Individual genotyping of red fox field stools from the analysis of 14 microsatellite targets and one sex marker and the research of Echinococcus multilocularis DNA presence.

Description

Individual genotyping of red fox stools sampled on the field, from the analysis of 14 microsatellite targets (AHT121, AHT137, C01.424, INU055, AHTh171, C08.618, CPH2, FH2010, C04.140, CXX0279, FH2848, REN169O18, CPH11, FH2457) and one sex marker (K9-AMELO). Em(+): Echinococcus multilocularis DNA detection in faeces, qPCRVv: qPCR for Vulpes Vulpes DNA detection on part of the CytB gene (Knapp et al., 2016) and Cq values, ND: no data. First the fox faeces were identified using a host faecal qPCR technique from total copro-DNA extracts, as previously described (Knapp et al., 2016). The study was conducted from March 2017 to January 2020 in a grassland area of 3.18 Km² in the village of Les Alliés (006°26’46”E, 46°56’53”N, Fig. 1) in eastern France, at an average altitude of 971 m above sea level. The QIAmp Fast DNA Stool kit (Qiagen, Hilden, Germany) was used to extract genomic DNA from faecal samples. Fragment size analyses were performed to obtained allele size quantification for the 14 microsatellites and the sex marker, by using a SeqStudio Genetic Analyzer (Applied Biosystems, Foster City, CA). Genetic profiles were determined using the Microsatellite Analysis module available on the Thermo Fisher cloud (https://apps.thermofisher.com/editor-web/#/app/app-microsatellites-web). Copro-DNA extracts from foxes were tested for the presence of E. multilocularis (Em(+)) using a specific E. multilocularis-qPCR, as previously described. References Breen, M., Jet al., 2001. Chromosome-specific single-locus FISH probes allow anchorage of an 1800-marker integrated radiation-hybrid/linkage map of the domestic dog genome to all chromosomes. Genome Res. 11, 1784–1795. Fredholm, M., Winterø, A.K., 1995. Variation of short tandem repeats within and between species belonging to the Canidae family. Mamm. Genome Off. J. Int. Mamm. Genome Soc. 6, 11–18. Holmes, N.G., et al., 1995. Eighteen canine microsatellites. Anim. Genet. 26, 132–133. Ichikawa, Y., et al., 2002. Identification and characterization of 40 dinucleotide microsatellites in the dog genome. Anim. Genet. 33, 400–401. Knapp, J., et al., 2016. Development of a Real-Time PCR for a Sensitive One-Step Coprodiagnosis Allowing both the Identification of Carnivore Feces and the Detection of Toxocara spp. and Echinococcus multilocularis. Appl. Environ. Microbiol. 82, 2950–2958. Moore, M., et al., 2010. Thirty-one short red fox (Vulpes vulpes) microsatellite markers. Mol. Ecol. Resour. 10, 404–408. Ostrander, E.A., et al., 1993. Identification and characterization of dinucleotide repeat (CA)n markers for genetic mapping in dog. Genomics 16, 207–213.

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Individual genotyping of red fox stools sampled on the field, from the analysis of 14 microsatellite targets (AHT121, AHT137, C01.424, INU055, AHTh171, C08.618, CPH2, FH2010, C04.140, CXX0279, FH2848, REN169O18, CPH11, FH2457) and one sex marker (K9-AMELO). Em(+): Echinococcus multilocularis DNA detection in faeces, qPCRVv: qPCR for Vulpes Vulpes DNA detection on part of the CytB gene (Knapp et al., 2016) and Cq values, ND: no data. First the fox faeces were identified using a host faecal qPCR technique from total copro-DNA extracts, as previously described (Knapp et al., 2016). The study was conducted from March 2017 to January 2020 in a grassland area of 3.18 Km² in the village of Les Alliés (006°26’46”E, 46°56’53”N, Fig. 1) in eastern France, at an average altitude of 971 m above sea level. The QIAmp Fast DNA Stool kit (Qiagen, Hilden, Germany) was used to extract genomic DNA from faecal samples. Fragment size analyses were performed to obtained allele size quantification for the 14 microsatellites and the sex marker, by using a SeqStudio Genetic Analyzer (Applied Biosystems, Foster City, CA). Genetic profiles were determined using the Microsatellite Analysis module available on the Thermo Fisher cloud (https://apps.thermofisher.com/editor-web/#/app/app-microsatellites-web). Copro-DNA extracts from foxes were tested for the presence of E. multilocularis (Em(+)) using a specific E. multilocularis-qPCR, as previously described. References Breen, M., Jet al., 2001. Chromosome-specific single-locus FISH probes allow anchorage of an 1800-marker integrated radiation-hybrid/linkage map of the domestic dog genome to all chromosomes. Genome Res. 11, 1784–1795. Fredholm, M., Winterø, A.K., 1995. Variation of short tandem repeats within and between species belonging to the Canidae family. Mamm. Genome Off. J. Int. Mamm. Genome Soc. 6, 11–18. Holmes, N.G., et al., 1995. Eighteen canine microsatellites. Anim. Genet. 26, 132–133. Ichikawa, Y., et al., 2002. Identification and characterization of 40 dinucleotide microsatellites in the dog genome. Anim. Genet. 33, 400–401. Knapp, J., et al., 2016. Development of a Real-Time PCR for a Sensitive One-Step Coprodiagnosis Allowing both the Identification of Carnivore Feces and the Detection of Toxocara spp. and Echinococcus multilocularis. Appl. Environ. Microbiol. 82, 2950–2958. Moore, M., et al., 2010. Thirty-one short red fox (Vulpes vulpes) microsatellite markers. Mol. Ecol. Resour. 10, 404–408. Ostrander, E.A., et al., 1993. Identification and characterization of dinucleotide repeat (CA)n markers for genetic mapping in dog. Genomics 16, 207–213.

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