Milk Polymorphonuclear Neutrophil Research Articles

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.

Neutrophil dynamics in the blood and milk of crossbred cows naturally infected with Staphylococcus aureus.

Aim:The present study was designed to evaluate the neutrophil dynamics in terms of the functional competence during subclinical mastitis (SCM) and clinical mastitis (CM).Materials and Methods:A total of 146 Karan fries cows were screened and were divided into three groups as control (n=12), SCM, n=12 and CM, n=12 groups on the basis of California mastitis test scoring, bacteriological evaluation, gross and morphological changes in milk and by counting milk somatic cell count (SCC). Both blood and milk polymorphonuclear neutrophils (PMNs) were isolated in the study. Phagocytic activity (PA) was studied by spectrophotometrically; neutrophil extracelluar traps (NETs) were studied by scanning electron microscopy (SEM); CD44 was quantified by flow cytometry and apoptosis was studied by fluorescent microscopy.Results:Significantly (p<0.05) higher SCC, PA was found in milk of CM cows as compared to SCM and control cows. Significantly lower (p<0.05) apoptosis was observed in PMNs isolated from both blood and milk of CM group of cows when compared to control and SCM group. The milk neutrophils of CM group of cows formed NETs as evidenced from the SEM images. Surface expression of CD44 revealed a significantly (p<0.05) lower expression in milk neutrophils of CM group of cows when compared to SCM and control group of cows.Conclusion:The study indicated a positive correlation between delayed neutrophil apoptosis, persistent staying of neutrophils at the site of infection along with formation of NETs as the strategies to fight against the pathogens in the udder during Staphylococcal mastitis. The study forms a strong base for future molecular research in terms of neutrophil recruitment and neutrophil removal from the site of infection.

Function of milk polymorphonuclear neutrophil leukocytes in bovine mammary glands infected with Corynebacterium bovis

Corynebacterium bovis is one of the most commonly isolated bacteria from aseptically collected bovine milk samples. The objective of the current study was to characterize the bovine innate immune response by evaluating milk polymorphonuclear neutrophilic leukocytes (PMNL) in mammary glands infected with C. bovis. Twenty quarters infected with C. bovis and 28 culture-negative quarters (with milk somatic cell count <1×105 cells/mL) were used. The percentages of milk PMNL and the PMNL expression of L-selectin (CD62L), β2-integrin (CD11b), and one of the endothelial-selectin ligands (CD44), as well as the levels of intracellular reactive oxygen species (ROS) and the phagocytosis of Staphylococcus aureus, were evaluated by flow cytometry. The apoptosis and necrosis rates of the PMNL were quantified using dual-color flow cytometry with fluorescein-labeled annexin and propidium iodide. The present study revealed a higher percentage of PMNL in the milk from C. bovis-infected quarters, although no significant differences were found in levels of CD44, CD62L, or CD11b expression among the PMNL. A lower percentage of apoptotic PMNL was observed in C. bovis-infected quarters, as well as higher percentages of viable PMNL and of PMNL that produced intracellular ROS. However, no alterations were observed in phagocytosis of Staph. aureus by the PMNL or in intensity of intracellular ROS production by PMNL. Thus, results from this investigation of the PMNL function support, at least in part, the fact that intramammary infections by C. bovis may offer protection against intramammary infections by other bacteria.

ROLE OF NEUTROPHILS IN PROTECTION OF UDDER FROM INFECTION IN HIGH YIELDING DAIRY COWS

Summary Protection of the mammary gland against mastitis-c ausing pathogens is mediated by many factors in the gland and blood circulation. The professional phago cytic cells of bovine udder, polymorphonuclear neutrophils (PMN) and macrophages, comprise the first line of defense against invading mastitis pathog ens. Most researchers now accept that the PMN is a key f actor in the cows’ defense against intramammary infections. The PMN are the only leukocytes in the milk compartment that are capable of producing large amounts of reactive oxygen species (ROS) to kill ph agocytosed bacteria. In this review, the role of PM N function as an effective defense against intramamma ry pathogens in dairy cows and physiopathological influencing factors on blood and milk PMN functions are discussed. Apart from playing a crucial role i n the first line of defense mechanism, the PMN can also, indirectly, interfere with the complex interactions of second line of defense against pathogens. To minimi ze mammary tissue damage caused by bacterial toxins and oxidative products released by PMN, elimination of invading bacteria should proceed quickly. This can provide balance among inflammation reactions, bactericidal activity and tissue damage. The good balanc e between host-pathogen interactions might be affecte d by the physiological (e.g., stage and number of lactation) and pathological (e.g., local-systemic e ffect of mastitis) status of dairy cows. Hormones, metabolites and acute phase proteins also influence PMN functions, thereby affecting the outcome of mastitis. This is especially the case around partur ition. PMN function in healthy cows after parturiti on is highly heritable and has been related to the cow’s susceptibility to clinical mastitis. Despite advanc es in veterinary science, nutrition and molecular biology , mastitis is still a very big problem in high yiel ding dairy cows. The long-term and fundamental solution for mastitis affecting high yielding dairy cows is to str engthen cows’ immune systems by means of attainable physio-immunological approaches. This requires a comprehensive study on the immunophysiological alterations throughout lactation and during mastitis. T his review focuses on some factors affecting PMN functions during the lactation cycle and mastitis in high yielding dairy cows.

Calcium homeostasis and its relationship to superoxide production in blood and milk neutrophils of lactating goats

Polymorphonuclear neutrophils (PMN), which comprise over 70% of the somatic cells in goat milk, are a major cellular component of innate immunity in the goat mammary gland. However, the function of milk PMNs is modified after diapedesis compared to PMNs in blood. As many aspects of PMN activity depend directly on intracellular Ca 2+ concentration ((Ca 2+) i), the present study aimed to determine the changes in Ca 2+ homeostasis of milk PMNs from lactating goats compared to autologous blood PMNs, and to examine the significance of these variations to the immuno-competency of milk PMNs. The intracellular Ca 2+ store of freshly prepared milk cells was estimated from the elevation of (Ca 2+) i after ionomycin treatment, which was found to be significantly less than blood PMNs. Replenishment of the intracellular Ca 2+ store in milk cells after intracellular Ca 2+ depletion by Bapta-AM followed by spiking with 2.5 mM Ca 2+ for 20 min was also compared to that of blood PMNs, showing that after depletion/spiking the intracellular Ca 2+ store in milk cells was much less than blood PMNs. The production of superoxide anion (O 2 −) in vitro in response to (Ca 2+) i-dependent or (Ca 2+) i-independent modulators was used to evaluate the relevance of altered Ca 2+ homeostasis on the immuno-competency of milk cells compared to blood PMNs. The results indicated that milk cells produced similarly low levels of O 2 − as blood PMNs when treated with ionomycin. However, the amount of O 2 − produced by milk cells in response to phorbol 12-myristate 13-acetate (PMA) stimulation, although greater than ionomycin treatment, was significantly less than that of blood PMNs. The capacity for O 2 − production by both cell types in response to PMA reverted to the resting state with use of the protein kinase C (PKC) inhibitor, staurosporine. In conclusion, the current study demonstrated an irreversible shortage of intracellular Ca 2+ in the milk PMNs of lactating goats compared to blood PMNs. It also showed that preliminary O 2 –production, primed by ionomycin treatment, remained unchanged in milk PMNs, despite the shortage in intracellular Ca 2+, but decreased O 2 − production capacity, mediated via the PKC pathway, in milk PMN. It is suggested that the defects in Ca 2+ homeostasis in milk PMNs of lactating goats is partially attributable for the post-diapedesis functionality modifications.

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.

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

Abstract 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.

High milk neutrophil chemiluminescence limits the severity of bovine coliform mastitis

Polymorphonuclear neutrophil (PMN) function changes during mastitis. To investigate the contribution of milk PMN to the severity of Escherichia coli (E. coli) mastitis, chemiluminescence (CL) of blood and milk PMN and their efficiency to destroy coliform bacteria in the mammary gland were examined following the induction of E. coli mastitis in early lactating cows. To better assess and define the degree of mastitis severity, cows were classified as moderate and severe responders according to milk production loss in the non-infected quarters at post-infection hour (PIH) 48. There was an inverse relationship between pre-infection milk PMN CL and colony-forming units at PIH 6. In moderate cows, the pre-infection blood and milk PMN CL was approximately 2-fold higher than that of severe cows. The probability of severe response increased with decreasing pre-infection PMN CL. At the beginning of the infection blood and milk PMN CL was consistently higher, and milk PMN CL increased faster after infection in moderate cows. At PIH > 48 milk PMN CL in severe cows exceeded that of moderate cows. The somatic cell count (SCC) in moderate cows increased faster than colony-forming units, whereas in severe cows the results were reversed. The kinetics of CL activity for blood and milk PMN before and during the early phase of infection confirmed an impairment in PMN CL activity for severe responding cows. High pre-infection blood and milk PMN CL and the immediate increase of milk PMN CL and SCC after infection limited bacterial growth thereby facilitating the recovery of E. coli mastitis in moderate cows. Our study strengthens the idea that pre-existing milk PMN (a static part of the udder's immune defense) functions as a "cellular antibiotic" before and during infection, and low milk PMN CL is a risk factor for bovine coliform mastitis.

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 12h 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.

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 β-, κ-, αs1-, and αs2-casein that were generated by somatic cell proteases. Although κ-casein is considered particularly resistant to endogenous proteolysis, a κ-casein peptide was electrophoretically isolated in association with a β-casein fragment. The in vitro kinetic studies of caseinate hydrolysis by elastase, one of the main polymorphonuclear neutrophil (PMN) proteases, suggested that the β-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.

Composition and Milk Cell Characteristics in Quarter Milk Fractions of Dairy Cows with Low Cell Count

Variation in milk composition and milk polymorphonuclear neutrophil leukocyte (PMN) characteristics and functions among quarter milk fractions were investigated in order to evaluate the optimal fraction for the determination of local immune response. Five fractions were classified during milking: foremilk (I), cisternal milk (II), main milk (III), strippings (IV) and residual milk (V). Somatic cell count (SCC), fat, protein, lactose, sodium, potassium, chloride, polymorphonuclear leukocyte necrosis, apoptosis and oxidative burst were analysed in each fraction.The logSCC and fat concentration were highest in residual milk (P<0.05), whereas protein and lactose concentration were highest in the earliest fractions (I, II, III) (P<0.05). Polymorphonuclear neutrophil leukocyte necrosis was lowest in strippings and residual milk (P<0.05), and PMN apoptosis was lowest in residual milk (P<0.001). The highest percentage of PMN with oxidative burst was found in residual milk (P<0.05), and was associated with the highest mean fluorescence intensity (MFI) (P<0.05).In conclusion, late fractions have more PMN and more active PMN. Nevertheless, it is suggested that each fraction is appropriate in the study of local immune response of the mammary gland, however the fraction used in the study has to be specified.

Enzymatic activities of bovine peripheral blood leukocytes and milk polymorphonuclear neutrophils during intramammary inflammation caused by lipopolysaccharide.

Leukocytes are recruited from peripheral blood into milk as part of the inflammatory response to mastitis. However, excessive accumulation of inflammatory cells alters the quality of milk and the proteases produced by polymorphonuclear neutrophils (PMNs) and macrophages may lead to mammary tissue damage. To investigate PMN recruitment and the kinetics of their intracytoplasmic enzymes in inflammation, we generated mastitis in six cows by intramammary infusion of lipopolysaccharide (LPS). Clinical signs of acute mastitis were observed in all of the cows, and normal status was resumed by 316 h. Intracytoplasmic elastase, collagenase, and cathepsin activities were measured within live cells by flow cytometry in peripheral blood leukocytes and milk PMNs before and during the inflammatory process (at 10 time points between 4 and 316 h). The proportion of immature PMNs was appreciated by CD33 surface labeling measured in flow cytometry. Leukopenia was observed in the peripheral blood 4 h postinfusion, concomitant to an increase in somatic cell counts in milk. CD33(+) PMNs were preferentially recruited from the peripheral blood to milk. Enzymatic activities were detected in PMNs, lymphocytes, and monocytes at levels depending on the cell type, sample nature, and time of collection. Milk PMNs had lower enzymatic activities than peripheral blood PMNs. This study showed that milk PMNs recruited during LPS-induced experimental mastitis have an immature phenotype and significantly lower enzymatic activities than peripheral blood PMNs. This suggests that CD33, an adhesion molecule, may be involved in the egress from blood to milk and that the enzymatic contents of PMNs are partly used during this process.

Phagocytic activity of bovine polymorphonuclear neutrophil leucocytes.

Two different investigations were conducted on the chemiluminescent activity of bovine milk polymorphonuclear neutrophil leucocytes (PMN) activated by different stimuli: zymosan, derived from the wall of Saccharomyces cerevisiae (Experiment A), and Streptococcus uberis and Escherichia coli (Experiment B). In Experiment A, a quarter with a phagocytic activity of PMN < 20 mV/1000 PMN following stimulation with zymosan was found to be 23 times more likely to be a clinical mastitis case than a quarter with phagocytic activity above this threshold value. In Experiment B, calculation of the odds ratio showed similar results following stimulation with Str. uberis or Esch. coli. These results provide evidence that immunocompromisation of mammary gland defences could predispose to clinical mastitis. They also support the need to challenge phagocytic cells with appropriate stimuli, and the Esch. coli test seems to be the most sensitive.

Flow Cytometric Measurement of Bovine Milk Neutrophil Phagocytosis

A flow cytometric method for the evaluation of the phagocytic capacity of bovine milk polymorphonuclear neutrophils (PMN) is described. Milk PMN were isolated from stripping milk collected from udder quarters fitted with abraded intramammary devices (AIMD). A significant increase in the milk somatic cell count was observed in the stripping milk after the insertion of AIMD (308×103 and 1447×l03 cells/ml milk before respectively after the insertion of the AIMD, p < 0.001). PMN were also isolated from blood by a discontinous gradient of Percoli. Blood and milk PMN were incubated for 15 min with FITC-labeled bacteria in a ratio of 1 PMN:20 bacteria and a final serum concentration of 10 %. The number of extracellular bacteria and the percentage of phagocytic cells were measured by a flow cytometer. Percentage of phagocytized bacteria by milk PMN was significantly lower than that by blood PMN (p < 0.05). A smaller number of active phagocytes was present among cells isolated from milk than among cells isolated from blood. The phagocytic capacity of milk PMN reflects that of blood PMN in the same animal. A large variation in the phagocytic capacity of blood and milk PMN among animals was observed.