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  • Author or Editor: José Cerón x
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Abstract

Objective—To develop and validate a time-resolved immunofluorometric assay (TR-IFMA) for measurement of C-reactive protein (CRP) in canine whole blood.

Animals—12 healthy dogs and 35 dogs with inflammatory processes.

Procedure—CRP was isolated from acute-phase serum by affinity chromatography and used as a standard for calibration. Analytic and functional limit of detection and intra-assay and interassay precision were calculated. Accuracy was evaluated by recovery assays and by comparison with results of a commercial ELISA. Correlation between CRP concentrations in whole blood and corresponding plasma fractions was tested by use of TR-IFMA. Stability of blood samples at 4°C was assessed during a 1-month period, and effects of anticoagulants were evaluated. Measurements of CRP in blood samples from 12 healthy dogs were compared with those of 35 dogs with inflammatory diseases.

Results—Analytic and functional limits of detection were 0.53 and 3.26 µg/mL, respectively. Intra-assay and interassay coefficients of variation varied between 2.1% to 8.9% and 8.0% to 12.3%, respectively. Mean recoveries of added CRP were 104% and 114%. Measurements of CRP by use of TR-IFMA and ELISA were highly correlated (R 2 = 0.97). Measurements of CRP in whole blood and in corresponding plasma fractions by use of TR-IFMA were also highly correlated (R 2 = 0.97). Neither storage nor use of anticoagulants disturbed measurement of CRP concentrations in whole blood. Concentrations of CRP in whole blood of dogs with inflammation were significantly higher than in healthy dogs.

Conclusions and Clinical Relevance—Determination of CRP concentrations in whole blood may provide a diagnostic test for inflammation in dogs. ( Am J Vet Res 2005;66:62–66)

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in American Journal of Veterinary Research

Abstract

Objective—To evaluate changes in serum concentrations of acute-phase proteins in dogs with leishmaniosis during short-term therapy in accordance with 2 treatment protocols and determine whether concentrations of acute-phase proteins could be used to monitor the initial response of dogs to treatment.

Animals—12 dogs naturally infected with Leishmania infantum.

Procedure—Dogs were allocated into 2 groups. Dogs of group 1 were treated by use of meglumine antimonate (100 mg/kg, SC, q 24 h) administered concurrently with allopurinol (15 mg/kg, PO, q 12 h) for 20 days and then with allopurinol alone at the same dosage for the subsequent 30 days. Dogs of group 2 were treated by administration of allopurinol alone (15 mg/kg, PO, q 12 h) for 60 days). Blood samples were obtained before and during treatment for measurement of serum concentrations of acute-phase proteins and determination of CBC counts, serum biochemical analyses, and electropherograms.

Results—All dogs evaluated in the study had increased concentrations of C-reactive protein, haptoglobin, and ceruloplasmin at the time of diagnosis of leishmaniosis. Mean concentration of serum amyloid A before treatment was also increased, but some of the dogs had concentrations of serum amyloid A that were within the reference range. Concentrations of C-reactive protein and ceruloplasmin decreased significantly in all dogs at the end of the study period.

Conclusions and Clinical Relevance—Measurement of concentrations of selected acute-phase proteins, such as C-reactive protein or ceruloplasmin, could be used to evaluate the initial response of dogs with leishmaniosis to treatment. (Am J Vet Res 2003;64:102–1026)

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in American Journal of Veterinary Research

Abstract

Objective—To evaluate changes in stability of haptoglobin and C-reactive protein (CRP) concentrations caused by freezing of saliva and meat juice samples.

Animals—16 specific-pathogen-free pigs and 16 pigs with clinical signs of disease.

Procedures—Saliva and diaphragmatic muscle were collected immediately before and after slaughter, respectively. Haptoglobin and CRP concentrations of pooled samples were measured before storage (day 0) and after 7, 15, 30, 60, 120, 210, and 365 days of storage at −20°C and after repeated freezing-thawing cycles (up to 7 times). In a second experiment, addition of a protease-inhibitor cocktail to saliva and storage of saliva samples at −80°C for up to 30 days were assessed for effects on CRP concentrations.

Results—Haptoglobin concentrations in saliva did not change for up to 120 days in samples stored at −20°C, but longer storage times and multiple freezing-thawing cycles increased haptoglobin concentrations. Salivary CRP concentrations decreased significantly after 7 days of storage at −20°C, and addition of a protease-inhibitor cocktail did not improve CRP stability. Lower temperatures limited salivary CRP degradation. In meat juice, haptoglobin and CRP concentrations were stable at −20°C up to 210 days.

Conclusions and Clinical Relevance—Acute-phase protein measurements in saliva should be performed as soon as possible after sample collection. When this is not possible, storage temperature of −80°C is recommended. Acute-phase protein concentrations appeared to be more stable in meat juice samples than in saliva samples. Saliva and meat juice could be used as alternatives to serum for haptoglobin and CRP analysis.

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in American Journal of Veterinary Research

Abstract

Objective—To evaluate 5 commercially available ELISAs for determination of leptin concentrations in serum samples from dogs.

Sample Population—Serum samples from overweight-obese and thin–ideal weight clientowned dogs.

Procedures—Serum samples with high and low leptin concentrations (n = 7 samples each) were used for validation of the assays. Intra- and interassay precision, linearity under dilution, spiking recovery, and limit of quantification were determined. In addition, leptin concentrations in thin–ideal weight (n = 8) and overweight-obese (37) dogs were quantified.

Results—Use of 2 of the 5 ELISAs (A and B) revealed reactivity with canine leptin. Intra-and interassay coefficients of variation were < 6.1% and 76%, respectively, for assay A and 14.0% and 13.7%, respectively, for assay B. In assays A and B, dilutions of canine serum pools were used to determine linear regression equations. Recoveries were 77% to 101% for assay A and 67% to 125% for assay B. Significant differences in leptin concentrations between thin–ideal weight and overweight-obese dogs were detected only when analyzed with assay A.

Conclusions and Clinical Relevance—Among 5 leptin ELISAs evaluated, a canine-specific leptin ELISA had adequate precision, linearity, and ability to discriminate between high and low leptin concentrations corresponding to overweight-obese and thin–ideal weight dogs, respectively.

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in American Journal of Veterinary Research

Abstract

Objective—To develop and evaluate an immunoassay based on time-resolved immunofluorometry (TR-IFM) for measurement of haptoglobin concentrations in samples of various body fluids of swine.

Animals—20 pigs without clinical signs of disease and seronegative for antibodies against major viruses that affect pigs and 30 pigs with clinical signs of disease.

Procedures—Haptoglobin concentrations were measured in samples of serum, saliva, and meat juice obtained from both groups of pigs to evaluate the ability of TR-IFM to differentiate between healthy and diseased pigs. Performance of TR-IFM was evaluated by means of its calibration curve and detection limit, analytic precision during routine operation, and linearity of results for serial dilutions for the 3 types of samples. In addition, performance of TR-IFM was compared with that of a commercial spectrophotometric assay.

Results—The TR-IFM assay involved only 1 step, and the results were obtained in 20 minutes, with good analytic sensitivity and reproducibility. The analytic limit of detection was 0.52 ng/mL. Intra-assay and interassay coefficients of variation ranged from 1.13% to 4.81% and 5.97% to 13.57%, respectively. The method yielded linear results for all sample types. Serum haptoglobin concentrations determined by use of TR-IFM and spectrophotometric assays were highly correlated (r = 0.96). Differences between healthy and diseased pigs with respect to median haptoglobin concentrations were significant for all types of samples.

Conclusions and Clinical Relevance—The 1-step TR-IFM assay accurately quantified haptoglobin concentrations in serum, saliva, and meat juice samples from swine and may be useful in laboratory and meat inspection settings.

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in American Journal of Veterinary Research

Abstract

Objective—To evaluate and validate 3 spectrophotometric assays for measuring serum activity of paraoxonase type-1 (PON1), an enzyme associated with high-density lipoproteins, in dogs.

Animals—22 healthy adult dogs and 10 dogs with eccentrocytosis.

Procedures—2 methods were adapted for use in 96-well microplates with phenyl acetate and 5-thiobutyl butyrolactonase as substrates, and 1 was adapted for use in an automated analyzer with p-nitrophenyl acetate as substrate. Blood samples were collected from all dogs, serum was harvested, and serum PON1 activity was measured with each method.

Results—Imprecision was low for all 3 methods, with the exception of interassay imprecision for 5-thiobutyl butyrolactonase, and results were linear across serial sample dilutions. The 3 methods were able to detect low PON1 activity when EDTA was used for blood sample collection, yielded lower PON1 values in sick dogs with eccentrocytosis than in healthy dogs, and yielded highly correlated results.

Conclusions and Clinical Relevance—The methods described here may allow a wider use of PON1 activity as a biomarker of oxidative stress in dogs in clinical and research settings. Results of each method were robust and precise (with the exception of the interassay values for the lactonase method), and the methods were easy to set up in a laboratory.

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in American Journal of Veterinary Research

Abstract

Objective—To evaluate the use of EDTA tubes for collection of blood samples for assays of secondary hemostasis in dogs.

Animals—108 dogs of various ages, breeds, and sexes (19 healthy and 89 with abnormalities of secondary hemostasis).

Procedures—Blood samples were collected via cephalic venipuncture and transferred to sodium citrate tubes and EDTA tubes. Plasma was harvested from each type of tube for assays of concentrations of fibrinogen and D-dimer as well as prothrombin time, activated partial thromboplastin time, and antithrombin activity. Intra-assay and interassay precision and correlation coefficients for all hemostatic tests were calculated for each type of plasma sample. The effect of storage conditions on assay results for the 2 types of plasma samples was also evaluated.

Results—Results of hemostatic tests were highly correlated between citrated and EDTA-treated plasma samples. Intra-assay imprecision for all hemostatic tests with the exception of D-dimer concentration was < 10% for both citrated and EDTA-treated plasma samples; interassay imprecision was higher for EDTA-treated versus citrated plasma samples. Storage of plasma samples for 1 hour did not result in significantly different assay results for either type of plasma sample, but storage for 2 hours significantly affected values for EDTA-treated plasma samples.

Conclusions and Clinical Relevance—Although evaluation of the sensitivity and specificity of hemostatic tests that use EDTA-treated plasma samples is required, EDTA may be a suitable alternative to sodium citrate as an anticoagulant for use in hemostatic testing in conditions in which tests could be performed within 1 hour after sample collection.

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in American Journal of Veterinary Research

Abstract

Objective—To determine serum concentrations of the selected acute-phase proteins (APPs) haptoglobin, serum amyloid A (SAA), and C-reactive protein (CRP) in pigs experimentally inoculated with classical swine fever (CSF) and African swine fever (ASF) viruses.

Animals—8 crossbred (Large White × Landrace) 10-week-old pigs.

Procedures—Pigs were allocated to 2 groups (4 pigs/group). One group was inoculated with the CSF virus Alfort 187 strain, whereas the other groupwas inoculated with the ASF virus Spain 70 isolate. Blood samples were collected at various time points. At the end of the study, pigs were euthanized and a complete necropsy was performed, including histologic and immunohistochemical analyses.

Results—Serum concentrations of APPs increased in pigs inoculated with CSF and ASF viruses, which suggested an acute-phase response in the course of both diseases. The most noticeable increase in concentration was recorded for SAA in both groups (up to a 300-fold increase for CSF virus and an approx 40-fold increase for ASF virus), followed by CRP and then haptoglobin, which each had only 3- to 4-fold increases.

Conclusions and Clinical Relevance—Serum concentrations of APPs increased significantly in pigs inoculated with CSF and ASF viruses. However, differences were evident in serum concentrations of the proteins evaluated in this study.

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in American Journal of Veterinary Research