SARS-CoV-2 infection outbreaks in minks have serious implications associated with animal health and welfare, and public health. In two naturally infected mink farms (A and B) located in Greece, we investigated the outbreaks and assessed parameters associated with virus transmission, immunity, pathology, and environmental contamination. Symptoms ranged from anorexia and mild depression to respiratory signs of varying intensity. Although the farms were at different breeding stages, mortality was similarly high (8.4% and 10.0%). The viral strains belonged to lineages B.1.1.218 and B.1.1.305, possessing the mink-specific S-Y453F substitution. Lung histopathology identified necrosis of smooth muscle and connective tissue elements of vascular walls, and vasculitis as the main early key events of the acute SARS-CoV-2-induced broncho-interstitial pneumonia. Molecular investigation in two dead minks indicated a consistently higher (0.3–1.3 log₁₀ RNA copies/g) viral load in organs of the male mink compared to the female. In farm A, the infected farmers were responsible for the significant initial infection of 229 out of 1,000 handled minks, suggesting a very efficient human-to-mink transmission. Subsequent infections across the sheds wherein animals were being housed occurred due to airborne transmission. Based on a R₀ of 2.90 and a growth rate equal to 0.293, the generation time was estimated to be 3.6 days, indicative of the massive SARS-CoV-2 dispersal among minks. After the end of the outbreaks, a similar percentage of animals were immune in the two farms (93.0% and 93.3%), preventing further virus transmission whereas, viral RNA was detected in samples collected from shed surfaces and air. Consequently, strict biosecurity is imperative during the occurrence of clinical signs. Environmental viral load monitoring, in conjunction with NGS should be adopted in mink farm surveillance. The minimum proportion of minks that need to be immunized to avoid outbreaks in farms was calculated at 65.5%, which is important for future vaccination campaigns.

Mutations resulting in amino-acid substitutions of the SARS-CoV-2 spike protein receptor-binding domain (RBD) have been associated with enhanced transmissibility and immune escape of the respective variants, namely Alpha, Beta, Gamma or Delta. Rapid identification of the aforementioned variants of concern and their discrimination of other variants is thus of importance for public health interventions. For this reason, a one-step real-time RT-PCR assay employing four locked nucleic acid (LNA) modified TaqMan probes was developed, to target signature mutations associated with amino-acid substitutions at positions 478, 484 and 501 present in the receptor-binding motif (RBM) of the spike protein RBD. This region contains most contacting residues of SARS-CoV-2 that bind to ACE2. A novel strategy employing the use of non-extendable LNA oligonucleotide blockers that can reduce non-specific hybridization of probes increased the number of different mutated sites examined in a multiplex PCR. The combinatory analysis of the different fluorescence signals obtained enabled the preliminary differentiation of SARS-CoV-2 variants of concern. The assay is sensitive with a LOD of 263 copies/reaction for the Delta variant, 170 copies/reaction for the Beta variant, amplification efficiencies > 91% and a linear range of >5 log₁₀ copies/reaction against all targets. Validation of the assay using known SARS-CoV-2-positive and negative samples from humans and animals revealed its ability to correctly identify the targeted mutations and preliminary characterize the SARS-CoV-2 variants. The novel approach for mutation typing using LNA oligonucleotide blockers can be modified to target signature mutations at four different sites in the RBM and further expand the range of variants detected.

Foodborne parasitic diseases represent a major threat to public health. Trichinellosis, caused by the nematode parasite Trichinella spp., is one of the most important foodborne diseases, while alariosis, caused by the trematode parasite Alaria spp., is less common in humans, and rare cases have been reported only in the USA and Canada. Both parasites can infect humans via the consumption of raw or undercooked wild boar meat. In order to investigate the prevalence of these parasites in wild boar meat in Greece, samples from the diaphragm pillars and the region of the mandibular angle from 128 wild boars, hunted in Greece, were collected. The samples were examined by classical parasitological (compression, artificial digestion, and Alaria spp. migration) and by molecular (real-time PCR) methods. For Trichinella spp. an existent real-time PCR detecting all species likely to be present in Greece was applied, while for Alaria spp. a real-time PCR was developed, employing an LNA TaqMan probe targeting the large subunit ribosomal RNA gene. All examined wild boar samples from Greece resulted negative for Trichinella and Alaria species, indicating a low prevalence of infection in the examined population. The novel real-time PCR for Alaria spp. has 81.5% amplification efficiency and is able to detect 0.12 larvae per 50 g of tissue and could be utilized as a complementary to AMT diagnostic tool in surveillance.

Small ruminant lentiviruses are a group of viruses infecting goat and sheep worldwide. These viruses exhibit an extraordinary degree of genetic and antigenic variability that severely influence in vivo and in vitro features, as well as diagnostic test results. Small ruminant farming is the most important animal farming business in Greece, with a high impact on the Greek primary economy. Although SRLV infection and its impact on animal production are well established in the country, little is known about the circulating SRLV strains and their prevalence. The aim of this study was to characterize SRLVs circulating in Greece with a combined serological and molecular approach, using the bulk milk matrix collected from 60 farms in different municipalities. This study allowed us to estimate a seroprevalence of around 52% at the herd level. The B1, B2 and A3 subtypes and a novel A viral cluster were identified. Moreover, the amplicon sequencing method allowed us to identify more than one viral subtype in a sample. These results again confirm the high variability of these viruses and highlight the importance of the constant monitoring of viral evolution, in particular in antigens of diagnostic interest.

The incidence of small ruminant pestivirus infections in Greece remains unknown as they have not been diagnosed in the country since 1974 when the most recent Border Disease Virus (BDV) outbreak was reported. The objective of our study was to explore the possible occurrence of pestiviral infections among sheep and goat farms in Greece and to further determine the variants of major concern. Thus, serum samples were collected from 470 randomly selected animals belonging to 28 different flocks/herds. ELISA on p80 antibody revealed the existence of seropositive animals in four out of the 24 studied sheep flocks, whereas all the goats in the four studied herds were seronegative. Viral RNA and antigens were detected in two sheep out of the four seropositive flocks by RT-PCR and ELISA, respectively. Sequencing and phylogenetic analysis showed that the newly identified Greek variants were closely related to the strains of the BDV-4 genotype. One of the BDV-positive sheep demonstrated the diagnostic profile of a persistently infected (PI) animal, providing additional information regarding the source of the infection. This is the first molecular identification of BDV isolates in Greece. Our findings indicate that BDV infections are likely to remain undiagnosed, highlighting the need for further epidemiological studies and active surveillance programs to determine the prevalence and impact of BDV infections on a countrywide level.