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.     

Hyperketonemia during lipopolysaccharide-induced mastitis affects systemic and local intramammary metabolism in dairy cows

Hyperketonemia interferes with the metabolic regulation in dairy cows. It is assumed that metabolic and endocrine changes during hyperketonemia also affect metabolic adaptations during inflammatory processes. We therefore studied systemic and local intramammary effects of elevated plasma β-hydroxybutyrate (BHBA) before and during the response to an intramammary lipopolysaccharide (LPS) challenge. Thirteen dairy cows received intravenously either a Na-dl-β-OH-butyrate infusion (n = 5) to achieve a constant plasma BHBA concentration (1.7 ± 0.1 mmol/L), with adjustments of the infusion rates made based on immediate measurements of plasma BHBA every 15 min, or an infusion with a 0.9% NaCl solution (control; n = 8) for 56 h. Infusions started at 0900 h on d 1 and continued until 1700 h 2 d later. Two udder quarters were challenged with 200 μg of Escherichia coli LPS and 2 udder quarters were treated with 0.9% saline solution as control quarters at 48 h after the start of infusion. Blood samples were taken at 1 wk and 2 h before the start of infusions as reference samples and hourly during the infusion. Mammary gland biopsies were taken 1 wk before, and 48 and 56 h (8 h after LPS challenge) after the start of infusions. The mRNA abundance of key factors related to BHBA and fatty acid metabolism, and glucose transporters was determined in mammary tissue biopsies. Blood samples were analyzed for plasma glucose, BHBA, nonesterified fatty acid, urea, insulin, glucagon, and cortisol concentrations. Differences were not different for effects of BHBA infusion on the mRNA abundance of any of the measured target genes in the mammary gland before LPS challenge. Intramammary LPS challenge increased plasma glucose, cortisol, glucagon, and insulin concentrations in both groups but increases in plasma glucose and glucagon concentration were less pronounced in the Na-dl-β-OH-butyrate infusion group than in controls. In response to LPS challenge, plasma BHBA concentration decreased in controls and decreased also slightly in the BHBA-infused animals because the BHBA concentration could not be fully maintained despite a rapid increase in BHBA infusion rate. The change in mRNA abundance of citrate synthase in LPS quarters was significant between the 2 treatment groups. The results indicate that elevated circulating BHBA concentration inhibits gluconeogenesis before and during immune response to LPS challenge, likely because BHBA can replace glucose as an energy source.     

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.     

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.     

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.     

Eucalcemia during lipopolysaccharide challenge in postpartum dairy cows: I. Clinical,inflammatory, and metabolic response

Hypocalcemia induced by immune activation is a conserved response across mammalian species; however, administration of Ca is discouraged in other species as it is associated with increased morbidity and mortality. Early postpartum cows experience a decrease in circulating Ca concentration following acute inflammation. Corrective Ca therapy during the transition period, particularly in dairy cows experiencing acute disease, is common practice. However, the effect of Ca administration on the inflammatory response during acute immune activation is unknown. Our objective was to compare the clinical, inflammatory, and metabolic response to an intravenous (IV) lipopolysaccharide (LPS) challenge between postpartum cows infused, or not, with IV Ca to maintain eucalcemia. Cows (n = 14, 8 ± 1 d in milk) were enrolled in a matched-pair randomized controlled design to receive IV Ca (IVCa) or sterile 0.9% NaCl (CTRL) during an IV LPS challenge (0.040 or 0.045 µg of LPS/kg of body weight over 1 h). Ionized Ca (iCa) was monitored cow-side, and IV Ca infusion was adjusted in a eucalcemic clamp for 12 h following the start of LPS infusion. Cows were monitored during the 24 h following challenge and serial blood samples were collected to quantify concentrations of glucose, β-hydroxybutyrate, nonesterified fatty acids, urea nitrogen, cytokines, acute-phase proteins, and cortisol. Blood iCa concentration decreased to 0.87 ± 0.03 mM in CTRL during challenge, and by design, iCa concentration was maintained within 3% of baseline in IVCa. Body temperature, heart rate, and respiratory rate were monitored for 24 h following the start of challenge and did not differ between groups. A treatment × time interaction was identified such that serum cortisol concentrations increased in both groups at 2 h but decreased to a greater extent at 6 h in IVCa compared with CTRL. Rumination time (min/h) over the first 12 h following challenge was greater in IVCa, but total rumination time in the 24 h following challenge did not differ from CTRL. Serum glucose and nonesterified fatty acid concentrations decreased, and β-hydroxybutyrate and urea nitrogen concentrations increased over time, but did not differ between groups. Acute leukopenia occurred in both groups at 4 h before leukocytosis was observed at 24 h with total white blood cell counts returning to baseline within 72 h. Plasma concentrations of tumor necrosis factor (TNF) and interleukin-10 (IL-10) increased within 1 h following the start of challenge and did not differ between groups. Serum haptoglobin and serum amyloid A concentrations increased within the 24 h following challenge and were elevated through 72 h but did not differ between groups. Eucalcemia during the acute systemic inflammatory response did not alter the TNF or IL-10 cytokine response, or the acute-phase protein SAA and haptoglobin response in this LPS challenge model; however, eucalcemia was associated with a more rapid decline in cortisol response and greater rumination time in the first 12 h following challenge. We did not find evidence that eucalcemia exacerbated the inflammatory response in early postpartum cows, but Ca administration may alter the clinical response to acute systemic inflammation.     

Serum concentration and mRNA expression in milk somatic cells of toll-like receptor 2, toll-like receptor 4, and cytokines in dairy cows following intramammary inoculation with Escherichia coli

The objective of the current study was to investigate the toll-like receptors (TLR), including the soluble forms sTLR2 and sTLR4, involved in innate immune responses of dairy cows to experimentally induced Escherichia coli mastitis. Six clinically healthy Holstein dairy cows received an intramammary inoculation of E. coli O111:K58 between 63 and 83 d postpartum. Concentrations of sTLR2 and sTLR4, the proinflammatory cytokines IL-6 and tumor necrosis factor-α (TNF-α), and acute phase proteins serum amyloid A (SAA) and haptoglobin (Hp) in blood were measured by ELISA. Furthermore, 10 mL of milk was collected from challenged quarters immediately before inoculation and at 6, 12, 24, 48, and 72 h after inoculation, and mRNA expression of selected genes, including TLR2, TLR4, IL-1β, IL-6, TNF-α, and IL-8, was quantified by real-time PCR. Escherichia coli intramammary infection elicited a decrease in the circulating levels of leukocytes. Rectal temperature was elevated at 6 h postinoculation (PI). Similarly, the serum concentrations of TNF-α, IL-6, and SAA increased at 6 h PI. However, serum concentrations of sTLR2, sTLR4, and Hp did not differ after challenge. The mRNA expression of TLR2, IL-1β, and IL-8 in milk somatic cells increased at 12 h PI, whereas a decreased IL-6 mRNA expression was detected from 6 to 48 h PI. In conclusion, we found that TLR2 mRNA expression increased in milk somatic cells collected from infected quarters of cows challenged with E. coli, whereas the concentrations of sTLR2 and sTLR4 remained unchanged after challenge. Thus, sTLR2 and sTLR4 may protect the host by sequestrating pathogen-associated molecular patterns during E. coli mastitis.     

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.     

Glucose requirements of an activated immune system in lactating Holstein cows

Accurately quantifying activated immune system energy requirements in vivo is difficult, but a better understanding may advance strategies to maximize animal productivity. Study objectives were to estimate whole-body glucose utilization following an i.v. endotoxin challenge. Lactating Holstein cows were jugular catheterized and assigned 1 of 3 bolus treatments: control (CON; 5 mL of saline; n = 6), lipopolysaccharide (LPS)-administered (LPS-C; 1.5 μg/kg of body weight; Escherichia coli 055:B5; n = 6), and LPS + euglycemic clamp (LPS-Eu; 1.5 μg/kg of body weight; 50% glucose solution infusion; n = 6). After LPS administration, blood glucose was determined every 10 min and glucose infusion rates were adjusted in LPS-Eu cows to maintain euglycemia for 720 min. Blood samples were obtained 180, 360, 540, and 720 min postbolus for further analysis. Cows were milked 360 and 720 min postbolus. Blood glucose was increased 84% in LPS-administered cows for up to 150 min postbolus; thereafter, circulating glucose was decreased 30% in LPS-C relative to LPS-Eu and CON cows. Mild hyperthermia (+0.5°C) occurred between 30 and 90 min postbolus in LPS-administered relative to CON cows; thereafter, rectal temperature did not differ between treatments. Milk yield and lactose percentage were decreased 80 and 11%, respectively, in LPS-administered relative to CON cows. Circulating insulin was increased 4 fold and nonesterified fatty acids, β-hydroxybutyrate, and ionized Ca were decreased ～50% in LPS-administered compared with CON cows. Plasma l-lactate, haptoglobin, and serum amyloid A increased ～160, 260, and 75%, respectively, in LPS-administered relative to CON cows. Overall, LPS-binding protein was increased 87% in LPS-administered relative to CON cows; however, at 720 min, it was decreased 25% in LPS-Eu compared with LPS-C cows. White blood cell count decreased ～90% in LPS-administered cows at 180 min and progressively increased to ～50% of CON values by 720 min. Total glucose deficit during the 720 min following LPS administration was calculated as the decrease in the amount of glucose required to synthesize milk (due to the decrease in milk yield relative to prebolus levels) plus the amount of glucose infused to maintain euglycemia (in LPS-Eu cows only) and was 461, 1,259, and 1,553 g for CON, LPS-C, and LPS-Eu cows, respectively. Our data indicate an acutely activated immune system uses >1 kg of glucose within 720 min and maintaining euglycemia did not rescue milk synthesis.     

Regulation of messenger ribonucleic acid expression for gluconeogenic enzymes during glucagon infusions into lactating cows.

The effects of glucagon infusions on expression of mRNA for enzymes that regulate gluconeogenesis were studied in lactating cows. Normal cows and cows with fatty liver that were susceptible to ketosis were assigned to either glucagon-treated or control groups. Glucagon at 0 or 10 mg/d was infused for 14 d beginning at d 21 postpartum. In normal cows, glucagon infusions increased concentrations of both plasma glucagon and glucose, which caused plasma insulin to increase. Consequently, hepatic phosphoenolpyruvate carboxykinase mRNA decreased during wk 1 of glucagon infusions. Glucagon infusions into cows with fatty liver also increased plasma glucagon and glucose, but concentrations of plasma insulin and hepatic phosphoenolpyruvate carboxykinase mRNA did not change. More phosphoenolpyruvate carboxykinase mRNA was present in the livers of cows with fatty liver than in livers of normal cows. In a follow-up experiment with midlactation cows, 3.5-h infusions of glucagon at 14 mg/d increased plasma glucose and insulin and decreased plasma nonesterified fatty acids and hepatic glycogen. Hepatic phosphoenolpyruvate carboxykinase mRNA was decreased 41%, pyruvate carboxylase mRNA was increased 50%, but fructose-1,6-bisphosphatase mRNA did not change. We conclude that the expression of the hepatic phosphoenolpyruvate carboxykinase gene in normal cows is inhibited by insulin to balance elevated carbohydrate status during glucagon infusions; however, inhibited expression of hepatic phosphoenolpyruvate carboxykinase mRNA probably is not involved in the pathogenesis of lactation ketosis.     

Influence of 4-week intraduodenal supplementation of quercetin on performance,glucose metabolism,and mRNA abundance of genes related to glucose metabolism and antioxidative status in dairy cows

Quercetin has been shown to be a potent antioxidant, acts hepatoprotectively, and affects glucose and lipid metabolism in monogastrics. If this is also true in ruminants, quercetin could be beneficial in periparturient high-yielding dairy cows by ameliorating the negative effects of free radical formation and reducing the severity of liver lipidosis and ketosis. In a first attempt to evaluate effects of a long-term quercetin treatment, we intraduodenally administered twice daily 18 mg of quercetin (Q)/kg of body weight to 5 late-lactation (215 d in milk) dairy cows over a period of 28 d. Frequent blood samples were taken before and during administration to determine plasma concentrations of flavonols and metabolites. Before and after 1 and 4 wk of Q administration, glycogen and fat content as well as mRNA expression of selected genes were measured in liver biopsies. Furthermore, euglycemic, hyperinsulinemic, and hyperglycemic clamp studies were conducted before and after 2 wk of Q administration. During the experiment, dry matter intake and most other zootechnical data remained unchanged. Milk protein content was increased in wk 2 and 4 of Q administration compared with basal values, whereas fat and lactose contents of milk remained unchanged. Plasma nonesterified fatty acids, γ-glutamyl transferase, cholesterol, glutamate dehydrogenase, triglyceride, and albumin concentrations, as well as liver fat and glycogen concentrations, were not affected by Q supplementation. Plasma glucose and β-hydroxybutyrate concentrations in plasma decreased and increased, respectively, under the influence of quercetin. During hyperglycemic clamp conditions, the relative increase of plasma insulin was higher after 2 wk of Q administration, and a tendency for an increased rQUICKI (revised quantitative insulin sensitivity check index) was observed. The relative mRNA expression levels of selected genes related to glucose metabolism, fat metabolism, and antioxidative status were not altered after 1 or 4 wk of Q supplementation. In conclusion, the effects on insulin release and sensitivity support the assumption that administration of Q could have positive effects on the metabolic adaption of high-yielding cows to early lactation. The increase of milk protein content in response to Q supplementation needs to be verified.     

Undernutrition modified metabolic responses to intramammary lipopolysaccharide but had limited effects on selected inflammation indicators in early-lactation cows

The objective was to assess effects of experimentally induced undernutrition on responses to an intramammary lipopolysaccharide (LPS) challenge in early-lactation cows. Starting at 24 ± 3 d in milk, multiparous Holstein cows either received a ration containing 48% straw for 96 h to restrict nutrient intake (REST, n = 8) or were allowed ad libitum intake of a lactation diet (CONT, n = 9). After 72 h on diet or after an equivalent period for CONT, 50 µg of LPS (Escherichia coli 0111:B4) was injected into one healthy rear mammary quarter to induce an acute inflammation response. Blood samples were collected weekly until 7 wk of lactation, daily during feed restriction (or control), before and at 1, 2, 4, 6, 10, and 24 h relative to LPS injection. Foremilk quarter samples were collected before and at 4, 6, 10, and 24 h after LPS injection. Dry matter intake, milk yield, energy balance, plasma glucose, nonesterified fatty acids (NEFA), and β-hydroxybutyrate (BHB) concentrations did not differ between CONT and REST immediately before nutrient restriction in REST (least squares means at d −1 were 21.8, 39.0 kg/d, −2.5 MJ/d, and 3.78, 0.415, 0.66 mM, respectively) but were significantly altered at 72 h of nutrient restriction (9.8, 28.3 kg/d, −81.6 MJ/d, and 2.77, 1.672, and 2.98 mM, respectively), when the LPS challenge was performed. The rectal temperature increment from baseline values in response to LPS did not differ, but cortisol increment was greater and cortisol response area under the curve (AUC) tended to be greater [202 vs. 122 (ng/mL) × 10 h] for REST than CONT. No treatment differences were observed in foremilk IL-8, IL-1β, tumor necrosis factor-α, and chemokine (C-X-C motif) ligand 3 concentrations in response to LPS injection. Composite milk somatic cell count per milliliter (6.919 × 10⁶ vs. 1.956 × 10⁶ cells/mL) and total number of somatic cells secreted in milk per day were greater for REST than CONT during the day following LPS. Plasma glucose, urea, and insulin concentrations increased after the LPS challenge, suggesting establishment of insulin resistance and modifications of glucose metabolism to support acute inflammation in both CONT and REST. Nonetheless, nutrient-restricted cows had delayed plasma insulin and glucose responses to LPS, smaller insulin AUC but greater glucose AUC compared with CONT, despite the limited nutrient availability to sustain an inflammation response. Undernutrition altered peripheral metabolic responses to an intramammary LPS challenge but had limited effects on selected indicators of inflammation response in early-lactation cows.     

Effect of enterotoxigenic Escherichia coli vaccine on innate immune function of bovine mammary gland infused with lipopolysaccharide

The effects of using an enterotoxigenic Escherichia coli vaccine on innate immune responses following intramammary infusion of lipopolysaccharide (LPS) were investigated in midlactation Holstein-Friesian cows. Seven out of 14 cows were inoculated with E. coli vaccine. Three weeks later, 100μg of LPS dissolved in 10mL of saline was infused into 1 quarter of all cows. Milk was collected every hour from infusion to 12h after infusion, and twice daily (at 0900 and 1600h) for 4d. Blood samples were collected 0, 4, 8, 24, 48, 72, and 96h after infusion. Rectal temperatures and milk yields were measured. The somatic cell count (SCC), lingual antimicrobial peptide concentration, lactoperoxidase (LPO) activity, and lactoferrin (LF) concentration in milk, and haptoglobin concentration in serum were determined. The mean rectal temperature in vaccinated cows was higher than in control cows at 10h. The mean milk yield was decreased significantly in the infused quarter of control cows at 24h compared with pretreatment, but not in vaccinated cows. The mean SCC in milk from vaccinated cows at 12 and 55h was significantly lower than that of control cows. The lingual antimicrobial peptide and LF concentrations were significantly lower at 8h and 55h, respectively, in vaccinated cows than in control cows. The mean antibody titer in the serum against the vaccine at the time of LPS infusion into vaccinated cows was significantly higher than in control cows. These antibody titers were positively correlated with the peak concentrations of LPO and LF in milk following challenge; therefore, cows with a high antibody titer were accompanied by high LPO activity and LF concentration in milk. These results suggest that vaccination suppresses the innate immune reaction after intramammary LPS infusion; however, the elevated antibody titer was unlikely to be responsible for the modification of the innate immune reaction.     

Jugular arginine infusion relieves lipopolysaccharide-triggered inflammatory stress and improves immunity status of lactating dairy cows

The objective of this study was to evaluate the effects of jugular l-Arg infusion on performance and immune function during lipopolysaccharide (LPS)-induced inflammation of lactating dairy cows. Eight Holstein cows (multiparous, 608.8 ± 31.5 kg) at mid-lactation were randomly assigned to 5-d jugular infusions of control (saline), Arg (3 g/h), LPS (0.033 μg/kg per h), and LPS + Arg (0.033 μg/kg per h of LPS and 3 g/h of Arg) in a replicated 4 × 4 Latin square design with 4 infusion periods separated by 10-d noninfusion periods. Jugular solutions of saline, Arg, LPS, and LPS + Arg were continuously infused using peristaltic pumps for approximately 6 h/d during infusion periods. Milk yield was measured on each day of the infusion period. Milk samples were obtained on the last 2 d of each infusion period, and blood samples were obtained on the last day of each infusion period before infusion (0 h) and at 3 and 6 h. We found that the jugular LPS infusion significantly increased serum concentrations of IL-1β, IL-6, tumor necrosis factor, inducible nitric oxide synthase, and lipopolysaccharide binding protein, whereas Arg attenuated the increase in IL-6 and inducible nitric oxide synthase levels and tended to decrease the lipopolysaccharide binding protein level. Arginine alleviated the decrease in dry matter intake and milk fat yield and the increase of somatic cell count induced by LPS. Total casein in milk was decreased during the LPS-induced inflammation period, and jugular Arg infusion significantly increased the content of total casein. In contrast, lactalbumin in milk increased during the LPS-induced inflammation period, whereas jugular Arg infusion significantly decreased the content of lactalbumin. The concentrations of plasma Gly, Thr, Ile, Leu, Arg, Phe, and total free AA were significantly decreased by LPS treatment, but Arg attenuated this tendency. These results indicated that jugular Arg infusion (18 g/d) has protective effects on relieving inflammatory stress and improving immunity status triggered by LPS. In conclusion, Arg could attenuate inflammatory stress and improve milk performance of lactating dairy cows. This protective effect may be due to the ability of Arg to suppress LPS effects and improve immunity status.     

Intramammary infusion of insulin or long R3 insulin-like growth factor-I did not increase milk protein yield in dairy cows

Two experiments investigated the regulation of milk protein synthesis in well-fed cows (n = 4) using 1) a hyperinsulinemic-euglycemic clamp and 2) intramammary infusion of insulin or long R3 insulin-like growth factor-I plus supplementary amino acids. In experiment 1, insulin was infused at 1.0 microg x kg BW(-1) x h(-1) to increase circulating levels fourfold, and euglycemia was maintained by infusion of glucose. An insulin clamp increased the yields of casein and whey protein both with and without supplementary amino acids. Plasma concentrations of insulin-like growth factor-I were increased and insulin-like growth factor binding protein-2 decreased during insulin clamp, while both insulin and insulin-like growth factor-I in milk were elevated by this treatment. Milk concentrations of insulin peaked on day 4, but insulin-like growth factor-I concentrations in milk peaked on day 1 of the insulin clamp. In experiment 2, intramammary infusion of insulin had no effects on any measured variables, while yields of milk, protein, and fat were slightly lower following long R3 insulin-like growth factor-I treatment. This could be associated with an increase in somatic cell count, which occurred following long R3 insulin-like growth factor-I treatment. Results from experiment 1 suggest insulin and insulin-like growth factor-I are likely candidates responsible for the increased milk protein yields during the insulin clamp. However, in experiment 2 neither hormone enhanced milk protein yield when administered using an intramammary technique.     

Intermittent parenteral administration of endotoxin triggers metabolic and immunological alterations typically associated with displaced abomasum and retained placenta in periparturient dairy cows

This study sought to investigate the effects of induced intermittent endotoxemia on plasma mediators of carbohydrate and lipid metabolism, humoral immunity, and clinical health status in periparturient dairy cows. Sixteen pregnant Holstein cows were blocked by parity and day of calving, and were randomly allocated to 1 of 2 different treatment groups. Eight cows were infused intravenously (i.v.) with 100 mL of sterile saline and served as the control group (CON). The other 8 cows were infused i.v. with 100 mL of sterile saline containing 3 increasing doses of lipopolysaccharide (LPS), from Escherichia coli O111:B4, for 3 consecutive weeks during the 2 wk before and 1 wk after parturition as follows: (1) 0.01 μg of LPS/kg of body weight (BW) on d −14 and −10; (2) 0.05 μg of LPS/kg of BW on d −7 and −3; and (3) 0.1 μg of LPS/kg of BW on d 3 and 7 postpartum. Nine blood samples were collected during the experimental period (i.e., from −14 to 28 d postpartum) and analyzed for calcium, zinc, iron, copper, glucose, lactate, β-hydroxybutyrate (BHBA), nonesterified fatty acids (NEFA), cholesterol, insulin, cortisol, serum amyloid A (SAA), lipopolysaccharide-binding protein (LBP), haptoglobin (Hp), and anti-LPS IgA, IgG, and IgM. Results showed that intermittently induced endotoxemia decreased feed intake and milk production and triggered alterations in plasma cholesterol, BHBA, Hp, Ca, Cu, and anti-LPS IgG and IgM. All of these changes were associated with a greater number of cows affected by metabolic disorders such as left displaced abomasum (LDA, 2 from 8 LPS cows vs. 0 from 8 CON cows) and retained placenta (RP; 4 from 8 LPS cows vs. 0 from 8 CON cows). In addition, the discriminant analysis differently clustered the cow responses within LPS group, each corresponding to LDA, RP, and the cows displaying no clinical health problems (LPS-NO). The stepwise selection procedure of the best discriminant variables revealed that plasma Ca and anti-LPS IgG, as well as glucose and cortisol, were the best discriminating variables for cows affected by LDA, whereas NEFA and cholesterol better discriminated for cows affected by RP. This analysis also revealed that the cluster of plasma variables including plasma Cu, SAA, BHBA, and anti-LPS IgA were the best discrimination for the LPS-NO group. In conclusion, our results indicate a role of endotoxemia, during the periparturient period, in development of metabolic and immune disturbances, as well as in the etiopathology of displaced abomasum and retained placenta in dairy cows.     

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.     

Between-cow variation in dermal fibroblast response to lipopolysaccharide reflected in resolution of inflammation during Escherichia coli mastitis

Effective response to mammary gland infection depends on efficient early innate immune response. The desired response would be one that is sufficient to clear the infection with a rapid return to the production of high-quality milk and limited tissue damage. In this study, 43 early lactation cows were ranked based on the ability of their fibroblasts to produce IL-8 in response to Escherichia coli lipopolysaccharide. Subsequently, the effect of a low or high response phenotype on the response to E. coli mastitis was determined. Untreated fibroblasts produced no detectable IL-8, whereas the range of IL-8 production in response to LPS (100 ng/mL) was approximately 7-fold between the lowest and highest responding cultures. Similar patterns of between-cow variation were observed in fibroblast production of IL-8 and IL-6 in response to IL-1β and Pam2CSK4 (a synthetic diacylated lipopeptide ligand). Four low and 4 high responder cows were challenged in late lactation with intramammary infusion of E. coli. All cows developed clinical mastitis in the challenged quarters and all cows cleared the infection within 8 d. However, somatic cell count began to decline earlier in the low responder group, and milk BSA concentration (an indicator of tissue damage) was also lower in low responders compared with high responders. Milk production from the challenged quarter was markedly depressed in both groups, but returned toward prechallenge values earlier in low responder cows. Dermal fibroblast cells appear predictive of a cow's response to mastitis. In this study, the low responder phenotype was sufficient to contain an E. coli infection with a more rapid return to the production of high quality milk.