Technical note: immunomagnetic procedure for positive selection of macrophages in ovine milk.

A simple immunomagnetic procedure was developed to select macrophages from ovine milk by using a non-specific magnetic positive separation technique. Samples of ewe bulk milk were collected during early, mid, and late lactation; milk samples were centrifuged at 2,000 x g for 30 min at 4 degrees C; the fatty fraction and supernatant were removed, and each pellet was dissolved in 500 microL of pH 7.4 phosphate buffered saline + 0.02% NaN(3). Cells were targeted for selection by using mouse-IgG anti-ovine macrophages. Several trials, testing 2 different fluorochrome-conjugated antibodies [i.e., mouse anti-human CD14:R-Phycoerythrin (RPE) (MCA1568PE, Serotec) and F(ab')2 rabbit anti-mouse IgG:RPE (STAR12A, Serotec)] and 3 different labeling procedures, were performed to evaluate the purity of samples by flow cytometry. A morphological test was carried out by direct microscopic count in enriched fraction smears stained with May-Grünwald-Giemsa stain to confirm the presence of macrophages. The method described in the present technical note can be considered an innovative application to obtain a single-cell population of high purity selected from all the somatic cells in milk

Changes occurring in immune responsiveness of single- and twin-bearing Comisana ewes during the transition period.

Changes induced by twin and single lambing in the immune response of 16 periparturient Comisana ewes were studied. Cell-mediated immune responses were evaluated by means of skin tests performed from 3 wk before and up to d 35 after parturition. At d 21 and 7 before lambing, the sheep received an intramuscular injection of the antigen keyhole limpet hemocyanin (KLH), to which the animals had not been previously exposed, to determine their humoral immune response. Starting 3 wk before lambing and up to d 35 postlambing, the ewes were sampled to determine the plasma concentrations of anti-KLH antibody (IgG), IL-6, and IL-1 beta. From parturition through d 35 postpartum, individual milk samples were collected for determination of anti-KLH IgG titers and IL-6 and IL-1beta concentrations by means of a capture ELISA. The number of lambs born affected IL-6 concentrations in ewe plasma; IL-6 secretion always was higher in ewes birthing twins than in single-lambing ewes. Apart from the number of lambs born, the concentrations of plasma IL-6 in ewes were higher at lambing than at d 21 antepartum and at d 35 postpartum. An interaction of number of lambs born x time of sampling was observed for plasma antibody titers to KLH. The IgG concentrations were significantly higher in single-bearing ewes than in twin-bearing ewes before parturition and were very similar across groups after parturition. A time effect was found for the cell-mediated immune response and for anti-KLH IgG concentrations in milk, such that at parturition, cellular responses were lowest, and the anti-KLH IgG concentration was highest. A significant correlation was found for IgG titers to KLH in plasma and milk. Results indicate that IL-6 concentrations in blood can be considered a reliable indicator of stress connected to lambing and that the mammary gland is a microenvironment unrelated to blood stream with respect to interleukins expression. In contrast, a relationship was found for the IgG secretions in milk and blood, which suggests that the assessment of humoral immune status may be combined with milking routine in dairy animals

Gentling and welfare of lambs

In traditional sheep production systems, lambs are suckled by their mothers and then gradually weaned at 35 days of age. However, the increased size of intensive dairy sheep flocks to obtain greater amounts of ovine milk for cheese making, is promoting the diffusion of artificial rearing programs involving early separation of lambs from their mothers. Maternal deprivation soon after birth can have detrimental effects on lamb immune functions, and lead to altered cortisol secretion and behavioural responses to isolation (Napolitano et al., 1995). Human-animal interactions have been documented to play a role in sustaining the welfare and production of domestic animals (Hemsworth, 2003). In addition, gentled animals are less difficult to be handled and less susceptible to the stress induced by management practices involving human contacts (Lensink et al., 2000). The aim of the present trial was to investigate the effects of gently handling newborn lambs on their behavioural, immune and endocrine responses, and on their growth rate when mothered or artificially reared

Influence of ventilation regimen on micro-environment and on ewe welfare and milk yield in summer

The effects of ventilation regimen on air quality, and on the welfare and production performance of thirty-six Comisanaewes were assessed in a 6-week trial conducted during the summer of 2002. Animals were divided into three groups of12, and subjected to the following treatments: low ventilation regimen providing a mean ventilation rate (VR) of 35 m3/hper ewe, split in 30 min ventilation cycles at an air speed of 2 m/s (LOV-30); moderate ventilation regimen (VR = 70m3/h per ewe) split in 30 min ventilation cycles at an air speed of 4 m/s (MOV-30); moderate ventilation regimen (VR =70 m3/h per ewe) split in 60 min ventilation cycles at an air speed of 2 m/s (MOV-60). Air concentrations of microorganisms,dust, and gaseous pollutants were measured twice weekly. Respiration rate (RR) and rectal temperature (RT)were monitored throughout the trial at 0830 and at 1400. Behavioral traits of ewes were recorded twice per week from0900 to 1200 and from 1500 to 1800. Cell-mediated immune response to phytohemagglutinin (PHA) and humoralimmune response to chicken egg albumin were determined. At d 37 ewes were injected with porcine ACTH, and subjectedto blood sampling for evaluation of cortisol concentrations immediately before and 1, 2 and 4 h after ACTH injection.Milk yield was recorded daily. Individual milk samples were analyzed for composition, renneting parameters, somaticcell count (SCC), and bacteriological characteristics. Averages of maximum THI were about 3 points higher in the LOV-30 and the MOV-30 than in the MOV-60 room, whereas no differences emerged in the air concentrations of dust, gaseouspollutants and microorganisms. Significant interactions of treatment x time (P < 0.05) were found for respiration rate,and for the time the ewes spent lying, idling and eating in the afternoon during weeks 2 and 3 of the study period.Significant effects of ventilation regimen x time (P < 0.05) were also observed for milk yield and milk renneting parameters,the LOV-30 ewes giving smaller volumes of milk with a deteriorated coagulating behavior than those of the MOV-60 group during the second half of the trial. No significant differences emerged in ewe immune and endocrine responses.Results show that ventilation regimen had a moderate impact on ewe behavior, physiology and production performance.This experiment suggests that the length of ventilation cycles and air speed, together with ventilation rate, arecritical for efficient ventilation regimens

Relationship between cortisol response to stress and behavior, immune profile, and production performance of dairy ewes.

The existence of a relationship between cortisol levels, after an acute stress, and behavioral activities, immunological profile, and production performance in sheep was studied. An initial flock of 30 Comisana ewes was involved in the experiment, and each of the 30 ewes was individually subjected to an isolation test in a novel environment. Subsequently, from the initial flock, 2 groups of 8 Comisana ewes were each retrospectively selected, and the animals were divided, according to their cortisol concentration 10 min after the isolation test, into high cortisol (HC) ewes, having a peak of cortisol concentration >90 ng/mL (average: 119.3 ng/mL +/- 11.8), and low cortisol (LC) ewes having a peak of cortisol concentration <80 ng/mL (average: 52.4+/-11.8). During the isolation test, the behavior of each animal was video-recorded and behavioral activities were registered. Blood samples were collected before the isolation test, immediately after the test (10 min), and at 60, 120, 300 min, 24 h, and 48 h after the test to evaluate percentages of T-helper (CD4(+)) and T-cytotoxic (CD8(+)) cells, CD4(+)/CD8(+) ratio, and IL-1beta and IL-6 levels. The ewes were milked for 3 d after the isolation test to determine cortisol levels and IL-1beta and IL-6 concentrations in whey. Milk yield was recorded at each milking, and milk samples were analyzed for pH, nutritional parameters, renneting properties, and somatic cell count. During the isolation test, HC ewes exhibited a shorter duration of movement and fewer bleats than LC ewes. The average plasma IL-1beta concentration was higher in HC than in LC ewes. The average whey IL-1beta and IL-6 concentrations were higher in whey from HC ewes than in LC ewes. A positive correlation emerged between plasma and whey IL-1beta concentrations. The average CD4(+)/CD8(+) ratio in blood was lower in HC than in LC ewes. Time from isolation affected the CD4(+)/CD8(+) ratio: at 120 min, the CD4(+)/CD8(+) ratio increased compared with that at 10 min after isolation and then decreased until 300 min after isolation. On average, ewes with low cortisol concentrations showed higher milk production and lower SCC than ewes with high cortisol concentrations. Results suggest that plasma cortisol concentration is connected to the behavioral response and immune competence of dairy ewes and cytokine concentrations. Both whey IL-1beta and IL-6 can be considered reliable indicators of the magnitude of hypothalamic-pituitary-adrenal axis activation. The stress-induced changes in CD4(+)/CD8(+) ratio are critical for controlling disease incidence and planning appropriate vaccination programs. High reactivity of the hypothalamic-pituitary-adrenal axis is also associated with a reduction in milk production and an increased predisposition to develop intramammary inflammatory processes

Contribution of macrophages to proteolysis and plasmin activity in ewe bulk milk.

A total of 225 bulk sheep milk samples were collected from 5 intensively managed flocks during early, mid, and late lactation to assess the contribution of macrophages to the regulation of the plasmin-plasminogen system. Samples were analyzed for composition, somatic cell counts, milk renneting characteristics, and for plasmin (PL), plasminogen (PG), and plasminogen activators (PA) activities. Isolation of macrophages from milk was performed using a magnetic positive separation and mouse antiovine macrophage antibody; separated cells were lysed by several freeze-thaw cycles, and activity of urokinase PA (u-PA) was determined. Plasmin activity decreased during lactation (42.06 +/- 0.66, early; 31.29 +/- 0.66, mid; 28.19 +/- 0.66 U/mL, late). The reduction in PL activity recorded in the mid and late lactation milk matched the increase in PG:PL ratio. The activity of PA increased throughout lactation; the highest value being recorded in the late lactation milk (260.20 +/- 8.66 U/mL). Counts of isolated and concentrated macrophages were higher in early and mid lactation milk (3.89 +/- 0.08 and 3.98 +/- 0.08 log10 cells/mL, respectively) than in late lactation milk (3.42 +/- 0.08 log10 cells/mL). Stage of lactation did not influence the activity of u-PA detected in isolated macrophages. The activity of u-PA associated with isolated milk macrophages only minimally contributed to total PA activity detected in milk. Proteolytic enzymes, associated with isolated macrophages, act on alpha-casein hydrolysis, as shown by urea-PAGE electrophoresis analysis. Somatic cell counts did not exceed 600,000 cells/mL, and this threshold can be considered a good index of health status of the flock and of the ability of milk to being processed. Our results lend support to the hypothesis that macrophages in ewe bulk milk from healthy flocks only slightly contribute to the activation of the PL-PG system