Suppression of milk production during endotoxin-induced mastitis.

Healthy, midlactation cows were given intramammary infusions of 10 micrograms of endotoxin in two homolateral quarters. Productive, inflammatory, and systemic responses were studied to investigate the pathophysiological effects of mastitis on lactational performance. Endotoxin suppressed milk yield in all quarters of treated cows. A more severe and prolonged suppression occurred in infused quarters compared with uninfused quarters. The fat percentage of milk from all quarters was increased with a greater increase occurring in infused quarters. The protein composition of milk was elevated, and the lactose concentration was depressed in infused quarters. Mammary inflammation--as measured by milk SCC, NAGase, serum albumin, and lactoferrin--was limited to infused quarters. Changes in milk NAGase closely paralleled changes in milk SCC. Daily feed intake was unaffected, and serum glucose levels did not decline following infusion. The lactose concentration of urine increased rapidly after infusion. Reduction in milk yield in all quarters, but varying changes in milk composition in infused versus uninfused quarters suggest that mastitic hypogalactia is mediated by multiple pathophysiological events and is not solely due to inflammatory damage to the mammary epithelium. Part of the reduced lactational performance may result from escape of milk components from the udder into the circulation.     

Experimental challenge of bovine mammary glands with Enterococcus faecium during early and late lactation

Mammary glands of early and late lactation cows were challenged with Enterococcus faecium of bovine origin to determine in vivo pathogenicity and milk somatic cell count (SCC) responses. A total of 20 early lactation and 18 late lactation mammary glands were challenged. Two isolates highly adaptive and 2 isolates poorly adaptive for in vitro growth in mammary secretion were used as challenge strains of bacteria. Challenged quarters of early lactation cows were more susceptible to intramammary infection caused by E. faecium than those of late lactation cows. Intramammary challenge with isolates poorly adaptive for in vitro growth in mammary secretions resulted in 94.7% of quarters infected compared with 36.8% of the quarters infused with the isolates highly adaptive for in vitro growth in mammary secretions. Milk from quarters infused with the isolates poorly adaptive for in vitro growth had higher SCC and bacterial counts compared with quarters challenged with the isolates highly adaptive for in vitro growth. A stage of lactation effect within treatment groups was measured when milk SCC were compared between early and late lactation cows. Milk SCC in uninfused (negative control) quarters were lower in early lactation cows compared with late lactation cows. Conversely, in quarters infused with isolates poorly adaptive for in vitro growth, SCC were higher in early lactation cows compared with late lactation cows on d 2, 3, 4, 15, 16, and 17 postchallenge. In quarters infused with isolates highly adaptive for in vitro growth, SCC response did not differ between early and late lactation cows. In vitro growth of E. faecium in mammary secretion was inversely related to in vivo pathogenicity in the mammary glands of early and late lactation cows.     

Milk composition and health status from mammary gland quarters adjacent to glands affected with naturally occurring clinical mastitis

Mammary gland quarters have usually been considered to be anatomically and physiologically independent, but some recent research has indicated more interdependence than previously reported. The objective of this study was to compare milk composition (fat, total protein, lactose, solids-not-fat, and chloride) and health status (somatic cell count, differential leukocyte count, and lactate dehydrogenase) of milk samples from unaffected mammary glands of an udder with a single clinically inflamed quarter to results of milk samples from healthy mammary glands of healthy cows. The study was designed as a prospective case control study with case and control cows matched by parity and days in milk. Cases were defined as cows (n = 59) experiencing clinical mastitis in a single mammary gland, and controls (n = 59) were defined as cows that had not experienced clinical mastitis during the current lactation. Quarter milk samples were collected from all mammary glands adjacent to clinically affected quarters of cases and from the same mammary glands of controls. Samples were used to assess concentration of chloride and lactate dehydrogenase, fat, total protein, solids-not-fat, somatic cell count, and differential leukocyte count. Microbiological analysis was also performed on milk samples obtained from clinically affected mammary glands (n = 59). Logistic regression models were used to assess possible associations among quarter somatic cell count (≥150,000 cells/mL) and quarter type (adjacent to case or control). Multivariate linear models were used to compare milk composition and health status between quarter types. A total of 170 quarters were enrolled per group. Milk obtained from adjacent quarters of cases contained a lesser concentration of total protein, lactose, and solids-not-fat, but had a greater concentration of fat and chloride. The somatic cell count, total leukocyte count, and absolute numbers of neutrophils, lymphocytes, and macrophages were all increased in milk obtained from adjacent quarters of case cows compared with milk obtained from quarters of control cows. The relative proportion of neutrophils was increased, whereas the proportion of macrophages was decreased in milk obtained from cases. Approximately 30% of milk samples obtained from adjacent quarters of cases had a somatic cell count ≥150,000 cells/mL compared with 12% of milk samples obtained from quarters of control cows. The position of the mammary gland was not associated with any outcomes. In conclusion, our results support previous research that indicates the immune response to intramammary infection in a single mammary gland quarter alters milk composition and health status throughout the udder.     

Effects of local or systemic administration of meloxicam on mammary gland inflammatory responses to lipopolysaccharide-induced mastitis in dairy cows

Nonsteroidal anti-inflammatory drugs (NSAID) are commonly used in combination with antimicrobial mastitis treatments to reduce pain. Little is known about whether meloxicam, an NSAID designed for the preferential inhibition of cyclooxygenase-2 over cyclooxygenase-1, affects the mammary immune response. The objective of this study was to analyze the mammary immune response to intramammary (local) or intravenous (systemic) administration of meloxicam with or without immune activation by lipopolysaccharide (LPS). We challenged 108 quarters of 30 cows with or without a low or high dose of LPS from Escherichia coli (0.1 or 0.2 µg/quarter), with or without meloxicam via intramammary administration (50 mg/quarter) or intravenous injection (0.5 mg/kg of body weight; ~300 mg/cow). Intramammary administration of meloxicam alone did not trigger an acute inflammatory response, verified by unchanged somatic cell count (SCC) and lactate dehydrogenase (LDH), BSA, and IgG concentrations in milk, which are normally augmented during mastitis due to an opening of the blood–milk barrier. Similarly, intramammary meloxicam did not change the mRNA abundance of inflammatory factors in mammary gland tissue. As expected, quarters challenged with either dose of LPS showed increased leukocyte infiltration (SCC); increased LDH, BSA, IgG, Na, and Cl concentrations; and diminished K concentrations in milk. In contrast to our hypothesis, the addition of intramammary or intravenous meloxicam did not reduce these markers of mastitis in milk. Instead, intramammary meloxicam appeared to accelerate the SCC response to LPS, but only at the lower LPS dose. Moreover, the mRNA expression of inflammatory factors in mammary tissue was not modified by the intramammary application of meloxicam compared with the contralateral quarters that were challenged with LPS only. We demonstrated for the first time that intramammary meloxicam at a dose of 50 mg/quarter did not trigger an immune response in the mammary glands of dairy cows. At the doses we used, meloxicam (intramammary or systemic) did not lower inflammatory responses. The intramammary administration of meloxicam seemed to stimulate leukocyte recruitment into the milk in quarters challenged with a low dose of LPS. The integrity of the blood–milk barrier was not protected by meloxicam in LPS-stimulated quarters. This study provides the first indications that meloxicam does not limit the inflammatory response in the mammary gland, although it does not impair the mammary immune system.     

Pathological changes in bovine mammary glands following intramammary infusion of recombinant interleukin-2.

Eighteen lactating dairy cows were used to evaluate the physiological response of mammary glands to increasing doses of recombinant bovine interleukin-2. Right front and rear quarters were intramammarily infused with five different doses (.1 to 100 micrograms per quarter) of interleukin-2 as either a single or multiple treatment. Left front and rear quarters were intramammarily infused with a saline placebo and served as within-animal controls. Milk secretion samples for compositional analysis were collected from each quarter prior to infusion and at 12, 24, 36, and 48 h following infusion. Animals were slaughtered by exsanguination immediately following the 48-h sampling period, and mammary gland tissue was obtained for morphometric analysis. No changes in milk composition were observed between control quarters and those infused with up to 10 micrograms of interleukin-2 per quarter, administered as either a single or multiple treatment. Quarters infused with a single 100-micrograms dose of interleukin-2 or three consecutive doses of 25 and 100 micrograms of interleukin-2 had significantly lower lactose concentrations; there was a concomitant increase in bovine serum albumin, pH, and SCC compared with preinfusion concentrations or with control quarters. Morphometric analysis of tissue demonstrated an increase in stroma, a decrease in lumenal area, and a marked increase in the number of infiltrating leukocytes in those quarters infused with the higher doses of interleukin-2. Results suggest that interleukin-2 can be intrammammarily infused at doses as high as 10 micrograms per quarter without adversely affecting milk quality or normal mammary gland function.     

Lipopolysaccharide and lipoteichoic acid induce different immune responses in the bovine mammary gland

Different pathogens, such as Escherichia coli and Staphylococcus aureus, can be responsible for different outcomes of mastitis; that is, acute and severe or chronic and subclinical. These differences in the disease could be related to different mammary responses to the pathogens. The objective of this study was to determine if intramammary challenge with the endotoxins lipopolysaccharide (LPS), from E. coli, and lipoteichoic acid (LTA), from Staph. aureus, induce different immune responses in vivo in milk cells and mammary tissue. To provide a reference level for comparing the challenge and to show the different stimulation of the mammary immune system on a quantitatively similar level, dosages of LPS and LTA were chosen that induced an increase of somatic cells in milk to similar maxima. One udder quarter in each of 21 lactating dairy cows was challenged with 0.2 μg of LPS or 20 μg of LTA. From these quarters and from respective control quarters, milk cells or tissue biopsies were obtained at 0, 6, and 12 h relative to the challenge to measure mRNA expression of tumor necrosis factor-α (TNFα), IL-1β, IL-8, lactoferrin, and RANTES (regulated upon activation, normal T-cell expressed and secreted). Furthermore, if no biopsies were performed, hourly milk samples were taken for measurement of somatic cell count, lactate dehydrogenase (LDH), and TNFα. Somatic cell count increased in all treatments to similar maxima with LPS and LTA treatments. Concentrations of TNFα in milk increased with LPS but not with LTA. The activity of LDH in milk increased in both treatments and was more pronounced with LPS than with LTA. The mRNA expression of TNFα, IL-1β, IL-8, and RANTES showed increases in milk cells, and LPS was a stronger inducer than LTA. Lactoferrin mRNA expression decreased in milk cells with LPS and LTA treatments. The measured factors did not change in either treatment in mammary tissue. Challenge of udder quarters with dosages of LPS and LTA that induce similar increases in SCC stimulate the appearance of different immune factor patterns. This dissimilar response to LPS and LTA may partly explain the different course and intensity of mastitis after infection with E. coli and Staph. aureus, respectively.     

Haptoglobin concentrations in blood and milk after endotoxin challenge and quantification of mammary Hp mRNA expression

Haptoglobin (Hp), an acute phase protein mostly secreted by the liver, is an inflammatory marker. To use the full diagnostic potential of Hp measurements for mastitis, we developed and validated an ELISA sensitive to quantify even basal and subclinical concentrations in both blood and milk. Bovine Hp was purified from serum and was used as a standard and to generate polyclonal antiserum. The limit of detection was 0.07 microg of Hp/mL. From 6 cows challenged by intracisternal injection of lipopolysaccharide (LPS) into one quarter, blood samples were collected 0, 3, 6, 9, and 12 h after LPS administration. Milk samples from the treated and from the contralateral quarters were collected 0, 3, 6, 9, 12, 24, 36, 48, and 60 h after LPS administration. Haptoglobin concentrations in blood were increased above basal at 9 h, whereas milk Hp concentration increased 3 h after LPS administration. We therefore evaluated Hp mRNA synthesis within the mammary gland and specifically demonstrated Hp mRNA expression in parenchymal tissue, in tissue around the cisternal milk ducts and also in teat tissue by RT-PCR. Haptoglobin mRNA expression was then quantitatively evaluated by real-time RT-PCR in mammary biopsies collected from the treated and the control quarter before, and 3, 6, 9, and 12 h after LPS challenge from 6 other cows. Haptoglobin mRNA expression in the treated vs. the control quarters was different. The relation between mammary Hp expression and milk Hp concentrations needs further investigation, but the results suggest good diagnostic potential of this parameter for mastitis.     

Endotoxin mastitis in cows milked four times daily

As part of a project to identify the pathophysiological cause or causes of mastitic hypogalactia, midlactation cows were infused in two homolateral quarters with 10 micrograms of endotoxin while being milked four times daily to resolve better the temporal changes in mammary synthetic activity during endotoxin mastitis. Milk fat was decreased by the first milking (5 h) postinfusion and then recovered rapidly. In contrast, milk yield and the yields of protein and lactose were not significantly inhibited until the second milking, and these yields recovered slowly thereafter. The decline in milk yield by infused quarters was only 20% greater than the decline by uninfused quarters in this experiment. Mammary inflammation developed rapidly in infused quarters as milk serum albumin concentration was maximal at the first milking. Milk SCC and NAGase were also elevated at this time, and maximal levels occurred at milkings 2 to 4. Increased temperature, increased cortisol, and a mild anorexia were apparent at the first milking only. Endotoxin treatment had no effect on serum prolactin or glucose. These data suggest that the delayed hypogalactia is consequent to the mammary inflammation and systemic responses following endotoxin infusion. The results indicate that different pathophysiological events may inhibit synthesis of the different milk components.     

Ketoprofen affects the mammary immune response in dairy cows in vivo and in vitro

Nonsteroidal anti-inflammatory drugs are commonly administered parenterally in addition to antimicrobial mastitis therapy to increase the well-being of the diseased animal. As mastitis is usually a localized infection of mammary tissue, we tested the hypothesis that a local administration of nonsteroidal anti-inflammatory drugs through the teat canal could have anti-inflammatory effects on the affected area. We investigated the effects of intramammarily administered ketoprofen (KET) during an LPS-induced immune response on somatic cell count (SCC) and blood–milk barrier integrity. In addition, we investigated the effects of KET on the mRNA abundance of immune factors and their prostaglandin E2 secretion in primary bovine mammary epithelial cells in vitro. Six cows received 0.2 µg of LPS (serotype O26:B6) together with 50 mg of KET into one quarter and LPS only in the opposing quarter. The increase of SCC and of serum albumin (SA) and IgG concentrations and the increase of lactate dehydrogenase (LDH) activity in milk induced by LPS were lower in quarters that received KET in addition. In 3 cows, intramammary KET (50 mg) without additional LPS did not affect SCC, SA, IgG, and LDH in milk. Effects of KET on the immune response of mammary epithelial cells in vitro were investigated in cells from 3 cows challenged with or without LPS (0.2 µg/mL) and with or without additional KET in 2 concentrations (1.25 or 2.5 mg/mL). Ketoprofen reduced the LPS-induced increase of mRNA abundance of tumor necrosis factor α, IL-8, serum amyloid A, and cyclooxygenase-2. The mRNA abundance of cyclooxygenase-1 and prostaglandin E synthase was reduced in cells without LPS challenge by addition of KET at 2.5 mg/mL. Furthermore, the LPS-induced secretion of prostaglandin E2 of mammary epithelial cells into the supernatant could not be detected if KET was added. The results demonstrate that intramammary KET diminishes the increase of SCC and reduces the impairment of the blood–milk barrier (based on SA and LDH in milk), leading to a reduced IgG concentration in milk during LPS-induced mastitis. In mammary epithelial cells, KET limits the expression of several immune factors that are increased during an immune response. In summary, intramammary administration of KET reduces the inflammatory response in the mammary gland. However, it remains unclear whether the inhibited transfer of immune cells and IgG from blood into milk after KET administration would reduce the success of the immune defense in infectious mastitis.     

Effect of mastitis on milk perchlorate concentrations in dairy cows

Recent surveys have identified the presence of perchlorate, a natural compound and environmental contaminant, in forages and dairy milk. The ingestion of perchlorate is of concern because of its ability to competitively inhibit iodide uptake by the thyroid and to impair synthesis of thyroid hormones. A recent study established that milk perchlorate concentrations in cattle highly correlate with perchlorate intake. However, there is evidence that up to 80% of dietary perchlorate is metabolized in clinically healthy cows, thereby restricting the available transfer of ingested perchlorate into milk. The influence of mastitis on milk perchlorate levels, where there is an increase in mammary vascular permeability and an influx of blood-derived components into milk, remains unknown. The present study examined the effect of experimentally induced mastitis on milk perchlorate levels in cows receiving normal and perchlorate-supplemented diets. Over a 12-d period, cows were ruminally infused with 1 L/d of water or water containing 8 mg of perchlorate. Five days after the initiation of ruminal infusions, experimental mastitis was induced by the intramammary infusion of 100 μg of bacterial lipopolysaccharide (LPS). Contralateral quarters infused with phosphate-buffered saline served as controls. A significant reduction in milk perchlorate concentration was observed in the LPS-challenged glands of animals ruminally infused with either water or perchlorate. In control glands, milk perchlorate concentrations remained constant throughout the study. A strong negative correlation was identified between mammary vascular permeability and milk perchlorate concentrations in LPS-infused glands. These findings, in the context of a recently published study, suggest that an active transport process is operative in the establishment of a perchlorate concentration gradient across the blood-mammary gland interface, and that increases in mammary epithelial and vascular endothelial permeability lead to a net outflow of milk perchlorate. The overall finding that mastitis results in lower milk perchlorate concentrations suggests that changes in udder health do not necessitate increased screening of milk for perchlorate.     

Local and systemic response to intramammary lipopolysaccharide challenge during long-term manipulated plasma glucose and insulin concentrations in dairy cows

The metabolic load during periods of high milk production in dairy cows causes a variety of changes of metabolite blood concentrations including dramatically decreased glucose levels. These changes supposedly impair the immune system. The goal of this study was, therefore, to evaluate adaptations of the cow's immune system in response to an intramammary lipopolysaccharide (LPS) stimulation during a 3-d modification of plasma glucose and insulin induced by different clamp infusions. Seventeen midlactating dairy cows received a hypoglycemic hyperinsulinemic clamp induced by insulin infusion (HypoG; n=5), a euglycemic hyperinsulinemic clamp induced by insulin and glucose infusion (EuG; n=6), or infusion of saline solution (NaCl; n=6) for 56 h. At 48 h of infusion, 2 udder quarters were challenged with 200 μg of Escherichia coli LPS. At 48 h of infusion (immediately before LPS challenge), tumor necrosis factor α, lactoferrin, and serum amyloid A (SAA) mRNA abundance was increased in HypoG and Il-1β mRNA abundance was decreased in EuG. After LPS challenge, plasma glucose concentration did not decrease, although plasma insulin increased simultaneously in all groups either due to enhanced endogenous release (NaCl) or due to increased insulin infusion rate (HypoG; EuG). Plasma cortisol, rectal temperatures, and milk somatic cell count of challenged quarters increased, whereas plasma nonesterified fatty acid concentrations were similarly decreased across treatments. In mammary biopsies, increased mRNA expression of tumor necrosis factor α, IL-1β, IL-8, and IL-10, and SAA were observed in LPS-treated quarters of all groups, with a more pronounced increase in IL-1β, IL-10, and SAA expression in EuG. Nuclear factor-κB mRNA expression was upregulated in NaCl and EuG but not in HypoG in response to LPS. Lactoferrin, toll-like receptor 4, and cyclooxygenase-2 mRNA expression was increased in LPS-treated quarters of EuG only, and 5-lipoxygenase mRNA expression was decreased in LPS-treated quarters only in treatments HypoG and NaCl. In conclusion, intramammary LPS induces local and systemic inflammatory responses, as well as systemic insulin resistance. The observed treatment differences of the mammary mRNA expression of several immune parameters both before and after LPS challenge indicate a direct influence of changed glucose and insulin concentrations during the course of lactation on the immune defense against mastitis pathogens.     

Induced hyperketonemia affects the mammary immune response during lipopolysaccharide challenge in dairy cows

Metabolic adaptations during negative energy and nutrient balance in dairy cows are thought to cause impaired immune function and hence increased risk of infectious diseases, including mastitis. Characteristic adaptations mostly occurring in early lactation are an elevation of plasma ketone bodies and free fatty acids (nonesterified fatty acids, NEFA) and diminished glucose concentration. The aim of this study was to investigate effects of elevated plasma β-hydroxybutyrate (BHBA) at simultaneously even or positive energy balance and thus normal plasma NEFA and glucose on factors related to the immune system in liver and mammary gland of dairy cows. In addition, we investigated the effect of elevated plasma BHBA and intramammary lipopolysaccharide (LPS) challenge on the mammary immune response. Thirteen dairy cows were infused either with BHBA (HyperB, n = 5) to induce hyperketonemia (1.7 mmol/L) or with a 0.9% saline solution (NaCl, n = 8) for 56 h. Two udder quarters were injected with 200 μg of LPS after 48 h of infusion. Rectal temperature (RT) and somatic cell counts (SCC) were measured before, at 48 h after the start of infusions, and hourly during the LPS challenge. The mRNA abundance of factors related to the immune system was measured in hepatic and mammary tissue biopsies 1 wk before and 48 h after the start of the infusion, and additionally in mammary tissue at 56 h of infusion (8 h after LPS administration). At 48 h of infusion in HyperB, the mRNA abundance of serum amyloid A (SAA) in the mammary gland was increased and that of haptoglobin (Hp) tended to be increased. Rectal temperature, SCC, and mRNA abundance of candidate genes in the liver were not affected by the BHBA infusion until 48 h. During the following LPS challenge, RT and SCC increased in both groups. However, SCC increased less in HyperB than in NaCl. Quarters infused with LPS showed a more pronounced increase of mRNA abundance of IL-8 and IL-10 in HyperB than in NaCl. The results demonstrate that an increase of plasma BHBA upregulates acute phase proteins in the mammary gland. In response to intramammary LPS challenge, elevated BHBA diminishes the influx of leukocytes from blood into milk, perhaps by via modified cytokine synthesis. Results indicate that increased ketone body plasma concentrations may play a crucial role in the higher mastitis susceptibility in early lactation.     

Decline in mammary translational capacity during intravenous glucose infusion into lactating dairy cows

The objective of this study was to determine effects of glucose on milk protein yield and mammary mammalian target of rapamycin (mTOR) activity in dairy cattle in early lactation. Eight multiparous cows at 73 ± 8 d in milk were randomly assigned to 2 treatments in a crossover design for two 6-d periods. Treatments were jugular infusion of either saline (Sal) or 896 g/d glucose (Glc). All cows were fed a total mixed ration with 42% neutral detergent fiber, had free access to water, and were milked twice a day. Within each period, blood samples were taken (d 5) and mammary tissue was collected by biopsy (d 6) from each hindquarter for Western blot analysis. In addition to Sal and Glc treatments, on d 6, rapamycin dissolved in 50% dimethyl sulfoxide was administered via the teat canals into the left quarters, with a control solution administered into the right quarters. Rapamycin had no effect on milk protein yields or phosphorylation state of mTOR signaling proteins. Infusions of Glc significantly increased milk yield but only tended to increase milk protein yields. Milk fat tended to be decreased in cows infused with Glc, whereas lactose yields were significantly increased. Glucose infusion did not increase plasma glucose levels, but insulin and nonessential AA concentrations increased by 21 and 16%, respectively, branched-chain AA concentrations decreased 24%, and essential AA concentrations tended to decrease by 14%. Infusion of Glc significantly decreased abundances of both phosphorylated and total ribosomal S6 kinase 1 (S6K1) in mammary tissue by 27 and 11%, respectively. Abundance of phosphorylated eukaryotic initiation factor 4E-binding protein 1 (4EBP1) decreased significantly by 25%, whereas total 4EBP1 exhibited a tendency to decrease by 16%. We conclude that the mTOR signaling pathway is not the only regulator of milk protein synthesis. Decreases in essential AA concentrations in plasma suggest that protein synthesis was stimulated in nonmammary tissues of the body, presumably skeletal muscle.     

Effects of intramammary infusions of casein hydrolysate,ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid,and lactose at drying-off on mammary gland involution

The transition from the lactation to the dry period in dairy cows is a period of high risk for acquiring new intramammary infections. This risk is reduced when involution of mammary glands is completed. Consequently, strategies that accelerate the involution process after drying-off could reduce the incidence of mastitis. The objective of this study was to assess the effect of 3 different treatments on mammary gland involution. Each quarter of 8 Holstein cows in late lactation was randomly assigned at drying-off to an intramammary infusion of casein hydrolysate (CNH; 70 mg), ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA; 5.7 g), lactose (5.1 g), or saline 0.9% (control) solutions. Milk samples were collected on the last 2 d before and 1, 3, 5, 7, 10, and 14 d after the last milking for determining concentrations of mammary gland involution markers. Lactoferrin, somatic cell counts (SCC), BSA, and Na⁺ concentrations, as well as matrix metalloproteinase-2 and matrix metalloproteinase-9 activities gradually increased in mammary secretions during the first 2 wk following the last milking, whereas milk citrate and K⁺ concentrations decreased. As involution advanced, the Na⁺:K⁺ ratio increased, whereas the citrate:lactoferrin ratio decreased. Compared with mammary secretions from control quarters, mammary secretions of quarters infused with CNH had higher SCC on d 1, 3, 5, and 7, and greater BSA concentrations on d 1, 3, and 5. Similarly, the CNH treatment induced a faster increase in lactoferrin concentrations, which were greater than in milk from control quarters on d 3, 5, and 7 after drying-off. Milk citrate concentrations were unaffected by CNH but the citrate:lactoferrin ratio was lower in CNH-treated quarters on d 3 and 5 than in control quarters. Moreover, CNH treatment hastened the increase in Na⁺ concentration and in the Na⁺:K⁺ ratio on d 1. Infusion of CNH also led to an increase in proteolytic activities, with greater matrix metalloproteinase 9 activities on d 1 and 3. The EGTA infusion increased SCC above that of control quarters on d 1 and 3 but it had no effect on the other parameters. Lactose infusion had no effect on any of the involution markers. In this study, intramammary infusions of CNH were the most efficient treatment to accelerate mammary gland involution, suggesting a potential role of CNH as a local milk secretion inhibitor during milk stasis.     

Acute experimental mastitis is not causal toward the development of energy-related metabolic disorders in early postpartum dairy cows

Twenty Holstein cows in early lactation (7 d in milk) were administered 100 μg of Escherichia coli lipopolysaccharide (LPS) dissolved in 10 mL of sterile 0.9% NaCl saline (treatment; TRT) or 10 mL of sterile saline (control) into both right mammary quarters to test the hypothesis that acute experimental mastitis would have negative impacts on aspects of energy metabolism that might lead to the development of metabolic disorders. A primed continuous intravenous infusion (14-μmol/kg of BW priming dose; 11.5-μmol/kg of BW per h continuous infusion) of 6,6-dideuterated glucose was used to determine pre- and posttreatment glucose kinetics using steady-state tracer methodologies. The LPS-treated cows displayed productive, clinical, and physiological signs of moderate to severe inflammation; control cows displayed no signs of immune activation. Pretreatment glucose rates of appearance (Ra) into plasma were similar (715 and 662 ± 33 mmol/h for TRT and control, respectively) between treatment groups. Intramammary LPS infusion into TRT cows resulted in increased glucose Ra relative to control cows (mean glucose Ra from 150 through 270 min after intramammary infusion were 815 and 674 ± 21 mmol/h for TRT and control cows, respectively). Furthermore, plasma concentrations of glucose increased, whereas plasma nonesterified fatty acids, glycerol, and β-hydroxybutyrate concentrations decreased, in TRT relative to control cows. Interestingly, plasma insulin concentration increased dramatically in TRT cows and occurred prior to the small increase in plasma glucose concentration. Although these results only represent the early stages of inflammation, they are not consistent with a causal relationship between mastitis and energy-related metabolic disorders and instead suggest a coordinated protective effect by the immune system on metabolism during the early stages of mammary insult.     

Effects of different nutrient supply on metabolism and mammary immune response to an LPS challenge in early lactation of dairy cows

Energy and nutrient deficiency in dairy cows in early lactation is considered to contribute to their increased susceptibility to mastitis. We have tested the hypothesis that feeding diets with high contents of either nitrogenic, glucogenic, or lipogenic components in early lactation affects both the endocrine and metabolic status, as well as the mammary immune competence. After calving, cows were fed increasing amounts of concentrate up to 10 kg/d rich in crude protein (nitrogenic, n = 10), glucogenic precursors (glucogenic, n = 11), or lipids (lipogenic, n = 11). In wk 3, one udder quarter was challenged with lipopolysaccharide (LPS) from Escherichia coli. Blood and milk were sampled on the day before LPS challenge (d −1), and on d 0, 1, 2, 3, and 9 after LPS challenge. On the day of LPS challenge additional samples were taken hourly for quarter milk and every 3 h for blood. Urea concentrations were higher in plasma and milk of cows fed the nitrogenic diet. However, plasma concentrations of glucose, cholesterol, triglycerides, β-hydroxybutyrate, nonesterified fatty acids, as well as insulin, glucagon, and insulin-like growth factor-1 were not affected by the different diets. The mammary immune challenge induced a substantial increase of somatic cell count (SCC) in the treated quarter, and a transient decrease of total milk yield and white blood cells similar in all diet groups for one day. The absolute phagocytosis of blood leukocytes was decreased; however, the phagocytosis per cell was increased in glucogenic-fed cows at 6 h after LPS challenge. During mammary inflammation an insulin resistance, shown by increased plasma glucose, insulin, and glucagon, developed similarly in all diet groups. β-hydroxybutyrate and nonesterified fatty acids were decreased at 1 d after LPS challenge in glucogenic-fed cows only. Cholesterol did not change, and triglycerides only decreased significantly in lipogenic-fed cows 6 h after challenge. On d 9 after LPS challenge, SCC and milk yield and metabolic factors were recovered in all groups. In conclusion, the endocrine and metabolic situation, and the immune response to intramammary LPS of dairy cows during early lactation was not substantially influenced by the elevated supply of nitrogenic, glucogenic, or lipogenic components due to the provided feed in this study.     

Increased levels of LPS-binding protein in bovine blood and milk following bacterial lipopolysaccharide challenge

Several species of gram-negative bacteria, including Escherichia coli, Klebsiella pneumoniae, and various species of Enterobacter, are common mastitis pathogens. All of these bacteria are characterized by the presence of endotoxin or lipopolysaccharide (LPS) in their outer membrane. The bovine mammary gland is highly sensitive to LPS, and LPS has been implicated, in part, in the pathogenesis of gram-negative mastitis. Recognition of LPS is a key event in the innate immune response to gram-negative infection and is mediated by the accessory molecules CD14 and LPS-binding protein (LBP). The objective of the current study was to determine whether LBP levels increased in the blood and mammary gland following LPS challenge. The left and right quarters of five midlactating Holstein cows were challenged with either saline or LPS (100 microg), respectively, and milk and blood samples collected. Basal levels of plasma and milk LBP were 38 and 6 microg/ml, respectively. Plasma LBP levels increased as early as 8 h post-LPS challenge and reached maximal levels of 138 microg/ ml by 24 h. Analysis of whey samples derived from LPS-treated quarters revealed an increase in milk LBP by 12 h. Similar to plasma, maximal levels of milk LBP (34 microg/ml) were detected 24 h following the initial LPS challenge. Increments in milk LBP levels paralleled a rise in soluble CD14 (sCD14) levels and initial rises in the levels of these proteins were temporally coincident with maximal neutrophil recruitment to the inflamed gland. Because LBP and sCD14 are known to enhance LPS-induced host cell activation and to facilitate detoxification of LPS, these data are consistent with a role for these molecules in mediating mammary gland responses to LPS.     

Deferoxamine reduces tissue damage during endotoxin-induced mastitis in dairy cows

The protective effects of 3 antioxidants on polymorphonuclear neutrophil-induced damage to mammary cells were evaluated in vivo using an endotoxin-induced mastitis model. Fifteen healthy, midlactation cows with no history of clinical Escherichia coli mastitis were randomly assigned to 1 of the 3 treatment groups corresponding to each modulator to be evaluated, that is, deferoxamine, catechin, and glutathione ethyl ester. Each cow had 1 quarter infused with saline and 1 quarter infused with the selected modulator; a third quarter was infused with lipopolysaccharides (LPS), whereas the fourth quarter received a combination of LPS and the modulator. Infusion of LPS caused acute mastitis as determined by visual observations and by large increases in milk somatic cell count, BSA, and proteolytic activity. These parameters were not affected by antioxidant administration. The extent of cell damage was evaluated by measuring milk levels of lactate dehydrogenase and N-acetyl-β-_D-glucosaminidase activity. Levels of these parameters were several times higher after LPS administration. Intramammary infusions of catechin or glutathione ethyl ester did not exert any protective effect, whereas infusion of deferoxamine, a chelator of iron, decreased milk lactate dehydrogenase and NA-Gase activity, suggesting a protective effect against neutrophil-induced damage. The protective effect of deferoxamine was also evidenced by a lower milk level of haptoglobin. The proteolytic activity of mastitic milk was not influenced by the presence of deferoxamine. Overall, our results suggest that local infusion of deferoxamine may be an effective tool to protect mammary tissue against neutrophil-induced oxidative stress during bovine mastitis.     

Whole-body glucose metabolism and mammary energetic nutrient metabolism in lactating dairy cows receiving digestive infusions of casein and propionic acid

This study analyzed the effect of propionate (C3) and casein (CN) on whole-body and mammary metabolism of energetic nutrients. Three multiparous Holstein cows fitted with both duodenal and ruminal cannulas were used in 2 replicated Youden squares with 14-d periods. Effects of CN (743 g/d in the duodenum) and C3 (1,042 g/d in the rumen) infusions, either separately or in combination as supplements to a grass silage diet, were tested in a factorial arrangement. The control diet provided 97% of energy and protein requirements. Within each period, blood samples were taken (d 11) from the carotid artery and the right mammary vein to determine net uptake of energetic nutrients. Plasma blood flow was calculated using the Fick principle (based on Phe and Tyr). On d 13, [6,6-²H₂]glucose was infused in the jugular vein to determine whole-body glucose rate of appearance (Ra) based on enrichments in arterial plasma. Both C3 and CN treatments increased whole-body Ra (17% and 13%, respectively) but only CN increased milk (18%) and lactose (14%) yields, suggesting no direct link between whole-body Ra and milk yield. When CN was infused alone, the apparent ratio of conversion of CN carbon into glucose carbon was 0.31 but, when allowance was made for the CN required to support the extra milk protein output, the ratio increased to 0.40, closer to the theoretical ratio (0.48). This may relate to the observed increases in arterial glucagon concentrations for CN alone. Conversely, the apparent conversion of infused C3 carbon alone to glucose was low (0.31). With C3, mammary plasma flow increased as did uptakes of lactate, Ala, and Glu whereas the uptake for β-hydroxybutyrate (BHBA) decreased. Mammary net carbon balance suggested an increase with C3 treatment in glucose, lactate, Ala, and Glu oxidation within the mammary gland. Mammary glucose uptake did not increase with CN treatment, despite an increase in glucose arteriovenous difference and extraction rate, because plasma flow decreased (−17%). Whereas CN, alone or in combination with C3, increased both lactose and protein yields, only mammary AA (and BHBA in CN alone) uptake increased because plasma flow decreased (−17%). These data suggest that the observed variations of milk lactose yield (and other milk components) are linked to metabolic interchanges between several energetic nutrients at both the whole-body and mammary levels and are not explained by increases in whole-body glucose availability.     

Variation in the composition of selected milk fraction samples from healthy and mastitic quarters, and its significance for mastitis diagnosis

Seven variables--electrical conductivity (EC), somatic cell count (SCC), N-acetyl-beta-D-glucosaminidase (NAGase), lactose, protein, fat and pH--were compared in four quarter milk fractions (MF1: strict foremilk; MF2: first 12-15 ml foremilk; MF3: subsequent 40-45 ml milk; MF4: strippings) and in one cow composite milk sample (CC) per cow. The study used 142 quarters from 37 lactating cows of the German Black Pied breed. To rule out any possible effect due to management, animal physiology and analytical procedures, the collection and processing of milk samples from each cow was repeated for three consecutive days, and the means of 3-d values were used. All variables were affected significantly by milk fraction and udder health. Compared with foremilk, EC, lactose and protein levels in strippings decreased, while SCC, NAGase and fat increased. The pH of foremilk and strippings did not differ significantly in healthy or in mastitic quarters. The difference between MF1 and MF2 was significant for EC in mastitic quarters, and for SCC in healthy quarters only. In general, mastitis resulted in a significant increase in EC, SCC, NAGase and protein but in a decrease in lactose and fat contents of milk in one or more of the milk fractions studied. Comparison of cow composite milk samples from healthy and mastitic cows revealed the significance (P < 0.01) of udder health for EC, SCC and lactose. Of the different parameters that can distinguish between healthy and mastitic quarters or cows, EC could be used to classify 76% of quarters and 73% of cows correctly, while the lactose content permitted correct identification of 81% of quarters and 76% of cows. NAGase and pH could be used to determine the status of 73% and 61% of quarters, respectively. In general, the correlation observed in strippings was higher than in foremilk for almost all the variables studied. Surprisingly, EC, SCC, NAGase and lactose in milk from healthy quarters of mastitic cows (with at least one mastitic quarter) differed significantly (P < 0.05) from those from healthy quarters of cows with all four healthy quarters, indicating an inconsistent effect of mastitic quarters on neighbouring healthy quarters (quarter interdependence).