Distinct behavior of bovine-associated staphylococci species in their ability to resist phagocytosis and trigger respiratory burst activity by blood and milk polymorphonuclear leukocytes in dairy cows

Mastitis affects a high proportion of dairy cows and is still one of the greatest challenges faced by the dairy industry. Staphylococcal bacteria remain the most important cause of mastitis worldwide. We investigated how distinct staphylococcal species evade some critical host defense mechanisms, which may dictate the establishment, severity, and persistence of infection and the outcome of possible therapeutic and prevention interventions. Thus, the present study investigated variations among distinct bovine-associated staphylococci in their capability to resist phagocytosis and to trigger respiratory burst activity of blood and milk polymorphonuclear neutrophil leukocytes (PMNL) in dairy cows. To do so, PMNL of 6 primiparous and 6 multiparous dairy cows were used. A collection of 38 non-aureus staphylococci (NAS) and 12 Staphylococcus aureus were included. The phagocytosis and intracellular reactive oxygen species (ROS) production by blood and milk PMNL were analyzed by flow cytometry. Phagocytosis, by both blood and milk PMNL, did not differ between S. aureus and NAS as a group, although within-NAS species differences were observed. Staphylococcus chromogenes (a so-called milk-adapted NAS species) better resisted phagocytosis by blood PMNL than the so-called environmental (i.e., Staphylococcus fleurettii) and opportunistic (i.e., Staphylococcus haemolyticus) NAS species. Otherwise, S. haemolyticus was better phagocytosed by blood PMNL than S. aureus, S. fleurettii, and S. chromogenes. No influence of the origin of the isolates within the staphylococci species in the resistance to phagocytosis by blood and milk PMNL was found. Overall, both S. aureus and NAS did not inhibit intracellular ROS production in blood and milk PMNL. Non-aureus staphylococci induced fewer ROS by milk PMNL than S. aureus, which was not true for blood PMNL, although species-specific differences in the intensity of ROS production were observed. Staphylococcus chromogenes induced more blood PMNL ROS than S. fleurettii and S. haemolyticus, and as much as S. aureus. Conversely, S. chromogenes induced fewer milk PMNL ROS than S. aureus. The origin of the isolates within the staphylococci species did not affect the ROS production by blood and milk PMNL. In conclusion, our study showed differences in staphylococci species in evading phagocytosis and triggering ROS production, which may explain the ability of some staphylococci species (i.e., S. aureus and S. chromogenes) to cause persistent infection and induce inflammation.     

Factors affecting the lactoferrin concentration in bovine milk

Lactoferrin (LF) concentrations in the milk with different levels of the somatic cell count score were examined using an ELISA to determine whether milk LF concentration is influenced by parity of the cow, stage of lactation, and the somatic cell count. The study animals were 198 Chinese Holstein cows randomly chosen from more than 1,600 cows in 4 dairy farms in the Beijing area. The cows had shown no sign of mastitis for 2 mo. Daily milk production was recorded, and milk samples were taken from individual cow samples. The LF concentration varied between 31.78 and 485.63 μg/mL in milk from normal animals. Lactoferrin was significantly associated with stage of lactation (r = 0.557) and daily milk production (r = −0.472). Nevertheless, there was no significant relationship with parity. Moreover, milk LF concentration tended to be correlated with the somatic cell count score (r = 0.375). This finding suggests that milk LF may be helpful as an indicator for intramammary infection in dairy cows.     

Factors affecting the concentration of sphingomyelin in bovine milk

Sphingomyelin is a phospholipid located in the outer leaflet of the plasma membrane of most cells and is a component of the milk fat globule membrane. Sphingomyelin and its digestion products participate in several antiproliferative pathways that may suppress oncogenesis. Although milk and dairy products are important sources of sphingomyelin in the human diet, little is known about factors that influence sphingomyelin concentrations in milk fat or whether concentrations can be modified via the nutrition of cows. Sphingomyelin concentrations were determined in milk from Holstein and Jersey cows matched for parity and stage of lactation. Sphingomyelin was more concentrated in milk fat from Holstein cows than in milk fat from Jersey cows (1,044 vs. 839 μg/g of fat). Concentrations in whole milk did not differ because of greater milk fat content for milk from Jerseys. Differences between breeds may be related to the greater fat globule size in milk from Jerseys. Sphingomyelin content in whole milk increased with increasing days in milk because of associated increases in milk fat content. Regardless of breed, primiparous cows had greater amounts of stearic acid and less palmitic acid in sphingomyelin than did older cows. The sphingomyelin concentration in milk fat of cows in a commercial Jersey herd was lower for cows in their fourth or greater parity. Sphingomyelin content in whole milk was greater for cows in late lactation because of greater milk fat content. Feed restriction of multiparous Holstein cows to 37% of ad libitum dry matter intake increased milk fat content but did not affect milk sphingomyelin content or milk fat globule size. Supplementation of the diet with 4% soybean oil did not affect milk composition, sphingomyelin content, or milk fat globule size. Milk was sampled seasonally from 7 herds throughout Illinois during a 2-yr period. Sphingomyelin concentration in milk fat was greatest during summer and least during winter, but whole milk concentrations did not vary across seasons. We conclude that 1) sphingomyelin content of milk fat is greater in milk from Holsteins than that from Jerseys, 2) sphingomyelin content in whole milk increases with stage of lactation, and 3) sphingomyelin content of milk fat is greater during summer. However, efforts to produce milk with a greater sphingomyelin content through altering management and nutrition are unlikely to be successful.     

Factors affecting insulin-like growth factor-I concentration in bovine milk.

To establish the naturally occurring range of insulin-like growth factor-I concentrations in bovine milk, samples from individual cows (n = 409) managed on five Missouri dairy herds were assayed. Parity, stage of lactation, and farm affected milk insulin-like growth factor-I concentration. Milk insulin-like growth factor-I concentration was higher in early lactation than mid and late lactation with concentrations in multiparous cows exceeding those in primiparous cows. Insulin-like growth factor-I concentration was negatively correlated to milk production the day of sample collection (r = -.15) and not correlated to predicted 305-d milk yields. Unprocessed bulk tank milk samples (n = 100) from a commercial processing plant had a mean concentration of insulin-like growth factor-I in milk of 4.32 ng/ml with a range of 1.27 to 8.10 ng/ml. This distribution was similar to the range detected in samples from individual cows, but values were lower than those reported for human milk. Concentration of insulin-like growth factor-I in milk was not altered by pasteurization (at 79 degrees C for 45 s). However, insulin-like growth factor-I was undetectable in milk heated to temperatures (121 degrees C for 5 min) required for infant formula preparation or in commercially available infant formula. These data indicated that insulin-like growth factor-I is a normal but quantitatively variable component of bovine milk that is not destroyed by pasteurization but is undetectable in infant formula. Concentration of insulin-like growth factor-I in bovine milk is lower than concentrations reported for human milk yet similar to those reported for human saliva.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of bovine skim milk and whey on monocyte function

Previous studies have documented the ability of bovine milk to inhibit lymphocyte proliferation in response to mitogens. It is not known whether inhibition of lymphocyte proliferation is mediated through the action of monocytes. To address this question, we examined the ability of bovine skim milk and whey to affect monocyte function with emphasis on expression of major histocompatibility class II antigens and production of interleukin-1 by monocytes. Data showed that expression of major histocompatibility complex class II molecules and production of interleukin-1 by monocytes were not altered when monocytes were cultured in the presence of bovine skim milk or whey. Thus, it is unlikely that the suppressive effect of milk on lymphocyte proliferation could be mediated through alterations in the expression of major histocompatibility class II molecules or in production of interleukin-1 by monocytes. The role of other monocyte antigens or secretory products, however, should also be evaluated.     

Effects of florfenicol, chloramphenicol, and thiamphenicol on phagocytosis, chemiluminescence, and morphology of bovine polymorphonuclear neutrophil leukocytes

Florfenicol, chloramphenicol, and thiamphenicol were tested in vitro to determine their potential toxic effects on bovine neutrophils. Antibiotics were tested at 4000, 2000, and 10 micrograms/ml of incubation mixture. Percentage phagocytosis was determined by incubations with neutrophils isolated from milk of five cows and 32P-labeled Staphylococcus aureus and 5% skim milk. The effect of 4000 micrograms of each antibiotic on chemiluminescence was determined on neutrophils isolated from mammary secretions of three nulliparous heifers. Morphological evaluation by transmission and scanning electron microscopy was performed on neutrophils isolated from two heifers at antibiotic concentrations of 4000 and 10 micrograms/ml. Chloramphenicol depressed phagocytosis at the high and medium doses and blocked chemiluminescence activity at the high dose. No effects were observed for florfenicol and thiamphenicol. Transmission electron microscopic examination showed that at the high concentration of drugs, 99, 99, 97, and 76% of the neutrophils treated with florfenicol, chloramphenicol, thiamphenicol, and dimethyl sulfoxide were abnormal. Examination by scanning electron microscopy showed that the percentage of neutrophils without pseudopodia averaged 67, 94, 32, and 16%, respectively. Results indicated that neither florfenicol nor thiamphenicol altered neutrophil function, but they did alter neutrophil morphology, although to a lesser extent than did chloramphenicol.     

Lipolytic activity with the membrane fraction of bovine skim milk.

Colloidal phosphate-free skim milk was subjected to gel filtration on Sepharose 4B. Lipolytic activity was observed in the membrane material eluted in the void volume fraction and in the protein fraction representing a broad range of molecular weights.     

Methods to assess the propensity of milk fat globules toward lipolysis and the ability of skim milk to inhibit lipolysis

Methods to quantitate factors in milk relevant to cold-induced lipolysis are described. Skim milk was incubated with milk fat globules isolated from a pool of normal milk and a fixed amount of purified lipoprotein lipase. The release of fatty acids in 24 h at 4 degrees C was determined. Most skim milk samples inhibited lipolysis, but the effect varied greatly. Skim milk from milk prone to spontaneous lipolysis was less inhibitory than skim milk from normal milk. In general, both the casein and the serum fractions of skim milk inhibited lipolysis. However, variation was greater in the effects of individual samples of milk serum. In a few extreme cases, with samples from milk with spontaneous lipolysis, the serum fraction actually stimulated lipolysis. Globules were isolated and then incubated with skim milk from normal milk and a fixed amount of purified lipoprotein lipase. This gave a measure of accessibility to lipolysis of milk fat globules in normal skim milk. There was a considerable variation in propensity toward lipolysis between milk fat globules from individual milk samples. Milk showing different levels of lipolysis obtained from five cows revealed that skim milk inhibition of lipolysis and the propensity of milk fat globules toward lipolysis were characteristic for each cow.     

Relationships between radioimmunoassays of alpha lactalbumin and prolactin in bovine skim milk

A radioimmunoassay developed for alpha-lactalbumin was sensitive between 5 and 140 ng alpha lactalbumin. Addition of increasing volumes of milk to assay tubes progressively decreased binding of iodine-125-labeled alpha-lactalbumin to the antisera in a manner which paralleled the binding curve generated by increasing concentrations of standard alpha-lactalbumin. The addition of 10, 20, 30, or 40 ng of alpha-lactalbumin to diluted milk samples gave 90, 100, 105, and 102% recoveries. Alpha-lactalbumin antisera did not crossreact with 1000 ng of bovine casein, blood serum albumin, beta-lactoglobulin, prolactin, or growth hormone. Milk from each of approximately 100 Holstein Friesian cows at different stages of lactation was sampled monthly for 12 mo. Concentrations of alpha-lactalbumin (1.63 mg/ml) and prolactin (24.9 ng/ml) in samples of skim milk collected in the 1st mo of lactation were greater than those in the remaining months of lactation. Monthly concentrations of alpha-lactalbumin and prolactin in skim milk did not change significantly during seasons of the year. The correlation between concentrations of prolactin and alpha-lactalbumin pooled within subclasses of month of lactation within month of year was .08 for 1125 pairs.     

Partitioning of nitroxynil,oxyclozanide and levamisole residues from milk to cream,skim milk and skim milk powder

Veterinary drugs are necessary to control fluke in animals, and if not properly used, residues of these drugs may be found in milk. The aim of this study was to determine whether residues of nitroxynil, levamisole and oxyclozanide in milk partition into skim milk powder during processing. Milk targeted to contain high, medium or low levels of residue was obtained following treatment of trial animals. On separation of cream and skim milk, > 90% of the residue partitioned with the skim milk in all cases. During powder processing, the residues were not degraded with almost 90% of the residue detected in the powder.     

Actions of bovine somatotropin on polymorphonuclear leukocytes and lymphocytes in cattle

Objectives were to determine 1) in vitro effects of bST on function of polymorphonuclear leukocytes and lymphocytes and 2) in vivo effects of bST on leukocyte function of heifers fed to maintain medium or high growth rates. When administered in vitro, bST did not affect function of polymorphonuclear leukocytes. [Methyl-3H]thymidine incorporation by resting lymphocytes was stimulated by 1000 ng/ml bST. When given in vitro, bST did not further enhance [methyl-3H]thymidine uptake by mitogen-stimulated lymphocytes cultured at 38.5 degrees C but reduced the depression of mitogen-stimulated proliferation caused by incubating cells at 42 degrees C. When bST was administered in vivo, phagocytosis and killing of Escherichia coli by polymorphonuclear leukocytes from bST-treated heifers were not different from cells of control heifers. As measured by [methyl-3H]thymidine uptake after stimulation with phytohemagglutinin, lymphocytes from bST-treated heifers responded similarly to those of control heifers when incubated at 38.5 degrees C, but the depression in [methyl-3H]thymidine uptake due to culture at 42 degrees C was less for lymphocytes obtained from bST-treated heifers. In conclusion, bST had little effect on function of polymorphonuclear leukocytes but could promote proliferation of lymphocytes in vitro and protect cells from effects of elevated temperature.     

Presence of lactoferrin in a bovine skim milk fraction with acid phosphatase activity

The aim of this study was to determine the principal active molecules in the acid phosphatase (AcP) fraction of skim milk origin using immunostaining and AcP staining. The AcP fraction was separated from skim milk at 0.38 m NaCl using carboxymethyl cellulose chromatography at pH 5.2. The molecular mass of the active molecule in AcP fraction was estimated to be 80 kDa by immunostaining and AcP staining. The 80 kDa protein was analyzed by a protein sequencer, using the automated Edman degradation method; the first thirteen N-terminal amino acid sequence obtained were shown to be APRKNVRWXTIXQ. For that amino acid sequence, there was 84% (11/13 residues) homology with the amino acid sequence of bovine lactoferrin (LF). The AcP fraction and commercial LF showed a similar AcP activity profile, having an optimum pH of 4.5 and temperature of 60 °C. Thus, the AcP fraction from bovine skim milk was isolated and the principal active molecule present was tentatively identified as LF.     

Factors influencing the production of acetoin and diacetyl by lactic acid bacteria in skim milk

Nine strains of starter cultures belonging to the genera Streptococcus, Leuconostoc and Lactobacillus were studied for the effect of pyruvate concentration, incubation period and the associative growth on the production of acetoin and diacetyl in skim milk. The absolute amount of acetoin and diacetyl and the amount pro μmol pyruvate increased with increasing the concentration of pyruvate. At the low concentration of pyruvate the maximum amount of these neutral carbonyl compounds was first reached after 34 days, whereas at the high concentration it was readily achieved after 6 days and then decreased. Production of acetoin and diacetyl by mixed-species cultures from added citrate was either increased or markedly decreased compared to the single-species culture.     

Fractionation of pasteurized skim milk proteins by dynamic filtration

This paper describes a two-stage process for separating milk proteins from pasteurized skim milk in three fractions: casein micelles, β-Lactoglobulin (β-Lg) and other large whey proteins, and α-Lactalbumin (α-La). Casein micelles were extracted in the retentate of a microfiltration using rotating ceramic disk membranes. α-La and β-Lg transmissions remained between 0.8 and 0.98. Their yields in permeate reached 81% for α-La and 76.6% for β-Lg at a VRR of 5.4. The separation between β-Lg and α-La was carried out by UF using a rotating disk module equipped with a 50 kDa PES circular membrane. Permeate fluxes were very high, remaining above 340 L h^?1 m^?2 at VRR = 5 and 40 °C. α-La transmission remained generally between 0.2 and 0.13 giving yields from 28% to 34%. β-Lg rejection was above 0.94, giving a maximum selectivity of 4.2. These data confirm the potential of dynamic membrane filtration for separating α-La and β-Lg proteins from skim milk.     

Factors affecting in vitro lipogenesis by bovine mammary tissue slices

An in vitro system of fatty acid synthesis by bovine mammary tissue slices was developed and characterized. Mamary tissue slices were prepared wih a hand microtome and 120 to 150-mg slices incubated in Krebs-Ringer bicarbonate buffer (pH 7.4) at 37 C. Acetate incorporation into fatty acids and acetate and glucose oxidation to carbon dioxide were linear for 3 h. Mammary tissue was stable and lost no lipogenic capacity for 60 min prior to slicing and incubation if maintained in isotonic sucrose at 5 or 25 C. Fatty acid synthesis from acetate was maximum at an acetate concentration of 10 mM and a glucose concentration of 5 to l0 mM. Insulin had no effect on acetate incorporation into fatty acids by cow mammary tissue slices when varied from physiological to pharmacological concentrations. An examination of lipids synthesized by bovine mammary slices indicated that over 70% of the synthesized fatty acids were located in triglycerides with the pattern of fatty acids similar to that produced in vivo. The use of the in vitro system of bovine mammary tissue slice incubations offer an excellent tool for investigation of cellular metabolism and biosynthesis of fatty acids.     

The binding ability of bovine milk caseins to mutagenic heterocyclic amines.

The binding ability of bovine milk caseins with mutagenic heterocyclic amines was investigated. Binding was determined with 2 mg of casein and 20 micrograms of heterocyclic amine in .40 ml of pH 7.4, 50 mM phosphate buffer, at 37 degrees C, in a shaker for 10 min. The unbound heterocyclic amine in protein-free ultrafiltrate was analyzed by HPLC. The binding ability of whole casein, alpha s-casein, beta-casein, and kappa-casein, respectively, was 90.08, 83.06, 90.92, and 96.70% with 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole; 85.48, 51.54, 63.62, and 82.71% with 3-amino-1-methyl-5H-pyrido[4,3-b]indole; and 87.03, 59.77, 97.04, and 88.30% with 2-amino-6-methyldipyrido[1,2-a:3',2'-d]-imidazole. Higher binding of 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole and 3-amino-1-methyl-5H-pyrido[4,3-b]indole to alpha s-casein, beta-casein, and kappa-casein was observed at pH above 7.4, and the binding was inhibited at pH below 6.5. The maximum binding of 2-amino-6-methyldipyrido[1,2-a:3',2'-d]imidazole to these caseins was at pH 6.6 and 7.4. The binding was inhibited at alkaline pH above 8.5 and acidic pH below 6.5.     

Advanced analytical tools for bovine lactoferrin identification and quantification in raw skim milk to finished lactoferrin powders

As the use of bovine lactoferrin (bLF) has increased, greater regulatory and commercial interest is being placed on bLF identification and quantitation and quality. Several methods can be used to quantitate bLF, but a robust accurate quantification method applicable as a standardised tool for feed milk, process monitoring, and evaluation of end products has been less of a concern. bLF concentrations increase through the bovine lactation cycle, so accurate and timely information is required that can increase both processing plant performance and product quality and output. In this article we evaluate analytical cation exchange, reverse phase high-performance chromatography and size exclusion chromatography methods for the analysis of bLF identification and quantification from whole milk, process intermediates, ingredients, milk and infant formula products. However, the methods described in this article to identify and quantify the bLF may not be directly assimilated to the biological activities of the bLF.     

Hexose shunt dehydrogenase activity in leukocytes isolated from bovine milk

A spectrophotometric method for measuring small changes in light absorption at specific wavelengths from highly turbid suspensions of biological material (dual-beam spectrophotometry) has been used to measure glucose-6-phosphate oxidation and nicotinamide adenine dinucleotide phosphate (oxidized) reduction catalyzed by lysates of milk leukocytes. This reduction was correlated .84 with cellular protein concentration and, therefore, with cell concentration.