Midori green advanced

DNA of four worms, recovered from two shot carcasses of A. aegyptiaca, was extracted using the DNAeasy Blood & Tissue kit (Qiagen (Hilden, Germany)) following the manufacturers’ instructions. Following this, a 768 base pair segment of 18S was amplified. Therefore, a PCR was performed, using the primers Sobo18SFWD 5′ TTTGGTTTTCGGATCTGAGG-3′ and Sobo18SREV 5′ GTACAAAGGGCAGGGACGTA-3′. The PCR conditions were the following: 45 µL reaction volume with 5 µL of the isolated template DNA, 5 µL 10x PCR buffer S (Peqlab, VWR, International GmbH, Erlangen, Germany), 1 µL of each primer, 1 µL dNTPs and 1 µL 5U/µL taq Polymerase (Peqlab, VWR, International GmbH, Erlangen, Germany). The PCR protocol started with an initial denaturation for 5 min at 94 °C followed by 35 cycles at 94 °C for 30 s, annealing at 51 °C for 45 s, extension for 90 s at 72 °C with a final extension for five min at 72 °C. The protocol was modified by the authors from a PCR protocol described by Koehler et al. [22 (link)].
The PCR product was visualised by a gel electrophoresis on 1.5% agarose gel with Midori Green Advanced^® (Nippon Genetics Europe, Düren, Germany) and extracted using the HiYield GelPCR^® DNA Extraction kit (Gauting, Germany). Sanger sequencing was performed by LGC genomics, Berlin, Germany. The sequence was added to the NCBI-GenBank with the accession number OQ561211.

Immediately upon isolation, DRGs were transferred to TRIzol (Thermo Fisher Scientific) and homogenized using a TissueLyser (Qiagen, Hilden, Germany). Subsequently, RNA was isolated according to manufacturer’s protocol (TRIzol, Thermo Fisher Scientific). The quality of RNA was assessed using a Nanodrop ND1000 Spectrophotometer (Thermo Fisher Scientific). Complementary DNA was synthesized from 500 ng to 1 µg RNA using oligo-dT primer and SCRIPT cDNA Synthesis Kit (Jena Bioscience, Jena, Germany). Quantitative real-time PCR was performed for 40 cycles at an annealing temperature of 60 °C using SensiFast Sybr No-ROX Kit (Bioline, London, UK) in a CFX Connect qPCR System (Bio-Rad Laboratories, Hercules, CA, USA). Primer sequences are listed in supplementary table S1. The detected quantification cycles (C_q) were normalized to C_q values of the housekeeping gene hypoxanthin-guanin-phosphoribosyltransferase (HPRT) using the 2^−ΔΔCT method^{63 (link)}. The amplified DNA products were separated by agarose gel electrophoresis on a 2% agarose gel supplemented with Midori Green Advanced (Nippon Genetics Europe, Dueren, Germany) and visualized using a Gel Doc XR + Gel Documentation System (Biorad, Hercules, CA, USA).

mP-BIT typing was performed using the KAPA multiplex PCR kit (Kapa Biosystems, USA) with master mix and amplification set according to the manufacturer’s instructions (Yamada et al. 2015 (link)). In detail, two multiplex PCRs were performed with 18 designed primer sets (Table S2). Detection of hipO gene was used for C. jejuni strain verification. PCR products were separated by 3% agarose gel electrophoresis (Bio-Rad Laboratories, USA) in Tris borate EDTA at 100 V for 90 min, stained using Midori Green Advanced (Nippon Genetics Europe GmbH, Germany), and then visualized with UV transilluminator (Quantum Vilber Lourmat, France). Band sizes were estimated by comparison with a 50 bp molecular size marker (New England Biolabs, USA). The results were then converted to a binary code, where the positive result was marked with “1” and the negative result with “0”. The binary codes were then converted into decimals representing the mP-BIT profiles.