In vitro Inhibition of Canine Complement‐Mediated Hemolysis

Background

Immune‐mediated hemolytic anemia (IMHA) is the most common hematologic immune‐mediated disease in dogs. Complement fixation on erythrocytes causes hemolysis. Complement inhibition decreases hemolysis in people with the hemolytic disease and also may prove effective in treating IMHA in dogs.

Hypothesis/Objectives

Evaluate the in vitro efficacy of 2 complement inhibitors used in humans against canine complement.

Methods

The inhibitory activity of the C3‐inhibitor compstatin and recombinant human C1‐esterase inhibitor (C1‐INH) was evaluated using an in vitro hemolytic assay and spectrophotometric measurement of released hemoglobin. Dose‐response curves for each inhibitor were generated.

Results

Compstatin decreased approximately 50% of canine complement‐mediated hemolysis in initial experiments. This inhibition largely was lost when a new lot of drug was purchased. C1‐INH showed a dose‐dependent inhibition. The highest concentration of C1‐INH tested (500 μg/mL) decreased >80% of canine complement‐mediated hemolysis, and the lowest concentration tested (31.25 μg/mL) decreased hemolysis >60%.

Conclusions and Clinical Importance

Human C1‐INH is a robust inhibitor of canine complement‐mediated hemolysis, whereas compstatin was minimally and variably effective. Human C1‐INH may substantially decrease complement‐mediated hemolysis in dogs with IMHA and warrants further investigation.     

Plasma‐Free Metanephrine and Free Normetanephrine Measurement for the Diagnosis of Pheochromocytoma in Dogs

Background

Measurement of plasma‐free metanephrines is the test of choice to identify pheochromocytoma in human patients.

Objectives

To establish the sensitivity and specificity of plasma‐free metanephrine (fMN) and free normetanephrine (fNMN) concentrations to diagnose pheochromocytoma in dogs.

Animals

Forty‐five client‐owned dogs (8 dogs with pheochromocytoma, 11 dogs with adrenocortical tumors, 15 dogs with nonadrenal disease, and 11 healthy dogs.)

Methods

A prospective study. EDTA plasma was collected from diseased and healthy dogs and submitted for fMN and fNMN measurement by liquid chromatography‐tandem mass spectrometry (LC‐MS/MS).

Results

Free MN concentration (median [range]) was significantly higher in dogs with pheochromocytoma (8.15 [1.73–175.23] nmol/L) than in healthy dogs (0.95 [0.68–3.08] nmol/L; P < .01) and dogs with adrenocortical tumors (0.92 [0.25–2.51] nmol/L; P < .001), but was not different from dogs with nonadrenal disease (1.91 [0.41–6.57] nmol/L; P ≥ .05). Free NMN concentration was significantly higher in dogs with pheochromocytoma (63.89 [10.19–190.31] nmol/L) than in healthy dogs (2.54 [1.59–4.17] nmol/L; P < .001), dogs with nonadrenal disease (3.30 [1.30–10.10] nmol/L; P < .001), and dogs with adrenocortical tumors (2.96 [1.92–5.01] nmol/L); P < 0.01). When used to diagnose pheochromocytoma, a fMN concentration of 4.18 nmol/L had a sensitivity of 62.5% and specificity of 97.3%, and a fNMN concentration of 5.52 nmol/L had a sensitivity of 100% and specificity of 97.6%.

Conclusions and Clinical Importance

Plasma fNMN concentration has excellent sensitivity and specificity for the diagnosis of pheochromocytoma in dogs, whereas fMN concentration has moderate sensitivity and excellent specificity. Measurement of plasma‐free metanephrines provides an effective, noninvasive, means of identifying dogs with pheochromocytoma.     

In vitro enantioselective pharmacodynamics of Carprofen and Flunixin‐meglumine in feedlot cattle

The activity of the anti‐inflammatory agents Flunixin‐meglumine (FLU), RS (±) Carprofen (CPF) and S (+) CPF on bovine cyclooxygenases (COXs) has been characterized in feedlot calves using an in vitro whole blood model. The drugs showed equivalent efficacy in their inhibitory activity on COXs, and the rank order of potency for both COX‐1 and COX‐2 inhibition was FLU > S (+) CPF > RS (±) CPF. Our results indicated that FLU is a nonselective inhibitor of bovine COXs, whereas RS (±) CPF and S (+) CPF exhibited different degrees of preferential inhibition of COX‐2 isoenzyme. The rank order of IC₅₀ COX‐1: IC₅₀ COX‐2 potency ratios was in fact S (+) CPF (51.882) > RS (±) CPF (13.964) > FLU (0.606), and the calculated percentage inhibition of COX‐1 corresponding to COX‐2 inhibition values comprised between 80% and 95% was comprised between 57.697 and 79.865 for FLU, 33.373 and 51.319 for RS (±) CPF, and 0.230 and 4.622 for S (+) CPF, respectively. These findings are discussed in relation to the prediction of the clinical relevance of COX inhibition by the test drugs in cattle.     

氟砜霉素和氯霉素与抗菌增效剂的体外联合抑菌效果比较

应用试管二倍稀释法测定了氟砜霉素、氯霉素、TMP和OMP对15株畜禽常见病原菌的最小柳菌浓度（MIC）。结果表明，氟砜霉素的体外抗菌活性强，优于或等于氯霉素；TMP和OMP的体外抗菌活性偏低，但OMP优于TMP。氟砜霉素和氯霉素与TMP、OMP对14株细菌的联合药敏试验经肉汤稀释棋盘法测定，结果表明，氟砜霉素和氯霉素分别与TMP、OMP体外联合，抑菌效果相同，呈协同作用的均占50%与28.6%，即TMP或OMP对氟砜霉素和氯霉素的增效作用相等，但TMP优于OMP。在14株细菌对氟砜霉素和氯霉素分别与TMP、OMP的总共56组体外联合试验中，呈协同作用的占39.3%，主要为大肠杆菌、多杀性巴氏杆菌、沙门氏菌及金黄色葡萄球菌；无关作用的点60.7%；无拮抗现象。故临床联合用药前，仍需进行联合药敏试验，呈协同作用者才可联用。     

In vitro fermentative characteristics of ruminant diets supplemented with fibrolytic enzymes and ranges of optimal endo‐β‐1,4‐glucanase activity

Effectiveness of fibrolytic enzymes supplementing a range of forage to concentrate (F:C) diets was assessed with goat (G) or cow (C) inoculum using the gas production (GP) technique. Four F:C diets were evaluated: forage (1:0), high forage (0.7:0.3), medium forage (0.5:0.5) and low forage (0.3:0.7) diets, supplemented or not with Promote^TM (PRO) at 1 or 2 ml/kg dry matter (DM). The GP kinetic was different between F:C (1:0 < 0.7:0.3 < 0.5:0.5 < 0.3:0.7) and inoculum. Responses to enzyme were positively related to forage level and differed with inoculum. The neutral detergent fibre and acid detergent fibre degradation were depressed by the concentrate in the substrates fermented with C and were not altered or even enhanced in G sets. Results confirm that increasing starch proportion modified the pattern of microbial fermentation, while no influences were detected in the improvement of cell wall degradation with fibrolytic enzymes. Another in vitro experiment was conducted to investigate factors by which endo‐β‐1,4‐glucanase activity (EA) of PRO is compromised in a factorial design (3 × 4 × 3) for three pH (4.0, 5.5 and 6.5), four temperatures (30, 40, 50 and 70 °C) and three doses (1, 2 and 3 ml/kg DM of substrate). Maximum EA were obtained for pH 4.0, 50 °C and 3 ml/kg DM. Optimal conditions for PRO proved to be outside the normal ranges in ruminal environment.     

In vitro anti‐tubulin effects of mebendazole and fenbendazole on canine glioma cells

Benzimidazole anthelmintics have reported anti‐neoplastic effects both in vitro and in vivo. The purpose of this study was to evaluate the in vitro chemosensitivity of three canine glioma cell lines to mebendazole and fenbendazole. The mean inhibitory concentration (IC₅₀) (±SD) obtained from performing the MTT [3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide] assay after treating J3T, G06‐A, and SDT‐3G cells for 72 h with mebendazole were 0.030 ± 0.003, 0.080 ± 0.015 and 0.030 ± 0.006 μM respectively, while those for fenbendazole were 0.550 ± 0.015, 1.530 ± 0.159 and 0.690 ± 0.095 μM; treatment of primary canine fibroblasts for 72 h at IC₅₀ showed no significant effect. Immunofluorescence studies showed disruption of tubulin after treatment. Mebendazole and fenbendazole are cytotoxic in canine glioma cell lines in vitro and may be good candidates for treatment of canine gliomas. Further in vivo studies are required.     

In vitro assessment of chloramphenicol and florfenicol as second‐line antimicrobial agents in dogs

The aim of this study was to evaluate the potential of chloramphenicol and florfenicol as second‐line antimicrobial agents for treatment of infections caused by methicillin‐resistant Staphyococcus pseudintermedius (MRSP) and extended‐spectrum β‐lactamase (ESBL)‐producing Escherichia coli in dogs, through a systematic in vitro assessment of the pharmacodynamic properties of the two drugs. Minimum inhibitory concentrations (MIC) and phenicol resistance genes were determined for 169 S. pseudintermedius and 167 E. coli isolates. Minimum bactericidal concentrations (MBC), time‐killing kinetics, and postantibiotic effect (PAE) of both agents against wild‐type isolates of each species were assessed. For S. pseudintermedius, the chloramphenicol MIC₉₀ was 32 μg/mL. No florfenicol resistance was detected in this species (MIC_90 = 4 μg/mL). The MIC₉₀ of both agents against E. coli was 8 μg/mL. Resistance genes found were cat_pC221 in S. pseudintermedius and catA1 and/or floR in E. coli. The phenicols displayed a time‐dependent, mainly, bacteriostatic effect on both species. Prolonged PAEs were observed for S. pseudintermedius, and no PAEs were detected for E. coli. More research into determination of PK/PD targets of efficacy is needed to further assess the clinical use of chloramphenicol and florfenicol as second‐line agents in dogs, optimize dosage regimens, and set up species‐specific clinical break points.     

Medium‐chain glycerides affect gut morphology,immune‐ and goblet cells in post‐weaning piglets: In vitro fatty acid screening with Escherichia coli and in vivo consolidation with LPS challenge

The influence of medium‐chain glycerides on performance and gastrointestinal well‐being in weaning piglets was assessed. First, caproic (C6), caprylic (C8) and capric (C10) acid activity against Escherichia coli was screened in vitro. Pig flora of the whole small intestine was used as inoculum. Seven in vitro incubations were done in duplicate at pH = 3 and 5: C10 (15 mM), C8 (12 mM), C6 (15, 12, 10 mM), a non‐incubated‐negative control and incubated negative control. Culture suspensions were plated on E. coli‐selective agar. Controls showed bacterial growth. C6 and C8 showed no growth at both pH‐values, where C10 showed growth at pH = 5. Secondly, an in vivo study was done with 80 weaned piglets over 42 days, housed in pens of eight animals (five pens/treatment), fed a basal diet containing broken rice/soya bean meal/fish meal and supplemented with C6 and C8 in medium‐chain glyceride form (MCT6/8, 0.175%) or antibiotic growth promoter (AGP, 0.020%) (Kasetsart University, Thailand) serving as control. Feed intake, daily gain and feed‐to‐gain ratio did not differ between MCT6/8 and AGP. Per replicate, two random selected piglets were challenged intravenously with E. coli‐lipopolysaccharide (LPS) or saline solution (S) at Days 21 and 28. All challenged animals were sacrificed; blood and digestive tract samples (jejunum/ileum) were collected at Day 35. LPS challenge consistently reduced villus height and crypt depth for MCT6/8 and AGP. However, LPS‐challenged piglets supplemented with MCT6/8 restored villus height, where AGP did not. MCT6/8 piglets had higher serum IgA, more jejunal IgA‐positive plasma cells and goblet cells than AGP. At the ileal level, results were similar, though less pronounced. The present study offers new insight in the benefits of MCT6/8 over AGP in the post‐weaning period. There is in vitro anti‐microbial action of C6 and C8 on E. coli. In vivo, MCT6/8 also has protective effects in the small intestine that may result in growth promotion.     

In vitro heparinization of canine whole blood with low molecular weight heparin (dalteparin) significantly and dose‐dependently prolongs heparinase‐modified tissue factor‐activated thromboelastography parameters and prothrombinase‐induced clotting time

Background: Low‐molecular‐weight heparin (LMWH) is being used increasingly in veterinary medicine for both treatment and prophylaxis of thromboembolic disease, but no predictable patient‐side method exists to monitor its effect. Objectives: The aim of this study was to evaluate thromboelastography (TEG) and prothombinase‐induced clotting time (PiCT) assays for detecting hemostatic alterations following in vitro heparinization of canine whole blood with dalteparin (Fragmin). Methods: Citrated whole‐blood samples were collected from 7 clinically healthy dogs. Dalteparin was added at concentrations of 0, 0.156, 0.625, 1.25, and 2.5 U/mL of whole blood. TEG was performed using heparinase cups with tissue factor (TF, 1:50,000) and kaolin as activators. Reaction time (R), clotting time (K), angle (α), and maximum amplitude (MA) were recorded. PiCT and anti‐FXa activity were measured in plasma. Results: With TF, increasing concentrations of dalteparin significantly prolonged R and K and significantly decreased α and MA. K, α, and MA ratios were significantly different from baseline at all dalteparin concentrations and R was significantly different from baseline at concentrations of 0.625, 1.25, and 2.5 U/mL. With kaolin, only R was significantly different from baseline at dalteparin concentrations of 0.625 and 2.5 U/mL. PiCT detected dalteparin concentrations ≤ 0.625 U/mL, with a good linear correlation (r²=.96, P<.0001). Conclusion: These results suggest that TF‐activated TEG and PiCT assays should be further evaluated as promising new methods for evaluating the effect of LMWH, using doses in the recommended clinical range and prospective clinical studies.