Hedgehog samples
From April, 2018 to June, 2019, hedgehogs were captured using traps baited with meat, and road killed or injured individuals were also permitted to be collected from Wuhan and Xianning cities, Hubei Province and Jiujiang city, Jiangxi Province of China (Fig.1). The hedgehogs were morphologically identified as Erinaceus amurensis as described previously [17]. Animals were classified into two age groups: young and adults. Age was estimated from the appearance of the animal, following the criteria set out by Robinson [18], gender was identified and recorded. Captured hedgehogs were anesthetized by an intramuscular injection (20 mg/kg) of Ketamine. One of the front feet was sterilized immediately after sedation, and one nail was clipped 3 mm short. Blood samples were immediately collected with 5ml centrifuge tubes and disposable plastic transfer pipettes. The sample volume ranged from 0.2-1ml. All blood samples were preserved with dry ice, and later stored in -80℃ for further use. All sampled individuals were later released back to the wild.
PCR amplification of Rickettsia, Anaplasma, Ca.Neoehrlichia and Ehrlichia
Hedgehog blood DNA was extracted with the Qiagen DNA Kit (Qiagen, Hilden, Germany). DNA concentration and purity were measured with an absorbance ratio of 260 to 280 nm by using DeNovix DS-11 spectrophotometer (DeNovix, Wilmington, DE, USA) and were stored at -20°C until used. Mean quantity of DNA obtained was 38 ± 9.7 ng/ul and the 260/280nm ratio of all samples were 1.66 ± 0.27. Blood DNA samples were used as templates for PCR amplification of Ehrlichia, Ca.Neoehrlichia, Rickettsia and Anaplasma DNA with primers described in Table 1. Anaplasma 16S rRNA (rrs) gene was amplified with a nested PCR by using the primers EHR1/EHR2 in the first-round reaction, and EHR3/EHR4 in the second-round reaction [19]. For rrs positive samples, groEL gene was targeted for further genotyping as described previously [20]. For Ehrlichia, nested PCR amplifications of rrs, heat sock protein gene (groEL) and citrate synthase gene (gltA) were performed [21, 22]. For detection of Rickettsia, nested PCR amplifications of Rickettsiarrs, gltA, and outer membrane protein B gene (ompB) were performed [23]. For Ca.Neoehrlichia detection, 16S rRNA were targeted [24] . DNA isolated from A.bovis, E. chaffeensis and R. japonica was served as positive controls. To avoid contamination, all steps were performed in separate rooms. Negative control with distilled water was run for each reaction.
PCR products were separated with 1.2% agarose gel electrophoresis and visualized with UV light after ethidium bromide staining. PCR products with expected sizes were excised from gels and extracted using a Gel Extraction Kit (Promega, Madison, WI, USA), which were then cloned into the pMD19-T vector (TaKaRa, Shiga, Japan). M13F-47,M13R-48 Universal Primers were used for Sanger dideoxy sequencing in TingKe Biotech Company (Wuhan, China) on both strands.
Phylogenetic analysis
All sequences were searched using BLAST in the GenBank database (http://blast.ncbi.nlm.nih.gov/Blast.cgi). After alignment by ClustalW with MEGA 7.0 [25], the datasets were analyzed by jModeltest2 and the best evolutionary models were chosen [26]. Phylogenetic trees were constructed using the Maximum Likelihood method with the best model in MEGA 7.0, and the robustness of the trees was tested with 1,000 bootstrap replications.