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Background: Bovine mastitis is a major challenge in dairy farms. Since the agents commonly used for pre- and post-dipping can affect the udder health by modifying milk microbiota, alternative products are needed. This study aimed to evaluate the effect of the use of pre- and post-dipping formulations containing the fermented broth of Nisin A-producing Lactococcus cremoris FT27 strain (treated group, TR) on the abundance and biodiversity of milk microbiota as compared to iodine-based commercial disinfectants (control group, CTR) during a three-month trial. The experiment was conducted on 20 dairy cows, divided into two groups (CTR and TR) of 10 lactating cows each. Milk samples were collected from two selected healthy quarters of each cow at 3 time-points. Microbial communities were investigated by cultural and sequence-based methods, and analyzed through bioinformatic and statistical approaches. Results: Clear differences in bacterial community composition were observed among groups, with higher species richness in TR, especially of Staphylococcus, Enterococcus, Lactococcus, and Streptococcus genera. The microbiota was dominated by Firmicutes, followed by Actinobacteriota, Proteobacteria, and Bacteroidota. Staphylococcaceae family was significantly higher in TR (p < 0.009), whereas Carnobacteriaceae, Mycobacteriaceae, and Pseudomonadaceae were significantly lower (p = 0.005, p = 0.001, and p = 0.040, respectively). CTR had considerably higher abundances of the genera Alkalibacterium (p = 0.011), Pseudomonas_E (p = 0.045), Corynebacterium (p = 0.004), and Alloiococcus (p = 0.004), and lower abundances of Staphylococcus (p < 0.009). Milk microbiota changed noticeably during the experimental period, regardless of treatment. A significant decrease was observed in both groups for Firmicutes_A phylum, with an increment in Actinobacteriota phylum, Propionibacteriaceae family, and Cutibacterium genus. Streptococcaceae significantly decreased in CTR (p = 0.013) and rose in TR (p = 0.001). Several differences were observed between the two groups during the experimental period. Streptococcus genus almost disappeared in CTR (p = 0.013), whereas it significantly increased in TR (p = 0.001). Three and twelve enriched groups were significantly identified respectively in CTR and TR using LEfSe. Conclusions: The use of Nisin A-based teat dip formulations could be linked to greater microbial diversity compared to commercial products. Despite the influence of seasonality, the experimental formulations maintained higher milk biodiversity, suggesting that lactic acid bacteria metabolites prevent alterations in the milk microbiota. [ABSTRACT FROM AUTHOR]

Simple Summary: In vitro digestibility tests are essential for the evaluation of the nutritional value of forages and feeds but they need rumen fluid collected from animals as inoculum to be performed. However, it is well known that the degradative capacity of the rumen fluid can fluctuate depending on the animal and the time of collection, probably as a function of the rumen microbes' populations. The enzymatic activity expressed by the microbes is expected to represent the rumen fluid's degradative potential. The objectives of the present study were to investigate the effects of the animal and sampling time and interval on rumen microbes' populations and degradative potential but also to investigate the relationship between different rumen characteristics. Rumen microbes differed by animal and week but not daytime. Rumen function indicators were not affected by the animal and only minor microbes' metabolites showed variations during the day. The enzymatic activity was more variable depending on the animal and time, indicating the need to mix rumen fluids and replicate the analysis to obtain trustable results. Rumen fluid (RF) is the greatest source of uncontrolled inter-assay variations in in vitro digestibility tests. This study aimed to investigate the effect of donor cow (DC) individual variability, time relative to feeding, and weekly sampling intervals on RF microbiota structure and metabolism, and on its degradative capacity expressed as enzymatic activity (EA). RF was collected using an esophageal probe from 4 Holstein hay-fed DCs before feeding, and 4 and 8 h post-feeding, over 3 days with one-week intervals, for a total of 3 weeks. Rumen bacteriome ß-diversity was affected by DCs and the one-week interval but not by time relative to feeding. However, volatile fatty acids and NDF and DM digestibility were not altered based on DCs. Propionic, lactic, and butyric acids were consistent between weeks, with some variations in lactic and butyric acids 8 h after feeding. Amylase and cellulase activities fluctuated between the weekly intervals, while xylanase activity increased in RF collected at 4 and 8 h after feeding (p = 0.030) and in DC3 compared to the other DCs (p < 0.001). Our results reinforce the importance of pooling RF from multiple cows and increasing the number of replicates to enhance the accuracy of the analysis. [ABSTRACT FROM AUTHOR]

Introduction: Legionnaires' disease is a severe pneumonia predominantly caused by Legionella pneumophila (Lp), whose major reservoirs are artificial water systems. As most human infections are caused by L. pneumophila serogroup 1 (Lp1), a reliable method for Lp distinction can be crucial for bacterial spread prevention. As the ability to withstand in environments and to cause the waterborne disease is strongly related to specific genes, the identification of virulent strains can be of great relevance to implement water environmental monitoring and to contain harmful outbreaks to public health. We aimed to test an assay for Lp identification among different Legionella species, and to determine the serogroups. Additionally, we investigated the carriage of virulence and antimicrobial resistance genes. Methods: A total of 90 Legionella spp. isolates identified by phenotypic tests were subjected to the designed quantitative PCR assay targeting specific mip for Lp, wzm for Lp1, pvcA and ahpD for biofilm production. Eleven serogroups were investigated in all our isolates tested positive for mip gene, subsequently analyzed for 12 virulence and 8 antimicrobial resistance genes. Results: Only the 70 Lp isolates were positive for mip. Out of 27 Lp isolates belonging to serogroup 1 based on agglutination test, 23 (85.2%) carried wzm. The presence of ahpD and pvcA was found in 94.3 and 98.6% of Lp isolates, respectively. By multiplex PCR, all 23 wzm-positive strains were confirmed as serogroup 1 that was the most predominant (33%). At least one virulence gene was detected in all Lp isolates. The most frequent gene was ispE (100%), followed by issD (96%), icmK and enhC (93%), cpxA (91%), rtxA2 (74%), lvhB8-B9 (61%), and prpA (54%). The other genes were less diffused in Lp strains (rtxA1, 44%; lvhB3-B4, 47%; pvcB, 27%; lvrE, 24%). Of the macrolide resistance genes, the ereA was found in 84% of Lp strains, while only 14 (20%) harbored the lpeAB among the efflux pump genes. Conclusion: The assays validated in this study enable the simultaneous Lp and Lp1 detection. The differentiation of Lp strains according to their virulence properties could be useful to predict the bacterial ability to survive and to cause the disease. [ABSTRACT FROM AUTHOR]

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).)