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.

Immunomodulatory molecules regulate adhesin gene expression in Staphylococcus aureus: Effect on bacterial internalization into bovine mammary epithelial cells.

Staphylococcus aureus is a major pathogen of subclinical bovine mastitis that usually is chronic and recurrent, which has been related to its ability to internalize into bovine mammary epithelial cells (bMECs). Previously, we reported that short and medium fatty acids and cholecalciferol reduce S. aureus internalization into pretreated-bMECs with these molecules suggesting a role as immunomodulatory agents. Hence, we assessed the role of sodium butyrate (NaB), sodium octanoate (NaO) and cholecalciferol on S. aureus adhesin expression and its internalization into bMECs. S. aureus pre-treated 2 h with 0.5 mM or 2 mM NaB showed a reduction in internalization into bMECs (∼35% and ∼55%; respectively), which coincided with a down-regulated expression of clumping factor B (ClfB). Also, the S. aureus internalization reduction by 2 mM NaB (2 h) agreed with a down-regulated expression of sdrC. Moreover, the 2 mM NaB (24 h) pre-treatment induced bacterial internalization (∼3-fold), which was related with an up-regulation of spa, clfB and sdrC genes. Also, NaO (0.25 mM and 1 mM) only reduced S. aureus internalization when bacteria were grown 2 h with this molecule but there was no relationship with adhesin expression. In addition, cholecalciferol (50 nM) reduced bacteria internalization at similar levels (∼50%) when bacteria were grown 2 and 24 h in broth supplemented with this compound, which correlated with spa and sdrC mRNA expression down-regulated at 2 h, and fnba and clfB mRNA expression decreased at 24 h. In conclusion, our data support the fact that fatty acids and cholecalciferol regulate adhesin gene expression as well as bacteria internalization in nonprofessional phagocytic cells, which may lead to development of anti-virulence agents for control of pathogens.

Mammary Gland Immunobiology and Resistance to Mastitis.

The ability of dairy cattle to prevent infectious pathogens from causing mastitis is related to the efficiency of the mammary immune system. The primary roles of the bovine immune system are to prevent bacterial invasion of the mammary gland, eliminate existing infections, and restore mammary tissues to normal function. Mammary gland immunity uses a multifaceted network of physical, cellular, and soluble factors to protect the cow from the diverse array of mastitis-causing pathogens. Strategies to optimize mammary gland defenses can be an effective way to prevent the establishment of new intramammary infections and limit the use of antimicrobials to treat mastitis.

Intramammary inoculation of Panax ginseng plays an immunoprotective role in Staphylococcus aureus infection in a murine model.

The immunoprotective effect of Panax ginseng (Pg) extract was investigated in a mouse mastitis model. Lactating female mice were intramammarily inoculated with Pg or placebo, and then were challenged with S. aureus, while other group was inoculated with S. aureus alone. The number of bacteria recovered from mammary glands was significantly lower in Pg-treated S. aureus-infected mice (group I) compared with placebo-treated S. aureus-infected mice (group II) and S. aureus-infected mice (group III). The mRNA expression of TLR2, TLR4, IL-1α and TNF-α was influenced by treatment; being the transcript levels for all genes higher in group I compared with group II and III. Activation of NF-κB and the number of monocytes-macrophages in mammary gland tissue was significantly increased in group I compared with group II and III. Pg extract was able to trigger an adequate immune response to confront an infection demonstrating its protective effect and potential for preventing bovine intramammary infections.

Oral supplementation of medium-chain fatty acids during the dry period supports the neutrophil viability of peripartum dairy cows.

A randomised clinical trial was conducted to explore the effect of orally supplemented medium-chain fatty acids (MCFA) to heifers and cows starting 6-8 weeks prior to expected calving date on blood and milk polymorphonuclear neutrophilic leucocyte (PMNL) apoptosis between 1 and 3 d in milk (DIM). The effects of MCFA-supplementation on the likelihood of intramammary infections (IMI) in early lactation, and test-day somatic cell count (SCC) and average daily milk yield (MY) during the first 4 months of lactation were evaluated as well. Twenty-two animals were included of which half were orally supplemented with MCFA starting 6-8 weeks prior to calving and half served as non-supplemented controls. The PMNL viability in both blood and milk was quantified using dual-colour flow cytometry with fluorescein-labelled annexin and propidium iodide. In non-supplemented animals, % blood PMNL apoptosis significantly increased between start of supplementation and early lactation, reflecting a potential reduction in innate immune capacity, whereas this was not true in the MCFA-supplemented animals. Similar results were seen in milk PMNL apoptosis. Overall, the % apoptotic milk PMNL between 1 and 3 DIM was significantly lower in the MCFA-supplemented group compared with the non-supplemented group. There was no substantial effect of oral MCFA-supplementation on the likelihood of quarter IMI nor on the composite test-day milk SCC or average daily MY. In conclusion, oral MCFA-supplementation starting 6-8 weeks before expected calving date supported the blood and milk neutrophil viability in early lactating dairy cows. Still, this was not reflected in an improvement of udder health nor MY in early and later lactation. The results should trigger research to further unravel the mechanisms behind the observed immunomodulating effect, and the potential relevance for the cows' performances throughout lactation.

Plant essential oils and mastitis disease: their potential inhibitory effects on pro-inflammatory cytokine production in response to bacteria related inflammation.

This paper highlights the role of plant volatile organic compounds, found in essential oils, for the treatment of bacteria related inflammation. This report is focused on tea tree oil, particularly its main compound terpinen-4-ol. Analysis of the published literature shows that many essential oils have significant antibacterial, antifungal and anti-inflammatory effects. Some of their major components, such as terpinen-4-ol, act by inhibiting pro-inflammatory cytokine expression while stimulating production of anti-inflammatory cytokines. Such observations may be exploited to encourage biotherapy against mastitis. The use of synthetic antibiotics is being increasingly discouraged because their presence in dairy milk may have potential downstream effects on population health and the agri-food chain. In the context of inflammation and related mammalian responses, understanding the interplay between volatile organic compounds, especially terpinen-4-ol, and cytokines during bacteria related inflammation should clarify their mode of action to control mastitis.

Effects of continuous lactation and short dry periods on mammary function and animal health.

The dry period is required to facilitate cell turnover in the bovine mammary gland in order to optimize milk yield in the next lactation. Traditionally, an 8-week dry period has been a standard management practice for dairy cows based on retrospective analyses of milk yields following various dry period lengths. However, as milk production per cow has increased, transitioning cows from the nonlactating state to peak milk yield has grown more problematic. This has prompted new studies on dry period requirements for dairy cows. These studies indicate a clear parity effect on dry period requirement. First parity animals require a 60-day dry period, whereas lactations following later parities demonstrate no negative impact with 30-day dry period or even eliminating the dry period when somatotropin (ST) is also used to maintain milk yields. Shortened dry periods in first parity animals were associated with reduced mammary cell turnover during the dry period and early lactation and increased numbers of senescent cells and reduced functionality of lactating alveolar mammary cells postpartum. Use of ST and increased milking frequency postpartum reduced the impact of shortened dry periods. The majority of new intramammary infections occur during the dry period and persist into the following lactation. There is therefore the possibility of altering mastitis incidence by modifying or eliminating the dry period in older parity animals. As the composition of mammary secretions including immunoglobulins may be reduced when the dry period is reduced or eliminated, there is the possibility that the immune status of cows during the peripartum period is influenced by the length of the dry period.

Bovine intramammary Escherichia coli challenge infections in late gestation demonstrate a dominant antiinflammatory immunological response.

Coliform mastitis that presents itself at parturition or in the early weeks of bovine lactation is often characterized by severe inflammation and impaired milk production and can lead to death of the animal. Chronic intramammary infections caused by persistent strains of Escherichia coli may result in high production losses. The aim of this study was to determine the inflammatory response to a teat-canal challenge of bovine mammary glands with a persistent strain of E. coli during late gestation (dry period) and into early lactation. Two weeks before parturition, animals were challenged in 2 quarters with 30 cfu of a persistent strain of E. coli; control quarters were vehicle-infused and not infused, respectively. Samples of dry cow secretions were taken from all quarters before challenge and at 6, 12, 18, 24, 48, 72, 96, and 120 h following challenge. Colostrum samples and milk samples were taken from all quarters at parturition and 6, 12, 18, 24, 48, 72, 96 and 120 h postpartum. Bacterial culture, combined with random amplified polymorphic DNA genetic strain-typing analysis, indicated recovery of the bacterial challenge strain until 48 to 96 h postchallenge, and again at parturition and up to 6 and 12h postpartum. One animal exhibited clinical mastitis and the bacterial challenge strain was evident to at least 12 d postpartum. During twice-daily milkings, production levels were lower in bacteria-challenged quarters compared with controls. Somatic cell counts decreased to normal levels at a slower rate in challenged quarters compared with control quarters. Cytokine analysis indicated a minimal proinflammatory cytokine response, including interleukin-1ß and tumor necrosis factor-α in challenged-quarter dry cow samples up to 120 h postchallenge. Interleukin-10 levels were significantly increased by 12h postchallenge in secretions from challenged and control quarters. These preliminary results in 2 cows indicate that proinflammatory signaling after intramammary bacterial infection may be actively suppressed during late gestation. We hypothesize that this immune-inhibitory response allows intramammary infections to become persistent in the dry period and cause clinical signs immediately after parturition.

Decrease in bovine CD14 positive cells in colostrum is associated with the incidence of mastitis after calving.

During the postpartum period there is a high incidence of mastitis in dairy cows. The reason for this increased risk of mastitis still remains unclear. Since leukocytes in colostrum have an important role in preventing the onset of mastitis, we investigated the leukocyte populations, which express CD4, CD8, CD14, CD21 or WC1, in colostrum as well as in blood obtained from 14 Holstein cows. Eight cows developed mastitis within a week after calving and the other 6 remained healthy. The percentage of CD14+ cells in colostrum was significantly lower in mastitic cows than in healthy cows. There were no significant differences in other marker positive cells either in the colostrum or in the blood. The CD14+ cells in colostrum play an important role of defense against invading microorganisms in the mammary glands. Our results suggested that the lower percentage of CD14+ cells in colostrum might predict the incidence of mastitis in the following period.

Evaluation of mammary gland immunity and therapeutic potential of Tinospora cordifolia against bovine subclinical mastitis.

Enhancement of the diseased mammary gland immunity and therapeutic potential of hydro-methanolic extract of Tinospora cordifolia (T. cordifolia; stem) in bovine subclinical mastitis was investigated. Somatic cell count (SCC), total bacterial count (TBC), phagocytic activity, and leukocyte lysosomal enzymes like myeloperoxidase and acid phosphatase activity and Interleukin-8 (IL-8) level were evaluated after intramammary infusion of hydro-methanolic extract (stem) of T.cordifolia in diseased cows. The qualitative analysis of the extract revealed the presence of polysaccharide, phenol, alkaloid, and protein. Intramammary infusion of hydro-methanolic extract of T. cordifolia treatment initially enhanced the SCC; thereafter, significant reduction in cell count (P < 0.05) was observed on day 15 of the treatment period, however, reduction in TBC was observed from day 3 onwards. The phagocytic activity of milk polymorphonuclear cells enhanced in the diseased cows treated with the T. cordifolia extract. Similarly, the lysosomal enzyme content of the milk polymorphonuclear cells enhanced significantly (P < 0.05) in diseased cows treated with T. cordifolia. The IL-8 level in milk serum also increased significantly (P < 0.05) in diseased cows treated with the herb extract. The results suggest that the hydro-methanolic extract of T.cordifolia (stem) possesses antibacterial and immunomodulatory properties. In the present study, the biological activity of the Tinospora cordifolia extract at standardized dose against bovine subclinical mastitis is reported for the first time. Development of alternative therapy with medicinal plants is an option for livestock farmers who are not allowed to use the conventional allopathic drugs under certain farming system or cannot afford to use allopathic drugs.

The role of dietary selenium in bovine mammary gland health and immune function.

Mastitis is not only a major cause of economic losses to the dairy industry but also a major problem in ensuring the quality and safety of the milk, associated with high somatic cell counts and residues of antibiotics used for treatment. One innovative approach to protection against mastitis is to stimulate the animal's natural defense mechanisms. Technological advances in immunological research have increased our ability to exploit the immunity of the bovine mammary gland during periods of high susceptibility to disease. The trace element selenium affects the innate and the adaptive immune responses of the mammary gland through cellular and humoral activities. Substantial research has been carried out on the effect of selenium (Se) on the immune function of the mammary gland and subsequent improvement in bovine udder health and mastitis control. Levels higher than current recommendations and Se-yeast can potentially be used to enhance our capacity to modulate the physiological mechanisms of the bovine mammary gland to respond to infection. This article provides an overview of the most recent research in this field.

Concentrations of ganglioside type M1 and immunoglobulin G in colostrum are inversely related to bacterial infection at early lactation in cows.

The levels of IgG and ganglioside type M1 in the colostrum of cows and heifers were analyzed to examine their utility in predicting acquisition of intramammary infection (IMI) during the first weeks postpartum. In general, high levels of IgG and ganglioside type M1 in cows were associated with lower new incidence of IMI, and linear discriminate analysis based on these 2 variables yielded 69.4% successful classification into cows that did or did not acquire new IMI. This analysis was less successful in heifers because a high proportion of them joined the herd when already infected with bacteria in their udders. It is suggested that application of a wider range of measures that reflect the immune status would enable the identification of most cows prone to new IMI.

Mastitis in post-partum dairy cows.

Transition from the dry period to lactation is a high risk period for the modern dairy cow. The biggest challenge at that time is mastitis. Environmental bacteria are the most problematic pathogens around parturition. Coliforms are able to cause severe infections in multiparous cows, and heifers are likely to be infected with coagulase-negative staphylococci. During the periparturient period, hormonal and other factors make the dairy cows more or less immunocompromised. A successful mastitis control programme is focused on the management of dry and calving cows and heifers. Clean and comfortable environment, proper feeding and adequate supplementation of the diet with vitamins and trace elements are essential for maintaining good udder health. Strategies which would enhance closure of the teat canal in the beginning of the dry period and would protect teat end from bacteria until the keratin plug has formed decrease the risk for mastitis after calving. Dry cow therapy has been used with considerable success. Yet, a selective approach could be recommended rather than blanket therapy. Non-antibiotic approaches can be useful tools to prevent new infections during the dry period, in herds where the risk for environmental mastitis is high. Vaccination has been suggested as a means to support the immune defence of the dairy cow around parturition. In some countries, implementation of Escherichia coli core antigen vaccine has reduced the incidence of severe coliform mastitis after calving.

Somatic cell count in milk of selenium-supplemented dairy cows after an intramammary challenge with Staphylococcus aureus.

The objective of this study was to evaluate the effect of selenium (Se) supplementation on milk somatic cell count (SCC) in dairy cows. Twelve multiparous Holstein-Friesian cows were fed a diet containing a suboptimal Se concentration (<0.05 ppm, dry basis) starting 2 months before calving. Supplemented cows (n=6) received a single s.c. injection of barium selenate (1 ml/50 kg BW) 45 days prior to calving, whereas control group was kept unsupplemented. Twenty weeks after calving, two mammary quarters (right side) of each cow were challenged with 205,000 cfu/ml of Staphylococcus aureus (strain Newbould 305). Blood was collected bi-weekly until day 150 of lactation for the analysis of blood glutathione peroxidase (GPx1; EC 1.11.1.9) activity. To re-isolate the challenging pathogen and to evaluate SCC, aseptic milk samples were collected daily starting on the day of challenge, and finishing 7 days after inoculation. Unsupplemented cows had a lower activity of GPx1 through the experiment (P<0.001). Natural log SCC (lnSCC) was higher in unsupplemented than Se-supplemented cows (P=0.04), showing evidence of significance after 5 days. Selenium supplementation of dairy cows fed a diet containing a suboptimal Se concentration, resulted in higher blood activity of GPx1, and lower mean lnSCC after an intramammary challenge with Staph. aureus.

Changes in leukocyte population after ozonated autohemoadministration in cows with inflammatory diseases.

In this study, we investigated whether ozonated autohemoadministration (OAHA) influences leukocyte populations in cows with clinical inflammatory disease. Eleven cows with inflammatory disease (Inflammatory Group) and three healthy cows (Control Group) were used for this study. The CD4(+)/CD8(+) ratio in the Inflammatory Group increased significantly compared to that in the Control Group 3 to 4 days after OAHA treatment. In the Inflammatory Group, the number of CD14(+) cells decreased gradually after OAHA, but CD14(+) levels remained stable in the Control Group. The number of MHC class-II(+) cells decreased gradually in the Inflammatory Group, but increased gradually in the Control Group, and the difference between the groups was significant on day 14 after OAHA. These findings suggest a possible difference in the activation of immune response after OAHA in infected cows compared to healthy cows.

The use of some immunomodulators and non-antibiotic drugs in a prophylaxis and treatment of mastitis.

Antibiotics are widely used in treatment and prophylaxis of mastitis, but their antibacterial and therapeutic efficacy continues to decrease. New methods of prophylaxis of mastitis with the use of vaccines and biological response modifiers are reviewed. Prophylactic activity of staphylococcal vaccines should be investigated in the future because of difficulties in successful treatment of infections caused by these organisms. Vaccination with J5 bacterin (0111:B4) reduced duration of IMI and local signs of clinical coliform-mastitis. Some immunomodulators proved to be effective in treatment of mastitis during lactating period. Promising results were obtained with the use of giseng saponin, herbal gel, herbal extracts, propolis, lysosubtilin, antibacterial proteins, and lysozyme dimer especially.

Immunity in the mammary gland.

The ruminant mammary gland is an extremely important economic organ in that it provides a major nutrition source for a significant portion of the world's human population. The ruminant mammary gland is also responsible for providing protective immunity to neonates and for defending itself from invading pathogens. A wide array of humoral and cellular immune mechanisms are present in the mammary gland and actively participate in providing immunity to newborns and the mammary gland per se. The acute inflammatory response is essential in determining the outcome of intramammary challenge, and factors affecting innate and adaptive immunity in the context of mammary health are reviewed in detail. The ruminant mammary gland is also unique in that lymphocyte trafficking, which is essential to adaptive immunity, is shared with the peripheral immune system rather than the common mucosal immune system.

Effect of subcutaneous injection of ginseng on cows with subclinical Staphylococcus aureus mastitis.

Cows with subclinical mastitis caused by Staphylococcus aureus were subjected to subcutaneous injections with either an extract from the root of Panax ginseng CA Meyer at a dose of 8 mg/kg body weight per day for 6 days, or with saline as a control. The injection areas were checked for adverse reactions. The daily milk production was measured before and after treatment. Blood was collected for total and differential leucocyte counts, identification of lymphocyte subpopulations using flow cytometry, lymphocyte proliferation test, and neutrophil phagocytosis and oxidative burst assay. Quarter milk samples were collected for bacteriological analysis and somatic cell counts (SCC). After the end of treatment, the numbers of S. aureus-infected quarters and milk SCC tended to decrease in ginseng-treated cows. Phagocytosis and oxidative burst activity of blood neutrophils were significantly increased 1 week after ginseng treatment, but the proliferative response of blood lymphocytes did not change significantly. The number of monocytes in ginseng-injected cows was significantly higher 1 week post-treatment than pre-treatment, and the number of lymphocytes was significantly higher than pre-infusion at 2 and 3 weeks after ginseng treatment. Similar changes were not observed in the control group. The present findings indicate that ginseng treatment can activate the innate immunity of cows and may contribute to the cow's recovery from mastitis. It is therefore suggested that ginseng has a potential as a stimulator of the immune system of dairy cows.

Immunological activities associated with milk.

Milk contains a multitude of components that can, or may, provide immune protection to the suckling offspring and that also may promote development of neonatal immune competence. In addition, these specialized factors are essential for the protection of the mammary gland, the offspring's food source, from pathogen colonization and lactation failure. Breast milk also facilitates the establishment of a gut flora that inhibits colonization by many pathogens and stimulates the growth of beneficial microorganisms. Maternal immunity can be transferred to the infant via antibodies, primarily of the sIgA type in humans, as well as by leukocytes including effector and memory T lymphocytes. In this way, protection is provided passively against the pathogens to which the mother has been exposed. Currently, there is much interest in determining the protective efficacy of oral supplementation with immunoglobulins from the milk of lactating animals hyperimmunized against specific pathogens. An array of immunostimulatory components in milk, notably cytokines, may be protected against intestinal proteolysis, thereby providing the offspring with a prepackaged immune response system. These components may help to boost the infant's immature immune system. At the same time, anti-inflammatory factors in breast milk help to modulate cytokine responses to infection, thereby facilitating defense while minimizing tissue damage such as that which occurs in infants with necrotizing enterocolitis. Undoubtedly, the many components constituting the repertoire of immune and immunomodulating agents in milk interact synergistically to protect both the mammary gland and the offspring from invading pathogenic microorganisms.

Presence of glutamine at position 74 of pocket 4 in the BoLA-DR antigen binding groove is associated with occurrence of clinical mastitis caused by Staphylococcus species.

Potential relationships between amino acid motifs in the antigen binding groove of various alleles of the bovine major histocompatibility complex DR (BoLA-DR) molecule and occurrence of clinical mastitis caused by Staphylococcus species (non-Staphylococcus aureus) were investigated in a case-control study. A significant association (P< or =0.05) was detected between the presence of glutamic acid at position beta 74 and occurrence of mastitis caused by Staphylococcus spp. with a relative risk of 11. This motif is present in BoLA-DRB3.2*22, *23 and *24 alleles. Presence of a positively charged residue (arginine or lysine) at position 13 also showed a tendency (P< o r=0.1) towards an association with a higher risk of clinical mastitis caused by the same bacteria. This motif is present in BoLA-DRB3.2*23 and *8 alleles. Similarly, presence of arginine at position beta 71 (present in alleles *23 and *22) was associated with occurrence of this disease. These positions (beta 13, beta 71 and beta 74) form pocket 4 of the antigen binding groove, which plays an instrumental role in antigen binding and recognition by T-lymphocytes. Thus, it can be concluded that pocket 4 of the BoLA-DR molecule is involved in conferring susceptibility to clinical mastitis caused by Staphylococcus spp.