Metagenomic Approach to Characterizing Disease Epidemiology in a Disease-Endemic Environment in Northern Thailand

Takhampunya, Ratree; Korkusol, Achareeya; Pongpichit, Chalermpol; Yodin, Komsan; Rungrojn, Artharee; Chanarat, Nitima; Promsathaporn, Sommai; Monkanna, Taweesak; Thaloengsok, Sasikanya; Tippayachai, Bousaraporn; Kumfao, Naruemon; Richards, Allen L.; Davidson, Silas A.

Ectoparasites (chiggers, ticks, fleas, and lice) collected from rodents and small mammals were morphologically identified and pooled by genus, the host species they were collected from, and ectoparasitic stage. Chiggers were identified to genus level using a taxonomic key (Nadchatram, 1974) . Other ectoparasites (fleas, ticks, and lice) were identified morphologically (Hopkins and Miriam, 1953; Tanskull and Inlao, 1989; Durden and Musser, 1994) and pooled by the host species they were collected from, type, stage, and gender of ectoparasites. Each pool was subjected to genomic DNA extraction using a modified protocol of QIAamp DNA Mini Kit (Qiagen). Briefly, ectoparasites in 180 µl of ATL buffer were punctured with a fine needle under a stereomicroscope to release the tissue from the hard chitin exoskeleton prior to adding 20 µl of Proteinase K solution (20 mg/ml). Samples were then incubated at 55 • C for 1 h or until the ectoparasites were homogenized. A volume of 200 µl of AL buffer was added to the sample and the sample mixed and incubated at 70 • C for 10 min. Then 100 µl of absolute ethanol was added to precipitate DNA. The solution was transferred to a QIAamp DNA column then centrifuged at 8,000 rpm for 1 min. The supernatant was discarded. DNA was washed twice with 500 µl of AW1 and AW2, respectively. The DNA was eluted at 50 µl of AE buffer and stored at −20 • C until used. Ultrapure DNA/RNA-free distilled water was also included as an extraction control.