In vitro aging alters the gene expression and secretome composition of canine adipose-derived mesenchymal stem cells.

Introduction: Canine adipose-derived mesenchymal stem cells (cAD-MSCs) hold therapeutic promise due to their regenerative potential, particularly within their secretome. However, concerns arise regarding the impact of in vitro cultivation necessitated for storing therapeutic doses, prompting this study to comprehensively explore the impact of in vitro aging on gene expression and secretome composition. Methods: The study involved collecting abdominal adipose tissue samples from nine healthy female dogs, from which cAD-MSCs were extracted and cultured. Stem cells were validated through trilineage differentiation assays and flow cytometry immunophenotyping. Gene expression profiling using RT-qPCR array, and cAD-MSCs secretome LC-MS/MS analysis, were conducted at passages 3 and 6 to reveal gene expression and protein composition alterations during in vitro culture. Results and Discussion: The results demonstrate that the gene expression and secretome composition of cAD-MSCs were impacted by in vitro aging. Among many alterations in gene expression between two passages, two significant downregulations were noted in the MSC-associated PTPRC and IL10 genes. While the majority of proteins and their functional characteristics were shared between passages, the influence of cell aging on secretome composition is highlighted by 10% of proteins being distinctively expressed in each passage, along with 21 significant up- and downregulations. The functional attributes of proteins detected in passage 3 demonstrated a greater inclination towards supporting the regenerative capacity of cAD-MSCs. Moreover, proteins in passage 6 exhibited a noteworthy correlation with the blood coagulation pathway, suggesting an elevated likelihood of coagulation events. To the best of our knowledge, this study presents the first original perspective on the changes in secretome composition that occur when cAD-MSCs age in vitro. Furthermore, it contributes to broadening the currently restricted knowledge base concerning the secretome of cAD-MSCs. In conclusion, our findings show that the regenerative potential of cAD-MSCs, as well as their secretome, may be compromised by in vitro aging. Therefore, our study suggests a preference for earlier passages when considering these cells for therapeutic applications.

The impact and complete genome characterisation of viruses involved in outbreaks of gastroenteritis in a farrow-to-finish holding.

Viral enteric pathogens continuously burden intensive pig farming, causing gastrointestinal diseases of epidemic and endemic nature. The present study investigated two diarrhoea outbreaks on a large farrow-to-finish holding and subsequent circulation of outbreak-related enteric viruses. These viruses were characterised by whole genome sequencing, and statistical evaluation of the impact on specific production metrics was performed. The results provided evidence that the Porcine epidemic diarrhoea virus-swine enteric coronavirus (PEDV-SeCoV) S gene recombinant strain was responsible for the first outbreak, whilst Rotavirus A (RVA) in a mixed infection with Rotavirus B (RVB) and porcine kobuvirus (PKV) probably caused the second diarrhoea outbreak. Whole genome characterisation revealed a porcine origin of all viruses involved and significant heterogeneity of RVB strain, proposing four novel genotypes and changes in RVB VP1 genotype classification. The statistical evaluation confirmed only a minor disturbance in the number of weaned pigs per sow, with statistical forecasting showing positive trends. A follow-up study corroborated the endemicity of RVA and PKV, in contrast to PEDV-SeCoV. Punctual, comprehensive and timely investigation of diarrhoea outbreaks is a prerequisite for applying adequate pig health and biosecurity management. Calculating such outbreaks' impact on production metrics can potentially shape future decisions on management improvements.

Interspecies transmission of porcine-originated G4P[6] rotavirus A between pigs and humans: a synchronized spatiotemporal approach.

As a leading viral cause of acute gastroenteritis in both humans and pigs, rotavirus A (RVA) poses a potential public health concern. Although zoonotic spillover of porcine RVA strains to humans is sporadic, it has been detected worldwide. The origin of chimeric human-animal strains of RVA is closely linked to the crucial role of mixed genotypes in driving reassortment and homologous recombination, which play a major role in shaping the genetic diversity of RVA. To better understand how genetically intertwined porcine and zoonotic human-derived G4P[6] RVA strains are, the present study employed a spatiotemporal approach to whole-genome characterization of RVA strains collected during three consecutive RVA seasons in Croatia (2018-2021). Notably, sampled children under 2 years of age and weanling piglets with diarrhea were included in the study. In addition to samples tested by real-time RT-PCR, genotyping of VP7 and VP4 gene segments was conducted. The unusual genotype combinations detected in the initial screening, including three human and three porcine G4P[6] strains, were subjected to next-generation sequencing, followed by phylogenetic analysis of all gene segments, and intragenic recombination analysis. Results showed a porcine or porcine-like origin for each of the eleven gene segments in all six RVA strains. The G4P[6] RVA strains detected in children most likely resulted from porcine-to-human interspecies transmission. Furthermore, the genetic diversity of Croatian porcine and porcine-like human G4P[6] strains was propelled by reassortment events between porcine and porcine-like human G4P[6] RVA strains, along with homologous intragenotype and intergenotype recombinations in VP4, NSP1, and NSP3 segments. Described concurrent spatiotemporal approach in investigating autochthonous human and animal RVA strains is essential in drawing relevant conclusions about their phylogeographical relationship. Therefore, continuous surveillance of RVA, following the One Health principles, may provide relevant data for assessing the impact on the protectiveness of currently available vaccines.

Rotavirus A in Domestic Pigs and Wild Boars: High Genetic Diversity and Interspecies Transmission.

Rotavirus A (RVA) is an important pathogen for porcine health. In comparison to humans, RVA in domestic animals and especially in wildlife is under researched. Therefore, the aim of the present study was to investigate the prevalence, genetic diversity, molecular epidemiology and interspecies transmission of RVA in domestic pigs and wild boars. During the three consecutive RVA seasons (2018-2021) we collected 445 and 441 samples from domestic pigs and wild boars, respectively. Samples were tested by real-time RT-PCR, and RVA-positive samples were genotyped in VP7 and VP4 segments. Our results report an RVA prevalence of 49.9% in domestic pigs and 9.3% in wild boars. Outstanding RVA genetic diversity was observed in VP7 and VP4 segments, especially in domestic pigs exhibiting a striking 23 different RVA combinations (G5P[13] and G9P[23] prevailed). Interspecies transmission events were numerous between domestic pigs and wild boars, sharing G3, G5, G6, G9, G11 and P[13] genotypes. Furthermore, our data indicate that such transmission events involved even bovines (G6, P[11]) and, intriguingly, humans (G1P[8]). This study contributes to the basic knowledge that may be considered important for vaccine development and introduction, as a valuable and currently missing tool for efficient pig health management in the EU.

An Outstanding Role of Adipose Tissue in Canine Stem Cell Therapy.

Adipose tissue, previously known as connective tissue with a role in energy storage, is currently changing the course of treatments in veterinary medicine. Recent studies have revealed one particularly impressive function among all the newly discovered functions of adipose tissue. The interactive cells hosted by adipose tissue, the stromal vascular fraction (SVF), and their role in treating numerous diseases have provided a prospective course of research with positive outcomes in regenerative veterinary medicine (RVM). This review describes the main features of adipose tissue, emphasizing an eclectic combination of cells within the SVF and its thus far researched therapeutic possibilities in canine RVM. An afterwards focus is on a highly researched component of the SVF, adipose-derived mesenchymal stem cells (ASCs), which were shown to have an extraordinary impact relying on several proposed mechanisms of action on mitigating pathologies in canines. Furthermore, ASC therapy showed the most significant results in the orthopaedics field and in neurology, dermatology, ophthalmology, gastroenterology, and hepatology, which elevates the possibilities of ASC therapy to a whole new level. Therefore, this review article aims to raise awareness of the importance of research on cellular components, within abundant and easily accessible adipose tissue, in the direction of regenerative therapy in canines, considering the positive outcomes so far. Although the focus is on the positive aspects of cellular therapy in canines, the researchers should not forget the importance of identifying the potential negative aspects within published and upcoming research. Safe and standardized treatment represents a fundamental prerequisite for positively impacting the lives of canine patients.