Rupert Bruckmaier

Tumor necrosis factor-α and nitrite/nitrate responses during acute mastitis induced by Escherichia coli infection and endotoxin in dairy cows

Concentrations of tumor necrosis factor-alpha (TNF-alpha) and of NO(x) (sum of nitrite and nitrate as indicators of endogenous nitric oxide production) in milk and blood plasma were measured in three mastitis models in dairy cows in early lactation. Escherichia coli P4:O37 bacteria or endotoxin O111:B4 were administered into both left quarters of 12 and 6 cows, respectively. Six of the E. coli-infected cows were treated with a bactericidal antibiotic (Enrofloxacin; Bayer AG, Leverkusen, Germany) i.v. at 10 hr and subcutaneously (sc) at 30 hr after infection. NO(x) concentrations transiently increased maximally 10- to 11-fold in milk of E. coli-infected quarters with or without antibiotic treatment at 24 hr and after endotoxin administration. NO(x) concentrations did not change in milk of unchallenged quarters and in blood plasma. Increases of NO(x) were proceeded by a transient (96- to 149-fold) rise of milk TNF-alpha concentrations, which in endotoxin-administered quarters was maximal at 6 hr and in infected quarters without or with Enrofloxacin treatment at 10 and 14 hr. In blood plasma TNF-alpha concentrations only moderately increased to peaks in endotoxin-administered cows at 6 hr and in E. coli-infected cows at 14 hr postchallenge. In one severely sick, nontreated E. coli-infected cow milk, TNF-alpha response at 14 hr was excessive and followed by a spectacular rise of NO(x) concentration in milk between 48 and 72 hr. In conclusion, a possible clinical relevance of nitric oxide production associated with a rise of intramammary and systemic TNF-alpha during acute mastitis by E. coli infection and endotoxin in lactating dairy cows is indicated, but could not be inhibited by antibiotic treatment.

Role of endotoxin and TNF-α 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.

Variation in hepatic regulation of metabolism during the dry period and in early lactation in dairy cows

The purpose of this study was to investigate variations in hepatic regulation of metabolism during the dry period, after parturition, and in early lactation in dairy cows. For this evaluation, cows were divided into 2 groups based on the plasma concentration of beta-hydroxybutyric acid (BHBA) in wk 4 postpartum (PP; group HB, BHBA >0.75 mmol/L; group LB, BHBA <0.75 mmol/L, respectively). Liver biopsies were obtained from 28 cows at drying off (mean 59 +/- 8 d antepartum), on d 1, and in wk 4 and 14 PP. Blood samples were collected every 2 wk during this entire period. Liver samples were analyzed for mRNA abundance of genes related to carbohydrate metabolism (pyruvate carboxylase, PC; phosphoenolpyruvate carboxykinase, PEPCK; citrate synthase, CS), fatty acid biosynthesis (ATP citrate lyase, ACLY) and oxidation (acyl-CoA synthetase long-chain, ACSL; carnitine palmitoyltransferase 1A, CPT 1A; carnitine palmitoyltransferase 2, CPT 2; acyl-coenzyme A dehydrogenase very long chain, ACADVL), cholesterol biosynthesis (3-hydroxy-3-methylglutaryl-coenzyme A synthase 1, HMGCS1), ketogenesis (3-hydroxy-3-methylglutaryl-coenzyme A synthase 2, HMGCS2), and of genes encoding the transcription factors peroxisome proliferator-activated receptor alpha (PPARalpha), peroxisome proliferator-activated receptor gamma (PPARgamma), and sterol regulatory element binding factor 1 (SREBF1). Blood plasma was assayed for concentrations of glucose, BHBA, nonesterified fatty acids, cholesterol, triglycerides, insulin, insulin-like growth factor-I, and thyroid hormones. In both groups, plasma parameters followed a pattern usually observed in dairy cows. However, changes were moderate and the energy balance in cows turned positive in wk 7 PP for both groups. Additionally, the energy balance and milk yield were similar for both groups after parturition onwards. Significant group effects were found at drying off, when plasma concentrations of triglycerides were higher in LB than in HB, and in wk 4 PP, when plasma concentrations of glucose and IGF-I were lower in HB than in LB. Similarly, moderate changes in mRNA expression of hepatic genes between the different time points were observed, although HB cows showed more adaptive performance than LB cows based on changes in mRNA expression of PEPCKc, PEPCKm, CS, CPT 1A, CPT 2, and PPARalpha. Part of the variation measured in this study was explained by parity. Significant Spearman rank correlation coefficients between the variables were not similar at each time point and were not similar between the groups at each time point, suggesting that metabolic regulation differs between cows. In conclusion, metabolic regulation in dairy cows is a dynamic system, and differs obviously between cows at different metabolic stages related to parturition.

Performance and metabolic profile of dairy cows during a lactational and deliberately induced negative energy balance with subsequent realimentation

Homeorhetic and homeostatic controls in dairy cows are essential for adapting to alterations in physiological and environmental conditions. To study the different mechanisms during adaptation processes, effects of a deliberately induced negative energy balance (NEB) by feed restriction near 100 d in milk (DIM) on performance and metabolic measures were compared with lactation energy deficiency after parturition. Fifty multiparous cows were studied in 3 periods (1=early lactation up to 12 wk postpartum; 2=feed restriction for 3 wk beginning at 98±7 DIM with a feed-restricted and control group; and 3=a subsequent realimentation period for the feed-restricted group for 8 wk). In period 1, despite NEB in early lactation [-42 MJ of net energy for lactation (NE(L))/d, wk 1 to 3] up to wk 9, milk yield increased from 27.5±0.7 kg to a maximum of 39.5±0.8 kg (wk 6). For period 2, the NEB was induced by individual limitation of feed quantity and reduction of dietary energy density. Feed-restricted cows experienced a greater NEB (-63 MJ of NEL/d) than did cows in early lactation. Feed-restricted cows in period 2 showed only a small decline in milk yield of -3.1±1.1 kg and milk protein content of -0.2±0.1% compared with control cows (30.5±1.1 kg and 3.8±0.1%, respectively). In feed-restricted cows (period 2), plasma glucose was lower (-0.2±0.0 mmol/L) and nonesterified fatty acids higher (+0.1±0.1 mmol/L) compared with control cows. Compared with the NEB in period 1, the decreases in body weight due to the deliberately induced NEB (period 2) were greater (56±4 vs. 23±3 kg), but decreases in body condition score (0.16±0.03 vs. 0.34±0.04) and muscle diameter (2.0±0.4 vs. 3.5±0.4 mm) were lesser. The changes in metabolic measures in period 2 were marginal compared with the adjustments directly after parturition in period 1. Despite the greater induced energy deficiency at 100 DIM than the early lactation NEB, the metabolic load experienced by the dairy cows was not as high as that observed in early lactation. The different effects of energy deficiency at the 2 stages in lactation show that metabolic problems in early lactating dairy cows are not due only to the NEB, but mainly to the specific metabolic regulation during this period.

Milk removal in familiar and unfamiliar surroundings: concentrations of oxytocin, prolactin, cortisol and β –endorphin

Eight cows were machine milked either in an operating theatre or in their familiar barn. During the experiments, milk flow curves were recorded and blood samples were taken for determination of concentrations of oxytocin, prolactin, cortisol and beta-endorphin. The milking cluster was attached without udder preparation. After cessation of milk flow, air was blown into the vagina for 2 min. When milk flow had stopped again, 1 i.u. oxytocin and finally 10 i.u. oxytocin were injected to remove the remaining milk. After the start of milking, oxytocin remained basal in unfamiliar, but increased in familiar surroundings. Therefore, during normal milking only 9% of total milk was removed in unfamiliar, whereas 79% was available in familiar surroundings. In response to subsequent vaginal stimulation in the operating theatre, oxytocin increased transiently in five cows and 15-71% of the milk was removed in these animals. In the other three cows in the operating theatre, oxytocin remained basal during vaginal stimulation, and no more milk was available. After injection of 1 i.u. oxytocin, 56 and 11%, and after injection of 10 i.u. oxytocin, 13 and 8% of milk was removed in unfamiliar and familiar surroundings respectively. Concentrations of prolactin increased during the course of milking in both treatments. Premilking concentrations of cortisol and beta-endorphin were elevated in unfamiliar as compared with familiar surroundings. During the course of milking, cortisol increased slightly and beta-endorphin decreased in unfamiliar, whereas both hormones increased markedly during milking in familiar surroundings.(ABSTRACT TRUNCATED AT 250 WORDS)

Milk ejection in dairy cows at different degrees of udder filling.

Occurrence of milk ejection and course of milk removal were investigated in 18 dairy cows at milking intervals of 4, 8 and 12 h in early, mid or late lactation. Milk ejection occurred fastest in early lactation at a milking interval of 12 h and was delayed at short milking intervals and in late lactation. Storage capacity of the udder was estimated and the actual milk yields of experimental milkings were calculated as a percentage of storage capacity, i.e. degree of udder filling. It was shown that the occurrence of milk ejection after the start of teat stimulation is a function of udder filling. The relationship between the degree of udder filling and the delay from the start of milking until commencement of milk ejection followed a linear regression curve. Changes in occurrence and course of milk ejection have to be considered in practical milking, mainly in late stages of lactation and after short milking intervals. In automatic milking systems where variable and sometimes extremely short milking intervals occur, the duration of pre-milking udder preparation should be adapted to the expected milk yield at each individual milking procedure.

Simultaneous recording of oxytocin release, milk ejection and milk flow during milking of dairy cows with and without prestimulation

The effects of manual teat stimulation compared with stimulation by the liner of a conventional milking machine and the influence of milk ejection on the course of milk flow during the entire milking were investigated. Twelve cows were milked with or without prestimulation on three quarters and milk flow was recorded while intramammary pressure (IMP) was recorded in the left front quarter and blood samples for the determination of oxytocin (OT) were taken at 1 min intervals. OT concentrations increased very similarly in response to manual teat stimulation or liner stimulation and remained elevated during the entire milking procedure. Milk flow curves were generally bimodal without stimulation and not bimodal during milking after prestimulation. Milk yield was not significantly lower in milkings without than with stimulation, whereas machine-on time was prolonged and peak milk flow rate was reduced during milking without stimulation. Time to reach milk flow plateau, time to reach peak flow rate and IMP profiles were similar in both treatments if time was calculated from the start of stimulation. IMP increased similarly in response to manual or liner teat stimulation. In milkings with and without prestimulation IMP did not reach its maximum until the start of milking. In bimodal milk flow curves the second increase of milk flow occurred concomitantly with the start of IMP increase; ejection IMP generally occurred concomitantly with the milk flow plateau. In some cases during milking without prestimulation only small and transient CT release was observed. In response, IMP did not increase to its maximum and increased further in response to a stripping-related CT release. During the course of milking IMP decreased slightly because of the emptying of the three milked quarters. After the end of milking, and with CT concentrations decreasing to the premilking values, IMP again decreased slightly to another plateau, probably owing to myoepithelial relaxation. Thus the course of milk ejection, i.e. the IMP profile, influenced milk removal markedly during early milking.

Continuously elevated concentrations of oxytocin during milking are necessary for complete milk removal in dairy cows

The importance of elevated concentrations of oxytocin (OT) during the entire milking period was investigated in seven primiparous dairy cows with inherent disturbed milk ejection and in sixteen healthy cows with disturbed milk ejection induced by placing them in an operating theatre. Disturbance of milk removal in both groups has previously been demonstrated to be exclusively due to central blockage of the expected OT release in response to teat stimulation and milking. However, milk ejection can be induced by exogenous OT. OT (0.2 i.u.) was injected i.v. before milking and 49 +/- 6% of the total milk was removed. When plasma OT decreased, milk flow stopped. In response to a second and third injection of 0.2 i.u. OT, 30 +/- 4 and 7 +/- 2% of the milk were removed respectively. The remaining milk was removed with 10 i.u. OT. The lag time from injection of OT to the start of milk flow was inversely correlated with the amount of milk actually removed in response to the OT injection. If 0.2 i.u. OT was injected during intramammary pressure (IMP) recording, IMP immediately increased to its maximum value. After 2.5 +/- 0.3 min, IMP decreased to an intermediate IMP (between preinjection and maximum IMP). After two additional injections of 0.2 i.u. OT and after injection of 0.5 i.u. OT, IMP increased to a similar maximum. However, after injection of 0.5 i.u. OT, maximum IMP lasted longer (2.9 +/- 0.3 min; P < 0.05) than after injections of 0.2 i.u. If OT was continuously infused (0.15 i.u./min) during milking, milk flow lasted until the udder was completely emptied. IMP increased during OT infusion to a maximum which remained stable until infusion was stopped after 10 min. The same IMP maximum was reached after the first individual OT injection (0.2 i.u.), but when plasma OT decreased towards basal concentrations, milk flow ceased and IMP decreased to an intermediate level. Thus continuously elevated OT concentrations such as those during infusion or during normal milking are necessary for complete milk removal.

Individual variability in physiological adaptation to metabolic stress during early lactation in dairy cows kept under equal conditions

This study was conducted to investigate individual metabolic and endocrine adaptation to lactation under conditions of identical housing and feeding conditions in high-yielding dairy cows. Forty-five cows were studied on a research farm under standardized but practical conditions. From wk 2 before calving until wk 14 postpartum, blood samples were collected at weekly intervals and assayed for blood chemistry and various metabolites and hormones. Body weight, BCS, and backfat thickness were also recorded weekly. Milk yield, milk composition, and feed intake and energy balance were accordingly measured during the postpartum phase. The animals were retrospectively classified according to their plasma concentration of beta-hydroxybutyrate (BHB): cows in which a BHB threshold of 1 mM was exceeded at least once during the experiment were classified as BHB positive (BHB+); cows with BHB values consistently below this threshold were classified as BHB negative (BHB -). Using this classification, differences for NEFA and glucose concentrations were observed, but the mean calculated energy balance did not differ between the groups during the experimental period (-22.2 MJ of NE(1)/d +/- 4.7 for BHB+ and -18.9 MJ of NE(1)/d +/- 4.9 for BHB-). In BHB+ cows, the peripartum decrease (P < 0.05) of BW, BCS, and backfat thickness was more pronounced than in BHB- cows. Mean milk yields did not differ between groups. However, BHB+ cows had greater milk fat and lesser milk protein contents (P < 0.05), resulting in a greater (P < 0.05) fat:protein ratio than in BHB- cows. Thus, to some extent, cows were able to compensate for the negative energy balance by adjustments in performance. Milk acetone concentrations followed BHB concentrations in blood. Insulin-like growth factor-I and leptin concentrations were greater (P < 0.05) in BHB- cows during the time of observation than in the BHB+ cows. Comparing the reproductive variables recorded (first increase of progesterone, first service conception rate, number of services per conception, interval from calving to first AI, interval from first AI to conception, and days open) between the 2 groups yielded no significant differences. Our findings imply that despite comparable energy balance, there is considerable individual variation of the adaptive ability of cows during early lactation based on a variety of metabolic and endocrine variables.

The innate immune response of the bovine mammary gland to bacterial infection.

Intra-mammary (IM) bacterial infection in cattle can result in clinical outcomes that range from being acute and life-threatening to those that are chronic and sub-clinical. The typical bacteria involved in IM bacterial infections activate the mammary immune system in different ways which can influence the severity of the outcome. A clear understanding of the mechanisms that activate and regulate this response is central to the development of effective preventative and treatment regimes. This review focuses on the different immune responses of the bovine mammary gland to common mastitis-causing pathogens. There is special emphasis on comparing the responses to Escherichia coli and Staphylococcus aureus infections, as these are typically associated, respectively, with acute/severe and chronic/sub-clinical forms of the disease.