Computational protocol: Survey on Helminths in the Small Intestine of Wild Foxes in Qinghai,China

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Protocol publication

[…] Study sites: This study was conducted in 5 counties located at northeast and south Qinghai, China (Fig. 1.). In all sites, Tibetan and other minority ethnic groups conduct nomadism of yak and sheep in a vast expanse of grassland. The average altitudes of the study sites are 3,000–3,800 m in Haiyan, Gangcha, Guinan and Xinghai counties and 4,500 m in Chengduo county.Hunting and dissection of foxes and parasite examination: Under the permission of the ministry of forestry, China for academic investigation on wild foxes, we asked local hunters to hunt foxes in Haiyan, Gangcha, Guinan and Chengduo counties in December 2010 to April 2011 and December 2011 to February 2012. Foxes were stored at −80°C for at least 10 days to inactivate Echinococcus eggs. Then, they were dissected and examined for their small intestinal helminths. The detected parasites were counted and stored in 70% ethanol for further examination. In addition, rectal feces were collected and stored at −20°C for fecal egg examination.Collection of fox feces in the field: Fox feces were collected at grassland within 100 km apart from Heka town in Xinghai county in September 2010, August 2011 and August 2012. Collected feces were stored at −80°C for at least 10 days to inactivate Echinococcus eggs and then stored at −20°C until use.Molecular identification of fox species by DNA barcording: A part of the liver and muscle of the dissected foxes were collected and stored in 70% ethanol. Then, DNA was extracted from the liver or muscle using QIAamp DNA Mini Kit (Qiagen, Tokyo, Japan). For the field-collected feces, fox rectum-derived cells distributed on the surface of feces were collected by washing the frozen feces with ASL buffer (QIAamp DNA Stool Mini Kit, Qiagen) following the method reported []. Then, DNA was extracted from the washing of frozen feces using QIAamp DNA Stool Mini Kit (Qiagen). On both DNA, PCR for the partial sequence of the D-loop region was performed with primers, prL (5’-CACCATTAGCACCCAAAGCT-3’) and prH (5’-CCTGAAGTAGGAACCAGATG-3’) as described previously [], and DNA sequences of PCR products were read with a DNA sequencer (Model 3100, Applied Biosystems of Life Technologies Corporation, Tokyo, Japan) using Big-Dye terminator cycle sequencing kit v3.1 (Applied Biosystems of Life Technologies Corporation). Sequences obtained were applied to BLAST similarity search (Basic Local Alignment Search Tool) for species identification.Fecal egg examination: Fecal egg examination was performed on 0.5 to 1.0 g of rectal feces or field-collected feces by the centrifugal sucrose flotation technique. When taeniid eggs were detected from the field-collected feces, the eggs were collected under a stereomicroscope for the molecular identification of their species.Molecular identification of detected parasites: DNA was extracted either from the worms detected in the intestine or from the eggs detected in feces. For cestodes and trematodes, PCR for the partial sequence of mitochondrial cytochrome oxidase c subunit 1 (CO1) gene was performed with primers, 2575 (5’-TTTTTTGGGCATCCTGAGGTTTAT-3’) and 3021 (5’-TAAAGAAAGAACATAATGAAAATG-3’) following the method reported []. For nematodes, PCR for the partial sequence of internal transcribed spacer 2 (ITS2) region was performed with primers, LC1 (5’-CGAGTATCGATGAAGAACGCAGC-3’) and HC2 (5’-ATATGCTTAAGTTCAGCGGG-3’) following the method reported []. DNA sequences of PCR products were read as described above.Statistics: The difference in the prevalence was statistically evaluated by the Fisher’s exact test and the Fisher-Freeman-Halton test (an extension of Fisher’s exact test from 2 × 2 tables to general row by column tables) using statistical software R []. P<0.05 was considered as significant. […]

Pipeline specifications

Software tools MUSCLE, BLASTN
Application Nucleotide sequence alignment
Organisms Vulpes ferrilata, Vulpes vulpes, Toxascaris leonina, Echinococcus shiquicus, Echinococcus multilocularis
Diseases Intestinal Diseases, Parasitic, Taeniasis