Due to SARS CoV-2 recombination rates, number of infected people and recent reports of environmental contamination, the possibility of SARS CoV-2 transmission to animals can be expected. We tested samples of dominant free-living and captive wildlife species in Croatia for the presence of anti-SARS CoV-2 antibodies and viral RNA. In total, from June 2020 until February 2021, we tested blood, muscle extract and fecal samples of 422 free-living wild boars (Sus scrofa), red foxes (Vulpes vulpes) and jackals (Canis aureus); blood and cloacal swabs of 111 yellow-legged gulls (Larus michahellis) and fecal samples of 32 zoo animals. A commercially available ELISA (ID.Vet, France) and as a confirmatory test, a surrogate virus neutralization test (sVNT; GenScript, Netherlands) were used. Fecal samples were tested for the presence of viral RNA by a real-time RT–PCR protocol. Fifteen out of 533 (2.8%) positive ELISA results were detected; in wild boars (3.9%), red foxes (2.9%) and jackals (4.6%). However, the positive findings were not confirmed by sVNT. No viral RNA was found. In conclusion, no spillover occurred within the investigated period (second COVID-19 wave). However, further investigation is needed, especially regarding wildlife sample features for serological tests.

Lumpy Skin Disease virus is a poxvirus from the genus Capripox that mainly affects bovines and it causes severe economic losses to livestock holders. The Lumpy Skin Disease virus is currently dispersing in Asia, but little is known about detailed phylogenetic relations between the strains and genome evolution. We reconstructed a whole-genome-sequence (WGS)-based phylogeny and compared it with single-gene-based phylogenies. To study population and spatiotemporal patterns in greater detail, we reconstructed networks. We determined that there are strains from multiple clades within the previously defined cluster 1.2 that correspond with recorded outbreaks across Eurasia and South Asia (Indian subcontinent), while strains from cluster 2.5 spread in Southeast Asia. We concluded that using only a single gene (cheap, fast and easy to routinely use) for sequencing lacks phylogenetic and spatiotemporal resolution and we recommend to create at least one WGS whenever possible. We also found that there are three gene regions, highly variable, across the genome of LSDV. These gene regions are located in the 5′ and 3′ flanking regions of the LSDV genome and they encode genes that are involved in immune evasion strategies of the virus. These may provide a starting point to further investigate the evolution of the virus.

Lumpy skin disease (LSD) is an emerging, transboundary viral pox disease affecting cattle of all ages and breeds. The serological assay for monitoring immunity following vaccination is a virus neutralization test (VNT/OIE) that determines the neutralization index (NI). The first validated enzyme-linked immunosorbent assay (ELISA; IDVet) has become commercially available, facilitating large-scale serosurveillance for LSD. Although the VNT is labor intensive and time consuming, it is still the recommended test by the OIE.

The objective of this paper is to provide an overview of the current status of rabies in Europe, with special emphasis on Croatia and Southeast and East Europe. Due to the systematic implementation of a rabies eradication program by oral vaccination of wild animals, by the end of the 20th century, most West and Central European countries were rabies-free. The EU goal was to eradicate rabies in wildlife and domestic animals by 2020. No matter how achievable the goal seemed to be, the disease is still present in the eastern part of the EU, as was notified in 2020 by two member states—Poland and Romania. Croatia has been rabies-free for the last seven years but given that it borders a non-EU country in which a case of rabies was confirmed in 2020, it will continue to contribute to the maintenance of the rabies-free region. A rabies-free EU can only be achieved by continuous oral vaccination, coordination and a regional approach. The prevention of reintroductions from bordering countries in which rabies has not been eradicated yet, and the support for the eradication efforts made by these countries, are goals still pending.

Although canine adipose derived stem cells (cASCs) morphology characteristics and differentiation ability are well documented, transcriptome alterations of undifferentiated cASCs during ex vivo cultivation remain unknown. Here we demonstrate, for the first time, the transcriptome composition of isolated cASCs in undifferentiated state originating from six donors. Transcriptome changes were monitored during ex vivo cultivation between passage 3 (P3) and P5, which are mostly used in therapy. Influence of donors’ age in given passage number on transcriptome composition was also investigated. Cultivation from P3 to P5 resulted in 16 differentially expressed genes with cooverexpression of pluripotency and self-renewal transcription factors genes SOX2 and POU5F1 dominant in old donors’ cells. Furthermore, cASCs demonstrated upregulation of IL-6 in young and old donors’ cells. In addition, ex vivo cultivation of cASCs revealed well-known morphological alterations accompanied with decrease in expression of CD90 and CD44 markers in P4 and higher monitored by flow cytometry and successful osteo- and chondrodifferentiation but inefficient adipodifferentiation in P3. Our results revealed the impact of ex vivo cultivation on nature of cells. Correlation of transcriptome changes with secretome composition is needed and its further impact on therapeutic potential of cASCs remains to be evaluated in clinical trials.

The influence of cultivation on the expression pattern of canine adipose-derived mesenchymal stem cells (cAD-MSCs) surface markers, contributing to, among others, the promotion of growth, proliferation, differentiation and immunomodulatory mechanisms of an excellent therapeutic, is still unknown. To fill the gap, we investigated CD90, CD44, CD73, CD29, CD271, CD105, CD45 and CD14 patterns of expression at the protein level with flow cytometry and mRNA level using a real-time polymerase chain reaction array. Gentle variations of expression occurred during cultivation, along with increased CD90, CD44 and CD29 expression, low and decreasing CD271 and CD73 expression and a decrease of initially high CD105. As expected, CD45 and CD14 were not expressed by cAD-MSCs. Interestingly, we discovered a significant decrease of CD73 expression, compared to early (P1–P3) to late (P4–P6) passages, although the CD73 gene expression was found to be stable. The percentage of positive cells was found to be higher for all positive markers up to P4. As CD73′s one important feature is a modulation from a pro-inflammatory environment to an anti-inflammatory milieu, the expression of CD73 in our conditions indicate the need to consider the time cells spend in vitro before being transplanted into patients, since it could impact their favourable therapeutical properties.