Tissue Residues and Pharmacokinetic/Pharmacodynamic Modeling of Tiamulin Against Mycoplasma anatis in Ducks

The pharmacokinetics of tiamulin were studied in 2 groups of ducks (n = 6) after its oral administration at 2 different doses (30 and 60 mg/kg, respectively). Plasma concentrations of tiamulin were measured by high performance liquid chromatography at different time points up to 24 h post-administration. The maximum plasma concentrations were 0.77 and 2.32 μg/mL attained at 2 h (Tmax) for 30 and 60 mg/kg, respectively. The elimination half-lives for these 2 doses were 3.54 and 6.34 h, respectively. The minimum inhibitory concentration for tiamulin against Mycoplasma anatis (M. anatis) strain 1340 was determined to be 0.06 μg/mL. The proper oral dose of tiamulin against M. anatis in ducks was calculated to be 35 mg/kg/day using the pharmacokinetic/pharmacodynamic modeling. Tiamulin was administered orally (40 mg/kg/day) to 30 ducks for 3 successive days to determine its residues in edible tissues and its preslaughter withdrawal time. The highest tiamulin residues were detected in the liver, followed by the muscle, whereas lower concentrations were detected in the skin and fat. The estimated withdrawal periods of tiamulin were 6, 5, 3, and 3 days for liver, muscle, skin, and fat, respectively. Therefore, an oral dosage regimen of 35 mg/kg/day should be adequate for tiamulin against M. anatis. We recommend a preslaughter withdrawal period of 6 days when ducks are treated with 40 mg tiamulin/kg/day, orally, for 3 days.


INTRODUCTION
Tiamulin was first isolated from the fungus Pleurotusmutilis in 1951 (1). It is available as a semisynthetic antibiotic which is a derivative of pleuromutilin. Tiamulin has high activity against Gram-positive bacteria as well as Mycoplasma spp. and Leptospirae (2)(3)(4). Tiamulin exhibits its bacteriostatic effect through blocking the 50S ribosomal subunit of bacteria, inhibiting peptidyl transferase (5). It is employed in veterinary medicine, particularly as a treatment for pneumonia, dysentery, gastrointestinal and mycoplasmal infections in swine and poultry (6)(7)(8). Tiamulin is also effective against mycoplasmal infection in ducks and geese (9).
Mycoplasma anatis affects predominantly ducks and eggs; the infection in ducks is usually associated with conjunctivitis, rhinitis, sinusitis, arthritis, growth retardation and reduced hatchability (17). The principal route of disease transmission is through eggs (vertical transmission) (18). Tiamulin has been utilized successfully as a treatment for mycoplasmosis in ducks and geese (9,19,20). It is crucial to optimize tiamulin dosage schedule to maximize clinical effectiveness, in addition to minimizing the development of drug resistance. Pharmacokinetic/pharmacodynamic (PK/PD) modeling points out the correlation between the pathogen, drug, and the treated animal, which in turn provides valuable information in the establishment of appropriate dose regimens for favorable clinical outcomes and for preventing the emergence of drug resistance (21,22). To the best of our knowledge, the PK-PD integration of tiamulin against M. anatis has not been studied yet.
Avoiding antibiotic residues in edible products obtained from medicated animals is essential to ensure consumer's safety (23). The committee for Veterinary Medicinal Product (CVMP) of the European Union (EU) announced the Maximal Residual Limit (MRL) for tiamulin in tissues of chickens and turkeys to be 0.1 µg/g for muscles, skin, and fat, 1 and 0.3 µg/g for liver of chickens and turkeys, respectively (24). The tissue residue depletion of tiamulin has been reported in chickens (25), rabbits (26), and pigs (27). To the best of our knowledge, no reports are available concerning the depletion of tiamulin residues from tissues of ducks.
Therefore, the goals of this study were: (a) to investigate the pharmacokinetic behavior of tiamulin in healthy ducks after oral administration; (b) to study antimicrobial efficacy of tiamulin against M. anatis, (c) to calculate the rational dose of tiamulin against M. anatis in ducks based on the AUC24 h/MIC (the ratio of area under the concentration-time profile from 0-24 h: minimum concentration of tiamulin inhibiting the tested strain of M. anatis) for PK/PD modeling, and (d) to determine tiamulin residues in liver, muscle, skin and fat after treatment with our suggested clinical dose of tiamulin in ducks in order to estimate its preslaughter withdrawal period in ducks designated for human consumption to guarantee food safety.

Materials
Tiamulin hydrogen fumarate solution (Denagard R , 12.5%, Elanco Co., Basel, Switzerland) was utilized in this study. The tiamulin standard was purchased from Sigma Aldrich Co. (St. Louis, MO. USA). The high-performance liquid chromatography (HPLC) grade methanol, acetonitrile, potassium dihydrogen phosphate, hexane, ethyl acetate, sodium carbonate, ammonium carbonate, phosphate acid, and tartaric acid were obtained from Merck (Darmstadt, Germany). Purified water was produced by Milli-Q system (Waters Corp., Milford, MA. USA). The M. anatis standard strain 1340 was supplied by Animal Health Research Institute, Giza, Egypt. M. anatis medium base was made according to a published method (28).

Animals
Forty-two healthy Muscovy ducks (10 ± 1 weeks, 2.0-2.5 kg, male: female, 1:1) were procured from Faculty of Agriculture, Mansoura University, Egypt. They were housed in adjusted climate at 23-27 • C and 45-65% relative humidity. The ducks were acclimated for 14 days before the start of the experiment. Drug-free feed and water were consumed ad libitum. Approval for the animal experiments was secured from the Animal Ethics Committee at the Faculty of Veterinary Medicine, Mansoura University (Approval No. R/44).

Experimental Design
In this research, two separate experiments were executed; in the 1st experiment, 12 ducks were randomly divided into two groups (n = 6, 3 males and 3 females). Group 1 and 2 were gavaged with tiamulin directly into the crop via a plastic tube linked to a syringe with a single dose of tiamulin at 30 and 60 mg/kg, respectively. These doses were chosen based on the suggested dosage of 30-60 mg/kg, orally, for 3-5 days (6). Food was withheld for 6 h before and after tiamulin administration, while water was consumed ad libitum. Blood samples of 1 mL were taken from a wing vein into EDTA tubes at time 0 (prior to tiamulin administration), and 0.5, 1, 2, 4, 6, 8, 10, 12, and 24 h post-tiamulin administration. Blood samples were centrifuged at 1,257 × g for 10 min to yield plasma which were immediately stored at −20 • C until analysis.
In the 2nd experiment, 30 ducks were administered tiamulin orally via gavage at 40 mg/kg daily for 3 successive days (6,16). After the 3rd day of treatment, 6 ducks each were slaughtered on 1st, 3rd, 5th, 7th, and 9th day after the tiamulin dosing. Tissue samples (liver, breast muscle, abdominal skin and subcutaneous fat) were collected from each duck and were frozen at −20 • C until analysis.

Extraction of Tiamulin From Plasma Samples
The preparation of plasma samples was performed based on a former published technique (29). Briefly, 0.5 mL plasma sample was mixed with 2.5 mL 1% aqueous solution sodium carbonate and then 2,5 hexane-ethyl acetate (3/1 v/v) was added for extraction. After shaking, the mixture was centrifuged at 2,360 × g for 20 min. Then, 1 ml of supernatant was evaporated under nitrogen stream. After that, 0.1% buffered aqueous solution tartaric acid (0.3 mL) was added to redissolve the residue. Ten microliters of the sample was injected into the HPLC system.

Chromatographic Conditions
Tiamulin concentrations in the standards and plasma samples were determined following a published HPLC method with few modifications (29). The HPLC Agilent Series 1200 quaternary gradient pump, Series 1200 Autosampler, Series 1200 UV VIS detector set at 210 nm, and HPLC 2D Chemstation software (Hewlett-Packard, Les Ulis, France) Data for recovery are presented as mean ± SEM. Intra-day RSD and Inter-day RSD % (n = 6, 0.025 µg/mL). Average recovery % (using spiked concentrations in the range of 0.01-5 µg/mL in triplicate analysis).
were operated. Chromatographic analysis was performed utilizing a Phenomenex C18 column (5 µm, 150 mm × 4.6 mm). Acetonitrile and potassium dihydrogen phosphate (pH = 2.8) (65:35, v/v) were used as a mobile phase under isocratic conditions. The flow rate was 1.5 mL/min. The retention time was 1.2 min. The method used for HPLC analysis was revalidated as described by a European Medicines Agency protocol (30) utilizing duck plasma ( Table 1). The calibration curve was established by depicting peak area vs. concentration of tiamulin using the data from 9 concentrations (ranging 0.01-5 µg/mL plasma). A linear correlation was identified in the calibration curve in the range of 0.01-5 µg/mL. The lower limits of detection and quantification of tiamulin were 0.003 and 0.01 µg/mL, respectively. The intra-and inter-day precision and accuracy of the assay was indicated in Table 1.

Extraction of Tiamulin From Tissue Samples
Tissue samples were prepared as reported (31). In brief, tissue samples were mixed with acetonitrile, purified by liquid partition separation. At last extraction was performed using n-hexane. Then the extract was concentrated and eluted via solid phase extraction cartridge column (Bond Elut C18, 3 mL/500 mg, Varian Company, Palo Alto, CA, USA) for HPLC analysis.

Chromatographic Conditions
The analysis of tiamulin in tissue samples (liver, muscle, skin, and fat) was undertaken utilizing a published method (31). Briefly, The HPLC Agilent Series 1200 quaternary gradient pump, Series 1200 Autosampler, Series 1200 UV VIS detector set at 210 nm, and HPLC 2D Chemstation software (Hewlett-Packard, Les Ulis, France) were employed. The HPLC column was a Phenomenex C18 (5 µm, 250 mm × 4.6 mm). The mobile phase comprised a mixture of 80% acetonitrile and 1% ammonium carbonate (90:10, v/v) at a flow rate of 1.0 mL/min. The retention time was 9.3 min. The calibration curve of peak area vs. tiamulin concentration was plotted with data from 8 concentrations [0.025-5 µg/g tissue (liver, muscle, skin, and fat)]. The analytical technique was validated using duck tissues ( Table 2). Linearity of this technique was observed (R 2 > 0.99) in the standard curve. The lower limits of detection and quantification of tiamulin were 0.008 and 0.025 µg/g, respectively. Data for recovery are shown as mean ± SEM. Intra-day RSD and Inter-day RSD % (n = 6, 0.05 µg/g). Average recovery % (using spiked concentrations in the range of 0.025-5 µg/g in triplicate analysis).

Evaluation of Minimum Inhibitory Concentration (MIC) of Tiamulin Against M. anatis
The determination of MIC of tiamulin against M. anatis strain 1340 was performed as reported (32). In brief, M. anatis titer of 10 7 CFU/mL in the mycoplasma medium was prepared. A series of tiamulin standard concentrations were made in the range of 0.03125-32 µg/mL using M. anatis culture medium as the diluent (1:1 dilution). The MIC was identified as the lowest concentration of tiamulin where no change in the color of culture medium was noticed.

In vitro Time-Killing Curve
The in vitro time-killing studies were conducted according to a reported technique (32). Different concentrations of tiamulin which were multiples of MIC were produced (0, 0

Determination of Pharmacokinetics Profile and Preslaughter Withdrawal Time
The pharmacokinetic profile of tiamulin was investigated utilizing the non-compartmental approach [WinNonlin 8.3 software (Certara, USA)] as reported (15,16). The values of the highest plasma concentration (C max ) and the time required to attain C max (T max ) were recorded from the plasma concentration-time plot. The area under the plasma concentration-time curve (AUC 0−∞ ) was computed using the linear-log trapezoidal method. The elimination half-life (T 1/2 λz) was calculated using the equation T 1/2 λz = 0.693/λz. The withdrawal time (WT) was calculated by applying WT 1.4 program which was established in Germany and approved by the CVMP of the EU. It was estimated utilizing the statistical approach (95% tolerance limit and 95% confidence interval) based on the EU MRL for tiamulin in chicken tissues, which were announced by the CVMP to be 0.1 µg/g for muscles, skin, and fat, and 1 µg/g for liver, respectively (24).

PK/PD Modeling and Dose Determination
The value of the area under the concentration-time profile over 24 h (AUC 24h ) for the ex vivo time killing curve was computed by multiplying each tiamulin concentration measured after the oral administration at 60 mg/kg by 24 h (incubation period). Subsequently, the AUC 24h value was divided by the MIC to estimate the AUC 24h /MIC (33)(34)(35).
The log 10 difference between M. anatis count (CFU/mL) after 24 h incubation and the initial count was correlated with the AUC24 h/MIC ratio using the Sigmoidal inhibitory E max model as illustrated by the equation: In which E reflects the antimycoplasmal effect identified as the variation between M. anatis count (log 10 CFU/mL) in the plasma sample after 24 h of incubation and the baseline log 10 CFU/mL (the growth without tiamulin); E max elucidates the highest antimycoplasmal activity; E 0 represents the difference in M. anatis count in the control sample (without tiamulin) between 0 and 24 h of incubation; EC 50 is the AUC/MIC ratio showing a 50% decline in M. anatis counts from the original count; C refers to the AUC/MIC in the effect compartment; γ is the Hill coefficient which describes the steepness of the AUC 24h /MIC effect curve. These PD parameters were computed with WinNonlin 8.3 software (Certara, USA).
The antimycoplasmal activity of tiamulin was evaluated by calculating the AUC/MIC desired for bacteriostatic action (no variation in bacterial count after 24 h incubation, E = 0), and bactericidal action (99 and 99.9% decrease in bacterial count, E = −2 and E = −3, respectively) by utilizing the sigmoid E max model.
Using the PK/PD modeling, the optimal daily dose for tiamulin in ducks was calculated based on this equation (36)  Where AUC 24h /MIC indicates the AUC 24h /MIC ratio for appropriate efficacy; MIC refers to the minimum inhibitory concentration; CL represents the clearance (L/h/kg); F reflects the bioavailability; fu is the free portion of tiamulin in plasma.

Statistical Analysis
Data are expressed as mean ± SEM. Normality of the data was examined by using Shapiro-Wilk test. Mann-Whitney test was used to compare the major pharmacokinetic parameters for the two different doses used (30 and 60 mg/kg). The counts of M. anatis of the time killing curves at 48 h were compared using one-way analysis of variance (ANOVA), followed by the Tukey's mean comparison test. Moreover, the residue levels of tiamulin were compared in various tissues using ANOVA, followed by the Tukey's mean comparison test. P < 0.05 was regarded as statistically significant. Statistical investigation was undertaken utilizing Statistical Package for Social Science (SPSS), version 20 (SPSS Inc., Chicago, IL, USA) for windows.

Tiamulin Pharmacokinetics
The plasma concentration-time curves of tiamulin following a single oral administration by gavage at 30 mg/kg and 60 mg/kg, respectively, are presented on a semilogarithmic graph in Figure 1. Tiamulin was detectable (≥3 ng/mL) in plasma up to 24 h after oral administration of two different doses of tiamulin. The main PK parameters of tiamulin are listed in Table 3. Statistical comparison of the parameters between the 2 doses showed no significant difference in the T max, T 1/2 λz, MRT, Vz_F_obs, and Cl_F_obs. Whereas, the differences in C max, AUC 0−last, and AUC 0−∞ were significant (p < 0.05) as their values increased in a dose-dependent manner.

Tiamulin PD
The MIC of tiamulin for M. anatis strain 1340 was estimated as 0.06 µg/mL. No growth of M. anatis was noted in the sterilizing Frontiers in Veterinary Science | www.frontiersin.org control and end-point control, whereas M. anatis growth was observed in the positive control. The time-kill curves for tiamulin in duck plasma against M. anatis strain 1340 were elucidated at 8 time points using samples harvested following oral dosing of tiamulin at 60 mg/kg  (Figure 3). Plasma samples collected between 0.5 and 12 h posttiamulin administration exhibited mycoplasmocidal action (≥3log 10 CFU/mL) after 24 h of incubation. Slight inhibition of Mycoplasma growth (2-log 10 CFU/mL reduction) was observed in 24 h samples. No M. anatis growth was detected after 48 h of incubation for samples collected between 1 and 10 h.

PK/PD Modeling and Dose Determination
The findings from PK/PD integration of time-killing curve are presented in Table 4 and Figure 4. The AUC 24h /MIC required to produce mycoplasmastasis (no alteration in the number of mycoplasma, E = 0), mycoplasmocidal (99% decline in mycoplasma count, E = −2), and mycoplasmocidal (99.9% decline in mycoplasma count, E = −3) were 6.75, 122.2 and 251.9 h, respectively. Based on the estimated AUC 24h /MIC from PK/PD modeling, MIC of M. anatis (0.06 µg/ml) obtained in this study, CL_F_obs (clearance divided by bioavailability after oral administration of tiamulin at 60 mg/kg; 2.61 L/h/kg) which reflects the unknown bioavailability (F), and the free fraction of tiamulin of 0.55 (10), the dose of tiamulin needed per 24 h for bacteriostatic, bacteriocidal action (99% decline in mycoplasma count, E =-2), and bacteriocidal action (99.9% decline in mycoplasma count, E = −3) against M. anatis in ducks were determined to be 2 mg/kg, 35 mg/kg/day, and 72 mg/kg, respectively . Table 5 illustrates the residue concentrations of tiamulin in duck tissues (liver, muscle, skin, and fat) after oral dosing at 40 mg/kg/day for 3 successive days. The highest tiamulin concentration was detected in the liver, followed by the muscle, whereas lowest concentrations were detected in the skin and fat. Furthermore, the present study showed that tiamulin was detected in the skin and fat up to the 3rd day post-dosing and up to the 5th and 7th day after the end of the treatment in muscle and liver, respectively. The withdrawal time was 5.7 days for liver   Emax, is the maximum difference n log 10 of bacterial number of sample incubated with tiamulin; EC 50 , is the PK/PD parameter of the drug that produces half of the maximal antibacterial effect; E 0 , the difference after 24 h of incubation in log 10 of number of bacteria in control samples (without tiamulin); AUC 24h /MIC, values required to achieve bacteriostatic effect and bactericidal effect.

Tissue Residues and Preslaughter Withdrawal Time of Tiamulin
(approximated to 6 days), and 5.4 days for muscle (approximated to 5 days) as elucidated in Figures 5A,B. Since the residues of tiamulin in skin and fat on the 3rd day after last dose were lower than EU MRL (0.1 µg/g), hence the 3-day withdrawal time for the skin and fat is suggested.

DISCUSSION
This study was the first research about PK of tiamulin in ducks. The findings of this study revealed that tiamulin, when administered orally to ducks, was rapidly absorbed with C max values [0.77 and 2.32 µg/mL attained at 2 h (T max ) for 30 and 60 mg/kg, respectively] and these values were similar to those reported for dogs, goats, and ewes, which were injected with tiamulin intramuscularly (i.m.) at 10 mg/kg (0.60, 0.56, and 0.64 µg/mL, respectively) (14,37), pig administered tiamulin orally at 10 mg/kg (0.69 µg/mL) (13), chickens received tiamulin orally at 40 mg/kg (0.73 µg/mL reached at 1.5 h) (16), and for   chickens injected with tiamulin i.m. at 5 mg/kg (2.05 µg/mL) and 40 mg/kg (8.8 µg/mL) (15). These results suggested that the oral absorption of tiamulin is potentially affected by intestinal factors. Moreover, the present study showed a second peak at 8 h post-tiamulin administration with the two different doses. This is likely due to the enterohepatic recycling of tiamulin during biliary elimination (15,16). In addition, following oral administration of tiamulin at 30 mg/kg, the AUC extrapolated to infinity was 5.85 µg * h/mL. This value was comparable to that reported for chickens (5.18 µg * h/mL) (16). Furthermore, our data revealed that after a single oral administration of tiamulin at 2 different doses, the T 1/2 λz (3.54 and 6.34 h) were short and these results suggested a rapid elimination of tiamulin in ducks. One potential reason for this short T 1/2 λz is the rapid clearance rate (2.61 and 5.59 L/h/kg, respectively, after the 2 doses). The relationship between the T 1/2 λz and Cl was elucidated by the following equation (37): Cl . These results were consistent with the findings in chickens (4.23 h) (16), dogs (2.23 h) (38), and pigs (2.14 h) (13). Additionally, the MRT (5.95 and 7.64 h for 30 and 60 mg/kg, respectively) observed in this study were comparable to the MRT of 7.1 h found in chickens treated with oral dose of tiamulin at 40 mg/kg (16). The Vz_F_obs for the 2 doses in ducks were 25.78 and 23.93 L/kg for 30 and 60 mg/kg, respectively. To the best of our knowledge, limited data are available about Vz of tiamulin in other species. The Cl_F_obs of tiamulin reported in this study were 5.59 and 2.61 L/h/kg for 30 and 60 mg/kg tiamulin doses, respectively. These values were nearly similar to the CL_F of 4.6 L/h/kg reported in Mycoplasma gallisepticum infected chickens injected i.m. with 40 mg/kg (15). In contrast, they were much lower than that reported in healthy chickens administered tiamulin at 40 mg/kg through the crop, drinking water, and feed (30.3, 26.4, and 14.7 L/h/kg, respectively) (16). However, these CL_F_obs values reported in chickens by Vinothini et al. (16) were much higher than that revealed in other species; for instance, in dogs the CL_F value was 1.36 L/h/kg after i.m. injection of tiamulin at 10 mg/kg (38). The interspecies variation in plasma clearance points out the differences in the relative importance of the multiple possible routes of drug excretion (liver and non-liver metabolism vs. biliary elimination vs. renal elimination) and the ability for any given method of elimination (39).
Moreover, in this study, tiamulin showed a time-and concentration-dependent effect against M. anatis in the in vitro time killing study. The antimycoplasmal action was augmented by the increment of drug concentration or the time of exposure. These results were in accordance with the work by Novak and Schlaes (7) in which the effects of most pleuromutilins were predominantly time-dependent. Moreover, Xiao et al. (40) reported that valnemulin was concentration-dependent against M. gallisepticum. It has been reported that the time-or concentration-dependent action of antibiotics may alter under various conditions (40).
The PK/PD index of pleuromutilin antibiotics is AUC 24 /MIC (41). This work elucidated that the MIC of tiamulin against M. anatis strain 1340 was 0.06 µg/mL. Using this MIC value, and the estimated AUC 24h /MIC for mycoplasmacidal action (122.2 h), the calculated daily dosage of tiamulin to produce mycoplasmpcidal effect (2 log 10 equivalent reduction in mycoplasma count) against M. antis in ducks would be 35 mg/kg/day. This dose was in the range of the recommended oral doses in poultry (30-60 mg/kg) (6).
In the present study, tiamulin showed a mycoplasmocidal effect. In the same line, valnemulin, a member of pleuromutilins, exhibited action against M. gallisepticum (15). On the contrary, Burch and Alvarz revealed that tiamulin did not achieve a mycoplasmocidal action against M. gallisepticum (42). This discrepancy may be due to the difference in the Mycoplasma species and/or the variation in the susceptibility to the drug. Xiao et al. reported that the bacteriostatic and bactericidal features may be varied for the same drug against different microorganisms (40).
The depletion of tiamulin residues from ducks' edible tissues was investigated after its administration at 40 mg/kg once daily for 3 successive days, as the recommended oral doses in poultry is 30-60 mg/kg bw for 3-5 days (6).
Based on the calculated withdrawal time of tiamulin in this study for liver, muscle, skin, and fat, it would be reasonable to suggest a preslaughter withdrawal time of 6 days after oral administration of 40 mg/kg tiamulin to ascertain a level of tiamulin lower than the MRL in duck tissues before being consumed by humans to ensure food safety. These results are in agreement with the report that after i.m. administration of tiamulin to pigs at 12 mg/kg once daily for 5 days, the most convenient preslaughter withdrawal period was 6 days (27). However, the withdrawal time calculated in the present study (6 days) was higher than that reported for chickens of 3-4 days (25,43).
The tiamulin dose calculation was performed using the data derived from a single strain of M. anatis. However, it would be better to determine the dose according to estimates of MIC90 (the 90th percentile of MIC distribution) values to accommodate the potential needs of the entire patient population. To the best of our knowledge, limited data are available about MIC distribution for tiamulin against M. anatis, the breakpoint and the epidemiological cut-off value (ECOFF) due to the difficulty of culturing M. antis and the long doubling time. Hence, further studies are needed to validate the calculated dosage in clinical circumstances to confirm its therapeutic efficacy.
In the present study, tiamulin was administered directly into the crop. Vinothini et al. concluded that tiamulin had equal efficacy after administration in chickens by the three different routes (in-crop, in-water, and in-feed) as no significant difference in the pharmacokinetics parameters among the three routes was noticeable (16).
In conclusion, the results of this research suggested a dose of 35 mg/kg/day orally for tiamulin to fulfill the desired effect against M. anatis in ducks. Furthermore, a 6-day preslaughter withdrawal period is proposed following oral administration of tiamulin to assure safe human consumption of duck tissues.

DATA AVAILABILITY STATEMENT
The original contributions presented in the study are included in the article/supplementary materials, further inquiries can be directed to the corresponding author/s.

ETHICS STATEMENT
The animal study was reviewed and approved by the Animal Ethics Committee at the Faculty of Veterinary Medicine, Mansoura University.