Search Results (76)

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 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

The objective of this work was to deliver a comprehensive genetic characterization of a collection of E. coli strains isolated from raw sheep milk. To complete our purpose, the technique of whole-genome sequencing, coupled with bioinformatics and phenotypic characterization of antimicrobial resistance, was performed. These Gram-negative, facultative anaerobic bacteria belong to the family Enterobacteriaceae, together with other intestinal pathogens, such as Shigella spp. and Salmonella spp. Genetic analysis was carried out on all strains (phylogram, sequence types, VFs, AMR genes, and pangenome). The results showed the presence of various genetic traits that are related to virulence factors contributing to their pathogenic potential. In addition, genes conferring resistance to antibiotics were also detected and confirmed using phenotypic tests. Finally, the genome of the E. coli strains was characterized by the presence of several mobile genetic elements, thus facilitating the exchange of various genetic elements, associated with virulence and antimicrobial resistance, within and beyond the species, through horizontal gene transfer. Contaminated raw sheep milk with pathogenic E. coli strains is particularly alarming for cheese production in artisan dairies. Full article

Background/Objectives: Staphylococcus aureus is a major pathogen of concern in raw milk due to its potential to cause foodborne illness and its increasing antimicrobial resistance (AMR). In Romania, data on the occurrence and resistance patterns of S. aureus in raw sheep milk from traditional farming systems remain limited. This study investigated the presence and antimicrobial resistance of S. aureus in 106 raw sheep milk samples collected from traditional farms in the Banat region of western Romania. Methods: Coagulase-positive staphylococci (CPS) were enumerated using ISO 6888-1:2021 protocols. Isolates were identified at the species level using the Vitek 2 system and molecularly confirmed via PCR targeting the 16S rDNA and nuc genes. Methicillin resistance was assessed by detecting the mecA gene. Antimicrobial susceptibility was determined using the Vitek 2 AST-GP79 card. Results: CPS were detected in 69 samples, with S. aureus confirmed in 34.9%. The mecA gene was identified in 13.5% of S. aureus isolates, indicating the presence of methicillin-resistant S. aureus (MRSA). Resistance to at least two antimicrobials was observed in 97.3% of isolates, and 33 strains (89.2%) met the criteria for multidrug resistance (MDR). The most frequent MDR phenotype involved resistance to lincomycin, macrolides, β-lactams, tetracyclines, and aminoglycosides. Conclusions: The high prevalence of S. aureus, including MRSA and MDR strains, in raw sheep milk from traditional farms represents a potential public health risk, particularly in regions where unpasteurized dairy consumption persists. These findings underscore the need for enhanced hygiene practices, prudent antimicrobial use, and AMR monitoring in small-scale dairy systems. Full article

The role of Enterococcus spp. in food is debated since this group of lactic acid bacteria contains opportunistic pathogenic strains, some of which exhibit a multidrug-resistant profile. In livestock farms, the use of antibiotics is the most common practice to deal with mastitis-causing bacteria. However, the heavy usage and/or misuse of antibiotics has led to the emergence of antibiotic resistance. This study aimed to genetically and phenotypically characterize Enterococcus strains isolated from raw sheep milk. Samples were collected over one year from the bulk tank of a dairy sheep farm and cultured on selective media. Isolates were purified and analyzed by whole-genome sequencing and antimicrobial susceptibility testing. The isolates were divided into clusters and the corresponding species were identified along with their genes related to virulence and antibiotic resistance. The pan-, core- and accessory-genomes of the strains were determined. Finally, the antibiotic-resistant profile of selected strains was examined and associated with their genomic characterization. These findings contribute to a better understanding of Enterococci epidemiology, providing comprehensive profiles of their virulence and resistance genes. The presence of antibiotic-resistant bacteria in raw sheep milk destined for the production of cheese should raise awareness. Full article

Sheep and goat milk authenticity is of great importance, especially for countries like Greece, where these products are connected to the country’s rural economy and cultural heritage. The aim of the study is to evaluate the effectiveness of Fourier Transform Infrared Attenuated Total Reflectance (ATR-FTIR) spectroscopy in combination with chemometric techniques for the classification of cow, sheep, and goat milk and consequently support fraud identification. A total of 178 cow, sheep and goat milk samples were collected from livestock farms in Thessaly, Greece. Sheep and goat milk samples were confirmed as authentic by applying a validated Enzyme Linked Immunosorbent Assay (ELISA), while all samples were analyzed using ATR-FTIR spectroscopy in both raw and freeze-dried form. Freeze-dried samples exhibited clearer spectral characteristics, particularly enhancing the signals from triglycerides, proteins, and carbohydrates. Partial Least Squares Discriminant Analysis (PLS-DA) delivered robust discrimination. By using the spectral range between 600 and 1800 cm⁻¹, 100% correct classification of all milk types was achieved. These findings highlight the potential of FTIR spectroscopy as a fast, non-destructive, and cost-effective tool for milk identification and species differentiation. This method is particularly suitable for industrial and regulatory applications, offering high efficiency. Full article

The production of Fiore Sardo cheese is regulated by the specification of the Protected Designation of Origin (PDO), which aims to guarantee the specific area of production, the know-how of local producers, and the specific use of raw milk from Sarda sheep. The thermization of milk is a sub-pasteurization process that is commonly used in cheese-making to lower the bacterial load and increase the shelf life of the product; it is therefore a cause of non-compliance with the PDO specification of Fiore Sardo cheese, allowing producers to gain practical and economic advantages. In this work, NIR spectroscopy coupled with multivariate discriminant analysis was used to identify the thermal treatment of milk in Fiore Sardo cheese samples. Cheeses were produced using raw milk (38 °C), low-thermized milk (57 °C for 30 s), and high-thermized milk (68 °C for 30 s). The NIR spectra of the cheeses were used to build discriminant models for individuating the thermal treatment of the processed milk. The obtained discriminant models were able to correctly classify about 90% of the Fiore Sardo cheese samples. This method could be suitable as a screening technique to authenticate Fiore Sardo PDO cheese. Full article

Paslahepevirus balayani (hepatitis E virus), a zoonotic pathogen transmitted primarily via the fecal–oral route, has undergone shifting transmission dynamics in China, with foodborne and zoonotic routes becoming increasingly significant. To assess the potential risk of HEV transmission through dairy products, this study investigated HEV RNA presence in raw milk from cows, sheep, and goats in Hebei Province, China. From March 2024 to April 2025, we collected 102 cow milk, 18 sheep milk, and 59 goat milk samples, analyzing them using RT-qPCR, with positive samples confirmed by RT-Nested PCR and partial ORF2 sequencing. While no HEV RNA was detected in cow milk, 3/18 (16.67%) sheep milk and 1/59 (1.69%) goat milk samples tested positive. Phylogenetic analysis of two sheep-derived and one goat-derived HEV isolate showed 99.43–100% nucleotide identity to local swine HEV strains (HEV/HB-SJZ158/CHN/2021 and HEV/HB-CD28/CHN/2021), all clustering within genotype 4d, the dominant subtype in the region. This study provided the first evidence of HEV RNA in sheep and goat milk in Hebei Province, suggesting possible cross-species transmission from pigs to ruminants. These findings highlighted the need for further research on HEV transmission risks through dairy products and emphasize the importance of monitoring zoonotic HEV strains in food safety assessments. Full article

Serpa cheese, a Portuguese Protected of Denomination Origin (PDO) cheese, known for its unique sensory attributes, is made from the raw milk of native sheep. In this preliminary work, ten samples of Serpa cheese were submitted for a sensory evaluation performed by an expert panel in a sensory laboratory accredited according to ISO 17025 for Serpa cheese parameters, and the panelists classified the cheeses based on texture, taste and odor scores, in accordance with the specifications for the classification of this type of cheese. All cheeses were analyzed by SPME-GC-MS. Following an exploratory unsupervised multivariate analysis, the supervised multivariate analysis by partial least squares—discriminant analysis (PLS-DA), associated the relative percent area of the identified volatiles with the classification of cheeses attributed by the sensory panel. Among the 144 compounds putatively identified, there was a pattern of compound distribution of some of them, such as acetoin, diacetyl, and 2,3-butanediol, leaning toward the cheese samples with high taste and odor scores, while other compounds, such as ethyl caprate, capric acid, and 3-methylindole, were more associated with the cheese samples rated with a low score. Despite the reduced number of samples that may have imposed some restrictions on the conclusions drawn, there was a clear trend in the volatiles’ distribution, allowing us to identify, based on the higher correlation loadings, potential candidates for the Serpa cheese sensory quality. This preliminary study presents, for the first time, an overview of the volatiles that are present in Serpa PDO cheese that may be responsible for the positive or negative sensory evaluation of this PDO cheese. Full article

Thermization is a sub-pasteurization heat treatment widely applied in traditional Greek hard cheese technologies. In this study, five bulk milk batches from two native Epirus sheep breeds were analyzed microbiologically before (raw milk; RM) and after thermization at 65 °C for 30 s (TM) followed by characterization of 125 presumptive LAB isolates from each of the counterpart RM and TM samples. Psychrotrophic Pseudomonas-like spoilage bacteria and mesophilic LAB, primarily of the genera Leuconostoc (48.4%) and Lactococcus/Streptococcus (32.8%), co-dominated in RM at mean levels 5.7–6.3 log CFU/mL, whereas thermophilic LAB, Enterococcus, Staphylococcus, coliforms, and yeasts were subdominant at mean levels 4.1 to 5.2 log CFU/mL. Coagulase-positive staphylococci were abundant (3–4 log CFU/mL) in all RM batches. Listeria monocytogenes was found in one batch. Both pathogens were diminished by thermization, which reduced all non-LAB contaminants below 100 CFU/mL. Enterococci (68.6%) were highly selected in all TM batches, followed by thermophilic streptococci (8.6%). Only 7.4% of the total RM microbiota survived in the resultant five TM batches. Leuconostoc showed the lowest (1.3%) survival. Thus, thermization improved the quality and safety of raw sheep milk, but reduced mesophilic LAB by ca. 2 log units in favor of enterococci in TM. Full article

Escherichia coli (E. coli) is one of the most common pathogens in both humans and livestock. This study aimed to investigate the prevalence of E. coli isolated from raw cow milk and evaluate its antimicrobial resistance rates. A total of 1696 milk samples were collected from Romanian dairy farms from 2018 to 2022. E. coli was isolated on various selective agar media, such as Cled agar and Columbia Agar with 5% Sheep Blood. The identification of E. coli was performed by MALDI-TOF MS. E. coli isolates were tested for their susceptibility against 18 commonly used antibiotics in a disk diffusion method. The overall prevalence of E. coli was 22.45% of all isolated pathogens. Antibiogram analysis revealed that 27.51% of E. coli isolates from milk were multidrug-resistant. Resistance was highest for penicillin–novobiocin (87.78%), followed by streptomycin (53.7%). Resistance to six drugs (amoxicillin, streptomycin, kanamycin–cephalexin, marbofloxacin, ampicillin) showed a significant increasing trend over time, while for two drugs (penicillin G-framycetin, doxycycline), a significant decrease was observed. Our results suggest that milk can be a reservoir of bacteria with the potential for infection in humans via the food chain. Furthermore, there is a need for surveillance and monitoring to control the increase in resistance to currently used antimicrobials in dairy farms because the occurrence of multidrug-resistant E. coli isolated from milk poses a health hazard to consumers. Full article

Although the highlands of East Africa lack the geo-ecological landmarks of Rift Valley fever (RVF) disease hotspots to participate in cyclic RVF epidemics, they have recently reported growing numbers of small RVF clusters. Here, we investigated whether RVF cycling occurred among livestock and humans in the central highlands of Kenya during inter-epidemic periods. A 2-year prospective hospital-based study among febrile patients (March 2022–February 2024) in Murang’a County of Kenya was followed by a cross-sectional human–animal survey. A total of 1468 febrile patients were enrolled at two clinics and sera tested for RVF virus RNA and antiviral antibodies. In the cross-sectional study, humans (n = 282) and livestock (n = 706) from randomly selected households were tested and questionnaire data were used to investigate sociodemographic and environmental risk factors by multivariate logistic regression. No human (n = 1750) or livestock (n = 706) sera tested positive for RVFV RNA. However, 4.4% livestock and 2.0% humans tested positive for anti-RVFV IgG, including 0.27% febrile patients who showed four-fold IgG increase and 2.4% young livestock (<12 months old), indicating recent virus exposure. Among humans, the odds of RVF exposure increased significantly (p < 0.05, 95% CI) in males (aOR: 4.77, 2.08–12.4), those consuming raw milk (aOR: 5.24, 1.13–17.9), milkers (aOR: 2.69, 1.23–6.36), and participants residing near quarries (aOR: 2.4, 1.08–5.72). In livestock, sheep and goats were less likely to be seropositive (aOR: 0.27, 0.12–0.60) than cattle. The increase in RVF disease activities in the highlands represents a widening geographic dispersal of the virus, and a greater risk of more widespread RVF epidemics in the future. Full article

Q fever is a widespread zoonotic disease caused by the obligate intracellular bacterium Coxiella burnetii, primarily transmitted through the inhalation of contaminated aerosols. This study aimed to detect C. burnetii in two Sicilian sheep flocks, with no better defined reproductive disorders reported by the farmers. Blood, individual and bulk milk, ticks, and conjunctival swabs were collected from both flocks (A and B). Real-time and traditional PCRs were carried out to detect C. burnetii DNA and anti-C. burnetii antibodies were searched using an ELISA. In terms of Farm A, C. burnetii DNA was detected in 7.1% of blood samples, 20% of individual milk samples, bulk milk, 66.6% of conjunctival swabs, and in all the examined tick pools. Anti-C. burnetii antibodies were found in 77.0% of sera, 92.5% of individual milk samples, and bulk milk. In terms of Farm B, C. burnetii DNA was detected in 3.8% of blood samples, 39.4% of individual milk samples, bulk milk, 100% of conjunctival swabs, and in all tick pools; anti-C. burnetii antibodies were present in 53.6% of sera, 73.2% of milk samples, and in bulk milk. Our results highlight the high diffusion of C. burnetii in the two outbreaks, with widespread pathogen circulation, significant shedding in dairy products, and high environmental contamination, highlighting the need for enhanced surveillance and control measures in dairy sheep farms. Full article

HEV mainly enters animal and human hosts through the orofecal route, which presents a critical health concern alongside the associated environmental variable. Among products of animal origin, milk (both ovine and bovine) can harbor HEV RNA, which can potentially be transmitted to consumers. In this study, a total of 220 raw ovine milk samples were collected from Apennine breed subjects farmed (transhumance method) in three different Italian provinces, L’Aquila, Pescara, and Teramo, located in the Abruzzo region (Central Italy). All the specimens were screened using one-step real-time RT-qPCR and nested RT-PCR assays. Among them, 5/220 or 2.27% harbored HEV RNA fragments belonging to the ORF1 and ORF2 codifying regions of the genotype 3c. The average viral amount discovered was 10² GE/mL. These subjects represented 2/57 or 3.51% of the Pescara herd, and 3/105 or 2.86% of the Teramo herd. Although HEV RNA was discovered in sheep fecal samples, the original data obtained in the present study represent the first HEV RNA detection in ovine raw milk from Italy. Full article