_0612) were amplified by PCR using Phusion DNA polymerase (Thermo Fisher Scientific). The PCR amplified products were purified from the agarose gel using Zymoclean Gel DNA Recovery kit (Zymo Research) and then digested with NdeI and XhoI. Subsequently, the digested fragments were ligated into the corresponding sites of pET-22b(+) to generate the expression vectors. In addition, for the target CBU_0937, two primers (CBU_0937_pelB_Fw and CBU_0937_pelB_Rev) was used to amplify the region encoding the mature protein without the first 23 amino acids according to the predicted signal peptide cleavage site. The resulting DNA fragment was cloned into the NcoI and XhoI sites of the pET-22b(+) vector using the InFusion ligation-independent cloning method (Takara Bio). All final constructs were verified by DNA sequencing (Eurofins Genomics) (Supplementary Figure 1).