Proteases involved in mammary tissue damage during endotoxin-induced mastitis in dairy cows

During and after diapedesis, milk polymorphonu-clear neutrophils (PMN) release many proteases that have the potential of degrading extracellular matrix proteins and milk proteins. However, the kinetics of milk proteolysis during inflammation and the underlying mechanisms are poorly defined. The enzymes involved in bovine mammary tissue destruction were investigated in this study using an endotoxin-induced mastitis model. Using zymography techniques, the proteolytic activity of milk and mammary tissue during mastitis was examined. Mastitic milk produced 6 caseolysis bands, 4 of which differed from the ones produced by plasmin. Peak proteolytic activity, bovine serum albumin contents, and mammary tissue damage occurred between 6 and 12 h postchallenge. Mastitic milk proteases hydrolyzed casein, gelatin, collagen, hemoglobin, mammary gland membrane proteins, and lactoferrin. These results confirm that mastitic milk proteases have a broad spectrum of activity. The hydrolytic activity of mastitic milk was partially inhibited by aprotinin, EDTA, 1,10-phenanthroline, leupeptin, and pefabloc. When cocultured with normal mammary tissue, mastitic milk, but not normal milk, caused mammary tissue degradation. In situ zymography of mammary gland showed increased proteolytic activity in mastitic tissue compared with normal tissue. The similarity of zymograms of mastitic milk, blood PMN, milk somatic cells, and PMN strongly suggests that proteases in mastitic milk mainly originate from milk PMN. These results suggest that proteases released by PMN are actively involved in udder tissue damage during mastitis.     

Milk trypsin-inhibitor capacity as an indicator of bovine mastitis--a novel principle which can be automated

As mastitis is associated with leakage of small molecular weight plasma proteins, such as alpha 1-antitrypsin, into milk, this antitrypsin can be used as an indicator of mastitis. A colorimetric procedure was developed for large scale monitoring of milk antitrypsin activity, using microtitration plates and the Multiskan system. The effect of stage of lactation and age of the cow on the antitrypsin concentration and its interrelationship with other mastitis indicators (bovine serum albumin (BSA), somatic cell count) was analysed by computer programs on 1029 cows. Milk antitrypsin activity was high after parturition owing to colostral inhibitors. After the first month of lactation the assay measures only blood-derived antitrypsin and is a good indicator for detecting an increased permeability between blood and milk due to mastitis. Increasing lactation number only slightly affected the antitrypsin and BSA concentrations whereas somatic cell content was markedly affected.     

Proteolytic and Milk Clotting Fractions in Milk Clotting Preparations

Five different milk clotting preparations were fractionated on Sephadex G-100 and then tested for milk clotting activity and for proteolysis of denatured hemoglobin. Two preparations were also tested for proteolytic activity on casein. Proteolytic activities on hemoglobin were correlated with clotting ability of bovine rennet, calf rennet, and rennin-pepsin mixture at pH 1.6 and with Mucor miehei protease at pH 5.2. Modified Mucor miehei protease activities on hemoglobin correlated equally well at pH 1.6 and 5.2. Gel filtration through Sephadex G-100 and elimination of nonclotting fractions reduced the proteolytic activities on hemoglobin at pH 5.2 of calf rennet, bovine rennet, Mucor miehei protease, modified Mucor miehei protease, and rennin-pepsin mixture by 68.6, 88.5, 3.7, 53.7, and 91.2%, respectively. At pH 1.6, proteolysis was reduced by 54.2, 41.2, 51.8, 59.5, and 60.8%. Proteolytic activities of bovine rennet and renninpepsin mixture on casein were reduced by 59.8 and 72%, respectively.     

Contribution of proteolytic activity associated with somatic cells in milk to cheese ripening

The effect of proteolytic enzymes from somatic cells on cheese quality was studied. In preliminary experiments, milk and two sodium caseinate systems (pH 6.5 and pH 5.2, the latter in the presence of 5% NaCl) were used as substrates to investigate the proteolytic activity of somatic cells recovered from mastitic milk. Urea-polyacrylamide gel electrophoretograms of hydrolysates suggested that somatic cell extracts contributed directly to proteolysis both in buffer and in milk, but that such activity was reduced by batch pasteurisation (63 °C for 30 min). Sodium caseinate was readily hydrolysed by somatic cell extracts; hydrolysis of α_s1-casein was greater at pH 5.2 and increased with level of somatic cells, suggesting that somatic cells contain proteolytic enzymes which are more active at acidic pH values. Subsequently, miniature Cheddar-type cheeses were made from batches of milk to which somatic cells were added (at levels of levels of 3×10⁵ or 6×10⁵ cells mL⁻¹), either before or after pasteurisation. Proteolysis during ripening of cheese (as measured by levels of pH 4.6-soluble nitrogen) increased with somatic cell addition, although this effect was reduced by pasteurisation after cell addition. Somatic cells may also have directly influenced cheese moisture content, which has been established as a principal indicator of quality of Cheddar-type cheese. Proteolytic enzymes of somatic cells from milk were shown to contribute directly to proteolysis in milk and cheese.     

Proteolytic and proteomic changes in milk at quarter level following infusion with Escherichia coli lipopolysaccharide

Mastitis is a major disease in dairy cattle, which causes significant economic losses due to decreased milk production, veterinary costs, and discarded milk. Escherichia coli is one of the most prevalent species of gram-negative bacteria that induce clinical mastitis. The objective of the present study was to characterize the proteolytic and proteomic changes in milk in response to infusion with lipopolysaccharide (LPS) at quarter level in a model mastitis system. One quarter of each of 2 cows was infused with 0.1 or 5 μg of LPS. The somatic cell count of the infused quarters reached a peak 6 h after infusion to a greater extent in the cow infused with 5 μg of LPS and changes in plasmin activity in milk differed between the 2 animals. Urea-polyacrylamide gel electrophoretograms of milk samples of the cow infused with 5 μg of LPS 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 due to indigenous proteolytic activity. Two-dimensional gel electrophoretograms of milk at 0, 6, or 12 h after infusion with LPS showed hydrolysis of α(s)-casein and β-casein as well as the appearance of lower molecular weight products. Eleven fragments from proteolysis of the caseins were identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and, in addition, proteolysis patterns of casein by the indigenous bovine milk proteases plasmin and cathepsin D were studied in model studies using 2-dimensional gel electrophoretograms. Twelve hours after infusion, lower abundance markers of inflammation were identified, including serotransferrin, fibrinogen β chain, protein S100 A12, and the antimicrobial polypeptide cathelicidin.     

Proteolytic patterns and plasmin activity in ewes' milk as affected by somatic cell count and stage of lactation

A total of 120 milk samples were collected from Comisana ewes throughout lactation. The ewes were ranked into two somatic cell count (SCC) categories: normal milk (N Milk) with SCC lower than 5.00x 10(5)/ml and high somatic cell milk (HSC Milk) with SCC higher than 1.00 x 10(6)/ml. Milk samples were analysed in triplicate for pH, fat and protein contents, renneting parameters, and plasmin and plasminogen activities. The peptide profile due to total proteolytic activity (endogenous and exogenous enzymes) on alpha- and beta-CNs were determined using urea-PAGE on sodium caseinate (pH 8.0 and pH 5.0) incubated at 37 degrees C for 4 d after sampling. The peptide profile due to non-plasmin enzyme activities at pH 5.0 was also determined using urea-PAGE. Plasmin activity was higher in the HSC milk than in the N milk throughout the study period. A decrease in plasmin activity was observed in the N milk during mid-lactation, which was probably related to decrease in pH, and in the HSC milk during late lactation, which may be ascribed to an enhanced influx of plasmin inhibitors from the blood stream. Proteolytic patterns in Comisana ewe milk were mainly affected by plasmin activity that increased with the SCC in milk. Also non-plasmin proteolytic activity was strongly enhanced by elevated SCC and resulted in a higher degradation of alpha-casein than of beta-casein. In general, plasmin activity did not increase with the advancement of lactation and exhibited a different trend in HSC and N milk, suggesting that physiological factors did not play a key role in regulating the plasminogen-plasmin system in ewes' milk. Plasmin activity, detected with the colorimetric assay was consistent with proteolytic activity on sodium caseinate shown in urea-PAGE electrophoregram.     

Lipoprotein lipase activity of milk from cows with prolonged subclinical mastitis

The influence of prolonged subclinical mastitis on bovine milk lipoprotein lipase activity was investigated. Nine cows with at least one quarter with prolonged subclinical mastitis and at least one nonmastitic quarter were selected in various stages of lactation. Milk from subclinical quarters had a mean somatic cell count of 5.7 X 10(6) cells/ml while milk from nonmastitic quarters had an average somatic cell count of 9.4 X 10(4) cells/ml. Quarters with a subclinical infection contained the same pathogenic organisms for a minimum of 6 wk. The average milk lipoprotein lipase activity of 108.7 units/ml milk from subclinical quarters was 27.1% higher than the average enzyme activity of 79.2 units/ml milk from nonmastitic quarters. Conditions present in the mammary gland during prolonged subclinical mastitis could lead to increased milk lipoprotein lipase activity in raw milk.     

Effects of Staphylococcus aureus on bovine mononuclear leukocyte proliferation and viability: modulation by phagocytic leukocytes

In vitro effects of killed Staphylococcus aureus cells on bovine blood mononuclear leukocytes from uninfected cows or cows with chronic staphylococcal mastitis were assessed using a lymphocyte proliferation assay and a [51Cr] release cytotoxicity assay. Killed S. aureus cells cultured with mononuclear leukocytes caused a concentration-dependent decrease in lymphocyte proliferation that was associated with a concomitant decrease in mononuclear leukocyte viability. Responses of mononuclear leukocytes from uninfected and infected cows to killed S. aureus were similar, indicating effects were independent of the infection status of the animal. Addition of blood polymorphonuclear leukocytes to blood mononuclear leukocyte cultures without S. aureus cells did not alter mononuclear leukocyte viability but suppressed lymphocyte proliferation at the highest polymorphonuclear leukocyte:mononuclear leukocyte ratios (4:1 and 8:1) tested. When S. aureus cells and polymorphonuclear leukocytes were cultured with mononuclear leukocytes, both blood and milk polymorphonuclear leukocytes protected against the loss of viability compared with leukocytes cultured with S. aureus cells alone but did not consistently restore proliferative responses of the lymphocytes. These observations demonstrate that lymphocyte proliferation and mononuclear leukocyte viability are detrimentally affected by S. aureus cells, an effect that can be modulated by blood or milk polymorphonuclear leukocytes.     

Deferoxamine reduces tissue damage during endotoxin-induced mastitis in dairy cows

The protective effects of 3 antioxidants on polymorphonuclear neutrophil-induced damage to mammary cells were evaluated in vivo using an endotoxin-induced mastitis model. Fifteen healthy, midlactation cows with no history of clinical Escherichia coli mastitis were randomly assigned to 1 of the 3 treatment groups corresponding to each modulator to be evaluated, that is, deferoxamine, catechin, and glutathione ethyl ester. Each cow had 1 quarter infused with saline and 1 quarter infused with the selected modulator; a third quarter was infused with lipopolysaccharides (LPS), whereas the fourth quarter received a combination of LPS and the modulator. Infusion of LPS caused acute mastitis as determined by visual observations and by large increases in milk somatic cell count, BSA, and proteolytic activity. These parameters were not affected by antioxidant administration. The extent of cell damage was evaluated by measuring milk levels of lactate dehydrogenase and N-acetyl-β-_D-glucosaminidase activity. Levels of these parameters were several times higher after LPS administration. Intramammary infusions of catechin or glutathione ethyl ester did not exert any protective effect, whereas infusion of deferoxamine, a chelator of iron, decreased milk lactate dehydrogenase and NA-Gase activity, suggesting a protective effect against neutrophil-induced damage. The protective effect of deferoxamine was also evidenced by a lower milk level of haptoglobin. The proteolytic activity of mastitic milk was not influenced by the presence of deferoxamine. Overall, our results suggest that local infusion of deferoxamine may be an effective tool to protect mammary tissue against neutrophil-induced oxidative stress during bovine mastitis.     

Proteomic and peptidomic study of proteolysis in quarter milk after infusion with lipoteichoic acid from Staphylococcus aureus

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 6 h after infusion, whereas plasmin activity of those quarters also increased, reaching a peak after 48 and 12 h 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 6 h 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 C₁₈ 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.     

Dairy management practices associated with incidence rate of clinical mastitis in low somatic cell score herds in France

An epidemiological prospective study was carried out in French dairy herds with Holstein, Montbéliarde, or Normande cows and with low herd somatic cell scores. The objective was to identify dairy management practices associated with herd incidence rate of clinical mastitis. The studied herds were selected on a national basis, clinical cases were recorded through a standardized system, and a stable dairy management system existed. In the surveyed herds, mean milk yield was 7420 kg/cow per yr and mean milk somatic cell score was 2.04 (132,000 cells/mL). Overdispersion Poisson models were performed to investigate risk factors for mastitis incidence rate. From the final model, the herds with the following characteristics had lower incidence rates of clinical mastitis: 1) culling of cows with more than 3 cases of clinical mastitis within a lactation; 2) more than 2 person-years assigned to dairy herd management; 3) balanced concentrate in the cow basal diet. Moreover, herds with the following characteristics had higher incidence rates of clinical mastitis: 1) milking cows loose-housed in a straw yard; 2) no mastitis therapy performed when a single clot was observed in the milk; 3) clusters rinsed using water or soapy water after milking a cow with high somatic cell count; 4) 305-d milk yield >7435 kg; 5) herd located in the South region; 6) herd located in the North region; 7) cows with at least 1 nonfunctional quarter; and 8) premilking holding area with a slippery surface. The underlying mechanisms of some highlighted risk factors, such as milk production level and dietary management practices, should be investigated more thoroughly through international collaboration.     

Polymorphonuclear neutrophils and Escherichia coli proteases involved in proteolysis of casein during experimental E. coli mastitis

An Escherichia coli mastitis model was used to characterize enzymes involved in bovine mammary tissue damage and proteolysis in milk. One-quarter each of four cows were inoculated with a suspension (10⁴ cfu mL⁻¹) of E. coli P4:O32. Blood and milk were collected before inoculation and for 216 h afterwards. Intracellular elastase, collagenase and cathepsin activities were measured by flow cytometry of peripheral blood leukocytes and milk polymorphonuclear neutrophils (PMNs). Leukopenia occurred in peripheral blood 9 h after infection, concomitant with an increase in somatic cell count in milk. Milk PMNs had lower activity of cathepsins and collagenase than peripheral blood PMNs. In parallel, milk samples were studied by zymography, and several proteases were detected in mastitic milk. These activities increased after infection, to reach a peak in 6 h. However, total protease profiles and plasmin activities differed. It was concluded that proteases released by PMNs and E. coli contribute to proteolysis of casein during mastitis, as well as plasmin.     

Effect of mastitis on proteolytic activity in bovine milk

Proteolytic activity of milk was studied before, during, and after experimental-induced mastitis. An inoculum of Streptococcus agalactiae was infused into one quarter of each udder of six cows to elicit an infection. Bacteriological cultures and SCC of milk were used to monitor infection status. Sodium dodecyl sulfate-PAGE was used to measure proteolytic activity of milk. Inhibitor 6-amino-n-hexanoic acid was used to determine the relative proportion of plasmin and nonplasmin proteolytic activity of milk. Somatic cell count, total milk proteolytic activity, and nonplasmin proteolytic activity were higher in infected quarters than in quarters preinfection. After elimination of infections, SCC and nonplasmin proteolytic activity decreased to preinfection amounts. Total proteolytic activity of milk decreased after infections were cured but remained significantly higher than preinfection activity. This postinfection proteolytic activity in milk may be due to an increase in milk plasmin activity. Our data suggest that detrimental effects of mastitis on milk quality can continue after infection has been eliminated and milk SCC have returned to low values.     

Economic optimization of selective dry cow treatment

The objective of this study was to develop a mathematical model to identify a scenario with the lowest costs for mastitis associated with the dry period while restricting the percentage of cows to be dried off with dry cow antimicrobials. Costs of clinical and subclinical mastitis as well as antimicrobial use were quantified. Based on data from a large field trial, a linear programming model was built with the goal to minimize the costs associated with antimicrobial use at drying off. To enable calculations on minimizing costs of dry cow treatment on herd-level by drying-off decisions in an “average” herd, we created an example herd. Cows were projected on 3 different types of herds, based on bulk tank somatic cell count, and were categorized in groups based on parity and somatic cell count from the last test recording before drying-off. Economically optimal use of antimicrobials was determined while restricting the maximum percentage of cows dried off with antimicrobials from 100 to 0%. This restriction reveals the relationship between the maximum percentage of cows dried off with antibiotics and the economic consequences. A sensitivity analysis was performed to evaluate the effect of variation in the most important input variables, with the effect of dry cow antimicrobials resulting in a lower or higher percentage of clinical and subclinical mastitis depending on being dried off with or without dry cow antimicrobials, respectively, and the milk price. From an economic perspective, blanket dry cow treatment seems not to be the optimal approach of dry cow therapy, although differences between approaches were small. With lower bulk tank somatic cell counts, more dry cow antimicrobials can be omitted without economic consequences. The economic impact of reducing the percentage of clinical mastitis was found to be much larger than reducing the bulk tank somatic cell count. The optimal percentage of cows to be dried off with antimicrobials depends on the udder health situation, expressed as the bulk tank somatic cell count and the incidence of clinical mastitis. For all evaluated types of herds, selective dry cow treatment was economically more beneficial than blanket dry cow treatment. Economic profits of selective dry cow treatment are greater if bulk tank somatic cell count and clinical mastitis incidence are lower. Economics is not an argument against reduction of dry cow antimicrobials by applying selective dry cow treatment.     

Cost benefit analysis of lactation therapy with somatic cell counts as indications for treatment

Lactating dairy cows (487) from five commercial herds were in a study of benefits from lactation therapy of sub-clinical mastitis. Bacterial isolations and composite milk samples for somatic cell counts were taken from each cow each month for 15 mo. Cows (254) in the experimental group were infused with cephapirin in all quarters for two consecutive milkings if somatic cell counts rose above 400,000 cells/ml; 103 cows were so treated. Stepwise regression showed that lactation number, somatic cell counts, days in milk, and percent quarters infected explained variation in milk production, but treatment group, herd, and season did not. Also, there were no significant differences between production of infected experimental and control cows with high somatic cell counts on test dates after treatment. With the experimental program, there was a net loss of +19.65/cow. Intramammary lactation therapy based on somatic cell counts less than 400,000 cells/ml is not recommended.     

Randomized controlled field trial comparing quarter and cow level selective dry cow treatment using the California Mastitis Test

Selective use of antibiotic dry cow treatment can be implemented at the cow or quarter level, with the latter having the potential to further reduce antibiotic use. Our objective was to compare these 2 approaches in 6 herds in the United Kingdom in which environmental mastitis predominated. Eight hundred seven cows were enrolled and categorized as having a high cell count (n = 401) or low cell count (n = 406) in the last 3 mo of lactation and clinical mastitis history. All quarters of all enrolled cows received an internal teat sealant. Within each category, cows were randomly allocated to 1 of 3 groups; in one group antibiotic treatment was allocated at cow level (i.e., all 4 quarters received antibiotic), whereas in the 2 remaining groups antibiotic treatment was allocated at quarter level, based on California Mastitis Test (CMT) findings. Two different thresholds, score 1 and 2, were used to determine likely infection status. Quarter milk samples were collected at dry off and postcalving for bacteriological culture and somatic cell count (SCC). Cows were monitored for clinical mastitis from dry off until 100 d in milk. Cow level SCC and milk yield data were collated from farm records. Within each category, the 2 quarter level treatment groups were compared with cow level treatment at dry off. Leaving quarters untreated with intramammary antibiotic in cows in the high cell count group, with a CMT <2 or <1, reduced antibiotic use by 55% and 31%, respectively, and resulted in no difference in the odds of being infected with any pathogen postcalving, but was associated with a higher SCC at the first test day. Intramammary antibiotic treatment of quarters with a CMT ≥1 in cows in the low cell count category at dry off was not associated with any reduction in the odds of being infected with a major pathogen postcalving but was associated with a decrease in the odds of being infected with a minor mastitis pathogen postcalving. The use of antibiotics in quarters of cows categorized as low cell count at dry off, increased the proportion of quarters treated with antibiotic from 0% at cow level to 31% (CMT ≥ 1) and 12% (CMT ≥ 2) at quarter level, only resulting in a reduction in SCC of around 20,000 cells/mL at the first test day, if all quarters with CMT score ≥1 were treated with antibiotic. No differences in clinical mastitis incidence and milk yield in the first 100 d in milk were detected between any of the treatment groups. These study findings support selective quarter level dry off treatment only in cows with cow level SCC >200,000 cells/mL at dry off.     

Bovine mastitis in Finland 2001--prevalence, distribution of bacteria, and antimicrobial resistance

A nationwide survey was conducted in Finland to estimate prevalence of bovine mastitis, distribution of mastitis pathogens, and in vitro antimicrobial susceptibility of different mastitis pathogens. In total, 12,661 quarter milk samples were collected from 3282 dairy cows at 216 farms. These were randomly selected from a database covering all Finnish dairy farms. Quarter milk samples collected by the dairy advisors were submitted for somatic cell counting, bacteriological examination, and testing for antimicrobial susceptibility. If the milk SCC of a cow or of a quarter exceeded 300,000/mL, the cow was defined as having mastitis. The results were compared with those of a previous survey done in 1995. The prevalence of mastitis continued to decrease from 38% in 1995 to 31% in 2001. Compared with the study from 1995, the number of quarters with bacterial growth in 2001 increased significantly from 21.0 to 33.5%. This mainly resulted from increased prevalence of Corynebacterium bovis. Coagulase-negative staphylococci remained the most common bacterial group, comprising almost one-half of the pathogens isolated, whereas the relative number of Staphylococcus aureus isolations decreased from the time of the previous study. According to in vitro antimicrobial susceptibility testing, the enterococci demonstrated the highest level of resistance. Compared with the other Nordic countries, penicillin resistance among the staphylococci was still at a relatively high level in Finland (52.1 and 32.0% for Staphylococcus aureus and coagulase-negative staphylococci, respectively). Streptococci isolated from mastitis were very susceptible to beta-lactam antibiotics, as also found in the previous survey in 1995.     

New aspects of naturally occurring proteases in bovine milk

The scientific literature on milk proteases, along with recent findings in the author's laboratory, are summarized and reviewed comprehensively. Emphasis is on detection of proteolytic enzymes and their activity, purification and kinetic characterization of the isolated enzymes, and technological problems associated with proteolytic enzymes in milk and milk products. Two serine proteinases isolated from milk are compared with plasmin of bovine blood serum. Results from these comparisons strongly suggest that milk proteinase I and plasmin are identical. Proteolysis studies with cold stored milk indicate a direct relationship between gamma-casein formation and milk proteinase association with casein micelles.     

Characterization and proteolytic origins of specific peptides appearing during lipopolysaccharide experimental mastitis

Based on the compositional change of the proteose peptone fraction, proteolysis was studied over time following lipopolysaccharide-induced experimental mastitis. Electrophoresis of the proteose peptone fraction revealed many degradation products. Five peptides were identified by amino-terminal sequencing as internal fragments of beta-, kappa-, alpha(s1)-, and alpha(s2)-casein that were generated by somatic cell proteases. Although kappa-casein is considered particularly resistant to endogenous proteolysis, a kappa-casein peptide was electrophoretically isolated in association with a beta-casein fragment. The in vitro kinetic studies of caseinate hydrolysis by elastase, one of the main polymorphonuclear neutrophil (PMN) proteases, suggested that the beta-casein peptide might be generated by elastase. In addition, elastase activity in milk PMN was higher during the inflammation of the mammary gland than prior to infusion.     

Peripartum changes of plasma and milk vitamin A and beta-carotene among dairy cows with or without mastitis

Over 12 mo we studied the relationship between peripartum concentrations of vitamin A and beta-carotene in blood plasma and milk of 93 Holsteins with or without subsequent mastitis. Blood was sampled daily from 7 days prepartum through 7 days postpartum and on alternate weeks through wk 10 of lactation. Milk samples were collected daily for 7 days postpartum and then biweekly for 10 wk. Somatic cell counts were on biweekly milk samples. Vitamin A and beta-carotene of blood plasma decreased rapidly prepartum to reach minimum concentrations at calving (vitamin A) or on day 4 to 6 postpartum (beta-carotene). Thereafter, both vitamin A and beta-carotene increased rapidly through 10 wk postpartum. Concentrations of vitamin A and beta-carotene in colostrum were higher than concentrations in milk. Cows with mastitis (somatic cells greater than 500,000 cells/ml milk) had lower vitamin A in blood plasma during days 0 to 7 and wk 2 and 4 postpartum than cows without mastitis. When data were analyzed with loge of somatic cell count as an independent regression variable, results were similar. In contrast to vitamin A, peripartum beta-carotene in blood plasma was higher among mastitic cows and was related to higher loge of somatic cell count. No significant difference was observed between mastitic and non-mastitic cows for vitamin A and beta-carotene in milk. Lower concentrations of plasma vitamin A and higher concentrations of beta-carotene during the immediate postpartum period were associated with higher milk somatic cell counts among dairy cows during lactation.