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.     

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.     

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.     

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.     

Changes of physicochemical indicators during mastitis and the effects of milk ejection on their sensitivity

We examined the relationship between physicochemical indicators and somatic cells in the milk of dairy cows during experimentally induced mastitis and their significance as indicators for use in controlling udder health. We were concerned particularly with the effect of alveolar milk ejection on the sensitivity of these indicators. In Expt 1, Escherichia coli lipopolysaccharide (Esch. coli LPS) was injected into the left rear quarter to induce an inflammatory reaction in one quarter in each of six cows. The contralateral control quarter was injected with a solution of NaCl (9 g/l). Nine milk samples were taken from both quarters until 60 h after injection. In Expt 2, repeated milk samples were taken every 20 s from one quarter during a 120-s teat stimulation in 20 cows with different somatic cell counts (SCC). Quarters were clustered for low (<5.0 log cells/ml), mid (5.0-5.7 log cells/ml) and high (>5.7 log cells/ml) SCC of the sample taken at t=0 s. Samples were analysed for SCC, electrical conductivity (EC) and Na+ and Cl- concentrations. During the experimental inflammation SCC, EC, Na+ and Cl- peaked at 12 h from LPS administration and values in treated quarters (T) at this time were elevated to 7900, 157, 501 and 169% of the values in untreated quarters, respectively. In Expt 2, SCC, EC, Na+ and Cl- in high SCC quarters were 2520, 121, 283 and 141% of low SCC quarters at the start of stimulation (t=0 s), respectively. Highly significant (P<0.001) differences in EC, Na+ and Cl- between high and low SCC quarters disappeared owing to the onset of alveolar milk ejection 100 s after the first contact with the teat. In conclusion, SCC in cows' milk provided the strongest amplitude in the case of an intramammary inflammation. EC, Na+ or Cl- were useful tools only if the measurements were performed in cisternal milk before the start of alveolar milk ejection.     

Intramammary challenge with Escherichia coli following immunization with a curli-producing Escherichia coli

Holstein and Jersey cattle were immunized with a curli-producing strain of Escherichia coli (pCRL65/A012) or a noncurli-producing strain (pUC18/HB101) to determine differences in resistance to establishment of experimental intramammary infection. Cows (n = 6 per group) were immunized at 14 d prior to drying off, 7 d of involution, and at calving with 3 x 10(10) E. coli in Freund's Incomplete Adjuvant. At 30 d of lactation, one mammary quarter of each cow was infused with a wild strain of E. coli (727). Escherichia coli 727 was isolated from a naturally occurring intramammary infection and produced curli. All challenged quarters became infected, and all cows developed acute clinical mastitis. Geometric mean duration of intramammary infections was 6 d for both immunization groups. All infections were spontaneously eliminated within 10 d. No differences occurred between immunization groups in blood selenium and glutathione peroxidase activity, plasma selenium, number of E. coli 727 isolated from secretion after challenge, rectal temperature and SCC response, clinical status of mammary quarters, or DMI. Reduction in milk production after challenge was greater for cows immunized with E. coli pCRL65/A012. Immunization of dairy cattle with a curli-producing strain of E. coli did not protect against experimental intramammary challenge during lactation.     

Comparison of probiotic and antibiotic intramammary therapy of cattle with elevated somatic cell counts.

The effects of treating subclinical mastitis with intramammary infusions of either a Lactobacillus or an antibiotic preparation on intramammary infection cure rate and on milk SCC were compared. Cows with two consecutive monthly DHIA composite SCC greater than 300,000 cells/ml (5.4771 log10/ml) were defined as high SCC cows. Twenty-six subclinical cows were randomly assigned to one of two treatments. Quarter foremilk samples were obtained from all quarters at d 0, 7, and 14 following infusion to determine the microbiological status and SCC. Composite milk SCC were determined monthly by DHIA and at d 0, 7, and 14 of the study. Coagulase-negative staphylococci were the predominantly isolated pathogens. Treatment of cows with Lactobacillus cured 21.7% of infected quarters, whereas 73.7% of infections treated with antibiotic were eliminated. Treatment of quarters with antibiotic did not reduce quarter SCC unless infected quarters were cured. Intramammary infusion of quarters with Lactobacillus increased quarter SCC, mainly because of an increase in SCC of initially uninfected, low SCC quarters. Monthly composite SCC were similar between treatments. The results indicate that administering Lactobacillus or antibiotic treatment to all quarters based on elevated composite SCC should not be adopted. Lactobacillus treatment increased SCC with no effect on infection rate.     

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.     

Effectiveness of intramammary antibiotic therapy based on somatic cell count

The Virginia Tech dairy herd was used in a 10-mo study to determine the effect of intramammary antibiotic therapy of quarters with elevated SCC on milk production, subsequent DHIA SCC, and infection status. Cows were assigned randomly to experimental or control groups. Animals in both the control and experimental groups with SCC scores greater than or equal to 5 for the first time during that lactation were quarter sampled, milk was cultured to detect presence of mastitis pathogens, and SCC was determined. All experimental cows with quarter SCC greater than or equal to 5 were treated with an intramammary cephapirin product only in those elevated quarters (DHIA SCC greater than or equal to 5), regardless of clinical symptoms. Control cows received antibiotic therapy when symptoms were clinical, regardless of SCC. Treatment group had no significant effect on milk production, SCC, or infection status of the cow. Treatment of cows in the experimental group cured 70% of infected quarters, whereas only 50% of infections in the control group were eliminated.     

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.     

Effects of prepartum intramammary antibiotic therapy on udder health, milk production, and reproductive performance in dairy heifers

Preparturient heifers (n = 561) from 9 herds in 6 US states and 1 Canadian province were enrolled in a study to test the hypothesis that prepartum intramammary therapy would cure existing intramammary infections (IMI) and lead to increased milk production, reduced linear somatic cell count (LSCC), and improved reproductive performance. Mammary secretions were collected 10 to 21 d before expected calving from each quarter. Heifers were then assigned by identification number to receive intramammary therapy consisting of infusion of one tube per mammary quarter of a lactating cow commercial antibiotic preparation containing cephapirin or to a nontreated control group. Overall, 34.1% of mammary quarters were infected with a mastitis pathogen before parturition and 63.4% of heifers had at least one mammary quarter infected. The coagulase-negative staphylococci (CNS) caused the majority (74.8%) of prepartum IMI. Coagulase-positive staphylococci, environmental streptococci, and coliforms accounted for 24.5% of prepartum infections. Treatment had a significant effect on the cure rate of infected mammary quarters. Mammary quarters that were infected prepartum and treated with antibiotics had a 59.5% efficacy of cure rate and the percentage reduction in heifers with IMI was 51.9. Control quarters had a spontaneous cure rate of 31.7%. Treatment did not significantly affect milk production or LSCC in the first 200 d of lactation; however, there was a significant treatment by herd interaction for milk production. Quarters cured of either CNS or major pathogens had a lower LSCC in the first 200 d of lactation. No significant effect on services per conception or days open between treatment and control groups was observed. This trial demonstrated that prepartum intramammary antibiotic therapy did reduce the number of heifer IMI postpartum. Milk production, LSCC, and reproductive performance during the first 200 d of the first lactation were not significantly affected by treatment. Given these results, use of prepartum intramammary antibiotic therapy in heifers as a universal strategy to increase milk production in first-lactation dairy cows may not be warranted.     

Comparative efficacy of local and systemic antibiotic treatment in lactating cows with clinical mastitis

The intramuscular administration of penethamate hydriodide over 3 consecutive days and the intramammary administration of an ampicillin/cloxacillin combination were compared in lactating cows suffering from infectious clinical mastitis in one quarter, through an open, randomized, controlled multicenter field trial. Clinical examinations were carried out on d 1 (immediately before treatment), 3, 8, 17, and 22. Milk samples were taken from affected quarters for bacteriological analysis on d 1, 17, and 22, and from all quarters for somatic cell count (SCC) determination on d 1, 8, 17, and 22. There was no significant difference in bacteriological and clinical cure rates between the 2 treatment groups. The systemic treatment with penethamate resulted more frequently in a reduction of the milk SCC below the threshold of 250,000 cells/mL. This also occurred in the adjacent quarters not affected by clinical mastitis but with an SCC above 250,000 cells/mL before treatment. These findings suggest that the parenteral treatment with penethamate provides collateral cure on the quarters of the cows affected by subclinical mastitis. The number of quarters per cow affected by clinical or subclinical mastitis should be considered when selecting an antibiotic treatment by the local or systemic route.     

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.     

Determination of milk and mammary tissue concentrations of ceftiofur after intramammary and intramuscular therapy

Twenty-five Staphylococcus aureus strains isolated from bovine mastitis were tested for their susceptibility to ceftiofur. Zone diameter for 30 micrograms disks averaged 39 mm, and minimum inhibitory concentrations ranged from .5 to 1 microgram/ml. Tissue and milk concentrations were determined from biopsy and quarter milk samples collected from eight cows treated with either intramammary infusion of 100 or 200 mg of ceftiofur, one or two intramuscular injections of 500 mg of ceftiofur, or combination therapy of intramammary infusion coupled with intramuscular injection. Three additional cows received two intramammary infusions of 200 mg of cephapirin at 24-h intervals. Intramuscular injections of ceftiofur resulted in tissue and milk concentrations below detectable limits. Staphylococcus aureus was not eliminated from infected mammary glands by infusion of 100 mg of ceftiofur or by injection of 500 mg of ceftiofur by 48 h after treatment. Combination therapy of 100 mg of ceftiofur infused and 500 mg injected reduced S. aureus numbers in milk and tissue markedly, as did infusion of 200 mg of ceftiofur. Cows receiving intramammary infusion of 200 mg of ceftiofur (two doses at 24-h intervals) had highest concentrations in milk (450 micrograms/ml at 4 and 6 h) and in tissue (.08 microgram/mg at 30 h). These concentrations are similar to those obtained with two 200-mg doses of cephapirin at 24-h intervals. Histologic analysis of mammary parenchymal tissues showed that combination therapy resulted in higher percentages of alveolar luminal area and lower percentages of interalveolar stroma compared with infusion or injection alone. Histology of quarters receiving combination therapy was not different from that of quarters receiving cephapirin infusion alone.     

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.     

Association of anatomical characteristics of teats with quarter-level somatic cell count

Mastitis occurs after bacteria successfully traverse the teat orifice and cause an intramammary infection. Anatomical characteristics of the teat are potential risk factors for infection. The objective of this study was to identify potential associations between anatomical characteristics of teats and quarter-level somatic cell count (QSCC) from cows on larger dairy farms in Wisconsin. Teat dimensions (length and diameter at the teat barrel and apex) were measured, and hyperkeratosis scores were assessed for 3,713 quarters of 959 cows on 9 dairy farms. The SCC of quarter milk samples obtained from those teats was determined. Multivariate models were used to determine associations of teat anatomical characteristics with QSCC. Subclinical mastitis was defined as a quarter milk sample with SCC of >150,000 cells/mL. Teat dimensions and milk components varied among farms. In the group of farms enrolled in this study, prevalence of subclinical mastitis in mammary gland quarters ranged from 13.6 to 28.9%. An interaction of teat apex diameter and quarter position (front or rear) was identified for QSCC. For both front and rear quarters, a tendency existed for narrower teat barrels to be associated with increased QSCC. However, for front quarters only, greater diameter of the teat apex was associated with increased QSCC. Teat shape (square or triangular teats) was not associated with QSCC. Milk samples obtained from teats with hyperkeratosis scores of very rough had greater QSCC compared with milk samples obtained from teats with hyperkeratosis scores of normal, smooth ring, or rough ring.     

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.     

Treatment of Staphylococcus aureus mastitis with penicillin and novobiocin: antibiotic concentrations and bacteriologic status in milk and mammary tissue

Cows with Staphylococcus aureus mastitis received either one or two intramammary infusions containing 100,000 U penicillin G and 150 mg novobiocin. Milk and tissue samples were obtained from each quarter. Peak mean penicillin and novobiocin concentrations from antibiotic-positive tissue samples were .013 U/mg and .06 microgram/mg, respectively, for quarters treated once. Quarters treated twice had peak mean penicillin and novobiocin concentrations of .057 U/mg and .06 microgram/mg, respectively. Viable Staphylococcus aureus were isolated intermittently from milk and tissue samples of quarters positive for penicillin and novobiocin for both treatment groups. Histological analysis of mammary parenchyma demonstrated marked decreases in luminal area and increases in connective tissue area and leukocyte infiltration in S. aureus-infected quarters compared with uninfected controls, suggesting that reduction in milk collecting space and presence of inflammatory changes may be responsible for poor drug distribution.     

Bovine mammary immune response to an experimental intramammary infection with a Staphylococcus aureus strain containing a gene for staphylococcal enterotoxin C1

Staphylococcal enterotoxin C (SEC), a superantigen, is the most frequently expressed enterotoxin by bovine strains of Staphylococcus aureus causing mastitis. To examine the possible impact of SEC on the immune response of the bovine mammary gland, we monitored changes in lymphocyte subpopulations in mammary glands of four lactating cows after intramammary instillation of S. aureus strain Rn4220 transformed with a plasmid containing a gene coding for SEC1. Four other lactating cows received the same strain transformed with the plasmid without the SEC1 gene (positive control), and four cows were untreated (negative control). Mammary quarter milk samples for somatic cell count (SCC) analysis and determination of N-acetyl-beta-D-glucosimindase (NAGase) activity levels were collected daily for 21 d postinstillation. Flow cytometry utilizing three-color analysis was used to phenotype lymphocyte subpopulations isolated from milk samples collected on d 0, 4, 7, 11, 14, 18, and 21 postinstillation from all the cows. Milk from mammary gland halves (positive control and experimental) or all mammary quarters (negative control) was collected for flow cytometric analysis. Increased NAGase activity, SCC, and isolated S. aureus demonstrated that infection was established in mammary quarters intrammarily instilled with bacteria. There were no significant differences (P > 0.05) in the proportions of BoCD4 helper T lymphocytes or BoCD8 cytotoxic T lymphocytes between the two infected treatment groups. There was a significant day x treatment difference of the proportion of a gammadelta T cell subpopulation that did not express BoCD2, but did express the ACT2 activation molecule and a significant treatment difference of a gammadelta T cell subpopulation that expressed BoCD2, but not the ACT2 activation molecule (P < 0.05). Results do not support the hypothesis that the presence of the gene for SEC1 alters the mammary BoCD4 or BoCD8 T lymphocyte response to infection.     

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.