In a previous study,1 Bacteroides fragilis strains isolated from horses with pleuropneumonia were shown to have in vitro susceptibility to clindamycin, imipenem, and metronidazole, suggesting that these drugs might be considered for treatment of horses with pleuropneumonia caused by Bacteroides spp. However, administration of lincomycin, which is in the same antimicrobial class (lincosamides) as clindamycin, should be avoided in horses because of an associated high risk of severe, possibly fatal, diarrhea.2,3 Also, although metronidazole is considered standard treatment for horses with anaerobic bacterial pleuropneumonia,4 how well metronidazole concentrates in the pleural fluid of horses has not yet been determined. Imipenem combined with cilastatin in a single formulation has been used for the treatment of people with anaerobic bacterial infections.5 However, imipenem is metabolized in the kidneys by dehydropeptidase I and can be nephrotoxic. Combining imipenem with cilastatin, a potent but reversible inhibitor of dehydropeptidase I, decreases the metabolism of imipenem and reduces the risk of imipenem-induced nephrotoxicosis.6 Still, how well imipenem concentrates in the pleural fluid of horses has not yet been determined, and little information on the pharmacokinetics of imipenem in horses has been published.7
The present study was designed to obtain additional information about possible use of metronidazole or imipenem for the treatment of horses with pneumonia or pleuropneumonia caused by Bacteroides spp. Specifically, the objective of the study reported here was to determine the pharmacokinetics of metronidazole and imipenem in healthy horses following administration of a single dose PO (metronidazole) or IV (imipenem). A computer simulation was then performed to estimate pleural fluid concentrations of metronidazole and imipenem following multiple dosing to determine whether concentrations obtained would be likely to inhibit growth of Bacteroides spp in the pleural cavity of horses, as determined on the basis of MICs for clinical isolates.
Materials and Methods
Animals
Four healthy 3-year-old Thoroughbreds (2 stallions and 2 mares) that had a median body weight of 470.5 kg (range, 450 to 480 kg) were used. The horses belonged to the Japan Racing Association and were housed indoors in individual stalls during the experiments with ad libitum access to water. Feed was withheld from all horses from 12 hours before to 2 hours after antimicrobial administration. Otherwise, the horses had ad libitum access to grass hay. The study was approved by the Animal Care and Use Committee of the Japan Racing Association Ritto Training Center Equine Hospital (protocol No. 12 303 T2 01 00106).
Study protocol
General design—A randomized crossover study design was used, with a washout period of 4 weeks between treatments. Injections, plasma and pleural fluid sample collection, and clinical monitoring were performed by 2 investigators (TK and NT). All horses were evaluated for signs of adverse effects (eg, diarrhea and anorexia) at 5, 10, 20, 30, 40, and 50 minutes and 1, 1.25, 1.5, 2, 3, 4, 6, 8, 12, and 24 hours after administration of each antimicrobial. For each horse, the abdomen was auscultated for borborygmi and fecal consistency and appetites were observed.
Administration of metronidazole and imipenem—Metronidazole tabletsa equivalent to 15 mg/kg were crushed, dissolved in 500 mL of water, and administered once through a nasogastric tube. The tube was then immediately flushed with 500 mL of water. A solution of imipenem and cilastatinb was administered IV at a dose of 10 mg of imipenem/kg. The calculated amount of solution was added to 500 mL of sterile saline (0.9% NaCl) solution and injected into the right jugular vein over 10 minutes. The dose of metronidazole was determined from that listed in an equine drug formulary,8 and the dose of imipenem was determined from that reported9 for people.
Blood sample collection—Blood samples were collected before antimicrobial administration and at 5, 10, 20, 30, 40, and 50 minutes and 1, 1.25, 1.5, 2, 3, 4, 6, 8, 12, and 24 hours after antimicrobial administration. Ten milliliters of blood was collected at each time point from the left jugular vein via a syringe connected to a preplaced 14-gauge catheterc and then transferred to a blood collection tubed that contained heparin. Samples were immediately centrifuged at 1,500 × g for 10 minutes, and plasma was withdrawn and stored in plastic tubes at −20°C until analysis. Analyses were performed ≤ 1 month after sample collection.
Pleural fluid collection—Pleural fluid was collected at 1, 3, and 8 hours after antimicrobial administration. In the area of the fifth to seventh intercostal spaces, the skin was shaved free of hair and a local anesthetic (lidocaine, SC) was administered. With the aid of ultrasonography, an 11-gauge, 64-mm steel catheter attached to a syringe was inserted with an aseptic technique into the thoracic cavity at the cranial aspect of the sixth, seventh, or eighth rib. After fluid collection, the catheter was removed and the punctured skin was stapled closed. The same technique was used to place a new catheter for each collection time point. The sampling site for each time point alternated between the left and right pleural cavities (left-right-left or right-left-right), and the first site was randomly selected for each horse. The objective was to collect at least 3 mL of pleural fluid each time. Fluid was stored in plastic tubes at −20°C until analysis, and analyses were performed ≤ 1 month after sample collection.
If 3 mL of pleural fluid could not be collected, 20 mL of sterile saline solution was injected into the thoracic cavity through the catheter and then collection was reattempted. For these samples, the concentration of metronidazole or imipenem in pleural fluid was estimated by comparing the concentration of urea nitrogen in the diluted pleural fluid sample with the concentration in plasma. For pleural fluid and plasma samples obtained from 10 horses that had been euthanized between January 2014 and July 2014 because of limb fractures sustained during racing, the concentration of urea nitrogen in pleural fluid samples (mean ± SD, 0.14 ± 0.02 mg/mL) was similar to the concentration of urea nitrogen in plasma samples (0.15 ± 0.02 mg/mL). Therefore, for study samples collected after injection of saline solution into the thoracic cavity, the dilution rate was determined by dividing the plasma urea nitrogen concentration by the urea nitrogen concentration in the diluted pleural fluid sample, and the plasma metronidazole or imipenem concentration was multiplied by the dilution rate to estimate the concentration in pleural fluid.
Determination of antimicrobial concentrations
High-performance liquid chromatography—High-performance liquid chromatography with UV detectione was used to measure metronidazole and imipenem concentrations in plasma and pleural fluid. Separation was performed at room temperature with a standard columnf (length, 250 mm; internal diameter, 4.6 mm; particle size, 5 μm).
Quality control samples—Quality control plasma samples were prepared by adding metronidazoleg or imipenemh to samples of plasma of a Thoroughbred that did not receive antimicrobials 1 month before plasma collection. Plasma samples were verified to be free from these antimicrobials with chromatographic analysis before preparation of the quality control samples. Because of difficulties to collect sufficient amounts of pleural fluid, quality control samples for pleural fluid were instead prepared by diluting metronidazole or imipenem in saline solution.
Validation of the detection method was performed only with plasma samples. The mean recovery rate for each antimicrobial was determined by analyzing 5 replicates of a single quality control sample. Precision and accuracy of the detection method were determined by analyzing 5 replicates each of 2 quality control samples.
Measurement of metronidazole concentration—For measurement of plasma and pleural fluid metronidazole concentrations, 1 mL of each plasma or pleural fluid sample was placed in a glass tube with 50 μL of an internal standard (ornidazole,g 100 μg/mL) and 3 mL of ethyl acetate. The mixture was stirred for 5 minutes and then centrifuged (1,940 × g at 4°C) for 5 minutes. The supernatant was evaporated, and the residue was dissolved in 0.5 mL of a 30% acetonitrile solution and transferred to a test tube for chromatographic analysis.
The mobile phase was a 70:30 (vol/vol) mixture of phosphate buffer (0.01 mol/L; pH 4.7) and acetonitrile, and the flow rate was set at 1 mL/min. The effluent was monitored at a wavelength of 318 nm.
A linear standard curve was created with metronidazole concentrations ranging from 0.5 to 20 μg/mL and a 1/Y weighting factor; the coefficient of correlation for the standard curve was > 0.99. The lower limit of quantification was 0.5 μg/mL. The mean recovery rate was 78.8% for a sample with a metronidazole concentration of 10 μg/mL. Precision and accuracy were 2.6% and 98.4%, respectively, at a metronidazole concentration of 1 μg/mL and 1.6% and 95.8%, respectively, at a metronidazole concentration of 10 μg/mL. Because no data were available regarding precision and accuracy of the chromatographic assay at the lower limit of quantification, inaccurate results were possible for metronidazole concentrations between 0.5 and 1 μg/mL.
Measurement of imipenem concentration—For measurement of plasma and pleural fluid imipenem concentrations, 0.2 mL of plasma or pleural fluid was mixed with 0.2 mL of 3-(N-morpholino)-propanesulfonic acid buffer (pH, 6.8) containing an internal standard (ceftazidimeh [500 μg/mL]) and 0.5 mL of acetonitrile in a glass tube. The mixture was stirred for 5 minutes and centrifuged (1,940 × g at 4°C) for 10 minutes. The supernatant was evaporated, and the residue was dissolved in 0.5 mL of 3-(N-morpholino)-propanesulfonic acid buffer and transferred to a test tube for chromatographic analysis.
The mobile phase consisted of 0.1mM phosphate buffer (pH, 6.8) and methanol, and the flow rate was set at 1 mL/min. A linear gradient of the mobile phase was used that started at 95% phosphate buffer and progressed to 70% phosphate buffer over 10 minutes. This composition was maintained for 2 minutes and then returned to the initial composition for equilibration over 8 minutes. The effluent was monitored at a wavelength of 298 nm.
A linear standard curve was created with imipenem concentrations ranging from 0.2 to 50 μg/mL and a 1/Y weighting factor; the coefficient of correlation for the standard curve was > 0.99. The lower limit of quantification was 0.2 μg/mL. The mean recovery rate was 98.2% for a sample with an imipenem concentration of 5.0 μg/mL. Precision and accuracy were 2.9% and 91.9%, respectively, at an imipenem concentration of 0.5 μg/mL and 1.4% and 100.4%, respectively, at an imipenem concentration of 5.0 μg/mL. Because no data were available regarding precision and accuracy of the chromatographic assay at the lower limit of quantification, inaccurate results were possible for imipenem concentrations between 0.2 and 0.5 μg/mL.
Pharmacokinetic analysis and simulations
Plasma pharmacokinetic parameters were estimated with a computer program.i The most appropriate models and weighting schemes for individual horse data were determined by visual inspection of curve fits and software analysis that incorporated the Akaike and Bayesian (Schwarz) information criteria. Data for metronidazole and imipenem concentrations were best fitted to 1- and 2-compartment models, respectively.
To evaluate the pharmacokinetics of each antimicrobial after multiple-dose administration, anticipated pleural fluid concentrations were modeled with a computer simulation program.i For the computer simulation, doses that were inputted were the same as those administered to study horses (15 mg of metronidazole/kg and 10 mg of imipenem/kg), but administration frequency was every 8 hours rather than once. These dosages represented standard dosages for metronidazole administration in horses8 and for imipenem administration in people.9 For metronidazole, the AUC24 and AUC24:MIC90 ratio for clinical Bacteroides isolates were calculated at steady state. For imipenem, the percentage of time per day at steady state that the pleural fluid concentration of imipenem exceeded the MIC90 for clinical Bacteroides isolates was determined.
MICs of clinical Bacteroides isolates
In a previous study,1 22 Bacteroides isolates, including B fragilis, obtained from Thoroughbreds with pneumonia and pleuropneumonia were tested for their susceptibilities to various antimicrobials in accordance with Clinical and Laboratory Standards Institute guidelines. All 22 isolates were reportedly susceptible to metronidazole and imipenem; however, the MICs of metronidazole and imipenem were not reported. For the present study, unpublishedj MIC data from the previous study1 were used to calculate the MIC90 for metronidazole (4 μg/mL) and imipenem (0.5 μg/mL).
Statistical analysis
Statistical analyses were conducted with commercially available software.k Mean ± SD and median and range were determined for all pharmacokinetic parameter estimates. Data for all parameters were normally distributed except for data for the distribution half-life of imipenem, as determined by use of the Shapiro-Wilk test. The Mann-Whitney U test was used to compare plasma concentrations with pleural fluid concentrations for each antimicrobial. Values of P < 0.05 were considered significant.
Results
No adverse effects of metronidazole or imipenem administration, including diarrhea and loss of appetite and borborygmi, were observed for any horse during the study.
After administration of the single dose of metronidazole, mean ± SD concentration of metronidazole in pleural fluid was 12.7 ± 3.3 μg/mL at 1 hour, 10.6 ± 0.96 μg/mL at 3 hours, and 4.9 ± 0.85 μg/mL at 8 hours. After administration of a single dose of imipenem, mean ± SD concentration of imipenem in pleural fluid was 12.1 ± 0.9 μg/mL at 1 hour, 5.9 ± 1.37 μg/mL at 3 hours, and 0.3 ± 0.08 μg/mL at 8 hours. Concentrations of metronidazole and imipenem in pleural fluid were not significantly different from those in plasma at each of the 3 time points (1, 3, and 8 hours; Figures 1 and 2). Ratios of pleural fluid to plasma metronidazole concentrations were 0.91 to 0.99; ratios of pleural fluid to plasma imipenem concentrations were 0.80 to 2.49.
Time versus mean ± SD concentrations of metronidazole in plasma (triangles) and pleural fluid (circles) collected from 4 healthy Thoroughbreds after PO administration of a single dose (15 mg/kg).
Citation: American Journal of Veterinary Research 81, 10; 10.2460/ajvr.81.10.783
Time versus mean ± SD concentrations of imipenem in plasma (triangles) and pleural fluid (circles) collected from 4 healthy Thoroughbreds after IV administration of a single dose (10 mg/kg).
Citation: American Journal of Veterinary Research 81, 10; 10.2460/ajvr.81.10.783
Pharmacokinetic parameters for metronidazole and imipenem in plasma following single-dose administration were summarized (Tables 1 and 2). Simulated pleural fluid concentrations of metronidazole and imipenem following multiple dosing were graphed (Figures 3 and 4). Computer simulation of multiple dosing of metronidazole (15 mg/kg, PO, q 8 h) indicated that at steady state, the AUC24 was 339.6 μg·h/mL and the AUC24:MIC90 ratio for the 22 clinical Bacteroides isolates (4 ug/mL) was 84.9. Computer simulation of multiple doses of imipenem (10 mg/kg, IV, q 8 h) indicated that at steady state, the percentage of time per day the pleural fluid imipenem concentration was higher than the MIC90 for the 22 clinical Bacteroides isolates (0.5 μg/mL) was 70.9%.
Estimated pharmacokinetic parameters for metronidazole in the plasma of 4 healthy adult Thoroughbreds after PO administration of a single dose (15 mg/kg).
| Parameter | Mean ± SD | Median | Range |
|---|---|---|---|
| AUC0-∞ (μg·h/mL) | 119 ± 27.7 | 118 | 87–152 |
| MRT (h) | 7.63 ± 1.53 | 7.73 | 6.01–9.08 |
| t1/2 (h) | 5.12 ± 1.09 | 5.20 | 3.98–6.13 |
| tmax (h) | 0.50 ± 0.23 | 0.42 | 0.33–0.83 |
| Cmax (μg/mL) | 16.3 ± 4.1 | 15.3 | 12.9–21.7 |
AUC0-∞ = Area under the concentration-versus-time curve from time 0 to infinity. Cmax = Maximum observed concentration. MRT = Mean residence time.
Estimated pharmacokinetic parameters for imipenem in the plasma of 4 healthy adult Thoroughbreds after IV administration of a single dose (10 mg/kg).
| Parameter | Mean ± SD | Median | Range |
|---|---|---|---|
| AUC0-∞ (μg·h/mL) | 48.8 ± 5.8 | 49.70 | 41.36–54.58 |
| AUMC0-∞ (μg·h2/mL) | 52.0 ± 5.8 | 51.86 | 45.20–59.23 |
| MRT (h) | 1.07 ± 0.03 | l.08 | 1.02–1.09 |
| Cl (L/kg/h) | 0.21 ± 0.03 | 0.20 | 0.18–0.24 |
| Vdarea (L/kg) | 0.33 ± 0.08 | 0.30 | 0.27–0.44 |
| Vdss (L/kg) | 0.22 ± 0.03 | 0.2l | 0.20–0.26 |
| t1/2α (h) | 0.39 ± 0.01 | 0.39 | 0.37–0.4 |
| t1/2β (h) | 1.09 ± 0.12 | l.07 | 0.96-l.26 |
AUC0-∞ = Area under the plasma concentration-versus-time curve from time 0 to infinity. AUMC0-∞ = Area under the first moment curve from time 0 to infinity. Cl = Clearance. MRT = Mean residence time. t1/2α = Distribution half-life. t1/2β = Elimination half-life. Vdarea = Apparent volume of distribution based on AUC. Vdss = Apparent volume of distribution at steady state.
Computer-based simulation of metronidazole concentrations in pleural fluid of healthy horses during administration at a dosage of 15 mg/kg, PO, every 8 hours. The dotted line represents the MIC90 of metronidazole (4 μg/mL) for 22 Bacteroides isolates obtained from Thoroughbreds with pneumonia and pleuropneumonia.10j
Citation: American Journal of Veterinary Research 81, 10; 10.2460/ajvr.81.10.783
Computer-based simulation of imipenem concentrations in pleural fluid of healthy horses during administration at a dosage of 10 mg/kg, IV, every 8 hours. The dotted line represents the MIC90 of imipenem (0.5 μg/mL) for 22 Bacteroides isolates obtained from Thoroughbreds with pneumonia and pleuropneumonia.10j
Citation: American Journal of Veterinary Research 81, 10; 10.2460/ajvr.81.10.783
Discussion
In the present study, we were able to estimate plasma pharmacokinetic parameters for metronidazole and imipenem in healthy horses following administration of a single dose PO (metronidazole) or IV (imipenem). On the basis of those estimates, we developed simulations of pleural fluid concentrations that could be expected following multiple dosing. By comparing those concentrations with the MIC90 for 22 clinical Bacteroides isolates, we were able to determine that metronidazole (15 mg/kg, PO, q 8 h) or imipenem (10 mg/kg, IV, q 8 h) would likely be effective for the treatment of pneumonia and pleuropneumonia caused by Bacteroides spp in horses.
Estimates of plasma pharmacokinetic parameters for metronidazole in horses have been reported previously,10–12 although dosages and fed state differed among those studies. Feeding affects the pharmacokinetics of metronidazole when it is orally administered, with feeding shortly after dosing decreasing the time to maximum concentration and half-life.11 In contrast, pharmacokinetic data for imipenem administered IV to horses are limited to 1 study.7 Compared with the results of that study,7 results of the present study indicated a similar half-life but lower clearance. The reasons for these differences were unclear. However, results of the present study were comparable to those from studies of healthy people,13,14 dogs,15 and cats,16 indicating that imipenem shows no major pharmacokinetic differences between these species and the horses of the present study.
Metronidazole has been reported17 to concentrate in the peritoneal and synovial fluids of horses, but no data have been reported for its ability to concentrate in the pleural fluid of horses. In the present study, ratios of the concentrations of metronidazole and imipenem in pleural fluid to those in plasma were near 1 at the 3 time points for which pleural fluid and plasma concentrations were concurrently determined. Therefore, penetration of metronidazole and imipenem into the pleural cavity was satisfactory. In people, ratios of imipenem concentrations in peritoneal fluid and skin to concentrations in plasma range from 0.73 to 1.18.18,19 The accumulation of an antimicrobial in tissue and fluid is associated with the amount of antimicrobial that is not bound to plasma proteins.20 Plasma protein binding of metronidazole and imipenem is low in people,9,21 but the extent of plasma protein binding of metronidazole and imipenem in horses has not been reported. Because concentrations of imipenem in pleural fluid and plasma in the present study were similar, plasma protein binding of imipenem is likely also low in horses. However, this conclusion may be premature because the sample size was small and pleural fluid was sampled at only 3 time points. Therefore, further research is needed to confirm the results reported here.
Knowledge of the MICs of antimicrobials for target bacteria and the pharmacokinetics of those antimicrobials can help with selection of the optimal antimicrobial and dosage.22–24 Metronidazole is a dose-dependent antimicrobial for which the AUC24:MIC90 ratio is useful to predict its effectiveness. In previous reports25,26 of people with B fragilis infections, the target AUC24:MIC90 ratio was ≥ 70. In the present study, the AUC24 was 339.6 μ·h/mL, and at steady-state conditions and on the basis of the MIC90 of metronidazole for 22 clinical Bacteroides isolates, including B fragilis, obtained from Thoroughbreds with pneumonia and pleuropneumonia, the AUC24:MIC90 ratio was 84.9. Because the ratio was ≥ 70, administration of metronidazole at a dosage of 15 mg/kg, PO, every 8 hours, the standard dosage for horses, may result in metronidazole concentrations sufficient to treat Bacteroides spp in the pleural fluid of horses. Because administering metronidazole every 8 hours while withholding feed for 12 hours before to 2 hours after its administration is impossible, client-owned horses will likely be fed when metronidazole is administered, and a fed state may affect the AUC24:MIC90 ratio and, therefore, the clinical response. A previous report11 indicated a 15% reduction of the area under the concentration-versus-time curve for horses in a fed state, compared with that for horses for which feed had been withheld. This suggests that the AUC24:MIC90 ratio for metronidazole may be lower for horses that are being fed. Further studies are needed to determine the effect of feeding on the area under the concentration-versus-time curve following PO administration of metronidazole and whether the effect is of clinical relevance.
In contrast to metronidazole, imipenem is considered a time-dependent antimicrobial. Therefore, the time when the concentration of imipenem exceeds the MIC90 is important. In people, the target percentage of time per day for which the concentration of an antimicrobial should be higher than the MIC90 is > 50%.27,28 With computer simulation performed in the present study, the estimated percentage of time per day for which the concentration of imipenem, at steady state, was higher than the MIC90 for Bacteroides spp was 70.9%. This indicated that IV administration of imipenem at a dosage of 10 mg/kg, every 8 hours, may result in imipenem concentrations sufficient to treat Bacteroides spp in the pleural fluid of horses.
In people, neuropathy and encephalopathy have been reported as adverse effects of metronidazole,29,30 but these have not been reported for horses. Instead, only poor appetite attributable to metronidazole administration has been reported and then only infrequently.31 Adverse effects of the combination of imipenem and cilastatin in people include seizures and diarrhea32; however, no adverse effects in horses have been reported. Although no adverse effects attributable to the combination of imipenem and cilastatin were observed in the present study, this finding should be interpreted with caution because only a single dose was administered to 4 healthy horses. Metronidazole may be a better choice of antimicrobial than imipenem for the treatment of pneumonia or pleuropneumonia caused by Bacteroides spp because the adverse effects of metronidazole in horses are known but infrequent31 and the administration of imipenem to veterinary patients is dissuaded to prevent the development of carbapenem-resistant bacteria.33
In the present study, pleural fluid concentrations of metronidazole and imipenem were determined for healthy horses. However, the amount of fibrin and volume of pleural fluid in the pleural cavity may affect the pleural fluid concentrations of these antimicrobials. The amount of fibrin and volume of pleural fluid will increase in horses with severe pleuropneumonia, and both are positively associated with an increased risk of death.34 Because the concentrations of these antimicrobials in the pleural fluid of horses with pleuropneumonia were not determined in the present study, the effects of accumulated fibrin and pleural fluid on their concentrations and antibacterial activity remain unknown. Fluid pockets created by accumulated fibrin and the presence of inflammatory cells and protein secondary to leaky blood vessels may impair antimicrobial penetration and, therefore, antimicrobial effectiveness. For horses with fluid pockets, surgical treatment is recommended in addition to antimicrobial treatment.35 Furthermore, intrapleural fibrinolytic treatment with recombinant tissue plasminogen activator may improve drainage.36
In conclusion, results of the present study suggested that the administration of metronidazole at a dosage of 15 mg/kg, PO, every 8 hours or imipenem at a dosage of 10 mg/kg, IV, every 8 hours resulted in their accumulation in the pleural fluid of healthy horses and concentrations were likely to be effective for the treatment of pneumonia and pleuropneumonia caused by Bacteroides spp. However, metronidazole should be selected as the first-line antimicrobial for the treatment of pneumonia and pleuropneumonia caused by Bacteroides spp because of its low risk of adverse effects and the concern of developing carbapenem-resistant bacteria with the administration of imipenem.
Acknowledgments
No external funding was used in this study. The authors declare that there were no conflicts of interest.
ABBREVIATIONS
| AUC24 | Area under the concentration-versus-time curve during 24 hours after drug administration |
| MIC | Minimum inhibitory concentration |
| MIC90 | Minimum concentration that inhibits growth of 90% of isolates |
Footnotes
Asuzol, Fuji Pharma, Saitama, Japan.
Sandoz, Tokyo, Japan.
BD Angiocath, Becton, Dickinson and Co, Franklin Lakes, NJ.
Venoject II VP-H100K, Terumo Corp, Tokyo, Japan.
Shimadzu prominence HPLC system, Shimazu Corp, Kyoto, Japan.
Mightysil RP-8 GP, Kanto Chemical Co, Tokyo, Japan.
Sigma-Aldrich Corp, St Louis, Mo.
LKT Laboratories Inc, Saint Paul, Minn.
Phoenix WinNonlin, Certara Inc, Princeton, NJ.
Kinoshita Y, Niwa H, Katayama Y, et al. Microbiology Division, Epizootic Research Center, Equine Research Institute, Japan Racing Association, Shimotuke, Tochigi, Japan: Unpublished data, 2013.
JMP, version 13.1.0, SAS Institute Inc, Cary, NC.
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