peer reviewed | OBJECTIVE: To compare sensitivity of the impulse oscillometry system (IOS) with that of the conventional reference technique (CRT; ie, esophageal balloon method) for pulmonary function testing in horses. ANIMALS: 10 horses (4 healthy; 6 with recurrent airway obstruction [heaves] in remission). PROCEDURE: Healthy horses (group-A horses) and heaves-affected horses (group-B horses) were housed in a controlled environment. At each step of a methacholine bronchoprovocation test, threshold concentration (TC(2SD); results in a 2-fold increase in SD of a value) and sensitivity index (SI) were determined for respiratory tract system resistance (R(rs)) and respiratory tract system reactance (X(rs)) at 5 to 20 Hz by use of IOS and for total pulmonary resistance (RL) and dynamic lung compliance (C(dyn)), by use of CRT. RESULTS: Bronchoconstriction resulted in an increase in R(rs) at 5 Hz (R(5Hz)) and a decrease in X(rs) at all frequencies. Most sensitive parameters were X(rs) at 5 Hz (X(5Hz)), R(5Hz), and R(5Hz):R(10Hz) ratio; RL and the provocation concentration of methacholine resulting in a 35% decrease in dynamic compliance (PC(35)C(dyn)) were significantly less sensitive than these IOS parameters. The TC(2SD) for X(rs) at 5 and 10 Hz was significantly lower in group-B horses, compared with group-A horses. The lowest TC(2SD) was obtained for X(5Hz) in group-B horses and R(5Hz) in group-A horses. CONCLUSIONS AND CLINICAL RELEVANCE: In contrast to CRT parameters, IOS parameters were significantly more sensitive for testing pulmonary function.The IOS provides a practical and noninvasive pulmonary function test that may be useful in assessing subclinical changes in horses.

peer reviewed | Increases of plasma concentrations of neutrophil myeloperoxidase (MPO) can be used as markers of polymorphonuclear leucocytes (PMN) activation in pathological situations (sepsis, acute lung injury, acute inflammation). To develop an assay for measurement of plasma MPO in horses during the above-mentioned infectious and inflammatory conditions, MPO was purified from equine PMN isolated from blood anticoagulated with citrate. PMN were extracted in a saline milieu (0.2 M Na acetate, 1 M NaCl, pH 4.7) to eliminate most of cellular proteins. Pellets were then extracted in the same buffer containing cationic detergent (1% cetyltrimethyl ammonium bromide). The supernatant was further purified by ion exchange chromatography (Hiload S Sepharose HP column 0.5 x 26 cm, equilibrated with 25 mM Na acetate, 0.2 M NaCl, pH 4.7) with a NaCl gradient (until 1 M). Most of the peroxidase activity of MPO (spectrophotometrically measured by the oxidation of orthodianisidine by hydrogen peroxide) was eluted at 0.65 M NaCl. MPO was further purified by gel filtration chromatography (Sephacryl S 200 column 2.6 x 42 cm with 25 mM Na acetate, 0.2 M NaCl, pH 4.7). MPO (specific activity: 74.3 U/mg) was obtained with a yield of 30% from the detergent extraction supernatant. Electrophoresis (non-reducing conditions) showed 3 bands identified, by comparison with human MPO, (i) the mature tetrameric enzyme (150 kDa) with 2 light and 2 heavy subunits, (ii) the precursor form (88 kDa) and (iii) a form of the heavy subunit without the prosthetic heme group (40 kDa). The mature enzyme and its precursor were glycosylated and possessed peroxidase activity. Equine MPO showed strong similarities with human and bovine MPO, with an absorption peak at 430 nm (Soret peak) characteristic of ferrimyeloperoxidase. Enzymatic activity was pH dependent (optimal value at pH 5.5).

Objective-To investigate the effects of IV administration of ergotamine and ergovaline and intraruminal administration of ergotamine on electromyographic (EMG) activity of reticuloruminal smooth muscle in conscious sheep. Animals-3 sheep with indwelling electrodes in the musculature of the reticulum and rumen. Procedure-In a crossover design study, reticuloruminal motility before and after IV administration of ergotamine (5, 10, 20, and 40 nmol/kg) or ergovaline (2.5, 5, and 10 nmol/kg) was evaluated; EMG effects were compared with those of corresponding control treatments (IV administration of saline 0.9% NaCl solution or acetone, respectively) in sheep. Ergotamine (800 nmol/kg) or water was also administered intraruminally and their effects compared. Results-After IV administration of ergopeptides, vagally dependent cyclical A and B sequences of contraction of the reticulorumen were immediately inhibited, preceding increases in baseline EMG activity (tonus). The return of cyclical contractions was associated with an increase in contraction amplitude. The effects were dose dependent; administration of 40 nmol of ergotamine/kg resulted in responses that continued for 3 to 4 hours. The effects of intraruminal administration of ergotamine were variable; after 8 hours, EMG activity was increased from baseline for < 2 hours in 1 sheep, 10 hours in another, and > 15 hours in the third. Conclusions and Clinical Relevance-In sheep, the effects of ergotamine and ergovaline on reticuloruminal motility after IV administration and the duration of responses following intraruminal administration suggest that disruption of digestion may occur in animals grazing endophyte-infected pasture that has a high ergopeptide content.

Bacampicillin hydrochloride is an ester prodrug that is hydrolyzed to ampicillin after its absorption from the gastrointestinal tract. It was administered intragastrically at a dose rate of 13.5 mg/kg of body weight to ponies and horses, and was highly bioavailable (F = 41.0%), compared with other penicillins in adult horses. The high peak ampicillin plasma concentration of 6.1 +/- 0.5 micrograms/ml achieved and persistence of the antibiotic at concentration of 0.3 +/- 0.1 micrograms/ml 6 hours after its intragastric administration, suggest that bacampicillin hydrochloride may reach suitable bactericidal concentrations for treatment of infections caused by susceptible microorganisms. In a separate experiment, dichlorvos, an organophosphate compound that inhibits some of the esterase activity in plasma, was administered orally to the same animals at a dose rate of 40 mg/kg followed by intragastric administration of bacampicillin hydrochloride at a dose of 13.5 mg/kg. Plasma pseudocholinesterase and erythrocyte acetylcholinesterase activities were reduced to < 5% of reference (predichlorvos) values after dichlorvos administration. However, rate of hydrolysis of bacampicillin into ampicillin was not affected. Consequently, the disposition and fate of bacampicillin when administered intragastrically 1 day after dichlorvos administration were similar to the values obtained after administration of bacampicillin alone. Intragastric coadministration of probenecid at a dose rate of 75 mg/kg and bacampicillin at 13.5 mg/kg limited absorption of the antibiotic from the gastrointestinal tract. This suggests existence of a common transport mechanism for bacampicillin and probenecid in the gastrointestinal wall, and precludes use of this combination for treatment. The bioavailable fraction of ampicillin after combination treatment indicated prolonged residence time in the plasma, presumably as a consequence of reduced renal tubular secretion.

A monoclonal antibody (MAB) against equine tumor necrosis factor-alpha (Eq TNF) was used to investigate the role of TNF in cytokine, eicosanoid, and metabolic responses of Miniature Horses given endotoxin. Plasma concentrations of interleukin 6 (IL-6), lactate, thromboxane A2 metabolite, and prostacyclin metabolite (6-keto-PGF(1 alpha)) were measured in 10 Miniature Horses given 0.25 micrograms of lipopolysaccharide (LPS; Escherichia coli O55:B5)/kg of body weight. Five horses were given Eq TNF MAB and 5 were given isotype-matched MAB as control. All horses were given 1.86 mg of antibody/kg by IV infusion, 5 minutes before LPS was given IV. Blood samples were taken 20 minutes before and at multiple intervals for 24 hours after LPS was given. Interleukin 6 bioactivity in plasma was measured, using IL-6-dependent cell line (B9). Eicosanoid activities were assessed by enzyme immunoassay, and plasma lactate concentration was determined enzymatically. Data were analyzed by ANOVA and Tukey's honest significant difference test for significant (P < 0.05) effect of treatment. Horses given Eq TNF MAB had significantly (P < 0.050) lower peak mean +/- SEM IL-6 (59 +/- 29 U/ml), lactate (16 +/- 2.00 mg/dl), and 6-keto-PGF(1 alpha) (254 +/- 79 pg/ml) values then did horses given control MAB (880 +/- 375 U/ml for IL-6; 26 +/- 0.04 mg/dl for lactate; and 985 +/- 290 pg/ml for 6-keto-PGF(1 alpha)). There was no effect of anti-TNF treatment on LPS-induced thromboxane A2 metabolite production. Tumor necrosis factor mediated IL-6, lactate, and prostacyclin responses, without affecting thromboxane production in horses given LPS.

We purified porcine whey lactoferrin by affinity chromatography on a heparin-sepharose column, followed by high-performance liquid chromatography. Molecular mass of purified lactoferrin (PLF) is 78,000 daltons. The iron-binding activity of PLF had a UV/ visible-light absorption spectrum indistinguishable from that of human and bovine lactoferrins (absorbance ratio [465 nm/280 nm] approx 0.046). The growth ratio of WIL-2 cells in PLF-supplemented medium is 70% of that in serum-containing medium. The aforementioned characteristics are similar to those of human and bovine lactoferrins. Immunoblot analysis, using polyclonal antibody raised in rabbits against porcine whey lactoferrin, revealed high specificity for PLF, and low cross-reactivity with commercial human and bovine lactoferrins.

Ouabain, a cardiac glycoside, binds to the Na+-K+i-adenosine triphosphatase (Na+ pump) and prevents active transport of Na+ and K+ across cell membranes. We used [3H]ouabain to quantify the number and affinity of Na+ pumps in skeletal muscle from Quarter Horses with the muscular disorder hyperkalemic periodic paralysis (HYPP). [3H]Ouabain-binding properties of gluteal muscle from clinically normal and affected horses were used to determine whether altered Na+ pump number or affinity could contribute to the pathologic features of muscle in affected horses. Foals and adult horses with HYPP were compared with age-matched clinically normal horses. The number of [3H]ouabain-binding sites in adult gluteal muscle was not different between the 2 types of horses (85.7 +/- 8.9 pmol of [3H]ouabain-binding sites/g [wet muscle weight] in horses with HYPP vs 100.2 +/- 8.8 pmol/g in clinically normal adult horses). Gluteal muscles in HYPP-affected and clinically normal foals also contained a similar number of [3H]ouabain-binding sites (222.3 +/- 21.0 pmol/g vs 225.3 +/- 24.2 pmol/g, respectively). The affinity of these binding sites for ouabain was not different, between adults or foals, in clinically normal or affected horses. Our results indicate that membrane events underlying the periodic episodes of paralysis in horses with HYPP are not attributable to quantitative changes in Na+ pump number or affinity. Our data cannot exclude the possibility that the specific activity of the Na+ pump is altered in muscle from HYPP-affected horses.

Stimulatory effects of 6 zymosan preparations on luminol-dependent chemiluminescence (CL) responses of isolated bovine neutrophils were compared. Unopsonized zymosan particles and zymosan particles opsonized with bovine IgG1, IgG2, fresh serum, or serum from which zymosan-specific antibodies, but not complement, had been removed (C3- serum) induced strong CL responses, with nearly equal maximal peaks in the presence of extracellular Ca2+ and Mg2+, whereas the response to fetal bovine serumopsonized zymosan particles was markedly low. Removal of extracellular divalent cations almost completely blocked the CL reaction triggered by unopsonized, IgG1-opsonized, C3-opsonized, and fetal bovine serum-opsonized zymosan particles. By contrast, no change in the respiratory burst activity induced by serum-opsonized zymosan and only partial reduction in the response to IgG2-opsonized zymosan were seen under these conditions. Further experiments were performed with 4 zymosan preparations on neutrophils isolated from 2 calves with a genetic deficiency of CD11/CD18 membrane antigens. The unopsonized zymosan-induced CL reaction was absent in these cells. A reduced, but clear, response was observed with C3-opsonized zymosan. Unexpectedly, in the absence of extracellular Ca2+ and Mg2+ , serum-opsonized zymosan failed to generate the respiratory burst, whereas response to IgG2-opsonized zymosan was normal in the CDll/CD18-deficient neutrophils. These findings indicate that unopsonized zymosan may act in a divalent cation-dependent manner at the receptor for C3bi in bovine neutrophils, as it has been shown to do in the human system. In addition, it seems that IgG2-Fc receptors capable of signaling the respiratory burst in the absence of extracellular Ca2+ and Mg2+ exist on bovine neutrophils.

Cell extracts that were prepared from blood mononuclear leukocytes from 66 samples obtained from 6 clinically normal calves contained mean 2',5'-oligoadenylate (2',5'-oligo[A]) synthetase activity sufficient to synthesize 186 +/- 82 pmol of 2',5'-oligo(A)/h/10(6) cells. Calves had no measurable serum interferon (IFN) activity. Five calves were given IM injections of 10(4), 10(5), 5 x 10(5), 10(6), and 10(7) U of bovine IFN-alpha 1/kg of body weight at 2-week intervals. Five dosing sequences were used with a 5 x 5 Latin square design so that each calf received each dose once. Activity of 2',5'-oligo(A) synthetase increased at 24 hours in response to all dosages of IFN and then declined following first-order kinetics, with an apparent half-life (t1/2) of 2.1 +/- 0.5 days. The area under the concentration-time curve for 2',5'-oligo(A) synthetase increased with dose of IFN more rapidly than did peak response. Serum IFN that was measured at 1-day intervals following administration of IFN was consistently measurable only at dosages above 10(6) U of IFN/kg. The t1/2 for circulating IFN was 12.4 +/- 1.0 hours. Over all dosages, increases in 2',5'-oligo(A) synthetase activity were measurable for 3.5 days longer than were increases in IFN following IM injection of IFN. None of the calves developed detectable anti-IFN antibodies.

Brain stem auditory-evoked potentials (BAEP) were recorded in 4 dogs to analyze the relationship between acoustic stimulus intensities and peak latencies of each wave, and to investigate the relative effects of xylazine-atropine-ketamine, and xylazine-atropine-pentobarbital combinations and the time-course effects of the latter 2 drug combinations on BAEP. Click stimulations fixed at a stimulus rate of 10/s and a frequency of 4 kHz were delivered at intensities ranging from 10- to 110-dB sound pressure level (SPL) in 10-dB steps for analyzing the relationship between the acoustic stimulus intensities and the peak latencies and at an intensity of 110-dB SPL for investigating the effects of the sedative and the anaesthetic drug combinations and their time-course effects on BAEP. Waves I and VI were identified with stimulus intensity of greater than or equal to 50-dB SPL. Wave VII was observed in some records, but was excluded from statistical analysis. As intensity was increased from 50- to 110-dB SPL, the latency decreased for all waves during xylazine-atropine-ketamine anesthesia. There were no statistically significant differences in the peak latencies of each wave in BAEP among xylazine-atropine, xylazine-atropine-ketamine, and xylazine-atropine-pentobarbital combinations 20 minutes after drug administration, except that the latency of wave VI during xylazine-atropine sedation was significantly (P < 0.01) shorter than that detected during xylazine-atropine-ketamine or xylazine-atropine-pentobarbital anesthesia. There were no significant changes in peak latencies of waves I, II, III, V, and VI for 90 minutes after administration of the xylazine-atropine-ketamine combination and for 120 minutes after administration of the xylazine-atropine-pentobarbital combination. It was concluded that BAEP did not change over time after xylazine-atropine-ketamine or xylazine-atropine pentobarbital administration.