Archived specimens from the Tropical Infectious Diseases Research and Education Centre (TIDREC), Universiti Malaya were utilized in this study. They consisted of tissues of small mammals from two sampling sites, viz. at UM Plantations Sdn. Bhd., Johor (an oil palm plantation) and Kampung Tumbuh Hangat, Perak (oil palm plantation bordering paddy fields and human settlements). These samples were collected at different times between December 2018 and December 2019 [21 (link)]. Ethical approval was obtained from the Universiti Malaya Institutional Animal Care and Use Committee (G8/01082018/24052018-01/R) and permission to conduct the study at Kampung Tumbuh Hangat, Perak was granted by the Department of Orang Asli Development (JAKOA), Malaysia (JAKOA/PP.30.052Jld13 (32)). Approval for small mammal trapping was also received from the University of Liverpool’s Animal Welfare and Ethics Review Body with reference no. AWC0127.
All small mammals captured were initially identified using morphological analysis [22 (link)]. Subsequently, tree shrew and rodent DNA barcoding was performed on DNA extracted from their spleens and other organs. Extracted rodent and tree shrew DNA was subjected to a polymerase chain reaction (PCR) targeting the cytochrome c oxidase I (COI) gene to determine the rodent and tree shrew species group [23 ]. The organs were stored at −80 °C immediately after harvesting and the extracted DNAs were aliquoted into three tubes to avoid multiple freeze-thawing. The primers used are listed inTable 1 . Positive controls used were genomic DNAs of O. tsutsugamushi strain UT176 received from University of Liverpool, United Kingdom, and Rickettsia roultii strain established from a tick cell line in TIDREC. Long oligo DNAs were synthesized for the positive controls of Borrelia spp. and Bartonella spp. The positive control fragments of the flagellin gene, flaB and the citrate synthase gene, gltA were obtained from Borrelia burgdorferi NC001318.1 (501 bp) and Bartonella quintana NC005955 (410 bp), respectively. Nuclease-free water was the negative control used in PCR protocols.
The remaining COI amplicons (approximately 20 µℓ each) were purified and subsequently sequenced (Apical Scientific Sdn. Bhd., Seri Kembangan, Malaysia). The DNA sequences obtained were trimmed and compared to those available in GenBank using the Basic Local Alignment Search Tool (BLAST). Each identified species was deposited into the GenBank accordingly.
All small mammals captured were initially identified using morphological analysis [22 (link)]. Subsequently, tree shrew and rodent DNA barcoding was performed on DNA extracted from their spleens and other organs. Extracted rodent and tree shrew DNA was subjected to a polymerase chain reaction (PCR) targeting the cytochrome c oxidase I (COI) gene to determine the rodent and tree shrew species group [23 ]. The organs were stored at −80 °C immediately after harvesting and the extracted DNAs were aliquoted into three tubes to avoid multiple freeze-thawing. The primers used are listed in
The remaining COI amplicons (approximately 20 µℓ each) were purified and subsequently sequenced (Apical Scientific Sdn. Bhd., Seri Kembangan, Malaysia). The DNA sequences obtained were trimmed and compared to those available in GenBank using the Basic Local Alignment Search Tool (BLAST). Each identified species was deposited into the GenBank accordingly.
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