Abstract
Echinococcus multilocularis is one of the most relevant zoonotic parasites with about 18,000 human cases per year. Its detection in wild host is crucial for disease prevention. The present study aimed to determine factors affecting the sensitivity of E. multilocularis detection by PCR using DNA extracted from fecal samples of coyotes (Canis latrans). Fecal samples were screened for the presence of Taeniidae eggs through centrifugation and sedimentation. DNA was extracted from fecal samples with and without prior freeze-thawing of the sample and then subjected to PCR targeting a mitochondrial gene (nad1) and a multi-loci microsatellite marker (EmsB). The presence of PCR inhibitors was determined through internal amplification control. Subjecting the sample to repeated freeze-thaw cycles significantly increased the sensitivity of the PCR by 20 %. Likewise, egg intensity had a significant effect on PCR, an effect which was more pronounced for samples not subjected to freeze-thawing. Two or more eggs per gram of feces significantly increased the odds for a positive PCR outcome. The presence of PCR inhibitors had no effect on PCR in samples subjected to freeze-thaw cycles, whereas in samples not subjected to freeze-thaw cycles, the presence of PCR inhibitors was associated with a 0.78 lower odds ratio of positive PCR outcome. Targeting a nuclear versus a mitochondrial gene did not have a significant effect on the sensitivity of PCR. We recommend freeze-thawing samples prior to DNA extraction to become a standard procedure for E. multilocularis detection in canid feces.
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Acknowledgments
We thank Drs. N Neuman and G. Banting for their support with the plasmid construct. We thank all the undergraduate students that participated in our study for their passion and commitment.
Canadian Council of Health Research CIHR (funding reference, 201011GIR-239508-IRG-CBBA-51314 dt 24/06/2011), Institute for Public Health of the Alberta Health, and Alberta Innovates Health Solutions (AIHS) contributed to operational funding. Student salary was covered by studentships of the University of Calgary, Department of Ecosystem & Public Health (Faculty of Veterinary Medicine, University of Calgary), andAlberta Innovates Health Solutions, The City of Calgary Parks covered part of the operational costs and provided in-kind contribution and logistic support.
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The authors declare that they have no competing interests.
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The material presented in this paper has not been published or submitted for publication elsewhere, and there is no overlap with other articles or abstracts published or in press in journals, books, or in preparation. The research has been approved by the Veterinary Science Animal Care Committee of the University of Calgary, AB, Canada (protocol number AC13-0199).
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Klein, C., Liccioli, S. & Massolo, A. Egg intensity and freeze-thawing of fecal samples affect sensitivity of Echinococcus multilocularis detection by PCR. Parasitol Res 113, 3867–3873 (2014). https://doi.org/10.1007/s00436-014-4055-x
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DOI: https://doi.org/10.1007/s00436-014-4055-x