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Abstract

Objective—To compare the effects of intra-articular (IA) versus IV administration of morphine on local and systemic inflammatory responses in horses with experimentally induced acute synovitis.

Animals—8 horses.

Procedures—Each horse received the following 2 treatments 4 hours after synovitis was induced: IA administration of morphine (0.05 mg/kg) with IV administration of 1 mL of saline (0.9% NaCl) solution/100 kg, and IA administration of 1 mL of saline solution/100 kg with IV administration of morphine (0.05 mg/kg). Treatments were administered in randomized order with a washout period of 3 weeks between treatments. Before each treatment, aseptic synovitis was induced by injection of lipopolysaccharide into a radiocarpal joint. For the second treatment, the contralateral radiocarpal joint was selected. Joint swelling and skin temperature over the treated joints were recorded. Clinical examinations were performed, and blood WBC count, serum amyloid A (SAA) concentration, serum cortisol concentration, synovial fluid WBC count, synovial fluid total protein (TP) concentration, and synovial fluid SAA concentration were measured before and repeatedly during each of the two 168-hour study periods. Data were analyzed by use of ANOVA with repeated measures.

Results—IA administration of morphine resulted in significantly less joint swelling and lower synovial fluid TP and serum and synovial fluid SAA concentrations, and blood WBC count than did IV administration of morphine.

Conclusions and Clinical Relevance—IA administration of morphine exerted anti-inflammatory properties in horses with experimentally induced acute synovitis, supporting its use as a part of a balanced analgesic protocol.

Full access
in American Journal of Veterinary Research

Abstract

Objective—To compare characteristics and enzymatic products of leukocytes detected in the skin and laminar tissues of horses administered black walnut heartwood extract (BWHE) and horses administered purified lipopolysaccharide (LPS).

Animals—25 healthy 5- to 15-year-old horses.

Procedures—Horses were randomly assigned to receive LPS (20 ng of O55:B5 Escherichia coli endotoxin/kg; n = 5) IV or 6 L of BWHE (10) or water (control group; 10) via nasogastric intubation. Horses were euthanatized 12 hours after treatment or at onset of Obel grade 1 lameness. Laminar tissue samples and skin samples from the middle region of the neck were harvested at the time of euthanasia. Leukocyte emigration (determined via CD13 immunohistochemical analysis) and matrix metalloproteinase (MMP)-2 and MMP-9 gene expressions and activities (determined via reverse transcription PCR assay and gelatin zymography, respectively) were measured in skin and laminar tissue samples.

Results—Tissues of horses receiving BWHE contained significantly higher numbers of CD13-positive cells and increased MMP-9 gene expression and activity, compared with findings in the other 2 groups. Values for laminar tissue and skin from LPS-treated horses were not increased, compared with findings in the control group, in any experiment.

Conclusions and Clinical Relevance—Results indicated that BWHE administration causes increases in CD13-positive leukocyte numbers and MMP-9 expression and activity in laminar tissue and skin in horses; similar effects were not detected following LPS administration. Leukocyte emigration in horses with experimentally induced endotoxemia and in horses administered BWHE differed markedly, thereby providing additional evidence that the development of laminitis involves more complex mechanisms than endotoxemia-induced leukocyte activation alone.

Full access
in American Journal of Veterinary Research

Abstract

Objective—To investigate effects of lidocaine hydrochloride administered IV on mucosal inflammation in ischemia-injured jejunum of horses treated with flunixin meglumine.

Animals—24 horses.

Procedures—Horses received saline (0.9% NaCl) solution (SS; 1 mL/50 kg, IV [1 dose]), flunixin meglumine (1 mg/kg, IV, q 12 h), lidocaine (bolus [1.3 mg/kg] and constant rate infusion [0.05 mg/kg/min], IV, during and after recovery from surgery), or both flunixin and lidocaine (n = 6/group). During surgery, blood flow was occluded for 2 hours in 2 sections of jejunum in each horse. Uninjured and ischemia-injured jejunal specimens were collected after the ischemic period and after euthanasia 18 hours later for histologic assessment and determination of cyclooxygenase (COX) expression (via western blot procedures). Plasma samples collected prior to (baseline) and 8 hours after the ischemic period were analyzed for prostanoid concentrations.

Results—Immediately after the ischemic period, COX-2 expression in horses treated with lidocaine alone was significantly less than expression in horses treated with SS or flunixin alone. Eighteen hours after the ischemic period, mucosal neutrophil counts in horses treated with flunixin alone were significantly higher than counts in other treatment groups. Compared with baseline plasma concentrations, postischemia prostaglandin E2 metabolite and thromboxane B2 concentrations increased in horses treated with SS and in horses treated with SS or lidocaine alone, respectively.

Conclusions and Clinical Relevance—In horses with ischemia-injured jejunum, lidocaine administered IV reduced plasma prostaglandin E2 metabolite concentration and mucosal COX-2 expression. Coadministration of lidocaine with flunixin ameliorated the flunixin-induced increase in mucosal neutrophil counts.

Full access
in American Journal of Veterinary Research

Abstract

Objective—To measure the effects of lowmolecular-weight inhibitors on the activity of bovine neutrophil matrix metalloproteinase 9 (MMP-9).

Sample Population—Bovine MMP-9 purified from bovine neutrophilconditioned medium.

Procedures—Neutrophils were degranulated by stimulation with phorbol ester. Enzyme purification was performed by use of gelatin affinity and gel-filtration chromatography. Activated enzyme was incubated with inhibitors prior to addition of substrate (gelatin fluorescein conjugate or fluorogenic peptide). Rates of enzymatic cleavage were determined by monitoring fluorescence as the reactions progressed. Values of IC50 (molar concentration of compound that inhibits specific activity by 50%) and KI (in vitro inhibition constant) were determined.

Results—Rates of enzymatic activity of monomeric and dimeric bovine MMP-9 measured by use of gelatin and peptide substrates were linear with respect to time and concentrations of enzyme and substrate. The MMP-9 was potently inhibited by hydroxamic acids (IC50 for gelatin, 29.2 to 55.7nM; IC50 for peptide, 4.8 to 24.6nM; KI, 0.2 to 0.5nM), whereas tetracyclines (IC50 for gelatin, 30.1 to 112.7MM; IC50 for peptide, 48.0 to 123.8MM; KI, 25.2 to 61.4µM) and chlorhexidine (IC50 for gelatin, 139.1MM; IC50 for peptide, 672.5MM to 1.7mM; KI, 495.0 to 663.0MM) had limited inhibition. Gelatinase-specific inhibitor SB-3CT had intermediate potency (IC50 for peptide, 185.0 to 290.0nM; KI, 66.5 to 86.0nM).

Conclusions and Clinical Relevance—Bovine MMP-9 was potently inhibited by hydroxamic acids and gelatinase inhibitor. These compounds may be useful as modulators of neutrophil-mediated protease activity in cattle.

Full access
in American Journal of Veterinary Research

Abstract

Objective—To examine effects of in vitro exposure to solutions of hay dust, lipopolysaccharide (LPS), or β-glucan on chemokine and cell-surface receptor (CSR) gene expression in primary bronchial epithelial cell cultures (BECCs) established from healthy horses and horses with recurrent airway obstruction (RAO).

Sample Population—BECCs established from bronchial biopsy specimens of 6 RAO-affected horses and 6 healthy horses.

Procedures—5-day-old BECCs were treated with PBS solution, hay dust solutions, LPS, or β-glucan for 6 or 24 hours. Gene expression of interleukin (IL)-8, chemokine (C-X-C motif) ligand 2 (CXCL2), IL-1β, toll-like receptor 2, toll-like receptor 4, IL-1 receptor 1, and glyceraldehyde 3–phosphate dehydrogenase was measured with a kinetic PCR assay.

Results—Treatment with PBS solution for 6 or 24 hours was not associated with a significant difference in chemokine or CSR expression between BECCs from either group of horses. In all BECCs, treatment with hay dust or LPS for 6 hours increased IL-8, CXCL2, and IL-1β gene expression > 3-fold; at 24 hours, only IL-1β expression was upregulated by > 3-fold. In all BECCs, CSR gene expression was not increased following any treatment. With the exception of a 3.7-fold upregulation of CXCL2 in BECCs from RAO-affected horses (following 6-hour hay dust treatment), no differences in chemokine or CSR gene expression were detected between the 2 groups. At 24 hours, CXCL2 gene expression in all BECCs was downregulated.

Conclusions and Clinical Relevance—Epithelial CXCL2 upregulation in response to hay dust particulates may incite early airway neutrophilia in horses with RAO.

Full access
in American Journal of Veterinary Research

Abstract

Objective—To evaluate the effects of cis–urocanic acid (cis-UCA) on mammary gland (MG) inflammation and injury associated with Escherichia coli–induced mastitis in dairy cows.

Animals—12 lactating dairy cows (36 MGs).

Procedures—At 7-week intervals, a different MG in each cow was experimentally inoculated with E coli. At 6-hour intervals from 6 to 36 hours after inoculation, the inoculated MG in each cow was infused with 40 mL of saline (0.9% NaCl) solution, 12.5mM cis-UCA, or 25mM cis-UCA (4 cows/group); ultimately, each cow received each treatment. Immediately prior to and at various time points after inoculation and treatment, milk samples were collected. Bacterial CFUs, somatic cell counts (SCCs), N-acetyl-beta-D-glucosaminidase (NAGase) and lactate dehydrogenase (LDH) activities, and concentrations of bovine serum albumin, tumor necrosis factor-α, and cis-UCA were quantified in each milk sample.

Results—Compared with findings in saline solution–treated MGs, NAGase and LDH activities in milk samples from cis-UCA–treated MGs were lower. Cis-UCA had no effect on milk SCCs and milk concentrations of bovine serum albumin and tumor necrosis factor-α. Furthermore, cis-UCA had no adverse effect on bacterial clearance; CFUs of E coli in MGs treated with saline solution or cis-UCA were equivalent.

Conclusions and Clinical Relevance—In cows, milk NAGase and LDH activities were both lower in E coli–infected MGs infused with cis-UCA than in those infused with saline solution, which suggests that cis-UCA reduced mastitis-associated tissue damage. Furthermore, these data indicated that therapeutic concentrations of cis-UCA in milk can be achieved via intramammary infusion.

Full access
in American Journal of Veterinary Research

Abstract

Objective—To evaluate perinuclear anti-neutrophilic cytoplasmic autoantibody (pANCA) status in Soft Coated Wheaten Terriers (SCWTs) and SCWT-Beagle crossbred dogs and to correlate pANCA status of dogs with clinicopathologic variables of protein-losing enteropathy (PLE), protein-losing nephropathy (PLN), or both.

Animals—13 SCWTs and 8 SCWT-Beagle crossbred dogs in a research colony and a control group comprising 7 dogs with X-linked hereditary nephropathy and 12 healthy SCWTs > 9 years old.

Procedures—Samples were obtained from dogs in the research colony every 6 months. At each sample-collection time point, serum concentrations of albumin, globulin, creatinine, and urea nitrogen; fecal concentration of α-proteinase inhibitor; and urinary protein-to-creatinine ratios were determined and correlated with pANCA status.

Results—20 of 21 dogs in the research colony had positive results for pANCAs at a minimum of 2 time points, and 18 of 21 dogs had definitive evidence of disease. None of the control dogs had positive results for pANCAs. A positive result for pANCAs was significantly associated with hypoalbuminemia, and pANCAs preceded the onset of hypoalbuminemia on an average of 2.4 years. Sensitivity and specificity for use of pANCAs to predict development of PLE or PLN were 0.95 (95% confidence interval, 0.72 to 1.00) and 0.8 (95% confidence interval, 0.51 to 0.95), respectively.

Conclusions and Clinical Relevance—Most dogs in this study affected with PLE, PLN, or both had positive results for pANCAs before clinicopathologic evidence of disease was detected. Thus, pANCAs may be useful as an early noninvasive test of disease in SCWTs.

Full access
in American Journal of Veterinary Research

Abstract

Objective—To determine the amount of colostral IgG required for adequate passive transfer in calves administered colostrum by use of oroesophageal intubation and evaluate the impact of other factors on passive transfer of colostral immunoglobulins in calves.

Animals—120 Holstein bull calves.

Procedures—Calves were randomly assigned to specific treatment groups on the basis of volume of colostrum administered and age of calf at administration of colostrum. Colostrum was administered once by oroesophageal intubation. Equal numbers of calves received 1, 2, 3, or 4 L of colostrum, and equal numbers of calves received colostrum at 2, 6, 10, 14, 18, or 22 hours after birth. Serum samples were obtained from calves 48 hours after birth for IgG determination by radial immunodiffusion assay. Effects of factors affecting transfer of colostral immunoglobulins were determined by use of a stepwise multiple regression model and logistic regression models.

Results—A minimum of 153 g of colostral IgG was required for optimum colostral transfer of immunoglobulins when calves were fed3Lof colostrum at 2 hours after birth. Substantially larger IgG intakes were required by calves fed colostrum > 2 hours after birth.

Conclusions and Clinical Relevance—Feeding 100 g of colostral IgG by oroesophageal intubation was insufficient for adequate passive transfer of colostral immunoglobulins. At least 150 to 200 g of colostral IgG was required for adequate passive transfer of colostral immunoglobulins. Use of an oroesophageal tube for administration of 3 L of colostrum to calves within 2 hours after birth is recommended.

Full access
in American Journal of Veterinary Research

Abstract

Objective—To evaluate the effect of lactoferrin on lipopolysaccharide (LPS)-induced proliferation of bovine peripheral blood mononuclear cells (PBMCs), gene expression of inflammatory mediators, and production of prostanoids in vitro.

Sample Population—PBMCs isolated from 15 Holstein bull calves.

Procedures—Mixed populations of PBMCs were isolated by differential centrifugation. Proliferation assays were conducted in 96-well plates designed to allow addition of lactoferrin (200 ng/mL) with and without LPS (1 μg/mL) in a checkerboard design. Incorporation of 3H-thymidine was used to determine proliferation of PBMCs. Prostaglandin E2 production was determined in culture-conditioned medium by use of enzyme immunoassay. Effects of lactoferrin on LPS-induced gene expression of cyclooxygenase (COX)-2 and matrix metalloproteinase (MMP)-9 were monitored by use of PCR assays.

Results—Lactoferrin supplementation significantly reduced LPS-induced incorporation of 3H-thymidine and production of prostaglandin E2 by PBMCs. Lactoferrin reduced LPS-induced expression of COX-2 and MMP-9 mRNA.

Conclusions and Clinical Relevance—Lactoferrin reduced LPS-induced cellular proliferation, inflammatory mediator gene expression, and prostaglandin E2 production by bovine PBMCs in vitro. These effects may be beneficial in reducing the impact of endotoxemia in neonates.

Full access
in American Journal of Veterinary Research

Abstract

Objective—To determine whether vaccinating cows during late gestation against Mycoplasma bovis will result in adequate concentrations of M bovis–specific IgG1 in serum, colostrum, and milk.

Animals—78 dairy cows.

Procedures—Serum samples were obtained 60 and 39 days prior to expected parturition in vaccinated and control cows from a single herd. Serum and colostrum samples were also obtained at parturition. Milk samples were obtained 7 to 14 days after parturition. Samples were analyzed for anti–M bovis IgG1 concentrations.

Results—Prior to vaccination, control and vaccinated cows had similar anti–M bovis IgG1 concentrations. After initial vaccination and subsequent booster and at parturition, there was a significant difference between the 2 groups, with vaccinated cows having higher IgG concentrations. Colostrum from vaccinated cows had higher anti–M bovis IgG1 concentrations, compared with control cows; however, IgG1 concentrations in milk did not differ between the 2 groups.

Conclusions and Clinical Relevance—Vaccination of late-gestation cows resulted in increased concentrations of anti–M bovis IgG1 in colostrum. However, ingestion of colostrum by calves may not guarantee protection against M bovis infection.

Full access
in American Journal of Veterinary Research