Knowledge on the presence of Cytauxzoon sp. and Hepatozoon spp. in Italy is scant and mostly limited to a few areas of Northern and Southern regions, respectively. The present study updated the current epidemiological scenario by investigating the occurrence of these protozoa in domestic cats from three broad regions of North-Eastern Italy. Blood samples from cats at risk of vector-borne diseases were processed by PCR to detect Cytauxzoon and Hepatozoon DNA. Blood smears were observed for haemoparasite inclusions. The influence of cat individual data (e.g., provenance, management, indoor/outdoor lifestyle) on the prevalence of haemoprotozoan infections was statistically evaluated. Among 158 cats, Cytauxzoon and Hepatozoon DNA were detected in 6 (3.8%) and 26 (16.5%) animals, respectively. No Hepatozoon gamonts were detected in blood smears, whereas all Cytauxzoon PCR-positive samples were microscopically positive, though with low levels of parasitaemia. Two species of Hepatozoon were identified, Hepatozoon felis (n = 10) and Hepatozoon silvestris (n = 16). Hepatozoon silvestris prevalence values were significantly (p < 0.05) higher in the region Friuli Venezia Giulia and in stray cats. Cytauxzoon sp. was detected in 6/39 (15.4%) stray cats from Friuli Venezia Giulia (Trieste province). These data add new information on the occurrence of these neglected protozoa in domestic cats’ populations.

Background: The Spirorchiidae is a family of blood flukes parasitizing turtles. Spirorchiids may cause a wide range of inflammatory reactions in the vascular system of their host being frequently implicated with stranding and death of sea turtles worldwide. Recent studies revealed the presence of two spirorchiid species in the Mediterranean basin. Our study presents comparative epidemiological data of spirorchiid infections in loggerhead turtles (Caretta caretta) stranded during an eight-year period from Adriatic and Tyrrhenian Seas, and the first report of Neospirorchis Neogen-11 in a green turtle (Chelonia mydas). Methods: We screened a total of 319 carcasses of loggerhead turtles stranded from January 2011 to December 2018 along the Tyrrhenian coast (n = 111) and the north-western Adriatic coast (n = 208) of Italy using traditional (copromicroscopy and histopathology) and molecular assays. Three green turtles from the Tyrrhenian coast were also included in the study. Results: A total of 56 (17.5%) loggerhead turtles and one green turtle (33.3%) were found to be infected with spirorchiid flukes. Amplification, sequencing of the ITS2 region of the ribosomal RNA gene cluster and BLAST analysis confirmed the presence of Hapalotrema mistroides and Neospirorchis Neogen-11 in 51 (16.0%) and 24 (7.5%) loggerhead turtles, respectively, and Neospirorchis Neogen-11 in an infected green turtle. Differences in prevalence of infection between the two sampling areas were found. Conclusions: The risk of spirorchiid infection in the Tyrrhenian Sea is lower than in the Adriatic Sea and in general the risk of infection in the Mediterranean is lower than in other geographical locations. Differences in the prevalence of infection between the two sampling areas were related to the differences of regional habitats supporting different abundance of spirorchiid intermediate hosts. A systematic monitoring to evaluate the progress of the infection is recommended, as well as studies on the occurrence and distribution of spirorchiid species from other Mediterranean areas.

Sulcascaris sulcata is a nematode parasite of sea turtles, widespread in neritic foraging grounds with variable prevalence, reaching 30% in loggerhead turtles Caretta caretta feeding in northern Adriatic Sea. Ulcerative gastritis associated to high intensity of infection is reported in this host species. The life cycle of S. sulcata has been elucidated in Australian and American waters, demonstrating the ability of the species of infecting a wide range of intermediate hosts, represented by bivalve and gastropod molluscs. During regular sanitary inspections, nematode larvae were found within the adductor muscle of Pecten jacobeus and Aequipecten opercularis collected from the Northern Adriatic Sea. Morphological and molecular analyses were performed for the identification of larvae, and molecular data were obtained from adult S. sulcata as well. Analysis of the sequences of ITS fragment, and mitochondrial genes cox1 and cox2, revealed high genetic similarity among all the samples, and no geographical clustering was observed between adult parasites collected in Adriatic and Tyrrhenian Seas. A common allele pool was detected also between the two developmental stages, included larvae from Pecten and Aequipecten. The comparison with other members of the family Anisakidae demonstrated that S. sulcata formed a clear monophyletic cluster. This study reports the first identification of intermediate hosts for S. sulcata within the Mediterranean Sea. Infection in edible scallops justifies the exclusion of the product from the market and zoonotic potential of larvae of this anisakid nematode are yet to be completely excluded. Fidelity of sea turtles to selected foraging grounds, such as the Northern Adriatic shelf, warrants the life cycle of S. sulcata to perpetrate in the area; at the same time, long distance migrations of individuals justify the dispersal of infecting elements over the Mediterranean basin, regardless of turtles' origin.

The red fox acts as reservoir for several helminthic infections which are of interest for both public and animal health. Huge efforts have been made for the assessment of the sensitivity of coprological tests for the detection ofEchinococcus multilocularis, while less attention has been paid to other helminthic species. This study aimed at assessing the performance of two copromicroscopic techniques in the detection and prevalence estimation of gastrointestinal helminths in the red fox. Helminths were isolated from the small intestines of 150 red foxes from Bolzano province, Italy, with a scraping, filtration and counting technique (SFCT) and morphologically identified. Rectal contents were collected and submitted to simple flotation (FT) and, only for Taenids, a method based on the concentration of eggs and identification with multiplex PCR (CMPCR). Using SFCT as a reference standard, we estimated the sensitivity of the copromicroscopic tests. Three species of nematodes (namely,Toxocara canis, Uncinaria stenocephalaandPterygodermatitessp.) and five species of cestodes (E.multilocularis, Taenia crassiceps, T. polycantha, Hydatigera taeniaeformis, Mesocestoidessp.) were identified with SFCT, whereas eggs referable to the same taxa were detected with fecal diagnostics, except forPterygodermatitessp. andMesocestoidessp. The sensitivity of FT was low for all taxa, ranging from 9.8 to 36.3%, with lower values for Taeniidae. CMPCR was confirmed to perform better for the detection of Taeniidae eggs (23.5%) and the multiplex PCR on retrieved eggs was effective in the identification of the species. A meta-analysis of literature also suggested that our results are consistent with existing data, indicating that copromicroscopy tends to underestimate the prevalence of helminthic infections. The extent of such underestimation varies with taxon, being higher at high prevalence levels, in particular for cestodes. Irregular dynamics of egg shedding, and routine deep freezing of red fox feces may explain the frequency of false negatives with copromicroscopy. Low sensitivity of copromicroscopic tests should be accounted for when estimating prevalence and when defining the correct sample size for the detection of the parasites.

Background: Parasites of the family Spirorchiidae cause disease and mortality in marine and freshwater turtles; two species, Hapalotrema mistroides and Neospirorchis sp., are reported in the resident population of loggerhead turtles of the Mediterranean Sea, with the first being the most widespread. In vivo diagnosis of spirorchidiasis can represent a challenge in guaranteeing prompt control and treatment of the disease and is currently limited to copromicroscopy. The aim of this study was the development of a real time PCR assay with TaqMan probe for the detection of H. mistroides infection in the blood of live loggerhead turtles, Caretta caretta, hospitalized in rehabilitation centres. Its potential use for in vivo diagnosis is explored. Results: The developed real time PCR successfully detected H. mistroides DNA from both positive controls and experimental blood samples of live loggerhead sea turtles, showing good specificity, sensitivity and good reaction efficiency. Two out of three turtles which had demonstrated positivity at copromicroscopy also tested positive to this blood assay; DNA of H. mistroides was detected within the blood of one sea turtle, which tested negative for copromicroscopy. Conclusions: This study describes a specific and rapid molecular assay to detect H. mistroides infection from live sea turtles and highlights for the first time the presence of DNA of this species in turtle blood samples. Since this assay is able to detect low amounts of the parasitic free DNA in blood samples, its application could be helpful for in vivo diagnosis of H. mistroides infection as well as for epidemiological purposes.