V. Cannella

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.

Extended Genetic Diversity of Bovine Viral Diarrhea Virus and Frequency of Genotypes and Subtypes in Cattle in Italy between 1995 and 2013

Genetic typing of bovine viral diarrhea virus (BVDV) has distinguished BVDV-1 and BVDV-2 species and an emerging putative third species (HoBi-like virus), recently detected in southern Italy, signaling the occurrence of natural infection in Europe. Recognizing the need to update the data on BVDV genetic variability in Italy for mounting local and European alerts, a wide collection of 5′ UTR sequences (n = 371) was selected to identify the frequency of genotypes and subtypes at the herd level. BVDV-1 had the highest frequency, followed by sporadic BVDV-2. No novel HoBi-like viruses were identified. Four distribution patterns of BVDV-1 subtypes were observed: highly prevalent subtypes with a wide temporal-spatial distribution (1b and 1e), low prevalent subtypes with a widespread geographic distribution (1a, 1d, 1g, 1h, and 1k) or a restricted geographic distribution (1f), and sporadic subtypes detected only in single herds (1c, 1j, and 1l). BVDV-1c, k, and l are reported for the first time in Italy. A unique genetic variant was detected in the majority of herds, but cocirculation of genetic variants was also observed. Northern Italy ranked first for BVDV introduction, prevalence, and dispersion. Nevertheless, the presence of sporadic variants in other restricted areas suggests the risk of different routes of BVDV introduction.

Evidence of zoonotic Poxviridae coinfections in clinically diagnosed papillomas using a newly developed mini-array test.

Our study describes a newly developed mini-array test for the rapid detection of poxviruses in animals and humans. The method is based on detection that combines target nucleic acid amplification by polymerase chain reaction and specific hybridization, using enzyme-linked antibodies, allowing identification of zoonotic orthopoxviruses and parapoxviruses in animal and human biological samples. With 100% specificity, the test rules out the possibility of cross-reactions with viral agents causing look-alike diseases. The assay was employed in the field to investigate the causes of several outbreaks of a malignant proliferative skin disease that affected domestic ruminants in Sicily during 2011–2014. Due to specific aspects of the lesions, the animals were clinically diagnosed with papillomatosis. The mini-array test allowed the identification of coinfections caused by more than 1 viral species belonging to the Parapoxvirus and Orthopoxvirus genera, either in goats or in cattle. Our study suggests that the so-called “papillomatosis” can be the result of multiple infections with epitheliotropic viruses, including zoonotic poxviruses that cannot be properly identified with classical diagnostic techniques.

Genotyping of bovine viral diarrhea viruses (BVDV) isolated from cattle in Sicily

Bovine Viral Diarrhea–Mucosal Disease (BVD–MD) is a widely spread infectious disease that causes important economic losses in farms. Several epidemiological studies indicate a high genetic heterogeneity among Bovine Viral Diarrhea Virus (BVDV) strains circulating in Italy. The aim of this study was to investigate the genotypes of BVDV in Sicily, a region in the South of Italy. For this purpose, 17 BVDV strains collected from cattle breed in Sicily between 2005 and 2008 were genetically typed by sequencing of the 5′-untraslated region (5′-UTR) of the viral genome. In this study, phylogenetic analysis showed that all 17 examined strains were clustered within the BVDV genotype 1. Particularly, 14 of them were clustered with the BVDV-1b subgroup, while the remaining three strains were clustered with the BVDV-1e. Moreover, the restriction analysis indicated a bovine origin for all of the 17 strains typed in this study. These results could be useful to carry out an epidemiological survey and to create vaccines that protect cattle against BVDV different subgroups.

Differentiation and characterization of rat adipose tissue mesenchymal stem cells into endothelial-like cells

In this study, mesenchymal stem cells were isolated from rat adipose tissue (AD‐MSCs) to characterize and differentiate them into endothelial‐like cells. AD‐MSCs were isolated by mechanical and enzymatic treatments, and their identity was verified by colony‐forming units (CFU) test and by differentiation into cells of mesodermal lineages. The endothelial differentiation was induced by plating another aliquot of cells in EGM‐2 medium, enriched with specific endothelial growth factors. Five subcultures were performed. The expression of stemness genes (OCT4, SOX2 and NANOG) was investigated. The presence of CD90 and the absence of the CD45 were evaluated by flow cytometry. The endothelial‐like cells were characterized by the evaluation of morphological changes and gene expression analysis for endothelial markers (CD31, CD144, CD146). Characterization of AD‐MSCs showed their ability to form clones, to differentiate in vitro and the OCT‐4, SOX‐2, NANOG genes expression. Immunophenotypic characterization showed the CD90 presence and the CD45 absence. The endothelial‐like cells showed morphological changes, the expression of CD31, CD144, CD146 genes and the presence of CD31 membrane receptor. Matrigel assay showed their ability to form network and vessels‐like structures. This study lays the foundations for future evaluation of the potential AD‐MSCs pro‐angiogenic and therapeutic role.