Listeriosis is a dangerous zoonosis caused by bacteria of the genus Listeria, with Listeria monocytogenes (LM) being the most pathogenic species. Listeria monocytogenes has been detected in various animal species and in humans, and its ability to evolve from an environmental saprophyte to a powerful intracellular pathogen is driven by the invasion mechanisms and virulence factors that enable cell invasion, replication and cell-to-cell spread. Key regulatory systems, including positive regulatory factor A (PrfA) and the stress-responsive sigma factor σ^B, control the expression of virulence genes and facilitate invasion of host cells. Listeriosis poses a significant threat to cattle, sheep and goat herds, leading to abortions, septicemia and meningoencephalitis, and ruminants are important reservoirs for Listeria, facilitating transmission to humans. Other Listeria species such as Listeria ivanovii and Listeria innocua can also cause disease in ruminants. Resilience of LM in food processing environments makes it an important foodborne pathogen that is frequently transmitted through contaminated meat and dairy products, with contamination often occurring along the food production chain. In humans, listeriosis primarily affects immunocompromised individuals, pregnant women and the elderly and leads to severe conditions, such as meningitis, septicemia and spontaneous abortion. Possible treatment requires antibiotics that penetrate the blood–brain barrier. Despite the relatively low antimicrobial resistance, multidrug-resistant LM strains have been detected in animals, food and the environment. Controlling and monitoring the disease at the herd level, along with adopting a One Health approach, are crucial to protect human and animal health and to minimize the potential negative impacts on the environment.

The aim of this study was to determine the effects of dietary vibroactivated clinoptilolite supplementation on the intramammary microbiological findings in dairy cows, causative agents of potential intramammary infection, and their sensitivity to antibiotics. Cows (n = 78) were randomly divided into two groups: CPL-treated group that received clinoptilolite (CPL) in-feed (n = 38), i.e., 50 g natural powdered zeolite CPL, twice daily from the seventh month of pregnancy to 75 days after calving, and the control group (CON) of untreated cows (n = 40). Milk samples were taken from each cow on days 7, 25, 45, and 75 postpartum. The following causative pathogens were isolated in 86 udder quarters: Staphylococcus aureus in 5.81% of positive samples, Staphylococcus spp. 9.32%, coagulase-negative Staphylococcus (CNS) 22.09%, Streptococcus uberis 13.95%, Streptococcus agalactiae 1.16%, Streptococcus sp. 3.49%, Escherichia coli 8.13%, Enterococcus spp. 6.98%, Corynebacterium spp. 11.63%, Pasteurella sp. 10.47%, Serratia spp. 2.33%, and Arcanobacterium pyogenes, Citrobacter sp., Prototheca sp., and yeasts each in 1.16% of samples. Additionally, 3.87% of environmental microflora samples (n = 47) and 89.06% of udder samples (n = 1083) were bacteriologically negative. The most effective antibiotics were cefoperazone and amoxicillin-clavulanic acid, while cloxacillin and tetracycline were the least effective antibiotics in both groups. In the clinoptilolite supplemented (CPL) group (n = 38) of 14 cows, nine causative agents of mastitis were isolated in 27 quarters, while in the control (CON) group (n = 40) of 24 cows, 13 causative agents of mastitis were isolated in 59 quarters. Cows from the CON group had a 1.96 times higher risk of intramammary infection than cows from the CPL group during the observation period (odds ratio = 1.96, p = 0.0031; 95% CI = 1.2570–3.0770).

Small ruminant lentiviruses (SRLVs), i.e., CAEV and MVV, cause insidious infections with life-long persistence and a slowly progressive disease, impairing both animal welfare and productivity in affected herds. The complex diagnosis of SRLVs currently combines serological methods including whole-virus and peptide-based ELISAs and Immunoblot. To improve the current diagnostic protocol, we analyzed 290 sera of animals originating from different European countries in parallel with three commercial screening ELISAs, Immunoblot as a confirmatory assay and five SU5 peptide ELISAs for genotype differentiation. A newly developed nested real-time PCR was carried out for the detection and genotype differentiation of the virus. Using a heat-map display of the combined results, the drawbacks of the current techniques were graphically visualized and quantified. The immunoblot and the SU5-ELISAs exhibited either unsatisfactory sensitivity or insufficient reliability in the differentiation of the causative viral genotype, respectively. The new truth standard was the concordance of the results of two out of three screening ELISAs and the PCR results for serologically false negative samples along with genotype differentiation. Whole-virus antigen-based ELISA showed the highest sensitivity (92.2%) and specificity (98.9%) among the screening tests, whereas PCR exhibited a sensitivity of 75%.

Background: Pathogenic strains of Escherichia coli ( E. coli ) cause colibacillosis in pre- and post-weaning piglets. Fimbrial and non-fimbrial adhesins, as well as heat-labile and heat-stable enterotoxins, are main virulence factors in enterotoxigenic (ETEC), enteroaggregative (EAEC), enteropathogenic (EPEC) and shigatoxigenic (STEC) pathotypes which cause colidiarrhea or colitoxemia in piglets. Methods: Fifty-five piglets submitted for necropsy were examined for gross and histological lesions. E. coli strains were isolated, biochemically confirmed, and tested by PCR for 15 virulence genes (VGs). Statistical analyses used appropriate parametric or non-parametric tests, depending on the distribution. The results with p values less than or equal to 0.05 (p ≤ 0.05) were considered statistically significant. Results: Overall, 84.48% of strains carried at least one VG. The occurrence of six VGs - astA , estII , faeG , estI , elt , and paa - was high, with frequencies of 67.24%, 63.97%, 55.18%, 50.00%, 48.27%, and 44.82%, respectively. ETEC predominated (63.79%), while 5.17% of strains carried EPEC or STEC genes; 15.52% were non-specific virotypes, and 15.52% were apathogenic. Lesions were most prominent in the small intestine. The virotype LT:STa:STb:EAST1:PAA:F4 was most common, whereas STa:Stx2:Stx2e was linked to the most severe lesions. Lesions varied depending on the pathotype involved and the VGs expressed. Severity of lesions differed significantly between suckling and weaned piglets (p = 0.0091) and between piglets with and without diarrhea (p = 0.0223), with suckling and diarrheic piglets showing more pronounced pathological changes. More extensive lesions in ETEC were associated with the acquired astA and paa genes. Pathoscores were significantly associated with faeG /F4 (p = 0.0001), eltA /LT (p = 0.0001), estII /STb (p = 0.0001), paa /PAA (p = 0.0002), and astA /EAST1 (p = 0.0029). Discussion and conclusions: Strong associations between specific VGs - particularly faeG , eltA , estII , paa , and astA - and higher lesion scores show that VG detection can help predict disease severity and guide interventions. Age-specific interpretation is crucial, as isolates from pre-weaned piglets often carried more VGs and were associated with more severe lesions. This study underscores the value of integrating bacteriological, molecular and histopathological data for accurate diagnosis, especially given the high prevalence of VG-positive and recombinant ETEC strains.

Owing to its hypoallergenic properties, nutritional value, and emerging therapeutic potential, donkey milk (DM) is attracting increasing scientific and commercial interest. However, its microbiome remains poorly characterised, with most studies relying on culture-dependent methods. Here, we conducted comparative high-throughput 16S rRNA amplicon sequencing of DM samples from two geographically and climatically distinct Croatian farms in Primorje-Gorski Kotar County (PGC) and Bjelovar-Bilogora County (BBC) to determine whether farm-associated factors significantly influence the microbial community composition of DM. To assess the impact of the lactation phase, two longitudinal samples per jenny were collected approximately two months apart, beginning within ten days postpartum. Across hierarchical clustering, α-diversity metrics (Observed, Chao1, and Fisher), and β-diversity ordination approaches (PCoA and NMDS), farm origin consistently emerged as the primary determinant of DM microbial community structure, surpassing the effects of lactation phase and other recorded variables under the conditions examined. Differential abundance analyses further revealed pronounced compositional differences associated with farm location across multiple taxonomic levels. These findings indicate that farm-associated factors are major determinants of DM microbiome composition and diversity, underscoring the importance of regional and environmental context in dairy microbiome research.

In this study, the concentrations of metals (arsenic (As), cadmium (Cd), copper (Cu), mercury (Hg) and lead (Pb)), and aflatoxin M1 were analysed in mare's milk samples taken from eight mares over a period of six months during lactation. The results showed that the concentrations of metals in milk occurred in the following order: Cu > Pb > Hg > Cd > As. Key findings of the study included the determined content of metals, depending on lactation stages and animal husbandry practices. Copper had the highest concentration among the metals analysed, while Cd and As were not detected. No statistically significant differences were found in the levels of heavy metals or aflatoxin M1 across different lactation stages. However, statistically significant differences in aflatoxin M1 concentrations were observed depending on the type of animal husbandry, illustrating the influence of environmental and husbandry conditions on milk contamination. The study concludes that there is no toxicological risk for consumers of Croatian Cold-Blooded mare's milk, which is an important finding for food safety and public health. The significance of this study lies in the provision of valuable data on the safety of mare’s milk, especially with regard to its possible contamination with toxic heavy metals and aflatoxin M1. The main objective was to determine the presence and concentrations of the listed contaminants, to evaluate the influence of lactation stage and husbandry, and to assess compliance with established safety standards.