SUMMARY: In order to describe the molecular epidemiology of Blastocystis infection in Italy, 189 isolates, which had been collected during the years 2012–2014 from mildly symptomatic patients, or those affected by inflammatory bowel disease (IBD), irritable bowel syndrome (IBS) or chronic diarrhoea, or otherwise immunosuppressed, were subtyped by sequence analysis of the SSU rRNA gene (536 bp). Six subtypes (STs) were detected: ST1 (15·3%), ST2 (13·8%), ST3 (46·0%), ST4 (21·7%), ST6 (3·2%) and ST8 (0·5%). They clustered in distinct clades, as inferred from Bayesian inference phylogenetic and median joining network analyses. A high genetic differentiation was found at the inter-subtype level; it ranged from Jukes–Cantor (JC) distance = 0·02 (between ST1 and ST4) to JC = 0·11 (between ST6 and ST2). At the intra-ST level, a high genetic homogeneity was registered in ST4, whereas higher genetic variation was found in isolates corresponding to ST1 and ST2. Accordingly, high values of haplotype and nucleotide diversity were observed in ST1, ST2 and ST3. No association was found between patient gender and ST, whereas ST3 and ST1 were significantly more prevalent in patients aged 15–50 years. A significant occurrence of Blastocystis ST4 in patients suffering from IBS, IBD or chronic diarrhoea was observed; in addition, a slight significant association between ST1 and ST3 and IBS patients was found. Multiple correspondence analysis showed some significant contribution of different variables (subtypes, haplotypes, age) in the observed pattern of ordination of the 189 patients in the symptom categories.

Blastocystis is a common intestinal protist distributed worldwide, infecting humans and a wide range of domestic and wild animals. It exhibits an extensive genetic diversity and, so far, 25 distinct small subunit ribosomal RNA (SSU rRNA) lineages termed subtypes (STs)) have been characterized; among them, 12 have thus far been reported in humans. The aims of the present study were to detect and genetically characterize Blastocystis sp. in synantropic animals to improve our current knowledge on the distribution and zoonotic transmission of Blastocystis STs in Italy. Samples were collected from N = 193 farmed animals and submitted to DNA extraction and PCR amplification of the SSU rRNA. Blastocystis was detected in 60 samples (31.08%) and successfully subtyped. Phylogenetic analysis evidenced that the isolates from fallow deer, goats, and pigs (N = 9) clustered within the ST5; those from pheasants (N = 2) in the ST6; those from chickens (N = 8) in the ST7; those from sheep (N = 6) in the ST10; and those from water buffaloes (N = 9) in the ST14 clade. The comparison between the present isolates from animals and those previously detected in humans in Italy suggested the animal-to-human spillover for ST6 and ST7. The present study represents the widest Blastocystis survey performed thus far in farmed animals in Italy. Further epidemiological studies using molecular approaches are required to determine the occurrence and distribution of Blastocystis STs in other potential animal reservoirs in Italy and to define the pathways of zoonotic transmission.

(1) Dirofilariosis is a vector-borne parasitic disease mainly in domestic and wild carnivores caused by Dirofilaria (Noctiella) repens, which is endemic in many countries of the Old World, and D. immitis, which has a worldwide distribution. In recent years, an increase in the number of human cases has been reported, suggesting that dirofilariosis is an emergent zoonosis. Here, we describe further cases (N = 8), observed in Central Italy during the years 2018–2019. (2) Molecular diagnosis was performed on: (i) live worms extracted from ocular conjunctiva, cheek, and calf muscle; (ii) histological sections of surgically removed nodules from parenchymal lung, coccyx, and breast. (3) Sequence analysis (650-bp) of the mitochondrial cytochrome oxidase subunit I gene (mtDNA cox1) showed a match of 100% with the sequences of D. repens previously deposited in GenBank. ELISA test to detect IgG against filarial antigens was performed on four patients’ sera and resulted positive in two patients who showed ocular and subcutaneous dirofilariosis, respectively. Microfilariae have been never detected in the peripheral blood of the patients. (4) The occurrence of N = 8 new cases of human D. repens-infections observed in a two-year period suggests an increased circulation of the parasite in Italy. Therefore, dirofilariosis should be included in differential diagnosis in patients presenting subcutaneous and/or pulmonary nodules. Molecular diagnosis of the etiological agents is fundamental. Specific serological diagnosis needs to be improved in future research work.