Abstract
Cystic echinococcosis, due to Echinococcus granulosus sensu lato (s. l.), currently affects three million people, especially in low-income countries and results in high livestock production loss. DNA-based methods demonstrated genetic variability of E. granulosus s. l., and five species were recognized to belong to the complex, including E. granulosus sensu stricto (s.s) (genotypes G1–G3), Echinococcus equinus (genotype G4), Echinococcus ortleppi (genotype G5), Echinococcus canadensis (genotypes G6–G10), and the lion strain Echinococcus felidis. The characterization of Echinococcus species responsible for human and animal echinococcosis is crucial to adapt the preventive measures against this parasitic disease. The sequencing approach is the gold standard for genotyping assays. Unfortunately, developing countries do not often have access to these techniques. Based on in silico RFLP tools, we described an accurate PCR-RFLP method for Echinococcus spp. characterization. The double digestion with the HaeIII and HinfI restriction enzymes of the PCR product from nad1 gene (1071 bp) led to a clear discrimination between E. granulosus s. l. and most closely related species (Echinococcus shiquicus and Echinococcus multilocularis).
Molecular procedures and phylogenetic analysis confirmed the efficiency and the reproducibility of this simple and fast PCR-RFLP method. This technique is proved useful for fresh/unfixed and FF-PET tissues and enables large-scale molecular epidemiological screening in developing countries.
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Acknowledgments
This study was supported by the Tunisian Ministry of Higher Education and Scientific Research. The authors thank the following members from the pediatric surgery team of the F. Bourguiba Teaching Hospital for providing the human cyst samples: Pr. Amine ksia, Pr. Rachida Laamiri, Pr. Mongi Mekki, and Pr. Abdelltif Nouri. We are grateful to the veterinaries from the Municipal Abattoirs of Sousse (Dr. Sofien Zmantar), Gafsa (Dr. Mabrouk Saii), Kasserine (Dr. Naiima Tlili), and Sidi Bouzid (Dr. Hamza Hajlaoui) for collecting the animal cyst samples. We thank Dr. Naglaa Abass (Friedrich-Loeffler-Institute, Greifswald, Germany) for providing the gDNA from the G6 genotype, Dr. Ghalia Boubaker and Pr. Bruno Gottstein (Institute of Parasitology, University of Bern, Switzerland) for the G4, G5, and G7 gDNAs, and Gérald Umhang and Pr. Franck Boué (Anses, National Reference Laboratory for Echinococcus spp., Nancy, France) for the E. multilocularis gDNA. We are grateful to Dr. Sara Mestiri and Pr. Moncef Mokni (Laboratories of Pathological Anatomy and Cytology, F. Hached Sousse Teaching Hospital) for giving the FF-PET isolates. We thank also Gérald Umhang (Anses, National Reference Laboratory for Echinococcus spp., Nancy, France) for critically reading the manuscript. We express our gratitude to Imen Younes for her linguistic assistance. We express our gratitude to Delphine Guillotin (Institute of Cancer Research in London) for the last check of the manuscript.
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This study was funded by the Tunisian Ministry of Higher Education and Scientific Research. The funding source is not involved in the study design; in the collection, analysis, and interpretation of the data; in the writing of the report; and in the decision to submit the article for publication.
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In this study, the human hydatid cyst sampling was approved by the ethic committee of the F. Bourguiba Teaching Hospital of Monastir (Tunisia). The cysts isolated from animals were collected during post-mortem examination following inspection by the veterinary officers at the respective slaughterhouses with their due consent. No experimentation was done on animals; therefore, no approval from institutional animal ethics committee was required.
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Chaâbane-Banaoues, R., Oudni-M’rad, M., M’rad, S. et al. A novel PCR-RFLP assay for molecular characterization of Echinococcus granulosus sensu lato and closely related species in developing countries. Parasitol Res 115, 3817–3824 (2016). https://doi.org/10.1007/s00436-016-5143-x
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DOI: https://doi.org/10.1007/s00436-016-5143-x