Epizootic Hemorrhagic Disease Virus Serotype 8, Italy, 2022.

We describe the detection of epizootic hemorrhagic disease virus (EHDV) serotype 8 in cattle farms in Sardinia and Sicily in October-November 2022. The virus has a direct origin in North Africa; its genome is identical (>99.9% nucleotide sequence identity) to EHDV serotype 8 strains detected in Tunisia in 2021.

Aujeszky's disease in hunting dogs after the ingestion of wild boar raw meat in Sicily (Italy): clinical, diagnostic and phylogenetic features.

BACKGROUND: Aujeszky's disease is caused by Suid Herpes Virus-1 and species belonging to the genus Sus scrofa are the main reservoir hosts. This virus, however, is capable of infecting and causing severe disease, with an almost constant fatal outcome in other species, both domestic and wild (carnivores, monogastric herbivores and ruminants). Moreover, the possibility of transmission to humans has been demonstrated. This study reports and describes the clinical, diagnostic, pathological and phylogenetic aspects of two cases of Aujeszky's disease in two hunting dogs following the ingestion of infected wild boar raw meat. These cases are contextualized in the province of Messina (Sicily), where a high prevalence of Aujeszky's disease has been recorded (average of 12,20% in the period 2010-2019) in farmed pig, and with evidence of spread to other species. A severe outbreak in cattle has recently been reported in these areas. Nevertheless, cases of Aujeszky's disease in dogs are rarely reported and this study represents the first well-documented report in this species in Sicily. CASE PRESENTATION: After a wild boar hunt, two dogs showed neurological symptoms and intense itching unresponsive to therapy. Diagnosis of Aujeszky's disease was made based on clinical suspicion, anamnestic information and confirmed by the isolation of the virus from the brain of both dogs. In addition, molecular typing, sequencing and phylogenetic analysis of the Real-Time PCR products were performed. The sequences studied were placed in the Italian Clade 1 along with the sequences obtained from wild boars and hunting dogs from Italy and France. CONCLUSIONS: The finding of this disease in non-natural hosts in Sicilian multi-host epidemiological contexts suggests that the risk of inter-species transmission is concrete and that attention should be paid to developing disease control programs in these territories. The data obtained from genome sequencing of the two SuHV-1 isolates contribute to the enrichment of the GenBank with unknown sequences and the phylogenetic analysis implementation.

Identification and Molecular Characterization of a Divergent Asian-like Canine Parvovirus Type 2b (CPV-2b) Strain in Southern Italy.

Canine parvovirus type 2 (CPV-2) is an infectious agent relevant to domestic and wild carnivorans. Recent studies documented the introduction and spread of CPV-2c strains of Asian origin in the Italian canine population. We investigated tissue samples from a puppy collected during necropsy for the presence of viral enteropathogens and all samples tested positive only for CPV-2. The full coding sequence of a CPV-2b (VP2 426Asp) strain was obtained. This virus was related to CPV-2c strains of Asian origin and unrelated to European CPV-2b strains. The sequence had genetic signatures typical of Asian strains (NS1: 60Val, 545Val, 630Pro; VP2: 5Gly, 267Tyr, 324Ile) and mutations rarely reported in Asian CPV-2b strains (NS1: 588N; VP2: 370Arg). Phylogenetic analyses placed this strain in well-supported clades, including Asian CPV-2c-like strains, but always as a basal, single-sequence long branch. No recombination was observed for this strain, and we speculate that it could have originated from an Asian CPV-2c-like strain that acquired the 426Asp mutation. This study reports the first evidence of an Asian-like CPV-2b strain in Italy, confirming the occurrence of continuous changes in the global CPV-2 spread. Since positive convergent mutations complicate data interpretation, a combination of phylogenetic and mutation pattern analyses is crucial in studying the origin and evolution of CPV-2 strains.

Molecular epidemiology of canine parvovirus type 2 in Italy from 1994 to 2017: recurrence of the CPV-2b variant.

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.

Novel dolphin morbillivirus (DMV) outbreak among Mediterranean striped dolphins Stenella coeruleoalba in Italian waters.

An unusual mortality event (UME) of striped dolphins Stenella coeruleoalba occurred in the period July to December 2016 along the Italian Ionian coastline. We conducted a complete postmortem examination on 28 specimens and detected dolphin morbillivirus (DMV), by means of biomolecular analyses, in the target tissues of 17 animals. Unlike previous outbreaks occurring in the Mediterranean Sea in 2011 and 2013, we observed typical pathological changes suggestive of morbilliviral infection in an acute/subacute phase and immunohistochemical reactivity. The same findings were observed in 13 other specimens beached along the Italian coastline during 2016 with no temporal and geographical relationship with the ongoing epidemic outbreak. Molecular characterization and phylogenetic analysis showed that DMV sequences detected in Italy in 2016 clustered with those identified in Portugal and Galicia (Spain), representing a novel DMV strain of Atlantic origin which entered the Mediterranean Sea and affected a naïve striped dolphin population. DMV sequences detected in the previous Mediterranean outbreaks exhibited a marked genetic relatedness and diverged from those detected in cetaceans stranded along the Galician and Portuguese coasts since 2007.

Investigation and control of a Norovirus outbreak of probable waterborne transmission through a municipal groundwater system.

During March 2011 an outbreak of gastroenteritis occurred in Santo Stefano di Quisquina, Agrigento, Sicily, Italy. Within two weeks 156 cases were identified among the 4,965 people living in the municipality. An epidemiological investigation was conducted to characterize the outbreak and target the control measures. A case was defined as a person developing diarrhea or vomiting during February 27-March 13, 2011. Stool specimens were collected from 12 cases. Norovirus (NoV) genotype GII.4 variant New Orleans 2009 was identified in stool samples from 11 of 12 cases tested (91.7%). Epidemiological investigations suggested a possible association with municipal drinking water consumption. Water samples from the public water system were tested for NoV and a variety of genotypes were detected during the first 3 months of surveillance, including GII.4 strains belonging to different variants from that involved in the gastroenteritis outbreak. Contamination of the well and springs supplying the public water network was eventually thought to be the source of the NoV contamination.

Malignant skin neoplasms in goats in Sicily, Italy: clinical, virological and pathological investigations.

Neoplasms in small ruminants are considered uncommon and their reported incidence is variable. The aims of this investigation were to characterize malignant skin neoplasms in adult goats reared in Sicily, Italy, and to evaluate potential correlations between gross and histopathology features of the tumours and signalment, tumour location and/or viral infections. A total of 75 malignant skin masses were examined. In selected animals with perineal masses (n = 28) virological and serological investigations on tissues and blood were also conducted. According to the histological features, the lesions were classified as 67 squamous cell carcinomas (SCCs) (of which 65 were located in the perineum), six melanomas and two fibrosarcomas. In three cases, neoplasms at the base of the horn were associated with nasal polyps. Among the selected perineal SCCs, papillomaviruses (PVs), caprine herpesvirus 1 and parapoxvirus were not detected on polymerase chain reaction or on serological examination. However, further investigation on a larger sample size is required to evaluate the potential role of PVs in the pathogenesis of skin tumours in goats.

Viral pathogens in domestic cats in southern Italy: A retrospective analysis in Sicily, 2020-2022.

A retrospective study was carried out on selected feline viral pathogens detected in domestic cat in Sicily, southern Italy. Samples from 64 cats, collected from 2020 to 2022, were analysed for the presence of feline panleukopenia virus, canine parvovirus type 2 (CPV-2), feline coronavirus (FCoV), feline calicivirus (FCV), feline herpesvirus type 1, norovirus (NoV), and rotavirus (RoV). Single (45 %) or mixed (38 %) viral infections were detected. FPV, related with other Italian FPV strains, remains the main viral cause of infection (66 %). CPV-2c Asian lineage strains (3 %) were detected for the first time in domestic cats in Europe. FCoV (29.6 %), either enteric or systemic, and systemic FCV (18.7 %) infections were detected in positive cats. Less commonly reported viruses (GIV.2/GVI.2 NoVs, RoV), potentially related to the animal/human interface, were detected at lower rates as well (5 %). The present epidemiological data suggest the need to improve disease prevention, immunization, and biosecurity strategies.

Molecular epidemiology of canine parvovirus type 2 in Sicily, southern Italy: A geographical island, an epidemiological continuum.

Since it emerged as a major dog pathogen, canine parvovirus type 2 (CPV-2) has featured a remarkable genetic and phenotypic heterogeneity, whose biological, epidemiological, and clinical impact is still debated. The continuous monitoring of this pathogen is thus of pivotal importance. In the present study, the molecular epidemiology of CPV-2 in Sicily, southern Italy, has been updated by analysing 215 nearly complete sequences of the capsid protein VP2, obtained from rectal swabs/faeces or tissue samples collected between 2019 and 2022 from 346 dogs with suspected infectious gastrointestinal disease. The presence of the original CPV-2 type (4%) and CPV-2a (9%), CPV-2b (18%), or CPV-2c (69%) variants was documented. Over the years, we observed a decrease in the frequency of CPV-2a/-2b and a rapid increase of CPV-2c frequency, with a progressive replacement of the European lineage of CPV-2c by the Asian lineage. The observed scenario, besides confirming epidemiological relevance of CPV-2, highlights the occurrence of antigenic variant shifts over time, with a trend toward the replacement of CPV-2a, CPV-2b, and the European lineage of CPV-2c by the emerging Asian CPV-2c lineage. The comparison with other Italian and international sequences suggests the occurrence of viral exchange with other Italian regions and different countries, although the directionality of such viral flows could not be often established with confidence. In several instances, potential CPV-2 introductions led to epidemiological dead ends. However, major, long-lasting clades were also identified, supporting successful infection establishment, local spreading, and evolution. These results, besides demonstrating the need for implementing more effective control measures to prevent viral introductions and minimize circulation, stress the relevance of routine monitoring activities as the only tool to effectively understand CPV-2 epidemiology and evolution, and develop adequate countermeasures.

Persistence of DNA from canine parvovirus modified-live virus in canine tissues.

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 × 102 and 9.95 × 104 DNA copies/10 µl of template. This study confirms that CPV vaccinal strains can be detected in canine tissues after vaccination, so post-mortem diagnosis of CPV infection needs further molecular analyses to assess the viral type (vaccine or field strains). The present study updates the current information on the persistence of CPV vaccine strains in canine tissues and their possible interference with molecular assays.

A SARS-CoV-2 full genome sequence of the B.1.1 lineage sheds light on viral evolution in Sicily in late 2020.

To investigate the influence of geographic constrains to mobility on SARS-CoV-2 circulation before the advent of vaccination, we recently characterized the occurrence in Sicily of viral lineages in the second pandemic wave (September to December 2020). Our data revealed wide prevalence of the then widespread through Europe B.1.177 variant, although some viral samples could not be classified with the limited Sanger sequencing tools used. A particularly interesting sample could not be fitted to a major variant then circulating in Europe and has been subjected here to full genome sequencing in an attempt to clarify its origin, lineage and relations with the seven full genome sequences deposited for that period in Sicily, hoping to provide clues on viral evolution. The obtained genome is unique (not present in databases). It hosts 20 single-base substitutions relative to the original Wuhan-Hu-1 sequence, 8 of them synonymous and the other 12 encoding 11 amino acid substitutions, all of them already reported one by one. They include four highly prevalent substitutions, NSP12:P323L, S:D614G, and N:R203K/G204R; the much less prevalent S:G181V, ORF3a:G49V and N:R209I changes; and the very rare mutations NSP3:L761I, NSP6:S106F, NSP8:S41F and NSP14:Y447H. GISAID labeled this genome as B.1.1 lineage, a lineage that appeared early on in the pandemic. Phylogenetic analysis also confirmed this lineage diagnosis. Comparison with the seven genome sequences deposited in late 2020 from Sicily revealed branching leading to B.1.177 in one branch and to Alpha in the other branch, and suggested a local origin for the S:G118V mutation.

Wastewater-Based Epidemiology as a Tool to Detect SARS-CoV-2 Circulation at the Community Level: Findings from a One-Year Wastewater Investigation Conducted in Sicily, Italy.

Wastewater-based epidemiology is a well-established tool for detecting and monitoring the spread of enteric pathogens and the use of illegal drugs in communities in real time. Since only a few studies in Italy have investigated the correlation between SARS-CoV-2 in wastewater and the prevalence of COVID-19 cases from clinical testing, we conducted a one-year wastewater surveillance study in Sicily to correlate the load of SARS-CoV-2 RNA in wastewater and the reported cumulative prevalence of COVID-19 in 14 cities from October 2021 to September 2022. Furthermore, we investigated the role of SARS-CoV-2 variants and subvariants in the increase in the number of SARS-CoV-2 infections. Our findings showed a significant correlation between SARS-CoV-2 RNA load in wastewater and the number of active cases reported by syndromic surveillance in the population. Moreover, the correlation between SARS-CoV-2 in wastewater and the active cases remained high when a lag of 7 or 14 days was considered. Finally, we attributed the epidemic waves observed to the rapid emergence of the Omicron variant and the BA.4 and BA.5 subvariants. We confirmed the effectiveness of wastewater monitoring as a powerful epidemiological proxy for viral variant spread and an efficient complementary method for surveillance.

Biomolecular Analysis of Canine Distemper Virus Strains in Two Domestic Ferrets (Mustela putorius furo).

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.

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.

GIV noroviruses and other enteric viruses in bivalves: a preliminary study.

We evaluated the presence of the enteric viruses: norovirus, adenovirus, enterovirus, astrovirus, hepatitis A virus, and hepatitis E virus in bivalves using nested PCR methods and cell culture assays. Noroviruses GII.4 and GIV.1, adenoviruses types 1 and 2, hepatitis A, and echovirus type 7 were detected in the shellfish tested, which were often co-infected. This is the first study to detect such a high level of viral contamination in Italian mussels (up to four different viral groups in a single sample), and the first to document the presence of GIV NoV in shellfish.

Antimicrobial Resistance (AMR) of Bacteria Isolated from Dogs with Canine Parvovirus (CPV) Infection: The Need for a Rational Use of Antibiotics in Companion Animal Health.

Canine parvovirus type 2 (CPV-2) represents a major viral threat to dogs. Considering the potential effects of pets on antimicrobial resistance, information on the CPV and associated bacterial co-infections is limited. The aim of this study was to analyze the antimicrobial susceptibility and multidrug-resistance profiles of bacterial species from tissue samples of dogs with canine parvovirus infection. A set of PCR assays and sequence analyses was used for the detection and the molecular characterization of the CPV strains and other enteric viruses. Bacterial isolation, the determination of antimicrobial susceptibility via the disk diffusion method, and the determination of the minimum inhibitory concentration were performed. The detection of ß-lactamase genes and toxin genes for specific bacteria was also carried out. CPV infection was confirmed in 23 dogs. Forty-three bacterial strains were isolated and all showed phenotypic resistance. Seventeen multidrug-resistant bacteria and bacteria with high resistance to third- and fourth-generation cephalosporins and metronidazole were detected. Almost 50% of the isolated Enterobacteriaceae were positive for at least one ß-lactamase gene, with the majority carrying more genes as well. The evidence for multi-resistant bacteria with the potential for intra- or cross-species transmission should be further considered in a One Health approach.

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.

Pilot Investigation of SARS-CoV-2 Variants in the Island of Sicily Prior to and in the Second Wave of the COVID-19 Pandemic.

After 2 years of the COVID-19 pandemic, we continue to face vital challenges stemming from SARS-CoV-2 variation, causing changes in disease transmission and severity, viral adaptation to animal hosts, and antibody/vaccine evasion. Since the monitoring, characterization, and cataloging of viral variants are important and the existing information on this was scant for Sicily, this pilot study explored viral variants circulation on this island before and in the growth phase of the second wave of COVID-19 (September and October 2020), and in the downslope of that wave (early December 2020) through sequence analysis of 54 SARS-CoV-2-positive samples. The samples were nasopharyngeal swabs collected from Sicilian residents by a state-run one-health surveillance laboratory in Palermo. Variant characterization was based on RT-PCR amplification and sequencing of four regions of the viral genome. The B.1.177 variant was the most prevalent one, strongly predominating before the second wave and also as the wave downsized, although its relative prevalence decreased as other viral variants, particularly B.1.160, contributed to virus circulation. The occurrence of the B.1.160 variant may have been driven by the spread of that variant in continental Europe and by the relaxation of travel restrictions in the summer of 2020. No novel variants were identified. As sequencing of the entire viral genome in Sicily for the period covered here was restricted to seven deposited viral genome sequences, our results shed some light on SARS-CoV-2 variant circulation during that wave in this insular region of Italy which combines its partial insular isolation with being a major entry point for the African immigration.

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.

The Effect of a 7 Year-Long Cryopreservation on Stemness Features of Canine Adipose-Derived Mesenchymal Stem Cells (cAD-MSC).

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.