Taeniidae, Ludwig, 1886

Poglayen, Giovanni, Gori, Francesca, Morandi, Benedetto, Galuppi, Roberta, Fabbri, Elena, Caniglia, Romolo, Milanesi, Pietro, Galaverni, Marco, Randi, Ettore, Marchesi, Barbara & Deplazes, Peter, 2017, Italian wolves (Canis lupus italicus Altobello, 1921) and molecular detection of taeniids in the Foreste Casentinesi National Park, Northern Italian Apennines, International Journal for Parasitology: Parasites and Wildlife 6 (1), pp. 1-7 : 3

2.5. Taeniidae identi fi cation

From the ISPRA genetic bank 130 specimens belonging to 54 wolves, chosen according to the genetic profile (not dog or wolf x dog hybrid) and to abundance of material, were examined for taeniidae eggs .

Up to 2 g of feces were sieved in a filter (mesh 150 M m) and washed several times in a cup. The filtrate was centrifuged (1600 × g) for ten minutes and the pellet collected. Taeniid eggs were isolated from the pellet using the flotation and sieving method described by Mathis et al. (1996) and subjected to morphological identification under an inverted microscope. In egg positive samples and in 14 of the negative samples, DNA extraction was carried out with the complete sieving fraction as described by ̆Stefani ć et al. (2004). The 14 negative samples were included as negative controls. A total of 69 samples were analyzed using a multiplex-PCR to discriminate between E. granulosus and E. multilocularis and other cestodes including Taenia spp. ( Trachsel et al., 2007). To obtain clear sequences of the E. granulosus positive samples, the PCR was repeated but only using E. granulosus primers (Cest5 and Cest4) keeping the same conditions as described above, and therefore amplicons were sequenced. Another PCR targeting nad1 gene ( Armua-Fernandez et al., 2011) was used in samples without clear sequencing results to confirm the species of those samples. The amplicons were directly sequenced after purification of the PCR products using the MinElute ® PCR purification kit (Qiagen). Sequencing was performed by Synergene Biotech GmbH, Biotech Center Zurich, Switzerland (http://www.synergenebiotech.com). Primer Cest5 and Cest5seq was used for non- Echinococcus cestodes including Taenia spp. and Cest5 for E. granulosus , while primer nad1 T-Rv for Taenia spp. for amplicons obtained with multiplex-PCR and nad1 PCR, respectively. Sequences were compared with the one present in GenBank using Blast tool (http:// www.blast.ncbi.nlm.nih.gov).