No abstract

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.

ABSTRACT:Mastitis is the inflammatory reaction of the udder to invading pathogens. One of the most apparent reactions is the increased influx of immunoreactive cells from blood into milk inducing a dramatic increase of milk somatic cell counts (SCC). We have investigated (i) the relationship between log SCC/ml in infected quarters being >6 (n = 8, group I) or varying between 5.4 and 6 (n = 8, group II) and concentration of dry ma�er (DM), fat, protein, lactose, insulin-like growth factor (IGF)-1, insulin, prolactin, tumor necrosis factor (TNF)-α, sodium, potassium, chloride, electrical conductivity and osmolarity as compared with the contralateral (healthy) quarter (log SCC/ml <5.2); and (ii) composition of fractionized milk [cisternal milk, quartiles of alveolar milk and residual milk (a�er i.v. injection of 10 u.i. oxytocin)] during machine milking of infected and healthy quarters. SCC were higher (P < 0.05) in infected than in healthy quarters. Concentrations of fat, sodium, chloride, and IGF-1 were higher (P < 0.05), while that of lactose was lower (P < 0.05) in infected than in healthy quarters (group I). Concentrations of fat and chloride in both groups, of DM (in group II), and electrical conductivity and sodium (in group I) increased from the cisternal to alveolar (100%) fractions in infected quarters, while fat and DM concentrations similarly increased in healthy quarters. In conclusion, several but not all milk traits changed in a different manner during the course of milking in infected and non-infected quarters.

Mastitic milk is associated with increased bovine protease activity, such as that from plasmin and somatic cell enzymes, which cause proteolysis of the caseins and may reduce cheese yield and quality. The aim of this work was to characterize the peptide profile resulting from proteolysis in a model mastitis system and to identify the proteases responsible. One quarter of each of 2 cows (A and B) was infused with lipoteichoic acid from Staphylococcus aureus. The somatic cell counts of the infused quarters reached a peak 6h after infusion, whereas plasmin activity of those quarters also increased, reaching a peak after 48 and 12h for cow A and B, respectively. Urea-polyacrylamide gel electrophoretograms of milk samples of cow A and B obtained at different time points after infusion and incubated for up to 7 d showed almost full hydrolysis of β- and α(S1)-casein during incubation of milk samples at peak somatic cell counts, with that of β-casein being faster than that of α(S1)-casein. Two-dimensional gel electrophoretograms of milk 6h after infusion with the toxin confirmed hydrolysis of β- and α(S1)-casein and the appearance of lower-molecular-weight products. Peptides were subsequently separated by reversed-phase HPLC and handmade nanoscale C(18) columns, and identified by matrix-assisted laser desorption/ionization time-of-flight tandem mass spectrometry. Twenty different peptides were identified and shown to originate from α(s1)- and β-casein. Plasmin, cathepsin B and D, elastase, and amino- and carboxypeptidases were suggested as possible responsible proteases based on the peptide cleavage sites. The presumptive activity of amino- and carboxypeptidases is surprising and may indicate the activity of cathepsin H, which has not been reported in milk previously.

Cytokines, eicosanoids, and lactoferrin are involved in the mammary gland's immune response to invading microorganisms. The goal of this work was to investigate the synthesis of these immunologically important factors in somatic milk cells, blood cells, and mammary tissue of cows with different somatic cell count levels, i.e., different immunological activity. On the level of mRNA expression, the cytokine tumor necrosis factor alpha (TNFalpha), lactoferrin (Lf), and specific key enzymes of leukotriene and prostaglandin biosynthesis, 5-lipoxygenase (5-LO), and cyclooxygenase-1 (COX-1) and -2 (COX-2), respectively, were determined. All 15 experimental cows were clinically healthy with no visible mammary disease. Eight cows were defined as control group with all quarters <150,000 cells/ml (C), whereas seven cows had partially elevated quarter somatic cell counts, with at least one quarter >150,000 cells/ml (H) and one quarter <150,000 cells/ml (L). Total quarter milk from one quarter of control group and from two quarters of cows with partially elevated cell counts (one of H and one of L) was collected at one milking and a blood sample was taken simultaneously. In addition, mammary tissue samples were taken from the respective quarters on the following day during slaughter. Total RNA from milk, blood, and tissue cells was isolated and reverse transcription and quantitative polymerase chain reaction was carried out. All factors investigated were not significantly different between groups in blood cells and between C and L quarters in milk cells and mammary tissue. TNFalpha and COX-2 mRNA expression was higher in milk cells and mammary tissue of H than in L quarters, except for COX-2 in mammary tissue. Generally, TNFalpha and COX-2 showed their highest expression in milk cells, 5-LO in blood cells, whereas lactoferrin was mainly expressed by the mammary tissue. COX-1 was similarly expressed in all tested samples.