Western Bluetongue virus serotype 3 in Sardinia, diagnosis and characterization.

Over the last 20 years, Italy has experienced multiple incursions of different serotypes of Bluetongue virus (BTV), a Culicoides-borne arbovirus, the causative agent of bluetongue (BT), a major disease of ruminants. The majority of these incursions originated from Northern Africa, likely because of wind-blown dissemination of infected midges. Here, we report the first identification of BTV-3 in Sardinia, Italy. BTV-3 circulation was evidenced in sentinel animals located in the province of Sud Sardegna on September 19, 2018. Prototype strain BTV-3 SAR2018 was isolated on cell culture. BTV-3 SAR2018 sequence and partial sequences obtained by next-generation sequencing from nucleic acids purified from the isolate and blood samples, respectively, were demonstrated to be almost identical (99-100% of nucleotide identity) to BTV-3 TUN2016 identified in Tunisia in 2016 and 2017, a scenario already observed in past incursions of other BTV serotypes originating from Northern Africa.

Brucella suis infection in domestic pigs in Sardinia (Italy).

During a 4-year (2007-2010) survey, the presence of Brucella suis infection in domestic pigs in Sardinia was investigated. Serum samples were collected from breeding pigs located on 108 commercial farms with documented reproductive problems and analysed using the Rose Bengal (RBT) and complement fixation (CFT) tests for screening and confirmation of Brucella, respectively. Of the 1251 serum samples analysed by RBT, 406 sera, originating from 36 farms, were positive for B. suis. CFT was positive in 292/748 sera analysed, confirming positivity in all 36 pig herds. Pigs with international complement fixation test units per ml (ICFTU/ml) values ⩾160 were slaughtered, and their organs collected for bacteriological examination and testing by polymerase chain reaction (PCR). Brucella spp. strains were isolated in culture from 13/502 organs analysed, and subsequently identified as B. suis biovar 2. PCR detected positivity to Brucella spp. in 19/285 organs analysed. These results confirm the presence and emergence of B. suis infection in domestic pigs in Sardinia.

Use of PCR-restriction fragment length polymorphism analysis for identification of yeast species isolated from bovine intramammary infection.

This study reports a rapid PCR-based technique using a one-enzyme RFLP for discrimination of yeasts isolated from bovine clinical and subclinical mastitis milk samples. We analyzed a total of 1,486 milk samples collected over 1 yr in south Sardinia and northern Italy, and 142 yeast strains were preliminarily grouped based on their cultural morphology and physiological characteristics. Assimilation tests were conducted using the identification kit API ID 32C and APILAB Plus software (bioMérieux, Marcy l'Etoile, France). For PCR-RFLP analysis, the 18S-ITS1-5.8S ribosomal(r)DNA region was amplified and then digested with HaeIII, and dendrogram analysis of RFLP fragments was carried out. Furthermore, within each of the groups identified by the API or PCR-RFLP methods, the identification of isolates was confirmed by sequencing of the D1/D2 region using an ABI Prism 310 automatic sequencer (Applied Biosystems, Foster City, CA). The combined phenotypic and molecular approach enabled the identification of 17 yeast species belonging to the genera Candida (47.9%), Cryptococcus (21.1%), Trichosporon (19.7%), Geotrichum (7.1%), and Rhodotorula (4.2%). All Candida species were correctly identified by the API test and their identification confirmed by sequencing. All strains identified with the API system as Geotrichum candidum, Cryptococcus uniguttulatus, and Rhodotorula glutinis also produced characteristic restriction patterns and were confirmed as Galactomyces geotrichum (a teleomorph of G. candidum), Filobasidium uniguttulatum (teleomorph of Crypt. uniguttulatus), and R. glutinis, respectively, by D1/D2 rDNA sequencing. With regard to the genus Trichosporon, preliminary identification by API was problematic, whereas the RFLP technique used in this study gave characteristic restriction profiles for each species. Moreover, sequencing of the D1/D2 region allowed not only successful identification of Trichosporon gracile where API could not, but also correct identification of misidentified isolates. In conclusion, the 18S-ITS1-5.8S region appears to be useful in detecting genetic variability among yeast species, which is valuable for taxonomic purposes and for species identification. We have established an RFLP database for yeast species identified in milk samples using the software GelCompar II and the RFLP database constitutes an initial method for veterinary yeast identification.

Trichinella britovi and Trichinella spiralis mixed infection in a horse from Poland.

Trichinella infections in horses continue to represent a health problem and, despite the rarity of infection, it is necessary to continue to control properly horse meat. In 2008, a 10-year-old horse imported from Poland to Italy for consumption found to have been positive at the digestion test. Both Trichinella britovi and Trichinella spiralis larvae in a proportion of 4:1 were detected in the horse muscles. This is the first report of a mixed Trichinella species infection in a horse. The epidemiological investigation revealed that the infected horse originated from a small farm about 120km from Warsaw and the horse owner had bought the horse at a horse market. The findings suggest that the horse was fed more than once with infected meat.

Evidence of hepatitis E virus (HEV) infection in human and pigs in Sardinia, Italy.

INTRODUCTION: The aim of this study was to determine the seroprevalence of anti-HEV antibodies in humans sera and to study HEV prevalence in swine from different Sardinian farms, testing viral HEV-RNA in bile samples. METHODS: In the first six months of 2008, 532 subjects of whom 402 blood donors and 130 workers at zoonotic risk, were enrolled. Anti-HEV were determined with an enzyme linked immunosorbent assay (ELISA). In positive subjects, RNA was extracted and tested by RT-Nested-PCR. From July 2006 to March 2007, 95 bile samples were collected from randomly selected pigs. RNA was extracted from 250 microl of bile and tested by RT-Nested-PCR. RESULTS: The overall prevalence of anti-HEV antibodies was 4.3%; 5.0% among blood donors and 2.3% among workers at zoonotic risk, with no statistically significant differences between sex, age classes and occupation. The search for HEV-RNA in the subjects positive for antibodies, gave negative results. HEV genome was detected in 6 of the 95 swine bile samples tested. Sequences were clustered within the genotype 3 and are edited on GenBank under accession number: from FJ850960 to FJ850962 and from FJ883000 to FJ883002. DISCUSSION: The overall prevalence of anti-HEV shows that the virus circulates without giving origin to cases of acute hepatitis. The low prevalence value found in workers at zoonotic risk do not apparently support the hypothesis of professional risk. In this study, HEV-RNA was isolated from pigs in Sardinia for the first time confirming the role of swine as HEV reservoir and the possibility of virus transmission to humans.