Search Results (545)

The effects of pelleting a total mixed ration (TMR) for dairy sheep were investigated in an experiment involving 24 lactating Assaf ewes, which were assigned to two groups and fed the same TMR ad libitum, offered either in pelleted (PTMR group, n = 12) or in unpelleted form (CTMR group, n = 12). The experiment lasted 28 days, during which feed intake, eating behavior (including meal frequency and size, meal duration, eating rate, between-meal interval), and milk yield were recorded daily. Body weight (BW) was recorded on days 1 and 28 and milk samples were collected on days 1, 8, 15, 22 and 28 for milk composition analysis. Blood acid-base status was determined at the beginning and at the end of the trial. Ewes fed the CTMR diet exhibited (p < 0.05) a higher meal frequency and longer meal duration, along with a smaller meal size and slower eating rate. However, feed intake in this group was less than that in ewes fed PTMR only during the final two weeks of the experimental period. Total eating time was also longer (p < 0.001) in the CTMR group, whereas the average time between meals was shorter (p < 0.002). No differences (p > 0.05) were observed between dietary treatments in blood acid-base status, milk yield or milk composition. However, a diet x day interaction (p < 0.05) was detected for milk yield, as during the last 2 weeks of the experimental period the ewes fed the PTMR yielded more milk than those fed the CTMR. Feed conversion ratio did not differ between groups (p > 0.05), but body weight loss was greater in ewes fed the CTMR diet (−3.00 vs. −0.58 kg; p < 0.05). A trend toward improved feed efficiency was observed in the PTMR group when calculated based on milk yield corrected for that theoretically derived from the mobilization of body reserves (1.98 vs. 1.41 g DMI/kg milk; p = 0.077), with estimated contributions from body reserves of 485 g/day in the CTMR group and 70 g/day in the PTMR group. In conclusion, the use of pelleted total mixed rations in high-yielding dairy ewes enhances feed intake, feed efficiency, milk yield, and energy balance without adversely affecting milk composition or animal health in the short term. Full article

This study evaluated the effects of varying dietary salt and boric acid addition doses on the milk quality of Savak Akkaraman sheep. A total of 120 animals were as-signed to six treatment groups (n = 20): control (C), rock salt (S; 10 g/day), boric acid 20 mg/day (B20), boric acid 40 mg/day (B40), BS20 (20 mg boric acid + 10 g/day rock salt), and BS40 (40 mg boric acid + 10 g/day rock salt). All analyses were performed in duplicate on six samples, taken on days 30 and 35 following the administration of the additives. Physicochemical analyses only showed significant variation in milk pH (p = 0.006), while acidity, dry matter, and ash remained unaffected. Strong positive correlations were found among protein, lactose, salt, and density (r > 0.95; p < 0.001). Coagulation times differed widely, with the longest being observed in BS20 (995.03 s) and the shortest in BS40 (141.73 s). Among mineral parameters, only selenium levels differed significantly between the treatment groups (p < 0.05). No significant differences were found for fat, solids-not-fat, lactose, freezing point, or electrical conductivity. Importantly, boron addition had a significant influence on total casein content (p < 0.001). Overall, dietary rock salt and boric acid did not markedly alter the basic milk composition but produced notable physicochemical changes, particularly in coagulation behavior and casein levels, which may influence the technological properties of sheep milk. Full article

In African countries and many developing countries, communal farmers rely on livestock such as cattle, goats, and sheep to support food security, income, and agricultural activities. Fertility in these animals is often limited by poor semen quality, which reduces sperm concentration, total motility, and morphology. Assisted reproductive biotechnologies, including semen cryopreservation and artificial insemination, are increasingly essential to enhance reproductive efficiency and productivity. Although cryopreservation preserves valuable genetic material, it can damage sperm cells, making high-quality extenders critical for protection. Common extenders, such as Tris-egg yolk glucose, citrate-sugar-based, and skimmed milk solutions, supply nutrients and protect sperm membranes. To further minimize oxidative stress, antioxidants are incorporated, with growing interest in plant-derived compounds. Many plants contain bioactive substances, including antioxidants and phytomelatonin, which can enhance sperm quality safely and effectively. This review examines the use of plant-based antioxidants during semen cryopreservation and highlights their potential to improve fertility in mammalian livestock. Full article

Precision livestock farming (PLF), by integrating multimodal sensor data, provides opportunities to enhance welfare monitoring and management in small ruminants. This study evaluated whether environmental, physiological, and behavioral measurements—including the temperature–humidity index (THI), carbon dioxide (CO₂) and ammonia (NH₃) concentrations measured at the barn level, body condition score (BCS), rectal and ocular temperatures, GPS-derived locomotion metrics, accelerometry data, and fixed animal traits—can serve as key predictors of welfare and productivity in dairy sheep. Data were collected from 90 ewes: all animals underwent the same repeated welfare assessments, while 30 of them were additionally equipped with GPS–accelerometer sensor collars; environmental conditions were continuously recorded for the entire flock, generating 773 complete multimodal records. All predictive models were developed using data from all 90 ewes; collar-derived behavioral variables were included only for individuals equipped with GPS–accelerometer collars. Nine regression methods (linear regression (LR), partial least square regression (PLSR), elastic net (EN), mixed-effects models, random forest (RF), extreme gradient boosting (XGBoost), support vector regression (SVR), neural networks (multilayer perceptron, MLP), and an ensemble of RF–XGBoost–EN were evaluated using a combination of nested cross-validation (CV) and leave-one-animal-out CV (LOAOCV) to ensure robustness and generalization at the individual animal level. Nonlinear models—particularly RF, XGBoost, SVR, and the ensemble—consistently delivered superior performance across traits. For behavioral (e.g., daily distance movement) and thermal indicators (e.g., medial canthus temperature), the highest predictive capacity (R² ≈ 0.60–0.70) was achieved, while moderate predictive capacity (R² ≈ 0.40–0.50 and ≈0.35–0.45), respectively, was observed for respiratory rate and milk yield, reflecting their multifactorial nature. Feature importance analyses underscored the relevance of THI, CO₂, NH₃, concentrations, and BCS across results. Overall, these findings demonstrate that multimodal sensor fusion can effectively support the prediction of welfare and productivity indicators in intensively reared dairy sheep and emphasize the need for larger and more diverse datasets to further enhance model generalizability and model transferability. Full article

Staphylococcus aureus (S. aureus)-induced mastitis poses a significant threat to animal husbandry. This condition triggers sustained mammary inflammation, oxidative stress, and disrupts mitochondrial homeostasis, ultimately impairing mammary gland function and milk yield. Conjugated linoleic acid (CLA) is a long-chain fatty acid found in meat and dairy products derived from ruminants. It exhibits multiple biological activities, including anti-cancer, anti-inflammatory, and antioxidative stress-alleviating effects. Thus, this study sought to determine whether CLA alleviates S. aureus-induced mastitis in Hu sheep through the PPARG-UCP2 axis. Fifteen lactating Hu sheep were randomly allocated into three groups (n = 5): control group, model group, and CLA group. The CLA group received 1 mg/mammary gland of CLA via intramammary infusion for seven days, followed by S. aureus challenge (5 × 10⁷ cells/mL, 2 mL/mammary gland) in the model and CLA groups, while the control group received saline. Venous blood and mammary tissue samples were collected at two days post-infection. The results demonstrated that S. aureus infection significantly upregulated the expression of inflammatory factors (IL-1β, IL-6, and NF-κB) in the mammary tissue of Hu sheep, p < 0.01. Relative to the control, the model group showed increased ROS and MDA levels, a diminished NAD+/NADH ratio, and downregulated expression of the antioxidant factors SOD, Nrf2, HO-1, and SIRT3, p < 0.01. Furthermore, the expression of p-AMPK and mitophagy-related factors (PARKIN, PINK1, and LC3b) showed a statistically significant increase in the model group than in the control group, p < 0.01. S. aureus infection also suppressed the expression of PPARG and UCP2, p < 0.01. In contrast, the CLA group showed lower levels of inflammatory factors (IL-1β, IL-6, and NF-κB), ROS and MDA, while the NAD+/NADH ratio and the expression of antioxidant factors (SOD, p-Nrf2, HO-1, and SIRT3) were elevated compared with the model group, p < 0.01. Moreover, the expression of p-AMPK and mitophagy-related factors (PARKIN, PINK1, and LC3b) was reduced in the CLA group relative to the model group, p < 0.05. Concurrently, the expression of PPARG and UCP2 was higher in the CLA group than in the model group, p < 0.001. These findings demonstrated that S. aureus infection induced mastitis in Hu sheep mammary tissue, whereas CLA alleviated the infection by upregulating the PPARG-UCP2 pathway, thereby reducing inflammation, oxidative stress, and mitophagy levels. This study offers a novel perspective on mammary tissue repair during mastitis and expands the understanding of UCP2’s biological role. Full article

Understanding the function of enzymes before their use as additives in ruminant diets is essential for achieving sustainable and efficient agricultural practices. Ruminants such as cattle, sheep, and goats are vital for global food production because of their ability to convert fibrous plant materials into high-quality proteins through enteric fermentation. Various datasets were carefully selected from four scientific databases: Science Direct, Scopus, PubMed, and Google Scholar. The rigorous Preferred Reporting Items for Systematic Review and Meta-Analysis (PRISMA) protocol was employed to ensure the eligibility of the selected articles used in the analysis. A systematic review showed that the administration of various types of enzymes can increase dry (DMD) and organic (OMD) matter, neutral (NDFD), and acid (ADFD) detergent fibre, and crude protein (CPD) digestibility in both in vitro and in vivo tests in individual studies. However, the pooled meta-analysis indicated that their overall effect on CPD was not significant (p > 0.05). The OpenMEE approach was used to calculate the effect size (Hedges’ g) for each experimental unit for various parameters. Across enzyme types and doses in the meta-analysis, the administration of enzymes did not have any significant effect (p > 0.05) on DMI, OMI, and CPD, but it did have a significant effect (p < 0.05) on DMD, OMD, ADFD, NDFD, pH and gas production at 24, 48, and 72 h, as assessed by in vitro experiments. Ruminant in vivo studies indicated that the administration of enzymes has significant impacts (p < 0.05) on digestibility parameters (DMD, OMD, NDFD, ADFD), milk production, milk lactose content, acetate, and propionate, but it had non-significant impacts on milk protein and rumen total volatile fatty acids and acetate: propionate ratio. Full article

Brucellosis remains a major zoonotic disease worldwide, with disproportionate burden in low- and middle-income countries where limited veterinary and healthcare infrastructure constrain effective control measures. However, its pathways of transmission are poorly understood. In pastoralist settings, we hypothesize that camels have a high burden of Brucella spp. and play a key role in spreading it to humans and other livestock. This manuscript presents a study protocol to quantify the relative contribution of various livestock species to brucellosis transmission and identify cost-effective control strategies in Kenya. Using probability-proportional-to-size sampling, we aimed to recruit a longitudinal cohort of 170 households and their herds per site in the Marsabit and Kajiado counties. Households rearing at least one livestock species (cattle, camels, goats, sheep) were eligible. Serum, milk, and vaginal swabs (from livestock), and serum (from humans) were collected for testing using Rose Bengal Test, ELISA, qPCR, and culture methods. Concurrently, surveillance for suspected brucellosis was conducted in study health facilities. A qualitative ethnographic study and livestock movement monitoring using GPS-collared animals were nested within the cohort. These data will be used to parameterize a multi-host, multi-species infectious disease model through Approximate Bayesian Computation. Through this One Health approach, our study will identify and optimize potential interventions and help inform the development of a comprehensive cost-effective national control program for brucellosis. Full article

Background/Objectives: Goat and sheep farming is an important agro-economic resource in Benin. However, their milk is both underutilized and insufficiently characterized, which limits the development of innovative dairy products and raises concerns about its safety. Against this backdrop, our pioneering study set out to investigate, for the first time in Benin and using an advanced metagenomic approach, the microbial diversity present in goat and sheep raw milk. The aim was to lay the groundwork for safer and more efficient dairy valorization. Methods: To achieve this, metagenomic DNA was extracted from 20 pooled milk samples representing both animal species, followed by shotgun sequencing. Results: Analyses revealed seven dominant phyla: Bacillota (17.44–27.23%), Pseudomonadota (12.39–15.55%), Campylobacterota (3.65–5.29%), Actinomycetota (1.47–6.03%), Spirochaetota (1.14–2.02%), Apicomplexa (0.28–0.50%), and Bacteroidota (0.17–0.22%) in the raw milk of both species. However, their proportions differ. Bacillota, which was the most dominant in both types of milk, was significantly more abundant in goat (27.23 ± 5.33) than in sheep milk (17.44 ± 8.44). In sheep milk, Enterobacteriaceae (11.36 ± 5.79) were the most predominant family, followed by Streptococcaceae (5.57 ± 2.29) and Staphylococcaceae (4.51 ± 3.63). Goat milk, on the other hand, presents a different hierarchy. Streptococcaceae (6.65 ± 2.19) and Staphylococcaceae (6.43 ± 2.33) were the most abundant families, surpassing Enterobacteriaceae (5.33 ± 1.66). The genus Escherichia was the most abundant in sheep milk (6.18 ± 5.33). The genera Staphylococcus (4.50 ± 3.63) and Streptococcus (5.05 ± 1.98) were also present. In contrast, in goat milk, the genera Streptococcus (6.54 ± 2.35) and Staphylococcus (6.42 ± 2.32) were the most dominant, while the average abundance of Escherichia was much lower (1.98 ± 1.28). In terms of species, Sheep milk was dominated by Escherichia coli (6.14 ± 5.28) and Staphylococcus aureus (5.17 ± 2.28) while Klebsiella pneumoniae (2.82 ± 1.72), Streptococcus pneumoniae (1.92 ± 1.36), and Campylobacter coli (1.52 ± 1.27) were also found. In addition to a relatively high abundance of Staphylococcus aureus (6.40 ± 2.45), goat milk was characterized by the presence of Corynebacterium praerotentium (5.32 ± 2.28) and Clostridium perfringens (3.39 ± 2.09). Additional pathogens identified included Clostridioides difficile (1.17–2.00%), Clostridium botulinum (0.27–0.43%), Listeria monocytogenes, Mycobacterium tuberculosis, Helicobacter pylori (0.36–0.62%), Salmonella enterica (0.22–0.26%). As for fungi, Ascomycota were predominant, with the presence of Aspergillus fumigatus, Saccharomyces cerevisiae, Trichophyton mentagrophytes, and Candida auris. Moreover, lactic acid bacteria with technological interest such as Oenococcus oeni (0.60–0.97%), Levilactobacillus namurensis (0.25–0.44%), Lactobacillus agrestimuris, and Lacticaseibacillus rhamnosus were also detected. Conclusions: These findings provide essential insights into the technological potential and health risks associated with these milks, which are key to developing safer and more efficient local dairy value chains. Full article

This study evaluated the microbiological quality, presence of virulence genes, and antimicrobial resistance of Staphylococcus aureus and Escherichia coli in sheep and goat milk and traditional Romanian dairy products (Telemea and Burduf cheeses). Raw milk contained high levels of coagulase-positive staphylococci (CPS; 5.2 log CFU/mL) and E. coli (4.3 log CFU/mL), with several samples exceeding legal limits. Sour cream exhibited moderate CPS (1.2–1.9 log CFU/g) and E. coli (0.4–3.12 log CFU/g) counts, with occasional non-compliance. Cheeses had low CPS (0.52–0.84 log CFU/g) and E. coli (0.37–0.89 log CFU/g) levels, all within permissible limits. Molecular analysis detected the nuc gene in 21 sheep milk samples, of which 85.7% also carried the sea gene and 14.3% carried seb. Additionally, two goat milk samples tested positive for both nuc and sea. Three Telemea cheese samples were also sea-positive. Among raw milk samples, 10 E. coli isolates carried stx1, and two additionally harbored hly, while dairy products were negative for E. coli virulence genes. Antimicrobial susceptibility testing showed high resistance of S. aureus to penicillin (90.5%) and ampicillin (85.7%), with multidrug resistance among sea- and seb-positive isolates. STEC isolates showed resistance to ampicillin (70%), tetracycline (50%), and trimethoprim–sulfamethoxazole (40%), but remained susceptible to amoxicillin–clavulanic acid. Full article

Autochthonous starter and adjunct cultures have gained increasing practical research interest for use in traditional cheese-making in recent years. This study evaluated the performance of a native starter comprising Streptococcus thermophilus ST1 and two wild Lactococcus lactis strains, including the M78 nisin A-producer, during the semi-industrial production of Kefalotyri cooked hard cheese from thermized sheep milk (TM; 65 °C, 30 s) in the absence (C-cheese) or presence of the native adjunct strains Lactiplantibacillus plantarum H25 and Leuconostoc mesenteroides KFM7 + KFM9 (N-cheese). The growth of the native starter was optimal in all three cheese trials within the first 24 h of processing, but afterward, ST1 failed to exceed 8 log CFU/g in favor of total mesophilic LAB, comprising both L. lactis starter strains, and the prevalent (ca. 8.5 log CFU/g) H25 adjunct in the ripening N-cheeses. Instead, in the C-cheeses, indigenous non-Enterococcus NSLAB survivors from TM prevailed, whereas enterococci failed to increase above 6 log CFU/g in all cheeses. Although the mature (90-day-old) N-cheeses presented no statistically significant differences regarding the pH value, gross composition, and hydrophobic (HO)/hydrophilic (HI) peptide ratios from the mature C-cheeses, they had lower total LAB counts and contained less residual lactose, more acetate, and an overall less diversified volatilome (VOC) profile. The most abundant VOCs in both cheeses were acetone, butyric acid, methyl butyrate, ethyl ether, and ethanol. All mature cheeses were safe and graded of ‘excellent quality’ (i.e., moisture < 35%; fat-in-dry-matter > 47%). Full article

Aflatoxins (Afs) belong to the most hazardous mycotoxins. Their detrimental effects on humans and higher animals are widely known, and actions are poised to avoid their synthesis already in the developing plant. However, this is often not effective enough or even not practicable, and hence, contaminated food and feed are consumed, resulting in severe health impairment. The use of adsorbents is one of the possibilities for the reduction or impediment of the venomous action after the intake of toxic food and feed. Purified clinoptilolite-tuff (PCT) was used as a binder of aflatoxins B1 and M1 in experiments with plant-based beverages as matrices for AfB1, while milk and dairy products of cow, sheep, and goat were the respective media for AfM1 contamination. Human gastrointestinal conditions were simulated by adequate temperature, movement, pH values, incubation times, and artificial juices. Analyses were implemented by appropriate ELISA assays for both toxins. PCT showed high affinity and kinetic velocity for AfB1 and AfM1. It neutralized irreversibly almost all toxins used with only traces detected after desorption experiments. PCT eliminated both plant-based beverages as well as milk and dairy products efficiently in a dose-dependent manner. This may offer a powerful method for minimizing the health risks of unavoidable aflatoxin exposure. Full article

In recent years, animal welfare has become a high priority in livestock production systems owing to the pressure to balance environmental sustainability, productivity, and ethics as demand continues to grow. This review presents the latest advances in small ruminant welfare, with emphasis on the effects of climate change, the main new innovative managerial and husbandry methods, and the use of precision livestock farming (PLF) technologies. In the first part, this review will examine how climate change is already re-shaping environmental and physiological conditions for farmed sheep and goats, with rising heat stress and negative impacts on both productive and reproductive performance. Secondly, more recent advances in small ruminant management will be presented, including improved housing systems, nutritional strategies, and behavioral monitoring, aimed at enhancing animal resilience and performance. Finally, particular focus will be given to the use of PLF tools for assessing milk quality and monitoring animal welfare. Evidence suggests that real-time monitoring technologies and sensor systems can accurately capture physiological and production parameters and provide an early sign of stress or health issues. Overall, the findings suggest that an integrated approach, combining climate adaptation strategies, welfare management, and the integration of precision technologies can serve as a key driver toward more ethical, sustainable, and resilient livestock production systems. Full article

Mastitis is a common mammary gland disease in mammals that severely impairs lactation function, with Staphylococcus aureus (S. aureus) being the primary pathogenic bacterium. However, the molecular mechanisms underlying S. aureus-induced mastitis in sheep remain incompletely elucidated. This study employed RNA sequencing (RNA-SEq) technology to systematically analyze the dynamic transcriptomic characteristics of mammary tissue in small-tailed sheep (SHT) after S. aureus infection, aiming to clarify the molecular regulatory mechanism of the host immune response and its relationship with the occurrence of mastitis. Twelve lactating STH were selected to establish an S. aureus-induced mastitis model. Blood, milk, and tissue samples were collected at 0, 24, 48, and 72 h post-infection (hpi). The infected sheep exhibited typical mastitis symptoms, including exacerbated breast swelling, reduced milk yield, elevated udder temperature, and darker, more viscous milk. Hematoxylin–eosin (HE) staining revealed significant pathological changes over time, such as stromal hyperplasia, extensive inflammatory cell infiltration, severe necrosis and sloughing of mammary epithelial cells, and compromised tissue integrity. RNA-Seq analysis identified 1299 differentially expressed genes (DEGs), among which 75 core genes maintained stable expression throughout the infection time (24 hpi, 48 hpi, and 72 hpi). Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses indicated that these DEGs were associated with metabolic processes, protein binding, Toll-like receptor signaling, and the NF-κB pathway. The PPI network analysis identified core hub genes including PTK2B, STAT3, and JAK1/3, providing critical evidence for therapeutic target screening. Furthermore, qPCR verification indicated that the expressions of innate immune receptors TLR2, TLR4, TLR7, and TLR10, as well as pro-inflammatory factors IL-1β, IL-16, TNF-α, type I interferon (IFN-α), and nuclear transcription factor NF-κB were significantly upregulated in a time-dependent manner (p < 0.05). In conclusion, this study delineated the dynamic response of ovine mammary tissue to S. aureus infection, systematically elucidated temporal gene expression patterns, and revealed the molecular mechanisms underlying the tissue’s initial defense against inflammatory challenges. Full article

Bioactive peptides (BAPs) from dairy products have garnered increasing attention as natural agents with health-promoting properties, including antihypertensive, antioxidant, antimicrobial, immunomodulatory, opioid, and antidiabetic activities. This systematic review synthesizes research published between 2014 and 2024, retrieved from Scopus and PubMed, and selected according to PRISMA guidelines. A total of 192 studies met the inclusion criteria, collectively reporting over 3200 distinct peptides, with antihypertensive sequences, predominantly angiotensin-converting enzyme (ACE) inhibitors, constituting the largest category (n = 1237). β-casein was the principal precursor across bioactivities, followed by αs1-casein, β-lactoglobulin, and α-lactalbumin. Peptides were primarily produced via enzymatic hydrolysis, microbial fermentation, and gastrointestinal digestion, with peptide profiles influenced by the type of milk, microbial strains, and processing conditions. While cow’s milk remained the dominant source, investigations into goat, sheep, camel, buffalo, and donkey milk revealed species-specific biopeptides. Recent advances in proteomics have enhanced peptide identification and bioactivity prediction, enabling the discovery of novel sequences. These findings underscore the significant potential of dairy-derived BAPs as functional food components and nutraceutical ingredients, while highlighting the need for further in vivo validation, bioavailability studies, and broader exploration of underrepresented milk sources. Full article

Autochthonous enterococci surviving mild thermization of raw milk (RM) before traditional Greek cheese processing may simultaneously comprise safe and virulent thermoduric strains with multiple antibiotic resistances (ARs). Therefore, this study biotyped and then compared the ARs of 60 Enterococcus isolates from two antilisterial sheep milks of native Epirus breeds before (RM) and after (TM) thermization at 65 °C for 30 s; the RM isolates were previously genotyped and evaluated for primary safety traits, namely, hemolytic activity, vanA/vanB, cytolysin, and virulence genes, by molecular methods. Biochemically typical and atypical strains of Enterococcus faecium (six biotypes), E. durans (five biotypes), E. faecalis (two biotypes), and E. hirae (one biotype), which were subdominant to other LAB species in RM (19 isolates), prevailed in TM (41 isolates). E. faecium biotypes 1A, 1D, and 1H included multiple-Ent+ (entA/entB/entP or entA/entB) strains with strong antilisterial CFS activity, whereas E. faecium 1X (entA), E. durans 2A, 2B, 2C, and 2X (entA/entP or entP), E. faecalis 3B, and E. hirae 4A (entA) biotypes displayed direct in vitro antilisterial activity only. Biotypes 1D, 1X, and 2A were selected in TM. All E. faecium/durans isolates were susceptible to vancomycin, but the m-Ent + E. faecium biotype 1A and 1D strains were resistant to penicillin, erythromycin, ciprofloxacin, and ampicillin. In contrast, all biotype 1X isolates were susceptible to all antibiotics tested. All E. faecalis and most E. durans isolates were resistant to penicillin but susceptible to erythromycin and ciprofloxacin. Biotype 2X isolates and one virulent (ace; gelE) E. faecalis isolate from RM were tetracycline-resistant. A sporadic RM isolate of E. hirae that was resistant to penicillin and vancomycin was not retrieved from the counterpart TM, but the inclusion of three vancomycin-resistant isolates from TM in the primary biotype 3B of E. faecalis was a cause for concern. In conclusion, based on the results, antibiotic-susceptible representatives of all strain biotypes of the E. faecium/durans group, as well as antagonistic m-Ent+ E. faecium strains from sheep milk that were susceptible to vancomycin and ampicillin and lacking virulence genes, can be included in safe complex natural starters to be developed for onsite use in traditional Greek hard cheese technologies. Full article