The main objective of the study reported here was to generate a panel of monoclonal antibodies (MAB) to bovine neutrophil surface antigens, and to identify MAB that modulate neutrophil chemotaxis, respiratory burst, and phagocytosis. A further objective was to study MAB reactivity with resting and activated neutrophils, to identify activation antigens and adhesion molecules. A panel of 14 MAB was generated by producing murine hybridomas. Neutrophils incubated with MAB at 4 C for 2 hours were used in chemotaxis, respiratory burst, and phagocytosis assays. Chemotaxis was evaluated in Boyden chambers, using Escherichia coli endotoxin-activated fetal bovine serum as the chemoattractant. Respiratory burst was determined by measuring chemoluminescence of neutrophils incubated with 5-amino-2,3-dihydro-1,4-phthalazinedione, and serum opsonized zymosan. Phagocytosis was determined by flow cytometry, using fluorescein-labeled Staphylococcus aureus. The MAB S7G8, S5F8G10, S7E10, and S5F8B8 enhanced chemotaxis (to > 125% of control). The MAB S7E10 and S8D9 enhanced respiratory burst activity (to > 125% of control), whereas MAB S2G8, S4G10, S8G10, and S5F8B8 caused inhibition (to < 75% of control). The MAB S2G8, S4G10, S8G10, and S5F8G10 enhanced phagocytosis (to > 125% of control). Chemotaxis, respiratory burst, and phagocytosis values of neutrophils not bound with MAB served as controls for comparison. The MAB binding for nonactivated neutrophils (at 4 C) ranged from 9 to 100%, and for activated neutrophils (at 37 C; at 37 C with phorbol myristate acetate) from 90 to 100%. Binding of MAB S4F5, S5F8B8, S6C6, S7E10, S8D9, and S5F8G10 increased when neutrophils had been incubated at 37 C. Binding of these MAB was further increased after incubation with phorbol myristate acetate (100 ng/ml) at 37 C, indicating recognition of activation antigens by MAB. The MAB generated in this study appeared to be potential candidates for studying mechanisms of neutrophil function and for enhancing neutrophil function in vitro and in vivo.

Sixteen Holstein cows were used to test the effect of postmilking teat treatment on colonization and intramammary infection by Staphylococcus aureus on chapped teats. Treatments were (1) chapping the teat and using 1% I2/10% glycerin postdip solution, (2) 1% I2/10% glycerin postdip solution on nonchapped teats, (3) chapping the teat and using 10% glycerin postdip solution, (4) chapping the teat and not using a postdip solution. All mammary glands were free of S aureus teat skin colonization and intramammary infection at the start of the study. Teats selected for chapping were dipped in 1N NaOH prior to 3 applications of S aureus broth culture; cultures were applied at 12-hour intervals on all teats. Treatments were applied after each milking for 30 days and were initiated after the second broth dip. Teat skin swab specimens and milk samples were collected before treatment application. Teat skin condition was scored daily. Nonchapped teats (treatment 2) did not support skin or orifice colonization by S aureus. Treatment-1 teats healed most rapidly and supported less colonization in skin and orifice than did treatment-3 and -4 teats. Teat skin scores and skin colonization were lower for treatment-3 than treatment-4 teats. A correlation between teat skin colonization and teat skin conditions was found. Two intramammary infections were found in treatment-4 quarters and 1 in a treatment-3 quarter. On the basis of our findings, we concluded that poor teat skin condition will more readily support S aureus colonization, that a dip of 1% I2 with glycerin helped reduce S aureus colonization and was associated with faster healing, and that glycerin in teat dips may be of value in preventing colonization by S aureus and in promoting healing.

Detection of capsular polysaccharide (CP) in milk of cows with natural intramammary infection caused by Staphylococcus aureus was attempted. Five quarters of 5 cows harboring S aureus strains that produce type-8 CP were selected. Using an ELISA with a monoclonal antibody, type-8 CP was not detected in extracts prepared from fresh milk collected aseptically. By contrast, CP was easily detectable after incubation of infected milk at 38 C for 20 hours. Quantitation of CP in extracts from incubated milk samples by use of ELISA indicated a great variation of CP expression by strains. Although an incubation step was necessary to detect CP, results of the study indicate that CP may be expressed in vivo during intramammary infection caused by S aureus.

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.

The tarsocrural joints of 11 horses were inoculated with 1.2 to 2.16 x 10(6) viable Staphylococcus aureus organisms susceptible to a trimethoprim-sulfadiazine (TMP-SDZ) combination with minimal inhibitory concentration (MIC) of 0.25 microgram of TMP/ml and 4.75 microgram of SDZ/ml. Antimicrobial treatment consisted of oral administration of a TMP-SDZ combination-30 mg/kg of body weight given once daily (group-1 horses) or 60 mg/kg given as 30 mg/kg every 12 hours (group-2 horses). Paired serum and synovial fluid samples were obtained before intra-articular inoculation with the S aureus, after inoculation with S aureus but before antimicrobial treatment, and after inoculation at various hourly intervals after oral administration of the TMP-SDZ combination. The TMP-SDZ combination was administered daily in the 2 dosages for 21 days. Samples were collected after day 3 of repetitive drug administration so that drug steady-state concentration would have been achieved. Serum and synovial fluid samples were analyzed for TMP and SDZ concentrations. Administration of the TMP-SDZ combination at a dosage of 30 mg/kg once daily was not effective in maintaining TMP or SDZ concentrations above the MIC of TMP-SDZ for the S aureus (0.25 and 4.75 microgram/ml for TMP and SDZ, respectively) in the infected synovial fluid or in maintaining adequate TMP concentration in the serum. The alternative use of the TMP-SDZ combination at a dosage of 60 mg/kg given as 30 mg/kg every 12 hours was effective in maintaining serum and synovial fluid concentrations of TMP and SDZ that were greater than the MIC for the infective organism. Sulfadiazine concentration was significantly (P less than or equal to 0.05) lower in the infected synovial fluid sample than that in the corresponding serum sample. We concluded that administration of 60 mg of TMP-SDZ/kg given as 30 mg/kg every 12 hours is more effective than 30 mg/kg given once daily for the treatment of equine infectious arthritis caused by organisms for which the MIC of TMP-SDZ is less than or equal to 0.25-4.75 microgram/ml.

OBJECTIVE To determine the concentration of tilmicosin in mammary gland secretions of dairy cows following administration of an experimental preparation once or twice during the dry period (45-day period immediately prior to calving during which cows are not milked) and to evaluate its efficacy for the treatment of cows with intramammary infections (IMIs) caused by Staphylococcus aureus at dry off (cessation of milking; first day of dry period), compared with that of an intramammary infusion of ceftiofur. ANIMALS 172 cows. PROCEDURES Milk samples were collected for microbiological culture 5 days before dry off and at calving and 15 and 30 days after calving. Cows with Staphylococcus IMIs were randomly assigned to receive an experimental preparation of tilmicosin (20 mg/kg, SC) once at dry off (n = 58) or at dry off and again 20 days later (56) or receive a long-acting intramammary preparation of ceftiofur (500 mg/mammary gland; 56) at dry off. Mammary gland secretions were collected from 5 cows in the tilmicosin-treated groups every 5 days after dry off until calving for determination of tilmicosin concentration. RESULTS Mean maximum concentration of tilmicosin in mammary gland secretions ranged from 14.4 to 20.9 μg/mL after the first dose and was 17.1 μg/mL after the second dose. The bacteriologic cure rate was 100% for all 3 treatments. Tilmicosin was detectable for 0 and 18 days after calving in the milk of cows treated with 1 and 2 doses of tilmicosin, respectively. CONCLUSIONS AND CLINICAL RELEVANCE Administration of an experimental preparation of tilmicosin (20 mg/kg, SC) once to dairy cows at dry off might be useful for the treatment of S aureus IMIs.

The aim of this study was to evaluate the antimicrobial activity and determine the minimum bactericidal concentration (MBC) of the essential oils derived from Origanum vulgare (oregano), Melaleuca alternifolia (tea tree), Cinnamomum cassia (cassia), and Thymus vulgaris (white thyme) against Salmonella Typhimurium, Salmonella Enteritidis, Escherichia coli, Staphylococcus aureus and Enterococcus faecalis. The study also investigated the ability of these different bacterial strains to develop adaptation after repetitive exposure to sub-lethal concentrations of these essential oils. The MBC of the essential oils studied was determined by disc diffusion and broth dilution methods. All essential oils showed antimicrobial effect against all bacterial strains. In general, the development of adaptation varied according to the bacterial strain and the essential oil (tea tree > white thyme > oregano). Therefore, it is important to use essential oils at efficient bactericidal doses in animal feed, food, and sanitizers, since bacteria can rapidly develop adaptation when exposed to sub-lethal concentrations of these oils.

Objective: To determine the pharmacokinetics of a commercial formulation of doxycycline hyclate after IM administration of a single dose to sheep. Animals: 11 healthy domestic sheep. Procedures: For each sheep, doxycycline was administered as a single dose of 20 mg/kg, IM. Blood samples were obtained prior to and for 84 hours after doxycycline administration. Plasma concentrations of doxycycline were determined via high-performance liquid chromatography with UV detection. Pharmacokinetic data were analyzed with noncompartmental methods. Results: Mean ± SD values for pharmacokinetic parameters included maximum plasma concentration (2.792 ± 0.791 μg/mL), time to reach maximum plasma concentration (0.856 ± 0.472 hours), mean residence time (91.1 ± 40.78 hours), elimination half-life (77.88 ± 28.45 hours), and area under the curve (65.67 ± 9.877 μg•h/mL). Conclusions and Clinical Relevance: Results indicated that doxycycline had prolonged absorption and elimination in sheep after IM administration. A daily dose of 20 mg/kg would be sufficient to reach effective plasma concentrations against Chlamydia spp (minimum inhibitory concentration, 0.008 to 0.031 μg/mL) and Staphylococcus aureus (minimum inhibitory concentration, 0.12 μg/mL). Doxycycline administered IM could be an option for therapeutic use in sheep, although further studies are needed.

The effect of a novel bovine mastitis trivalent vaccine, containing Staphylococcus aureus capsular polysaccharide type 5 (T5), 8 (T8), and 336 (T336), on lymphocyte subpopulations, antibody production, and neutrophil phagocytosis was evaluated. Twenty pregnant heifers were immunized with either the trivalent alone, trivalent emulsified in Freund's incomplete adjuvant (FICA), trivalent in aluminum hydroxide, or adjuvant only (FICA). Immunization was done 30 d before the expected calving date followed by 2 boosts in a 2-week interval. Compared to FICA, serum antigen-specific immunoglobulin (Ig)G1 and IgG2 were significantly increased in all the vaccinated groups before parturition and sustained until 3 wk postpartum. In comparison with the trivalent alone, formulation with either adjuvant enhanced production of IgG2, but not IgG1. Immune sera, which contained the highest amount of antibodies, slightly increased neutrophil phagocytosis to the 3 serotypes of killed S. aureus, but most of the differences were not significant due to large variation between the cows. The percentage of CD4+ lymphocyte was significantly higher in vaccinated groups than that of FICA 4 wk after the primary immunization. In comparison with FICA, cows inoculated with trivalent vaccine and adjuvants had an increased percentage of CD8+ lymphocytes at 2 time points, 2 wk before and after calving. Our results indicated that the whole cell trivalent vaccine, with or without adjuvants, is able to elicit antibody responses specific to the 3 capsular polysaccharide antigens. The increase of T8-specific IgG2 was more noticeable when the vaccine was emulsified with adjuvants.

The effects of 3 days of glucocorticoid administration on bovine blood neutrophil expression of L-selectin and CD18, and on the health status of mammary glands subclinically infected with Staphylococcus aureus were measured in 9 lactating Holsteins. The experiment was a 3 x 3 Latin square cross-over design, with 3 glucocorticoid treatments switched among groups of 3 cows/treatment during 3 periods. Treatments consisted of a vehicle (control, 10 ml of excipient/cow/d), cortisol (7.5, 15, and 7.5 mg/cow on days 1, 2, and 3, respectively), and dexamethasone (0.04 mg/kg of body weight/cow/d for total daily dosages that ranged from 21.6 to 33.2 mg). Blood samples for immunostaining and flow cytometric analysis of L-selectin and CD18 and leukograms, as well as foremilk samples for determination of S aureus shedding somatic cell counts, protein and fat percentages, and daily milk yields were collected repeatedly before, during and after treatment days. Dexamethasone caused a profound, acute, short-lived down-regulation of L-selectin on neutrophils, which correlated in time to leukocytosis, mature and immature neutrophilias, increased shedding of S aureus in infected glands, and onset of high percentages of fat and protein and decreased milk yields. Dexamethasone also caused profound but delayed down-regulation of neutrophil CD18, which reached nadir simultaneously with reappearance of L-selectin-bearing neutrophils, normalized blood neutrophil counts, markedly high foremilk somatic cell counts and protein percentage, decreased S aureus shedding in milk, and finally, expression of clinical mastitis in some infected quarters. Each of these variables had returned to control (vehicle) values by the ninth (and last) sample collection day. Although cortisol treatment also decreased expression of L-selectin and CD18 on neutrophils, dosages used in this study were not sufficient to alter the number of circulating cells or to convert subclinical mammary gland infections to clinical mastitis. These results suggest that mammary gland health status can be altered by sudden exposure of blood neutrophils to glucocorticoids, because these steroid hormones caused profound down-regulation of the adhesion molecules that direct neutrophil margination and migration through the vascular endothelium. The results also reinforce the potential disease risk of treating infected animals with potent synthetic glucocorticoids, such as dexamethasone.