Species diversity and molecular insights into phlebotomine sand flies in Sardinia (Italy)-an endemic region for leishmaniasis.

This study provides updated information on the distribution of the phlebotomine sand fly species and their genetic characterization in Sardinia, a Mediterranean island where leishmaniasis is endemic. From April to November 2017, sand flies were trapped in five different capture sites using sticky traps and light traps as collection methods, operated nearby sheep, poultry, cat, and dog shelters. Phlebotomine specimens (n = 513) collected were morphologically and molecularly identified as Phlebotomus perniciosus (249/513, 48.5%), Phlebotomus perfiliewi (236/513; 46%), and Sergentomyia minuta (28/513, 5.5%). Sand flies were collected from the second half of May to October confirming the well-defined seasonal activity, which peaks in August in Sardinia. Overall, correlation analyses indicated a significant positive association between the monthly number of sand flies collected and the mean temperature (r = 0.88, rho = 0.87, and tau = 0.69, P < 0.05), while there was non-significant, moderately negative correlation between the monthly number of sand flies collected and the monthly mean relative humidity and wind (r = - 0.22, rho = 0.02, and tau = 0.04, P > 0.05). This study provides the first data on the molecular characterization of phlebotomine sand flies in this region and confirms the presence of three sand fly species. Molecular results suggest that the morphological features used for analysis represent synapomorphic-derived characters which are shared among descendant taxa and the common ancestor.

Gongylonema infection of wild mammals in Japan and Sardinia (Italy).

The gullet worms, classical Gongylonema pulchrum and newly differentiated Gongylonema nepalensis, are prevalent in various mammals in Japan and Sardinia, Italy, respectively. The former species is cosmopolitan in distribution, dwelling in the mucosa of the upper digestive tract of a variety of domestic and wild mammals, and also humans. At present, the geographical distribution of G. nepalensis is known in Nepal and Sardinia, with the nematode having been recorded from the oesophagus of water buffaloes (Nepal), cattle, sheep, goats and wild mouflon (Sardinia). To clarify their natural transmission cycles among domestic and wild mammals, the present study analysed the ribosomal RNA gene (rDNA) and mitochondrial cytochrome c oxidase subunit 1 gene (cox1) of worms of various origins: G. pulchrum worms from sika deer, wild boars, Japanese macaques, and feral alien Reeves's muntjacs in Japan, and G. nepalensis worms from a red fox and a wild boar in Sardinia. Although the internal transcribed spacer (ITS) regions of rDNA and partial cox1 nucleotide sequences of G. pulchrum from native wild mammals in Japan were distinct from those of the worms in cattle, the worms from feral alien Reeves's muntjacs showed the cattle-type ITS genotype and cox1 cattle-I and II haplotypes. The rDNA and cox1 nucleotide sequences of G. nepalensis from a red fox in Sardinia were almost identical to those of the worms from domestic and wild ruminants on the island. The ecological interaction between domestic and wild mammals and their susceptibility to different Gongylonema spp. must be considered when trying to elucidate this spirurid's transmission dynamics in nature.

First record of Gongylonema nepalensis in domestic and wild ruminants in Europe.

The gullet worm, Gongylonema pulchrum, is cosmopolitan in distribution, infecting a variety of mammals including domestic and wild ruminants. Gongylonema nepalensis recently collected from the esophageal epithelium of water buffaloes in Nepal was separated from G. pulchrum based on its distinctly shorter left spicule relative to body length and unique nucleotide sequences of the ribosomal RNA gene (rDNA) and the mitochondrial cytochrome c oxidase subunit 1 gene (cox-1). During meat inspections at four abattoirs on Sardinia Island, Italy, 25 Gongylonema worms were collected from one each individual of cattle (n=8), sheep (n=7), goats (n=4), and mouflon (Ovis aries musimon; n=6), and characterized morphologically and genetically. Intriguingly, all of the collected worms from these ruminants were G. nepalensis, exhibiting comparable body lengths to G. pulchrum in cattle from other regions but with significantly shorter left spicules like G. nepalensis (less than 20.9% of the entire body length in contrast to 21.8-65.6%, the reported proportion of G. pulchrum). Furthermore, the rDNA nucleotide sequences of these worms from different ruminant species on Sardinia Island were almost identical to each other and to Nepalese G. nepalensis isolates. With the exception of one worm from a sheep (displaying a single nucleotide substitution), the 369-bp cox-1 nucleotide sequences of all the Sardinian G. nepalensis isolates from the different host sources were absolutely identical, forming a clade with Nepalese G. nepalensis isolates and not G. pulchrum isolates. The present study reveals that G. nepalensis is not a local parasite in the Indian subcontinent (Nepal), but instead has a certain geographical distribution in Europe and takes several ruminant species as a definitive host.

Trichinella britovi from domestic to wild animals of Sardinia, Italy.

The zoonotic nematode Trichinella britovi has been documented in animals and/or humans of the Mediterranean islands of Corsica and Sardinia since 2004. From 2005 to 2007 in the Sardinia island, several surveys had shown that T. britovi was circulating among backyard and free-ranging pigs reared in the Orgosolo municipality but all attempts had failed to detect this parasite in wild susceptible animals. The aim of the present work was to investigate the circulation of T. britovi in pigs and wildlife of the Orgosolo municipality, and of surrounding municipalities and provinces in the 2010-2014 slaughtering/hunting seasons. The results show that the T. britovi circulation was still restricted to the Orgosolo municipality with a prevalence of 2.6% in free-ranging pigs and 0.2% in backyard pigs but, for the first time, this parasite was detected also in 0.4% of wild boar, and 27.6% of red foxes. No infection was detected in backyard pigs, wild boar, and red foxes of the other municipalities and provinces. Since 1978, African swine fever is endemic in Sardinia and foci of this virus are still active in the investigated areas favoring cannibalism and, consequently, the T. britovi transmission, due to the high mortality rate caused by this virus. This is the first documented report on the transmission of T. britovi between the domestic and the sylvatic cycle. The health authority of the island must provide a service to dispose animal carcasses and offal, stamping out illegal free-ranging pigs, and train hunters and pig owners to manage waste and by-products according to the EU regulations.

Simultaneous infection by four feline lungworm species and implications for the diagnosis.

Besides Aelurostrongylus abstrusus, other parasites belonging to the superfamily Metastrongyloidea, namely Oslerus rostratus, Troglostrongylus brevior and to the family Trichuridae, i.e. Eucoleus aerophilus (syn. Capillaria aerophila), have also been reported as agents of respiratory infection in domestic cats. A case of simultaneous infection by four feline lungworm species in Sardinia is herein described. An adult female cat (Felis silvestris catus), road-killed in the southeast part of Sardinia (municipality of Villacidro, province of Cagliari), Italy, was referred to the Laboratory of Parasitology of the Veterinary Teaching Hospital in Sassari. At necropsy, the lungs were examined and dissected under a stereomicroscope for the presence of parasites, and first-stage larvae (L1) of broncho-pulmonary nematodes were searched for in a faecal sample using the Baermann method. Parasites collected in the lungs were morphologically identified as A. abstrusus, E. aerophilus, and O. rostratus. In addition to the above species, L1s of Troglostrongylus spp. were detected at coproscopy but no adult specimen was found in the lungs. The morphological identification was confirmed by the molecular amplification and sequencing of cox1 mitochondrial gene, 18S and ITS2 ribosomal DNA. This finding stands as the first simultaneous infection by four feline lungworm species in the same animal, and as the first report of O. rostratus and E. aerophilus in Sardinia.