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A novel bovine astrovirus genotype species (BoAstV-CH13/NeuroS1) was recently identified in brain tissues of cattle as a plausible cause of encephalitis. The purpose of the present study was to develop and validate real time RT-PCR assays for the detection of BoAstV-CH13/NeuroS1 in brain tissues of cattle. Three different primer-probe combinations were designed based on BoAstV-CH13/NeuroS1 full-genome sequences of 11 different strains identified in cattle, and established in three distinct one-step real time RT-PCR protocols. These protocols were compared regarding their diagnostic performance using brain tissues of cattle with and without astrovirus associated encephalitis. The limit of detection (LOD) of all three assays was between 1.34 × 10 1 and 1.34 × 10 2 RNA copies, leading to an analytical sensitivity two orders of magnitude superior compared to a conventional pan-astrovirus RT-PCR protocol (LOD 1.31 × 10 4 RNA copies). Amplification efficiency was in the range of 97.3% to 107.5% with linearity (R 2 ) > 0.99. The diagnostic sensitivity and specificity of the assays was determined as 100%, and all three revealed good intra- and inter-test repeatability. In conclusion, the newly developed RT-qPCRs are sensitive, specific, and reliable test formats that will facilitate BoAstV-CH13/NeuroS1 detection in routine diagnostics as well as in research settings.

Brucellosis in small ruminants, primarily caused by B. melitensis, remains a significant threat to public health in many regions. Although early-age vaccination of breeding stocks was expected to facilitate infection control, this approach did not meet expectations, while in some cases, late vaccination of animals has been associated with an increased number of human cases. Therefore, in this field study, we investigated the immune response and bacteremia cases in ten apparently healthy hoggets vaccinated at the age of nine months. Before vaccination, the hoggets were seronegative and negative in blood cultures, although B. melitensis DNA was detected in three animals using PCR. After vaccination, twelve Brucella spp. strains were isolated from the blood cultures of nine hoggets at different time points. Whole genome analysis identified eleven of them as identical to three B. melitensis strains previously isolated in Greece. The tested animals completed their gestation without any adverse outcomes. According to our results, late vaccination, despite extending animal exposure to B. melitensis, apparently protects against disease and abortion but not against infection. The onset of post-vaccination immune response may be influenced by transient infections by field strains.

Aujeszky’s disease, or pseudorabies, is a viral infection caused by Suid herpesvirus 1 (pseudorabies virus), with swine as its natural host. Although eradicated in domestic pigs in many European countries, PRV remains endemic in wild boar populations, posing a risk to other species, including carnivores. In this study, we report eight fatal cases of PRV infection in hunting dogs from Epirus and Thessaly, Greece, all of which followed direct contact with hunted wild boars. Postmortem brain samples tested positive for PRV via PCR targeting the glycoprotein C (gC) gene. Partial sequencing and phylogenetic analysis of the amplified gC fragments revealed genetic divergence among the examined isolates. The Epirus-derived strains formed a distinct cluster, closely related to previously reported Greek strains from the region of Central Macedonia as well as to the French strain FRA 527 and the German isolate GER614BW. In contrast, the two Thessaly sequenced isolates were phylogenetically distant from all other Greek strains, potentially representing an independently evolving lineage, and clustered more closely with the Kaplan strain. These findings underscore the persistent threat of PRV transmission from wild to domestic species and highlight the genetic heterogeneity of PRV strains circulating in Greece. Veterinary practitioners should consider PRV in the differential diagnosis of encephalitic symptoms in hunting dogs. Enhanced molecular surveillance and public awareness are critical to mitigating the risks posed by this emerging threat.

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.

European sporadic bovine encephalitis is a frequent diagnosis in neurologically diseased cattle, but its etiology remained unresolved. Using hybridization, we have detected a recently discovered neurotropic bovine astrovirus in historical tissues in a high proportion of brain samples of affected cattle. Our results suggest that astroviruses were already involved in the pathogenesis of the disease several decades ago, but have gone undetected.

The objective of this study is to prospectively evaluate the seroepidemiology of maedi-visna (MV) infections in intensively reared dairy sheep. A total of 407 purebred Chios and Lacaune ewes from four farms were surveyed for two consecutive years and were serologically tested semiannually with an indirect ELISA at pre-mating and pre-lambing. The farms’ structure and management practices were similar and animal traits (age, breed, and production stage) were recorded. Based on the serological status, morbidity frequency measures were estimated, and ewes were categorized as constantly seronegative, constantly seropositive, seroconverted, seroreverted, or as animals with an intermittent presence of antibodies. During the study, period seroprevalence, incidence rate, and cumulative incidence were 84.8%, 33.6 new cases per 100 sheep-semesters, and 64.2%. Point-seroprevalence ranged from 48.5% to 96.0% among the studied farms and sampling occasions, and they increased by age. Increased morbidity frequency measures indicate the significance of horizontal transmission in intensive dairy sheep farms. A remarkable percentage of infected animals seroreverted (8.1%) or presented an intermittent presence of antibodies (10.3%) during the study, confirming the risk of misdiagnosis in cross-sectional studies and in the currently implemented testing and elimination programs. The serological patterns observed in our study need to be considered when studying MV epidemiology and for the designing of efficient MV elimination programs.

The high genetic heterogeneity of small ruminant lentiviruses (SRLV) renders the genetic characterization of the circulating strains crucial for the epidemiological investigation and the designation of effective diagnostic tools. In Greece, research data regarding the genetic diversity of the circulating SRLV strains is scarce, hindering the implementation of efficient surveillance and control programs. The objective of the study was to genetically characterize SRLV strains isolated from intensive dairy sheep farms in Greece and evaluate the variability of the immunodominant regions of the capsid protein. For this reason, a total of 12 SRLV-infected animals from four intensive dairy sheep farms with purebred Chios and Lacaune ewes were used for the amplification and sequencing of an 800 bp gag-pol fragment. The phylogenetic analyses revealed a breed-related circulation of strains; Chios ewes were infected with strains belonging exclusively to a separate group of genotype A, whereas strains belonging to subtype B2 were isolated from Lacaune ewes. Immunodominant epitopes of capsid protein were quite conserved among the strains of the same genotype, except for the Major Homology Region which showed some unique mutations with potential effects on viral evolution. The present study contributes to the extension of the current knowledge regarding the genetic diversity of SRLV strains circulating in sheep in Greece. However, broader genetic characterization studies are warranted for the exploration of possible recombinant events and the more comprehensive classification of the circulating strains.

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.

A two-year longitudinal cohort study was conducted on a total of 407 purebred Chios and Lacaune ewes from four intensive dairy sheep farms to assess potential risk factors for small ruminant lentiviruses (SRLVs) seropositivity. Ewes were serologically tested semiannually at pre-mating and pre-lambing, and their age, breed, and body condition score (BCS) were recorded. Εwes were categorized as constantly seronegative, constantly seropositive, seroconverted, seroreverted, or animals with an intermittent presence of antibodies. Mixed binary logistic regression models were used to estimate the adjusted relative risks of the studied risk factors for (i) the individual ewes’ seropositivity, (ii) the manifestation of specific serological patterns, and (iii) the occurrence of seroconversion and seroreversion incidents. Increased age was associated with seropositivity and constantly seropositive status (p < 0.001 in both cases). On the other hand, age was negatively associated with constantly seronegative pattern, seroconversion incident, and the intermittent presence of antibodies (p < 0.05 in all cases). Moreover, breed was recognized as a risk factor: Lacaune ewes demonstrated increased seropositivity, whereas Chios ewes were more likely to demonstrate an intermittent presence of antibodies (p < 0.01 in both cases). Seropositive status (p < 0.001), seropositivity in animals with an intermittent presence of antibodies (p = 0.001), and seroconversion incidents (p < 0.001) were significantly increased at pre-lambing compared to pre-mating. The risk factors recognized in our study contribute to a better understanding of SRLVs epidemiology and the evidence-based designation of SRLVs’ control programs in intensive dairy sheep farms in Greece.

Background The occurrence of paratuberculosis in Ugandan cattle has recently been reported but there is no information on the strains of Mycobacterium avium subspecies paratuberculosis (MAP) responsible for the disease. The aim of this study was to isolate and characterise MAP from seropositive cattle and paratuberculosis lesions in tissues obtained from slaughtered cattle in Uganda. Results Twenty one isolates of MAP were differentiated into 11 genotype profiles using seven genotyping loci consisting of Insertion Sequence 1311(IS 1311 ), Mycobacterial interspersed repeat units (MIRU) (loci 2, 3), Variable number tandem repeats (VNTR) locus 32 and Short sequence repeats (SSR) (loci 1, 2 and 8). Three different IS 1311 types and three MIRU 2 profiles (7, 9, 15 repeats) were observed. Two allelic variants were found based on MIRU 3 (1, 5 repeats), while VNTR 32 showed no polymorphism in any of the isolates from which it was successfully amplified. SSR Locus 1 revealed 6 and 7 G1 repeats among the isolates whereas SSR locus 2 revealed 10, 11 and 12 G2 repeats. SSR locus 8 was the most polymorphic locus. Phylogenetic analysis of SSR locus 8 sequences based on their single nucleotide polymorphisms separated the isolates into 8 genotypes. We found that the use of Ethylene glycol as a PCR additive improved the efficiency of the PCR reactions for MIRUs (2, 3), VNTR 32 and SSR (loci 1 and 2). Conclusions There is a high strain diversity of MAP in Uganda since 21 isolates could be classified into 11 genotypes. The combination of the seven loci used in this study results into a very precise discrimination of isolates. However analysis of SNPs on locus alone 8 is very close to this combination. Most of the genotypes in this study are novel since they differed in one or more loci from other isolates of cattle origin in different studies. The large number of MAP strains within a relatively small area of the country implies that the epidemiology of paratuberculosis in Uganda may be complicated and needs further investigation. Finally, the use of Ethylene glycol as a PCR additive increases the efficiency of PCR amplification of difficult templates.