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Essential oils (EO) are known for their antibacterial and anti-inflammatory properties and can be used as an alternative to reduce the reliance on antimicrobials in dairy cattle. While many studies have explored the beneficial properties of EO in vitro, their effects on milk quality and milk microbiota, when applied directly to the udder skin, remain relatively unknown. This study aimed to investigate the impact of Thymus capitatus essential oil (TCEO), known for its high antibacterial and antioxidant properties, on milk microbiota using 16S rRNA sequencing, the lipidomic profile via liquid chromatography-quadrupole time-of-flight mass spectrometry, udder skin microbiota, and inflammatory biomarkers of dairy cows at the end of lactation. Sixteen-quarters of 12 Holstein cows were selected, and TCEO was topically applied to the udder skin twice a day for 7 days. Milk was collected aseptically on days 0, 7, 21, and 28 before morning farm milking. The results showed no significant changes in microbiota composition after the EO treatment in alpha and beta diversity or taxonomical composition at the phylum and genus levels. TCEO induced limited changes in the milk lipidome, primarily affecting diacylglycerols at T21. The treatment did not affect inflammatory biomarkers, milk sensory properties, or quality. Our study is the first to demonstrate that a local application of 10% TCEO on cow's quarters does not significantly alter milk quality or microbiota composition in milk and skin. More studies should be conducted to ensure the safe use of TCEO in dairy cows and explore its potential benefits on antibiotic-resistant bacteria as an alternative or support for antibiotic therapy., Competing Interests: Declarations. Competing interests: The authors declare no competing interests. Ethics approval and consent to participate: The present study was approved by the French Ministry for Higher Education, Research and Innovation and the local institutional animal care and use committee (approval number: #2020011709229229). The authors confirm that: (1) the reported experiments were conducted at Mejusseaume INRAE experimental farm under agreement for animal housing no. C-35–275–23; (2) all methods and animal treatment used in the study were performed according to the guidelines and regulations stated in Directive 2010/63/EU on the protection of animals used for scientific purposes ( http://data.europa.eu/eli/dir/2010/63/oj ). The study reported in the manuscript follows the recommendations in the ARRIVE guidelines (PLoS Biol 18(7): e3000411. https://doi.org/ https://doi.org/10.1371/journal.pbio.3000411 )., (© 2025. The Author(s).)

Intramammary dry-off treatment is widely considered an effective method for preventing and curing intramammary infection (IMI) in lactating cows; however, it is not commonly used in small ruminants like goats. Therefore, this study was designed to evaluate the effect of an approved cefazolin-based intramammary treatment on the milk microbiota of Alpine dairy goats during the dry and early lactation periods. Sixty goats were randomly selected based on bacteriological results and randomly allocated into the control group (CG) or the treatment group (TG). Cefazolin 250 mg (Cefovet A, Dopharma, Firenze, Italy) was administered to the TG group at dry-off, whereas the CG received no treatment. Pooled milk samples were collected at dry-off (T1; 52 samples), colostrum (T2; 46 samples), and 5-10 days in milk (T3; 55 samples) for bacteriological analysis, somatic cell count (SCC), and 16 S rRNA gene sequencing. SCC levels were initially high in both groups at T1 (TG: 1,588,000 cells/mL; CG: 1,629,000 cells/mL), which significantly decreased at T3 (TG: 148,000 cells/mL; CG: 153,000 cells/mL). Notably, the TG had fewer infected mammary glands than the CG at T3 (p = 0.0248), while no differences were found at T1 or T2. Despite the reduction in SCC and infection rates, cefazolin-based treatment did not significantly affect the alpha- and beta-diversity between the TG and CG. On the other hand, shifts in microbial composition, including fluctuations in Firmicutes, Proteobacteria, and Actinobacteria, were primarily due to the lactation stage rather than the treatment. Differential abundance analyses identified non-pathogenic genera, such as Acinetobacter, Bacteroides, and Paracoccus, that varied between groups at different timepoints. The study provided insights into the effects of cefazolin-based dry goat treatment on goat milk microbiota and its changes during the lactation cycle, demonstrating its potential to reduce SCC and mammary gland infections without significant alterations to the milk microbiota., Competing Interests: Declarations. Competing interests: The authors declare no competing interests. Ethics approval and consent to participate: All experiments were approved by the Ethics Committee for Animal Welfare of the University of Milan, Italy (documentation no. OPBA 118–2023)., (© 2025. The Author(s).)

Staphylococcus aureus is the main etiologic agent of contagious dairy cow mastitis, while non-aureus staphylococci and mammaliicocci (NASM) are the bacteria most frequently isolated from milk. Beyond their impact on animal health, NASM can harbor antimicrobial resistance (AMR) genes with potential for bidirectional transfer with S. aureus, and methicillin-resistant (MR) staphylococci (MRS) can raise significant One Health concerns. In our study, we evaluated the prevalence and characteristics of MRS in the bulk tank milk (BTM) of 88 dairy farms in the livestock-dense province of Lodi, Lombardy, northern Italy. S. aureus was isolated from 32.95 % of BTM samples, with the Ribosomal Spacer PCR (RS-PCR) genotype B being the most prevalent, identified in 37.93 % of S. aureus positive farms. All isolates carried the ica genes (icaA, icaB, icaC, icaD) indicating the potential to produce biofilm. MRS were isolated in 56.81 % of farms. According to MALDI-TOF MS analysis, the most prevalent MR species included S. epidermidis (MRSE, 35.59 %) followed by S. aureus (MRSA, 18.64 %), M. sciuri (15.25 %), S. saprophyticus (11.86 %), S. borealis (6.78 %), S. haemolyticus (5.08 %), M. fleurettii, (3.39 %), S. cohnii, and S. pettenkoferi (1.70 % each). Most MR isolates carried the mecA gene, while none carried mecC. The staphylococcal cassette chromosome mec (SCCmec) was predominantly type V in MRSA (45.45 %) and type IV in MRSE (61.90 %). Given their relevance to One Health, monitoring AMR in all staphylococci and mammaliicocci isolated from milk is essential for understanding the prevalence, characteristics, and transmission dynamics of MR gene pools within dairy herds., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Controlling foodborne pathogens in buffalo milk is crucial for ensuring food safety. This study estimated the prevalence of nine target genes representing seven critical foodborne bacteria in milk and milk products, and identified factors associated with their presence in buffalo milk chain nodes in Bangladesh. One hundred and forty-three milk samples from bulk tank milk (n = 34), middlemen (n = 37), milk collection centers (n = 37), and milk product shops (n = 35) were collected and analyzed using RT-PCR. Escherichia (E.) coli, represented through yccT genes, was the most prevalent throughout the milk chain (81-97%). Chi-squared tests were performed to identify the potential risk factors associated with the presence of foodborne bacteria encoded for different genes. At the middleman level, the prevalence of E. coli was associated with the Mymensingh, Noakhali, and Bhola districts (P = 0.01). The prevalence of Listeria monocytogenes, represented through inlA genes, and Yersinia (Y.) enterocolitica, represented through yst genes, were the highest at the farm level (65-79%). The prevalence of both bacteria in bulk milk was associated with the Noakhali and Bhola districts (P < 0.05). The prevalence of Y. enterocolitica in bulk milk was also associated with late autumn and spring (P = 0.01) and was higher in buffalo-cow mixed milk than in pure buffalo milk at the milk collection center level (P < 0.01). The gene stx2 encoding for Shiga toxin-producing (STEC) E. coli was detected in 74% of the milk products. At the middleman level, the prevalence of STEC E. coli was associated with the use of cloths or tissues when drying milk containers (P = 0.01). Salmonella enterica, represented through the presence of invA gene, was most commonly detected (14%) at the milk collection center. The use of plastic milk containers was associated with a higher prevalence of Staphylococcus aureus, represented through htrA genes, at milk product shops (P < 0.05). These results suggest that raw milk consumers in Bangladesh are at risk if they purchase and consume unpasteurized milk., (© 2024. The Author(s).)

The bovine udder is colonized by a huge quantity of microorganisms that constitute the intramammary ecosystem, with a specific role in modulating not only udder homeostasis and mastitis susceptibility, but also the quality of the dairy products. However, generating high-quality bacterial DNA can be critical, especially starting from a complex biological matrix like milk, characterized by high fat, protein, and calcium contents. Here, bacterial DNA was recovered from a commercial ultra-high-temperature (UHT) milk sample artificially spiked with a predetermined mock community composition and from three bulk tank milk (raw milk) samples. The DNA was isolated using three different protocols to evaluate the effect of the extraction procedures on the milk microbiota composition. In the mock community experiment, the bacterial profiles generated by the three DNA extraction protocols were profoundly different, with the genera Staphylococcus, Lactobacillus , Listeria , and Salmonella underestimated by all the protocols. Only one protocol revealed values close to the expected abundances for Escherichia/ Shigella spp., Bacillus spp., Enterococcus spp., and Pseudomonas spp. On the other hand, the nonspiked UHT milk sample exhibited a similar microbiota composition, revealing the prevalence of Acinetobacter spp., for all the DNA extraction protocols. For the raw milk samples, the three DNA extraction kits performed differently, revealing significant separations in both the microbial richness (alpha diversity) and composition (beta diversity). Our study highlights the presence of significant differences among these procedures, probably due to the different DNA extracting capacities and to the different properties of the milk samples, revealing that the selection of DNA extraction protocol is a critical point. IMPORTANCE The advance of high-throughput technologies has increased our knowledge of the world of microorganisms, especially of microbial populations inhabiting living animals. This study provides evidence that milk, as other complex sources, could be critical for generating high-quality DNA for microbiota analysis. In addition, it demonstrates that the microbial population highlighted by metagenomic studies changes in relation to different DNA extraction procedures, revealing that attention should be paid especially when comparing different studies.

Mastitis represents one of the major economic and health threats to the livestock sector associated with reduction in milk quality, loss of production and is a major reason for culling. Somatic cell score (SCS) is used as a criterion in breeding programmes to select cows genetically less susceptible to mastitis. The relevance of SCS as a predictor of udder health and susceptibility to mastitis is still untested in goats. In this study, two lines of French Alpine goats selected for extreme breeding values for somatic cell scores, one line with high SCS (HSCS) and the other with low SCS (LSCS), were used to test the hypothesis that the mammary response and function differed between the lines. The aim of the present study was to investigate differences in the early immune response in caprine mammary gland tissues challenged with Staphylococcus aureus, one of the main pathogens responsible for the intra-mammary infection in small ruminants, using transcriptomic and histopathology analyses. The comparison between HSCS and LSCS goat lines, showed differences in the response at the histological level for inflammation, presence of neutrophils and micro-abscess formation, and at the molecular level in the expression of CXCL8, IL-6, NFKBIZ and IL-1β. CXCL8 and CXCL2 genes, which showed a higher level of expression in the experimentally infected HSCS line. The molecular data and histopathology both suggested that following S. aureus infection, mobilization, recruitment, infiltration, and chemotaxis of neutrophil, leads to a more severe inflammation in the HSCS compared to LSCS animals. Our results represent an initial basis for further studies to unravel the genetic basis of early mastitis inflammatory responses and the selection of dairy animals more resistant to bacterial mastitis., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Dry and early lactation periods represent the most critical phases for udder health in cattle, especially in highly productive breeds, such as the Holstein Friesian (HF). On the other hand, some autochthonous cattle breeds, such as the Rendena (REN), have a lower prevalence of mastitis and other transition-related diseases. In this study, milk microbiota of 6 HF and 3 REN cows, all raised on the same farm under the same conditions, was compared. A special focus was placed on the transition period to define bacterial groups' prevalence with a plausible effect on mammary gland health. Four time points (dry-off, 1 d, 7-10 d and 30 d after calving) were considered. Through 16S rRNA sequencing, we characterized the microbiota composition for 117 out of the 144 milk samples initially collected, keeping only the healthy quarters, in order to focus on physiological microbiome changes and avoid shifts due to suspected diseases. Microbial populations were very different in the two breeds along all the time points, with REN milk showing a significantly lower microbial biodiversity. The taxonomic profiles of both cosmopolitan and local breeds were dominated by Firmicutes, mostly represented by the Streptococcus genus, although in very different proportions (HF 27.5%, REN 68.6%). Large differences in HF and REN cows were, also, evident from the metabolic predictive analysis from microbiome data. Finally, only HF milk displayed significant changes in the microbial composition along the transition period, while REN maintained a more stable microbiota. In conclusion, in addition to the influence on the final characteristics of dairy products obtained from milk of the two breeds, differences in the milk microbiome might, also, have an impact on their mammary gland health., Competing Interests: The authors have declared that no competing interests exist.

A strain of an achlorophyllic alga, named PR24 T , was isolated from cow milk samples from the state of Minas Gerais, Brazil. Based on 18S rDNA, 28S rRNA, D1/D2 region of the LSU rDNA and SSU rRNA gene sequence similarities, this strain was found to be a member of the genus Prototheca and closely related to Protothecablaschkeae SAG2064 T . However, the novel strain could easily be distinguished from recognized Prototheca species by internal transcribed spacer, species-specific PCR, single-strand conformation polymorphism-PCR analysis and phenotypic characteristics. The inability to grow in Sabouraud broth at pH 4.0 and the different cellular fatty acid composition clearly distinguished PR24 T from the reference strain of P. blaschkeae. The combination of genotypic and phenotypic data indicates that strain PR24 T represents a subspecies of P. blaschkeae, for which the name Prototheca blaschkeae subsp. brasiliensis subsp. nov. is proposed. The respective type strain is PR24 T (=DSM 103592 T =IHEM 26958 T ).

Clostridium beijerinckii, Clostridium sporogenes and Clostridium tyrobutyricum are considered the leading bacteria implicated in late blowing defects affecting semi-hard and hard cheese production. The aim of this study was to develop a multiplex Real-Time PCR (qPCR) analysis for a rapid and simultaneous detection of C. beijerinckii, C. sporogenes and C. tyrobutyricum, using specific primers respectively targeting the nifH, gerAA and enr genes. The limits of detection in raw milk were 300 CFU/50 mL in the case of C. beijerinckii, 2 CFU/50 mL for C. sporogenes and 5 CFU/50 mL for C. tyrobutyricum spores. The qPCR method was applied to artificially contaminated raw milk samples, and molecular quantification showed good correlation (R(2) = 0.978) with microbiological counting. Our results demonstrate that this method, combined with a DNA extraction protocol optimized for spore lysis, could be a useful tool for the direct quantification of the considered clostridia species., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Most of the acute intestinal diseases are caused by foodborne pathogens with infants and elderly people being at major risk. The aim of this study was to develop a procedure to simultaneously detect 20 foodborne pathogens in complex alimentary matrices such as milk, cheese and meat. The list of targets include, among the others, Listeria spp., Salmonella spp., Shigella spp., Escherichia coli spp., Campylobacter spp., Clostridium spp. and Staphylococcus aureus. The accuracy of detection was determined by using ATCC strains as positive and negative controls. The achieved sensitivity of each of assays was 1 pg of genomic DNA, which was equivalent to ∼1 cfu. The working ranges of the TaqMan(®) Real-time PCR assays, when used quantitatively on cheese and meat samples inoculated with serial dilution of Listeria spp., Listeria monocytogenes, S. aureus, Salmonella enterica, Shigella boydii, E. coli O157:H7, Bacillus cereus, Campylobacter coli, Yersinia enterocolitica, Enterobacter sakazakii and Pseudomonas aeruginosa was 10(8) cfu/g to 10(4) cfu/g. No matrix interferences were observed., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Late blowing, caused by the outgrowth of clostridial spores present in raw milk and originating from silage, can create considerable product loss, especially in the production of hard and semi-hard cheeses. The conventional method for the isolation of Clostridium spp. from cheeses with late-blowing symptoms is very complicated and the identification of isolates is problematic. The aim of this work was the development of a multiplex PCR method for the detection of the main dairy-related clostridia such as: Cl. beijerinckii, Cl. butyricum, Cl. sporogenes, Cl. tyrobutyricum. Samples derived from silage, raw milk and hard cheese were analysed by the most probable number (MPN) enumeration. Forty-four bacterial strains isolated from gas positive tubes were used to check the reliability of the multiplex PCR assay. The specificity of the primers was tested by individually analysing each primer pair and the primer pair combined in the multiplex PCR. It was interesting to note that the samples not identified by the multiplex PCR assay were amplified by V2-V3 16S rRNA primer pair and the sequencing revealed the aligned 16S rRNA sequences to be Paenibacillus and Bacillus spp. This new molecular assay provides a simple promising alternative to traditional microbiological methods for a rapid, sensitive detection of clostridia in dairy products.

A multiplex PCR for the simultaneous detection of Staphylococcus aureus 23S rRNA, the coagulase and thermonuclease genes as well as the enterotoxin genes sea, sec, sed, seg, seh, sei, sej, sel was developed. The method was used to determine the presence of enterotoxigenic types for 93 S. aureus strains isolated from milk and dairy products. The data obtained by mPCR resulted comparable to those obtained by immunoassay methods. In addition, the mPCR assays also amplified some se genes, whose toxins are undetectable by immunoassay. Multiplex amplification can be obtained starting from 1 pg of DNA, showing the excellent specificity and high sensitivity of the assay.

Recently, the use of antimicrobials on dairy farms has been significantly limited from both the legislative and consumer points of view. This study aims to check the efficacy of selective dry cow therapy (SDCT) versus blanket dry cow therapy (BDCT) on bovine udder in healthy animals. SDTC is when an antibiotic is administered only to infected cows, compared with BDCT, where all cows receive an antimicrobial, regardless of their infection status. The milk samples were collected from enrolled Holstein Friesian cows 7 days before dry-off (T0) and 10 days after calving (T1) to assess somatic cell count (SCC), intramammary infections (IMIs), and milk microbiota variation. After pre-drying sampling, cows are randomly assigned to the following treatments: internal teat sealant alone (ITS; 24 cows), which is a treatment in a cow that does not receive antibiotics in SDTC, or in combination with intramammary antibiotic treatment (A+ITS; 22 cows). Non-statistically significant results are found between the two treatment groups at T1 for SCC, milk yield, and alpha diversity in milk microbiota. A statistically (p < 0.033) T1 IMI decrease is reported in the A+ITS group, and a significant beta diversity analysis is shown between the two timepoints (p = 0.009). This study confirms the possibility of selective drying without new IMI risk or increased SCC at calving, considering healthy cows without contagious infections and SCC values >200,000 cells/mL in the previous lactation. [ABSTRACT FROM AUTHOR]