Goldview

The commercial E.Z.N.A.^® Stool DNA Kit (Omega Biotek Inc., Norcross, GA, USA) was used to extract genomic DNA, following the manufacturer’s instructions, and extracted DNA was stored at −20 °C. PCR targeting the ITS region was used to explore the prevalence and genotypes of E. bieneusi. All the PCR operations have been described in a previous study [31 (link)]. In each trial, positive and negative controls were present. The amplification products were observed using UV light after electrophoresis in a 1.5% agarose gel containing GoldView^TM (Solarbio, Beijing, China).

Genomic DNA was extracted from faeces using an EZNAR Stool DNA kit (OMEGA, USA) following the manufacturer’s instructions and stored at -20 °C until PCR analysis. Cryptosporidium species/genotypes were identified by nested PCR amplification of the small subunit ribosomal RNA (SSU rRNA) gene [3 (link)]. Every amplification included positive and negative controls. Amplification products were visualised on  1.5 % agarose gels containing GoldView™ (Solarbio, China).

Genomic DNA was isolated from individual mosquitoes using the QIAamp® DNA Mini Kit (QIAGEN, Hilden, Germany) following the manufacturer’s instructions. Approximately 650 bp of the COII gene and a 550-bp PCR product of the ITS2 region was amplified using primer pairs LYS-R (5′-ACTTGCTTTCAGTCATCTAATG-3′) and LEU-F (5′-TCTAATATGGCAGATTAGTGCA-3′) and ITS2-R (5′-TATGCTTAAATTCAGGGGGT-3′) and ITS2-F (5′-TGTGAACTGCAGGACACAT-3′), respectively. ITS2 was amplified in a PCR reaction volume of 25 µl with the following cycling parameters: 94 ℃, 2 min; then 94 ℃/30 s, 50 ℃/30 s, 72 ℃/40 s for 40 cycles; and a final extension at 72 ℃ for 10 min. COII was amplified in a PCR reaction volume of 25 µl with the following cycling parameters: 95 ℃, 5 min; then 95 ℃/1 min, 51 ℃/1 min, 72 ℃/ 2 min for 35 cycles; wiath a final extension 72 ℃ for 10 min. The PCR products were then analyzed by 1.5% agarose gel electrophoresis stained with GoldView (Beijing Solarbio Science & Technology Co., Ltd., Beijing, China), under UV transillumination. The sequencing reaction proceeded in both directions with the assistance of an ABI Big Dye Terminator Kit v.3.1 (Applied Biosystems, Thermo Fisher Scientific, Waltham, MA, USA); analysis was conducted using ABI Prism 3500xL-Avant Genetic Analyzer (Applied Biosystems, Thermo Fisher Scientific) in Shanghai (Sangon Biotech).

Polymerase chain reactions were carried out to screen the samples for detection of 18S rRNA sequences of piroplasm; 16S rRNA sequences of A. phagocytophilum, Anaplasma bovis, and Ehrlichia spp.; msp4 sequences of Anaplasma marginale and Anaplasma ovis; and ompA sequences of spotted fever group (SFG) rickettsiae, according to previously described (Table 1). The PCR amplifications were performed in an automatic thermocycler (Bio-Rad, Hercules, CA, USA) with a final volume of 25 μl containing 2.5 μl of 10× PCR buffer (Mg²⁺ Plus), 2.0 μl of each deoxyribonucleotide triphosphate (dNTP) at 2.5 mM, 1.0 μl of each primer (10 pmol), 2.0 μl of DNA samples (blood DNA, 18.4–24.3 ng/μl; liver DNA, 94.8–134.2 ng/μl), 1.25 U of Taq DNA polymerase (TaKaRa, Dalian, China), and 16.25 μl of distilled water. Positive (containing DNA of the corresponding organisms) and negative controls were included in all amplifications. PCR products were analyzed by agarose gel electrophoresis (1.2%) and visualized under UV transillumination after Goldview staining (Solarbio, Beijing, China).

Total DNA was extracted from the legs or abdominal tissue of 560 individuals (297 individuals of E. aenea; 263 individuals of L. perrisi) using the Chelex protocol (Casquet, Thebaud & Gillespie, 2012 (link)), followed by PCR amplification of ca. 650 bp-long barcoding fragment of the mitochondrial cytochrome c oxidase subunit I (COI) using the primer pair LCO1490 and HCO2198 (Folmer et al., 1994 (link)). The PCR was performed in a total volume of 25 μl containing 5 μl of 5× DreamTaq^™ Buffer, 1.5 μl of Mg⁺² (25 mM), 0.5 μl of each primer (concentration 5 mM), 0.5 μl of dNTP Mix (20 mM), 0.125 μl (0.625 U) DreamTaq TMDNA Polymerase, 11.875 μl ultra-pure H₂O and 5 μl of DNA template. The PCR cycling consisted of a 2-min initial denaturation at 94 °C, followed by 40 cycles of 94 °C (40 s) denaturation, 46 °C (40 s) annealing and 72 °C (1 min) extension and termination at 72 °C (10 min) for a final extension. A 4 μl aliquot of the PCR products were visualized by GoldView (Solarbio) in electrophoresis on a 1% agarose gel and GelLogic imaging equipment to check PCR product quality and length. The PCR products were purified with Exo-FastAP Thermo Scientific and were sent for sequencing to Macrogen Europe Inc., Amsterdam.