Pharmacokinetic/Pharmacodynamic Modeling of Spiramycin against Mycoplasma synoviae in Chickens

This research aimed to assess the pharmacokinetics/pharmacodynamics (PK/PD) and tissue residues of spiramycin in chickens. The PK of spiramycin were determined in 12 chickens using a parallel study design in which each group of chickens (n = 6) received a single dose of spiramycin at 17 mg/kg intravenously (IV) or orally. Plasma samples were collected at assigned times for up to 48 h to measure spiramycin concentrations. Additionally, a tissue depletion study was performed in 42 chickens receiving spiramycin at 17 mg/kg/day orally for 7 days. The area under the plasma concentration–time curve values were 29.94 ± 4.74 and 23.11 ± 1.83 µg*h/mL after IV and oral administrations, respectively. The oral bioavailability was 77.18%. The computed withdrawal periods of spiramycin were 11, 10, and 7 days for liver, muscle, and skin and fat, respectively. The minimum inhibitory concentration for spiramycin against Mycoplasma synoviae (M. synoviae) strain 1853 was 0.0625 µg/mL. Using the PK/PD integration, the appropriate oral dose of spiramycin against M. synoviae was estimated to be 15.6 mg/kg. Thus, we recommend an oral dose of 15.6 mg spiramycin/kg against M. synoviae in chickens and a withdrawal period of 11 days following oral treatment with 17 mg spiramycin/kg/day for 7 days.

Similar to other macrolides, spiramycin is recognized by its high tissue distribution [9]. The pharmacokinetics (PK) of spiramycin have been assessed in cattle [7,10], pigs [11], ewes [12], and monkeys [13]. To date, no reports exist concerning the PK of spiramycin in chickens, despite the fact that it is efficacious in the control of mycoplasmosis in this species [8].
Mycoplasma synoviae (M. synoviae) is a widespread and economically significant pathogen of birds. It causes subclinical respiratory disease and the infection may spread systemically throughout the body if accompanied by a secondary infection to invade the joints and tendons, resulting in synovitis in chickens and turkeys [14][15][16]. Affected birds with infectious synovitis usually exhibit depression, lameness, and swollen hock joints [17]. Horizontal and vertical transmission are the main routes of M. synoviae dissemination [18]. M. synoviae causes considerable economic losses to the poultry industry as a result of reduced hatchability, drop in egg production, and carcass condemnation [15,19,20]. Spiramycin has been recommended as an effective remedy for mycoplasmosis in chickens [8]. Adjustment and optimization of the spiramycin dosage regimen is essential to maximize its antimicrobial activity and to prevent the development of drug resistance.
Pharmacokinetic/pharmacodynamic (PK/PD) integration provides a powerful tool for establishing rational dosage schedules for antibacterial drugs that achieve the desirable therapeutic outcomes and avoid toxicity and the emergence of resistant bacterial strains [21,22]. It has been reported that high doses of spiramycin may cause gastrointestinal disturbances and allergic reactions [23]. To the best of our knowledge, the PK/PD modeling of spiramycin against M. synoviae has not been established.
The indiscriminate usage of antimicrobial agents and/or disregarding the withdrawal period instructions after treatment lead to accumulation of drug residues in edible products obtained from treated animals [24,25]. Such drug residues may be associated with adverse reactions in consumers such as allergies, direct toxicity, imbalance of commensal intestinal microflora, bone marrow depression, various organ diseases, teratogenicity, or cancer [26]. In particular, the existence of macrolide antibiotics residues in the edible tissues has been implicated in the occurrence of hypersensitivity manifestations in sensitive persons, in addition to the alterations of gut microflora [27]. Moreover, the antibiotic residues may enhance the resistance of pathogens [28]. Hence, the World Health Organization identifies the Maximum Residual Limits (MRL) for all antimicrobials in various food products to guarantee consumers' safety. The Committee for Veterinary Medicinal Products (CVMP) of the European Union (EU) reports the MRL for spiramycin in tissues of chickens to be 0.2 µg/g for muscles, 0.3 µg/g for skin and fat, and 0.4 µg/g for liver [29]. The depletion of spiramycin residues from tissues has been studied in cattle [10], buffalo [30], and fish [31]. However, limited data are available about the depletion of spiramycin from chicken tissues [32].
Thus, this study was undertaken to: (a) investigate the PK characteristics of spiramycin in healthy chickens after oral and intravenous (IV) administrations; (b) investigate the antimicrobial activity of spiramycin against M. synoviae; (c) estimate the dose of spiramycin against M. synoviae in chickens using the AUC 24h /MIC (the ratio of the area under the concentration-time curve from 0-24 h: minimum concentration of spiramycin inhibiting the examined strain of M. synoviae) for PK/PD integration, and (d) evaluate spiramycin residues in tissues (muscle, skin and fat, and liver) of chickens treated orally with the clinical dose of spiramycin and calculate its withdrawal period in chickens.

Spiramycin PK
Spiramycin was quantifiable in the plasma for 36 h after IV and oral administrations, respectively, at a single dose of 17 mg/kg. No spiramycin concentration could be detected thereafter. Concentration vs. time profiles of spiramycin in plasma after both routes of administration in chickens are depicted on a semilogarithmic graph in Figure 1. The mean (±SEM) PK parameters of spiramycin are illustrated in Table 1. The values of the area under the plasma concentration-time curve (AUC 0-last ) were 29.94 ± 4.74 and 23.11 ± 1.83 µg*h/mL after IV and oral administrations, respectively. The oral bioavailability (F) was 77.18%. No statistical difference was noticed in the PK parameters between IV and oral routes. (±SEM) PK parameters of spiramycin are illustrated in Table 1. The values of the area under the plasma concentration-time curve (AUC0-last) were 29.94 ± 4.74 and 23.11 ± 1.83 µg*h/mL after IV and oral administrations, respectively. The oral bioavailability (F) was 77.18%. No statistical difference was noticed in the PK parameters between IV and oral routes.  3.30 ± 0.52 --------Vz_F_obs (L/kg) ---------6.58 ± 0.89

Plasma Protein Binding
The mean value of plasma protein binding ratio of spiramycin was 34.60 ± 1.28%.

Plasma Protein Binding
The mean value of plasma protein binding ratio of spiramycin was 34.60 ± 1.28%.
The in vitro anti-mycoplasmal activities of multiples of MIC (0.5-64 MIC) of spiramycin against M. synoviae through a period of 48 h is shown in Figure 2. On the whole, a time-dependent feature of spiramycin against M. synoviae was noticed from the killing profiles as the rate of killing increased by increasing the time that M. synoviae was subjected to spiramycin application. In addition, the killing curves revealed a concentration-dependent The minimum inhibitory concentration (MIC) of spiramycin against M. synoviae strain (MS WVU-1853) was 0.0625 µg/mL. The in vitro anti-mycoplasmal activities of multiples of MIC (0.5-64 MIC) of spiramycin against M. synoviae through a period of 48 h is shown in Figure 2. On the whole, a time-dependent feature of spiramycin against M. synoviae was noticed from the killing profiles as the rate of killing increased by increasing the time that M. synoviae was subjected to spiramycin application. In addition, the killing curves revealed a concentrationdependent effect of spiramycin against M. synoviae as the anti-mycoplasmal effect increased in response to the rise in the spiramycin concentration , where 64 MIC displayed the maximal activity (7.56 log10CFU/mL) decline during the 48 h incubation.   Figure 3 shows the ex vivo time-killing curves. For plasma samples obtained from chickens between 0.5 and 8 h, spiramycin produced a ≥3 log 10 CFU/mL reduction in the M. synoviae count after 24 h of exposure. Meanwhile, for samples collected at 12, 15, 24, and 36 h, spiramycin application resulted in a ≤2 log 10 CFU/mL decline in the count following 24 h of incubation.

PK/PD Integration and Dose Estimation
The correlation between the PK/PD parameters derived from dynamic model assays and anti-mycoplasmal activity are presented in Figure 4. The parameters obtained from the PK/PD modeling of ex vivo spiramycin data for plasma are illustrated in Table 2.

PK/PD Integration and Dose Estimation
The correlation between the PK/PD parameters derived from dynamic model assays and anti-mycoplasmal activity are presented in Figure 4. The parameters obtained from the PK/PD modeling of ex vivo spiramycin data for plasma are illustrated in Table 2.

PK/PD Integration and Dose Estimation
The correlation between the PK/PD parameters derived from dynamic model assays and anti-mycoplasmal activity are presented in Figure 4. The parameters obtained from the PK/PD modeling of ex vivo spiramycin data for plasma are illustrated in Table 2.
The estimated AUC24h/MIC needed to obtain mycoplasmastasis (E = 0, no difference in bacterial count after incubation for 24 h), mycoplasmocide (E = −2 and E = −3, 99 and 99.9% reduction in the M. synoviae count, respectively) and the virtual mycoplasmal eradication (E = −4) were 10, 185, 405, and 780 h, respectively. Hence, the dose of spiramycin required daily for the bacteriostatic action (E = 0), bactericidal action (E = −2 and E = −3) and bacterial elimination (E = −4) against M. synoviae in chickens were calculated to be 0.84, 15.60, 34.18, and 65.83 mg/kg, respectively.  h 780 E max , is the maximum antibacterial effect identified as difference in log 10 CFU/mL in samples over 24 h of incubation with spiramycin; EC 50 , is the PK/PD parameter of the drug that induced 50% of the maximal antibacterial effect; E 0 , is the corresponding mycoplasmal growth in the absence of spiramycin (control samples); AUC 24h /MIC, values needed to produce bacteriostatic effect and bactericidal effect.

Tissue Residues and Withdrawal Periods of Spiramycin
Results from tissue residue analysis of chickens receiving spiramycin orally at 17 mg/kg for 7 consecutive days revealed that liver samples had the highest spiramycin concentration, followed by the muscle, while the lowest levels were found in skin and fat samples. The residue concentrations of spiramycin were above the limit of quantification (LOQ) up to the eleventh, ninth, and seventh days after the last dose in liver, muscle, and skin and fat, respectively. The mean spiramycin residue levels in chicken tissues are listed in Table 3.
The computed withdrawal periods were 11, 10, and 7 days for liver, muscle, and skin and fat, respectively, as displayed in Figure 5A-C.

Discussion
Despite extensive marketing of spiramycin in poultry medicine, PK data in birds are absent. To the best of our knowledge, this is the first report about PK/PD modeling of spiramycin against M. synoviae in chickens. The PK analysis in the present study demonstrated that following IV injection of spiramycin in chickens at 17 mg/kg, the apparent volume of distribution (Vz_obs) was 3.3 L/kg which illustrated its wide distribution in the intracellular and extracellular compartments of the body. This finding coincides with the view of Riviere [33] who indicated that a Vz higher than 1 L/Kg represents a wide diffusion of the drug throughout the tissues. The high lipid solubility and the moderate plasma protein binding features of spiramycin are probably the main reasons beyond its high Vz. However, the value found for Vz in this study was lower than that reported in pigs (5.6 L/Kg) receiving IV spiramycin at 10 mg/kg [11], ewes (16.56 L/kg) receiving IV spiramycin at 20 mg/kg [12], calves (23.5 L/kg) receiving spiramycin IV at 15 mg/kg [34], and cattle (11.97 L/Kg) receiving spiramycin IV at 15.6 mg/kg [35].
In addition, the clearance (Cl_obs) value of spiramycin obtained from this research (0.68 L/h/kg) was in line with that found in ewes (0.757 L/h/kg) [12]. However, this value

Discussion
Despite extensive marketing of spiramycin in poultry medicine, PK data in birds are absent. To the best of our knowledge, this is the first report about PK/PD modeling of spiramycin against M. synoviae in chickens. The PK analysis in the present study demonstrated that following IV injection of spiramycin in chickens at 17 mg/kg, the apparent volume of distribution (Vz_obs) was 3.3 L/kg which illustrated its wide distribution in the intracellular and extracellular compartments of the body. This finding coincides with the view of Riviere [33] who indicated that a Vz higher than 1 L/Kg represents a wide diffusion of the drug throughout the tissues. The high lipid solubility and the moderate plasma protein binding features of spiramycin are probably the main reasons beyond its high Vz. However, the value found for Vz in this study was lower than that reported in Pathogens 2021, 10, 1238 7 of 14 pigs (5.6 L/Kg) receiving IV spiramycin at 10 mg/kg [11], ewes (16.56 L/kg) receiving IV spiramycin at 20 mg/kg [12], calves (23.5 L/kg) receiving spiramycin IV at 15 mg/kg [34], and cattle (11.97 L/Kg) receiving spiramycin IV at 15.6 mg/kg [35].
In addition, the clearance (Cl_obs) value of spiramycin obtained from this research (0.68 L/h/kg) was in line with that found in ewes (0.757 L/h/kg) [12]. However, this value was lower than that published for cows (1.18 L/h/kg) [7], and pigs (2.6 L/h/kg) [11] receiving IV spiramycin at 10 mg/kg. These variations may be due to the species difference.
The findings of the present study displayed a short elimination half-life (T 1/2λz ) (3.97 h) for spiramycin after IV injection, which reflected its rapid distribution into tissues or elimination in chickens. This T 1/2λz was shorter than that reported in cows (8.61 h) [7]. This disparity may be explained by the species difference in the metabolic rate. Poultry are known to have a higher metabolic rate than mammals, which may account for the rapid metabolism of spiramycin in birds [36]. The AUC extrapolated to infinity was 30.25 µg*h/mL after IV administration. A similar value was observed in ewes (28.22 µg*h/mL) [11]. Following oral administration, spiramycin was rapidly absorbed from the gut with a peak plasma concentration (C max ) of 4.78 µg/mL achieved at 2 h (the time to accomplish C max (T max )). These results are consistent with Nielsen and Gyrd-Hansen [11], who reported a C max of 5.2 µg/mL attained at 2 h after oral administration of spiramycin at 55 mg/kg in pigs. Moreover, in pigs receiving spiramycin in feed at 85 mg/kg. a C max of 4 µg/mL was reported [37]. On the basis of the calculated AUC 0-last from the present study, the oral bioavailability of spiramycin in chickens was calculated as 77.18%. This bioavailability is comparable to that reported for pigs (60%) [11]. Furthermore, the mean residence time (MRT) (6.63 h) was similar to that observed in pigs (7.2 h) [11].
Previous reports have elucidated the importance of plasma protein binding in PK as it plays a pivotal role in the distribution and the excretion of drugs [38,39]. Since the ratio of plasma protein binding differs between animal species, it is crucial to consider protein binding in the target species in the adjustment of volume and clearance [40,41]. A considerable variation was noticed in plasma protein binding among members of the macrolide antibiotic family [42]. Herein, spiramycin was 34.6% bound to plasma proteins in chickens. This ratio was comparable to that previously recorded for tylosin (32.40%), a macrolide antibiotic, in duck plasma [43]. However, this percentage was higher than that reported for tylvalosin (13%) in turkey plasma [44]. To the best of our knowledge, this is the first report regarding the plasma protein biding of spiramycin.
Although macrolides have been recognized as time-dependent drugs [45,46], recent studies have proved a concentration dependency for some members of this class. Tulathromycin expressed a concentration-dependent action against Pasteurella multocida [47]. Moreover, Huang et al. [48] reported a concentration-dependent activity for tilmicosin against Mycoplasma gallisepticum in vitro. Moreover, a few macrolides (e.g., azithromycin) exhibit both a time-and concentration-dependent killing effect [49]. Prior investigation revealed that the kind of bacterial killing effect (either time-or concentration-dependent activity) of antibiotics may vary under different circumstances [50]. In the present study, the dynamics of the bactericidal effect exerted by spiramycin were both time-and concentrationdependent. These observations are in agreement with our previous study [43] in which tylosin, a macrolide antibiotic, displayed a time-and concentration-dependent activity against Mycoplasma anatis in vitro. Furthermore, Renard et al. [51] demonstrated a timedependent activity for spiramycin against Staphylococcus aureus.
The time above the MIC (%T > MIC) index is markedly correlated with the antimicrobial efficacy of macrolides [52,53]. Nevertheless, for azithromycin, the AUC 24h /MIC ratio is the favorable PK/PD parameter utilized to elucidate its antibacterial activity [54]. In the present study, the PK/PD findings demonstrated that the AUC 24h /MIC had the best correlation with the anti-mycoplasmal activity (correlation coefficient (R 2 ) = 0.9628). In accordance with that of Emam et al. [55], the MIC of spiramycin against M. synoviae strain (MS WVU-1853) was estimated to be 0.0625 µg/mL. On the basis of this MIC, the obtained AUC 24h /MIC for mycoplasmacidal activity (185 h), the evaluated unbound portion of spiramycin in chicken plasma (65.4%), and the oral bioavailability (77.1%), the appropriate daily dosage of spiramycin needed to achieve a mycoplasmacidal effect (2 log 10 decline in the Mycoplasma count) against M. synoviae in chickens was calculated to be 15.6 mg/kg/day. This calculated dose is comparable to the one recommended by the manufacturer (17 mg/kg/day) for the treatment of mycoplasmosis in poultry.
As presented in Figure 4, the administered spiramycin dose (17 mg/kg) could achieve mycoplasmastasis, mycoplasmacide, and mycoplasmal elimination. These results are consistent with those of Renaudin et al. [56] who reported a bactericidal activity of spiramycin against Mycoplasma pneumoniae in vitro. In addition, tilmicosin produced a mycoplasmacidal effect against M. gallisepticum in vitro [48].
In this research, the tissue depletion study for spiramycin after oral administration at 17 mg/kg/day for 7 successive days showed that spiramycin was highly concentrated in the liver, followed by the muscle, whereas the fat and skin tissue had the lowest level. Spiramycin was quantifiable in the liver, muscle, and skin and fat tissue up to eleventh, ninth, and seventh day post-medication, respectively. The persistence of spiramycin residues in these tissues for a long time may be attributable to its high tissue penetration property [11]. Our findings are in concordance with the data exhibited in the CVMP report [57], which revealed that after providing spiramycin at 0.8/L in drinking water for chickens for 3 days, spiramycin became undetectable in muscle and skin and fat; spiramycin was undetectable in liver at the 10th day post-last dose.
Considering the computed withdrawal period of spiramycin in the present study for the liver, muscle, and skin and fat tissue, a preslaughter clearance time of 11 days is recommended after oral administration of spiramycin at 17 mg/kg for 7 days in chickens to ensure consumers' safety. Along the same lines, Alsafi and Kafi [32] recommended that chickens not be slaughtered until 8 days after the end of the treatment with spiramycin at 15 mg/kg orally for 5 consecutive days.
In conclusion, our present findings suggested a dose of 15.6 mg/kg/day for spiramycin to be used clinically against M. synoviae in chickens. Additionally, a note should be added on the package of spiramycin indicating that an 11-day withdrawal period is required after its administration orally to ascertain the secure consumption of chicken edible tissues. However, this recommended dose is dependent on the findings of the MIC of one strain of M. synoviae; it would be preferable to incorporate many diverse strains of M. synoviae to take into account the variation in the sensitivity to spiramycin. Moreover, further studies are warranted to assess the PK and the activity of spiramycin in M. synoviaeinfected chickens as the PK characteristics of antimicrobial agents may vary between the healthy and infected chickens.

Materials
The current research was executed using spiramycin injectable solution (Mayco Spira ® , 540,000 IU/mL corresponding to 168.75 mg/mL spiramycin as spiramycin adipate, Laboratorios Maymó, S.A., Barcelona, Spain); it was diluted with sterile 0.9% NaCl to make 56 mg/mL spiramycin for the animal experiment. The reference standard of spiramycin was procured from Sigma-Aldrich Co. (St. Louis, MO, USA). Phosphoric acid, methanol, n-hexane, acetonitrile, and potassium monobasic phosphate were manufactured by Thermo Fisher Scientific (Waltham, MA, USA). Ammonium acetate and chloroform were bought from Merck (Darmstadt, Germany). All utilized chemicals were of high-performance liquid chromatography (HPLC) analytical grade. Purification of water for HPLC was performed employing a Milli-Q system (Waters Corp., Milford, MA, USA). M. synoviae reference strain (MS WVU-1853) was provided by Animal Health Research Institute, Giza, Egypt. M. synoviae medium base was produced following the method of Frey et al. [58].

Experimental Animals
This investigation was performed using 54 healthy Hubbard broiler chickens of both sexes, nearly 4 weeks old and weighing 1-1.5 kg. Chickens were purchased from the Faculty of Agriculture, Mansoura University, Egypt. They were housed individually in cages under hygienic conditions at 25-27 • C and 60 ± 10% relative humidity with a 12-h light/dark cycle.
The birds were offered a medication-free diet with ad libitum access to water. They underwent a 2-week acclimation period preceding the study. The Research Ethics Committee of the Faculty of Veterinary Medicine, Mansoura University, Mansoura, Egypt, approved all animal procedures (Approval No. R/88).

Experimental Design
For the PK study, 12 chickens were assigned randomly to 2 groups, with 6 chickens in the oral group and 6 chickens in the IV group: 3 females and 3 males. Spiramycin was administered at a single dose of 17 mg/kg [32, 35, and as recommended by the manufacturer] directly into the crop by gavage and into the right wing vein of chickens in the oral and IV groups, respectively. Blood (1 mL) was collected from the left wing vein into heparinized tubes before spiramycin dosing (0 h) and at 0.5, 0.75, 1, 1. For tissue residue study; 42 chickens were given spiramycin orally by gavage at 17 mg/kg once daily for 7 consecutive days [59]. After the 7th day of dosing, 6 (3 females and 3 males) chickens were slaughtered on 1st, 4th, 7th, 9th, 11th, 13th, and 15th days, respectively, post-spiramycin administration. Tissue samples (breast muscle, abdominal skin with fat, and liver) were collected from each chicken and were kept at −20 • C until analysis.

Extraction of Plasma Samples
The extraction procedures of spiramycin from plasma samples were performed as previously described [11] with minor modification. In brief, a 0.5 mL plasma sample was mixed with 250 µL of 0.2 M potassium phosphate buffer (pH 5.8) and 1.5 mL chloroform, and was vortexed for 1 min. Thereafter, centrifugation of the mixture was performed for 20 min at 2600× g. Then, the upper aqueous phase was discarded. After evaporation of the chloroform layer to dryness at 40 • C under air stream, the residue was redissolved in the mobile phase (0.05 M phosphoric acid-acetonitrile (75: 25, pH 3.0, v/v)). After the extraction of the analyte, 100 µL of the sample was injected into the HPLC device.

Extraction of Tissue Samples
Spiramycin was extracted from tissue samples as previously described [31]. After extraction, 100 µL of the sample was injected into the HPLC apparatus.

Chromatographic Conditions
The analysis of spiramycin in plasma and tissue samples was conducted by a method adapted from Lee et al. [31] with minor modifications. Spiramycin concentrations were measured using HPLC Agilent Series 1200 quaternary pump, Series 1200 autosampler, Series 1200 UV VIS detector set at 232 nm, and HPLC 2D ChemStation software (Hewlett-Packard, Les Ulis, France). Separation was achieved using a Phenomenex C18 column (5 µm, 250 × 4.6 mm) at 35 • C and 0.05 M phosphoric acid-acetonitrile in the ratio of 75: 25 (pH 3.0, v/v) as the mobile phase at a flow rate of 1.5 mL/min. The retention time was 3.17 min. The HPLC technique was validated (by determining recovery, precision, sensitivity, and linearity) as stated in the European Medicines Agency report [60] (Table 4). The lower limits of detection and quantification of spiramycin were 0.003 and 0.01 µg/mL in plasma, and 0.02 and 0.05 µg/g in tissue samples, respectively. Data for recovery are presented as mean ± SEM. Average recovery % for plasma (using spiked concentrations in the range of 0.01-20 µg/mL in triplicate analysis). Average recovery % for muscle, skin, fat, and liver tissues (using spiked concentrations in the range of 0.05-20 µg/g in triplicate analysis. Intra-day relative standard deviation (RSD) and inter-day RSD % for plasma (0.01 µg/mL, n = 6). Intra-day RSD and Inter-day RSD % for muscle, skin and fat, and liver tissues (0.05 µg/g, n = 6).

Plasma Protein Binding Determination
In vitro protein binding assay was performed utilizing plasma samples obtained from chickens before spiramycin administration. The ultrafiltration method was employed as previously reported [61]. Five replicates of 1 mL plasma samples were spiked with spiramycin at concentrations of 0.05, 0.1, 0.5, 1, and 2.5 µg/mL. One mL of each sample was placed in a Pierce protein concentrator PES, 30 MWCO (Thermo Fisher Scientific, USA). Centrifugation of samples was carried out at 2000× g for 10 min. Plasma samples and ultrafiltrates were injected into HPLC column for analysis. The ratio of plasma protein binding was determined using the following equation: Protein binding (%) = (Total concentration − Ultrafiltrate concentration) × 100 Total concentration (1)

Determination of MIC of Spiramycin against M. synoviae
The MIC of spiramycin against M. synoviae strain (MS WVU-1853) was determined utilizing a previously described method [48,62]. In brief, cultures of M. synoviae were diluted with the mycoplasma medium to 10 7 CFU/mL. A series of spiramycin concentrations were prepared in 96-well plate (final concentration, 0.03125 to 32 µg/mL). M. synoviae at 10 7 CFU/mL was added into each well in a similar amount of medium (100µL). In addition, a sterility control (sterile M. synoviae broth at pH 7.8), a growth control (M. synoviae inoculum without antimicrobial), and an end point control (blank medium at pH 6.8) were also utilized. The humidified incubator was set at 37 • C, and 5% CO 2 for the cultured plates. The MIC was evaluated as the lowest concentration of spiramycin that caused no change in the color of culture medium. All the experiments were conducted in triplicate.

In Vitro Time-Killing Curve
The in vitro time-killing investigations were carried out as reported [63]. Briefly, spiramycin was used at 0, 0.5, 1, 2, 4, 8, 16, 32, and 64 MIC. In a penicillin bottle, the culture system consisted of 3.5 mL blank medium, 0.1 mL of spiramycin solution, and 0.4 mL suspension of M. synoviae in the exponential phase. The final inoculum was 10 7 CFU/mL. The penicillin bottles were kept in the incubator for 48 h with 5% CO 2 , and at 37 • C. Samples (100 µL) were obtained from each bottle at 0, 2, 12, 15, 18, 24, 36, and 48 h, to quantify the viable counts of M. synoviae. Each spiramycin concentration was examined thrice. For establishing the in vitro time-killing plot, the mean values of log 10 CFU/mL (n = 3) were drawn against time (h) with various spiramycin concentrations.

Ex Vivo Time-Killing Curve Determination
The determination of the ex vivo anti-mycoplasmal activity of spiramycin was conducted utilizing plasma samples collected from healthy chickens at 0, 0.5, 0.75, 1, 1.5, 2, 3, 4, 6, 8, 12, 15, 24, 36 h post-oral spiramycin administration. The viable counts of M. synoviae were determined using the above-mentioned technique for the in vitro time-killing experiments. The ex vivo time-killing curve illustrated the relationship between the mean values of log 10 CFU/mL (n = 6) in plasma samples collected at various time points from chickens receiving spiramycin and the time of incubation (h).

Assessment of PK Properties and Withdrawal Time Determination
Plasma spiramycin concentrations vs. time plots were investigated for each chicken using a non-compartmental approach [11]. The PK analysis was conducted utilizing WinNonlin 8.3 software (Certara, USA). Following oral administration of spiramycin, the C max and the T max ¬ were determined. The T 1/2λz was calculated via the equation T 1/2λz = 0.693/λz, where λz, the first order rate constant, was defined from the slope of the terminal phase of the concentration vs. time curve. The linear trepazoidal rule and the linear-up log-down method were used to calculate AUC 0-∞ after IV and oral administrations of spiramycin, respectively. The Cl-obs was determined as Cl-obs = dose/AUC. In addition, the Vz-obs was calculated as Vz = dose/(λz × AUC). The F was obtained by dividing mean AUC oral by mean AUC IV .
The concentrations of spiramycin in the collected tissues were utilized to determine preliminary withdrawal times using linear regression approach by applying WT 1.4 software, designed in Germany and licensed by the CVMP of EU as previously reported [64]. The withdrawal time (WT) was defined to be the time when the upper one-sided tolerance limit (95%) with 95% confidence interval was below the MRL of spiramycin which was determined by CVMP in chicken tissues to be 0.2 µg/g for muscle, 0.3 µg/g for skin and fat, and 0.4 µg/g for liver [29].

PK/PD Integration and Dose Computation
For PK/PD modeling, the AUC 24h /MIC values were computed based on the area under the plasma concentration curve over 24 h divided by the MIC. The AUC 24h values were determined by multiplying the plasma spiramycin concentration at each time point of collection after oral administration by the incubation period (24 h) [47,65].
The sigmoidal inhibitory E max model [WinNonlin 8.3 software (Certara, USA)] was applied to correlate the ex vivo AUC 24h /MIC with the log 10 difference between the initial M. synoviae count and that after 24 h of incubation. This model was elucidated by the following equation: where E denotes the anti-mycoplasmal activity measured as the change in the M. synoviae count (log 10 CFU/mL) in the plasma sample after 24 h, compared to the baseline log 10 CFU/mL (the growth without spiramycin; E max points out the maximum antimycoplasmal effect; E 0 , reflects the variation in M. synoviae count in the control samples (without spiramycin) between 0 and 24 h. EC 50 indicates AUC/MIC ratio producing 50% reduction in M. synoviae counts from the initial count; C represents AUC/MIC in the effect compartment, and γ is the Hill coefficient which accounts for the steepness of the AUC 24h /MIC effect curve. Three levels of anti-mycoplasmal effect of spiramycin were assessed by estimating the values of AUC 24h /MIC required for bacteriostatic action (E = 0), bactericidal action (E = −2 and E = −3), and for bacterial elimination (E = −4).
The appropriate daily dose of spiramycin in chickens was calculated on the basis of the following equation: In which AUC 24h /MIC elucidates the ex vivo AUC 24h /MIC value for optimal activity; MIC indicates the minimum inhibitory concentration; CL reflects the clearance; F is the bioavailability, and fu refers to the free part of spiramycin in plasma

Statistical Analysis
Data are presented as mean ± SEM. A Shapiro-Wilk test was used to check the normality of the data. The main PK parameters after oral and IV administrations were compared using Wilcoxon's rank sum test. Furthermore, the concentrations of spiramycin were statistically analyzed in different tissues utilizing ANOVA. Mean comparisons were performed using Tukey's test. Differences were considered significant when p < 0.05. Statistical comparison was carried out by Statistical Package for Social Science (SPSS), version 20 (SPSS Inc., Chicago, IL, USA).

Data Availability Statement:
The data sets generated for this study are available from the corresponding author upon reasonable request.