Estimating the Sensitivity and Specificity of Real‐Time Quantitative PCR of Fecal Samples for Diagnosis of Rhodococcus equi Pneumonia in Foals          下载免费PDF全文

S.D. Shaw  N.D. Cohen  M.K. Chaffin  G.P. Blodgett  M. Syndergaard  D. Hurych 《Journal of veterinary internal medicine / American College of Veterinary Internal Medicine》2015,29(6):1712-1717

Background

Real‐time, quantitative PCR (qPCR) methods for detecting Rhodococcus equi in feces have been developed as a noninvasive, rapid diagnostic test for R. equi pneumonia, but have not been evaluated in a large population of foals.

Objective

The objective of this study was to evaluate the clinical utility of fecal PCR as a diagnostic test for R. equi pneumonia in foals using receiver operating characteristic (ROC) methods.

Animals

186 foals born in 2011 at an R. equi‐endemic ranch in Texas.

Methods

Fecal samples were collected at the time of onset of clinical signs for pneumonic foals (n = 31). Foals with pneumonia were matched by age and birth date to healthy (n = 31) and subclinical (n = 124) control foals; fecal samples were collected from these controls. DNA was extracted from feces using commercial kits and concentration of virulent R. equi in feces was determined by qPCR.

Results

Concentration of R. equi in feces differed significantly (P < .05) among groups. The area under the ROC curve for fecal qPCR for diagnosis of R. equi pneumonia was 89% (95% CI, 83–99), with a sensitivity of 94% and specificity of 72%.

Conclusions and Clinical Importance

qPCR of feces can be useful as an alternative to tracheobronchial aspiration for the diagnosis of R. equi in foals with clinical signs of pneumonia. Caution should be used in extrapolating results of this study to other populations because fecal concentration of R. equi might vary by geographic location or management practices.  相似文献   

Baicalein induction of hydroxyl radical formation via 12-lipoxygenase in human platelets: an ESR study     

Chou DS  Lee JJ  Hsiao G  Hsieh CY  Tsai YJ  Chen TF  Sheu JR 《Journal of agricultural and food chemistry》2007,55(3):649-655

The pro-oxidant activities of baicalein, morin, myricetin, quercetin, and rutin were examined in various cell-containing systems including human platelets, rat vascular smooth muscle cells, human umbilical vein endothelial cells (HUVECs), human THP-1 cells, and fibroblast cells. Electron spin resonance (ESR) results showed that only baicalein generated hydroxyl radicals in a resting human platelet suspension, whereas the other flavonoids showed no effects on any of the resting cell systems. A low concentration of arachidonic acid (AA) increased the intensity of hydroxyl radicals, but a high concentration inhibited it. Collagen and thrombin, platelet aggregatory agents that can cause the release of AA by platelets, enhanced baicalein-induced hydroxyl radical formation, whereas ADP and U44619 showed no significant effects. Quinacrine and 5,8,11,14-eicosatetraenoic trifluoromethyl ketone, both PLA2 inhibitors, significantly attenuated baicalein-induced hydroxyl radical formation. These results suggest that baicalein-induced hydroxyl radical formation is associated with AA metabolite enzymes in human platelets. The formation of hydroxyl radicals was significantly inhibited by lipoxygenase inhibitors including nordihydroguaiaretic acid, (-)-epicatechin, (-)-epicatechin gallate, and hinokitiol, but was not affected by desferroxamine or the heme protein inhibitors KCN and NaN3. On the other hand, semiquinone free radicals were generated when baicalein was incubated with horseradish peroxidase/H2O2 or platelets/AA. The semiquinone radicals formed in the platelets/AA system could be extensively inhibited by desferroxamine, diethylenetriaminepentaacetic acid, KCN, and NaN3, indicating that prostaglandin H synthase (PGHS)-peroxidase may be involved. The results of this study led to the proposal that baicalein induces hydroxyl radical formation via 12-lipoxygenase and induces semiquinone radical formation via PGHS-peroxidase in human platelets.  相似文献   

Altering the substrate specificity of Candida rugosa LIP4 by engineering the substrate-binding sites     

Lee LC  Chen YT  Yen CC  Chiang TC  Tang SJ  Lee GC  Shaw JF 《Journal of agricultural and food chemistry》2007,55(13):5103-5108

Candida rugosa (formerly Candida cylindracea) lipase (CRL) is an important industrial enzyme that is widely used in biotechnological applications such as the production of fatty acids and the synthesis of various esters. CRL comprises at least seven isozymes (LIP1-LIP7), which share a similar amino acid sequence but with different specificities for substrates. Previously, LIP4 was reported to have higher esterase activity toward long acyl-chain ester and lower lipase activity toward triglycerides. A296 and V344 of LIP4 were predicted to play decisive roles in its substrate specificity. In this study, site-specific saturation mutagenesis has been employed to study the substrate specificity of LIP4. Point mutations were separately introduced into A296 and V344 positions using degenerate primer sets containing 32 codons to generate two libraries of variants. LIP4 variants were heterologously expressed in the yeast Pichia pastoris. A specific plate assay was used to identify lipase-producing P. pastoris clones in a medium containing tributyrin. LIP4 variants with high activity toward short fatty acyl-chain triglyceride (tributyrin) were screened. Specificity analysis and biochemical characterization indicated that the recombinant variants A296I, V344Q, and V344H had properties remarkably different from those of wild-type LIP4. All three variant enzymes had significantly higher specific activities toward tributyrin than LIP4. In addition to short-chain triglyceride, A296I and V344Q also improved hydrolytic activities of triglycerides toward medium- and long-chain triglycerides tested. The results suggested that A296 played an important role in lipase activity and high-temperature dependence of LIP4, whereas it had no effect on the chain-length specificity in lipolytic reaction. The V344 residue had a significant effect on the substrate chain-length specificity of LIP4.  相似文献   

Improved HPLC-MS/MS method for determination of isoxaflutole (balance) and its metabolites in soils and forage plants     

Lin CH  Lerch RN  Garrett HE  Li YX  George MF 《Journal of agricultural and food chemistry》2007,55(10):3805-3815

A robust multi-residue procedure is needed for the analysis of the pro-herbicide isoxaflutole and its degradates in soil and plant materials at environmentally relevant (<1 microg kg-1) levels. An analytical method using turbo-spray and heat-nebulizer high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) was developed for the analysis of isoxaflutole (IXF) and its two metabolites, diketonitrile (DKN) and the benzoic acid metabolite (BA), at sub-microgram per kilogram levels in soil and plant samples. The average recoveries of the three compounds in spiked soil and plant samples ranged from 84 to 110% and 94 to 105%, respectively. The limits of quantification were validated at 0.06 microg kg-1 for soil and 0.3 microg kg-1 for plant samples. The limits of detection (LOD) for soil analysis were 0.01, 0.002, and 0.01 microg kg-1 for IXF, DKN, and BA, respectively. Corresponding LOD for the plant analysis method were 0.05, 0.01, and 0.05 microg kg-1. The developed method was validated using forage grass and soil samples collected from a field lysimeter study in which IXF was applied to each of four forage treatments. Forage plants and soils were sampled for analyses 25 days after IXF application to the soil. In soils, IXF was not detected in any treatment, and DKN was the predominant metabolite found. In forage plants, the concentrations of DKN and BA were 10-100-fold higher than that in soil samples, but IXF was not detected in any forage plants. The much higher proportion of BA to DKN in plant tissues (23-53%), as compared to soils (0-5%), suggested that these forages were capable of detoxifying DKN. The developed methods provided LODs at sub-microgram per kilogram levels to determine the fate of IXF and its metabolites in soils and forage plants, and they also represent considerable improvements in extraction recovery rates and detection sensitivity as compared to previous analytical methods for these compounds.  相似文献   

Effect of dips in a 1-methylcyclopropene-generating solution on 'Harrow Sun' plums stored under different temperature regimes     

Manganaris GA  Vicente AR  Crisosto CH  Labavitch JM 《Journal of agricultural and food chemistry》2007,55(17):7015-7020

The effect of postharvest dips in a 1-methylcyclopropene-generating solution of the formulation AFxRD-038 (Rohm & Haas) on plum fruit (Prunus salicina Lindell cv. 'Harrow Sun') quality and ripening during storage was determined. Fruit weight loss, tissue firmness, soluble solids content (SSC), titratable acidity (TA), ethylene production, respiration, and the activities of the cell wall modifying enzymes polygalacturonase (PG), 1,4-beta-D-glucanase/glucosidase (EGase), beta-galactosidase (beta-gal), and pectin methylesterase (PME) were measured. Fruit reddening, anthocyanin content, and phenylalanine ammonia-lyase (PAL) activity were also analyzed. The 1-MCP-treated fruit showed reduced ethylene production and respiration rate and delayed softening, which was associated with the reduction in the activity of PG, EGase, and beta-gal. The immersion in 1-MCP-generating solutions also decreased weight and acidity loss without modifying the fruit SSC. The immersion treatment was particularly effective in the fruit stored at 5 degrees C, keeping higher overall quality, maintaining lower levels of anthocyanins and PAL activity, and preventing flesh reddening. The present data show that beneficial effects in delaying plum fruit ripening and controlling chilling injury can be obtained by dipping the fruits in a solution of this novel 1-MCP-generating formulation.  相似文献   

Determination of anabolic steroids in muscle tissue by liquid chromatography-tandem mass spectrometry   总被引：1，自引：0，他引：1  

Kaklamanos G  Theodoridis G  Papadoyannis IN  Dabalis T 《Journal of agricultural and food chemistry》2007,55(21):8325-8330

A specific and sensitive method based on liquid chromatography-tandem mass spectrometry using atmospheric pressure chemical ionization (LC-APCI-MS/MS) has been developed for the determination of four anabolic steroids [trenbolone, methylboldenone, methyltestosterone, and norethandrolone] in bovine muscle. Methyltestosterone- d 3 was used as internal standard. The procedure involved enzymatic hydrolysis, extraction with tert-butyl methyl ether, defattening, and final cleanup with solid-phase extraction with Oasis HLB cartridges. The analytes were analyzed by reversed-phase LC-MS/MS, acquiring two diagnostic product ions from the chosen precursor [M + H] (+) for the unambiguous confirmation of hormones. The method was validated according to the European Commission Decision 2002/657/EC for the detection and confirmation of residues in products of animal origin. The limits of detection (LOD) and limits of quantitation (LOQ) were found to be 0.3 ng/g and 1.0 ng/g, respectively. The accuracy and precision have been determined, with recoveries ranging from 83% to 104% and the CV factor not exceeding the value of 7%. The decision limits CCalpha were calculated and ranged from 0.05 to 0.15 ng/g while the detection capabilities CCbeta ranged from 0.09 to 0.25 ng/g. The method proved to be sensitive and reliable and thus renders an appropriate means for residue analysis studies.  相似文献   

Autoimmune hepatitis-specific antibodies against soluble liver antigen and liver cytosol type 1 in patients with chronic viral hepatitis     

Eirini I Rigopoulou  Maria Mytilinaiou  Ourania Romanidou  Christos Liaskos  George N Dalekos 《Journal of Autoimmune Diseases》2007,4(1):2

  相似文献   

Synthesis and characterization of canola oil-stearic acid-based trans-free structured lipids for possible margarine application     

Lumor SE  Jones KC  Ashby R  Strahan GD  Kim BH  Lee GC  Shaw JF  Kays SE  Chang SW  Foglia TA  Akoh CC 《Journal of agricultural and food chemistry》2007,55(26):10692-10702

Incorporation of stearic acid into canola oil to produce trans-free structured lipid (SL) as a healthy alternative to partially hydrogenated fats for margarine formulation was investigated. Response surface methodology was used to study the effects of lipozyme RM IM from Rhizomucor miehei and Candida rugosa lipase isoform 1 (LIP1) and two acyl donors, stearic acid and ethyl stearate, on the incorporation. Lipozyme RM IM and ethyl stearate gave the best result. Gram quantities of SLs were synthesized using lipozyme RM IM, and the products were compared to SL made by chemical catalysis and fat from commercial margarines. After short-path distillation, the products were characterized by GC and RPHPLC-MS to obtain fatty acid and triacylglycerol profiles, 13C NMR spectrometry for regiospecific analysis, X-ray diffraction for crystal forms, and DSC for melting profile. Stearic acid was incorporated into canola oil, mainly at the sn-1,3 positions, for the lipase reaction, and no new trans fatty acids formed. Most SL products did not have adequate solid fat content or beta' crystal forms for tub margarine, although these may be suitable for light margarine formulation.  相似文献   

Effect of protein source and level on growth and performance of the captive freshwater prawn,Macrobrachium rosenbergii     

George H. Balazs  Ernest Ross 《Aquaculture (Amsterdam, Netherlands)》1976,7(4):299-313

Growth, feed conversion and survival were determined for juvenile Macrobrachium rosenbergii held in tanks under semi-controlled environmental conditions. Feeding trials incorporated water-stable diets at three levels of protein (15, 25 and 35%). The principal protein source combinations consisted either of soybean and tuna meal, or of soybean, tuna and shrimp meal. In a 244-day comparison of these diets, higher protein content produced larger prawns (P < 0.01), but differences between sources were not significant. No significant differences existed between feed conversion ratios (range 1.36–1.72) or percentage survival (range 90.3–93.6%). Trials of several other diets were also conducted, including soybean and Tilapia meal, and copra and Tilapia meal (25% protein level) as principal protein source combinations. After 167 days on these diets, growth was inferior to that obtained with soybean and tuna meal or soybean, tuna and shrimp meal combinations. No significant differences existed between feed conversion ratios or percentages of survival.For the 244 days, a control group of prawns received no formulated diet. Growth and survival in this group during the first 110 days suggested that naturally occurring algae contributed substantially to the prawns' nutrition.Mean prawn length after 244 days on the best diet (35% protein from soybean and tuna meal) was 73 mm, and growth rate was equivalent to that achieved under pilot pond conditions.  相似文献   

The Chinese grass carp, Ctenopharyngodon idella, its biology,introduction, control of aquatic macrophytes and breeding in the Sudan     

T.T. George 《Aquaculture (Amsterdam, Netherlands)》1982,27(3):317-327

The paper describes the distribution and biology of the grass carp following its introduction into Sudan. It provides aspects on the culture of the grass carp and its breeding under Sudanese conditions with emphasis on future research.  相似文献