Characterization of the bovine innate immune response to intramammary infection with Klebsiella pneumoniae

Gram-negative bacteria are responsible for almost one-half of the clinical cases of mastitis that occur annually. Of those gram-negative bacteria that induce mastitis, Klebsiella pneumoniae remains one of the most prevalent. Detection of infectious pathogens and the induction of a proinflammatory response are critical components of host innate immunity. The objective of the current study was to characterize several elements of the bovine innate immune response to intramammary infection with Klebsiella pneumoniae. The inflammatory cytokine response and changes in the levels of soluble CD14 (sCD14) and lipopolysaccharide (LPS)-binding protein (LBP), 2 proteins that contribute to host recognition of gram-negative bacteria, were studied. The contralateral quarters of 7 late-lactating Holstein cows were challenged with either saline or K. pneumoniae, and milk and blood samples were collected. Initial increases in the chemoattractants C5a and IL-8, as well as TNF-alpha, were evident in infected quarters within 16 h of challenge and were temporally coincident with increases in milk somatic cells. Augmented levels of TNF-alpha and IL-8 were observed in infected quarters until >48 h postchallenge, respectively. Elevated levels of IL-12, IFN-gamma, and the antiinflammatory cytokine, IL-10, which were first detected between 12 and 20 h postinfection, persisted in infected quarters throughout the study (>96 h). Initial increases in milk LBP and sCD14 were detected 16 and 20 h, respectively, after challenge. Together, these data demonstrate that intramammary infection with K. pneumoniae elicits a host response characterized by the induction of proinflammatory cytokines and elevation of accessory molecules involved in LPS recognition.     

Elevated milk soluble CD14 in bovine mammary glands challenged with Escherichia coli lipopolysaccharide

The purpose of this study was to determine whether soluble CD14 (sCD14) in milk was affected by stage of lactation, milk somatic cell count (SCC), presence of bacteria, or lipopolysaccharide (LPS)-induced inflammation. Milk samples from 100 lactating cows (396 functional quarters) were assayed for sCD14 in milk to determine effects of stage of lactation, SCC, and intramammary infection. The concentration of sCD14 was highest in transitional milk (0 to 4 d postpartum) and in milk with high SCC (> 750,000 cells/ml). Most of the infected quarters (> 80%) were infected by coagulase-negative staphylococci and yeast. No difference was found between noninfected and infected quarters. One quarter of six healthy lactating cows was challenged with 100 microg LPS in order to study the kinetics of sCD14 during an LPS-induced inflammation. Milk samples were collected at various intervals until 72 h after injection. Rectal temperature, milk tumor necrosis factor-alpha, and interleukin-8 increased immediately after challenge. The increase in sCD14 paralleled the increase in SCC, peaked at 12 h, and started to decline after 24 h. Serum leakage, as characterized by the level of bovine serum albumin in milk, peaked at 4 h and then gradually decreased. All parameters remained at basal levels in control quarters throughout the study. In vitro experiments indicated that neutrophils released sCD14 in response to LPS in a dose-dependent manner. The results indicate that the concentration of sCD14 was significantly increased in milk after LPS challenge. The increase was not likely due to serum leakage. Instead, infiltrated neutrophils might be the main source of increased sCD14 in milk during inflammation.     

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.     

Increase of Escherichia coli inoculum doses induces faster innate immune response in primiparous cows

The objective of the current study was to evaluate the dynamics of infection and the immunological response to varying numbers of Escherichia coli injected into the mammary glands of primiparous cows during the periparturient period. Primiparous cows have been shown to be more resistant to intramammary E. coli challenge, and an increase of the inoculum dose by 2 log10 units induced a more rapid clinical response and clearance of the organisms. Recognition of lipopolysaccharide (LPS) is a key event in the innate immunity response to gram-negative infection and is mediated by the accessory molecules CD14 and LPS-binding protein (LBP). Primiparous cows were inoculated with 1 x 10(4) (Group A; n=8) or 1 x 10(6) (Group B; n=8) cfu E. coli P4:O32 in their 2 left quarters during the periparturient period. Clinical examination and analysis of blood and milk parameters, including IL-8, complement fragment 5a (C5a), LBP, and soluble CD14 (sCD14), were performed from d -4 to d +3 relative to infection. Primiparous cows in Group B initiated a more rapid clinical response following intramammary infection (IMI), resulting in typical clinical signs and changes in blood and milk parameters approximately 3 h earlier compared with primiparous cows in Group A. Based on average milk production in the noninfected quarters on d +2 postinoculation, all heifers reacted as moderate responders. Distinct differences in the kinetic patterns of rectal temperature, somatic cell count (SCC), IL-8, C5a, LBP, and sCD14 were observed between both groups during the early phase of inflammation. Both C5a and IL-8 increased before cellular influx into the infected glands, followed by increases in sCD14 and LBP. In conclusion, primiparous cows are able to clear an intramammary E. coli infection efficiently. Moreover, increasing the inoculum dose induces a more rapid inflammatory reaction, mainly because of early activation of the innate host immune response.     

Increased milk levels of transforming growth factor-alpha, beta1, and beta2 during Escherichia coli-induced mastitis

Among the gram-negative bacteria that cause mastitis, Escherichia coli are the most prevalent. The innate immune system provides initial protection against E. coli infection by detecting the presence of the foreign pathogens and by mounting an inflammatory response, the latter of which is mediated by cytokines such as IL-1β, IL-8, and tumor necrosis factor (TNF)-α. Although changes in these cytokines during mastitis have been well-described, it is believed that other mediators moderate mammary gland inflammatory responses as well. The growth factors/cytokines transforming growth factor (TGF)-α, TGF-β1, and TGF-β2 are all expressed in the mammary gland and have been implicated in regulating mammary gland development. In other tissues, these growth factors/cytokines have been shown to moderate inflammation. The objective of the current study was to determine whether TGF-α, TGF-β1, and TGF-β2 milk concentrations were altered during the course of E. coli-induced mastitis. The contralateral quarters of 11 midlactating Holstein cows were challenged with either saline or 72 cfu of E. coli, and milk samples were collected. Basal milk levels of TGF-α, TGF-β1, and TGF-β2 were 98.81 ± 22.69 pg/mL, 3.35 ± 0.49 ng/mL, and 22.36 ± 3.78 ng/mL, respectively. Analysis of whey samples derived from E. coli-infected quarters revealed an increase in milk levels of TGF-α within 16 h of challenge, and these increases persisted for an additional 56 h. Elevated TGF-β1 and TGF-β2 milk concentrations were detected in E. coli-infected quarters 32 h after challenge, and these elevations were sustained throughout the study. Because TGF-α, TGF-β1, and TGF-β2 have been implicated in mediating inflammatory processes, their induction during mastitis is consistent with a role for these molecules in mediating mammary gland host innate immune responses to infection.     

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.     

Lipoprotein lipase activity of milk from cows with prolonged subclinical mastitis

The influence of prolonged subclinical mastitis on bovine milk lipoprotein lipase activity was investigated. Nine cows with at least one quarter with prolonged subclinical mastitis and at least one nonmastitic quarter were selected in various stages of lactation. Milk from subclinical quarters had a mean somatic cell count of 5.7 X 10(6) cells/ml while milk from nonmastitic quarters had an average somatic cell count of 9.4 X 10(4) cells/ml. Quarters with a subclinical infection contained the same pathogenic organisms for a minimum of 6 wk. The average milk lipoprotein lipase activity of 108.7 units/ml milk from subclinical quarters was 27.1% higher than the average enzyme activity of 79.2 units/ml milk from nonmastitic quarters. Conditions present in the mammary gland during prolonged subclinical mastitis could lead to increased milk lipoprotein lipase activity in raw milk.     

Lipopolysaccharide challenge of the mammary gland in bovine induced a transient glandular shift to anaerobic metabolism

Support of milk production in modern dairy cows demands a large proportion of its own metabolic resources, such as glucose, which might be required under stressful situations. The aim of the experiment was to test the hypothesis that acute immune stress shifts oxidative metabolism to glycolysis. Two mammary quarters in 6 Holstein cows were infused with lipopolysaccharide (LPS), whereas the 2 counter quarters served as controls to the treatment. An additional 6 cows were infused with saline and served as running controls. The LPS challenge induced dramatic transient increases in milk lactate (75-fold) and malate (11-fold) concentrations (both markers of glycolysis) at 24 h posttreatment. No significant changes in lactate and malate concentrations were recorded in control quarters and control animals, indicating that the effect of LPS was restricted to the treated gland. The LPS challenge induced a dramatic transient decrease in milk yield, and lactose and citrate (a marker of mitochondrial metabolism) secretion at 24 h posttreatment. The kinetics were inversely proportional to those of lactate and malate concentrations. Thus, our data suggest that LPS challenge induces acute conversion of epithelial cell metabolism from principally mitochondrial-oxidative to principally cytosolic (glycolytic), which allows the diversion of metabolic resources normally used to synthesize milk to support the immune system. An in vitro bacterial growth test showed that concentrations of lactate, malate, and lactose equivalent to those found in the in vivo experiment delayed and reduced the growth of a pathogenic Escherichia coli strain, suggesting that they play a role in diminution of bacterial multiplication in the mammary gland.     

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.     

Determination of milk and blood concentrations of lipopolysaccharide-binding protein in cows with naturally acquired subclinical and clinical mastitis

Blood and milk concentrations of the acute phase protein lipopolysaccharide-binding protein (LBP) were evaluated in cows with naturally occurring mastitis. Blood and milk samples were collected from 101 clinically healthy dairy cows and 17 dairy cows diagnosed with clinical mastitis, and the LBP concentrations of the samples were measured by an ELISA. Concentrations of LBP were greater in the blood and milk of cows with clinical mastitis than in those with healthy quarters. Concentrations of LBP also differed between uninfected and subclinically infected quarters with low somatic cell count. Blood concentrations of LBP in cows with subclinical intramammary infections could not be differentiated from those of cows with all healthy quarters. Together, these data demonstrate that increased blood and milk concentrations of LBP can be detected in dairy cows with naturally acquired intramammary infections that cause clinical mastitis.     

Effect of carprofen treatment following experimentally induced Escherichia coli mastitis in primiparous cows

Acute Escherichia coli mastitis is one of the major sources of economic loss in the dairy industry due to reduced milk production, treatment costs, discarded milk, and occasional fatal disease. Nonsteroidal anti-inflammatory drugs (NSAIDs) are frequently used as adjunctive therapy to antibiotics. The objective of the current study was to evaluate the effect of carprofen treatment following infusion of Escherichia coli into the mammary glands of primiparous cows during the periparturient period. Severity of mastitis was scored based on the average milk production in the uninfected quarters on d +2 postinoculation and a clinical severity score. Carprofen was administered intravenously at 9 h postchallenge, when clinical signs of mastitis appeared. In previous work, efficacy of NSAIDs was mainly evaluated using clinical symptoms. In the present study, the effect of carprofen on innate immune response was also assessed by quantification of inflammatory mediators. All primiparous cows reacted as moderate responders throughout the experimental period. Primiparous cows were intramammarily inoculated with 1 × 10⁴ cfu of E. coli P4:O32 in 2 left quarters. Analysis of blood and milk parameters, including IL-8, complement component C5a, lipopolysaccharide-binding protein (LBP), soluble CD14, prostaglandin E₂, and thromboxane B₂ was performed from d 0 to d +6 relative to intramammary inoculation. Rectal temperature in carprofen-treated animals was lower than in control animals at 3 and 6 h posttreatment. Treatment also restored the decreased reticulorumen motility that occurs during E. coli mastitis to preinfection levels faster than in control animals. Carprofen treatment resulted in an earlier normalization of the clinical severity score. Eicosanoid (prostaglandin E₂ and thromboxane B₂) production in milk tended to be inhibited by carprofen. No significant differences in the kinetic patterns of somatic cell count, IL-8, complement component C5a, LBP, and soluble CD14 were observed. In conclusion, carprofen treatment improved general clinical condition by effective antipyrexia and restoration of reticulorumen motility but did not significantly inhibit eicosanoid production. Carprofen treatment did not result in a significant decrease of chemotactic inflammatory mediators, IL-8 and C5a, and early innate immune molecules, sCD14 and LBP. Therefore, major modulatory effects from NSAID administration were not observed in this mastitis model, although a larger study might confirm some apparent trends obtained in the present results.     

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.     

Relationship between milk lactoferrin and etiological agent in the mastitic bovine mammary gland

Bovine mastitis is one of the most deleterious diseases for dairy herds and is mainly caused by contagious and environmental bacterial pathogens. Among contagious bacteria, Staphylococcus aureus is the most prevalent, whereas the main environmental mastitis pathogens are Streptococcus uberis and Escherichia coli. Bovine lactoferrin (bLF) is an approximately 80-kDa glycoprotein present in milk that participates in the innate response of the mammary gland against bacterial infection. The objectives of the current study were to analyze potential changes in bLF milk concentration, which would constitute a response of the mammary gland toward mastitis induced by different etiologic agents, and to evaluate a possible relation between this response and pathogen susceptibility to bLF. Microbiology analysis and bLF quantification in milk from different bovine mammary gland quarters were performed. Infected quarters presented greater concentrations of bLF compared with those from microbiologically negative quarters. Analysis of individual pathogen contributions showed that most of this increase was attributable to Strep. uberis intra-mammary infection. The ability of mammary gland cells to synthesize bLF in response to Strep. uberis challenge was demonstrated by immunodetection of the protein in in vitro infection experiments. Susceptibility of Strep. uberis, E. coli, and Staph. aureus to the antimicrobial activity of bLF was determined by growth inhibition assays conducted with 4 different isolates of each species. Whereas Staph. aureus and E. coli were shown to be susceptible to this protein, Strep. uberis appeared to be resistant to the antimicrobial activity of bLF. Molecular typing of the 4 Strep. uberis isolates used throughout this study showed that this result was representative of the species and not exclusive of a particular strain. Results presented herein suggest that different bacteria species may elicit different mammary gland responses mediated by bLF secretion and that Strep. uberis has probably adapted to this immune reaction by developing resistance to bLF inhibitory action.     

Role of endotoxin and TNF-alpha in the pathogenesis of experimentally induced coliform mastitis in periparturient cows

Twelve cows were experimentally infected in two quarters with 1 x 10(4) cfu Escherichia coli per quarter and six cows were infused with 500 microg endotoxin into two quarters. Six cows infected intramammarily with Esch. coli were treated intravenously with a bactericidal antibiotic 10 h after infection and subcutaneously 20 h later. Blood and milk samples were collected from all cows at regular time intervals. Milk production decreased more rapidly, but was less pronounced, after endotoxin infusion than (during Esch. coli mastitis. The milk production losses in the noninflamed quarters were negligible in endotoxin mastitis, but were substantial during Esch. coli mastitis, probably due to more pronounced systemic effects. Reticulorumen motility was inhibited only during Esch. coli mastitis. Changes in plasma haptoglobin were more pronounced during Esch. coli mastitis, although they occurred sooner during endotoxin mastitis. No changes in plasma activities of enzymes such as lactate dehydrogenase, glutamic-oxaloacetic transaminase and gamma-glutamyl transpeptidase were observed. Concentrations of tumour necrosis factor-alpha increased in both types of mastitis. Absorption of these cytokines into the circulation was highest during Esch. coli mastitis, especially in the untreated control group. We found only minor differences between the treated and untreated Esch. coli groups, but there were larger differences between the Esch. coli groups and the endotoxin group. These differences were probably due to differences in kinetics, composition and amounts of different cytokines released in the mammary gland and subsequently absorption into the circulation. Endotoxin is probably not directly responsible for the systemic changes during coliform 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.     

Efficacy of an Escherichia coli J5 bacterin administered to primigravid heifers.

The efficacy of an Escherichia coli J5 bacterin for reducing the incidence of intramammary infections and clinical signs of mastitis was tested in first lactation heifers. Ten primigravid heifers were immunized with an E. coli J5 bacterin. Four heifers received a placebo. The bacterin and placebo were injected subcutaneously approximately 60 d prior to calving, 28 d later, and within 48 h after calving. Vaccinated and placebo-injected heifers were challenged by intramammary infusion of E. coli 727 in one mammary gland between 23 and 37 d after calving. All challenged quarters were diagnosed with an intramammary infection within 6 h after bacteria were infused. The severity and duration of local signs of clinical mastitis were reduced in vaccinated heifers compared with placebo-injected heifers. Systemic signs of clinical mastitis were limited and did not differ between treatment groups. Bacteria counts in milk from challenged quarters were lower in vaccinated heifers than in control heifers at 12, 15, and 48 h after challenge. Serum immunoglobulin G titers against whole-cell E. coli J5 antigen at calving were higher in vaccinated heifers than they were in controls. Vaccinated heifers had higher immunoglobulin G titers than did controls in mammary secretions at calving and immediately prior to challenge. Immunization of primigravid heifers with an E. coli J5 bacterin during the last trimester of gestation and at calving reduced the severity and duration of clinical signs following intramammary challenge with a heterologous strain of E. coli.     

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.     

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.     

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.