The objective of this bovine peripheral blood study was a comparative assessment of the phagocytic activity of neutrophils and monocytes and of the intracellular killing capacity of neutrophils from cows given no probiotic and from cows which were administered a probiotic consisting of Saccharomyces cerevisiae, Lactobacillus acidophilus, Lactobacillus plantarum and Rhodopseudomonas palustris. These activity types were compared during different lactation periods.

Objective—To evaluate immunomodulatory properties of all-trans retinoic acid and a fully oxidized β-carotene dietary product in calves with Mannheimia haemolytica–induced pneumonia. Animals—Twenty-five 6- to 10-week-old male Holstein calves for experimental inoculations and three 8- to 30-week-old Angus heifers for blood donations. Procedures—In vitro, neutrophils and monocyte-derived macrophages isolated from blood of healthy Angus heifers were treated with all-trans retinoic acid (1μM) or fully oxidized β-carotene (8.3 μg/mL) for various times and assessed for markers of cellular death, antimicrobial function, and production of proinflammatory leukotriene B4. Following 28 days of dietary supplementation with fully oxidized β-carotene, Holstein calves were experimentally inoculated with M haemolytica. Bronchoalveolar lavage fluid was collected at 3 and 24 hours after challenge inoculation and analyzed for markers of apoptosis. Results—In vitro, all-trans retinoic acid and fully oxidized β-carotene induced cell-selective, caspase-3–dependent apoptosis in neutrophils, which subsequently enhanced efferocytosis in macrophages. Conversely, neither treatment altered phorbol 12-myristate 13-acetate–induced oxidative burst, phagocytosis of nonopsonized zymosan (complement or antibody independent), or M haemolytica–induced leukotriene B4 production in bovine neutrophils. In vivo, fully oxidized β-carotene enhanced leukocyte apoptosis in bronchoalveolar lavage fluid as well as subsequent efferocytosis by macrophages without altering numbers of circulating leukocytes. Conclusions and Clinical Relevance—Neutrophil apoptosis and subsequent efferocytosis by macrophages are key mechanisms in the resolution of inflammation. Findings for the present study indicated that all-trans retinoic acid and fully oxidized β-carotene could be novel nutraceutical strategies that may confer anti-inflammatory benefits for cattle with respiratory tract disease.

Marked differences in bone marrow cellularity were observed between cattle affected with leukocyte adhesion deficiency (LAD) and control cattle. The number of nucleated cells in bone marrow was 2.9 to 8.8 times higher in cattle affected with LAD, compared with controls. The myeloid-to-erythroid ratio of bone marrow from 3 cattle affected with LAD ranged from 2.4 to 12. Deficient CD18 expression on neutrophils isolated from bone marrow of cattle with LAD was clearly detected by flow cytometric analysis. Neutrophils from bone marrow of cattle affected with LAD appeared round and not flat, after adherence to plastic wells under agarose, whereas neutrophils from bone marrow of clinically normal cattle were firmly spread on the surface of plastic wells. In the chemotaxis under-agarose assay, many pseudopodia were detected on bone marrow neutrophils from clinically normal cattle, but were not detected on bone marrow neutrophils from cattle with LAD. Activities of chemotactic movements and phagocytosis of neutrophils isolated from bone marrow of cattle affected with LAD were documented to be severely impaired.

Cultures of bovine alveolar macrophages were inoculated with type-1 and type-8 adenoviruses, initially isolated from calves with respiratory tract disease, and functional properties of the cells were observed over a period of 10 to 11 days. Both viruses replicated in macrophages; viral titers were low (< 3.75 log10 TCID50/0.1 ml), and intranuclear inclusions were detected by indirect immunofluorescence in 5 to 10% of the cells from 3 days after inoculation. Highest titers were induced by type-1 adenovirus, which also induced the greatest functional changes. Expression of Fc and complement receptors was reduced by both viruses, although the greatest effects were seen with type 1. Phagocytosis of Candida krusei cells was reduced following type 1 infection, whereas phagocytosis in type-8-infected cells was not different from that of noninfected macrophages. Ability to kill ingested Candida cells also was reduced following type-1 infection, whereas type-8-infected macrophages had lower killing ability only at 2 to 4 days after inoculation. Neither virus had substantial effects on the production of neutrophil chemotactic factors by the macrophages.

Six primiparous Holstein cows were fed a Se-deficient diet, beginning at least 90 days before their first calving, and 6 other primiparous cows were given the same diet plus a supplement of 2 mg of Se/cow/d as sodium selenite. All cows were fed their diets for the duration of the experimental period. One uninfected quarter of each cow was injected with 25 microgram of Escherichia coli endotoxin at postpartum week 5. Leukocytes were isolated by centrifugation from milk collected at postinjection hour 16. Isolated cells were 92 +/- 3% neutrophils and were incubated with Staphylococcus aureus or E coli in a 1:300 ratio. Phagocytosis and intracellular killing by neutrophils were assessed after 0, 30, 60, and 90 minutes by a fluorochrome assay, using acridine orange. Viability of neutrophils was assessed by use of trypan blue. Superoxide anion production and hydrogen peroxide production by neutrophils also were determined. Cows fed Se-deficient diets had significantly (P < 0.05) lower blood Se concentration and blood glutathione peroxidase activity than cows fed Se-supplemented diets. Selenium status had no effect on the phagocytic capacity of neutrophils. Neutrophils obtained from cows fed Se-supplemented diets killed a significantly (P < 0.05) higher percentage of ingested bacteria than did neutrophils from cows fed the Se-deficient diet. Viability was significantly (P < 0.05) reduced by incubation with S aureus in neutrophils from both groups of cows, with neutrophils from Se-deficient cows having lower viability. Superoxide anion production did not differ significantly between neutrophils from the 2 groups, but extracellular hydrogen peroxide concentration was significantly (P < 0.05) higher in neutrophils harvested from milk of cows fed the Se-deficient diet.

Phagocytic and oxidative metabolic activities of bovine blood neutrophils were determined in the presence of glycolytic (NaF) and cytoskeletal (colchicine, cytochalasin B, and prostaglandin E1) inhibitors. Phagocytosis and postphagocytic oxidative metabolic activity, measured by nitroblue tetrazolium reduction, were determined using zymosan, Escherichia coli, Staphylococcus aureus, or Streptococcus agalactiae. Sodium fluoride (1.25 micromolar to 1.25 mM concentrations) did not significantly (P greater than 0.05) inhibit phagocytosis of S aureus and Str agalactiae, whereas phagocytosis of zymosan and E coli was significantly (P less than 0.05) inhibited only at 1.25 mM concentration. Colchicine at 1.25 nM to 1.25 micromolar conce ntrations significantly inhibited phagocytosis of zymosan and E coli, but not of S aureus and Str agalactiae. Cytochalasin B at 1.25 nM to 1.25 micromolar concentrations significantly inhibited phagocytosis of zymosan and all 3 bacteria, whereas prostaglandin E1 was noninhibitory at similar concentrations. Nitroblue tetrazolium reduction, in general, was not significantly affected by NaF and cytoskeletal inhibitors.

Functional capabilities of morphologically mature (segmented) and immature granulocytes (neutrophils and eosinophils) from bone marrow from cows were studied and compared with similar activities of segmented granulocytes from blood. Phagocytosis of Escherichia coli and postphagocytic oxidative metabolic stimulation, measured by nitroblue tetrazolium (NBT) reduction, were evaluated simultaneously. Phagocytosis was observed readily in segmented neutrophils, neutrophilic bands, and metamyelocytes and rarely in myelocytes. Phagocytosis was not seen in promyelocytes and myeloblasts. Neutrophilic bands and metamyelocytes were phagocytically less active than were segmented neutrophils. Washed segmented bone marrow neutrophils possessed phagocytic activity similar to that of blood neutrophils, whereas the activity of unwashed segmented bone marrow neutrophils was markedly less than that of blood neutrophils. Reduction of NBT was observed only in blood segmented neutrophils and bone marrow segmented neutrophils; the magnitude of NBT reduction was significantly (P = less than 0.005) less in bone marrow neutrophils than in blood neutrophils. Eosinophils were phagocytically less competent than were neutrophils. The NBT reduction was observed only in eosinophils from blood, but not in eosinophils from bone marrow.

Autologous canine neutrophils were labeled with technetium 99m and reinjected in 7 dogs with experimentally induced focal abscessess to determine the ability of scintigraphy to localize a focus of sepsis (abscess). Good localization of labeled cells in an abscess was achieved; however, a large portion of the technetium 99m eluted from the neutrophils.

A study was conducted to examine sources of variation introduced into a phagocytosis assay as a result of the isolation of neutrophils from bovine blood, including variation attributable to isolation of neutrophils from blood, variation between duplicate determinations of percentage phagocytosis, and the variation in the ability of neutrophils isolated from blood (over repeated collections from the jugular vein) to phagocytose. For the phagocytosis assay, jugular venous blood from each of 4 cows was divided into 2 equal portions. The neutrophils were isolated by lysis of red blood cells with 0.2% sodium chloride. The neutrophils (2 X 10(7)) were incubated in duplicate with 32P-labeled Staphylococcus aureus ([32P]SA; 2 X 10(8)) inskimmed milk samples (2.5% final concentration) prepared from 4 cows. This process was repeated thrice on neutrophils isolated from 4 cows at 2-week intervals. The proportions of variation in percentage of 32P-labeled S aureus phagocytosed between duplicate neutrophil isolations and between duplicate assay determinations were 0 and 1%. Differences among skimmed milk sources and among runs, using blood neutrophils taken at different times from the same donor cow, accounted for 62 and 36% of the total variation. The results indicated that variation arising from blood neutrophil isolation introduced into a phagocytosis assay within a single-day trial is of no concern. The large variation among skimmed milk sample sources indicated differences among cows in the ability of their milk to support phagocytosis. The variation in neutrophil isolations over time for any cow was considered too large to allow for evaluation of physiologic and environmental effects on phagocytosis of neutrophils isolated from blood.