Flow cytofluorometric studies on the alteration of leukocyte populations in blood and milk during endotoxin-induced mastitis in cows

Alterations in the various leukocyte populations in milk, blood, and mammary lymph were studied by use of the flow cytometric method during acute mastitis episodes induced by endotoxin infusion (50 micrograms of lipopolysaccharide of Salmonella typhimurium SH 4809) via the teat canal. Lymph samples were collected via a semipermanent catheter from an afferent duct to the supramammary lymph node. Milk somatic cell count increased at 4 hours after infusion of endotoxin. Neutrophils were the predominant cell population for up to 59 hours after infusion. Numbers of lymphocytes and monocytes-macrophages in milk also increased after the endotoxin infusion. The total cell count in milk started to decrease during the third postinfusion day and returned to preinfusion values during the fourth day. Lymphocyte numbers remained high for about 1 week after the infusion, and lymphocytes were the predominant cell population between postinfusion days 4 and 8. Total blood leukocyte count decreased during the first 6 hours after infusion, followed by an increase until postinfusion hour 31. The proportion of neutrophils in blood increased during the first day, whereas that of lymphocytes decreased. Lymph flow rate and leukocyte numbers in lymph increased after endotoxin infusion. The proportion of neutrophils in the lymph increased during the first 6 hours, whereas that of lymphocytes decreased. After postinfusion hour 6, the inverse course of events was seen.     

Phagocytosis of bovine blood and milk polymorphonuclear leukocytes after ozone gas administration in vitro

To determine the effects of ozone on the phagocytosis of bovine polymorphonuclear leukocytes (PMNs), ozone gas was administered in vitro on the blood and milk of healthy lactating cows, cows with acute mastitis, and cows with milk fever. In the blood of healthy dairy cattle, although there was no significant effect of ozone gas on the viability of the leukocytes, phagocytosis of PMNs significantly decreased. In contrast, ozone gas administration in vitro significantly increased phagocytosis of PMNs from the blood of cows with acute mastitis and milk fever, and from mastitic milk. These findings showed that ozone administration in vitro has positive and negative effects on bovine PMN phagocytosis, depending on the health status of the animal.     

Regulation of mitogenic responses by bovine milk leukocytes

Bovine milk lymphocytes are less responsive to in vitro mitogen stimulation than peripheral blood lymphocytes (PBL). In this study, milk leukocytes (ML) or their soluble products, were co-cultured with mitogen stimulated PBL to determine if suppression could be transferred to normally responsive cells. Addition of either ML (treated with mitomycin C to prevent cell division), or supernatant from ML cultures to cultures of autologous PBL resulted in a reduction of mitogenesis by the PBL, but no suppression was seen with addition of treated PBL or PBL supernatant. Suppression was greater when the ML were from animals with chronic staphylococcal infection. Suppression by ML supernatant was not due to toxicity to the responders, since addition at the latter stages of culture had no effect on the response. These results indicate that reduced mitogenesis by milk lymphocytes may be due to the presence of suppressor cells or molecules.     

Flow cytometric characterization of bovine blood neutrophil phagocytosis of fluorescent bacteria and zymosan particles

A Flow Cytometric method for the evaluation of the phagocytic capacity of bovine blood neutrophils is described. The neutrophils were isolated from bovine blood by a one step discontinuous gradient of Percoll. By this technique of isolation, 90 ± 2.8 % (mean ± s) of the granulocytes in the whole blood were recovered.Isolated neutrophils were incubated with FITC labeled S. aureus or zymosan particles in a ratio of 1:20 and 1:10, respectively, and a final serum concentration of 10 %. Phagocytosis was terminated after 15 min and the number of extracellular bacteria or zymosan particles and the percentage of phagocytic granulocytes were registered by Flow Cytometry (FCM). FCM and microscopic studies revealed that eosinophils play a minor role in the phagocytosis of bacteria. The neutrophils were the main population of the granulocytes which were actively phagocytic. Variation among cows in the ability of their blood neutrophils to phagocytize bacteria was evident.     

Flow cytometric detection of bovine viral diarrhea virus in peripheral blood leukocytes of persistently infected cattle.

Flow cytometry was investigated for detection of bovine viral diarrhea virus (BVDV) in peripheral blood mononuclear leukocytes of persistently infected cattle. The mononuclear leukocytes were purified by sedimentation in a gradient of Ficoll-Paque, fixed, permeabilized, and then labelled by indirect immunofluorescence using biotinylated immunoglobulins from a porcine antiserum to BVDV. Flow cytometric analysis of blood samples obtained from persistently infected cattle revealed virus in 3.0-21.0% (mean +/- SD, 11.2% +/- 6.4%) of the mononuclear leukocytes. Fluorescent cells were not observed in controls. Flow cytometric detection of BVDV in blood cells of persistently infected bovines is a rapid and objective technique which does not require cell culture facilities.     

Preparation, purification and characterization of bovine Peyer''s patch leukocytes.

An enzymatic technique for isolating bovine Peyer's patch leukocytes was developed. Enzymatic dissociation of Peyer's patches yielded a cell population rich in T and B lymphocytes containing 6-10% adherent accessory cells, as defined by morphology and nonspecific esterase staining. Analysis of Peyer's patch lymphocytes, using sheep erythrocyte rosetting and immunofluorescence with conventional antisera and monoclonal antibodies, showed that leukocytes were approximately 45% T cells and 25% Ig-positive B cells. The cell population contained functionally active T and B lymphocytes which proliferated in response to T and B cell mitogens and to exogenous human recombinant interleukin-2. The observed differences in patterns of Peyer's patch leukocyte responsiveness to these mitogens suggest some cellular heterogeneity of the bovine Peyer's patch.     

Assays and activities of glycosidic enzymes in bovine peripheral blood leukocytes

Methods are described for the quantitative measurement of N-acetyl-beta-D-glucosaminidase, alpha-mannosidase and beta-glucuronidase in peripheral blood leukocytes of the bovine. Enzyme kinetics and stability were determined. Activities of the glycosidases in polymorphonuclear leukocytes and mononuclear leukocytes were determined using the optimized assays. Polymorphonuclear leukocytes had greater activities of N-acetyl-beta-D-glucosaminidase and alpha-mannosidase, and similar levels of beta-glucuronidase, when compared to mononuclear leukocytes.     

Plasminogen activator production by bovine milk macrophages and blood monocytes

The type of plasminogen activator (PA) produced by bovine milk macrophages has been determined. Macrophages produce a PA protein with molecular weight of 28,000 and isoelectric point of 8.5, and with enzymatic activity independent of fibrin. These characteristics are identical to those reported for bovine urokinase-PA. Although blood monocytes and milk macrophages produce PA after stimulation with lipopolysaccharide, mammary macrophages are clearly limited in their ability to release PA. At maximal stimulation, 78% of the PA produced by milk macrophages remained cell-associated. In marked contrast, blood monocytes released 76% of the PA produced into the culture medium. Macrophages isolated from mastitic quarters produced higher (2.5 times) amounts of PA, compared with those produced by macrophages isolated from healthy quarters. However, in both cases, macrophages were unable to secrete the protein already produced. The limited PA secretion by milk macrophages might be a residual function of a differentiated macrophage population.     

Flow cytometric analysis of intracellular complexity and CD45 expression for use in rapid differentiation of leukocytes in bovine blood samples.

OBJECTIVE: To develop an efficient and reliable method that accurately differentiates bovine lymphocytes from monocytes in leukograms. SAMPLE POPULATION: Blood samples from 30 healthy cows and 1 calf with bovine leukocyte adhesion deficiency. PROCEDURE: Flow cytometric analysis of intracellular complexity and CD45 expression on bovine leukocytes was compared with results for conventional light microscopy methods. Verification of leukocyte subpopulations determined by intracellular complexity and CD45 expression was conducted, using 2-color phenotypic analysis with selected monoclonal antibodies. RESULTS: The CD45 and side-scatter properties of bovine leukocytes clearly differentiated cell types, including neutrophils, eosinophils, monocytes, and lymphocytes. CONCLUSIONS AND CLINICAL RELEVANCE: This is a rapid assay that is simple to use. More importantly, it is more accurate than the conventional method that involves the use of blood slides and light microscopy, because of the ability of the assay to readily distinguish bovine monocytes and lymphocytes. Rapid preparation of samples and short analysis times allow for efficient and reliable examination of a large number of samples, and the task of viewing slides by light microscopy is eliminated. The labor-savings benefit of this procedure is most apparent in research environments that require frequent processing of batches of blood samples.     

The bovine neutrophil: Structure and function in blood and milk

Migration of polymorphonuclear neutrophil leukocytes (PMN) into the mammary gland provide the first line of defense against invading mastitis pathogens. Bacteria release potent toxins that activate white blood cells and epithelial cells in the mammary gland to secrete cytokines that recruit PMN that function as phagocytes at the site of infection. While freshly migrated PMN are active phagocytes, continued exposure of PMN to inhibitory factors in milk such as fat globules and casein, leads to altered PMN morphology and reduced phagocytosis. In the course of phagocytosing and destroying invading pathogens, PMN release chemicals that not only kill the pathogens but that also cause injury to the delicate lining of the mammary gland. This will result in permanent scarring and reduced numbers of milk secretory cells. The life span of PMN is limited by the onset of apoptosis. To minimize damage to mammary tissue, PMN undergo a specialized process of programmed cell death known as apoptosis. Macrophages quickly engulf and phagocytose apoptotic PMN, thereby minimizing the release of PMN granular contents that are damaging to tissue. The PMN possess an array of cell surface receptors that allow them to adhere and migrate through endothelium and to recognize and phagocytose bacteria. One receptor found on phagocytes that is receiving considerable attention in the control of infections by Gram-negative bacteria is CD14. Binding of lipopolysaccharide (LPS) to membrane bound CD14 causes release of tumor necrosis factor-alpha and sepsis. Binding of LPS to soluble CD14 shed from CD14-bearing cells results in neutralization of LPS and rapid recruitment of PMN to the site of infection. Recent advances in the fields of genomics and proteomics should greatly enhance our understanding of the PMN role in controlling intramammary infections in ruminants. Further, manipulation of PMN, through either recombinant proteins such as soluble CD14 that enhance PMN response or agents that mediate PMN apoptosis, may serve as novel therapeutics for the treatment of mastitis.     

Inability to detect a K cell in bovine peripheral blood leukocytes

Antibody-dependent cellular cytotoxicity to viral-infected cells, chicken red blood cells, and tumor cells was tested using different effector cell populations: polymorphonuclear cells, mononuclear cells, and mononuclear cells separated into adherent and nonadherent populations by Sephadex G-10. Polymorphonuclear cells were the most efficient mediators of antibody-dependent cellular cytotoxicity against most targets, although a combination of G-10 adherent and polymorphonuclear cells was more efficient in killing infectious bovine rhinotracheitis virus-infected cells than either single cell population. Removal of G-10 adherent cells from the mononuclear cell population removed all antibody-dependent cellular cytotoxicity from that population, indicating the lack of a typical K cell in bovine peripheral blood.     

Molecular characterization of Streptococcus agalactiae and Streptococcus uberis isolates from bovine milk

Streptococci are one among the major mastitis pathogens which have a considerable impact on cow health, milk quality, and productivity. The aim of the present study was to investigate the occurrence and virulence characteristics of streptococci from bovine milk and to assess the molecular epidemiology and population structure of the Indian isolates using multilocus sequence typing (MLST) and pulsed-field gel electrophoresis (PFGE). Out of a total of 209 bovine composite milk samples screened from four herds (A–D), 30 Streptococcus spp. were isolated from 29 milk samples. Among the 30 isolates, species-specific PCR and partial 16S rRNA gene sequence analysis identified 17 Streptococcus agalactiae arising from herd A and 13 Streptococcus uberis comprising of 5, 7, and 1 isolates from herds B, C, and D respectively. PCR based screening for virulence genes revealed the presence of the cfb and the pavA genes in 17 and 1 S. agalactiae isolates, respectively. Similarly, in S. uberis isolates, cfu gene was present in six isolates from herd C, the pau A/skc gene in all the isolates from herds B, C, and D, whereas the sua gene was present in four isolates from herd B and the only isolate from herd D. On MLST analysis, all the S. agalactiae isolates were found to be of a novel sequence type (ST), ST-483, reported for the first time and is a single locus variant of the predicted subgroup founder ST-310, while the S. uberis isolates were found to be of three novel sequence types, namely ST-439, ST-474, and ST-475, all reported for the first time. ST-474 was a double locus variant of three different STs of global clonal complex ST-143 considered to be associated with clinical and subclinical mastitis, but ST-439 and ST-475 were singletons. Unique sequence types identified for both S. agalactiae and S. uberis were found to be herd specific. On PFGE analysis, identical or closely related restriction patterns for S. agalactiae ST-483 and S. uberis ST-439 in herds A and B respectively, but an unrelated restriction pattern for S. uberis ST-474 and ST-475 isolates from herds D and C respectively, were obtained. This signifies that the isolates of particular ST may exhibit related PFGE patterns suggesting detection of a faster molecular clock by PFGE than MLST. Since all the isolates of both the species belonged to novel sequence types, their epidemiological significance in global context could not be ascertained, however, evidence suggests that they have uniquely evolved in Indian conditions. Further research would be useful for understanding the role of these pathogens in bovine sub-clinical mastitis and implementing effective control strategies in India.     

The chemiluminescent response of bovine polymorphonuclear leucocytes isolated from milk and blood

Polymorphonuclear leucocytes (PMN) were isolated from milk and blood of healthy cows, and the generation of reactive oxygen by the two cell populations was compared by measuring chemiluminescence (CL) after stimulation with zymosan. The ratio of milk to blood PMN CL was relatively constant in a given animal, but varied widely between different cows, ranging from 0.3 to 1.3.The relative contributions of various oxygen species to CL was studied by measuring quenching using different oxygen scavengers. While the relative contributions of H₂O₂, ^?O₂ and ′O₂ seemed to be similar in both milk and blood PMN, the OH· radical was clearly more prominent in PMN isolated from milk than from blood. In addition, blood PMN CL was more dependent on the presence of glucose in the reaction medium than milk PMN CL. Furthermore, the CL response to phorbol myristate acetate, to the Ca ionophore A23187 and to Sendai virus was different in the two cell types. The results suggest that CL generation in milk PMN differs from that in blood PMN in quantitative as well as qualitative aspects.     

Chemotactic requirements of bovine leukocytes

A system was described for studying bovine leukotaxis. Chemotaxis was readily observed toward bovine serum activated by zymosan of agarose. However, bacterial culture filtrates and peptides, which were potent chemotaxins for leukocytes from other species, failed to affect bovine leukotaxis. Using conditions suitable for studying binding to leukocytes from other species, specific binding of a radiolabeled chemotactic peptide to bovine leukocytes was not apparent. These data indicate that bovine leukocytes have chemotactic requirements distinct from those of other species.     

A method for separation of bovine blood leukocytes for in vitro studies.

Various methods have been developed for separation of the cellular components of blood. Size and density differences in cellular blood elements of various animal species necessitate the use of specific separative procedures. This study describes a method which combines isopycnic density-gradient centrifugation with erythrocyte aggregation as a means of isolating leukocytes from bovine blood. The procedure yields a viable population of mixed leukocytes which has proven useful for cell culture and in vitro tests of cell-mediated immunity.     

Correlation of blood and milk components with the milk yield response to bovine somatotropin in dairy cows

Our aim was to correlate the individual variation in the milk yield response (MYR) of Holstein dairy cows to bovine somatotropin (bST), with changes in milk plasmin and plasminogen activities as well as with plasma hormone and metabolite levels. Thirty-two housed multiparous Holstein cows (90 +/- 3.8 days post partum) received daily subcutaneous injections of saline for 1 week followed by subcutaneous injections of 20 mg/day of bST for 2 weeks. Blood samples were taken at approximately 4h intervals over 24 h at the end of the saline and bST treatment periods. Milk samples were also taken at the end of the saline and bST treatment periods. The difference in milk yield between the saline and the second week of bST treatment (MYR) varied considerably between animals (from -0.2 to +8.6 kg/day, relative to the saline treatment week). Low milk yield before bST treatment was associated with a high MYR. The plasma growth hormone response to treatment was negatively correlated with MYR. Plasma insulin-like growth factor-1 response to treatment was positively correlated with MYR. Furthermore, a high MYR to bST was associated with a lower milk plasminogen level before treatment and a greater reduction in the level of plasminogen in milk following treatment.     

Apoptosis and necrosis of blood and milk polymorphonuclear leukocytes in early and midlactating healthy cows.

Increased milk somatic cell counts (SCC) are used as an indicator for bovine mastitis. During mastitis, polymorphonuclear leukocytes (PMN) become the predominant cell type. Shortly after parturition, the severity of mastitis is increased and several PMN functions are downregulated. Apoptotic and necrotic processes of PMN could influence SCC and PMN functions. In this study, the percentages of apoptotic and necrotic PMN in blood and milk from early and midlactating healthy cows were compared. Apoptosis and necrosis of PMN were quantified using a dual-color flow cytometric procedure with fluorescein labeled annexin-V (green) and propidium iodide (red). Using this technique three different subpopulations of bovine PMN could be detected: apoptotic cells (high intensive green fluorescence), necrotic cells (high intensive green and high intensive red fluorescence) and viable cells (low intensive green and low intensive red fluorescence). Following a 4 h incubation of blood from both groups of cows at 37 degrees C to induce apoptosis, the mean percentage of apoptotic blood PMN was significantly higher (P < 0.01) in early lactating cows (15.1%, n = 9) compared with midlactating cows (5.3%, n = 10). The mean percentage of necrotic PMN remained lower than 5% in all cows. In contrast to blood, no significant difference was found between the percentage of apoptotic PMN in milk from early (41.2%, n = 7) and midlactating cows (34.0%, n = 8). The percentage of necrotic PMN in milk from early lactating cows (25.9%, n = 7) was significantly higher than that in midlactating cows (14.2%, n = 8) (P < 0.05). Higher percentages of apoptotic as well as necrotic PMN were consistently found in milk compared to blood in all cows. From these results, it can be concluded that spontaneously induced apoptosis was higher in blood PMN from early lactating cows than in blood PMN from midlactating cows. The higher percentage of necrotic milk PMN in early lactating cows than in midlactating cows could be explained by the induction of secondary necrosis.     

Occurrence and characterization of methicillin-resistant staphylococci from bovine mastitis milk samples in Finland

Background

Methicillin-resistant staphylococci (MRS) are increasingly being isolated in bovine mastitis. The aim of our study was to evaluate the occurrence of MRS in Finnish mastitis milk samples and characterize the MRS isolates using molecular methods.

Results

Methicillin-resistant S. aureus (MRSA) was a rare finding in bovine mastitis in Finland. Only two out of 135 (1.5%) S. aureus isolates were positive for mec genes. One of these carried mecA and was of spa type t172, SCCmec type IV and ST375, and the other harboured mecC, being spa type t3256, and ST130. MRSA ST375 is common among human MRSA isolates in Finland, but this is the first report in the country of bovine mecC MRSA. In coagulase-negative staphylococci (CoNS) originating from bovine mastitis, methicillin resistance was more common. In the two CoNS collections studied, 5.2% (17/324) and 1.8% (2/110) of the isolates were mecA positive. Eighteen of these were methicillin-resistant S. epidermidis (MRSE), which were divided into 6 separate PFGE clusters. One pulsotype was detected in different parts of the country, indicating clonal spread. Most MRSE (13/18) were of SCCmec type IV, one was of type V and four were non-typeable. Comparison with a human staphylococcal database indicated that bovine MRSE strains were not closely related to human MRSE isolates.

Conclusions

The occurrence of MRS, especially MRSA, in bovine mastitis in Finland was low. Most methicillin-resistant bovine CoNS are MRSE, and we found evidence of a bovine MRSE strain that may spread clonally. This is the first report of a Finnish bovine isolate of MRSA_mecC ST130. The study provides a baseline for further MRS monitoring.