Evaluation of the efficacy of a new inactivated vaccine against Staphylococcus aureus, Echerchia coli and Mycoplasma bovis mastitis in cows.

Staphylococcus aureus, Escherichia coli and Mycoplasma bovis are the most commonly isolated mastitis pathogens. The aim of this study was to evaluate the efficacy of a new mixed vaccine against mastitis caused by  Staphylococcus aureus, Escherichia coli, and Mycoplasma bovis. For this purpose, a mixed inactivated vaccine was administered subcutaneously to 24 heifers as one dose (2 mL) on the 45th day before birth and the second dose 21 days later. In 9 heifers, 2 mL of PBS was administered as placebo instead of vaccine. Then, heifers were divided into 3 groups as 7 vaccinated and 3 unvaccinated animals. Staphylococcus aureus, Escherichia coli, and Mycoplasma bovis were administered to the groups through intramammary route. Three vaccinated heifers were considered the common control without bacteria in all groups. The parameters considered to assess the effect of vaccination were clinical findings, bacterial count in milk, somatic cell count, and antibody titers. Clinical signs were observed only in the unvaccinated placebo group. Bacteria count and somatic cell count in milk increased in vaccinated and unvaccinated heifers. However, this increase was less in vaccinated animals and gradually returned to the normal level. In the unvaccinated heifers, it was ever high. Serum antibody titers were measured before and after vaccination. Antibody titers were high in vaccinated heifers after vaccination and were negative in unvaccinated heifers. In conclusion, the mixed vaccine had beneficial effect against Staphylococcus aureus, Escherichia coli, and Mycoplasma bovis mastitis and stimulated the immune response of vaccinated heifers.

Immunotherapy in mastitis: state of knowledge, research gaps and way forward.

Mastitis is an inflammatory condition that affects dairy cow's mammary glands. Traditional treatment approaches with antibiotics are increasingly leading to challenging scenarios such as antimicrobial resistance. In order to mitigate the unwanted side effects of antibiotics, alternative strategies such as those that harness the host immune system response, also known as immunotherapy, have been implemented. Immunotherapy approaches to treat bovine mastitis aims to enhance the cow's immune response against pathogens by promoting pathogen clearance, and facilitating tissue repair. Various studies have demonstrated the potential of immunotherapy for reducing the incidence, duration and severity of mastitis. Nevertheless, majority of reported therapies are lacking in specificity hampering their broad application to treat mastitis. Meanwhile, advancements in mastitis immunotherapy hold great promise for the dairy industry, with potential to provide effective and sustainable alternatives to traditional antibiotic-based approaches. This review synthesizes immunotherapy strategies, their current understanding and potential future perspectives. The future perspectives should focus on the development of precision immunotherapies tailored to address individual pathogens/group of pathogens, development of combination therapies to address antimicrobial resistance, and the integration of nano- and omics technologies. By addressing research gaps, the field of mastitis immunotherapy can make significant strides in the control, treatment and prevention of mastitis, ultimately benefiting both animal and human health/welfare, and environment health.

Soluble CD14 produced by bovine mammary epithelial cells modulates their response to full length LPS.

Bovine mastitis remains a major disease in cattle world-wide. In the mammary gland, mammary epithelial cells (MEC) are sentinels equipped with receptors allowing them to detect and respond to the invasion by bacterial pathogens, in particular Escherichia coli. Lipopolysaccharide (LPS) is the major E. coli motif recognized by MEC through its interaction with the TLR4 receptor and the CD14 co-receptor. Previous studies have highlighted the role of soluble CD14 (sCD14) in the efficient recognition of LPS molecules possessing a full-length O-antigen (LPSS). We demonstrate here that MEC are able to secrete CD14 and are likely to contribute to the presence of sCD14 in milk. We then investigated how sCD14 modulates and is required for the response of MEC to LPSS. This study highlights the key role of sCD14 for the full activation of the Myd88-independent pathway by LPSS. We also identified several lncRNA that are activated in MEC in response to LPS, including one lncRNA showing homologies with the mir-99a-let-7c gene (MIR99AHG). Altogether, our results show that a full response to LPS by mammary epithelial cells requires sCD14 and provide detailed information on how milk sCD14 can contribute to an efficient recognition of LPS from coliform pathogens.

Innate immune response of bovine mammary epithelial cells in Mycoplasma bovis mastitis using an in vitro model of bovine mammary gland infection.

Mycoplasma bovis mastitisis highly contagious and disrupts lactation, posing a significant threat to the dairy industry. While the mammary gland's defence mechanism involves epithelial cells and mononuclear cells (MNC), their interaction with M. bovis remains incompletely understood. In this study, we assessed the immunological reactivity of bovine mammary epithelial cells (bMEC) to M. bovis through co-culture with MNC. Upon co-culture with MNC, the mRNA expression levels of interleukin (IL)-1ß, IL-6, IL-8 and tumor necrosis factor (TNF)-α in bMEC stimulated by M. bovis showed a significant increase compared to monoculture. Additionally, when stimulated with M. bovis, the culture supernatant exhibited significantly higher concentrations of IL-6 and interferon (IFN)-γ, while IL-1ß concentration tended to be higher in co-culture with MNC than in monoculture. Furthermore, the mRNA expression levels of toll-like receptor (TLR) 2 in bMEC stimulated with M. bovis tended to increase, and TLR4 significantly increased when co-cultured with MNC compared to monocultures. However, the surface expression levels in bMEC did not exhibit significant changes between co-culture and monoculture. Overall, our research indicates that the inflammatory response of bMEC is increased during co-culture with MNC, suggesting that the interaction between bMEC and MNC in the mammary gland amplifies the immune response to M. bovis in cows affected by M. bovis mastitis.

Intramammary lipopolysaccharide challenge in early- versus mid-lactation dairy cattle: Immune, production, and metabolic responses.

Study objectives were to compare the immune response, metabolism, and production following intramammary LPS (IMM LPS) administration in early and mid-lactation cows. Early (E-LPS; n = 11; 20 ± 4 DIM) and mid- (M-LPS; n = 10; 155 ± 40 DIM) lactation cows were enrolled in an experiment consisting of 2 periods (P). During P1 (5 d) cows were fed ad libitum and baseline data were collected, including liver and muscle biopsies. At the beginning of P2 (3 d) cows received 10 mL of sterile saline containing 10 µg of LPS from Escherichia coli O111:B4/mL into the left rear quarter of the mammary gland, and liver and muscle biopsies were collected at 12 h after LPS. Tissues were analyzed for metabolic flexibility, which measures substrate switching capacity from pyruvic acid to palmitic acid oxidation. Data were analyzed with the MIXED procedure in SAS 9.4. Rectal temperature was assessed hourly for the first 12 h after LPS and every 6 h thereafter for the remainder of P2. All cows developed a febrile response following LPS, but E-LPS had a more intense fever than M-LPS cows (0.7°C at 5 h after LPS). Blood samples were collected at 0, 3, 6, 9, 12, 24, 36, 48, and 72 h after LPS for analysis of systemic inflammation and metabolism parameters. Total serum Ca decreased after LPS (26% at 6 h nadir) but did not differ by lactation stage (LS). Circulating neutrophils decreased, then increased after LPS in both LS, but E-LPS had exaggerated neutrophilia (56% from 12 to 48 h) compared with M-LPS. Haptoglobin increased after LPS (15-fold) but did not differ by LS. Many circulating cytokines were increased after LPS, and IL-6, IL-10, TNF-α, MCP-1, and IP-10 were further augmented in E-LPS compared with M-LPS cows. Relative to P1, all cows had reduced milk yield (26%) and DMI (14%) on d 1 that did not differ by LS. Somatic cell score increased rapidly in response to LPS regardless of LS and gradually decreased from 18 h onwards. Milk component yields decreased after LPS. However, E-LPS had increased fat (11%) and tended to have increased lactose (8%) yield compared with M-LPS cows throughout P2. Circulating glucose was not affected by LPS. Nonesterified fatty acids (NEFA) decreased in E-LPS (29%) but not M-LPS cows. ß-Hydroxybutyrate slightly increased (14%) over time after LPS regardless of LS. Insulin increased after LPS in all cows, but E-LPS had blunted hyperinsulinemia (52%) compared with M-LPS cows. Blood urea nitrogen increased after LPS, and the relative change in BUN was elevated in E-LPS cows compared with M-LPS cows (36% and 13%, respectively, from 9 to 24 h). During P1, metabolic flexibility was increased in liver and muscle in early lactating cows compared with mid-lactation cows, but 12 h after LPS, metabolic flexibility was reduced and did not differ by LS. In conclusion, IMM LPS caused severe immune activation, and E-LPS cows had a more intense inflammatory response compared with M-LPS cows, but the effects on milk synthesis was similar between LS. Some parameters of the E-LPS metabolic profile suggest continuation of metabolic adjustments associated with early lactation to support both a robust immune system and milk synthesis.

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.

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.

Memory CD4+ and CD8+ T lymphocyte proliferation in vaccinated dairy cows with different histories of Staphylococcus aureus mastitis.

Staphylococcus aureus mastitis constitutes a serious threat to dairy cows. The reasons why available vaccines are not fully effective remain poorly understood; thus, in the present study, we investigated CD4+ and CD8+ T lymphocyte proliferation in dairy cows vaccinated with a polyvalent mastitis vaccine that had distinct precedent Staphylococcus aureus mastitis. We studied 17 S. aureus-infected dairy cows (11 vaccinated and six unvaccinated) and eight vaccinated healthy dairy cows with no previous S. aureus mastitis infections. Flow cytometry was used to assess lymphocyte proliferation using an anti-Ki67 antibody, and monoclonal antibodies were used to identify T cell subsets. S. aureus-infected cows exhibited reduced overall lymphocyte proliferation, including CD4+ T lymphocyte proliferation, and memory lymphocyte proliferation in response to S. aureus isolate stimulus. Immunization did not influence the expansion of blood lymphocyte populations. Furthermore, CD8+ T cells, memory CD8+ T lymphocytes, and effector memory CD8+ T lymphocytes displayed reduced proliferation 21 days after the third vaccine dose compared with before vaccination at time zero. The present data demonstrates an overall negative regulation of the T-cell response suggesting its detrimental impact leading to the persistence of S. aureus intramammary infections. Furthermore, the lack of vaccination effect on T-cell mediated immunity (e.g., proliferation) may be related to poor vaccine efficacy.

The Rumen Microbiota Contributes to the Development of Mastitis in Dairy Cows.

Mastitis, a highly prevalent disease in dairy cows, is commonly caused by local infection of the mammary gland. Our previous studies have suggested that the gut microbiota plays an important role in the development of mastitis in mice. However, the effects of rumen microbiota on bovine mastitis and the related mechanisms remain unclear. In this study, we assessed the effects and mechanisms of rumen microbiota on bovine mastitis based on the subacute rumen acidosis (SARA) model induced by feeding Holstein Frisian cows a high-concentrate diet for 8 weeks. Then, the inflammatory responses in the mammary gland and the bacterial communities of rumen fluid, feces, and milk were analyzed. The results showed that SARA induced mastitis symptoms in the mammary gland; activated a systemic inflammatory response; and increased the permeability of the blood-milk barrier, gut barrier, and rumen barrier. Further research showed that lipopolysaccharides (LPS), derived from the gut of SARA cows, translocated into the blood and accumulated in the mammary glands. Furthermore, the abundance of Stenotrophomonas was increased in the rumen of SARA cows, and mastitis was induced by oral administration of Stenotrophomonas in lactating mice. In conclusion, our findings suggested that mastitis is induced by exogenous pathogenic microorganisms as well as by endogenous pathogenic factors. Specifically, the elevated abundance of Stenotrophomonas in the rumen and LPS translocation from the rumen to the mammary gland were important endogenous factors that induced mastitis. Our study provides a foundation for novel therapeutic strategies that target the rumen microbiota in cow mastitis. IMPORTANCE Mastitis is a common and frequently occurring disease of humans and animals, especially in dairy farming, which has caused huge economic losses and brought harmful substance residues, drug-resistant bacteria, and other public health risks. The traditional viewpoint indicates that mastitis is mainly caused by exogenous pathogenic bacteria infecting the mammary gland. Our study found that the occurrence of mastitis was induced by the endogenous pathway. Evidence has shown that rumen-derived LPS enters the mammary gland through blood circulation, damaging the blood-milk barrier and then inducing inflammation of the mammary gland in cows. In addition, a higher abundance of Stenotrophomonas in the rumen was closely associated with the development of mastitis. This study provides a basis for novel therapeutic strategies that exploit the rumen microbiota against mastitis in cows.

Regulatory network of miRNA, lncRNA, transcription factor and target immune response genes in bovine mastitis.

Pre- and post-transcriptional modifications of gene expression are emerging as foci of disease studies, with some studies revealing the importance of non-coding transcripts, like long non-coding RNAs (lncRNAs) and microRNAs (miRNAs). We hypothesize that transcription factors (TFs), lncRNAs and miRNAs modulate immune response in bovine mastitis and could potentially serve as disease biomarkers and/or drug targets. With computational analyses, we identified candidate genes potentially regulated by miRNAs and lncRNAs base pair complementation and thermodynamic stability of binding regions. Remarkably, we found six miRNAs, two being bta-miR-223 and bta-miR-24-3p, to bind to several targets. LncRNAs NONBTAT027932.1 and XR_003029725.1, were identified to target several genes. Functional and pathway analyses revealed lipopolysaccharide-mediated signaling pathway, regulation of chemokine (C-X-C motif) ligand 2 production and regulation of IL-23 production among others. The overarching interactome deserves further in vitro/in vivo explication for specific molecular regulatory mechanisms during bovine mastitis immune response and could lay the foundation for development of disease markers and therapeutic intervention.

Colonization and local host response following intramammary Staphylococcus chromogenes challenge in dry cows.

Although extensive research has been performed on bovine non-aureus staphylococci (NAS), several aspects such as bacteria-host interaction remain largely unstudied. Moreover, only a few mastitis pathogen challenge studies in cows have been conducted in the dry period, an important period that allows intramammary infection (IMI) to cure and new IMI to occur. We challenged 16 quarters of 4 Holstein Friesian cows at dry off with 100; 100 000 or 10 000 000 CFU of the udder-adapted S. chromogenes IM strain. Four quarters from one cow served as negative controls. Internally sealed quarters remained untouched, whereas non-sealed quarters were sampled 3 times during the dry period. After parturition, colostrum and daily milk samples were taken during the first week of lactation of all quarters. In total, 8 quarters appeared to be colonized, since S. chromogenes IM was recovered at least once during the experiment, as substantiated using Multilocus Sequence Typing. S. chromogenes IM shedding was highest in dry quarters inoculated with 10 000 000 CFU. Colonized quarters had the highest quarter somatic cell count (qSCC) in early lactation. Inoculated quarters (both colonized and non-colonized) had lower IL-6 and IL-10 concentrations in the dry period, whilst IFN-γ levels tended to be higher in colonized quarters compared to non-inoculated quarters. Also, IgG2 levels were higher in inoculated compared to non-inoculated quarters and the IgG2/IgG1 ratio was on average above 1. To conclude, we showed that dry quarters can be colonized with S. chromogenes IM, resulting in a shift towards a Th1 response in late gestation and early lactation characterised by an increased IgG2 concentration. However, further research is needed to confirm our findings.

Going further post-RNA-seq: In silico functional analyses revealing candidate genes and regulatory elements related to mastitis in dairy cattle.

This study aimed to obtain a better understanding of the regulatory genes and molecules involved in the development of mastitis. For this purpose, the transcription factors (TF) and MicroRNAs (miRNA) related to differentially expressed genes previously found in extracorporeal udders infected with Streptococcus agalactiae were investigated. The Gene-TF network highlighted LOC515333, SAA3, CD14, NFKBIA, APOC2 and LOC100335608 and genes that encode the most representative transcription factors STAT3, PPARG, EGR1 and NFKB1 for infected udders. In addition, it was possible to highlight, through the analysis of the gene-miRNA network, genes that could be post-transcriptionally regulated by miRNAs, such as the relationship between the CCL5 gene and the miRNA bta-miR-363. Overall, our data demonstrated genes and regulatory elements (TF and miRNA) that can play an important role in mastitis resistance. The results provide new insights into the first functional pathways and the network of genes that orchestrate the innate immune responses to infection by Streptococcus agalactiae. Our results will increase the general knowledge about the gene networks, transcription factors and miRNAs involved in fighting intramammary infection and maintaining tissue during infection and thus enable a better understanding of the pathophysiology of mastitis.

Transcriptome Profiling of m6A mRNA Modification in Bovine Mammary Epithelial Cells Treated with Escherichia coli.

Mastitis is a common disease in dairy cows that is mostly caused by E. coli, and it brings massive losses to the dairy industry. N6-Methyladenosine (m6A), a methylation at the N6 position of RNA adenine, is a type of modification strongly associated with many diseases. However, the role of m6A in mastitis has not been investigated. In this study, we used MeRIP-seq to sequence the RNA of bovine mammary epithelial cells treated with inactivated E. coli for 24 h. In this in vitro infection model, there were 16,691 m6A peaks within 7066 mRNA transcripts in the Con group and 10,029 peaks within 4891 transcripts in the E. coli group. Compared with the Con group, 474 mRNAs were hypermethylated and 2101 mRNAs were hypomethylated in the E. coli group. Biological function analyses revealed differential m6A-modified genes mainly enriched in the MAPK, NF-κB, and TGF-ß signaling pathways. In order to explore the relationship between m6A and mRNA expression, combined MeRIP-seq and mRNA-seq analyses revealed 212 genes with concomitant changes in the mRNA expression and m6A modification. This study is the first to present a map of RNA m6A modification in mastitis treated with E. coli, providing a basis for future research.

Connecting Metabolism to Mastitis: Hyperketonemia Impaired Mammary Gland Defenses During a Streptococcus uberis Challenge in Dairy Cattle.

ß-hydroxybutyrate (BHB) has been associated with disease incidence in early lactation dairy cattle, but such associations do not demonstrate causation. Therefore, the objective of this study was to examine the effects of BHB during an intramammary Streptococcus uberis challenge. A secondary objective was to elucidate the mechanisms behind BHB effects on cytokine transcript abundance using the RAW 264.7 cell line. Late lactation multiparous dairy cows (n = 12) were continuously infused intravenously with either BHB to induce hyperketonemia (target concentration: 1.8 mM) or with saline (CON) for 72 h during a S. uberis intramammary challenge. Body temperature, dry matter intake (DMI), milk production, and milk S. uberis cfu were measured daily until one week post-challenge. Blood samples were collected during infusion to assess changes in metabolism (glucose, insulin, glucagon, NEFA, and cortisol) and systemic inflammation (IL-1ß and SAA). Mammary biopsies were conducted at 72 h post-challenge to assess transcript abundance of inflammation-associated genes. BHB-infused cows exhibited a delayed febrile response, noted by a lesser vaginal temperature during the final day of infusion, followed by a greater vaginal temperature 6 d post-challenge. Consequently, BHB-infused cows had greater S. uberis cfu on d 4, 6, and 7 as compared to CON. Accordingly, BHB-infused cows consumed less DM, produced less milk, had reduced blood glucose, and had increased cortisol concentrations, however, no effects were seen on other systemic parameters or transcript abundance of inflammation-related genes in mammary tissue. To elucidate mechanisms behind the impaired immune defenses, RAW 264.7 cells were transfected with a GPR109A siRNA for 24 h and then treated with or without 1.8 mM BHB and challenged or left unchallenged with S. uberis for an additional 3 h. Transfection with siRNA reduced Gpr109a by 75%. Although BHB treatment did not significantly increase Il10, GPR109A knockdown as compared to the scrambled control reduced Il10 by 90% in S. uberis challenged macrophages treated with BHB, suggesting that macrophage immune responses to S. uberis can be altered via a GPR109A-dependent mechanism. Taken together, these data suggest that BHB altered the immune response promoting tolerance toward S. uberis rather than resistance.

Mechanisms by which mastitis affects reproduction in dairy cow: A review.

Reproductive performance is a key factor in determining the profitability of dairy farm, which is affected by many factors such as environment and diseases. Mastitis is a common and important disease, which has caused huge economic losses to the dairy industries worldwide. Mammary gland infection causes immune responses, resulting in the abnormal secretion of cytokines and hormones and abnormal function of the reproductive system such as the ovary, corpus luteum, uterus and embryo. Cows with mastitis have delayed oestrus, decreased pregnancy rate and increased risk of abortion. The adverse effects of mastitis on reproductive performance are affected by many factors, such as occurrence time, pathogen and cow factors. This paper primarily reviews the progress in the effects and mechanisms of mastitis on reproductive performance, with emphasis on maternal transcriptome, genomic analysis, epigenetic modification, microbiota, inflammatory regulation and immune evasion mechanism of mastitis, aiming to provide directions for the prevention and control of mastitis in the future.

Intramammary infection caused by Staphylococcus aureus increases IgA antibodies to iron-regulated surface determinant-A, -B, and -H in bovine milk.

The aim of this study was to identify virulence factors that have high immunogenicity. An in vivo-expressed Staphylococcus aureus antigen was identified by probing bacteriophage expression libraries of S. aureus with antibodies in bovine mastitis milk. Eighteen clones were isolated, and their proteins were identified as 5 characterised proteins (IsdA, Protein A, IsdB, autolysin, and imidazole glycerol phosphate dehydratase) and 13 hypothetical proteins. We focused on IsdA, IsdB, and IsdH as virulence factors that have a high immunogenicity and are capable of inducing a specific humoral immune response in S. aureus-infected quarters. The optical density (OD) values of IsdA and IsdB IgA and IgG antibodies in milk affected by naturally occurring mastitis caused by S. aureus increased significantly compared to those in healthy milk. In the experimental infection study, the OD values of IsdA- and B-specific IgA and IgG antibodies were significantly increased from 2 to 4 weeks after S. aureus infection compared to day 0 (P < 0.05). On the other hand, we demonstrated that milk from natural and experimental intramammary infections caused by S. aureus are associated with significantly higher IgA levels against IsdH (P < 0.05), but no significant change in IgG levels. Our findings facilitated our understanding of the pathogenicity of S. aureus in bovine mastitis, as well as the mechanisms by which specific humoral immune responses to S. aureus infection are induced. In addition, the results obtained could provide insight into how bovine mastitis can be controlled, for example, through vaccination.

Heat inactivation partially preserved barrier and immunomodulatory effects of Lactobacillus gasseri LA806 in an in vitro model of bovine mastitis.

Probiotics could help combat infections and reduce antibiotic use. As use of live bacteria is limited in some cases by safety or regulatory concerns, the potential of inactivated bacteria is worth investigating. We evaluated the potential of live and heat-inactivated Lactobacillus gasseri LA806 to counteract Staphylococcus aureus and Escherichia coli infection cycles in an in vitro model of bovine mastitis. We assessed the ability of live and inactivated LA806 to impair pathogen colonisation of bovine mammary epithelial cells (bMECs) and to modulate cytokine expression by pathogen-stimulated bMECs. Live LA806 induced a five-fold decrease in S. aureus adhesion and internalisation (while not affecting E. coli colonisation) and decreased pro-inflammatory cytokine expression by S. aureus-stimulated bMECs (without interfering with the immune response to E. coli). The ability of inactivated LA806 ability to diminish S. aureus colonisation was two-fold lower than that of the live strain, but its anti-inflammatory properties were barely impacted. Even though LA806 effects were impaired after inactivation, both live and inactivated LA806 have barrier and immunomodulatory properties that could be useful to counteract S. aureus colonisation in the bovine mammary gland. As S. aureus is involved in various types of infection, LA806 potential would worth exploring in other contexts.

Immune response during the onset of coliform mastitis in dairy cows vaccinated with STARTVAC®.

The immune response during the onset of coliform mastitis in vaccinated cows was investigated by measuring lactoferrin (LF), interleukin-8 (IL-8), and interleukin-1ß (IL-1ß) concentrations and somatic cell counts in 28 milk samples at the onset of acute coliform mastitis (ACM) and 73 milk samples at the onset of peracute coliform mastitis (PCM). Vaccinated ACM, unvaccinated ACM, and vaccinated PCM showed significantly higher values for LF and IL-1ß levels than unvaccinated PCM (p < .01). The IL-8 concentration was lower in vaccinated PCM than in unvaccinated PCM (p < .05). There was no significant difference in somatic cell counts for each parameter. There were no significant differences in the parameters between vaccinated and unvaccinated ACM cows, or vaccinated ACM and PCM cows. From the above results, it is suggested that mastitis vaccination improved the early immune response, particularly at the onset of PCM, and played a large role in host defense against the initial infection.

Intramammary inoculation with lactic acid bacteria at dry-off triggers an immunomodulatory response in dairy cows.

The use of antibiotics to prevent bovine mastitis is responsible for the emergence and selection of resistant strains. Lactic acid bacteria (LAB) could be introduced into animal feed as an alternative prevention method that would bypass the risk of resistance development. In previous research, we demonstrated that two probiotic LAB strains isolated from bovine milk were capable of stimulating the production of antibodies and the host's immune cellular response in the udder. The present study aimed to elucidate whether the antibodies of animals inoculated with these strains were able to increase phagocytosis by neutrophils and inhibit the growth of different mastitis-causing pathogens. Moreover, the effect of LAB on the expression of pro-inflammatory cytokines was assessed. Ten animals were inoculated intramammarily with 106 cells of the two strains at dry-off. The blood serum was tested for its ability to opsonize bovine mastitis pathogens, the in vitro bactericidal activity of bovine blood and milk against these pathogens was determined, and cytokine mRNA expression was quantified in milk somatic cells. The inoculated animals did not show abnormal signs of sensitivity to the LAB. Their blood serum significantly enhanced the phagocytosis of Staphylococcus spp. and the LAB. Escherichia coli and Streptococcus uberis were inhibited by the milk serum but not the blood serum, whereas Staphylococcus aureus and Staphylococcus haemolyticus were inhibited by both. In regard to cytokine expression, interleukin (IL)-1ß increased markedly for up to 4 h post-inoculation, and an increase in IL-8 was observed 4, 12 and 24 h after inoculation. Tumour necrosis factor-α mRNA increased 1 and 2 h after inoculation and a significant difference was registered at 6 h for interferon-γ. This rapid immunomodulatory response shows that inoculating animals with LAB at dry-off, when they are especially susceptible, could be a useful strategy for the prevention of bovine mastitis.

Staphylococcal enterotoxin M induced inflammation and impairment of bovine mammary epithelial cells.

Staphylococcus aureus is one of the major etiological pathogens of bovine mastitis. Its invasion into mammary epithelial cells has been proven to be a key event in the pathogenesis of mastitis. However, the specific pathogenic factors have not been clearly identified. Staphylococcus aureus often triggers infections by releasing virulence factor. Recent several studies reported that staphylococcal enterotoxin M was one of the most frequently found enterotoxin genes associated with bovine mastitis. Thus, the effect of staphylococcal enterotoxin M on inflammation and damage of the bovine mammary epithelial bovine mammary gland epithelial cell line (MAC-T) cells with 48 h treatment was explored in the present study. First, staphylococcal enterotoxin M protein was purified by a Ni-NTA spin column (GE Life Science, Westborough, MA). The levels of tumor necrosis factor-α, IL-6, and monocyte chemoattractant protein 1 (MCP-1) secretion were measured with the corresponding ELISA kits (R&D Systems, Abingdon, UK). Second, cell viability was assessed with a Cell Counting Kit-8 (Bioswamp, Wuhan, China) and the apoptotic percentage of cells was determined by annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI; Beyotime, Nanjing, China) staining. Third, ATP concentration, reactive oxygen species (ROS) generation and lactate dehydrogenase (LDH) release were assayed with commercial kits, then mitochondrial membrane potential (ΔΨm) was estimated using fluorescent probe JC-1 (Beyotime). Finally, the production intercellular cell adhesion molecule-1 (ICAM-1), microtubule-associated protein 1A/1B-light chain 3 I/II (LC3 I/II), p62 (Proteintech, Rosemont, IL), and phosphorylation of IκBα, caspase 3, and mammalian target of rapamycin were detected by Western blot. The results showed that staphylococcal enterotoxin M induced inflammation of epithelial cells (upregulating tumor necrosis factor-α, IL-6, MCP-1, and ICAM-1 production) and activated NF-κB (promoting phosphorylation of IκBα). Furthermore, staphylococcal enterotoxin M impaired MAC-T cells via cell necrosis (enhancing LDH release), apoptosis (annexin V-FITC/PI stain, exacerbating oxidative stress, decreasing ΔΨm and intracellular ATP concentration, and activating caspase 3), but independent of autophagy (nonsignificantly increasing LC3-II, decreasing p62 expression, and activating mammalian target of rapamycin). Thereby, staphylococcal enterotoxin M induced the inflammatory property of bovine mammary epithelial cells by boosting cytokine, chemokine, and adhesion molecule production. Furthermore, it caused epithelial cell dysfunction via depressing cell viability and initiating cell necrosis and apoptosis. Because epithelial cells played important roles in orchestrating the inflammatory response and protecting bovine mammary tissue from mastitis, our results indicated that staphylococcal enterotoxin M may be associated with mastitis.