Opsonin-Dependent Stimulation of Bovine Neutrophil Oxidative Metabolism by Brucella Abortus Peter C
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Veterinary Microbiology and Preventive Medicine Veterinary Microbiology and Preventive Medicine Publications 2-1988 Opsonin-dependent stimulation of bovine neutrophil oxidative metabolism by Brucella abortus Peter C. Canning U.S. Department of Agriculture Billy L. Deyoe U.S. Department of Agriculture James A. Roth Iowa State University, [email protected] Follow this and additional works at: https://lib.dr.iastate.edu/vmpm_pubs Part of the Veterinary Microbiology and Immunobiology Commons, Veterinary Preventive Medicine, Epidemiology, and Public Health Commons, and the Veterinary Toxicology and Pharmacology Commons The ompc lete bibliographic information for this item can be found at https://lib.dr.iastate.edu/ vmpm_pubs/190. For information on how to cite this item, please visit http://lib.dr.iastate.edu/ howtocite.html. This Article is brought to you for free and open access by the Veterinary Microbiology and Preventive Medicine at Iowa State University Digital Repository. It has been accepted for inclusion in Veterinary Microbiology and Preventive Medicine Publications by an authorized administrator of Iowa State University Digital Repository. For more information, please contact [email protected]. Opsonin-dependent stimulation of bovine neutrophil oxidative metabolism by Brucella abortus Abstract Nonopsonized Brucella abortus and bacteria treated with fresh antiserum, heat-inactivated antiserum, or normal bovine serum were evaluated for their ability to stimulate production of superoxide anion and myeloperoxidasemediated iodination by neutrophils from cattle. Brucella abortus opsonized with fresh antiserum or beat-inactivated antiserum stimulated production of approximately 3 nmol of 0 2 -/106 neutrophils/30 min. Similarly treated bacteria also stimulated the binding of approximately 4.3 nmol of Nal/ 107 neutrophils/h to protein. Significant CP < 0.05) production of 0 2 - and iodination activity by neutrophils were not stimulated by nonopsonized bacteria or organisms treated with normal bovine serum. Seemingly, B abortus stimulated oxidative metabolism in bovine neutrophils; however, the stimulation was dependent on the presence of bacterial usociated opsonins. Disciplines Veterinary Microbiology and Immunobiology | Veterinary Preventive Medicine, Epidemiology, and Public Health | Veterinary Toxicology and Pharmacology Comments This article is published as Canning, P.C., B.K. Deyoe, and J.A. Roth. 1988. Opsonin-dependent stimulation of bovine neutrophil oxidative metabolism by Brucella abortus. Am J Vet Res, 49:160-163. Rights Works produced by employees of the U.S. Government as part of their official duties are not copyrighted within the U.S. The onc tent of this document is not copyrighted. This article is available at Iowa State University Digital Repository: https://lib.dr.iastate.edu/vmpm_pubs/190 Opsonin-dependent stimulation of bovine neutrophil oxidative metabolism by Brucella abortus Peter C. Canning, PhD,- Billy L. Deyoe, DVM, PhD; James A Roth, DV'.M, PhD SUMMARY · Virulent B abortus inhibits the myeloperoxidase (MPO) H202-helide antibacterial reaction of PMN.3 Fractions 3b Nonopsonized Brucella abortus and bacteria treated with fresh antiserum, heat-inactivated antiserum, or normal and 10, responsible for this inhibition, were isolated from the < 1,000-dalton fraction of supernatant preparations bovine serum were evaluated for their ability to stimu of heat-killed B abortus. The inhibition of the ~iro-H 0 - late production of superoxide anion and myeloperoxidase 2 2 halide activity is the result of specific suppression of per mediated iodination by neutrophils from cattle. Brucella oxidase-positive granule degranulation.7 Fraction 3b has abortus opsonized with fresh antiserum or beat-inacti been identified as 5' -guanosine monophosphate, and frac· vated antiserum stimulated production of approximately tion 10 has been identified as adenine.8 The purpose of 3 nmol of 0 -/106 neutrophils/30 min. Similarly treated 2 the study reported here was to determine whether differ bacteria also stimulated the binding of approximately 4.3 ent opsonins (C3b, antibody) altered the oxidative meta nmol of Nal/107 neutrophils/h to protein. Significant CP bolic response of bovine PMN to ingestion of B Obortus. < 0.05) production of 0 2- and iodination activity by neu trophils were not stimulated by nonopsonized bacteria or organisms treated with normal bovine serum. Seemingly, Materials and Methods B abortus stimulated oxidative metabolism in bovine Preparation of bacteria-Procedures used to grow B abortus neutrophils; however, the stimulation was dependent on 1 the presence of bacterial usociated opsonins. have been described. Briefly, smooth virulent strain 2308 of B abortua was grown in a fermentor. After 48 hours' incubation at 37 C, bacteria were harvested in log phase, washed 3 times with .saline solution (0.85% NaCl), and suspended in Earle bal· anced salt solution {Eess)• without phenol red at a concentra· tion of 5 x 10' bacteria/ml. The etiologic agent of bovine brucellosis is the facul A culture of S aureus 1:train 305 was obtained" for use a& a tative intracellular bacterium Brucella abortua. Mecha control organism that is known to be killed readily on ingestion nisms responsible for the organism's ability to survive by bovine PMN. Bacteria were inoculated into flasks containing 100 ml of Trypticaae eoy broth. c After 18 hours' incubation at within polymorphonuclear neutro;>hihi (PMN) are not fully 37 C with agitation, cells were harvested by centrifugation, understood. Smooth and rough strains of B abortus are 1 2 washed 3 times with aaline solution, and suspended in EBSS at readily ingested by bovine and human PMN. • Virulent a density of 5 x 10" bacteria/ml. B abortus also does not possess surface-associated sub stances that inhibit the ability of PMN to ingest radiola Opsonization of bacteria and zymosan-Suspensions of B beled Staphylococcus aureus.3 Ingestion of B abortus by abortus were opsonized with 1 of 3 11erum preparations. Serum human PMN did not appear to stimulate hexose mono containing anti.-B abortua 2308 antibody (stand&rd aggiutina· phosphate shunt activity, as measured by oxidation of tion titer :s 1:3,200) was obtained from a B abortus-infected 14 11 [ C]filucose. Bacteria used in that study were treated cow. This antiserum preparation also was used after heating at with heat-inactivated homologous serum. Ingestion of 56 C for 30 minutes to inactivate complement components. A nonopsonized B abortus did not stimulate production of pool of normal bovine serum was prepared from freshly collected blood obtained from 3 steers seronegative for anti-Brucella an· 0 - , as measured by quantitative nitroblue tetrazolium 2 tibodiee. Suspensions of S aureus were opsonized with a sub· dye reduction, because of a lack of stimulation of oxida agglutinating titer of a pool of fresh antiserum preparations tive metabolism on ingestion.• Seemingly, a lack of stim from 6 cows infected with S aureus 305. ulation of the oxidative metabolic burst enhances the Aliquot.a ( 1 ml) of standardized bacterial suspensions were intracellular survival of B abortus. However, this inter added to 1.5-ml microcentrifuge tubes and were mixed with 0.25 pretation may apply only to nonopsonized B abortus, be ml of the desired opsonizing serum. Tubes were placed in a 37 cause neither of previous studies5•6 used specific antibody C water bath and were incubated for 15 minutes. After incu· as an opsonin. Opsonized B abortus is capable of eliciting bation, bacteria were pelleted by centrifugation at 9,000 x g an oxidative response by be-vine mammary giand mac for 5 minutes. The supernatant was discarded, and bacteria were rophages.-1 The observation that opsonized zyniosan-stim suspended in 1 ml of EBSS. ulated production of 0 - by bovine PMN is not inhibited Opsonization of zymosan particles with a pool of normal bo 2 9 in the presence of B abortus indicates that the bacteria vine serum from 3 steers was performed as described. This procedure results in the depoaition of C3b on the surface of the do not specifically inhibit 0 - production by neutrophils. 3 2 zymosan particles via the alternate pathway of complement (uc· _Received for publication Nov 26, 1986. ation. From the Brucelloeia Reeean:h Unit (Canning, Deyoe), National Animal Di& ease Center, l!SDA, Agricultural Resean:h Service, PO Box 70, Ames, IA 50010, • Grand bland Biological Co, Grand bland, NY. and the Department or Veterinaty MierobioloaY Md Preventive Medicine, Iowa ~ Obtained £rom the MMtiU. Rele8l'ch Unit, National Animal Diaeue Center, State University, Amn, IA 50010 (Roth). ......,,_ The authors thank Karen Halloum 8lld Katie Meredith for t.echnical aan.tance. • BBL MicrobioJoo Symtem&. Cockeygyille, Md. 160 Am J Vet Res, Vol 49, No. 2, February 1988 PJfN preparation-Eight healthy adult mixed-breed steers cubatioo at 37 C for 20 minutes with agitation, cells were washed free of detectable anti-Brucella antibOdies, as determined by the twice by filtration with 0.25 ml of EBSS, and filters were trans standard tube agglutination assay, were used as a source of ferred to 12 x 75-mm tubes. The amount of entrapped radio PY!N. These steers were housed in outdoor pens and were fed activity was determined, uaing a gamma counter," and results hay and grain once daily. Neutrophils were isolated as described9 were expressed as nmoles of Nal/10' PMNlh. and were suspended in RPMI-1640 medium" to a concentration of 1 x 10~ cells. ml. Stalistical eualuation-To determine effects of different pref)" arations of opsonized bacteria on each PMN function, the value Complement fixation by B abortus-The ability of live B abor obtained when a bacterial prep.aration was added to PMN WllS t us 2308 to fL-,; complement was evaluated by an indirect flu compared with control tEBSS-treated) P!.tN from the same steer. orest-ent antibody technique. Suspensions of B abortus were An 1111aJyais of variance procedure (blocked by dnyl or a Student treated with 1 of the 3 serum preparation!!' used to opsonize the t teet was used to determine significance of differences in PMN bacteria as described.