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.

Utilizing intramammary Melaleuca alternifolia as an organic internal sealant for dry-off therapy in Murrah buffaloes.

The effects of intramammary dry cow therapy based on the administration of 5% Melaleuca alternifolia tea tree essential oil (TTO) as an internal teat sealant to Murrah cows were evaluated. A longitudinal prospective and retrospective negative control study was performed using 12 buffaloes from a total of 20 Murrah buffaloes on an organic farm, with the cow used as a control for herself. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) for treatments with pure oil (TTO) and medication containing 5% TTO (O5) were determined. The buffaloes were clinically examined, and the teats were evaluated using thermography and ultrasound. Udder health was monitored during the first 100 days in milk (DIM) using milk somatic cell count (SCC) and California mastitis test (CMT). Laboratory tests against standard strains Staphylococcus aureus ATCC®25,923™, Escherichia coli ATCC®25,922™, and wild bacterial strains showed maximum MIC values of 50 µL/mL for the TTO and O5 treatments. One wild-type S. aureus strain showed no MBC. No adverse effects were observed after the intramammary application of TTO. The CMT and SCC values were similar (P > 0.05) for all observations. The medication containing 5% TTO was effective in vitro and compatible with the intramammary tissue in vivo of Murrah buffaloes. TTO was safe, not inducing inflammatory processes or other modifications of the teat detectable by thermography or ultrasound. It was able to protect buffaloes during the dry period under field conditions, demonstrating potential use as a teat sealant for organic farms.

Ozone use in the treatment of subclinical mastitis in dairy cows.

This research communication paper addresses the hypothesis that the use of therapeutic alternatives for mastitis, such as intramammary ozone, can cure the disease with lower costs and without harmful residues for human consumption and without formation of microbial resistance like the ones caused by indiscriminate use of antibiotics in dairy farms. The study was performed in 36 mammary quarters from 12 dairy cows with subclinical mastitis grade three. The experimental units were randomly assigned into four groups and each group received a treatment. Treatments comprised (a) 20 µg/ml ozone gas; (b) 40 µg/ml ozone gas; (c) negative control treatment of 12.5 µg/ml ozonated saline and (d) positive control treatment of 100 mg of cephalexin + 100 mg of neomycin + 10 mg of prednisolone, all by intramammary injection. In all quarters, milk was collected before and after the application of treatments for California mastitis test and evaluation of milk composition, somatic cell count, and bacterial cultures. The results indicated that the use of intramammary ozone did have a therapeutic effect, and whilst this was less than that of antibiotics, ozone does confer some advantages. Treated milk had a good composition, the treatment cost was low, milk withdrawal may not be necessary and there is no risk of antibiotic resistance.

Genetic diversity and iron metabolism of Staphylococcus hominis isolates originating from bovine quarter milk, rectal feces, and teat apices.

Staphylococcus hominis, a member of the non-aureus staphylococci (NAS) group, is part of the human and animal microbiota. Although it has been isolated from multiple bovine-associated habitats, its relevance as a cause of bovine mastitis is currently not well described. To successfully colonize and proliferate in the bovine mammary gland, a bacterial species must be able to acquire iron from host iron-binding proteins. The aims of this study were (1) to assess the genetic diversity of S. hominis isolated from bovine quarter milk, rectal feces, and teat apices, and (2) to investigate the capacity of bovine S. hominis isolates belonging to these different habitats to utilize ferritin and lactoferrin as iron sources. To expand on an available collection of bovine S. hominis isolates (2 from quarter milk, 8 from rectal feces, and 19 from teat apices) from one commercial dairy herd, a subsequent single cross-sectional quarter milk sampling (n = 360) was performed on all lactating cows (n = 90) of the same herd. In total, 514 NAS isolates were recovered and identified by MALDI-TOF mass spectrometry; the 6 most prevalent NAS species were S. cohnii (33.9%), S. sciuri (16.7%), S. haemolyticus (16.3%), S. xylosus (9.6%), S. equorum (9.4%), and S. hominis (3.5%). A random amplified polymorphic DNA (RAPD) analysis was performed on 46 S. hominis isolates (19 from quarter milk, 8 from rectal feces, and 19 from teat apices). Eighteen distinct RAPD fingerprint groups were distinguished although we were unable to detect the presence of the same RAPD type in all 3 habitats. One S. hominis isolate of a distinct RAPD type unique to a specific habitat (8 from quarter milk, 3 from rectal feces, and 4 from teat apices) along with the quality control strain Staphylococcus aureus ATCC 25923 and 2 well-studied Staphylococcus chromogenes isolates ("IM" and "TA") were included in the phenotypical iron test. All isolates were grown in 4 types of media: iron-rich tryptic soy broth, iron-rich tryptic soy broth deferrated by 2,2'-bipyridyl, and deferrated tryptic soy broth supplemented with human recombinant lactoferrin or equine spleen-derived ferritin. The growth of the different strains was modified by the medium in which they were grown. Staphylococcus chromogenes TA showed significantly lower growth under iron-deprived conditions, and adding an iron supplement (lactoferrin or ferritin) resulted in no improvement in growth; in contrast, growth of S. chromogenes IM was significantly recovered with iron supplementation. Staphylococcus hominis strains from all 3 habitats were able to significantly utilize ferritin but not lactoferrin as an iron source to reverse the growth inhibition, in varying degrees, caused by the chelating agent 2,2'-bipyridyl.

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.

AIMS: 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. METHODS AND RESULTS: 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. CONCLUSIONS: 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. SIGNIFICANCE AND IMPACT OF STUDY: 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.

Consumption of Supplementary Inulin Modulates Milk Microbiota and Metabolites in Dairy Cows with Subclinical Mastitis.

The milk microbiota and mediated metabolites directly affect the health of the udder in dairy cows. Inulin, a dietary prebiotic, can modulate the profile of gastrointestinal microbiota. However, whether the inulin intake affects the milk microbial population and metabolites remains unknown. In this study, 40 subclinical mastitis (SCM) cows were randomly divided into 5 groups. Five inulin addition doses, 0, 100, 200, 300, and 400 g/day per cow, based on the same basal diet, were supplemented. The experiments lasted for 8 weeks. The results showed lower relative abundance of mastitis-causing and proinflammation microbes in milk (i.e., Escherichia-Shigella, Pseudomonas, Rhodococcus, Burkholderia-Caballeronia-Paraburkholderia, etc.) and higher abundances of probiotics and commensal bacteria, such as Lactobacillus, Bifidobacterium, etc., in the cows fed 300 g/day inulin compared to that in the control group. Meanwhile, the levels of arachidonic acid proinflammatory mediators (leukotriene E3, 20-carboxy-leukotriene B4, and 12-Oxo-c-LTB3) and phospholipid metabolites were reduced, and the levels of compounds with antibacterial and anti-inflammatory potential (prostaglandin A1, 8-iso-15-keto-prostaglandin E2 [PGE2], etc.) and participating energy metabolism (citric acid, l-carnitine, etc.) were elevated. These data suggested that inulin intake might modulate the microflora and metabolite level in extraintestinal tissue, such as mammary gland, which provided an alternative for the regulation and mitigation of SCM. IMPORTANCE The profile of the microbial community and metabolic activity in milk are the main determinants of udder health status and milk quality. Recent studies have demonstrated that diet could directly modulate the mammary gland microbiome. Inulin is a probiotic dietary fiber which can improve the microbiota population in the gastrointestinal tract. However, whether inulin intake can further regulate the profile of the microbiota and metabolic activities in milk remains unclear. In subclinical mastitic cows, we found that inulin supplementation could reduce the abundance of Escherichia-Shigella, Pseudomonas, Rhodococcus, and Burkholderia-Caballeronia-Paraburkholderia and the levels of (±)12, 13-DiHOME, leukotriene E3 and 20-carboxy-leukotriene B4 etc., while it elevated the abundance of Lactobacillus, Bifidobacterium, and Muribaculaceae, as well as the levels of prostaglandin A1 (PGA1), 8-iso-15-keto-PGE2, benzoic acid, etc. in milk. These data suggest that inulin intake affects the profile of microorganisms and metabolites in milk, which provides an alternative for the regulation of mastitis.

Short communication: Effects of moringa extract on adhesion and invasion of Escherichia coli O55 in bovine mammary epithelial cells.

The objective of this study was to evaluate the antibacterial activities of extract derived from moringa leaves. In particular, the effect of moringa extract (Mor) on adhesion and invasion of Escherichia coli O55, Enterococcus faecalis, Staphylococcus simulans, and Serratia liquefaciens was evaluated in bovine mammary epithelial cells (MAC-T). Broth microdilution method, minimum inhibitory concentration and minimum bactericidal concentration assays, adhesion and invasion assays, and real-time PCR were performed. The minimum inhibitory concentration and minimum bactericidal concentration of Mor ranged from 12.5 to 50 mg/mL on 18 out of 27 tested isolates. Treatment of E. coli O55 with Mor (100 and 200 µg/mL) inhibited the adhesion and invasion on MAC-T cells via downregulation of adhesion factors (i.e., papC, f17c-A, and eaeA). Also, when MAC-T cells were pretreated with Mor (200 µg/mL, 12 h) and incubated with E. coli O55, Enterococcus faecalis, Staphylococcus simulans, or Serratia liquefaciens, both E. coli O55 and Enterococcus faecalis showed a significant decrease in adhesion and invasion. Staphylococcus simulans exhibited decreased adhesion and increased invasion. Serratia liquefaciens showed increased adhesion and decreased invasion. In addition, Mor increased mRNA expression of antioxidant enzymes (e.g., heme oxygenase-1, NAD(P)H:quinone oxidoreductase-1, and thioredoxin reductase 1) in MAC-T cells. In conclusion, 12.5 to 50 mg/mL of Mor exhibited antibacterial activity against 18 out of 27 tested isolates. Also, pretreatment of 200 µg/mL of Mor to MAC-T cells modulated adhesion and invasion of E. coli O55 and other mastitis-associated pathogens. Furthermore, Mor increased antioxidant capacities in MAC-T cells, but further in vivo studies are needed.

Dynamics of the within-herd prevalence of Mycoplasma bovis intramammary infection in endemically infected dairy herds.

We aimed to identify the dynamics of the within-herd prevalence of Mycoplasma (M.) bovis intramammary infection (IMI) in four dairy herds, estimate prevalence of M. bovis in colostrum and clinical mastitis cases and compare M. bovis strains from calves' respiratory and cow clinical mastitis samples. Within a six-month study period, cow composite milk samples (CMS) were collected three times during routine milk recording, first milking colostrum samples from all calving cows and udder quarter milk samples from clinical mastitis cases. Calf respiratory samples were collected from calves with respiratory disease. Pooled milk samples were analysed for M. bovis with the Mastitis 4B polymerase chain reaction (PCR) test kit (DNA Diagnostic A/S). Prevalence estimates were calculated with Bayesian framework in R statistical programme. cg-MLST was used for M. bovis genotyping. In Herd I and II first testing M. bovis IMI within-herd prevalence (95 % credibility interval (CI)) was 4.7 % (2.9; 6.8) and 3.4 % (2.3; 4.6), changing to 1.0 % (0.1; 1.7) and 0.8 % (0.1; 1.4) in Herd I and 0.4 % (0.0; 0.7) in Herd II at the next samplings. In Herd III and IV first testing M. bovis IMI within-herd prevalence was 12.3 % (9.7; 15.2) and 7.8 % (6.2; 9.5), changing to 4.6 % (3.0; 6.4) and 3.2 % (1.9; 4.8) in Herd III and to 2.8 % (1.9; 3.8) and 4.9 % (3.6; 6.4) in Herd IV at the next samplings. The estimated prevalence of M. bovis in colostrum ranged between 1.7 % (0.2; 2.8) and 4.7 % (2.7; 7.1) and in clinical mastitis cases between 3.7 % (1.7; 6.4) and 11.0 % (7.5; 15.2) in the study herds. M. bovis strains isolated from cows and calves clustered within herds indicating possible transmission of M. bovis between dairy cows and calves. Prevalence of M. bovis in colostrum and clinical mastitis cases as well as the within-herd prevalence of M. bovis IMI was low in endemically infected dairy herds.

Safety and efficacy of a mesenchymal stem cell intramammary therapy in dairy cows with experimentally induced Staphylococcus aureus clinical mastitis.

Although, antibiotics are effective in the treatment of bovine mastitis, they do not address the regeneration of mammary glandular tissue and have been associated to the increment in antimicrobial resistance worldwide. Considering the necessity of alternative therapies for this disease of high economic impact and the reported regenerative and antibacterial effects of mesenchymal stem cell (MSCs), we evaluated the safety and efficacy of an allogenic MSC-based intramammary therapy in dairy cows with experimentally induced Staphylococcus aureus clinical mastitis. In a safety trial, heifers were inoculated intramammarily with a 2.5 × 107-suspension of bovine fetal AT-MSCs on experimental days 1 and 10. Animals were evaluated clinically on a daily basis during a 20-day experimental period and blood samples were collected for hemogram determination and peripheral blood leukocytes (PBLs) isolation. In an efficacy trial, Holstein Friesian cows were inoculated with S. aureus and treated intramammarily with vehicle (NEG; days 4 and 10), antibiotics (ATB; days 4 and 5) or a suspension of 2.5 × 107 AT-MSCs (MSC; days 4 and 5). Cows were clinically evaluated daily and milk samples were collected for somatic cell count (SCC) and colony forming units (CFU). Blood samples were collected for serum haptoglobin and amyloid A determination. Intramammary administration of two doses of bovine fetal AT-MSCs in healthy cows did not induce changes in clinical or hematological variables, and gene expression profiles in PBLs associated to activation (CD4, CD8, CD25, CD62L and CD69) and proinflammatory cytokines (CCL2, CCL5, IL2, CXCL3, IFNγ, and TNFα). Quarters of MSC group of cows had similar SCC log/mL in milk compared to infected quarters of ATB or NEG cows. However, quarters of MSC cows had lower CFU log/mL in milk compared to quarters of NEG cows. Intramammarily inoculation of repeated doses of 2.5 × 107 allogenic AT-MSCs did not induce clinical or immunological response in healthy cows. Moreover, MSC-intramammary treatment reduced bacterial count in milk of cows with S. aureus clinical mastitis compared to untreated cows. This work provides initial evidence for the safety and efficacy of an allogenic MSC-based intramammary therapy for the treatment of bovine mastitis.

Noninferiority field trial for evaluation of efficacy of ciprofloxacin associated with internal teat sealant as dry-off protocol.

The aim of this study was to compare the efficacy of two dry-off protocols: (a) dry cow therapy using ciprofloxacin hydrochloride 400 mg followed by the administration of an internal teat sealant composed of 4 g of bismuth subnitrate, and (b) a positive control using dry cow therapy with 250 mg cephalonium followed by the administration of 2.6 g bismuth subnitrate internal teat sealant. A total of 578 Holstein cows selected from 7 commercial herds were randomly allocated into two groups at drying off: (a) ciprofloxacin hydrochloride 400 mg (CH) associated with ITS, n = 1112 mammary quarters/296 cows, or (b) positive control (PC) = cephalonium (250 mg) associated with ITS, n = 1058 mammary quarters/282 cows). A total of 1787 out of 2170 mammary quarters (82%) had negative culture at drying off. The microorganisms most frequently isolated at drying off were CNS (5.62%), Strep. uberis (1.9%), Corynebacterium spp. (1.8%), and Staphylococcus aureus (1.01%). A total of 465 mammary quarters experienced new intramammary infections (NIMIs), and the main microorganisms causing NIMI were CNS (21.94%), Strep. uberis (17.2%), and Pseudomonas spp. (9.7%). The CH protocol was not inferior to PC, as the cure risk of mammary quarters CH-treated was at the noninferiority limit. However, the mammary quarters treated by CH protocol had 24% and 31% lower risk of overall NIMI and NIMI caused by major pathogens, respectively, than mammary quarters dried with the PC protocol. In addition, the mammary quarters treated with CH protocol had a lower risk of CM through the first 60 DIM than those treated with PC protocol. Both DCT protocols showed similar odds of microbiological cure, but the CH protocol had greater prevention against NIMI during dry-off period.

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.

Nanocurcumin ameliorates Staphylococcus aureus-induced mastitis in mouse by suppressing NF­κB signaling and inflammation.

Mastitis is the inflammation of the mammary glands caused by bacteria. It causes severe economic loss to dairy industry. Curcumin, a polyphenol obtained from turmeric, has considerable anti-inflammatory effect. Since it is rapidly eliminated from the body, its oral bioavailability is low. However, nanoformulation of curcumin significantly enhances its therapeutic efficiency by improving its oral bioavailability. We evaluated whether nanocurcumin could be more effective than normal curcumin against bovine Staphylococcus aureus mastitis in mouse model. Curcumin-loaded PLGA nanoparticles (CUR-NP) were prepared by solid-in-oil-in-water emulsion method. The mouse model of mastitis was induced by inoculation of a field strain of S. aureus (bovine mastitis isolate) on the 9th day of parturition through the duct of the mammary gland. CUR-NP and curcumin were given orally for 7 days (day 2 to day 8 of parturition) prior to S. aureus inoculation. We determined the levels of inflammatory cytokines and the mRNA expression of NF­κB. S. aureus infection increased the levels of tumor necrosis factor­α, interleukin­1ß and myeloperoxidase in mammary tissues and C-reactive protein in serum. Both CUR-NP and curcumin significantly attenuated the levels of these cytokines. However, comparatively, the ameliorative efficiency of CUR-NP was better than normal curcumin. S. aureus infection-induced NF­κB mRNA expression was significantly reduced to the healthy control level by CUR-NP. Our study demonstrates that the nanoformulation of curcumin can reduce pro-inflammatory mediators in S. aureus-infected mammary tissues by improving NF­κB signaling. Besides, compared to normal curcumin, this nanoformulation appears to be a better alternative against murine mastitis.

Short communication: Effect of 3 phytoceutical products on elimination of bacteria in experimentally induced Streptococcus uberis clinical mastitis.

Our objective was to assess the ability of 3 herbal products to eliminate experimentally induced Streptococcus uberis mastitis. These herbal products, also known as phytoceuticals, are used in organically managed dairy cattle to maintain or promote udder health. The products tested were an intramammary product, a topical product, and a product applied to the vulvar area. These products are not approved by the US Food and Drug Administration for treatment of mastitis but they are sold to enhance milk quality or for maintenance or improvement of udder health. Each of the products contains at least one component shown to have antibacterial activity. In this study, we successfully challenge-inoculated 25 lactating dairy cows maintained under organic conditions with an isolate of S. uberis. All challenged cows were positive for S. uberis by milk culture after challenge. When cows met predefined criteria indicating the presence of clinical mastitis, treatment with 1 of the 3 products was initiated based upon a predetermined random allocation. Culture of aseptically collected quarter milk samples was performed before, during, and following challenge with S. uberis. Eight, 8, and 9 cows received the intravulvar, intramammary, and topical treatments, respectively. Milk from all cows that were treated with phytoceuticals were culture-positive for S. uberis at every time point following treatment through 168 h following the last phytoceutical treatment. Based upon the presence of clinical signs and for humane reasons, 2 intravulvar-treated cows, 1 topical-treated, and 4 intramammary-treated cows received intramammary antibiotic therapy. We concluded that the phytoceuticals tested, as dosed and used in this trial, did not produce bacterial cures in S. uberis-induced mastitis.

Composition of the teat canal and intramammary microbiota of dairy cows subjected to antimicrobial dry cow therapy and internal teat sealant.

Antimicrobial dry cow therapy (DCT) is an important component of mastitis control programs aimed to eliminate existing intramammary infections and prevent the development of new ones during the dry period. However, to what extent the microbiota profiles of different niches of the udder change during the dry period and following administration of DCT remains poorly understood. Therefore, the main objective of the present study was to qualitatively evaluate dynamics of the microbiota of teat canal (TC) and mammary secretions (i.e., milk and colostrum) of healthy udder quarters subjected to DCT using a long-acting antimicrobial product, containing penicillin G and novobiocin, in combination with internal teat sealant. To this end, TC swabs (n = 58) and their corresponding milk (n = 29) and colostrum samples (n = 29) were collected at the time of drying off and immediately after calving from clinically healthy udder quarters of Holstein dairy cows from a commercial dairy farm. All samples were subjected to DNA extraction and high-throughput sequencing of the V1-V2 hypervariable regions of bacterial 16S rRNA genes. Overall, shifts were more pronounced within the microbiota of mammary secretions than the TC. In particular, microbiota of colostrum samples collected immediately after calving were less species-rich compared with the pre-DCT milk samples. Proportions of several bacterial genera belonging to the phylum Proteobacteria, including Pseudomonas, Stenotrophomonas, and unclassified Alcaligenaceae, were enriched within the microbiota of colostrum samples, whereas Firmicutes genera, including Butyrivibrio, unclassified Clostridiaceae, and unclassified Bacillales, were overrepresented in pre-DCT milk microbiota. Apart from shifts in the proportion of main bacterial genera and phyla, qualitative analysis revealed a high degree of commonality between pre-DCT and postpartum microbiota of both niches of the udder. Most importantly, a considerable number of bacterial genera and species commonly regarded as mastitis pathogens or opportunists (or both), including Staphylococcus spp., unclassified Enterobacteriaceae, and Corynebacterium spp., were shared between pre-DCT and postpartum microbiota of mammary secretions. Percentage of shared bacterial genera and species was even higher between pre-DCT and postpartum microbiota of TC samples, suggesting that the DCT approach of the present study had limited success in eliminating a considerable proportion of bacteria during the dry period.

Use of a culture-independent on-farm algorithm to guide the use of selective dry-cow antibiotic therapy.

An algorithm using only computer-based records to guide selective dry-cow therapy was evaluated at a New York State dairy farm via a randomized field trial. DairyComp 305 (Valley Ag Software, Tulare, CA) and Dairy Herd Improvement Association test-day data were used to identify cows as low risk (cows that might not benefit from dry-cow antibiotics) or high risk (cows that will likely benefit). Low-risk cows were those that had all of the following: somatic cell count (SCC) ≤200,000 cells/mL at last test, an average SCC ≤200,000 cells/mL over the last 3 tests, no signs of clinical mastitis at dry-off, and no more than 1 clinical mastitis event in the current lactation. Low-risk cows were randomly assigned to receive intramammary antibiotics and external teat sealant (ABXTS) or external teat sealant only (TS) at dry-off. Using pre-dry-off and postcalving quarter-level culture results, low-risk quarters were assessed for microbiological cure risk and new infection risk. Groups were also assessed for differences in first-test milk yield and linear scores, individual milk weights for the first 30 d, and culling and mastitis events before 30 d in milk. A total of 304 cows and 1,040 quarters in the ABXTS group and 307 cows and 1,058 quarters in the TS group were enrolled. Among cows to be dried, the proportion of cows that met low-risk criteria was 64% (n = 611/953). Of cultures eligible for bacteriological cure analysis (n = 171), 93% of ABXTS cured, whereas 88% of TS cured. Of the non-cures, 95% were contributed by the minor pathogens coagulase-negative staphylococci (n = 19/20). These organisms also accounted for 57.5% of new infections (n = 77/134). We found no statistical differences between treatment groups for new infection risk (TS = 7.3% quarters experiencing new infections; ABXTS = 5.5%), milk production (ABXTS = 40.5 kg; TS = 41.2 kg), linear scores (ABXTS = 2.5; TS = 2.7), culling events (ABXTS, n = 18; TS, n = 15), or clinical mastitis events (ABXTS, n = 9; TS, n = 5). Results suggest that the algorithm used decreased dry-cow antibiotic use by approximately 60% without adversely affecting production or health outcomes.

The influence of the rearing period on intramammary infections in Swiss dairy heifers: A cross-sectional study.

Healthy replacement heifers are one of the foundations of a healthy dairy herd. Farm management and rearing systems in Switzerland provide a wide variety of factors that could potentially be associated with intramammary infections (IMI) in early lactating dairy heifers. In this study, IMI with minor mastitis pathogens such as coagulase-negative staphylococci (CNS), contagious pathogens, and environmental major pathogens were identified. Fifty-four dairy farms were enrolled in the study. A questionnaire was used to collect herd level data on housing, management and welfare of young stock during farm visits and interviews with the farmers. Cow-level data such as breed, age at first calving, udder condition and swelling, and calving ease were also recorded. Data was also collected about young stock that spent a period of at least 3 months on an external rearing farm or on a seasonal alpine farm. At the quarter level, teat conditions such as teat lesions, teat dysfunction, presence of a papilloma and teat length were recorded. Within 24h after parturition, samples of colostral milk from 1564 quarters (391 heifers) were collected aseptically for bacterial culture. Positive bacteriological culture results were found in 49% of quarter samples. Potential risk factors for IMI were identified at the quarter, animal and herd level using multivariable and multilevel logistic regression analysis. At the herd level tie-stalls, and at cow-level the breed category "Brown cattle" were risk factors for IMI caused by contagious major pathogens such as Staphylococcus aureus (S. aureus). At the quarter-level, teat swelling and teat lesions were highly associated with IMI caused by environmental major pathogens. At the herd level heifer rearing at external farms was associated with less IMI caused by major environmental pathogens. Keeping pregnant heifers in a separate group was negatively associated with IMI caused by CNS. The odds of IMI with coagulase-negative staphylococci increased if weaning age was less than 4 months and if concentrates were fed to calves younger than 2 weeks. This study identified herd, cow- and quarter-level risk factors that may be important for IMI prevention in the future.

Defensin γ-thionin from Capsicum chinense has immunomodulatory effects on bovine mammary epithelial cells during Staphylococcus aureus internalization.

ß-Defensins are members of the antimicrobial peptide superfamily that are produced in various species from different kingdoms, including plants. Plant defensins exhibit primarily antifungal activities, unlike those from animals that exhibit a broad-spectrum antimicrobial action. Recently, immunomodulatory roles of mammal ß-defensins have been observed to regulate inflammation and activate the immune system. Similar roles for plant ß-defensins remain unknown. In addition, the regulation of the immune system by mammalian ß-defensins has been studied in humans and mice models, particularly in immune cells, but few studies have investigated these peptides in epithelial cells, which are in intimate contact with pathogens. The aim of this work was to evaluate the effect of the chemically synthesized ß-defensin γ-thionin from Capsicum chinense on the innate immune response of bovine mammary epithelial cells (bMECs) infected with Staphylococcus aureus, the primary pathogen responsible for bovine mastitis, which is capable of living within bMECs. Our results indicate that γ-thionin at 0.1 µg/ml was able to reduce the internalization of S. aureus into bMECs (∼50%), and it also modulates the innate immune response of these cells by inducing the mRNA expression (∼5-fold) and membrane abundance (∼3-fold) of Toll-like receptor 2 (TLR2), as well as by inducing genes coding for the pro-inflammatory cytokines TNF-α and IL-1ß (∼14 and 8-fold, respectively) before and after the bacterial infection. γ-Thionin also induces the expression of the mRNA of anti-inflammatory cytokine IL-10 (∼12-fold). Interestingly, the reduction in bacterial internalization coincides with the production of other antimicrobial products by bMECs, such as NO before infection, and the secretion into the medium of the endogenous antimicrobial peptide DEFB1 after infection. The results from this work support the potential use of ß-defensins from plants as immunomodulators of the mammalian innate immune response.

Somatic cell counts as an indicator of mammary infection in periparturient cows / Contagem de células somáticas como indicador de infecção mamária em vacas periparturientes

The somatic cell count (SCC) is a screening test for the evaluation of intramammary infection; however, changes in mammary secretion during colostrogenesis can promote a physiological increase in the SCC, potentially reducing its reliability in the diagnosis of mastitis. Therefore, the aim of this study was to evaluate bovine colostrum SCC as an indicative parameter of breast infection in Holstein periparturient cows. A total of 80 samples were harvested from the first milking colostrum of 20 cows and were subjected to manual SCC and bacteriological examination. Bacterial growth was present in 36.62% of the crops; coagulase-negative Staphylococcus (CNS) was the predominant microorganism (76.92%). The median SCC in infected cows (1.8 × 106 cells/mL) was significantly higher than in uninfected cows (0.9 × 106 cells/mL) (p = 0.0451). The sensitivity (10015%), specificity (1002.2%), and false positive (1002.2%) of the SCC decreased gradually when thresholds of 0.210.0 × 106 cells/mL were adopted. In contrast, the proportion of false negatives (084.6%) revealed an opposite trend. The threshold of greatest concordance between SCC and bacteriological examination was 10.0 × 106 cells/mL; however, the sensitivity rates (15.4%), specificity (2.2%), and false positive (2.2%) were very low. Based on these results, we conclude that SCCs increased prior to the infectious processes of the mammary gland, particularly in the CNS group. However, physiological changes caused by colostrogenesis resulted in poor concordance between the SCC and bacteriological examination of the colostrum...

A contagem de células somáticas (CCS) é um teste de triagem para avaliação de infecção intramamária, porém, as alterações na composição do colostro observadas durante a colostrogênese podem promover o aumento fisiológico da CCS e inviabilizar o uso desta prova para diagnóstico da mastite. Assim, o objetivo desta pesquisa foi avaliar a CCS do colostro como parâmetro indicativo de infecção mamária em vacas Holandesas periparturientes. Para tanto, foram colhidas 80 amostras de colostro de primeira ordenha, provenientes de 20 vacas, para a CCS manual e exame bacteriológico (EB). Observou-se crescimento bacteriano em 36,62% dos cultivos, com predomínio de Staphylococcus coagulase-negativa (SCN) (76,92%). O valor mediano da CCS de vacas infectadas (1,8 x106 células/mL) foi maior do que o obtido para as vacas sadias (0,9 x106 células/mL) (P=0,0451). A sensibilidade (100 a 15%), especificidade (100 a 2,2%) e falso-positivo (100 a 2,2%) diminuíram gradativamente quando os limiares de 0,2 a 10,0 x106 células/mL foram adotados. Em contrapartida, a proporção de falso-negativo (0 a 84,6%) apresentou perfil inverso. O limiar de maior concordância entre a CCS e EB foi de 10,0 x106 células/mL, porém os índices de sensibilidade (15,4%), especificidade (2,2%) e falso-positivo (2,2%) foram muito baixos. Com base nos resultados encontrados, conclui-se que a CCS apresentou elevação diante dos processos infecciosos da glândula mamária (GM), causados especialmente por bactérias do grupo SCN. No entanto, as alterações fisiológicas decorrentes da colostrogênese resultaram em baixa concordância entre a CCS e o exame bacteriológico do colostro...

Effects of Rhodomyrtus tomentosa Leaf Extract on Staphylococcal Adhesion and Invasion in Bovine Udder Epidermal Tissue Model.

Bovine mastitis is one of the most important infectious diseases in dairy herds, and staphylococci are the most important etiologic agents of this disease. Antibiotics and chemical agents used in livestock for prevention and cure of the disease can accumulate in milk and give rise to food safety concerns. Rhodomyrtus tomentosa leaf extract was studied as an alternative approach to reduce the bacterial infections. The ethanolic extract of this plant demonstrated antibacterial activity with minimum inhibitory concentration (MIC) values as low as 16-64 µg/mL against staphylococcal isolates. In addition, the extract had an effect on the bacterial cell surface properties by increasing its hydrophobicity in a concentration dependent manner. To further extend the antibacterial efficacy, silver nanoparticles synthesized with the extract, a pure rhodomyrtone, and liposomal encapsulated rhodomyrtone were applied and their inhibitory effects on bacterial adhesion and invasion were determined by ex vivo study in a bovine udder epidermal tissue model. These agents exerted remarkable antibacterial activity against staphylococci and decreased the adhesion of the bacterial cells to the tissues. These results supported that R. tomentosa ethanolic extract could be applied as an alternative agent for bovine udder care in dairy farms.

Development of intramammary delivery systems containing lasalocid for the treatment of bovine mastitis: impact of solubility improvement on safety, efficacy, and milk distribution in dairy cattle.

BACKGROUND: Mastitis is a major disease of dairy cattle. Given the recent emergence of methicillin-resistant Staphylococcus aureus as a cause of bovine mastitis, new intramammary (IMA) treatments are urgently required. Lasalocid, a member of the polyether ionophore class of antimicrobial agents, has not been previously administered to cows by the IMA route and has favorable characteristics for development as a mastitis treatment. This study aimed to develop an IMA drug delivery system (IMDS) of lasalocid for the treatment of bovine mastitis. METHODS: Minimum inhibitory concentrations (MICs) were determined applying the procedures recommended by the Clinical and Laboratory Standards Institute. Solid dispersions (SDs) of lasalocid were prepared and characterized using differential scanning calorimetry and Fourier transform infrared spectroscopy. IMDSs containing lasalocid of micronized, nano-sized, or as SD form were tested for their IMA safety in cows. Therapeutic efficacy of lasalocid IMDSs was tested in a bovine model involving experimental IMA challenge with the mastitis pathogen Streptococcus uberis. RESULTS: Lasalocid demonstrated antimicrobial activity against the major Gram-positive mastitis pathogens including S. aureus (MIC range 0.5-8 µg/mL). The solubility test confirmed limited, ion-strength-dependent water solubility of lasalocid. A kinetic solubility study showed that SDs effectively enhanced water solubility of lasalocid (21-35-fold). Polyvinylpyrrolidone (PVP)-lasalocid SD caused minimum mammary irritation in treated cows and exhibited faster distribution in milk than either nano or microsized lasalocid. IMDSs with PVP-lasalocid SD provided effective treatment with a higher mastitis clinical and microbiological cure rate (66.7%) compared to cloxacillin (62.5%). CONCLUSION: Lasalocid SD IMDS provided high cure rates and effectiveness in treating bovine mastitis with acceptable safety in treated cows.