Endocrine changes during the peripartal period related to colostrogenesis in mammalian species.

This review discusses endocrine and functional changes during the transition from late gestation to lactation that are related to the production of colostrum in different mammalian species. Species covered in this article include ungulate species (cattle, sheep, goats, pigs, horses), rodents (rat, mouse), rabbits, and carnivores (cats, dogs), as well as humans. An immediate availability of high quality colostrum for the newborn after birth is crucial in species where a transfer of immunoglobulins (Ig) does not or only partially occur via the placenta during pregnancy. Declining activity of gestagens, in most species progesterone (P4), is crucial at the end of pregnancy to allow for the characteristic endocrine changes to initiate parturition and lactation, but the endocrine regulation of colostrogenesis is negligible. Both, the functional pathways and the timing of gestagen withdrawal differ considerably among mammalian species. In species with a sustaining corpus luteum throughout the entire pregnancy (cattle, goat, pig, cat, dog, rabbit, mouse, and rat), a prostaglandin F2α (PGF2α)-induced luteolysis shortly before parturition is assumed to be the key event to initiate parturition as well as lactogenesis. In species where the gestagen production is taken over by the placenta during the course of gestation (e.g., sheep, horse, and human), the reduction of gestagen activity is more complex, as PGF2α does not affect placental gestagen production. In sheep the steroid hormone synthesis is directed away from P4 towards estradiol-17ß (E2) to achieve a low gestagen activity at high E2 concentrations. In humans the uterus becomes insensitive to P4, as parturition occurs despite still high P4 concentrations. However, lactogenesis is not completed as long as P4 concentration is high. Early colostrum and thus Ig intake for immune protection is not needed for the human newborn which allows a delayed onset of copious milk secretion for days until the placenta expulsion causes the P4 drop. Like humans, horses do not need low gestagen concentrations for successful parturition. However, newborn foals need immediate immune protection through Ig intake with colostrum. This requires the start of lactogenesis before parturition which is not fully clarified. The knowledge of the endocrine changes and related pathways to control the key events integrating the processes of colostrogenesis, parturition, and start of lactation are incomplete in many species.

This manuscript reviews and compares hormonal and functional changes occurring in the conceptus (embryo and its extra-embryonic membranes) and their effects on the mammary gland during development from pregnancy to colostrum formation and milk production in multiple mammalian species. Declining activity of gestagens at the end of pregnancy is crucial to allow for both parturition and onset of milk production in most mammals. Strategies to achieve this state of low gestagen activity are different among species. In species where the corpus luteum is sustained throughout the entire pregnancy, luteolysis is the key event to initiate parturition and onset of milk secretion (cattle, goat, pig, cat, dog, rat, mouse, rabbit). However, in species where the placenta takes over gestagen production during the course of pregnancy, the achievement of a state of low gestagen activity is more complex. It ranges from redirection of the hormone production pathway away from gestagens in sheep, to decreasing sensitivity of the uterus towards gestagens in humans. In the horse, there is evidence pointing towards redirection of the hormone production as well as a decrease in sensitivity towards gestagens, but the exact mechanisms are still not clarified.

Inhibition of lipopolysaccharide-induced suppression of luteal function in isolated perfused bovine ovaries.

Recently, we observed that lipopolysaccharide (LPS) suppresses corpus luteum (CL) function in isolated perfused ovaries. It remained unclear if this suppression was due to increased luteal PGF2α secretion or LPS-induced apoptosis. Therefore, possible impacts of PGF2α and LPS were inhibited by a non-steroidal anti-inflammatory drug (flunixin) and an endotoxin-binding agent (polymyxin B), respectively. Bovine ovaries with a mid-cycle CL were collected immediately after slaughter and perfused for 240 min. After 50 min of equilibration, either flunixin or polymyxin B (5 µg/ml of each) were added to the perfusion medium of six ovaries, respectively. All ovaries (n = 12) were treated with E. coli LPS (0.5 µg/ml) 60 min after the onset of perfusion, and received 500 I.U. of hCG after 210 min of perfusion. Progesterone and PGF2α were measured in the effluent perfusate every 10 and 30 min, respectively. Biopsies of the CL were collected every 60 min to determine the mRNA expression of the cytokine TNFA and factors of apoptosis (CASP3, -8). Flunixin-treatment inhibited the increase of PGF2α after LPS-challenge that was observed in the polymyxin B-treated (PX-LPS) ovaries. After hCG-stimulation, progesterone secretion increased (P < 0.05) in group PX-LPS but not in the flunixin-treated (F-LPS) ovaries. Compared to initial values before LPS-challenge, luteal mRNA expression of TNFA and CASP3 was increased (P < 0.05) in group F-LPS at 120 and 180 min, respectively, and those of CASP8 was decreased (P < 0.05) in PX-LPS at 60 and 120 min after LPS-treatment. In conclusion, although flunixin managed to inhibit PGF2α, it did not suffice to successfully prevent LPS-induced apoptosis. However, endotoxin-binding polymyxin B resulted in luteal responsiveness to hCG after LPS-challenge.

Effects of an oral hydro-ethanolic purple coneflower extract on performance, clinical health and immune parameters in calves.

The objective of this randomized, placebo-controlled, double-blinded field trial was to investigate the effects of oral administration of purple coneflower (Echinacea purpurea L. (EP)) on performance, health and immune parameters in calves. Calves (n = 27) were enrolled to three groups (9 calves per group): 0.5 g EP/calf per day (ECL), 5 g EP/calf per day (ECH) or placebo. Calves were vaccinated with Bluetongue-Virus (BTV) serotype 4 vaccine to investigate EPs effects on seroconversion. Clinical and performance parameters, inter alia body weight, health and milk intake were recorded for 57 days. Blood samples were analyzed for BTV antibodies and IgG by ELISA, white and red blood cell counts by flow cytometry and mRNA abundance of various inflammatory markers in leukocytes (IL-1ß, IL-8, tumor necrosis factor α (TNFα), cyclooxygenase 2 (Cox-2) and prostaglandin E synthase) was studied. The findings demonstrated no differences between groups regarding performance parameters. In all groups, calves suffered from diarrhea for a minimum of 2 days, but EP reduced the number of diarrhea days by 44% in ECL and increased the body temperature. Interestingly, ECL resulted in an increased number of respiratory disease days during the follow-up period. EP did not change blood cell and IgG counts, whereas eosinophil granulocytes were reduced in ECL. Decreased levels of hemoglobin and hematocrit were found in ECH. Prostaglandin E synthase levels in leukocytes were higher in ECL and ECH, whereas no differences were obtained for IL-1ß, IL-8, TNFα and Cox-2. Due to the unexpected occurrence of BTV seropositive calves before the first vaccination, 13 calves were excluded from the evaluation on seroconversion and no statistical analyses could be performed regarding antibody production. BTV-4 antibodies were not produced in 4 placebo-calves, whereas 4 of 5 and 1 of 6 ECL- and ECH-calves produced antibodies. Further investigations are needed to draw final conclusions on mode of action and efficacy of EP in calves.

Increased teat wall thickness in response to machine milking.

Transiently increased teat wall thickness in response to machine milking has been documented by various methods, including ultrasound. However, correlative ultrasonography and histology to detect the origin of this phenomenon is lacking. The first goal of the present study was to evaluate and compare milking-related changes of the teat tissue in 2 breeds of dairy cows (11 Simmental and 3 Holstein) using B-mode ultrasonography. Additionally, the observed changes were compared with ultrasonographic findings in a Holstein cow with periparturient udder edema. Finally, corresponding histological sections of the Simmental teats were analyzed and compared with those from a lactating nonmilked Angus cow. We hypothesized that the mechanical load of both stretching by the vacuum during phases of open teat cup liner and compression by the closed liner during machine milking results in a transient congestion of blood vessels in the teat wall. The barrel of 1 front teat of each cow was scanned immediately before and after machine milking (system vacuum: 42 kPa; pulsation rate: 60 cycles/min; pulsation ratio: 65:35). Shortly after milking (33 ± 6 min), the Simmentals were slaughtered, and their scanned teat was immediately removed and processed for investigation by light microscopy. Ultrasonography after milking revealed anechoic tubular structures mainly in the inner half of the teat wall. Histological examination revealed these structures to be thick-walled veins. The left front and hind teats of the nonmilked lactating cow, collected and prepared identically to those from the Simmental cows, showed the same histological features. Ultrasonographic measurements showed that the diameter of these veins significantly increased after milking compared with matching images before milking. This effect was most pronounced in the Holstein cows. Similarly, these veins were very prominent in the periparturient cow. However, neither the milked cows, including the periparturient cow, nor the lactating nonmilked cow provided any evidence of edematous extravasation on ultrasonography or histology. These findings corroborated our hypothesis that the increase in size of thick-walled veins in the teat tissue is the main reason for the thickening of the teat walls in response to machine milking.

Colostrogenesis: Role and Mechanism of the Bovine Fc Receptor of the Neonate (FcRn).

Colostrogenesis is a separate and unique phase of mammary epithelial cell activity occurring in the weeks before parturition and rather abruptly ending after birth in the bovine. It has been the focus of research to define what controls this process and how it produces high concentrations of specific biologically active components important for the neonate. In this review we consider colostrum composition and focus upon components that appear in first milked colostrum in concentrations exceeding that in blood serum. The Fc Receptor of the Neonate (FcRn) is recognized as the major immunoglobulin G (IgG) and albumin binding protein that accounts for the proteins' long half-lives. We integrate the action of the pinocytotic (fluid phase) uptake of extracellular components and merge them with FcRn in sorting endosomes. We define and explore the means of binding, sorting, and the transcytotic delivery of IgG1 while recycling IgG2 and albumin. We consider the means of releasing the ligands from the receptor within the endosome and describe a new secretion mechanism of cargo release into colostrum without the appearance of FcRn itself in colostrum. We integrate the insulin-like growth factor family, some of which are highly concentrated bioactive components of colostrum, with the mechanisms related to FcRn endosome action. In addition to secretion, we highlight the recent findings of a role of the FcRn in phagocytosis and antigen presentation and relate its significant and abrupt change in cellular location after parturition to a role in the prevention and resistance to mastitis infections.

Different vacuum levels, vacuum reduction during low milk flow, and different cluster detachment levels affect milking performance and teat condition in dairy cows.

Traditionally, machine milking is performed at a constant vacuum supply. The system vacuum has to be set high enough to allow a sufficiently high vacuum at the teat end, despite the inevitable vacuum drop caused by milk flow. This leads to an increased vacuum load on the teat, especially when milk flow ceases at the end of milking. We tested the hypothesis that a milk flow-controlled adaptation of vacuum settings during milking allows even higher vacuum levels than are usually recommended during the period of high milk flow if the vacuum is reduced during low milk flow. Combined with a high cluster detachment flow rate level, increased milking performance is expected without an increased effect on teat tissue. Ten Holstein dairy cows were milked with a bucket milker with the claw vacuum adjusted in the absence of milk flow at a regular (43 kPa) and high (48 kPa) claw vacuum, with and without vacuum reduction during low milk flow (<2 kg/min), and combined with different cluster detachment levels (0.2, 0.6, and 1 kg/min). Each treatment was applied in each cow during 4 subsequent milkings in a randomized crossover design. Both claw vacuum and milk flow were continuously recorded throughout milking. Teat tissue thickness was measured using a cutimeter and teat wall diameter was measured by B-mode ultrasonography at 5 min after the end of milking. Milk yield was not affected by either vacuum settings or detachment levels. Machine-on time in treatments with vacuum reduction was shorter at high than at low vacuum and decreased with increasing detachment levels. Average milk flow was higher at high than at low vacuum and reached highest values in milkings without vacuum reduction at both vacuum levels. The average milk flow was higher at a cluster detachment of 1 kg/min than at 0.2 kg/min. However, both teat tissue thickness and (as a tendency) teat wall diameter at 5 min after cluster detachment were higher in milkings at high vacuum without vacuum reduction compared with all other treatments. In conclusion, high claw vacuum up to 48 kPa increases milking performance because of higher milk flow and reduced machine-on time. Negative effects of high vacuum on teat tissue are prevented by reducing vacuum during low milk flow (<2 kg/min) at the start and end of milking. Additionally, using a high cluster detachment level reduces machine-on time without a loss of harvested milk.

Short communication: Teat wall diameter and teat tissue thickness in dairy cows are affected by intramammary pressure and by the mechanical forces of machine milking.

The objective of this study was to validate and apply 2 different methods to record changes in teat tissue related to machine milking. Teat wall diameter was measured via B-mode ultrasound cross sectioning with a 7.5-MHz linear probe. Teat tissue thickness was measured using a cutimeter (spring-loaded caliper, spring constant 6.5 N/cm, 0.5 N at closed jaws). Both methods were applied at the teat barrel, 2 cm above the teat tip. In experiment 1, 24 teats from freshly slaughtered cows were used to perform ultrasound imaging (12 teats) or cutimeter measurements (12 teats) while the teat cisterns were filled with water to increase the intracisternal pressure from 0 to 30 kPa in steps of 1 kPa. Teat tissue thickness did not change at an intracisternal pressure from 0 to 10 kPa but increased with intracisternal pressure at levels >10 kPa. In contrast, teat wall diameter decreased with intracisternal pressure between 0 and 7 kPa but did not significantly change at a pressure ≥7 kPa up to 30 kPa. Significant Pearson correlation coefficients between intracisternal pressure and teat wall diameter were observed from 0 to 7 kPa (r = -0.38), and between intracisternal pressure and teat tissue thickness from 10 to 30 kPa (r = 0.45). In experiment 2, ultrasound and cutimeter measurements were performed in 12 lactating Holstein cows. Measurements before and during milking, immediately after cluster removal, with normal milking or with a 5-min overmilking, were performed and continued at 5-min intervals for 60 min and at 10-min intervals until 120 min. Additionally, with the 5-min overmilking treatment, measurements were continued at 60-min intervals up to 10 h after milking. Teat wall diameter decreased in response to milk ejection, followed by a continuous increase during the course of milking, with highest values after 5 min overmilking. Teat tissue thickness did not change during milking but was significantly increased after overmilking. Teat wall diameter and teat tissue thickness recovered to premilking levels within 35 min after normal milking and within 60 min after overmilking. Until 10 h after overmilking, the teat wall diameter decreased steadily, whereas teat tissue thickness was unfluctuating. In the physiologically relevant range of intramammary pressure, ultrasound measurements of the teat wall were affected by both intramammary pressure and mechanical forces, whereas cutimeter measurements were not affected by the intramammary pressure.

Effect of Different Sources of Supplemental Zinc on Performance, Nutrient Digestibility, and Antioxidant Enzyme Activities in Lambs.

Zinc (Zn) is an essential element in the growth of all animals and plays structural and catalytic roles in many enzymes and functional proteins. Two completely randomized trials were conducted to evaluate the effects of different sources of zinc on performance, nutrient digestibility, blood mineral profile, and antioxidant enzyme activities in male growing lambs on a barley-based diet. The first trial was conducted for 70 days and consisted of 30 lambs (30.8 ± 2.8 kg mean body weight, 4-5 months of age) which were randomly allocated to five treatments consisting of a basal diet (19.72 mg Zn/kg DM), or the basal diet supplemented with 30 mg Zn/kg DM, added as either zinc-sulfate (ZnSulf; inorganic), zinc-methionine (ZnMet), zinc-proteinate (ZnProt) or zinc-glycinate (ZnGly). For the second trial, to measure the effects of dietary Zn on nutrient digestibility, four lambs from each group of the first experiment were randomly allocated to individual digestibility cages for 12 days (first 7 days as an adaptation period followed by 5 days of sample collection). Among the groups, dietary Zn supplementation above basal level significantly improved average daily gain, average daily feed intake, feed/gain ratio, and superoxide dismutase activity of red blood cells (P < 0.05). Glutathione peroxidase activity of lambs supplemented with organic Zn was significantly (P < 0.05) higher than inorganic and control groups. At the end of the trial, the concentration of plasma Zn, tri-iodothyronine (T3), thyroxine (T4), and the activity of alkaline phosphatase was increased (P < 0.05) in all groups receiving Zn as compared with controls (P < 0.05). In addition, thyroxine level in animals supplemented with Zn-methionine and Zn-proteinate was greater than in animals receiving Zn-glycine and Zn-sulfate. The results of the second trial revealed that the supplementation with Zn-methionine and Zn-proteinate increased the digestibility of crude protein (CP) and acid detergent fiber (ADF) compared to groups supplemented with Zn sulfate and control (P < 0.05). All organic sources of Zn improved organic matter (OM) digestibility compared to inorganic and control (P < 0.05). Results indicated that, regardless of source, supplementation of Zn in growing lambs improved growth performance, blood antioxidants, and thyroid hormone levels. Furthermore, Zn-methionine and Zn-proteinate supplementation appeared to improve the digestibility of CP, OM, and ADF more effectively than Zn-sulfate.

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.

Differential somatic cell count in milk before, during, and after lipopolysaccharide- and lipoteichoic-acid-induced mastitis in dairy cows.

Intramammary infections induce the initiation of the inflammatory response, resulting in an increase in somatic cell count (SCC) in milk. The SCC includes several different types of cells but does not differentiate between them. On the contrary, the new differential somatic cell count (DSCC) parameter allows for the differentiation between 2 groups of cells: polymorphonuclear neutrophils (PMN) and lymphocytes versus macrophages. Therefore, the aim of this paper was to describe the changes of both DSCC and SCC during mastitis induced by cell wall components from typical mastitis-causing pathogens [lipopolysaccharide (LPS), Escherichia coli; lipoteichoic acid (LTA), Staphylococcus aureus] known to trigger different severities of mastitis. In addition, the effect the glucocorticoid prednisolone (PRED), which is known to attenuate the immune response in the mammary gland, was investigated. Twenty dairy cows were equally divided into 5 groups and treated with LPS, LTA, LPS+PRED, LTA+PRED, or a saline control. Milk samples were taken at the following time points: baseline (d -3, -2, and -1), right before treatment (d 0), 5 h after treatment (d 0.2), early cure phase (d 1 and 2), and late cure phase (d 3, 4, 5, 6, 7, and 14) and analyzed for DSCC and SCC. Mean DSCC values increased significantly from <60% at baseline and right before treatment to >81% 5 h after treatment and the early cure phase in all groups, except for the groups control and LTA+PRED. This increase clearly reflects a shift in cell populations to predominantly PMN. The SCC increased significantly following the stimulation, too, as expected. Interestingly, we observed cases where SCC increased moderately only whereas DSCC showed an evident increase, meaning that the shift in cell populations occurred even at low SCC levels. The PRED clearly lowered the cell migration in group LTA+PRED. This is the first ever study investigating DSCC during induced mastitis under controlled conditions. The combination of DSCC and SCC could be employed for the earlier detection of mastitis by revealing the shift in cell population independent from the SCC level. Furthermore, combining DSCC and SCC information could help to determine the stage of mastitis because we observed high DSCC and SCC results in the early stage of mastitis but evidently lower DSCC and high SCC in the cure phase. Hence, our results offer the first fundamental insights on how mastitis monitoring could be improved in the frame of dairy herd improvement programs.

Short communication: Differential loss of bovine mammary epithelial barrier integrity in response to lipopolysaccharide and lipoteichoic acid.

In the mammary gland, the blood-milk barrier prevents an uncontrolled intermixture of blood and milk constituents and hence maintains the osmotic gradient to draw water into the mammary secretion. During mastitis, the permeability of the blood-milk barrier is increased, which is reflected by the transfer of blood constituents into milk and vice versa. In this study, we aimed to investigate changes in the barrier function of mammary epithelial cells in vitro as induced by cell wall components of different pathogens. Primary bovine mammary epithelial cells from 3 different cows were grown separately on Transwell (Corning Inc., Corning, NY) inserts. The formation of tight junctions between adjacent epithelial cells was shown by transmission electron microscopy and by immunofluorescence staining of the tight junction protein zona occludens-1. The integrity of the epithelial barrier was assayed by means of transepithelial electrical resistance, as well as by diffusion of the fluorophore Lucifer yellow across the cell layer. The release of lactate dehydrogenase (LDH) was used as an indicator for cytotoxic effects. In response to a 24-h challenge with bacterial endotoxin, barrier integrity was reduced after 3 or 7h, respectively, in response to 0.5mg/mL lipopolysaccharide (LPS) from Escherichia coli or 20mg/mL lipoteichoic acid (LTA) from Staphylococcus aureus. No paracellular leakage was observed in response to 0.2mg/mL LPS or 2mg/mL LTA. Although LPS and LTA affected barrier permeability, most likely by opening the tight junctions, only LPS caused cell damage, reflected by increased LDH concentrations in cell culture medium. These results prove a pathogen-specific loss of blood-milk barrier integrity during mastitis, which is characterized by tight junction opening by both LPS and LTA and by additional epithelial cell destruction through LPS.

Assessment of the expression of biomarkers of uremic inflammation in dogs with renal disease.

OBJECTIVE: To assess the expression of inflammatory cytokines and enzymes in venous whole blood of dogs with impaired renal function attributable to various causes. ANIMALS: 46 dogs with acute kidney injury (AKI), 8 dogs with chronic kidney disease (CKD), and 10 healthy dogs. PROCEDURES: Dogs with AKI and CKD were prospectively enrolled during 2010 if they met inclusion criteria. Demographic and laboratory characteristics were evaluated for each dog, and expression of inflammatory cytokines (interleukin [IL]-1α, IL-1ß, IL-8, tumor necrosis factor [TNF]-α, IL-10, and transforming growth factor [TGF]-ß) and enzymes (inducible nitric oxide synthase [iNOS] and 5-lipoxygenase [5-LO]) was measured in venous whole blood obtained at initial evaluation. RESULTS: Dogs with impaired renal function had markedly higher expression of the cytokines IL-1α, IL-1ß, and TGF-ß and the enzyme 5-LO, compared with expression in healthy dogs. Additionally, 17 of 46 AKI dogs (but none of the CKD dogs) had higher IL-8 mRNA expression and 3 of 8 CKD dogs (but only 2/46 AKI dogs) had higher TNF-α expression, compared with results for healthy dogs. No significant difference between renal disease groups was detected for inflammatory markers and laboratory variables, degree of azotemia, or cause of impaired renal function. CONCLUSIONS AND CLINICAL RELEVANCE: In this study, expression of the cytokines IL-1α, IL-1ß, and TGF-ß and the enzyme 5-LO was clearly increased in dogs with renal disease, which suggested that these markers were part of an inflammatory response in animals with AKI or CKD.

Expression Profile of Cytokines and Enzymes mRNA in Blood Leukocytes of Dogs with Leptospirosis and Its Associated Pulmonary Hemorrhage Syndrome.

BACKGROUND: Dogs with leptospirosis show similar organ manifestations and disease course as human patients, including acute kidney injury and pulmonary hemorrhage, making this naturally-occurring infection a good animal model for human leptospirosis. Expression patterns of cytokines and enzymes have been correlated with disease manifestations and clinical outcome in humans and animals. The aim of this study was to describe mRNA expression of pro- and anti-inflammatory mediators in canine leptospirosis and to compare it with other renal diseases to identify patterns characterizing the disease and especially its pulmonary form. METHODOLOGY AND PRINCIPAL FINDINGS: The mRNA abundance of cytokines (IL-1α, IL-1ß, IL-8, IL-10, TNF-α, TGF-ß) and enzymes (5-LO, iNOS) was measured prospectively in blood leukocytes from 34 dogs with severe leptospirosis and acute kidney injury, including 22 dogs with leptospirosis-associated pulmonary hemorrhages. Dogs with leptospirosis were compared to 14 dogs with acute kidney injury of other origin than leptospirosis, 8 dogs with chronic kidney disease, and 10 healthy control dogs. Canine leptospirosis was characterized by high 5-LO and low TNF-α expression compared to other causes of acute kidney injury, although the decreased TNF-α expression was also seen in chronic kidney disease. Leptospirosis-associated pulmonary hemorrhage was not characterized by a specific pattern, with only mild changes noted, including increased IL-10 and decreased 5-LO expression on some days in affected dogs. Fatal outcome from pulmonary hemorrhages was associated with low TNF-α, high IL-1ß, and high iNOS expression, a pattern possibly expressed also in dogs with other forms of acute kidney injury. CONCLUSION: The patterns of cytokine and enzyme expression observed in the present study indicate a complex pro- and anti-inflammatory response to the infection with leptospires. The recognition of these signatures may be of diagnostic and prognostic relevance for affected individuals and they may indicate options for newer therapies targeting the identified pathways.

The immune response of bovine mammary epithelial cells to live or heat-inactivated Mycoplasma bovis.

Mycoplasma bovis is an emerging bacterial agent causing bovine mastitis. Although these cell wall-free bacteria lack classical virulence factors, they are able to activate the immune system of the host. However, effects on the bovine mammary immune system are not yet well characterized and detailed knowledge would improve the prevention and therapy of mycoplasmal mastitis. The aim of this study was to investigate the immunogenic effects of M. bovis on the mammary gland in an established primary bovine mammary epithelial cell (bMEC) culture system. Primary bMEC of four different cows were challenged with live and heat-inactivated M. bovis strain JF4278 isolated from acute bovine mastitis, as well as with the type strain PG45. The immune response was evaluated 6 and 24h after mycoplasmal challenge by measuring the relative mRNA expression of selected immune factors by quantitative PCR. M. bovis triggered an immune response in bMEC, reflected by the upregulation of tumor necrosis factor-α, interleukin(IL)-1ß, IL-6, IL-8, lactoferrin, Toll-like receptor-2, RANTES, and serum amyloid A mRNA. Interestingly, this cellular reaction was only observed in response to live, but not to heat-inactivated M. bovis, in contrast to other bacterial pathogens of mastitis such as Staphylococcus aureus. This study provides evidence that bMEC exhibit a strong inflammatory reaction in response to live M. bovis. The lack of a cellular response to heat-inactivated M. bovis supports the current hypothesis that mycoplasmas activate the immune system through secreted secondary metabolites.

Increasing serotonin concentrations alter calcium and energy metabolism in dairy cows.

A 4×4 Latin square design in which varied doses (0, 0.5, 1.0, and 1.5 mg/kg) of 5-hydroxy-l-tryptophan (5-HTP, a serotonin precursor) were intravenously infused into late-lactation, non-pregnant Holstein dairy cows was used to determine the effects of serotonin on calcium and energy metabolism. Infusion periods lasted 4 days, with a 5-day washout between periods. Cows were infused at a constant rate for 1 h each day. Blood was collected pre- and 5, 10, 30, 60, 90, and 120 min post-infusion, urine was collected pre- and post-infusion, and milk was collected daily. All of the 5-HTP doses increased systemic serotonin as compared to the 0 mg/kg dose, and the 1.0 and 1.5 mg/kg doses increased circulating glucose and non-esterified fatty acids (NEFA) and decreased beta-hydroxybutyrate (ßHBA) concentrations. Treatment of cows with either 1.0 or 1.5 mg/kg 5-HTP doses decreased urine calcium elimination, and the 1.5 mg/kg dose increased milk calcium concentrations. No differences were detected in the heart rates, respiration rates, or body temperatures of the cows; however, manure scores and defecation frequency were affected. Indeed, cows that received 5-HTP defecated more, and the consistency of their manure was softer. Treatment of late-lactation dairy cows with 5-HTP improved energy metabolism, decreased loss of calcium into urine, and increased calcium secretion into milk. Further research should target the effects of increasing serotonin during the transition period to determine any benefits for post-parturient calcium and glucose metabolism.

Effect of intramammary administration of prednisolone on the blood-milk barrier during the immune response of the mammary gland to lipopolysaccharide.

OBJECTIVE: To investigate effects of intramammary administration of prednisolone on the immune response of mammary glands in cows. ANIMALS: 5 lactating Red Holsteins. PROCEDURES: Cows received a different intramammary infusion in each mammary gland (10 mg of prednisolone, 100 µg of lipopolysaccharide [LPS], 100 µg of LPS and 10 mg of prednisolone, or saline [0.9% NaCl] solution). Milk samples were collected before (time 0) and 3, 6, 9, 12, 24, and 36 hours after treatment. Somatic cell count (SCC), lactate dehydrogenase (LDH) activity, and concentrations of serum albumin (SA) and tumor necrosis factor (TNF)-α in milk and mRNA expression of TNF-α, interleukin (IL)-8, and IL-1ß in milk somatic cells were analyzed. RESULTS: Saline solution or prednisolone did not change SCC, LDH activity, and SA and TNF-α concentrations in milk and mRNA expression of TNF-α, IL-1ß, and IL-8 in milk somatic cells. The SCC and TNF-α concentration in milk increased similarly in glands infused with LPS, independent of prednisolone administration. However, the increase of LDH activity and SA concentration in milk after LPS infusion was diminished by prednisolone administration. The mRNA expression of TNF-α, IL-8, and IL-1ß in milk somatic cells increased after LPS infusion and was unaffected by prednisolone. CONCLUSIONS AND CLINICAL RELEVANCE: Intramammary administration of prednisolone did not induce an immune response and did not change mRNA expression of TNF-α, IL-8, and L-1ß during the response to intramammary administration of LPS. However, prednisolone reduced disruption of the blood-milk barrier. This could influence the severity and cure rate of mastitis.

The inflammatory response of primary bovine mammary epithelial cells to Staphylococcus aureus strains is linked to the bacterial phenotype.

Staphylococcus aureus is a major mastitis-causing pathogen in dairy cows. The latex agglutination-based Staphaurex test allows bovine S. aureus strains to be grouped into Staphaurex latex agglutination test (SLAT)-negative [SLAT(-)] and SLAT-positive [SLAT(+)] isolates. Virulence and resistance gene profiles within SLAT(-) isolates are highly similar, but differ largely from those of SLAT(+) isolates. Notably, specific genetic changes in important virulence factors were detected in SLAT(-) isolates. Based on the molecular data, it is assumed that SLAT(+) strains are more virulent than SLAT(-) strains. The objective of this study was to investigate if SLAT(-) and SLAT(+) strains can differentially induce an immune response with regard to their adhesive capacity to epithelial cells in the mammary gland and in turn, could play a role in the course of mastitis. Primary bovine mammary epithelial cells (bMEC) were challenged with suspensions of heat inactivated SLAT(+) (n = 3) and SLAT(-) (n = 3) strains isolated from clinical bovine mastitis cases. After 1, 6, and 24 h, cells were harvested and mRNA expression of inflammatory mediators (TNF-α, IL-1ß, IL-8, RANTES, SAA, lactoferrin, GM-CSF, COX-2, and TLR-2) was evaluated by reverse transcription and quantitative PCR. Transcription (ΔΔCT) of most measured factors was induced in challenged bMEC for 6 and 24 h. Interestingly, relative mRNA levels were higher (P<0.05) in response to SLAT(+) compared to SLAT(-) strains. In addition, adhesion assays on bMEC also showed significant differences between SLAT(+) and SLAT(-) strains. The present study clearly shows that these two S. aureus strain types cause a differential immune response of bMEC and exhibit differences in their adhesion capacity in vitro. This could reflect differences in the severity of mastitis that the different strain types may induce.

Colostrogenesis: IgG1 transcytosis mechanisms.

Biological transport of intact proteins across epithelial cells has been documented for many absorptive and secretory tissues. Immunoglobulins were some of the earliest studied proteins in this category. The transcellular transport (transcytosis) of immunoglobulins in neonatal health and development has been recognized; the process is especially significant with ungulates because they do not transcytose immunoglobulins across the placenta to the neonate. Rather, they depend upon mammary secretion of colostrum and intestinal absorption of immunoglobulins in order to provide intestinal and systemic defense until the young ungulate develops its own humoral defense mechanisms. The neonatal dairy calf's ability to absorb immunoglobulins from colostrum is assisted by a ~24 h "open gut" phenomenon where large proteins pass the intestinal epithelial cells and enter the systemic system. However, a critical problem recognized for newborn dairy calves is that an optimum mass of colostrum Immunoglobulin G (IgG) needs to be absorbed within that 24 h window in order to provide maximal resistance to disease. Many calves do not achieve the optimum because of poor quality colostrum. While many studies have focused on calf absorption, the principal cause of the problem resides with the extreme variation (g to kg) in the mammary gland's capacity to transfer blood IgG1 into colostrum. Colostrum is a unique mammary secretory product that is formed during late pregnancy when mammary cells are proliferating and differentiating in preparation for lactation. In addition to the transcytosis of immunoglobulins, the mammary gland also concentrates a number of circulating hormones into colostrum. Remarkably, the mechanisms in the formation of colostrum in ungulates have been rather modestly studied. The mechanisms and causes of this variation in mammary gland transcytosis of IgG1 are examined, evaluated, and in some cases, explained.

Effects of a latency period between pre-stimulation and teat cup attachment and periodic vacuum reduction on milking characteristics and teat condition in dairy cows.

The goal of the present study was to examine the suitability of a short pre-stimulation (P) for 15 s followed by a latency period (L) of 30 s before cluster attachment for machine milking. In addition we tested the effect of a periodic reduction of the vacuum under the teat (VR) during the massage phase from 43 kPa to 12-15 kPa on milking characteristics and teat tissue condition. The study was carried out in 9 cows in a cross-over design. Animals were milked twice daily, and each of the 4 treatment combinations was used for six subsequent milkings (P+L vs. continuous P, and standard pulsation vs. VR, respectively). Milk flow was recorded during all experimental milkings. Longitudinal ultrasound cross sections of the teat were performed by B-mode ultrasound after the last milking of each treatment at 0, 5, and 15 min after the end of milking, respectively. None of the evaluated milking characteristics (total milk yield, main milking time, peak flow rate, average milk flow) differed between treatments. Teat measures as obtained by ultrasound cross sections showed no significant difference if individual treatments were compared at the three time points individually. However, teat wall thickness (TWT) tended to be smaller in VR vs. non-VR treatments at 5 min after milking (P=0·05). In conclusion, teat preparation consisting of a short stimulation followed by a latency period represents a similarly efficient pre-stimulation as a continuous pre-stimulation. VR seems to reduce the load on the teat tissue during milking and thus reduces the development of oedema and hence a less pronounced increase of TWT while milking characteristics are similar with or without VR.

Comparison of the effect of a CIDR-Select Synch versus a long-term CIDR based AI protocol on reproductive performance in multiparous dairy cows in Swiss dairy farms.

BACKGROUND: Synchronization programs have become standard in the dairy industry in many countries. In Switzerland, these programs are not routinely used for groups of cows, but predominantly as a therapy for individual problem cows. The objective of this study was to compare the effect of a CIDR-Select Synch and a 12-d CIDR protocol on the pregnancy rate in healthy, multiparous dairy cows in Swiss dairy farms. METHODS: Cows (N = 508) were randomly assigned to CIDR-Select Synch (N = 262) or 12-d CIDR (N = 246) protocols. Cows in the CIDR-Select Synch group received a CIDR and 2.5 ml of buserelin i.m. on d 0. On d 7, the CIDR insert was removed and 5 ml of dinoprost was administered i.m.. Cows in the 12-d CIDR group received the CIDR on d 0 and it was removed on d 12 (the routine CIDR protocol in Swiss dairies). On d 0 a milk sample for progesterone analysis was taken. Cows were inseminated upon observed estrus. Pregnancy was determined at or more than 35 days after artificial insemination. As a first step, the two groups were compared as to indication for treatment, breed, stud book, stall, pasture, and farmer's business using chi square tests or Fisher's exact test. Furthermore, groups were compared as to age, DIM, number of AI's, number of cows per farm, and yearly milk yield per cow using nonparametric ANOVA. A multiple logistic model was used to relate the success of the protocols to all of the available factors; in particular treatment (CIDR-Select Synch/12-d CIDR), milk progesterone value, age, DIM, previous treatment of the uterus, previous gynecological treatment, and number of preceding inseminations. RESULTS: The pregnancy rate was higher in cows following the CIDR-Select Synch compared to the 12-d CIDR protocol (50.4% vs. 22.4%; P < 0.0001). CONCLUSION: The CIDR-Select Synch protocol may be highly recommended for multiparous dairy cows. The reduced time span of the progesterone insert decreased the number of days open, improved the pregnancy rate compared to the 12-d CIDR protocol and the cows did not to have to be handled more often.