Investigation of persistent infection of bovine viral diarrhea virus (BVDV) in Holstein dairy cows.

The aim of this study was to investigate the persistent infection (PI) of bovine viral diarrhea virus (BVDV) along with its coexistence between BVDV antibody titer and BVD virus in blood of Holstein dairy cows. Only large commercial farms (each contained < 1000-3000 unvaccinated cows) were included. There were 11 dairy cattle herds. They included nearly 20,000 dairy cows. Totally, 140 cows, > 3 months to almost 10 years old, were randomly sampled. Indirect enzyme-linked immunosorbent assay (ELISA) and reverse transcription-polymerase chain reaction (RT-PCR) were used to detect BVDV antibody and virus, respectively. The percent positivity (PP) < 14 and ≥ 14 values are interpreted negative and positive, respectively. Simultaneously, whole blood samples pooled in groups of 10 animals were used for molecular detection of BVDV. The results revealed that 138 (98.56%) out of 140 cows were positive for BVDV antibody, while the BVDV antigen was detected only in 2 (1.42%) cows, which were negative for BVDV antibody and so were considered as persistent infection (PI) cows. They were also retested 3 weeks apart. Since the results showed the strong coexistence between seropositivity and BVD virus, in the infected dairy cattle herds, the combination of simple ELISA and pooled whole blood RT-PCR strategy could be an achievable approach to detect PI animals.

Effect of culture and maturation on human monocyte-derived dendritic cell surface markers, necrosis and antigen binding.

Dendritic cells (DC) are antigen-presenting cells (APC) that are important for innate and acquired immune responses. Owing to their involvement in autoinflammation, autoimmunity and cancer, DC are useful cellular models for biomedical research. Appropriate DC production in vitro could aid the study of DC in many human diseases. We used fluorochrome-based flow cytometry assays to analyze the effects of culture period and maturation of monocyte-derived DC (MoDC) on their viability and necrosis, purity, CD11c expression and phagocytic capacity. The morphological changes that occur as purified monocytes become DC were assessed at 24 and 72 h, and 6 and 9 days in culture. The dynamics of certain cell surface markers of monocytes and mature MoDC (mMoDC) also were assessed using fluorescence-based assays. We found that day 6 of culture yielded the most functional immature MoDC (iMoDC) with maximal viability, purity, CD11c expression and appropriate phagocytic capacity. Mass production of viable MoDC could be useful for immunotherapy.

In silico study of effects of polymorphisms on biophysical chemical properties of oxidized N-terminal domain of X-ray cross-complementing group 1 protein.

Base excision repair (BER) is the major pathway involved in removal of endogenous and mutagen-induced DNA damage. The X-ray cross-complementing group 1 protein (XRCC1), which participates in BER, is a scaffolding protein. The oxidized XRCC1 N-terminal domain (NTD) forms additional interactions with DNA polymerase ß (Pol ß). Any change in the residues of a protein (XRCC1, XRCC4, etc.) may alter its stability and function. Many coding regions of genes have single nucleotide polymorphisms (SNPs) that change the conformation of their products, and they are probably involved in some diseases. The R7L and R107H mutations are located in the XRCC1-NTD. In the present study, biophysical chemical properties of oxidized XRCC1-NTD (wild type or mutants) were investigated at different temperatures (290, 295, 298, 301, 304, 309, 310, 311, and 312 K) in water using in silico molecular mechanic computational methods. Comparison of the average calculated potential energies of oxidized XRCC1-NTD reveals that the R7L mutation increases stability, but the R107H and R7L&R107H mutations are destabilizing. Therefore, mutant types of this protein (R107H or R7L&R107H) may not function correctly. Furthermore, quantitative structure-activity relationship (QSAR) of oxidized XRCC1-NTD and docking assay showed that the R7L mutation is advantageous but the R107H and R7L&R107H mutations are disadvantageous for XRCC1-NTD, and in the latter cases it cannot interact with Pol ß as well as the wild type does. Hence, DNA repair may be defective. Also, using the equation dE = ∂Ε/(∂Τ)V·dT + ∂Ε/(∂V)T·dV, it was determined that the best temperature for normal activity of oxidized XRCC1-NTD is exactly the natural body temperature (310 K).

In vitro effects of very low levels of aflatoxin B1 on free radicals production and bactericidal activity of bovine blood neutrophils.

As one of the most potent and hazardous feed/food-originated mycotoxins, aflatoxin (AF) B1 is regarded as a potent immunosuppressor in dairy cows. Neutrophils (PMN), as key effector cells against pathogens, have a high potential to kill engulfed microbes. To investigate the in vitro effects of very low doses of AFB1 on blood PMN functions, we examined the effects of biologically relevant concentrations of AFB1 on the phagocytosis and non-phagocytosis dependent luminol, representative of mainly intracellular free radicals, and isoluminol, representative of mainly extracellular free radicals, chemiluminescence (CL), necrosis and apoptosis of PMN. Isolated blood PMN from healthy dairy cows (n=12) were exposed to 0, 0.01, 0.05 and 0.5 ng/ml of AFB1 for 0.5 and 18 h depending on the assay. Further, blood PMN of healthy dairy cows (n=8) were exposed to 0.5 ng/ml of AFB1 for 3h and myeloperoxidase (MPO) activity, superoxide anion (O2⁻) production, phagocytosis and killing activities against Staphylococcus (S.) aureus and Escherichia (E.) coli, were examined. Though the effect of extremely low doses of AFB1 were less pronounced, at 0.5 ng/ml the production of free radicals was greatly enhanced, especially extracellularly. In contrast to isoluminol CL, the AFB1-treated PMN showed a remarkably impaired phagocytosis-depended luminol CL. PMN necrosis and apoptosis were not affected by AFB1. MPO activity, O2⁻ production, phagocytosis rates and killing of E. coli and S. aureus by AFB1-treated PMN were significantly lower than those of non-treated ones. Our results show the extracellularly pro-oxidant and antiphagocytic properties of very low doses of AFB1 for bovine PMN. The scope of the suppressive effects of the in vitro AFB1 levels on cellular innate immune functions should be considered for high yielding dairy cows.

Effect of bovine viral diarrhoea virus biotypes on adherence of sperm to oocytes during in-vitro fertilization in cattle.

Bovine viral diarrhoea virus (BVDV), a member of the Pestivirus genus, is one of the most important pathogens of dairy cattle; it can cause several clinical syndromes, ranging from subclinical to severe disease. The objectives of the current studies were to assess the effects of two biotypes of BVDV on sperm attachment to the zona pellucida (ZP) of oocytes and on fertilization rate in bovine in vitro fertilization (IVF). In two experiments, sperm at two concentrations (105 and 106/mL) and oocytes were incubated with 106 TCID50/mL cythopatic (CP) or noncythopatic (NCP) BVDV. In the first experiment, with the lower sperm concentration (105/mL), male and female gametes were infected with CP or NCP BVDV, whereas in the second experiment, the sperm concentration was 106/mL, and sperm and oocytes were also infected with CP or NCP BVDV. The number of sperm attached to the ZP and the fertilization rate were evaluated with fluorescence microscopy on the ZP of fertile and infertile oocytes. In the first experiment, compared to the control group (n = 97), oocytes infected with CP BVDV and incubated at the lower (105/mL) sperm concentration positively affected sperm attachment (n = 123) to the ZP of fertile oocytes (P < 0.05). In comparison with the control group (n = 115), sperm infected with CP BVDV negatively affected sperm binding (n = 93) to the ZP of infertile oocytes (P < 0.05). In the second experiment (106 sperm/mL), for both fertile and infertile oocyte groups, sperm attachment in the control group was very high and deemed uncountable. However, in treated groups, the number of sperm attached to the ZP was countable. Only sperm infected with CP BVDV negatively affected sperm binding capacity (n = 81) to the ZP of fertile oocytes (P < 0.05). Although CP and NCP BVDV significantly reduced the fertilization rate of oocytes incubated with a higher sperm concentration, with the lower sperm concentration, only NCP BVDV significantly diminished fertilization rate with contaminated sperm and oocytes (P < 0.05). In conclusion, this study supported the detrimental impacts of sperm or ooctyes infected with CP or NCP BVDV on sperm attachment to the ZP of bovine oocytes and on fertilization rate during bovine IVF.

Phagocytic and bactericidal activity of blood and milk-resident neutrophils against Staphylococcus aureus in primiparous and multiparous cows during early lactation.

To examine the effect of parity on polymorphonuclear neutrophils (PMN) function, phagocytic and bactericidal activity of the PMN isolated from blood and milk against Staphylococcus aureus was compared between groups of 6 primiparous and 6 multiparous healthy dairy cows during early lactation using bacteriological and PMN-pathogen interaction assays. Latex-stimulated luminol-amplified chemiluminescence (CL) and viability of these PMN were also investigated. The phagocytosis and killing of S. aureus by blood were remarkably higher than those of milk PMN. Similarly, the CL and viability in blood PMN were markedly higher than in milk PMN. Both in blood and in milk the phagocytosis of S. aureus by PMN in primiparous cows was substantially higher than in multiparous cows. The killing activity of blood PMN against S. aureus was 42.3+/-3.4% and 23.2+/-1.7% in primiparous and multiparous, respectively. Milk PMN killed only 20.7+/-2% S. aureus in primiparous and 10.2+/-1.3% in multiparous cows. Blood and milk PMN CL and milk PMN viability were significantly higher in primiparous cows. The pronounced reduction in phagocytic and bactericidal activity in blood and milk-resident PMN from multiparous cows, in part, resulted from the pronounced decrease of PMN viability and free radicals production capacity; this suggests that heifers' udders could be more protected against S. aureus, which remains to be tested in the field.

The effect of selenium sources and supplementation on neutrophil functions in dairy cows.

Selenium (Se), an essential micronutrient, is believed to enhance neutrophil functions. This study aimed to compare the effects of supplemented organic (Sel-Plex®) and inorganic (sodium selenite) Se on neutrophil functions in high-producing dairy cows, during the periparturient period. Twenty-five Holstein cows were randomly allocated to five dietary treatments as follows: control diet (basal diet without Se supplementation), IN 0.3 (basal diet supplemented with inorganic Se at 0.3 mg/kg dry matter (DM)), IN 0.5 (inorganic Se at 0.5 mg/kg DM), OR 0.3 (organic Se at 0.3 mg/kg DM) and OR 0.5 (organic Se at 0.5 mg/kg DM). Some evaluated parameters included neutrophil functions and plasma Se concentrations in cows and plasma Se concentrations in calves. Neutrophil phagocytosis did not significantly differ among the five groups. However, organic Se supplementation significantly increased (P < 0.01) the respiratory burst of neutrophils when compared to cows fed IN 0.3 and the control diet. In comparison to inorganic Se, neutrophil apoptosis was decreased (P < 0.01) when cows were fed organic Se or the control diets. These effects of organic Se on respiratory burst activities and apoptosis of neutrophils were in a dose-dependent manner. Calf plasma Se concentrations were higher (P < 0.05) when cows were fed OR 0.5 and IN 0.5.

Increase in Escherichia coli inoculum dose accelerates CD8+ T-cell trafficking in the primiparous bovine mammary gland.

Although migration of leukocytes into the mammary gland is pivotal for a cow's response against intramammary invading pathogens, the contribution of lymphocyte subsets to this response remains unclear. To investigate the dynamics of lymphocyte populations during Escherichia coli mastitis, T-lymphocyte subsets, CD4+/CD8+ ratio, CD21+ cells, and lymphoproliferation were studied in blood and milk of primiparous cows exposed to different quantities of bacteria. The cows were intramammarily inoculated with 10(4) cfu of E. coli (group A) and 10(6) cfu (group B). Compared with group A, a much greater number of lymphocytes migrated into the infected quarters at postinfection hour (PIH) 6 to 24 in group B, and the CD8+ cells were the first-recruited T cells in the milk. There was a significant decline in the CD4+/CD8+ ratios at PIH 6 to 24 in group B. The decrease of CD4+/CD8+ ratios at PIH 6 to 24 resulted mainly from greater CD8+ cell concentrations in milk. In contrast, at PIH 72, CD4+/CD8+ ratios increased about 2-fold in both groups. This increase was mainly due to the increase in CD4+ cell concentration. The increased concentration of CD4+ cells coincided with an increase in the CD21+ cell population in the milk. In blood, the increase of CD8+ cells appeared much faster in group B (PIH 6 and 12) than in group A. The results from lymphoproliferation also indicated a greater increase in the proliferative response in both blood and milk lymphocytes of group B. Our study demonstrates for the first time that an increase of E. coli inoculum dose accelerates the trafficking of CD8+ cells during initiation of E. coli mastitis, and these cells are the predominant T cells in milk during the early hours of bovine E. coli mastitis.

Effects of short dry periods on performance and metabolic status in Holstein dairy cows.

To evaluate effects of different dry period lengths on milk yield, milk composition, and energy balance of dairy cows, 122 multiparous and primiparous Holstein dairy cows were used in a completely randomized experimental design with 56-, 42-, and 35-d dry period lengths. Actual dry period lengths for respective treatments (TRT) were 56 +/- 5.1 d, 42 +/- 2.1 d, and 35 +/- 2.7 d. Overall, cows in the 42- and 56-d TRT gained more body condition than those in 35-d TRT during the dry period; however, postpartum body condition score did not change substantially among the TRT. Although from 3 to 210 DIM, differences were not detected in the milk yield of multiparous cows between the 35- and 56-d TRT, primiparous cows in the 35-d TRT produced less milk than those in 56-d TRT. In primiparous cows, the milk production at wk 9, 10, and 11 of lactation was lower in the 35-d compared with the 56-d TRT. Primiparous cows in the 35-d compared with the 56-d TRT produced less milk protein. In the 35-d TRT, serum triglyceride concentration was greater in primiparous cows than in multiparous cows during the peripartum period. Among primiparous cows, those in the 56-d TRT had greater concentrations of nonesterified fatty acids than those in the 35-d TRT during the peripartum period. No significant differences were observed in concentrations of serum glucose, insulin, and insulin-like growth factor-I during early lactation among TRT. There was also no difference among TRT for incidence of metabolic disorders. Thus, this study indicates that shortening the dry period to 35 d may be beneficial in multiparous and overconditioned cows, but not in primiparous cows.

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.

Viability of milk neutrophils and severity of bovine coliform mastitis.

To study the host-pathogen interactions during Escherichia coli mastitis, we first determined whether E. coli infection would change blood and milk polymorphonuclear neutrophil (PMN) chemiluminescence (CL) and viability. We then hypothesized that when E. coli invade the mammary gland, the viable PMN in milk would efficiently phagocytose and destroy E. coli before establishment of infection. We observed that the phagocytosis-dependent and independent CL were closely linked to PMN viability and were crucial to the outcome of mastitis. Maximal PMN influx and colony-forming units in infected quarters appeared at postinfection hours (PIH) 6 to 24. This further boosted PMN recruitment through bone marrow-blood barrier as well as blood-milk barrier. The survival of recruited PMN in the E. coli-infected quarters was much higher than that of noninfected quarters. Chemiluminescence activity of PMN from the infected quarters significantly increased following E. coli infection, even exceeding that of blood at PIH 6, 12, and 18 to 24; no such increase was observed in noninfected quarters, suggesting that the various responses of milk PMN to stimuli resulted largely from PMN viability. The highest CL intensity and durability was observed in milk PMN from infected quarters at PIH 12. Whereas an increased viability of PMN in the noninfected quarters was only significant at PIH 6, the viability of PMN in infected quarters was long lasting and significantly higher at PIH 6 to 72. Importantly, higher preinfection milk PMN viability correlated with bacterial clearance, which was accompanied by faster recovery. Our study strongly supports the hypothesis that boosting milk PMN viability could be a strategy with which to prevent or reduce the severity of coliform mastitis in dairy cows. This strategy might be achieved through strengthening bone marrow functionality.

Role of neutrophil polymorphonuclear leukocytes during bovine coliform mastitis: physiology or pathology?

The review compiles some major findings concerning the inflammatory reaction in the mammary gland of dairy cows within the physiological context of the lactation cycle. The dual role of the PMN leukocyte in defense and tissue damage during experimentally induced coliform mastitis, especially around parturition and during early lactation, is highlighted. This disease affects many high producing cows in dairy herds and may cause several cases of death per year in the most severe cases. Most researchers now accept that the PMN is a key factor in the cows' defense against intramammary infection with E coli. During diapedesis of PMN into the mammary gland, several functionally important receptors are up-regulated, allowing for a more efficient phagocytosis and killing of invading pathogens. While PMN are phagocytosing and destroying the invading pathogens, they inadvertently release chemical mediators which induces swelling of secretory epithelium cytoplasm, sloughing of secretory cells, and decreased secretory activity. Permanent scarring will result in a loss of milk production. PMN's act as friends and as foes and are important components in the balance between mammary defense and damage. The mammary gland is a complex open self-regulatory system with a continuous flow of matter, energy and information. Metabolically, it has absolute priority over many other tissues except the brain. Self-regulation with change over time is characterized by a dynamic equilibrium between two mechanisms: homeostatic and homeorhetic. The defense against invaders by innate immunity and auto-repair of the damaged tissues are covered by homeostatic mechanisms while colostrogenesis and maintenance of milk secretion are controlled by mainly homeorhetic mechanisms. However, also innate immunity has to function and develop in time, depending on the lactation cycle, and its behavior and evolution in time in such a dynamical system is a challenge and a problem at the same time. In such a complex dynamic situation it is not surprising that physiology is not far away from pathology. E. coli mastitis can be a severe problem during the beginning of lactation whereas it is completely self-curing after peak lactation (8 weeks). The approach to focus on the PMN doesn't mean that the defense of the mammary gland is more simple than in other tissues. The defense of mammary gland is characterized by its complexity and over the last years many data show that there are tight connections with the mononuclear cells in mammary gland tissue. Today it is known that T cells play a central role in orchestrating the immune response. However, because of the peculiar interest in the PMN of the authors during the last 10 years, the immunobiology of the mononuclear cells in the mammary gland is not covered.

Differential leukocyte count method for bovine low somatic cell count milk.

Whereas many differential leukocyte count methods for high somatic cell count (SCC) milk from mastitic cows are available, only a few have been developed for low SCC milk. We have developed a flow cytometric differential leukocyte count method for low SCC milk. The procedure consists of 1) 1.5 ml of diluted milk sample (30%, vol/vol dilution with PBS), 2) centrifugation, 3) leukocyte labeling with SYTO 13 and 4) flow cytometric analysis. Four major leukocyte populations can be clearly identified in the green fluorescence-side scatter dot plot: lymphocytes and monocytes (LM), polymorphonuclear neutrophils (PMN), mature macrophages (Mphi), and cells with apoptotic features based on chromatin condensation and nuclear fragmentation. The optimal processing temperature was 20 degrees C. Significant differences among samples with similar differential leukocyte counts were found. Storage of milk samples during 2 d at 7 degrees C had no effect on differential leukocyte count. Using the new method, differential leukocyte count was performed in low SCC milk samples from cows in early, mid, and late lactation. In accordance with previous studies, PMN and Mphi percentages were lower and LM percentages were higher in early lactation than in the other stages of lactation. The percentage of cells with apoptotic features was higher in early lactation than in mid and late lactation. In conclusion, a rapid, simple, accurate, and reproducible standard procedure was developed to determine the differential leukocyte count (Mphi, PMN, LM, and cells with apoptotic features) of bovine low SCC milk.

A comparative study of bovine blood and milk neutrophil functions with luminol-dependent chemiluminescence.

In this study, a technique was developed for the chemiluminescence (CL) measurement of bovine milk polymorphonuclear leukocytes (PMN). In the first study, the effects of cell number and the concentration of phorbol-12-myristate-13-acetate (PMA), luminol, latex bead particles, dimethyl sulphoxide (DMSO) and gelatin on the luminol-dependent cellular CL (LDCL) response were assessed with healthy cows in different stages of lactation. In the second study, the LDCL and in vitro bactericidal activity of blood and milk PMN towards Staphylococcus aureus was investigated. In general, the CL activity of blood PMN was consistently higher than that of milk PMN. We found that (a) the optimal cell density in blood and milk cells for maximal LDCL response ranged from 1.5 x 10(6) to 5 x 10(6) cells/mL; (b) the optimal concentrations of PMA, latex beads and luminol for maximal LDCL response were 100-200 ng/ml, 500 particles/PMN and 0.1 mmol/L, respectively. Concentrations of DMSO of 0.5-1% (v/v) did not significantly affect the maximal CL response of PMN. Gelatin concentrations of 0.1 -0.5 mg/ml had no effect on the LDCL of PMN. In addition, the LDCL of PMN was significantly correlated with bactericidal activity towards S. aureus (r = 0.78, p < 0.001 for blood PMN and r = 0.66, p < 0.01 for milk PMN). Under the optimal experimental conditions for measurement of CL produced by bovine blood and milk PMN defined in this study, LDCL assay is an accurate and reproducible technique for the rapid quantification of PMN bactericidal activity in physiological and pathological conditions of high-yielding dairy cows.

Respiratory burst activity of blood and milk neutrophils in dairy cows during different stages of lactation.

The non-stimulated and phorbol 12-myristate 13-acetate (PMA)-stimulated luminol-augmented cellular chemiluminescence (CL) response and viability of milk and blood polymorphonuclear leukocytes (PMN) were determined in lactating dairy cows during different stages of lactation. In the first study, ten healthy cows each in early, mid and late lactation were compared. In a second study, the same measurements as in the first study were evaluated longitudinally in 12 cows during 1 month following parturition. The CL activity and myeloperoxidase (MPO) content of milk PMN and macrophages (M) were also compared. Milk M did not possess MPO activity and were devoid of any luminol-enhanced CL. The CL activity of milk and blood PMN was significantly lower in early lactation than in mid and late lactation (P < 0.001). Whereas little changes were observed in viability of blood PMN, the viability of milk PMN was lower in early lactation than in mid and late lactation (P < 0.001). The percentage of PMN in isolated milk cells was also lower during early lactation than during mid and late lactation (P < 0.001). The CL activity in response to PMA during early, mid and late lactation increased 13, 59 and 42-fold in blood PMN and 1.7, 2.6 and 2.4-fold in milk PMN, respectively, in comparison with non-stimulated PMN. The CL activity, both in milk and blood PMN. the milk PMN viability and the percentage of milk PMN were lowest between 3 d and 11 d post partum. These observed changes immediately after calving could contribute to a higher susceptibility to mastitis in that period.

Local and systemic effects of endotoxin mastitis on the chemiluminescence of milk and blood neutrophils in dairy cows.

The local and systemic effects of intramammary lipopolysaccharide (LPS) injection on the chemiluminescence (CL) of milk and blood polymorphonuclear leukocytes (PMN) were investigated in six healthy early lactation cows. Clinical signs of acute mastitis such as fever, increased heart rate and a decreased milk production were observed in all cows. Before LPS challenge, the CL activity of milk PMN was significantly lower than that of blood PMN (P < 0.01). A significant negative correlation was found between pre-challenge milk and blood PMN CL and, the decreased milk production in unchallenged quarters. The CL activity of milk PMN from LPS-injected quarters increased following LPS challenge, whereas it remained unchanged in control quarters. The CL activity of blood PMN showed a biphasic increase, with two peaks and a valley below pre-challenge CL activity (P < 0.01). At post-challenge hours (PCH) 6 and 12, the CL activity of milk PMN from LPS-injected quarters exceeded that of blood PMN (P < 0.05 and P < 0.001, respectively). The decreased CL activity of blood PMN and the enhanced CL activity of milk PMN during endotoxin-induced mastitis was reflected by changes in the shape of the CL curve. In blood PMN, a decrease of the second peak of the CL curve suggests that the myeloperoxidase (MPO)-H2O2 system is impaired during endotoxin-induced mastitis. In contrast, the MPO-H2O2 system was enhanced in milk PMN from challenged quarters. The highest duration and intensity of reactive oxygen intermediate (ROI) production was observed in milk PMN from LPS-injected quarters at PCH 12. The increased viability of PMN in LPS-injected quarters and to a lesser extent in control quarters suggests possible effects of both facilitated diapedesis and inflammatory mediators on milk PMN survival. In conclusion, our results suggest that a combination of local and systemic action of E. coli endotoxin is involved in the priming of milk PMN during mastitis.

Effect of milk sampling techniques on milk composition, bacterial contamination, viability and functions of resident cells in milk.

Three different milk sampling techniques were evaluated during milk sampling: a direct aseptic collection from the udder through a sterile cannula was used as the reference technique, compared with either a manual or a mechanical sampling method. In this study 30 high-yielding Holstein-Friesian dairy cows at different stages of lactation and free of udder infection were used. For each milk sample, the influence of milk sampling techniques was determined for the following parameters: somatic cell count, milk composition, bacterial contamination, viability, in vitro phagocytosis and overall killing of Staphylococcus aureus Newbould 305, and cellular chemiluminescence. Because milk sampling occurred throughout lactation, the differences between early, mid- and late lactation were estimated. It was concluded that bacterial contamination was not significantly different in manual milking samples and the reference technique; bacterial contamination was, however, significantly (P < 0.001) higher in machine milking samples than in the reference technique. Among the different sampling techniques, no significant effects on SCC, milk composition, viability and functions of the cells isolated from milk were observed. It was found that viability, intracellular killing and cellular chemiluminescence of milk PMN were significantly lower (P < 0.05) in early lactation compared to mid-lactation. Phagocytosis was significantly (P < 0.05) higher in early lactation compared to mid- and late lactation, and no significant differences were observed between mid- and late lactation. From this study, it can be concluded that despite a higher bacterial contamination obtained with the mechanical sampling method, the 3 milk sampling techniques described in this study can be used for the evaluation of milk cell functions.