When incubated with solutions of hydrogen peroxide, erythrocytes of stress-susceptible pigs produced more by-products of lipid peroxidation (as measured as thiobarbituric acid-reactive substances [TBARS]) than did erythrocytes from stress-resistant pigs. Using this technique, discrimination between the 2 pig types was absolute at hydrogen peroxide concentrations of 0.9 and 1.5%. This was in contrast to other methods of identifying stress-susceptible pigs, such as osmotically induced erythrocyte lysis and the determination of plasma pyruvate kinase and creatine kinase activities, for which considerable overlap of data was observed between pig types. The increased TBARS production by erythrocytes was further evidence for the existence of an antioxidant abnormality in stress-susceptible pigs. However, because there were no discernible differences in the major blood antioxidant-related values between stress-susceptible and stress-resistant pigs, the nature of the defect remains unclear. The production of TBARS by erythrocytes when incubated with hydrogen peroxide provides an improved method for identifying stress-susceptible pigs.

OBJECTIVE To evaluate the effects of vaporized hydrogen peroxide (VHP) sterilization on the in vitro antimicrobial efficacy of meropenem-impregnated polymethyl methacrylate (M-PMMA) beads. SAMPLE 6-mm-diameter polymethyl methacrylate beads that were or were not impregnated with meropenem. PROCEDURES Meropenem-free polymethyl methacrylate and M-PMMA beads were sterilized by use of an autoclave or VHP or remained unsterilized. To determine the antimicrobial efficacy of each bead-sterilization combination (treatment), Mueller-Hinton agar plates were inoculated with 1 of 6 common equine pathogens, and 1 bead from each treatment was applied to a sixth of each plate. The zone of bacterial inhibition for each treatment was measured after 24 hours. To estimate the duration of antimicrobial elution into a solid or liquid medium, 1 bead from each treatment was transferred every 24 hours to a new Staphylococcus aureus–inoculated agar plate or a tube with PBS solution, and an aliquot of the eluent from each tube was then applied to a paper disc on an S aureus–inoculated agar plate. All agar plates were incubated for 24 hours, and the zone of bacterial inhibition was measured for each treatment. RESULTS In vitro antimicrobial efficacy of M-PMMA beads was retained following VHP sterilization. The duration of antimicrobial elution in solid and liquid media did not differ significantly between unsterilized and VHP-sterilized M-PMMA beads. CONCLUSIONS AND CLINICAL RELEVANCE Results indicated that M-PMMA beads retained in vitro antimicrobial activity and eluted the drug for up to 2 weeks after VHP sterilization.

In May of 2013, porcine epidemic diarrhea virus (PEDV) was detected in swine for the first time in North America. It spread rapidly, in part due to contaminated livestock trailers. The objective of this study was to test the efficacy of an accelerated hydrogen peroxide disinfectant for inactivating PEDV in the presence of feces on metal surfaces, such as those found in livestock trailers. Three-week-old barrows were inoculated intragastrically with 5 mL of PEDV-negative feces for the negative control, 5 mL of untreated PEDV-positive feces for the positive control, and 5 mL or 10 mL of PEDV-positive feces that was subjected to treatment with a 1:16 or 1:32 concentrations of accelerated hydrogen peroxide disinfectant for a contact time of 30 min at 20°C. These pigs served as a bioassay to determine the infectivity of virus following treatment. Rectal swabs collected from the inoculated pigs on days 3 and 7 post-inoculation were tested by using PEDV-specific real-time reverse transcription polymerase chain reaction and the proportion of pigs in each group that became infected with PEDV was assessed. None of the pigs used for the bioassay in the 4 treatment groups and the negative control group became infected with PEDV, which was significantly different from the positive control group (P < 0.05) in which all pigs were infected. The results suggest that the application of the accelerated hydrogen peroxide under these conditions was sufficient to inactivate the virus in feces found on metal surfaces.

Objective—To evaluate the effect of multiple hydrogen peroxide gas plasma (HPGP) sterilizations on the rate of closure of ameroid constrictors. Sample—Thirty-six 5.0-mm ameroid constrictors. Procedures—Ameroid constrictors were randomly allocated to 6 groups. Each group underwent 1, 2, 3, 4, 5, or 6 HPGP sterilizations. Ameroid constrictors were then incubated for 35 days in canine plasma and digitally imaged at predetermined times during incubation. One individual, who was unaware of the group to which each ameroid constrictor was assigned, measured the lumen area of the constrictor on each digital image. Mean lumen area was compared among groups. Results—No ameroid constrictors were completely closed after 35 days of incubation in canine plasma. Mean lumen area after incubation did not differ among constrictors that underwent 1, 2, and 3 sterilizations. Constrictors that underwent 4 sterilizations were closed significantly more than were those that underwent 1, 2, or 3 sterilizations. Mean lumen area after incubation did not differ significantly between constrictors that underwent 5 and 6 sterilizations, although the final lumen areas for those constrictors were significantly smaller than those for constrictors that underwent 1, 2, 3, and 4 sterilizations. Conclusions and Clinical Relevance—Ameroid constrictors that underwent 5 and 6 HPGP sterilizations had a 9% to 12% decrease in lumen area, compared with that of constrictors that underwent ≤ 4 plasma sterilizations, and the use of such constrictors could increase the risk of portal hypertension and secondary acquired shunting or decrease the risk of persistent shunting.

Objective-To determine whether oxidative stress could be induced in canine chondrocytes in vitro. Sample-Chondrocytes obtained from healthy adult mixed-breed dogs. Procedures-Harvested chondrocytes were maintained at 37°C with 5% CO2 for 24 hours. To assess induction of oxidative stress, 2 stimuli were used: hydrogen peroxide and a combination of interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α). To determine the effect of hydrogen peroxide, a set of chondrocyte-seeded plates was incubated with control medium alone or hydrogen peroxide (100, 200, or 300μM) for 24 hours. For inhibition of oxidative stress, cells were incubated for 24 hours with N-acetylcysteine (NAC; 10mM) before exposure to hydrogen peroxide. Another set of chondrocyte-seeded plates was incubated with control medium alone or with IL-1β (10 ng/mL) and TNF-α (1 ng/mL) for 24 hours. Supernatants were obtained for measurement of prostaglandin E2 production, and cell lysates were used for measurement of superoxide dismutase (SOD) activity and reduced-glutathione (GSH) concentration. Results-Chondrocytes responded to the oxidative stressor hydrogen peroxide with a decrease in SOD activity and GSH concentration. Exposure to the antioxidant NAC caused an increase in SOD activity in hydrogen peroxide-stressed chondrocytes to a degree comparable with that in chondrocytes not exposed to hydrogen peroxide. Similarly, NAC exposure induced significant increases in GSH concentration. Activation with IL-1β and TNF-α also led to a decrease in SOD activity and increase in prostaglandin E2 production. Conclusions and Clinical Relevance-Canine chondrocytes responded to the oxidative stress caused by exposure to hydrogen peroxide and cytokines. Exposure to oxidative stress inducers could result in perturbation of chondrocyte and cartilage homeostasis and could contribute to the pathophysiology of osteoarthritis. Use of antioxidants, on the other hand, may be helpful in the treatment of arthritic dogs.

Objective: To compare effects of sterilization with hydrogen peroxide gas plasma (HPGP), ethylene oxide, and steam on bioadhesive properties of nylon and polyethylene lines used for stabilization of canine stifle joints. Sample: Samples of a 36.3-kg test nylon leader line, 57.8-kg test nylon fishing line, and 2-mm ultrahigh–molecular weight polyethylene (UHMWPE) were used. Procedures: In this in vitro study, samples of nylon leader line, fishing line, and UHMWPE sterilized by use of HPGP, ethylene oxide, and steam or unsterilized samples were used. Bacterial adherence on unsterilized and sterilized samples was tested with Staphylococcus epidermidis and Escherichia coli. Five samples were examined for each line type and sterilization condition, and final colony counts were obtained. Results: Bacterial adherence was significantly affected by method of sterilization for all 3 line types. For most of the samples, bacterial adherence was similar or lower when HPGP sterilization was used, compared with results for sterilization via ethylene oxide and steam, respectively. Bacterial adherence was significantly higher for UHMWPE, compared with adherence for the nylon line, regardless of the sterilization method used. Bacterial adherence was higher for nylon fishing line than for nylon leader line for S epidermidis after ethylene oxide sterilization and for E coli after HPGP and ethylene oxide sterilization. Conclusions and Clinical Relevance: Effects of HPGP sterilization on bioadhesive properties of nylon and polyethylene lines compared favorably with those for ethylene oxide and steam sterilization. Also, nylon line may be a more suitable material than UHMWPE for suture prostheses on the basis of bacterial adherence properties.

During acute bouts of recurrent airway obstruction (heaves) in horses, neutrophils that are capable of increased production of reactive oxygen species accumulate in the airways. In the study reported here, the effect of hydrogen peroxide (H2O2; 1 micromolar to 0.1M), one of these reactive oxygen species products, on the responses of isolated trachealis muscle of horses was determined. Before and after incubation with H2O2, contractile responses to acetylcholine, electrical field stimulation (EFS), 127 mM KCl, and relaxation responses to isoproterenol and activation of the nonadrenergic noncholinergic inhibitory response (iNANC) were evaluated. Beginning at 1 mM, H2O2 contracted trachealis muscle in a concentration-dependent manner. This contraction was unaffected by atropine (1 micromolar), tetrodotoxin (1 micromolar), or 1 micromolar meclofenamate. Contraction of trachealis muscle in response to H2O2 is, therefore, not attributable to release of prostaglandins, acetylcholine, or other neurotransmitters. Above a concentration of 0.1 mM, H2O2 depressed the responses to EFS. acetylcholine, and KCl in a concentration-dependent manner. At 0.1M, H2O2 decreased the maximal responses to EFS, acetylcholine, and KCl by 62.7 +/- 7.2, 60.58 +/- 6.12, and 37.8 +/- 9.54%, respectively. In the presence of meclofenamate (1 micromolar), partial but significant protection against 1 to 100 mM H2O2 was observed. In tracheal strips contracted with 0.3 micromolar methacholine, H2O2 had no effect on the isoproterenol concentration-response curve. Up to a concentration of 100 mM, H2O2 had no effect on iNANC response. However, in the presence of 100 mM H2O2, this response was abolished in 2 of 4 horses. We conclude that high concentrations of H2O2 affected the responses of airway smooth muscle by actions on neurotransmission, muscarinic receptors, and downstream from receptors; some of the H2O2 effects were in part mediated by cyclooxygenase products; and H2O2 had no effect on beta-adrenergic- or iNANC-induced relaxation.

A flow cytometric procedure was evaluated to measure the oxidative burst activity (hydrogen peroxide formation) of bovine neutrophils. The method involves measuring the oxidation of intracellular dichlorofluorescin to fluorescent dichlorofluorescein (DCF). Phorbol myristate acetate (PMA) was used to perturb the neutrophil plasma membrane. The sources of variation introduced into the DCF assay were also examined. The sources of variation were attributable to the isolation of neutrophils from blood, variation between duplicate assays and duplicate flow cytometric determinations of oxidative product formation, variation in neutrophil oxidative product formation among cows, and the variation (over repeated daily and weekly neutrophil isolations) in neutrophil oxidative product formation. A final objective was to determine effects of dexamethasone on oxidative product formation, and whether differences existed between blood and mammary neutrophils in oxidative product formation. There was an increasing trend in the formation of DCF with increasing time of incubation and with increasing PMA concentration. Increasing the concentration of PMA decreased lag time and increased the rate of oxidative product formation. The increase in DCF formation was statistically significant up to a PMA concentration of 10 ng/ml. This concentration was considered optimal for bovine neutrophils. Examination of the sources of variation indicated that (i) the neutrophil isolation technique was a major source of variation (17.2 to 28.4% of the total variation), and that more than one neutrophil isolation within a cow would be required to obtain an accurate estimation of DCF formation in neutrophils; (ii) duplicate assays and duplicate readings on the flow cytometer accounted for < 0.05% of the total variation and would not be necessary when performing the DCF assay; (iii) large variation (62.4 to 70.8%) existed among cows in neutrophil oxidative product formation, indicating that any treatment being compared should be done either within or preferably repeated across a large number of cows; and (iv) the variation over repeated daily (0.3%), but not weekly (19.6%) determinations of neutrophil oxidative product formation, were small enough to allow for the evaluation of major physiologic and environmental effects. Intramuscular administration of dexamethasone (50 microgram/ kg of body weight) resulted in an approximate 80% decrease in neutrophil oxidative product formation. Oxidative product formation was 75% less for neutrophils isolated from mammary secretions when compared with neutrophils from blood. These results indicated that the DCF procedure was responsive to factors known to interfere with oxidative metabolism of bovine neutrophils.

Avian peritoneal exudate macrophages, when exposed to phagocytic stimuli, produced an appreciable oxidative burst as measured by production of chemiluminescence, superoxide anion, and hydrogen peroxide. Metabolic inhibitors of the oxidative burst and scavengers of oxygen radicals clearly inhibited macrophage chemiluminescence, but had no significant effect on macrophage bactericidal activity against Escherichia coli or fungistatic activity against Candida tropicalis. Therefore, avian macrophages were capable of oxygen-independent bactericidal and fungistatic activities.

Canine circulating neutrophils, isolated by a blood lysing technique, were incubated with 7-S nerve growth factor (NGF), at final concentrations between 12.5 and 800 ng/ml, for 30 minutes at 37 C. Neutrophil cytosolic H2O2 production, measured by flow cytometry, after 7-S NGF incubation was not significantly different from that produced at 37 C (baseline temperature controls) alone. Phorbol myristate acetate (PMA; 100 ng/ml) stimulation of neutrophils produced cytosolic H2O2 concentrations almost 13 times that of baseline temperature control neutrophils. Preincubation of neutrophils with 7-S NGF (100 to 800 ng/ml, 30 minutes, 37 C) and subsequent stimulation by PMA resulted in augmented H2O2 production in excess of twice that of neutrophils treated with PMA alone, and almost 30 times that of baseline temperature controls.