Study on the Canine Adenovirus Type 1 (CAdV-1) Infection in Domestic Dogs in Southern Italy.

Canine adenovirus type 1 (CAdV-1) is the causative agent of a systemic and potentially fatal viral disease of domestic and wild canids. In Italy, CAdV-1 infection has also been occasionally described in dogs, but information on the epidemiology and its genomic features is still limited. A study was conducted on 291 dogs suspected of infectious gastrointestinal disease. Samples collected from dogs in southern Italy between 2017 and 2020 were analyzed. Virological and histopathological assays were carried out. The presence of CAdVs and other canine viral enteropathogens was investigated, and sequence and phylogenetic analyses were performed. CAdV-1 was detected in six (2.1%) dead stray dogs alone or in mixed infections with other viruses. Gross lesions and histopathological findings referred to CAdV infection were observed, also involving the central nervous system tissues. All inoculated samples were successfully isolated. Sequence analysis evidenced divergences with the circulating strains previously described in Italy and a closer relation with older CAdV-1 strains collected from other countries, suggesting a genetic heterogeneity of CAdV-1 in Italy. The evidence of the circulation of CAdV-1 and its genomic features allows us to have more in-depth knowledge of the epidemiology and evolution of the CAdV-1 genomic variants.

Detection and Molecular Characterization of Two Gammaherpesviruses from Pantesco Breed Donkeys during an Outbreak of Mild Respiratory Disease.

Equid and asinine gammaherpesviruses (GHVs; genus Percavirus) are members of the Herpesviridae family. Though GHVs have been reported in horse populations, less studies are available on gammaherpesviral infections in donkeys. This study reports the co-infection with two GHVs in Pantesco breed donkeys, an endangered Italian donkey breed. Samples (n = 124) were collected on a breeding farm in Southern Italy from 40 donkeys, some of which were healthy or presented erosive tongue lesions and/or mild respiratory signs. Samples were analysed by using a set of nested PCRs targeting the DNA polymerase, glycoprotein B, and DNA-packaging protein genes, and sequence and phylogenetic analyses were performed. Twenty-nine donkeys (72.5%) tested positive, and the presence of Equid gammaherpesvirus 7 and asinine herpesvirus 5 was evidenced. In 11 animals, we found evidence for co-infection with viruses from the two species. Virions with herpesvirus-like morphology were observed by electron microscopic examination, and viruses were successfully isolated in RK-13-KY cell monolayers. The histological evaluation of tongue lesions revealed moderate lympho-granulocytic infiltrates and rare eosinophilic inclusions. The detection of GHVs in this endangered asinine breed suggests the need long-life monitoring within conservation programs and reinforces the need for further investigations of GHV's pathogenetic role in asinine species.

Occurrence of Human Enteric Viruses in Shellfish along the Production and Distribution Chain in Sicily, Italy.

Contamination of bivalve mollusks with human pathogenic viruses represents a recognized food safety risk. Thus, monitoring programs for shellfish quality along the entire food chain could help to finally preserve the health of consumers. The aim of the present study was to provide up-to-date data on the prevalence of enteric virus contamination along the shellfish production and distribution chain in Sicily. To this end, 162 batches of mollusks were collected between 2017 and 2019 from harvesting areas, depuration and dispatch centers (n = 63), restaurants (n = 6) and retail stores (n = 93) distributed all over the island. Samples were processed according to ISO 15216 standard method, and the presence of genogroup GI and GII norovirus (NoV), hepatitis A and E viruses (HAV, HEV), rotavirus and adenovirus was investigated by real-time reverse transcription polymerase chain reaction (real-time-RT PCR), nested (RT)-PCR and molecular genotyping. Our findings show that 5.56% of samples were contaminated with at least one NoV, HAV and/or HEV. Contaminated shellfish were sampled at production sites and retail stores and their origin was traced back to Spain and several municipalities in Italy. In conclusion, our study highlights the need to implement routine monitoring programs along the whole food chain as an effective measure to prevent foodborne transmission of enteric viruses.

Molecular characterization of human enteric viruses in food, water samples, and surface swabs in Sicily.

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.

Evaluation of a commercial enzyme-linked immunosorbent assay (ELISA) for detecting antibodies against Toxoplasma gondii from naturally and experimentally infected pigs.

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, bioMérieux, 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.

Canine mesenchymal stem cells (MSCs): characterization in relation to donor age and adipose tissue-harvesting site.

Adipose tissue as a stem cell source is ubiquitously available and has several advantages compared to other sources, for example it is easily accessible in large quantities with minimal invasive harvesting procedure, and isolation of adipose-derived mesenchymal stem cells (MSCs) yields a high amount of stem cells, essential for stem cell-based therapies and tissue engineering. We have explored the effect of donor age, and the anatomical origin of the adipose tissue on several aspects of MSCs in dogs, such as cell yield, proliferative ability, multi-differentiation potential, colony-forming capacity, stemness marker expression. We also assessed the effect of cell passaging on the MSCs stemness. We found that the anatomical origin of the adipose tissue and the age of donors have effects only on the proliferative capacity of the MSCs. Moreover, cells show a progressive loss of the stemness characteristics with passages. Cell therapies need a suitable number of cells to use in clinical applications. Characterization of MSCs at different passages, allowed us to demonstrate that, under our culture conditions, the best quantitative and qualitative characteristics are obtained at early passages. Adult MSCs are of particular interest for the therapeutic approach to musculoskeletal diseases, and the dog provides an excellent preclinical model for the development of new approaches in regenerative medicine that might be applied to humans.

Modeling human osteosarcoma in mice through 3AB-OS cancer stem cell xenografts.

Osteosarcoma is the second leading cause of cancer-related death for children and young adults. In this study, we have subcutaneously injected-with and without matrigel-athymic mice (Fox1nu/nu) with human osteosarcoma 3AB-OS pluripotent cancer stem cells (CSCs), which we previously isolated from human osteosarcoma MG63 cells. Engrafted 3AB-OS cells were highly tumorigenic and matrigel greatly accelerated both tumor engraftment and growth rate. 3AB-OS CSC xenografts lacked crucial regulators of beta-catenin levels (E-cadherin, APC, and GSK-3beta), and crucial factors to restrain proliferation, resulting therefore in a strong proliferation potential. During the first weeks of engraftment 3AB-OS-derived tumors expressed high levels of pAKT, beta1-integrin and pFAK, nuclear beta-catenin, c-Myc, cyclin D2, along with high levels of hyperphosphorylated-inactive pRb and anti-apoptotic proteins such as Bcl-2 and XIAP, and matrigel increased the expression of proliferative markers. Thereafter 3AB-OS tumor xenografts obtained with matrigel co-injection showed decreased proliferative potential and AKT levels, and undetectable hyperphosphorylated pRb, whereas beta1-integrin and pFAK levels still increased. Engrafted tumor cells also showed multilineage commitment with matrigel particularly favoring the mesenchymal lineage. Concomitantly, many blood vessels and muscle fibers appeared in the tumor mass. Our findings suggest that matrigel might regulate 3AB-OS cell behavior providing adequate cues for transducing proliferation and differentiation signals triggered by pAKT, beta1-integrin, and pFAK and addressed by pRb protein. Our results provide for the first time a mouse model that recapitulates in vivo crucial features of human osteosarcoma CSCs that could be used to test and predict the efficacy in vivo of novel therapeutic treatments.

Production of canine mesenchymal stem cells from adipose tissue and their application in dogs with chronic osteoarthritis of the humeroradial joints.

Autologous AD-MSC [adipose-derived MSC (mesenchymal stem cell)] therapy involves harvesting fat from the patient by isolating the stem and regenerative cells and administering the cells back to the patient. This study evaluated the production of canine AD-MSCs and their possible application in cellular therapy for dogs. To assess whether cellular therapy can replace drug therapy, the clinical effect of a single intra-articular injection of AD-MSCs was evaluated on 4 dogs with lameness associated with OA (osteoarthritis) of the humeroradial joints. MSCs were readily isolated from adult dog adipose tissue, and their ability to form colony and differentiate into various phenotypes was confirmed. AD-MSCs expressed OCT4, NANOG and SOX2 at the mRNA level, pluripotency markers usually ascribed to embryonic stem cells. The results suggest the stemness of the cells isolated from canine fat, and good quality control made them available for both experimental and clinical use. Follow-up studies to evaluate the effects of AD-MSC therapy showed that OA of the elbow joints improved with time, indicating significant potential for clinical use in the treatment of lameness, particularly when administered before the injury becomes severe.