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Canine parvovirus (CPV-2) modified-live virus vaccine strain can replicate in lymphoid tissues and intestinal mucosa after administration, being shed through canine faeces. Detection of vaccine strains has been reported in the bloodstream and faeces, potentially interfering with molecular diagnostic tests. The persistence of these strains in canine tissues has not yet been described. With this aim, canine tissues were tested during a molecular survey to screen for the presence of canine enteric viruses. Tissue samples from 165 dead dogs were tested by a conventional PCR assay. Positive samples and five commercial vaccines were subjected to sequence analysis. Vaccinal strains were detected and virus load was measured by using a set of real-time PCR assays using minor-groove binder (MGB) probes. Seventy-five dogs (45.4%) tested positive for CPV-2. Strains from 70 dogs were characterised as field variants. The presence of CPV sequences of vaccine origin was observed in the spleen, intestine, and mesenteric lymph nodes of five young dogs. Vaccinal strains were detected from 12 to 24 days after the last vaccine administration. Viral loads comprised between 6.3 × 10

Some parvoviruses of carnivorans can infect multiple host species. Since many canine parvoviruses were only discovered recently, their host-range is still unexplored. We examined the host distribution and diversity of five dog parvoviruses in four canine populations from Newfoundland and Labrador, Canada, and investigated the potential for these viruses to cross the species barriers. Canine bocavirus 2 (CBoV-2) and the minute virus of canines were detected in stool from free-roaming dogs from Labrador (5/48 [10.4%] and 3/48 [6.3%], respectively) and two different CBoV-2 variants were identified. Canine bufavirus was identified in stool from free-roaming dogs (1/48, 2.1%) and foxes (3/80, 3.8%) from Labrador, but two different variants were observed in the two host species. The variant found in foxes was highly divergent from previously identified strains. Two cachavirus 1 variants, genetically similar to those circulating in other Canadian wildlife, were found in spleens from Newfoundland coyotes (3/87, 3.5%). Canine parvovirus type 2 (CPV-2) was found in stool from free-roaming dogs from Labrador (2/48, 4.2%) and in spleens from Newfoundland coyotes (3/87, 3.5%). Comparing CPV-2 sequences from these hosts to those retrieved from local symptomatic domestic dogs revealed the presence of a highly heterogeneous viral population as detected strains belonged to five different clades. The close relationship between CPV-2a strains from a dog and a coyote suggests the occurrence of viral transfer between wild and domestic canids. The identification of highly related strains with a similar molecular signature characteristic of older CPV-2 strains in free-roaming and domestic dogs suggests a probable common ancestry and that older CPV-2 strains, which have not been identified in dogs since the 1990s, persist in this part of Canada. Follow-up studies should evaluate samples from a larger number of animals and host species to extensively investigate the possible occurrence of cross-species transmission for recently discovered parvoviruses.

Canine parvovirus type 2 (CPV‐2) emerged as dog pathogen in the late 1970s, causing severe and often fatal epizootics of gastroenteritis in the canine population worldwide. Although to date CPV‐2 is circulating in all continents, most of the current studies have analysed the amino acid changes accounted in the VP2 gene sequence, with limited information on virus introductions from other countries. The aim of this study was to analyse the genetic features of CPV‐2c strains currently spreading in Italy. Swabs and tissue samples were collected from dogs suspected of CPV infection. The nearly complete genome sequence from the CPV‐positive samples was obtained. The co‐circulation of two different but related CPV‐2c strains, with amino acid changes characteristic of CPV strains of Asian origin (NS1: 60V, 544F, 545F, 630P – NS2: 60V, 151N, 152V ‐ VP2: 5A/G, 267Y, 297A, 324I, 370R), were observed. The phylogenetic analyses inferred from the NS1 and VP2 gene sequences confirmed the relationship with Asian CPV‐2c strains. This study reports the spread of novel CPV‐2c mutants in Italy and supports further studies to evaluate the coexistence of genetically divergent CPV strains in the same geographical environment.

Background Toxoplasmosis is one of the most frequent parasitic infections in animals causing reproductive disorders and thus notable economic losses in productivity. Among food animals, pigs along with sheep and goats possess the highest incidence of Toxoplasma gondii cysts in meat, and play a role as a source of human infection. Methods The commercial ELISA kit (PrioCHECK® Toxoplasma Ab SR, Prionics Schlieren-Zurich, Switzerland) for the detection of specific antibodies against T. gondii in swine serum was compared with a commercial IFAT (indirect fluorescent antibody test) (Toxo-Spot IF, bioMerieux, France), used as the reference test. Results The kappa value obtained comparing the results performed on sera by ELISA with the by IFAT was 1. By a receiver operating characteristics curve analysis, the commercial ELISA had a relative sensitivity of 100%, and a relative specificity of 100% respect to IFAT. Conclusions The commercial ELISA showed a very good agreement with the commercial IFAT in the detection of serum antibodies to Toxoplasma in pigs. Our study confirmed the usefulness of the commercial ELISA kit for the detection of antibodies against T. gondii in pigs, representing a valuable tool to improve the diagnostic activity for T. gondii in swine populations at the farm level or at the slaughterhouse, contributing to the control of this widespread infection.

Objective. Biomaterial research for soft tissue augmentation is an increasing topic in aesthetic medicine. Hyaluronic acid (HA) fillers are widely used for their low invasiveness and easy application to correct aesthetic defects or traumatic injuries. Some complications as acute or chronic inflammation can occur in patients following the injection. Biocompatibility assays are required for medical devices intended for human use, in order to prevent damages or injuries in the host. In this study, nine HA fillers were tested in order to evaluate their cytotoxicity and their effects on L929 cell line, according to the UNI EN ISO 10993 regulation. Methods. Extracts were prepared from nine HA fillers, and MTS viability assay was performed after 24 h, 48 h, and 72 h of exposure of cells to extracts. Cells cultured with HA filler extracts were monitored for up to 72 h, counted, and stained with haematoxylin/eosin in order to evaluate the cell proliferation rate and morphology. Results. None of the filler tested showed a cytotoxic effect. Two samples showed a higher vitality percentage and higher cell number while two samples showed a lower vitality percentage and lower cell number at 72 h. Conclusion. Data obtained suggest that although examined fillers are not cytotoxic, they show different effects on the in vitro cell proliferation rate. In vitro studies of medical devices could lead to important implications since these could aid to predict effects about their in vivo application. These easy and rapid assays could be useful to test new materials intended for human use avoiding animal tests.

Objective. The aim of this study was to evaluate the cytotoxic potential of a type of endodontic pin on L929 cell line according to the UNI EN ISO 10993/2009 rule. Methods. L929 cells were used for the assays; extracts were prepared from three different-diameter endodontic pins, made of epoxy resin and fiberglass matrix and from Reference Materials (ZDEC, ZDBC, and HDP films). MTS assay was performed after 24 h, 48 h, and 72 h of exposure of L929 cells to pin and Reference Material extracts, 5% phenol solution, and control reagent. Cells cultured with different media containing extracts were monitored for up to 72 h and stained with haematoxylin/eosin. Results. Pins of different diameters had no cytotoxic effects on L929 cells at 24 h, 48 h, and 72 h (all values >70%). Cells cultured in medium containing pin extracts grew without any differences compared to the control cells. Conclusion. The endodontic pins tested showed no cytotoxic effects and did not induce changes in morphology for up to 72 h.

Since its emergence in Nigeria, canine parvovirus type 2 (CPV-2) infection has posed problems to dog breeding and requires constant awareness and monitoring. In this study, the status, the assessment of extrinsic risk factors of parvoviral infection in dog kennels in North Central Nigeria, and isolation of the CPV-2 were carried out. Potential risk factors were considered during sampling: age, breed, sex, location, vaccination and health status, using well-structured questionnaires on dog owners with experience of CPV-2 infection. There was high prevalence which depended on age, breed, location, clinical status of the dog while vaccination status of the dogs did not influence the prevalence. CPV-2 vaccination compliance by the breeders and management system of the kennels were also observed as risk factors. Isolation of CPV-2a and -2c strains from Nigeria for further study has been reported. The spread of CPV-2 in Nigeria is increasing, hence needs for continual epidemiological monitoring and review.

Background Canine parvovirus type 2 (CPV-2) is the most important enteric virus infecting canids. It is a rapidly evolving virus; after its emergence in the 1970s, new antigenic variants (called CPV-2a, 2b and 2c) emerged and replaced the original antigenic type. The three antigenic variants are globally distributed with different frequencies and levels of genetic variability. This study focused on VP2 gene sequence analysis and the phylodynamics of CPV-2 which were detected in 123 dogs showing clinical signs of gastroenteritis collected in Italy from 1994 to 2017. Results For the most part, the sick dogs were young, and a third of them (32.5%) had been vaccinated. No statistical association was found between the CPV-2 antigenic variants, and sex, age, breed and vaccination status. Sequence analysis showed that all three antigenic types circulated in Italy; the CPV-2a type was the prominent genotype, followed by CPV-2c and CPV-2b, with notable differences regarding regional bases and significant fluctuations over time. Nucleotide sequence data showed high genetic heterogeneity with 67 nucleotide sequence types (ntSTs) identified, corresponding to 21 amino acid sequence types (aaSTs). The aaSTs and ntSTs obtained were distributed differently among the three CPV-2 antigenic variants: CPV-2a grouped 12/21 (57.1%) aaSTs and 41/67 (61.2%) ntSTs; CPV-2b grouped 5/21 (23.8%) aaSTs and 6/67 (8.9%) ntSTs, and CPV-2c grouped 4/21 (19.1%) aaSTs and 20/67 (29.9%) ntSTs. Canine parvovirus 2a was characterised by the highest genetic variability while CPV-2c was characterised by notable stability with a predominant amino acid profile during the entire sampling time. Canine parvovirus 2b re-emerged in recent years, showing a new and distinctive amino acid profile of the VP2 protein. Conclusions The findings of the present study provided new insights regarding the phylodynamics and evolution of CPV-2 in Italy, pointing out notable differences at the local level in the distribution of the CPV-2 variants and the selection of genetic subtypes. The evolution of CPV-2 has raised questions regarding the efficacy of vaccination; therefore, continuous monitoring regarding the evolution and spread of new CPV-2 variants should be a key aim of ongoing research.

Objectives: Enteric viruses are responsible for foodborne and waterborne infections affecting a large number of people. Data on food and water viral contamination in the south of Italy (Sicily) are scarce and fragmentary. The aim of this study was to evaluate the presence of viral contamination in food, water samples, and surface swabs collected in Sicily Methods: The survey was conducted on 108 shellfish, 23 water samples (seawater, pipe water, and torrent water), 52 vegetables, one peach and 17 berries, 11 gastronomic preparations containing fish products and/or raw vegetables, and 28 surface swabs. Hepatitis A virus (HAV), genogroup GI, GII, and GIV norovirus (NoV), enterovirus (EV), rotavirus (RoV), hepatitis E virus (HEV), adenovirus (AdV), and bocavirus (BoV) were detected by nested (RT) PCR, real-time PCR, and sequence analysis. Results: The most frequently detected viruses in shellfish were HAV (13%), NoV (18.5%), and EV (7.4%). Bocavirus was found in 3.7%, HEV in 0.9%, and AdV in 1.9% of the molluscs. Of the 23 water samples, 21.7% were positive for GII NoV and 4.3% for RoV and HEV genotype 3. Of the 70 vegetable samples, 2.9% were positive for NoV GI (GI.5 and GI.6), 2.9% for EV, and 1.4% for HEV. In the gastronomic preparations, only one EV (9%) was detected. No enteric viruses were detected in the berries, fruit, or swabs analyzed. Conclusions: Molecular surveillance of water and food samples clearly demonstrated that human pathogenic viruses are widely found in aquatic environments and on vegetables, and confirmed the role of vegetables and bivalve molluscs as the main reservoirs. Keywords: Enteric virus, PCR, Genotyping, Shellfish, Waters, Vegetables

The genus Pestivirus, which belongs to the Flaviviridae family, includes ssRNA+ viruses responsible for infectious diseases in pigs, cattle, sheep, goats and other domestic and wild ruminants. Like most of the RNA viruses, pestivirus has high genome variability with practical consequences on disease epidemiology, diagnosis and control. In addition to the officially recognized species in the genus Pestivirus, such as BVDV-1, BVDV-2, BDV and CSFV, other pestiviruses have been detected. Furthermore, most of the ruminant pestiviruses show low or absent species specificity observed in serological tests and are able to infect multiple species. Particularly, small ruminants are receptive hosts of the most heterogeneous group of pestiviruses. The aim of this study was to carry out the molecular characterization of pestiviruses isolated from sheep and goats in Sicily, Italy. Phylogenetic analysis of two viral genomic regions (a fragment of 5'-UTR and the whole Npro regions) revealed the presence of different pestivirus genotypes in the analysed goat and sheep herds. Two of five viral isolates were clustered with BVDV-1d viruses, a strain widespread in Italy, but never reported in Sicily. The other three isolates formed a distinct cluster with high similarity to Tunisian isolates, recently proposed as a new pestivirus species. This represents the first evidence for Tunisian-like pestivirus presence in small ruminants in Italy. Furthermore, one of the isolates was collected from a goat, representing the first isolation of Tunisian-like pestivirus from this species.

Background: West Nile Virus (WNV) is a virus included in the Japanese encephalitis sero-complex within the genus Flavivirus. In August 2010, cases of West Nile disease were reported for the first time in Sicily. Neurological symptomswere observed in native horses resident in the rural areas around the province of Trapani, in the western part of the island. During the epidemic, important critical questions onwhen the viruswas introduced in the area and aboutwhether the virus had circulated/was circulating in theurban area, emerged and needed to be answered. A retrospective study using dog serum samples was designed to answer these questions. Methods: Between January 2009 and September 2010, 182 sera were collected by public and private vets and examined for the presence of WNV neutralising antibodies by using neutralisation assays. All samples belonged to urban dogs hosted in animal shelters or staying in vet surgeon located in towns of the western area of Sicily. Results: Out of the 182 samples tested, 11 (6.0%) had WNV neutralising antibodies with titres ranging from 1:10 to 1:80. A prevalence of 2.7% (n=74) was found in dogs sampled in 2009 whereas a prevalence of 8.3% (n=108) in the animals sampled in 2010. The highest prevalence (12.9%) was found in samples collected in the moth of September 2010 (n=31). WNV neutralizing titers were also found in a dog sampled in May 2009. There was a complete correspondence between areas with positive dogs and areas where the WND horse cases occurred. Conclusion: WNV infected dogs have been frequently detected in serological surveys. Even though not playing a major role in the epidemiology of WNV, dogs have been shown to be incidentally involved in the maintenance of the virus. This WNV retrospective study on dog sera was extremely useful to prove that WNV was in Sicily far before the first reported clinical case and that the virus had circulated/was circulating in the urban areas. It confirms the importance of these domestic animals as sentinels for either early detection or monitoring the virus activity in urban and sub-urban areas with possible threat to human health.

Summary Canine parvovirus (CPV) is an important infectious agent of domestic and wild carnivores, responsible for severe and often fatal haemorrhagic gastroenteritis and leukopenia. This paper reports the genomic characterization of a CPV strain collected from a dog recently imported to Italy from Thailand. The virus was detected in all tissue samples collected. The whole genome encompassing the two open reading frames encoding for non‐structural (NS1/NS2) and structural (VP1/VP2) proteins was amplified and sequenced. On the basis of genetic analysis of the VP2 gene, the isolate was characterized as CPV‐2c, but it presented genetic signatures typical of Asian strains. Sequence analysis revealed the presence of amino acid changes never observed in European CPV‐2c strains (NS1: Ile60Val, Tyr544Phe, Glu545Val, Leu630Pro; VP2: Ala5Gly, Phe267Tyr, Tyr324Ile, Gln370Arg). By phylogenetic analysis of full‐length VP2 gene, the analysed strain clustered together with Asian viruses. Therefore, a possible introduction of the virus from Asia through the imported dog was suggested, thus confirming the important role of movement of dogs in the global spread of viruses. In addition, full‐length genome analysis could help better trace the spread of canine viruses through different continents.