Tilmicosin was administered intravenously and subcutaneously at a dose rate of 10 mg/kg bwt to determine its concentration in blood and bronchial secretion as well as its kinetic behavior in healthy and Pasteurella haemolytica type A1-infected calves. Sever acute bronchopneumonia was induced in 10 calves by inoculating them intra-tracheally with P. haemolytica type A1. The calves were treated with tilmicosin; 5 of these received the drug intravenously and the other 5 were injected subcutaneously. After a slow intravenous injection, the serum concentration-time curve indicated a two compartment open model with a mean elimination half-lives (t1/2bs) of 22.09 and 22.14 hours before and after infection, respectively. The mean residence time (MRT) corrected for a bolus injection was 2.25 and 2.20 hours and the mean MRTinf was 25.27 and 25.46 hours in healthy and P. haemolytica-infected calves, respectively. After subcutaneous injection, the drug was eliminated more slowly (before and after infection) from serum and bronchial secretions, with t1/2bs of (24.60 and 25.85 hours) and (33.74 and 31.78 hours), respectively. The apparent volume of distribution (Vd(area)) of tilmicosin was more than 1 litre·kg-1. The peak serum and bronchial secretions of tilmicosin concentration were (1.33 and 1.36 mg·ml-1) and (1.40 and 1.70 mg·ml-1) attained at (7.21 and 7.15 hours) and 7.11 and 7.10 hours) after subcutaneous injection, respectively. Tilmicosin was good secreted into bronchial secretions having AUCbronchial secretion/ AUCserum ratio of approximately 1:1.24 and 1:1.22 in healthy and P. haemolytica-
Bronchopneumonia is one of the most economically important respiratory diseases of calves reared indoors [
Micotil injection, 300 mg tilmicosin per ml, is administered as a single subcutaneous injection at a dose rate of 10 mg/kg body weight. Tilmicosin has proven particularly effective in the treatment of bovine respiratory disease (BRD) [
This study was undertaken to investigate the efficacy, disposition, distribution pattern and the penetration of tilmicosin into respiratory tract secretions of clinically healthy and experimentally P. haemolytica-infected calves after its subcutaneous and intravenous administration.
Tilmicosin (Micotil injection, 300 mg/ml) was supplied by Elanco Animal Health, England).
Ten calves aged one-and-a-half months, and weighing 60 to 70 kg were used in the experimental work. The calves were housed individually in adjacent boxes which had a floor area of 4 m2 and a volume of 6 m3. The animals were bedded on straw. They were fed on milk replacer (free of antimicrobial substances twice daily at 09.00 and 18.00) and alfalfa with drinking water available ad libitum. The animals were shaved over jugular vein to facilitate the collection of blood samples.
The calves were allocated, randomly to two groups of 5 animals each. The first group was treated with tilmico- sin by the intravenous route and the second group was treated with tilmicosin subcutaneously. Tilmicosin solu- tion (300 mg/ml) was diluted in saline to 2.5 mg/ml and the diluted solution was administered by slow intra- venous infusion into the left jugular vein for 20 minutes at the rate of 0.5 mg/kg/min at a total dose of 10 mg/kg [
Two weeks later after the intravenous and subcutaneous injection of tilmicosin, the calves were subjected to physical stress of a two-hour journey before they were inoculated with the pathogen. On day 0, approximately six hours after journey, all the calves were inoculated intratracheally with P. haemolytica type A1 (LPB 1419) [
The first and second groups were treated with tilmicosin (10 mg/kg body weight) by the intravenous infusion and subcutaneous injections as mentioned before. Treatment was initiated when the calves had a body tempera- ture of more than 39.5˚C and a respiration rate of more than 52/min.
The calves were examined twice a day for 10 days (5 days before and 5 days after infection) at 09.00 and 18.00. Body temperature and respiratory rate were recorded daily at 10.00.
Blood samples were obtained from the catheterized right jugular vein before and after infection. Samples were collected in heparinized tubes immediately before and at 1, 2, 4, 6, 8, 12, 24, 48, 72 and 96 hours following i.v. and s.c injections. Samples were centrifuged at 3000 rpm for 15 minutes and the obtained sera were used for the estimation of tilmicosin concentration. The serum samples were stored at −80˚C until analysis, and the assay was performed within a week of obtainment.
Bronchial secretions were collected from the calves at the same intervals of the blood samples following tilmi- cosin administration before and after infection. Bronchial secretions (0.5 ml) were collected by a tampon-device introduced into the principal bronchi through the tracheal tube [
Another blood samples were collected in vacutainer tubes (Venoject, Terumo) containing EDTA as an anti- clotting agent, once daily on the day of the inoculation (day 0) and on the following five days at 10.00 after drug administration. Total white cell counts and granulocyte/agranulocyte ratios were determined by standard me- thods.
BAL fluid samples were obtained from infected animals on day 0, 3, 4 and 5 after tilmicosin administration to infected animals. A polyethylene tube (Intramedic, VEL) with an external diameter of 4.8 mm was inserted through the right nostril and advanced into trachea and the bronchial tree until an elastic resistance felt. Fifty ml of a sterile 0.9% solution of sodium chloride at 37˚C was injected and aspirated immediately by gentle suction; approximately 75% of the infused fluid could be retrieved [
From each BAL fluid sample, 10 ml was stored in sterile plastic tubes at 4˚C and examined for the presence of bacteria and mycoplasmas within two hours after collection. All the samples were inoculated onto three me- dia: Colombia blood agar (Oxoid), PPLO agar (Difco) enriched with 25% inactivated horse serum, 7% yeast ex- tract, 400 mg/ml ampicillin, 0.05% thallium acetate and 1% glucose and Tween 80 PPLO agar (the same en- riched and selective medium with 0.1% Tween 80) [
The free tilmicosin concentrations in serum and bronchial secretions were measured by a microbiological assay technique [
The concentrations of tilmicosin in serum and bronchial fluid were subjected to kinetic analysis and the phar- macokinetic parameters were calculated for each animal by classical methods [
Obtained data was analyzed by analysis of variance, and the mean values were compared by Duncan’s Multiple Range test and student’s “t” test (LSD) using the SAS statistical analysis program [
The slow intravenous infusion of a dilute solution of tilmicosin resulted in clinical signs suggesting acute car- diac toxicity which disappeared 25 minutes later. No side effects were observed after the subcutaneous injection.
The mean concentrations of tilmicosin in serum and bronchial secretions after tilmicosin injection in healthy and P. haemolytica-infected calves are shown in
Values for the kinetic parameters describing the disposition of the drug are given in
The serum and bronchial secretions concentration (µg/ml)-time profile of tilmicosin (10 mg/kg bw) after IV administration after tilmicosin injection in healthy and P. haemolytica-infected calves. Data are presented as the mean ± SD
The serum and bronchial secretions concentration (µg/ml)-time profile of til- micosin (10 mg/kg bw) after SC administration after tilmicosin injection in healthy and P. haemolytica-infected calves. Data are presented as the mean ± SD
. Mean (SD) pharmacokinetic parameters of tilmicosin in serum (S) and bronchial secretions (BS) after a slow intravenous infusion of 10 mg∙kg−1 body weight in healthy and Pasteurella haemolytica-infected calves (n = 5)
Parameter | Healthy S BS | Infected S BS | LSD (Pr > F) | ||
---|---|---|---|---|---|
C˚ (μg∙ml−1) | 6.47 (0.38)c | 7.43 (0.12)a | 6.96 (0.14)b | 7.47 (0.24)a | 0.323 (0.0001) |
A (μg∙ml−1) | 5.27 (0.29)c | 6.12 (0.10)a | 5.81 (0.07)b | 5.79 (0.16)b | 0.234 (0.0001) |
α (h−4) | 0.71 (0.03)a | 0.64 (0.02)b | 0.70 (0.02)a | 0.56 (0.01)c | 0.026 (0.0001) |
t1/2(a) (h) | 2.70 (0.10)c | 2.99 (0.07)b | 2.76 (0.09)c | 3.49 (0.05)a | 0.105 (0.0001) |
B (μg∙ml−1) | 1.20 (0.12)bc | 1.29 (0.07)b | 1.15 (0.08)c | 1.69 (0.09)a | 0.122 (0.0001) |
b (h−2) | 0.32 (0.03)a | 0.32 (0.01)a | 0.32 (0.02)a | 0.34 (0.01)a | 0.025 (0.2311) |
t1/2(b) (h) | 22.09 (1.96)a | 21.72 (0.99)a | 22.14 (1.10)a | 20.53 (0.33)a | 1.658 (0.1821) |
Vc (Litre∙kg−1) | 1.55 (0.09)a | 1.35 (0.02)c | 1.44 (0.03)b | 1.34 (0.04)c | 0.071 (0.0001) |
Vd(area) (Litre∙kg−1) | 4.28 (0.12)a | 3.29 (0.08)c | 3.83 (0.06)b | 2.84 (0.10)d | 0.124 (0.0001) |
Vd(ss) (Litre∙kg−1) | 1.90 (0.09)a | 1.63 (0.02)c | 1.72 (0.02)b | 1.72 (0.05)b | 0.090 (0.0001) |
Cl(B) (Litre∙kg−1∙h−1) | 0.14 (0.01)a | 0.11 (0.004)c | 0.12 (0.01)b | 0.10 (0.003)d | 0.009 (0.0001) |
AUC (μg∙h−1∙Litre−1) | 73.95 (6.18)d | 95.19 (3.66)b | 83.50 (3.43)c | 104.35 (2.76)a | 5.643 (0.0001) |
AUMC (μg∙h−1∙Litre−1) | 155.1 (44.01)a | 185.1 (51.78)a | 169.6 (32.65)a | 202.0 (18.53)a | 52.04 (0.2934) |
AUMCinf (μg∙h−1∙Litre−1) | 2941 (240.17)a | 2118 (525.58)bc | 2525 (186.16)ab | 1860 (189.82)c | 427.5 (0.0004) |
MRT (h) | 2.25 (0.44)a | 2.11 (0.49)a | 2.20 (0.33)a | 2.11 (0.18)a | 0.508 (0.9107) |
MRTinf (h) | 25.27 (1.45)a | 26.60 (4.72)a | 25.46 (1.41)a | 28.36 (1.48)a | 3.58 (0.2752) |
a,b,c,dMeans with the same letter in the same row are not significantly different. LSD = Least significant difference.
more than 1 L/kgindicating good penetration into tissues and bronchial secretions. The total body clearance of the drug was 0.14 and 0.12 L/kg/hin healthy and P. haemolytica-infected calves, respectively. The mean MRT corrected for a bolus injection was 2.25 and 2.20 hours; the mean MRTinf was 25.27 and 25.46 hours and the mean Vd(ss) was 1.90 and 1.72 L/kg in healthy and P. haemolytica-infected calves, respectively.
The mean peak concentration of tilmicosin in serum (Cmax, 1.33 and 1.36 mg/ml) and bronchial secretions (1.4 and 1.7 mg/ml) was reached in (7.21 and 7.15 hours) and (7.11 and 7.1 hours) after subcutaneous injection in healthy and P. haemolytica-infected calves, respectively. P. haemolytica infection significantly increased the mean peak concentration of tilmicosin in bronchial secretions as compared with pre-infection values.
The mean body temperature of the calves is shown in
The mean respiratory rate of the calves is shown in
The means and ranges of the total WBC counts recorded in the two groups of calves on the day of the intratra- cheal challenge and on the five days after inoculation are shown in
. Mean (SD) pharmacokinetic parameters of tilmicosin in serum (S) and bronchial secretions (BS) after a single subcutaneous injection of 10 mg·kg−1 body weight in healthy and Pasteurella haemolytica-infected calves (n = 5)
Parameter | Healthy S BS | Infected S BS | LSD (Pr > F) | ||
---|---|---|---|---|---|
t1/2(ab) (h) | 7.72 (1.07)a | 7.37 (0.19)ab | 6.87 (0.38)b | 8.11 (0.27)a | 0.791 (0.0256) |
Cmax (μg∙ml−1) | 1.33 (0.25)b | 1.40 (0.04)b | 1.36 (0.05)b | 1.70 (0.08)a | 0.180 (0.0016) |
tmax (h) | 7.21 (0.11)a | 7.11 (0.21)a | 7.15 (0.16)a | 7.10 (0.08)a | 0.198 (0.6369) |
t1/2(b) (h) | 24.60 (0.43)b | 33.74 (1.86)a | 25.85 (1.39)b | 31.78 (2.05)a | 2.096 (0.0001) |
AUC (μg∙h−1∙l−1) | 67.58 (4.16)c | 83.7317.53)ab | 70.24 (8.24)bc | 84.91 (8.14)a | 14.36 (0.0397) |
AUCBS/AUCS | 1.24 (0.26)a | 1.22 (0.20)a | 0.338 (0.9196) | ||
F (AUCSC/AUCIV) | 0.916 (0.02)a | 0.875 (0.16)a | 0.839 (0.07)a | 0.813 (0.06)a | 0.122 (0.3401) |
a,b,cMeans with the same letter in the same row are not significantly different. LSD = Least significant difference.
. Mean (SD) and [ranges] of body temperature (TEMP, ˚C) and respiratory rates (RESP, min−1) in groups of 5 calves infected experimentally with Pasteurella haemolytica type A1 and treated with tilmicosin either slow intravenously or by subcutaneously
Time (days) | Measurement | Intravenous | Subcutaneous |
---|---|---|---|
0 | TEMP RESP | 38.00 (0.10) [37.9 - 38.1]e 29.80 (1.48){28 - 32]e | 38.00 (0.14) [37.8 - 38.2]e 30.00 (1.23) [29 - 32]d |
1 | TEMP RESP | 40.16 (0.11) [40.0 - 40.3]a 82.00 (1.58) [80 - 84]a | 40.10 (0.10) [40.0 - 40.2]a 79.80 (2.39) [77 - 83]a |
2 | TEMP RESP | 39.72 (0.19) [39.5 - 40.0]b 59.80 (3.83) [55 - 64]b | 39.28 (0.13) [39.1 - 39.4]b 49.80 (2.86) [46 - 53]b |
3 | TEMP RESP | 39.40 (0.10) [39.3 - 39.5]c 50.20 (2.39) [47 - 53]c | 39.00 (0.16) [38.8 - 39.2]c 39.80 (2.86) [36 - 43]c |
4 | TEMP RESP | 39.30 (0.16) [39.1 - 39.5]cd 44.40 (3.36) [40 - 48]d | 38.88 (0.19) [38.6 - 39.1]c 31.60 (1.14) [30 - 33]d |
5 | TEMP RESP | 39.18 (0.15) [39.0 - 39.4]d 42.60 (1.67) [40 - 44]d | 38.70 (0.70) [38.6 - 38.8]d 29.80 (1.92) [27 - 32]d |
MSE | TEMP RESP | 0.020 6.533 | 0.019 4.767 |
a,b,c,dMeans with the same letter in the same column measurement are not significantly different at (P > 0.0001). MSE = Minimum significant error.
counts returned to the pre-inoculation values on day 2 after the infection in the calves treated subcutaneously and on day 3 after inoculation in the slow intravenously-infused calves.
Severe granulocytosis occurred on the first day after the infection in comparison with the pre-inoculation data, but it persisted for only two days after the inoculation in intravenously treated calves.
The results of the mean Neutrophil/Macrophage (N/M) ratio in the BAL fluid are shown in
P. haemolytica type A1 was not isolated from the BAL fluid samples before the intratracheal challenge. Fur- thermore, no other respiratory pathogens were isolated from of the calves before the infection. P. haemolytica type A1 was not isolated from BAL fluid samples of the subcutaneously-treated calves. Mycoplasma bovis was isolated from one of the five intravenously-treated calves on day 5 after inoculation. On all occasions the strains of P. haemolytica type A1 isolated were the same as that inoculated.
The intrinsic antibacterial activity of tilmicosin together with its pharmacokinetic properties after subcutaneous injection suggests that they should be explored in field efficacy trials against P. haemolytica lung infection.
The results of this study showed that the serum and bronchial secretions concentrations of tilmicosin in expe- rimentally P. haemolytica-infected calves remained above the minimum inhibitory concentrations (MICs) for the most sensitive bacteria (A. pyogenes and S. aureus) isolated from cattle, which range from 0.04 to 0.78 mg/ml [
. Mean (SD) and [ranges] of total blood cell counts (WBC, ×103/mm3), blood granulocyte/agranulocyte ratios (G/AG) and BAL fluid neutrophil/macrophage ratios (N/M) in groups of 5 calves infected experimental- ly with Pasteurella haemolytica type A1 and treated with tilmicosin either slow intravenously or by subcuta- neously
Time (days) | Measurement | Intravenous | Subcutaneous |
---|---|---|---|
0 | WBC G/AG N/M | 12.40 (2.41) [9.0 - 15.0]b 0.92 (0.24) [0.6 - 1.2]b 0.15 (0.04) [0.10 - 0.21]b | 12.80 (2.39) [10.0 - 16.0]bc 0.84 (0.27) [0.5 - 1.1]bc 0.31 (0.33) [0.12 - 0.90]a |
1 | WBC G/AG N/M | 27.20 (5.07) [21.0 - 34.0]a 2.62 (0.51) [2.0 - 3.3]a NS | 25.60 (4.45) [20.0 - 31.0]a 2.80 (0.54) [2.1 - 3.5]a NS |
2 | WBC G/AG N/M | 26.20 (4.66) [21.0 - 32.0]a 2.20 (0.57) [1.5 - 2.9]a NS | 15.00 (4.74) [9.0 - 21.0]b 1.30 (0.51) [0.7 - 1.9]b NS |
3 | WBC G/AG N/M | 17.80 (6.01) [10.0 - 25.0]b 1.20 (0.46) [0.4 - 1.6]b NS | 11.20 (2.59) [5.0 - 15.0]bc 0.68 (0.30) [0.3 - 1.1]c NS |
4 | WBC G/AG N/M | 14.60 (2.07) [12.0 - 17.0]b 0.78 (0.24) [0.5 - 1.1]b 0.36 (0.08) [0.26 - 0.47]a | 9.20 (1.04) [8.0 - 10.5]c 0.60 (0.16) [0.4 - 0.8]c 0.23 (0.04) [0.18-0.29]a |
5 | WBC G/AG N/M | 14.00 (1.58) [12.0 - 16.0]b 0.96 (0.21) [0.6 - 1.1]b 0.40 (0.07) [0.30 - 0.49]a | 10.20 (1.30) [9.0 - 12.0]c 0.70(0.16) [0.5 - 0.9]c 0.27 (0.06) [0.20 - 0.36]a |
MSE LSD | WBC G/AG N/M | 16.033 0.159 0.005 | 9.577 0.127 0.038 |
(a,b,c)Means with the same letter in the same column measurement are not significantly different at (P > 0.0001 and P > 0.805 for N/M only). NS = Not sampled. MSE = Minimum significant error. LSD = Least significant difference.
The ion (pH) trapping of such basic molecules in areas of the body with a pH lower than the blood (7.4) can lead to high tissue concentration [
The subcutaneous administration of tilmicosin at 10 mg/kg resulted in free drug concentrations in serum and bronchial secretions which were under the MIC of the drug for P. haemolytica {0.78 to 6.25 or £2 mg/ml [
Following intravenous administration, the serum concentration data were best fitted to a two-compartment pharmacokinetic model. The slow intravenous infusion of a dilute solution of tilmicosin resulted in clinical signs suggesting acute cardiac toxicity but these side-effects were not observed after the subcutaneous injection. Sim- ilar clinical signs were also reported in cattle and goats [
The apparent volume of distribution at steady-state of a drug (Vd(ss)) is an indication of its diffusion into body tissues [
Following subcutaneous administration, the mean apparent elimination half-lives of tilmicosin in serum and bronchial secretions were (24.6 and 25.85 hours) and (33.74 and 31.78 hours) in healthy and P. haemolytica- infected calves, respectively. A similar half-lives (29.3 and 41.4 hours) was reported in serum and milk of healthy goats [
During the clinical efficacy experiments no calves died in the treated groups. Body temperature, respiratory rate, haematological and broncho-alveolar-lavage fluid parameters returned to normal significantly faster in subcutaneously treated group than in the intravenously treated calves. Similar findings were also observed for tilmicosinin pneumonia of calves or pigs [
The results described in this paper are similar to earlier observations that P. haemolytica type A1 is capable of producing severe acute pneumonia in calves [
These preliminary results suggest that subcutaneous injection of tilmicosin can be effectively used in the treat- ment of acute P. haemolytica bronchopneumonia in calves.