Bovine Udder Research Articles

Improved ability to utilize lactose and grow in milk as a potential explanation for emergence of the novel bovine Staphylococcus aureus ST5477

Staphyloccous aureus belonging to sequence type 5477 have recently been identified as a predominant clone causing bovine mastitis in Rwanda and Tanzania. We compared nine S. aureus ST5477 to 17 isolates belonging to other sequence types by their biochemical profile and ability to acidify milk and grow in minimum media containing lactose. We found that ST5477 isolates all were positive in ONPG (o-nitrophenyl-β-D-galactopyranoside) test and negative for mannitol fermentation potentially challenging the correct identification of this sequence type as S. aureus. In addition, ST5477 isolates were all much faster in acidifying milk and grew faster in minimal media with lactose compared to other strains suggesting an increased lactose utilization and thereby adaptation to the bovine udder environment as a possible reason for the recent successful emergence. Comparison of the lac gene region of the genome of a recently sequenced ST5477 and that of S. aureus reference genome showed that both strains contained the known lacABCD genes involved in the lactose degradation, but that ST5477 had a 12 amino-acid deletion and two amino-acid differences in the lac gene transcription regulator, suggesting that increased transcription might play a role. In conclusion, these preliminary data suggests that improved lactose utilization and the ability to grow faster in milk may have been a key feature for the recent success of ST5477 as a bovine adapted clone.

An in vitro evaluation of the effect of antimicrobial treatment on bovine mammary microbiota

Antimicrobial-resistant bacteria have been an increasing problem in human medicine and animal husbandry since the introduction of antimicrobials on the market in the 1940s. Over the last decades, efforts to reduce antimicrobial usage in animal husbandry have been shown to limit the development of resistant bacteria. Despite this, antimicrobial-resistant bacteria are still commonly detected and isolated worldwide. In this study, we investigated the presence of antimicrobial-resistant bacteria in bovine milk samples using a multiple approach based on culturing and amplicon sequencing. We first enriched milk samples obtained aseptically from bovine udders in the presence of two antimicrobials commonly used to treat mastitis and then described the resistant microbiota by amplicon sequencing and isolate characterization. Our results show that several commensal species and mastitis pathogens harbor antimicrobial resistance and dominate the enriched microbiota in milk in presence of antimicrobial agents. The use of the two different antimicrobials selected for different bacterial taxa and affected the overall microbial composition. These results provide new information on how different antimicrobials can shape the microbiota which is able to survive and reestablish in the udder and point to the fact that antimicrobial resistance is widely spread also in commensal species.

Light-activated conjugated polymer nanoparticles to defeat pathogens associated with bovine mastitis

Bovine mastitis (BM) represents a significant challenge in the dairy industry. Limitations of conventional treatments have prompted the exploration of alternative approaches, such as photodynamic inactivation (PDI). In this study, we developed a PDI protocol to eliminate BM-associated pathogens using porphyrin-doped conjugated polymer nanoparticles (CPN). The PDI-CPN protocol was evaluated in four mastitis isolates of Staphylococcus and in a hyper-biofilm-forming reference strain. The results in planktonic cultures demonstrated that PDI-CPN exhibited a bactericidal profile upon relatively low light doses (∼9.6 J/cm2). Furthermore, following a seven-hour incubation period, no evidence of cellular reactivation was observed, indicating a highly efficient post-photodynamic inactivation effect. The successful elimination of bacterial suspensions encouraged us to test the PDI-CPN protocol on mature biofilms. Treatment using moderate light dose (∼64.8 J/cm2) reduced biofilm biomass and metabolic activity by up to 74% and 88%, respectively. The impact of PDI-CPN therapy on biofilms was investigated using scanning electron microscopy (SEM), which revealed nearly complete removal of the extracellular matrix and cocci. Moreover, ex vivo studies conducted on bovine udder skin demonstrated the efficacy of the therapy in eliminating bacteria from these scaffolds and its potential as a prophylactic method. Notably, the histological analysis of skin revealed no signs of cellular degeneration, suggesting that the protocol is safe and effective for BM treatment. Overall, this study demonstrates the potential of PDI-CPN in treating and preventing BM pathogens. It also provides insights into the effects of PDI-CPN on bacterial growth, metabolism, and survival over extended periods, aiding the development of effective control strategies and the optimization of future treatments.

An update on non-aureus staphylococci and mammaliicocci in cow milk: unveiling the presence of Staphylococcus borealis and Staphylococcus rostri by MALDI-TOF MS

Non-aureus staphylococci and mammaliicocci (NASM) are the microorganisms most frequently isolated from milk. Given their numerosity and complexity, MALDI-TOF MS is one of the preferred species identification approaches. Nevertheless, reference mass spectra for the novel species Staphylococcus borealis were included only recently in the Bruker Biotyper System (MBT) library, and other species of veterinary interest such as S. rostri are still absent. This work provides an updated picture of the NASM species found in milk, gained by retrospectively analyzing the data relating to 21,864 milk samples, of which 6,278 from clinical mastitis (CM), 4,039 from subclinical mastitis (SCM), and 11,547 from herd survey (HS), with a spectrum library including both species. As a result, S. borealis was the second most frequently isolated NASM (17.07%) after S. chromogenes (39.38%) in all sample types, with a slightly higher percentage in CM (21.84%), followed by SCM (17.65%), and HS (14.38%). S. rostri was also present in all sample types (3.34%), reaching 8.43% of all NASM in SCM and showing a significant association (p < 0.01) with this condition. Based on our findings, the presence of S. borealis and S. rostri in milk and their potential association with mastitis might have been overlooked, possibly due to the difficulties in differentiating these species from other closely related NASM. Our results indicate that S. borealis could be a more frequent contributor to bovine udder infections than previously thought and that S. rostri should also not be underestimated considering its significant association with SCM.

The impact of kit, environment, and sampling contamination on the observed microbiome of bovine milk.

In low-microbial biomass samples such as bovine milk, contaminants can outnumber endogenous bacteria. Because of this, milk microbiome research suffers from a critical knowledge gap, namely, does non-mastitis bovine milk contain a native microbiome? In this study, we sampled external and internal mammary epithelia and stripped and cisternal milk and used numerous negative controls, including air and sampling controls and extraction and library preparation blanks, to identify the potential sources of contamination. Two algorithms were used to mathematically remove contaminants and track the potential movement of microbes among samples. Results suggest that the majority (i.e., >75%) of sequence data generated from bovine milk and mammary epithelium samples represents contaminating DNA. Contaminants in milk samples were primarily sourced from DNA extraction kits and the internal and external skin of the teat, while teat canal and apex samples were mainly contaminated during the sampling process. After decontamination, the milk microbiome displayed a more dispersed, less diverse, and compositionally distinct bacterial profile compared with epithelial samples. Similar microbial compositions were observed between cisternal and stripped milk samples, as well as between teat apex and canal samples. Staphylococcus and Acinetobacter were the predominant genera detected in milk sample sequences, and bacterial culture showed growth of Staphylococcus and Corynebacterium spp. in 50% (7/14) of stripped milk samples and growth of Staphylococcus spp. in 7% (1/14) of cisternal milk samples. Our study suggests that microbiome data generated from milk samples obtained from clinically healthy bovine udders may be heavily biased by contaminants that enter the sample during sample collection and processing workflows.IMPORTANCEObtaining a non-contaminated sample of bovine milk is challenging due to the nature of the sampling environment and the route by which milk is typically extracted from the mammary gland. Furthermore, the very low bacterial biomass of bovine milk exacerbates the impacts of contaminant sequences in downstream analyses, which can lead to severe biases. Our finding showed that bovine milk contains very low bacterial biomass and each contamination event (including sampling procedure and DNA extraction process) introduces bacteria and/or DNA fragments that easily outnumber the native bacterial cells. This finding has important implications for our ability to draw robust conclusions from milk microbiome data, especially if the data have not been subjected to rigorous decontamination procedures. Based on these findings, we strongly urge researchers to include numerous negative controls into their sampling and sample processing workflows and to utilize several complementary methods for identifying potential contaminants within the resulting sequence data. These measures will improve the accuracy, reliability, reproducibility, and interpretability of milk microbiome data and research.

1,25 dihydroxyvitamin D3-mediated effects on bovine innate immunity and on biofilm-forming Staphylococcus spp. isolated from cattle with mastitis

Mastitis is one the most widespread and serious diseases in dairy cattle. Recurrent and chronic infections are often attributable to certain pathogenicity mechanisms in mastitis-causing pathogens such as Staphylococcus spp. These include growing in biofilm and invading cells, both of which make it possible to resist or evade antimicrobial therapies and the host’s immune system. This study tested the effects of active vitamin D3 (i.e., calcitriol or 1,25-dihydroxyvitamin D3) on the internalization and phagocytosis of biofilm-forming Staphylococcus spp. isolated from animals with mastitis. Two established bovine cell lines were used: MAC-T (mammary epithelial cells) and BoMac (macrophages). Calcitriol (0–200 nM) did not affect the viability of MAC-T cells nor that of BoMac cells after 24 and 72 h. Concentrations of 0–100 mM for 24 h upregulated the expression of 24-hydroxylase in MAC-T cells, but did not alter that of VDR. Pre-treatment of the cells with calcitriol for 24 h decreased the internalization of S. aureus V329 into MAC-T cells (0–100 nM), and stimulated the phagocytosis of the same strain and of S. xylosus 4913 (0–10 nM). Calcitriol and two conditioned media, obtained by treating the cells with 25–200 nM of the metabolite for 24 h, were also assessed in terms of their antimicrobial and antibiofilm activity. Neither calcitriol by itself nor the conditioned media affected staphylococcal growth or biofilm formation (0–200 nM for 12 and 24 h, respectively). In contrast, the conditioned media (0–100 nM for 24 h) decreased the biomass of preformed non-aureus staphylococcal biofilms and killed the bacteria within them, without affecting metabolic activity. These effects may be mediated by reactive oxygen species and proteins with antimicrobial and/or antibiofilm activity. In short, calcitriol could make pathogens more accessible to antimicrobial therapies and enhance bacterial clearance by professional phagocytes. Moreover, it may modulate the host’s endogenous defenses in the bovine udder and help combat preformed non-aureus staphylococcal biofilms (S. chromogenes 40, S. xylosus 4913, and/or S. haemolyticus 6). The findings confirm calcitriol’s potential as an adjuvant to prevent and/or treat intramammary infections caused by Staphylococcus spp., which would in turn contribute to reducing antibiotic use on dairy farms.

Evaluation of the Antimicrobial, Cytotoxic, and Physical Properties of Selected Nano-Complexes in Bovine Udder Inflammatory Pathogen Control.

Mastitis in dairy cows is a worldwide problem faced by dairy producers. Treatment mainly involves antibiotic therapy, however, due to widespread antibiotic resistance among bacteria, such treatments are no longer effective. For this reason, scientists are searching for new solutions to combat mastitis, which is caused by bacteria, fungi, and algae. One of the most promising solutions, nanotechnology, is attracting research due to its biocidal properties. The purpose of this research was to determine the biocidal properties of nanocomposites as a potential alternative to antibiotics in the control of mastitis, as well as to determine whether the use of nanoparticles and what concentration is safe for the breeder and the animal. In this study, the effects of Ag, Au, Cu, Fe, and Pt nanoparticles and their complexes were evaluated in relation to the survival of bacteria and fungi isolated from cattle diagnosed with mastitis, their physicochemical properties, and their toxicity to bovine and human mammary epithelial cells BME-UV1 and HMEC (human microvascular endothelial cells). Moreover, E. coli, S. aureus, C. albicans, and Prototheca sp. invasion was assessed using the alginate bead (bioprinted) model. The NPs were tested at concentrations of 25, 12.5, 6.25, 3.125, 1.56 mg/l for Au, Ag, Cu and Fe NPs, and 10, 5, 2.5, 1.25, 0.625 mg/l for Pt. With the exception of Fe and Pt, all exhibited biocidal properties against isolates, while the AgCu complex had the best effect. In addition, nanoparticles showed synergistic effects, while the low concentrations had no toxic effect on BME-UV1 and HMEC cells. Synergistic effects of nanoparticles and no toxicity to bovine and human cells might, in the future, be an effective alternative in the fight against microorganisms responsible for mastitis, and the implementation of research results in practice would reduce the percentage of dairy cows suffering from mastitis. The problem of increasing antibiotic resistance is posing a global threat to human's and animal's health, and requires comprehensive research to evaluate the potential use of nanoparticles - especially their complexes - as well as to determine whether nanoparticles are safe for the breeders and the animals. The conducted series of studies allows further consideration of the use of the obtained results in practice, creating a potentially new alternative to antibiotics in the treatment and prevention of mastitis in dairy cattle.

The coexistence of several microbial species at the same site of bovine mammary gland parenchyma infection and their mixed infections

&lt;b&gt;The purpose of this study was to analyse both negative results of bacteriological cultures and the coexistence of several pathogens at the same intramammary inflammatory focus within bovine udder parenchyma in order to compare the health status of cows’ udders in 1985 and 2021. Mastitis was diagnosed on the basis of anamnesis, clinical inspection of the udder, milk quality, and somatic cell counts (SCC on the Fossomatic 5000-FC). In 1985, joint bacteriological and mycological diagnosis was based on the guidelines established for bovine mastitis by FIL-IDF (1971), while for 2021, it was based on the National Mastitis Council (NMC) laboratory Handbook on bovine mastitis (Hogan et al., 1999). In the first study (1985), mainly the genus Staphylococcus was isolated, but at present other species may be involved in cases of mastitis. In both periods, both subclinical and clinical cases were registered. In addition, the milk yield of cows was observed to increase over time, with concomitant deterioration of mammary gland immunity. The phenomenon of biofilms was documented in the study, which may be explained not only by the low efficacy of antibiotic therapy against udder disease but also by impaired immunity in cows, as the lymphocytes trapped in the biofilm matrix were inactive with respect to both cellular and humoral responses.

Effect of the Selective Dry Cow Therapy on Udder Health and Milk Microbiota.

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.

Bovine Udder Health: From Standard Diagnostic Methods to New Approaches-A Practical Investigation of Various Udder Health Parameters in Combination with 16S rRNA Sequencing.

Bovine udder health is an important factor for animal wellbeing and the dairy farm economy. Thus, researchers aim to understand factors causing mastitis. The gold standard for diagnosing mastitis in cows is the conventional culturing of milk samples. However, during the last few years, the use of molecular methods has increased. These methods, especially sequencing, provide a deeper insight into the diversity of the bacterial community. Yet, inconsistent results regarding the mammary microbiome have been published. This study aimed to evaluate the udder health of eight dairy cows at seven days postpartum with the standard methods in veterinary practice. Additionally, swabs from the teat canal and milk samples were analyzed using 16S rRNA gene amplicon sequencing. The sensitive low-biomass milk samples displayed only a few contaminations even though they were sampled in a field environment. In healthy udders, no bacterial communities were detected by the bacterial culture nor the 16S rRNA gene amplicons. The results from the standard examination of the cows, the cell count, and the bacteriological examination were comparable with the results from 16S rRNA gene amplicon sequencing when cows displayed subclinical or latent mastitis. Besides the pathogen detected in bacterial culturing, a second bacterial strain with low but significant abundance was detected by sequencing, which might aid in the understanding of mastitis incidence. In general, molecular biological approaches might lead to promising insights into pathological events in the udder and might help to understand the pathomechanism and infection source via epidemiological analyses.

Metataxonomic analysis and host proteome response in dairy cows with high and low somatic cell count: a quarter level investigation

Host response to invasive microbes in the bovine udder has an important role on the animal health and is essential to the dairy industry to ensure production of high-quality milk and reduce the mastitis incidence. To better understand the biology behind these host-microbiome interactions, we investigated the somatic cell proteomes at quarter level for four cows (collected before and after milking) using a shotgun proteomics approach. Simultaneously, we identified the quarter microbiota by amplicon sequencing to detect presence of mastitis pathogens or other commensal taxa. In total, 32 quarter milk samples were analyzed divided in two groups depending on the somatic cell count (SCC). The high SCC group (>100,000 cell/mL) included 10 samples and significant different proteome profiles were detected. Differential abundance analysis uncovers a specific expression pattern in high SCC samples revealing pathways involved in immune responses such as inflammation, activation of the complement system, migration of immune cells, and tight junctions. Interestingly, different proteome profiles were also identified in quarter samples containing one of the two mastitis pathogens, Staphylococcus aureus and Streptococcus uberis, indicating a different response of the host depending on the pathogen. Weighted correlation network analysis identified three modules of co-expressed proteins which were correlated with the SCC in the quarters. These modules contained proteins assigned to different aspects of the immune response, but also amino sugar and nucleotide sugar metabolism, and biosynthesis of amino acids. The results of this study provide deeper insights on how the proteome expression changes at quarter level in naturally infected cows and pinpoint potential interactions and important biological functions during host-microbe interaction.

Traditional and emerging Fusarium mycotoxins disrupt homeostasis of bovine mammary cells by altering cell permeability and innate immune function.

High incidence of traditional and emerging Fusarium mycotoxins in cereal grains and silages can be a potential threat to feed safety and ruminants. Inadequate biodegradation of Fusarium mycotoxins by rumen microflora following ingestion of mycotoxin-contaminated feeds can lead to their circulatory transport to target tissues such as mammary gland. The bovine udder plays a pivotal role in maintaining milk yield and composition, thus, human health. However, toxic effects of Fusarium mycotoxins on bovine mammary gland are rarely studied. In this study, the bovine mammary epithelial cell line was used as an in-vitro model of bovine mammary epithelium to investigate effects of deoxynivalenol (DON), enniatin B (ENB) and beauvericin (BEA) on bovine mammary gland homeostasis. Results indicated that exposure to DON, ENB and BEA for 48h significantly decreased cell viability in a concentration-dependent manner (P<0.001). Exposure to DON at 0.39μmol/L and BEA at 2.5μmol/L for 48h also decreased paracellular flux of FITC-40 kDa dextran (P<0.05), whereas none of the mycotoxins affected transepithelial electrical resistance after 48h exposure. The qPCR was performed for assessment of expression of gene coding tight junction (TJ) proteins, toll-like receptor 4 (TLR4) and cytokines after 4, 24 and 48h of exposure. DON, ENB and BEA significantly upregulated the TJ protein zonula occludens-1, whereas markedly downregulated claudin 3 (P<0.05). Exposure to DON at 1.35μmol/L for 4h significantly increased expression of occludin (P<0.01). DON, ENB and BEA significant downregulated TLR4 (P<0.05). In contrast, ENB markedly increased expression of cytokines interleukin-6 (IL-6) (P<0.001), tumor necrosis factor α (TNF-a) (P<0.05) and transforming growth factor-β (TGF-β) (P<0.01). BEA significantly upregulated IL- 6 (P<0.001) and TGF-β (P=0.01), but downregulated TNF-α (P<0.001). These results suggest that DON, ENB and BEA can disrupt mammary gland homeostasis by inducing cell death as well as altering its paracellular permeability and expression of genes involved in innate immune function.

Identification of bacterial pathogens in dry bovine udder and their antimicrobial susceptibility

Mastitis is a multi-etiological production disease of dairy cows worldwide. The likelihood of developing clinical mastitis in subsequent calving is enhanced by intramammary infections with pathogens during the late dry and post-calving period. There is an increase in the occurrence of intramammary infections during early and late dry period. Thirty apparently healthy, pregnant cross-bred dairy cows were screened for the presence of contagious mastitis pathogens in the secretions taken after 48 h of cessation of milking. A total of 118 bacterial isolates were obtained from the secretions collected from 108 quarters, and identified using morphological and cultural characteristics. Among them, 50.8 per cent were coagulase negative Staphylococci (CNS), 16 per cent were coagulase positive Staphylococci (CPS) and 27.1 per cent were Streptococci isolates. Micrococci and E. coli constituted 2.5 per cent each and Klebsiella spp. formed 0.8 per cent of the total isolates obtained. The CNS isolates identified included 22 S. chromogens, 14 S. xylosus, 12 S. saprophyticus, six S. hominis and S. epidermidis each. Among the CPS isolated 11.8 per cent were S. aureus and 4.2 per cent were non-aureus coagulase positive Staphylococcus which included three S. hyicus and two S. pseudointermedius. Molecular confirmation of S. aureus, CNS isolates, St. agalactiae were done by targeting nuc, cns and 16SrRNA gene respectively. Identification of bacterial pathogens that are present during dry period can help in formulating an effective dry cow therapy. Antibiogram studies of the isolates obtained were done using in vitro antibiotic sensitivity testing method.

Balın GSBL ve Biyofilm Üreten Enterobacterales Üzerindeki Antibakteriyel Etkisinin Değerlendirilmesi

Mastitis is a mammary gland inflammatory disease that causes milk yield reduction and economic losses. Mastitis is bacteriological and antibiotics are usually used for treatment. Alternative natural treatment methods such as bee products, phytotherapy, and essential oils were evaluated to reduce the use of antibiotics in the treatment of mastitis. In this study, the in vitro antibacterial effect of flower and oak honey samples dissolved in distilled water and boric acid (2%) on ESBL and biofilm-producing Enterobacterales pathogens was investigated. The aim was to establish the usability of honey/boric acid solution against mastitis as a natural antiseptic solution for bovine udder surfaces. Honey samples were studied by dissolving in distilled water and boric acid (2%) solvents. There was no significant statistical difference between honey solutions using distilled water and boric acid (p&gt;0.05). Antibacterial effects were increased according to the increasing honey proportion in flower honey solutions. However, the antibacterial activity of oak honey dissolved in boric acid solution was higher than dissolved in distilled water. As a result of the statistical correlation analysis between flower and oak honey samples, antibacterial effects of flower honey samples were determined to be higher than oak honey samples (p

Role of micronutrients in managing bovine udder health in changing climate scenario of Indian subtropical regions

Role of micronutrients in managing bovine udder health in changing climate scenario of Indian subtropical regions

Investigation of the pathophysiology of bacterial mastitis using precision-cut bovine udder slices

Mastitis in cattle is a major health problem as well as incurring high costs for the dairy industry. To assess the suitability of precision-cut bovine udder slices (PCBUS) for bovine mastitis studies, we infected PCBUS with 2 different Staphylococcus aureus strains. Accordingly, we investigated both the tissue response to infection based on immune mediators at the mRNA and protein levels and the invasion of bacteria within the tissue. The studied proteins represent immune mediators of early inflammation [IL-1β, tumor necrosis factor-α (TNF-α), prostaglandin E2 (PGE2)] and showed a time-dependent increase in concentration. Infection of PCBUS with S. aureus resulted in increased expression of proinflammatory cytokines and chemokines such as TNF-α, C-C motif chemokine ligand 20 (CCL20), IL-1β, IL-6, and IL-10, but not C-X-C motif chemokine ligand 8 (CXCL8), lingual antimicrobial peptide (LAP), or S100 calcium binding protein A9 (S100A9) at the mRNA level. To compare the data acquired with this model, we carried out investigations on primary bovine mammary epithelial cells. Our results showed that the immune responses of both models-PCBUS and primary bovine mammary epithelial cells-were similar. In addition, investigations using PCBUS enabled us to demonstrate adherence of bacteria in the physiological cell network. These findings support the use of PCBUS in studies designed to further understand the complex pathophysiological processes of infection and inflammation in bovine mastitis and to investigate alternative therapies for mastitis.

Lactococcus lactis subsp lactis CRL1655 and Schleiferilactobacillus perolens CRL1724 inhibit the adherence of common bovine mastitis pathogens to mammary gland cells, without causing histological changes in the mammary gland.

The present work assessed the ability of two selected lactic acid bacteria (LAB) strains (Schleiferilactobacillus perolens CRL1724 and Lactococcus lactis subsp. lactis CRL1655) to inhibit the adherence of bovine mastitis pathogens to mammary epithelial cells (MAC-T) and their effects (if any) on the structure of the gland after intramammary inoculation at dry-off. Established bovine mammary epithelial cells (MAC-T) were used to assess the LAB strains' ability to inhibit the adherence of bovine mastitis pathogens. Monolayers of MAC-T cells were co-cultured with the LABs and then individual pathogen was added. Both strains prevented the adherence of S. aureus RC108, S. chromogenes, S. uberis UT102 and E. coli ATCC 35218. Adherence of the latter two pathogens was inhibited most strongly in vitro. To evaluate the effect of the LAB on the structure of the bovine udders, quarters were intramammary inoculated with the LAB mixture at dry-off. After slaughtering, the teats were dissected and histopathologically analysed. No modifications were identified post-inoculation in the structure of the epithelial, subepithelial and connective tissues of the mammary gland. Probiotic strains L. lactis subsp lactis CRL1655 and S. perolens CRL1724 were both able to inhibit the adherence of a number of bovine mastitis pathogens in vitro, and that the intramammary inoculation of these strains at the established dose and concentration did not cause significant alterations in the mammary epithelium nor had undesirable effects on tissues, and may therefore be considered harmless. The promising findings demonstrated in this work support the potential of probiotic micro-organisms as a natural and effective alternative to prevent bovine mastitis during the dry-off period.

Testing Two Somatic Cell Count Cutoff Values for Bovine Subclinical Mastitis Detection Based on Milk Microbiota and Peripheral Blood Leukocyte Transcriptome Profile.

Simple SummarySetting a proper SCC cut-off value for determining the intramammary infection status of each individual cow is essential to early mastitis detection and prevention. In this study, we compared the effect of two commonly used SCC thresholds on distinguishing milk microbiota and host gene expression patterns, and demonstrated that the microbial composition and peripheral blood leukocyte transcriptome profiles did have conspicuous differences between cows with SCC above and below 100,000 cells/mL, respectively, which may help establish why 100,000 cells/mL is a more suitable cow-level cut-off value of subclinical mastitis diagnosis than 200,000 cells/mL from the perspective of microbiota and transcriptomic responses. Somatic cell count (SCC) is an important indicator of the health state of bovine udders. However, the exact cut-off value used for differentiating the cows with healthy quarters from the cows with subclinical mastitis remains controversial. Here, we collected composite milk (milk from four udder quarters) and peripheral blood samples from individual cows in two different dairy farms and used 16S rRNA gene sequencing combined with RNA-seq to explore the differences in the milk microbial composition and transcriptome of cows with three different SCC levels (LSCC: <100,000 cells/mL, MSCC: 100,000–200,000 cells/mL, HSCC: >200,000 cells/mL). Results showed that the milk microbial profiles and gene expression profiles of samples derived from cows in the MSCC group were indeed relatively easily discriminated from those from cows in the LSCC group. Discriminative analysis also uncovered some differentially abundant microbiota at the genus level, such as Bifidobacterium and Lachnospiraceae_AC2044_group, which were more abundant in milk samples from cows with SCC below 100,000 cells/mL. As for the transcriptome profiling, 79 differentially expressed genes (DEGs) were found to have the same direction of regulation in two sites, and functional analyses also showed that biological processes involved in inflammatory responses were more active in MSCC and HSCC cows. Overall, these results showed a similarity between the milk microbiota and gene expression profiles of MSCC and HSCC cows, which presented further evidence that 100,000 cells/ml is a more optimal cut-off value than 200,000 cells/mL for intramammary infection detection at the cow level.

Comparison of Dynamics of Udder Skin Microbiota From Grazing Yak and Cattle During the Perinatal Period on the Qinghai-Tibetan Plateau.

The perinatal period has an important impact on the health of ruminants, and the imbalance of udder skin microbiota might be an important inducement of bovine mastitis. However, it is not clear how the perinatal period affects the microbial structure and stability of the udder skin of yak and cattle. Here, we used 16S rRNA gene high-throughput sequencing to analyze the udder skin microbiota of yak and cattle during the perinatal period. We found that the diversity and richness of microbiota of bovine udder skin during 1–2 weeks postpartum were significantly lower than those in the 1–2 weeks prenatal and 1-month postpartum period (Wilcoxon, p < 0.05). Besides, we found sharing of 2,533 OTUs in the udder skin microbiota of yak and cattle during the perinatal period, among which the core microbiota at the genera level was mainly composed of Staphylococcus, Moraxella, and Acinetobacter. However, the genus Acinetobacter was significantly abundant in the udder skin of cattle during 1–2 weeks postpartum. The NMDS and LEfSe results showed that the perinatal period had more effects on the composition and stability of microbial community in the udder skin of cattle compared to yak, particularly during 1–2 weeks postpartum. In addition, the average content of total whey proteins and immunoglobulin G of whey protein were significantly higher in the yak colostrum when compared to those found in the cattle (p < 0.05). In conclusion, the structure of udder skin microbiota of yak during the perinatal period is more stable than that of cattle in the same habitat, and 1–2 weeks postpartum may be a potential window period to prevent cattle mastitis.

Longitudinal dynamics of the bovine udder microbiota

BackgroundIn recent years, the number of studies concerning microbiota of the intramammary environment has increased rapidly due to the development of high-throughput sequencing technologies that allow mapping of microbiota without culturing. This has revealed that an environment previously thought to be sterile in fact harbours a microbial community. Since this discovery, many studies have investigated the microbiota of different parts of the udder in various conditions. However, few studies have followed the changes that occur in the udder microbiota over time. In this study, the temporal dynamics of the udder microbiota of 10 cows, five with a low somatic cell count (SCC, SCC < 100,000 cells/mL) and five with a high SCC (SCC > 100,000 cells/mL), were followed over 5 months to gather insights into this knowledge gap.ResultsAnalysis of the temporal changes in the microbial composition of milk from udders with a low SCC revealed a dynamic and diverse microbiota. When an imbalance due to one dominating genus was recorded, the dominant genus quickly vanished, and the high diversity was restored. The genera dominating in the samples with a high SCC remained the dominant genera throughout the whole sampling period. These cows generally displayed a heightened SCC or an intramammary infection in at least one quarter though-out the sampling period.ConclusionOur results show that the bovine udder has a diverse microbiota, and that the composition and diversity of this community affects udder health with regards to SCC. Understanding what influences the composition and stability of this community has important implications for the understanding, control, and treatment of mastitis.