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Abstract Background West Nile Disease (WND) is a zoonotic mosquito‐borne infection involving viral pathogens, human and animal hosts, vectors and environment. Cooperation among medical, veterinary and entomological fields has been promoted by the Italian Public Health Authorities, and an integrated West Nile Virus (WNV) Surveillance Plan has been in force in Italy since 2016 to prevent the transmission risk of WND to humans through an early detection of viral circulation by animal and entomological surveillance. This managing model is unique in Europe. Objectives This survey aimed at presenting the ‘One Health’ approach applied in 2016 to the first autochthonous human case of West Nile Neuroinvasive Disease (WNND) in Sicily (Southern Italy). Methods Serological (anti‐WNV IgM and IgG ELISA, anti‐WNV neutralizing antibodies) and molecular tests were conducted on blood, liquor and urine of a 38‐year‐old man with encephalitis and meningitis. Overall, 2704 adult culicides from 160 mosquito catches were morphologically identified. Female mosquitoes were analysed in pools for WNV RNA detection. Serological (anti‐WNV IgM and IgG ELISA) and molecular analyses for WNV were carried out in 11 horses, 271 chickens and two dogs sampled in farms around the man's residence. Results and conclusions WNND was confirmed by serological analysis on patient's liquor and serum. Collected mosquito species included Culex pipiens (93.56%, CI95% 92.64%–94.49%), Aedes albopictus (5.25%, CI95% 4.41%–6.09%), Culex hortensis (0.59%, CI95% 0.30%–0.88%), Culiseta longiareolata (0.55%, CI95% 0.27%–0.83%) and Anopheles maculipennis s.l. (0.04%, CI95% –0.04% to 0.11%). Mosquito pools were negative for WNV RNA. Two dogs (100%) and two horses (18.18%, CI95% –4.61 to 40.97%) resulted positive for anti‐WNV specific antibodies. The ‘One Health’ approach allowed to report the first human neuroinvasive WND in Sicily and to confirm the local circulation of WNV in animals of the same area where the clinical case occurred, defining the autochthonous origin of the infection.

Canine distemper is a contagious and severe systemic viral disease that affects domestic and wild carnivores worldwide. In this study, two adult female ferrets (Mustela putorius furo) were evaluated for cutaneous lesions. Scab, fur, and swab samples from the external auditory canal, cutaneous lesions, and scrapings were analyzed. Canine distemper virus (CDV)-positive samples underwent RT-PCR/RFLP with the restriction enzyme PsiI, and the hemagglutinin gene sequence was obtained. According to the restriction enzyme and sequence analyses, the viral strains were typed as CDV field strains that are included within the Europe lineage and distinct from those including vaccinal CDV strains. The sequence analysis showed the highest nucleotide identity rates in older Europe lineage CDV strains collected from dogs and a fox in Europe. This study is the first to report on CDV infection in ferrets in southern Italy and contributes to the current knowledge about natural CDV infection in this species. In conclusion, vaccination remains crucial for preventing the disease and counteracting cross-species infection. Molecular biology techniques can enable the monitoring of susceptible wild animals by ensuring the active surveillance of CDV spread.

Peste des petits ruminants virus (PPRV) is the aetiologic agent of Peste des petits ruminants (PPR), an important viral disease of sheep and goats. PPR is endemic in Nigeria and leads to social and economic losses. The objective of this study was to determine the prevalence of PPR infection and genetically characterize PPRV strains obtained from sheep and goats in three States of Southeast Nigeria. A total of 285 nasal swab samples collected in 2017‑2018 were processed for PPRV genome detection by RT‑PCR. Sixty‑five (22.81%) of the samples were positive for PPRV. Sequence and phylogenetic analyses revealed that the PPRV belonged to lineages II (11/38, 28.9%) and IV (27/38, 71.1%). The N gene fragment sequence showed a 99.77%‑100% and 99.98%‑100% identity among the strains of lineages II and IV, respectively. Fourteen amino acid substitutions, previously unreported in PPRV strains from Nigeria, were recorded. This study confirms the circulation of PPRV lineages II and IV in Southeast Nigeria, the dominance of strains belonging to lineage IV in recent years, and their close genetic relationship with those previously reported in other parts of Nigeria and neighboring countries.

Abstract 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.

Canine parvovirus type-2 (CPV-2) is a DNA virus that causes a very common worldwide diffused infectious disease in dogs. Since its appearance, the CPV-2 evolved generating novel genetic and antigenic variants (CPV-2a/2b/2c) which are distributed throughout the world. In the present study, the frequency of CPV-2 in a canine population in Sicily (Italy) was investigated, using a polymerase chain reaction (PCR) for a fragment of the VP2 gene. Out of a total of 673 samples from 370 dogs, submitted to the laboratory from July 2009 to August 2015, 265 samples (39.38%) were positive and these were further analyzed by restriction fragment length polymorphism (RFLP) and DNA sequence analysis. A high prevalence of the CPV-2c variant (79.56% of CPV-2 positive dogs) was observed in this survey, underlining that this type of CPV field isolate is prevalent circulating in Sicily. Sequence and phylogenetic analysis showed a close relationship with CPV-2a and CPV-2c strains from Europe and non-European countries. Considering that CPV-2c is reaching a worldwide distribution and that this variant is also affecting vaccinated dogs, efforts should be made towards the development of new CPV vaccines.

To evaluate the immunological response following vaccination, 40 WNV serologically negative horses were selected and divided in two groups of 20 animals. One group was vaccinated (booster after 28 days) with a whole inactivated viral strain and the second group with a live recombinant canarypox virus expressing the genes coding for the WNV preM/E viral proteins. IgM, IgG and neutralizing antibodies were monitored by class specific ELISAs and serum neutralization assay for 360 days. In both groups, IgM antibodies were first detected 7 days post vaccination (dpv). However, in the group vaccinated with inactivated vaccine, IgM antibodies were detected until day 42 pv, whereas in the group vaccinated with the recombinant vaccine, they were detected up to day 52 pv. A similar (P > 0.05) proportion of horses showed IgM antibodies after vaccination with either recombinant [30%; 95% confidence interval (CI): 14.59%-52.18%] or inactivated (32%; 95% CI: 15.39-54.28%) vaccine. Both vaccines induced in vaccinated horses a detectable IgG antibody response starting from day 7 pv and lasting till the end of the trial. Analogously, both products elicited WNV specific neutralizing antibodies. The response induced by the live canarypox virus-vectored vaccine was higher (mean titres 1:298 vs 1:18.9) and lasted longer than did that induced by the killed-virus vaccines. In fact, one year after the vaccination, neutralizing antibodies were still detectable in the horses which received the canarypox virus-based vaccine but not in the group vaccinated with the killed product. The use of vaccines is a valuable tool to prevent WNV disease in horses and the availability of different products facilitates the control of the disease in endemic areas.

Mesenchymal stem cells (MSCs) are used in therapy in animal models and veterinary medicine, due to their capacity of inducing tissue regeneration and immunomodulation. Their clinical application requires a ready off-the-shelf amount of viable therapeutics doses. For this purpose, it is useful to cryopreserve MSCs to gain a ready and controlled source of abundant autologous stem cells. We evaluated the effect of 7 years cryopreservation using 10% dimethyl sulfoxide (DMSO) with different fetal bovine serum (FBS) concentrations (from 10 to 90%) on different passages of MSCs isolated from canine adipose tissue (cAD-MSCs). The study aimed to evaluate the most adequate cell passage and FBS percentage for the long-term cryopreservation of cells by maintaining the stemness features. Phenotype morphology, cell viability, osteogenic and adipogenic differentiation potentials, proliferative potential and expression of pluripotency markers were analyzed in thawed cells and compared with fresh ones. We demonstrated that cells cryopreserved with at least 80% FBS maintain unaltered the stemness characteristics of the freshly isolated cells. In particular, cells of P0–P1 passages have to be expanded in vitro and subsequently cryopreserved and cells of P2–P4 passages should be considered in the studies on therapeutic application and in vitro study of cAD-MSCs.

Vector-borne pathogens such as Erlichia canis and Rickettsia conorii are widespread in the Mediterranean basin. Rhipicephalus sanguineus, is considered the main vector in Mediterranean climatic areas. Seroprevalence in dogs is variable in relation to environmental factors, presence of vectors, and dogs’ management. We investigated the seroprevalence in Sicilian dogs during 2017–2019, considering temporal as well as spatial variations, and different canine population. A total of 11,009 sera were analyzed: 7568 and 3441 sera were tested to detect antibodies to E. canis and to R. conorii, respectively, by immunofluorescence assay. The rainfall average in the sampling sites during the three-year period was also considered. Statistical analyses were performed using chi-square tests for association between two or more variables. We reported a prevalence of 29.6% and 53.6% for E. canis and R. conorii, respectively. Significant temporal variation was found in R. conorii, while significant difference was found considering canine population and spatial variation regarding both pathogens. Our study updates the previous results of E. canis and R. conorii seroprevalence in dogs in Sicily, and confirms the wide distribution of these pathogens. In addition, we considered, for the first time, three different variables to identify the areas and the canine populations most exposed to risk.

Canine distemper virus (CDV) is the etiologic agent of distemper in dogs. It exhibits an elevated potential of crossing species barriers, infecting a wide range of wild and domestic carnivores. Of its encoding genes, hemagglutinin (H) shows high heterogeneity, and it was used to determine the relationship between CDV strains due to its variability and key role in determining cell tropism, host shift, and in eliciting a protective immune response. This study analysed the full-length H gene sequence of Arctic-like CDV strains collected from dogs in Italy during a period in which an increased activity of CDV diffusion was observed. The common amino acid changes and features of Arctic-like CDV strains collected from 2011 to 2016 in Europe were described, providing an updated analysis of the genomic features. A comparison with CDV vaccine strains was carried out to evaluate the increased genomic difference with CDV Arctic-like field strains. This study provides a complete and updated analysis of the current spreading strains of Arctic-like lineage and the main amino acid variations in the hemagglutinin gene sequence circulating in Italy. Moreover, it provides novel information regarding the evolution of the most recent CDV Arctic-like lineage strains collected in Europe.