• biomolecular techniques
• diagnosis
• dog
• epidemiology
• fox
• helminths
• SEM
• Vulpes vulpes

An epidemiological survey on the helminths of 165 foxes and 450 rural dogs from N-W Italy (Imperia and Savona districts) was conducted between 2010 and 2012. Foxes’ cardiorespiratory system, gastrointestinal tract, liver, urinary apparatus, muscle tissue and rectal faecal samples were examined. For each dog faecal and blood samples were collected: feacal samples were examined by centrifugal floatation and by Baermann technique, blood samples were Knott test, serological examination for antigens of Dirofilaria immitis, histochemical staining and PCR. Dogs’ serum samples were also tested with two newly developed ELISA tests for the detection of circulating A. vasorum antigens and specific antibodies. Several species of Trichuridae nematodes found during the study were examined by scanning electron microscopy (SEM) (applied on eggs) and were subjected to biomolecular techniques to characterize parts of the ribosomal and mitochondrial DNA (for adults and eggs), in order to investigate morphological and genetic aspects of this complex group of nematodes.
A wide variety of parasites were found in foxes at necropsy: Angiostrongylus vasorum (78%), Eucoleus aerophilus (42%), Eucoleus boehmi (1 of 2 foxes), Crenosoma vulpis (16%) and Filaroides sp. (5%) in the cardiopulmonary tract; Spirocerca lupi (23.5%), Physaloptera spp. (2.5%), Uncinaria stenocephala (70%), Toxascaris leonina (27%), Toxocara canis (25%), Molineus legerae (24%), Trichuris vulpis (21%), Aonchoteca putorii (9%), Pterygodermatites affinis (5%), cestodes of the genus Mesocestoides (82%), of the genus Taenia (5.5%), of the family Dilepididae (27%) and one trematode species (Plagiorchidae) (0.6%) in the gastrointestinal tract and Pearsonema plica (57%) in the urinary bladder. Echinococcus multilocularis, Trichinella spp. and filarial infections were not found. In dogs’ coprology larvae of A. vasorum (0.6%) and C. vulpis (0.3%) and eggs of E. aerophilus (9.6%), E. boehmi (1.6%), T. canis (20%), T. vulpis (18%), Ancylostomatidae (12%), Capillaria spp. (1.8%), Physaloptera spp. (0.2%), Hymenolepis diminuta (1.1%) and Dipylidium caninum (0.2%) were observed. D. immitis (1.7%), D. repens (1%) and Acanthocheilonema reconditum (7%) were found in dogs, in a region historically free from filariosis.In a subset of dogs (n=347) which were contemporaneously tested with the Baermann method and with the ELISA tests for A. vasorum, a higher prevalence was found with serology (0.3% with the Baermann method, 0.6% with the ELISA tests). No cross reactions were observed with the serological tests in those dogs known to be positive for other parasitic infections; serology proved to be a valid alternative diagnostic technique for A. vasorum.
SEM allowed distinguishing differences in the external structure of the eggs of several Trichuridae nematodes (E. boehmi, E. aerophilus, P. plica, Calodium hepaticum and T. vulpis). A part of the small subunit rRNA (18S rRNA) gene and of the mitochondrial cytochrome oxidase subunit I (mt COI) gene was amplified from specimens of T. vulpis, E. aerophilus, E. boehmi, P. plica, A. putorii, C. hepaticum (found in the liver of a fox from Cuneo and of a vole from Switzerland) and Calodium splenaecum (in the spleen of a Crocidura sp. from Switzerland), yielding the first 18S rRNA gene sequences of all the capillariid nematodes and the first mt COI sequences of E. boehmi, P. plica, C. hepaticum, A. putorii and T. vulpis. The 18S rRNA gene is highly conserved among the different species, while interspecies divergence was much higher at the mtCOI gene locus, making it suitable for resolving closely related species and for the future development of Trichuridae species-specific primers.