This study was undertaken to develop new analytical methods for assessment of anticoccidials. High-performance liquid chromatography (HPLC) was found to be a fast, reliable, and practical method. The anticoccidials used in this experiment were toltrazuril and diclazuril, and the analysis factors were specificity, linearity, accuracy, repeatability, and intermediate precision. The linearity of each anticoccidial was better than 0.99, and the accuracies were 99.5% and 99.1% with relative SD of 0.5 and 0.4, respectively. To assess whether the developed HPLC method could be effectively applied, toltrazuril and diclazuril post-market veterinary products (five products) that are currently sold were tested. The results revealed no non-compliant items and the method was applied successfully. Therefore, the newly developed HPLC method for anticoccidial assessment described in this study may be useful as a reference method in the Korean Standards of Veterinary Pharmaceuticals for the analysis of toltrazuril and diclazuril.
Chromatography, High Pressure Liquid ; Chromatography, Liquid* ; Coccidiostats ; Methods* ; Sensitivity and Specificity ; Veterinary Drugs

Escherichia coli (E. coli) strains were collected from young diarrheic calves in farms and field. Strains that expressed the K99 (F5) antigen were identified by agglutination tests using reference antibodies to K99 antigen and electron microscopy. The K99 antigen from a selected field strain (SAR-14) was heat-extracted and fractionated on a Sepharose CL-4B column. Further purification was carried out by sodium deoxycholate treatment and/or ion-exchange chromatography. Monoclonal antibodies to purified K99 antigen were produced by the hybridoma technique, and a specific clone, NEK99-5.6.12, was selected for propagation in tissue culture. The antibodies, thus obtained, were affinity-purified, characterized and coated onto Giemsastained Cowan-I strain of Staphylococcus aureus (S. aureus). The antibody-coated S. aureus were used in a coagglutination test to detect K99+ E. coli isolated from feces of diarrheic calves. The specificity of the test was validated against reference monoclonal antibodies used in co-agglutination tests, as well as in ELISA. Specificity of the monoclonal antibodies was also tested against various Gram negative bacteria. The developed antibodies specifically detected purified K99 antigen in immunoblots, as well as K99+ E. coli in ELISA and co-agglutination tests. The co-agglutination test was specific and convenient for large-scale screening of K99+ E. coli isolates.
Agglutination Tests/methods/*veterinary ; Animals ; *Animals, Newborn ; Antibodies, Monoclonal/*immunology ; Antigens, Surface/immunology/isolation & purification ; Bacterial Toxins/immunology/isolation & purification ; Cattle ; Cattle Diseases/*immunology/*microbiology ; Chromatography, Gel/veterinary ; Chromatography, Ion Exchange/veterinary ; Chromatography, Liquid/veterinary ; Diarrhea/immunology/*veterinary ; Electrophoresis, Polyacrylamide Gel/veterinary ; Enzyme-Linked Immunosorbent Assay/veterinary ; Escherichia coli/*immunology ; Escherichia coli Infections/immunology/*veterinary ; Immunoblotting/veterinary ; Staphylococcus aureus

The present study was planned to evaluate the bioequivalence of two commercial formulations of enrofloxacin, which have been marketed as 10% injectable solution after intramuscular administration at a single dose of 2.5 mg/kg body weight to 12 clinically healthy goats The study was carried out on the basis of crossover design. The two formulations were: Baytril as a reference product and Spectrama Vet as a test product. The plasma concentrations of enrofloxacin were measured by high performance liquid chromatography (HPLC) with UV detector. The pharmacokinetics of that data was performed using non-compartmental analysis. The maximum plasma concentration (Cmax), time to reach peak concentration (Tmax), area under concentration-time curve (AUC), elimination half-life (t0.5el) were 1.14 and 1.05 microg/mL, 0.79 and 0.83 h, 5.70 and 5.79 microg.h/mL, 5.19 and 5.39 h for Baytril and Spectrama Vet, respectively. The 90% confidence interval for the mean ratio of Tmax, Cmax and AUC were 94.72~116.2, 87.88~97.16 and 86.44~118.72%, respectively. These values falls within the European Medicines Agency bioequivalence acceptance range of 80~125% for both Tmax and AUC and between 75~133% for Cmax. In conclusion, Spectrama-Vet is bioequivalent to Baytril and both products can be used as interchangeable drug in veterinary medicine practice.
Area Under Curve ; Body Weight ; Chromatography, High Pressure Liquid ; Chromatography, Liquid ; Confidence Intervals ; Cross-Over Studies ; Fluoroquinolones ; Goats ; Half-Life ; Plasma ; Therapeutic Equivalency ; Veterinary Medicine

Despite the development of new technologies, new challenges still remain for large scale proteomic profiling when dealing with complex biological mixtures. Fractionation prior to liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis is usually the preferred method to reduce the complexity of any biological sample. In this study, a gel LC-MS/MS approach was used to explore the stage specific proteome of Cryptosporidium (C.) parvum. To accomplish this, the sporozoite protein of C. parvum was first fractionated using SDS-PAGE with subsequent LC-MS/MS analysis. A total of 135 protein hits were recorded from 20 gel slices (from same gel lane), with many hits occurring in more than one band. Excluding all non-Cryptosporidium entries and proteins with multiple hits, 33 separate C. parvum entries were identified during the study. The overall goal of this study was to reduce sample complexity by protein fractionation and increase the possibility of detecting proteins present in lower abundance in a complex protein mixture.
Chemical Fractionation/methods ; Chromatography, Liquid/methods/veterinary ; Cryptosporidium parvum/*chemistry/growth & development/metabolism ; Electrophoresis, Polyacrylamide Gel/methods/veterinary ; Gene Expression Profiling/*methods/veterinary ; Proteome/analysis ; Proteomics/*methods ; Protozoan Proteins/*analysis ; Sporozoites/chemistry/metabolism ; Tandem Mass Spectrometry/methods/veterinary

Autoantibodies against thyroxin (T4AA) and triiodothyronine (T3AA) are present in dogs with autoimmune thyroiditis and have been reported to interfere with immunoassays. The objectives of this study were to determine the frequency of autoantibodies and to determine whether interference occurs by T4AA, using a non-immunological method (high performance liquid chromatography, HPLC) for thyroxin (T4) measurement. Based on clinical symptoms, T4 and thyroid stimulating hormone (TSH) concentration, 1,339 dogs were divided into six groups: Group 1: hypothyroid (n = 149); Group 2: subclinical thyroiditis (n = 110); Group 3: suspicious for non thyroidal illness (n = 691); Group 4: biochemical euthyroid (n = 138); Group 5: hypothyroid dogs under substitution therapy (n = 141); Group 6: healthy dogs (n = 110). The incidence of T4AA and T3AA, determined using radiometric assay, was low (0.5% and 3.8%) and higher in hypothyroid dogs compared to dogs suspicious for hypothyroidism (Group 2-4) (p<0.05). T4AA was not detected in dogs with normal T4 and elevated TSH. T4 concentrations of T4AA positive samples determined using HPLC were comparable to results obtained by chemiluminescence immunoassay. These findings indicate that the probability of interference of T4AA leading to falsely elevated T4 concentration in the T4 assay seems to be low.
Animals ; Autoantibodies/*immunology ; Chemiluminescent Measurements/methods/*veterinary ; Chromatography, High Pressure Liquid/veterinary ; Dog Diseases/*diagnosis/*immunology ; Dogs ; Immunoassay/methods/*veterinary ; Thyroid Hormones/*immunology ; Thyroiditis, Autoimmune/diagnosis/immunology/*veterinary ; Thyroxine/*blood

A study of amoxicillin pharmacokinetics was conducted in healthy goats and goats with chronic lead intoxication. The intoxicated goats had increased serum concentrations of liver enzymes (alanine aminotransferase and gamma-glutamyl transferase), blood urea nitrogen, and reactivated delta-aminolevulinic acid dehydratase compared to the controls. Following intravenous amoxicillin (10 mg/kg bw) in control and lead-intoxicated goats, elimination half-lives were 4.14 and 1.26 h, respectively. The volumes of distribution based on the terminal phase were 1.19 and 0.38 L/kg, respectively, and those at steady-state were 0.54 and 0.18 L/kg, respectively. After intramuscular (IM) amoxicillin (10 mg/kg bw) in lead-intoxicated goats and control animals, the absorption, distribution, and elimination of the drug were more rapid in lead-intoxicated goats than the controls. Peak serum concentrations of 21.89 and 12.19 microg/mL were achieved at 1 h and 2 h, respectively, in lead-intoxicated and control goats. Amoxicillin bioavailability in the lead-intoxicated goats decreased 20% compared to the controls. After amoxicillin, more of the drug was excreted in the urine from lead-intoxicated goats than the controls. Our results suggested that lead intoxication in goats increases the rate of amoxicillin absorption after IM administration and distribution and elimination. Thus, lead intoxication may impair the therapeutic effectiveness of amoxicillin.
Amoxicillin/blood/*pharmacokinetics/urine ; Animals ; Anti-Bacterial Agents/blood/*pharmacokinetics/urine ; Area Under Curve ; Chromatography, High Pressure Liquid/veterinary ; Goat Diseases/*chemically induced/metabolism ; Goats ; Half-Life ; Injections, Intramuscular/veterinary ; Injections, Intravenous/veterinary ; Lead Poisoning/etiology/metabolism/*veterinary ; Male

Macrolides are frequently used in veterinary medicine as therapeutic and preventive agents for various diseases. It is difficult to determine macrolides simultaneously with conventional methods due to their similar structures. A simultaneous analysis for erythromycin, roxithromycin, tiamulin and tylosin with LC/MS has been developed. Separation was performed on C18 reversed phase column. Mobile phase was gradiently flowed with 10 mM ammonium acetate and methanol. The mass spectrometer was run in the positive mode and selective ion monitoring mode. The molecular ions were [M+H]+ form at m/z 837.5 for erythromycin, at m/z 859.5 for roxithromycin, at m/z 494.2 for tiamulin and at m/z 916.7 for tylosin. Limits of detection were in the range from 0.001 to 0.01 microgram/g lower than their MRLs.
Anti-Bacterial Agents/*analysis ; Chromatography, Liquid/*methods ; Diterpenes/*analysis ; Erythromycin/*analysis ; Mass Spectrometry/*methods ; Molecular Structure ; Roxithromycin/*analysis ; Sensitivity and Specificity ; Tylosin/*analysis ; Veterinary Medicine

Ofloxacin was administered to six male goats intravenously (5 mg/kg) to determine its kinetic behavior, tissue residue, in vitro plasma protein binding and to compute a rational dosage regimen. The concentration of ofloxacin in plasma and tissue samples collected at prescheduled time were estimated by using HPLC. The pharmacokinetic parameters were determined by non-compartmental model and plasma protein binding was estimated by equilibrium dialysis technique. The therapeutic concentration (> or = 0.5 microgram/ml) was maintained up to 36 h and the initial concentration at 2.5min (14.76 +/- 0.47 microgram/ml) declined to 0.05 +/- 0.03 microgram/ml at 96 h with a secondary peak (0.64 +/- 0.15 microgram/ml) at 24 h. The mean AUC, AUMC, t1/2, MRT, Cl and Vd were calculated to be 58.94 +/- 19.43 microgram h/ml, 1539.57 +/- 724.69 microgram h2/ml, 15.58 +/- 1.87 h, 22.46 +/- 2.71 h, 135.60 +/- 31.12 ml/h/kg and 2.85 +/- 0.74 L/kg respectively. Significantly high concentration of drug was detected in different tissues after 24 h of intravenous dosing of 5mg/kg, at 24 h interval for 5 days. The in vitro plasma protein binding of ofloxacin was found to be 15.28 +/- 0.94%. Based on these kinetic parameters, a loading dose of 5mg/kg followed by the maintenance dose of 3mg/kg at 24 h dosing interval by intravenous route is recommended.
Animals ; Anti-Infective Agents/*pharmacokinetics ; Blood Proteins/*metabolism ; Chromatography, High Pressure Liquid/veterinary ; Goats/*metabolism ; Male ; Ofloxacin/*pharmacokinetics ; Protein Binding ; Tissue Distribution

An improved method to determine meloxicam (MEL) concentrations in koala plasma using reversed phase high performance liquid chromatography equipped with a photo diode array detector was developed and validated. A plasma sample clean-up step was carried out with hydrophilic-lipophilic copolymer solid phase extraction cartridges. MEL was separated from an endogenous interference using an isocratic mobile phase [acetonitrile and 50 mM potassium phosphate buffer (pH 2.15), 45:55 (v:v)] on a Nova-Pak C18 4-microm (300 x 3.9 mm) column. Retention times for MEL and piroxicam were 8.03 and 5.56 min, respectively. Peak area ratios of MEL to the internal standard (IS) were used for regression analysis of the calibration curve, which was linear from 10 to 1,000 ng/mL (r2 > 0.9998). Average absolute recovery rates were 91% and 96% for MEL and the IS, respectively. This method had sufficient sensitivity (lower quantitation limit of 10 ng/mL), precision, accuracy, and selectivity for routine analysis of MEL in koala plasma using 250-microL sample volumes. Our technique clearly resolved the MEL peak from the complex koala plasma matrix and accurately measured MEL concentrations in small plasma volumes.
Animals ; Anti-Inflammatory Agents, Non-Steroidal/*blood ; Chromatography, High Pressure Liquid/methods/*veterinary ; Molecular Structure ; Phascolarctidae/*blood ; Piroxicam/chemistry ; Quality Control ; Reproducibility of Results ; Sensitivity and Specificity ; Thiazines/*blood ; Thiazoles/*blood

An improved method to determine meloxicam (MEL) concentrations in koala plasma using reversed phase high performance liquid chromatography equipped with a photo diode array detector was developed and validated. A plasma sample clean-up step was carried out with hydrophilic-lipophilic copolymer solid phase extraction cartridges. MEL was separated from an endogenous interference using an isocratic mobile phase [acetonitrile and 50 mM potassium phosphate buffer (pH 2.15), 45:55 (v:v)] on a Nova-Pak C18 4-microm (300 x 3.9 mm) column. Retention times for MEL and piroxicam were 8.03 and 5.56 min, respectively. Peak area ratios of MEL to the internal standard (IS) were used for regression analysis of the calibration curve, which was linear from 10 to 1,000 ng/mL (r2 > 0.9998). Average absolute recovery rates were 91% and 96% for MEL and the IS, respectively. This method had sufficient sensitivity (lower quantitation limit of 10 ng/mL), precision, accuracy, and selectivity for routine analysis of MEL in koala plasma using 250-microL sample volumes. Our technique clearly resolved the MEL peak from the complex koala plasma matrix and accurately measured MEL concentrations in small plasma volumes.
Animals ; Anti-Inflammatory Agents, Non-Steroidal/*blood ; Chromatography, High Pressure Liquid/methods/*veterinary ; Molecular Structure ; Phascolarctidae/*blood ; Piroxicam/chemistry ; Quality Control ; Reproducibility of Results ; Sensitivity and Specificity ; Thiazines/*blood ; Thiazoles/*blood