In dairy cattle, the purpose of dry cow treatment is to treat and prevent IMIs during the 45- to 60–day period immediately prior to calving in which cows are not milked (dry period).1–3 Dry cow treatment minimizes the number of clinical cases of mastitis during the subsequent lactation.4–6 Thus, the dry period is considered a key intervention point for the prevention of future cases of mastitis and an opportunity to lessen the economic impact of mastitis on dairy farms.7,8 Although it is not without controversy, the most accepted method for the prevention of IMIs in dairy cows during the dry period is the administration of antimicrobials, generally by the intramammary route,3,9 and most commercially available dry-cow treatments contain an antimicrobial. Anecdotal reports suggest that up to 71% to 85% of IMIs present at the time of dry cow treatment (first day of dry period; dry off) will be resolved; however, results of a meta-analysis1 indicate that 37% to 56% of IMIs present at dry off will resolve spontaneously without antimicrobial administration. The cure rate for IMIs caused by Staphylococcus aureus following antimicrobial administration is much lower than that for IMIs caused by other bacterial pathogens and ranges from 27% to 80%.10 The breadth of that range is understandable given the various antimicrobials administered and treatment protocols used and the varying health status of treated cows. Factors that affect the cure rate for IMIs include, but are not limited to, the age of the cow being treated, whether the fore or rear mammary glands (quarters) are infected, overall size of the mammary gland, duration of the current infection, and the IMI history and immune status of the cow being treated.11,12 Intramammary infections caused by S aureus are particularly difficult to treat because the pathogen often becomes sequestered within neutrophils, macrophages, and epithelial cells where it can replicate and remain viable even in the face of antimicrobial administration.11,13
Tilmicosin, a macrolide, has been used as an intramammary infusion (1,500 mg/quarter) to treat cows with IMIs caused by S aureus at dry off, and reported cure rates range from 62% to 74.2%.10,14 Conversely, parenteral administration of tilmicosin (5 mg/kg, SC) to cows with IMIs caused by S aureus at dry off and again 4 days later resulted in a cure rate of only 9.1%.10
A new experimental polymer-based 40% tilmicosin preparation has been described.15 This preparation releases tilmicosin from the thermoreversible poloxamer 407 with zero-order kinetics, which results in a mean t1/2β of 39.8 hours.15 In cows, clinically relevant serum concentrations of tilmicosin are achieved for 8 to 10 days following administration of 1 dose (20 mg/kg, SC). The constant release of tilmicosin from the poloxamer 407 gel formed in situ prevents the buildup of toxic concentrations of the drug in the serum and prevents adverse changes in the heart rate and other signs of cardiac toxicosis. The pharmacokinetics of this experimental preparation of tilmicosin, in addition to the antimicrobial spectrum of tilmicosin (particularly against Staphylococcus spp),16 its immunomodulating capabilities,17 and its ability to readily diffuse into the bovine mammary gland,18 suggests that parenteral administration of this experimental preparation to dairy cows at dry off might be a beneficial dry cow treatment. The purpose of the study reported here was to determine the concentration of tilmicosin in mammary gland secretions of dairy cows following administration of this experimental preparation of tilmicosin (20 mg/kg, SC) once or twice during the dry period and to evaluate its efficacy for the treatment of cows with IMIs caused by S aureus at dry off, compared with that of an intramammary infusion of ceftiofur.
Materials and Methods
Animals
All study procedures and animal care activities were conducted in accordance with Mexican Official Regulations19 under the oversight of the Institutional Committee of Research, Care and Use of Experimental Animals of the National Autonomous University of Mexico. The study was conducted on a commercial dairy farm located in the State of Mexico from January through November 2015. The herd consisted of 1,500 Holstein-Friesian cows with a mean milk production of 23 kg/cow/d. To be eligible for the study, cows had to be clinically normal as determined on the basis of results of a physical examination, have 4 functioning quarters with teats free of substantial lesions (eg, cuts and deformities), have an IMI caused by Staphylococcus spp on the basis of results of microbiological culture of milk samples obtained 5 days prior to dry off, and not have been treated with an antimicrobial during the 30 days prior to dry off. During the 11-month observation period, 360 cows were screened for the study, of which 170 had an IMI caused by Staphylococcus spp and were enrolled in the study at dry off, which was approximately 45 days prior to the expected calving date.
Study design
Cows were divided into 3 groups on the basis of the pathogen causing the IMI (S aureus, Staphylococcus intermedius, or coagulase-negative staphylococci). Within each group, cows were randomly assigned by means of a random number generator to 1 of 3 treatment groups. Cows assigned to the tilmicosin-1 group (n = 58) received the experimental preparation of tilmicosina (20 mg/kg, SC) once at dry off. Cows assigned to the tilmicosin-2 group (n = 56) received the experimental preparation of tilmicosina (20 mg/kg, SC) at dry off and again 20 days later. Cows assigned to the ceftiofur group (n = 56; control) received a commercially available, long-acting, intramammary preparation of ceftiofurb (500 mg/quarter) at dry off. Three 20-mL milk (or colostrum) samples (ie, samples that contained an approximately equal amount of milk from each of the 4 quarters) were obtained for microbiological culture from each cow 5 days prior to dry off, within 24 hours after calving, and 15 and 30 days after calving. Additionally, a sample (5 to 10 mL) of mammary gland secretions was obtained for determination of tilmicosin concentration from each quarter of each of 5 randomly selected cows from each treatment group once every 5 days after dry off until calving.
Microbiological culture and determination of SCC
Milk or colostrum samples for microbiological culture and determination of SCC were collected aseptically as described by the NMC.20 Briefly, each teat was disinfected with chlorhexidine solution. Then, 20 to 30 mL of milk was stripped and discarded from each teat before approximately 8 mL of milk from each teat was stripped into each well of a CMT paddle for performance of the CMT. Each teat was disinfected with chlorhexidine again, and three 20-mL milk samples were collected into sterile 50-mL sample collection tubes. Milk samples were immediately submitted for microbiological culture. Each sample was stirred with a vortex for 5 minutes to achieve a homogenous sample. Then, 15 to 20 μL of the sample was streaked onto a blood agar culture plate and MacConkey agar culture plate with the quadrant streak method. The plates were incubated in aerobic conditions at 37°C for 48 hours.21 Intramammary pathogens were distinguished from contamination or microbiota by the presence of at least 3 grossly similar colonies in each of at least 3 of the 4 quadrants of the culture plate as described by Carter.22 Bacterial colonies were initially categorized on the basis of their morphological features, gram stain result, catalase reaction result, and hemolytic ability. Gram-negative bacilli underwent an oxidase test, and then further species identification was performed as described22 by the conduction of the triple sugar iron test and the citrate-urea-sulfide-indole motility test. Gram-positive cocci suspected of being S aureus were subjected to coagulase, CAMP, and esculin tests. Samples with > 3 bacterial species identified were considered contaminated and were excluded from further analyses. An electronic cell counterc was used to determine the SCC in each milk sample. A CMT test was not performed on cows within 24 hours after calving because colostrum causes false-positive results. Likewise, the SCC was not determined for colostrum samples collected within 24 hours after calving.
An IMI at dry off was defined as the isolation of ≥ 3 CFUs of a Staphylococcus spp/15 to 20 μL of inoculum (milk) in at least 2 of the 3 samples cultured. An IMI was considered cured when a previously identified pathogen was not cultured in any of the samples collected on the subsequent 3 sampling days (within 24 hours after calving and 15 and 30 days after calving). A new IMI was defined as the isolation of a Staphylococcus spp from a gland that had previously cultured negative for that organism.
Dry cow treatment administration
At dry off, the cows assigned to the tilmicosin-1 and tilmicosin-2 groups received a polymer-based preparation of 40% tilmicosin phosphate designed for extended actiona (experimental preparation; 20 mg/kg, SC). The drug was administered in the lateral aspect of the neck just cranial to the shoulder. The volume of the required dose for each cow ranged between 20 and 30 mL; therefore, each dose was divided into 3 approximately equal volumes and was injected by use of a 10-mL syringe with a 16-gauge 3-cm needle. No more than 10 mL of the drug was administered per injection site. The cows assigned to the tilmicosin-2 group received a second dose of the experimental preparation 20 days later. The cows assigned to the ceftiofur group received an intramammary infusion of ceftiofur hydrochloride sterile suspensionb (500 mg/quarter) at dry off. Each quarter was infused in an aseptic manner after the cow was milked. Briefly, the entire udder was washed with warm water that contained a chlorhexidine-based antiseptic,d with particular attention paid to cleansing each teat. Each teat was dried with a paper towel. Then a separate alcohol pad was used to wipe the end of each teat immediately before the ceftiofur suspension was infused into the streak canal. Aside from dry cow treatment administration and collection of mammary gland secretion samples for determination of tilmicosin concentration, all study cows were managed in accordance with the herd's standard husbandry practices for cows during the dry period, which included isolation of cows immediately after dry off for daily observation to detect mastitis or other diseases and a diet change.
Determination of tilmicosin concentration
Mammary gland secretions (5 to 10 mL) were aseptically collected from each quarter from each of 5 randomly selected cows from each treatment group every 5 days after dry off until calving. No cow was sampled more than once during the dry period. Mammary gland secretions (5 to 10 mL) were collected from each quarter of all study cows 1 day after calving. Each teat was sealed with a barrier teat sealere after each sample collection.
Tilmicosin concentration in mammary gland secretions was determined by use of HPLC as described.23 All solvents used were HPLC grade; tilmicosin phosphatef and tylosin tartaratef were used as internal standards. An HPLC systemg equipped with a quaternary pump,g 3.5μM-C18 column (4.6 × 100 mm),h C18 precolumn (4.6 × 20 mm),h and UV and diode-array detectorsg was used, and analysis was performed with proprietary software.i Tilmicosin was extracted from samples on the basis of its chemical characteristics. During the initial liquid-liquid extraction, tilmicosin remained in a weakly acidic aqueous solution, and matrix impurities were removed in a nonpolar organic phase. A tissue blank and a recovery tube were included for the analysis of each test sample. Three milliliters of each mammary gland secretion sample was placed in a 50-mL polypropylene centrifuge tube and homogenized. Then, 10 to 30 mL of methanol was added to the tube, and the contents were stirred for 5 minutes. Samples were centrifuged for 20 minutes at 3,000 × g, and the supernatant was decanted into a 250-mL beaker. An additional 30 mL of methanol was added to the centrifuge tube, and the contents were stirred with a stirring rod for 5 minutes to resuspend the pellet in the methanol. The tube was centrifuged again for 10 minutes at 3,000 × g, and the resulting supernatant was combined with that in the beaker. Thirty milliliters of a 10% sodium chloride solution was added to the beaker. The pH of the beaker contents was adjusted as necessary by the use of 1N hydrochloric acid in a drop-wise manner until a pH of 4 was achieved. Fifty milliliters of hexane was added to the aqueous extract, which was then stirred for 30 seconds with regular venting; the partitioned hexane layer was discarded. An additional 25 mL of hexane was added to the extract, the partitioning process was repeated, and the partitioned hexane layer was again discarded. The pH of the aqueous layer was adjusted to 9.0 with 0.5M sodium carbonate solution. Then, 20 mL of a 2:1 chloroform-hexane solution was added to the beaker; the sample was stirred and phases were allowed to separate. The chloroform-hexane layer was decanted, and the procedure was repeated. The organic phase was then evaporated to dryness, and the residue was resuspended in 5 mL of methanol and 5 mL of 0.05M dibutylammonium phosphate. The sample was sonicated for 1 minute to ensure complete dissolution. The sample was passed through solid-phase extraction, then through a 0.22-µm filterj for analysis.
For the HPLC procedure, 50 μL of the extracted and filtered sample was injected into the system at a flow rate of 1.5 mL/min. Separation was achieved by use of a triple gradient analysis with 50% acetonitrile in water (pH, 2.5), water (pH, 2.5), and 0.08M dibutylammonium phosphate. The run time was 30 minutes with a post time of 10 minutes. Tilmicosin was detected at a wavelength of 280 nm. The limit of detection was 0.015 μg/mL, and the limit of quantification was 0.045 μg/mL. Robustness and tolerance tests indicated that the assay had an absolute difference of 1.54 (< 2.0) and a coefficient of variance of 1.2% (< 2.0%).
Data for tilmicosin concentration in mammary gland secretions were based on individual concentration-versus-time curves and were processed by means of specialized software,k which generated results for the following parameters: Cmax, tmax, t1/2β, AUC, and MRT. Model 13 (r = 0.925) was used with this general formula: concentration (time) = Ae−a(time) + Be−b(time) + Ce−KAB(time)
Additionally, for all study cows, milk samples were analyzed for tilmicosin residues with a commercially available qualitative test for antimicrobialsl on a daily basis after calving until a negative result was obtained.
Statistical analysis
At each mammary gland secretion acquisition time, pharmacokinetic parameters for tilmicosin were compared among the 3 treatment groups by use of a 1-way ANOVA. Although all bacterial isolates identified following culture of milk or colostrum samples were recorded, the efficacy of tilmicosin for the treatment of IMIs caused only by S aureus was assessed. The efficacy was determined for each individual gland (quarter), and the cure rate for IMIs caused by S aureus was compared among the 3 treatment groups by use of a Kruskal-Wallis test. A mixed model was used as described24,25 to compare the SCC among the 3 treatment groups. Fixed effects included in the model were treatment group, sample acquisition time (time), and the interaction between treatment group and time. The model also included a random effect for cow nested within treatment group to account for repeated measures within cows. The model had the following form:
where Yijkl is the SCC for an individual cow in a particular treatment group at a particular time, μ is overall mean response for the study population, treatmenti is the fixed effect for treatment group (i = tilmicosin-1, tilmicosin-2, or ceftiofur), timej is the fixed effect for time (j = prior to dry off and 15 and 30 days after calving), treatment × timeij is the fixed effect for the interaction between treatment group and time, and Eijkl is the random error term for cow nested within treatment group. All analyses were performed with a commercially available software program,m and values of P < 0.05 were considered significant.
Results
Cows
Descriptive statistics for cows within each treatment group were summarized (Table 1). Review of herd records revealed that most of the cows enrolled in the study had at least 2 IMIs caused by Staphylococcus spp during the lactation immediately prior to dry off.
Descriptive data for 170 Holstein-Friesian cows that were treated with 1 (tilmicosin-1 group; n = 58) or 2 (tilmicosin-2 group; 56) doses (20 mg/kg, SC) of an experimental preparation of tilmicosin or were treated with a long-acting intramammary preparation of ceftiofur (500 mg/mammary gland; ceftiofur group; 56).
| Lactation | |||||||
|---|---|---|---|---|---|---|---|
| Treatment group | Age (y) | No. of days since last calving | Milk production for current lactation (kg) | Mean | No. | No. (%) of cows | No. of previous IMIs* |
| Tilmicosin-1 | 4.86 ± 1.43 | 285 ± 20 | 7,284 ± 1,333 | 2.64 ± 1.17 | 1 | 10 (17) | 2.2 ± 0.45 |
| 2 | 18 (31) | 2.8 ± 0.67 | |||||
| 3 | 16 (28) | 3.1 ± 0.64 | |||||
| 4 | 10 (17) | 3.6 ± 0.89 | |||||
| 5 | 4 (7) | 3.7 ± 0.95 | |||||
| Tilmicosin-2 | 5.07 ± 1.44 | 290 ± 19 | 6,916 ± 1,006 | 2.75 ± 1.26 | 1 | 8 (14) | 2.5 ± 0.58 |
| 2 | 20 (36) | 2.7 ± 0.67 | |||||
| 3 | 14 (25) | 3 ± 0.82 | |||||
| 4 | 6 (11) | 3.3 ± 0.58 | |||||
| 5 | 8 (14) | 3.8 ± 0.96 | |||||
| Ceftiofur | 4.40 ± 1.17 | 293 ± 16 | 6,791 ± 1,205 | 2.25 ± 1.04 | 1 | 14 (25) | 2.3 ± 0.49 |
| 2 | 22 (39) | 2.8 ± 0.75 | |||||
| 3 | 14 (25) | 3 ± 0.82 | |||||
| 4 | 4 (7) | 3.5 ± 0.71 | |||||
| 5 | 2 (4) | 3 ± 0.5 | |||||
Values represent the mean ± SD unless otherwise indicated. Cows in both the tilmicosin-1 and tilmicosin-2 groups received 1 dose of tilmicosin at dry off (ie, day that the cows were administered dry cow treatment, and the first day of the dry period [45-day period immediately before calving during which cows are not milked]), and cows in the tilmicosin-2 group received a second dose of tilmicosin 20 days later. Cows in the ceftiofur group were treated once at dry off.
Intramammary infections caused by Staphylococcus spp during the current lactation.
Tilmicosin concentration in mammary gland secretions
For cows in the tilmicosin-1 and tilmicosin-2 groups, the mean concentration of tilmicosin in mammary gland secretions over time (Figure 1) and pharmacokinetic parameters (Table 2) were summarized. The mean ± SD Cmax for cows in the tilmicosin-2 group after injection of the first dose (20.9. ± 4.9 μg/mL) was greater than that after injection of the second dose (17.1. ± 3.1 μg/mL). Similarly, the mean ± SD tmax for cows in the tilmicosin-2 group after injection of the first dose (4.1 ± 0.7 days) was greater than that after injection of the second dose (3.9 ± 0.4 days). However, the mean ± SD t1/2β for cows in the tilmicosin-1 group (4.7 ± 2.3 days) and tilmicosin-2 group after injection of the first dose (4.9 ± 1.4 days) was less than that for cows in the tilmicosin-2 group after injection of the second dose (7.7 ± 1.6 days). For cows in the tilmicosin-2 group, the mean ± SD AUC after the second dose of tilmicosin (358.1 ± 51.4 μg/mL•d) was significantly (P < 0.01) greater than that after the first dose of tilmicosin (242.1 ± 70.6 μg/mL•d), and those values were in agreement with the AUC that was calculated by the trapezoidal rule. Also, the MRT after the second dose of tilmicosin (8.3 ± 0.4 days) was significantly (P < 0.01) greater than that after the first dose of tilmicosin (5.8 ± 0.9 days).
Mean ± SD pharmacokinetic parameters for an experimental preparation of tilmicosin in mammary gland secretions of dairy cows following administration of 1 (tilmicosin-1 group; n = 58) or 2 (tilmicosin-2 group; 56) doses (20 mg/kg, SC) of the preparation.
| Timicosin-2 group | |||
|---|---|---|---|
| Parameter | Tilmicosin-1 | First dose | Second dose |
| Cmax (µg/mL) | 14.4 ± 2.1a | 20.9 ± 4.9b | 17.1 ± 3.1ab |
| tmax (d) | 3.0 ± 0.5a | 4.1 ± 0.7b | 3.9 ± 0.4a,b |
| t1/2β (d) | 4.7 ± 2.3a | 4.9 ± 1.4a,b | 7.7 ± 1.6b |
| AUC (µg/mL•d) | 177.3 ± 2.3a | 242.1 ± 70.6a | 358.1 ± 51.4b |
| AUCt (µg/mL•d) | 109.8 ± 16.1a | 146.2 ± 36.1a | 177.0 ± 28.0b |
| AUMC (µg/mL•d2) | 1,194.4 ± 274.6a | 1,365.2 ± 194.9a | 1,477.6 ± 194.7a |
| MRT (d) | 6.8 ± 1.7a | 5.8 ± 0.9a | 8.3 ± 0.4b |
AUCt = AUC calculated by the trapezoidal rule. AUMC = Area under the first moment of the concentration-time curve.
Within a row, values with different superscript letters differ significantly (P < 0.05).
See Table 1 for remainder of key.
Mean ± SD concentrations of tilmicosin over time in mammary gland secretions obtained from clinically normal Holstein-Friesian cows with subclinical IMIs caused by Staphylococcus spp that were treated with an experimental preparation of tilmicosin (20 mg/kg, SC) once at dry off (tilmicosin-1 group; solid line; n = 58) or at dry off and again 20 days later (tilmicosin-2 group; dashed line; 56). The horizontal dotted line represents the minimum concentration of tilmicosin necessary to inhibit 90% of Staphylococcus aureus isolates (1.0 μg/mL) determined in another study.26 Mammary gland secretion samples were collected from 5 randomly selected cows from each treatment group every 5 days after dry off until calving, and no cow was sampled more than once during the dry period. Therefore, the respective mean values represent the mean for 5 different cows at each sampling day.
Citation: American Journal of Veterinary Research 77, 9; 10.2460/ajvr.77.9.922
Results of daily qualitative testing of milk samples indicated that tilmicosin was detectable in the milk of cows in the tilmicosin-2 group for up to 18 days after calving, whereas tilmicosin was not detectable in the milk of cows in the tilmicosin-1 or ceftiofur groups after calving.
Efficacy of tilmicosin for treatment of IMIs
The numbers of cows with IMIs caused by Staphylococcus spp before and after administration of the assigned treatment and the associated cure rates for each treatment group were summarized (Table 3). The cure rate for all pathogens was 100% for all 3 treatments. The numbers of cows that developed new IMIs caused by Staphylococcus spp in each treatment group were likewise summarized (Table 4).
Number of dairy cows with an IMI caused by Staphylococcus aureus, Staphylococcus epidermidis, or coagulase-negative Staphylococcus spp (CNS) before and after parenteral administration of 1 (tilmicosin-1 group; n = 58) or 2 (tilmicosin-2 group; 56) doses (20 mg/kg, SC) of an experimental preparation of tilmicosin or administration of a long-acting intramammary preparation of ceftiofur (500 mg/mammary gland; 56).
| Treatment group | |||||||||
|---|---|---|---|---|---|---|---|---|---|
| Tilmicosin-1 | Tilmicosin-2 | Ceftiofur | |||||||
| Pathogen | Before treatment | After treatment | Cure rate (%) | Before treatment | After treatment | Cure rate (%) | Before treatment | After treatment | Cure rate (%) |
| S aureus | 24 | 0 | 100 | 24 | 0 | 100 | 22 | 0 | 100 |
| S epidermidis | 8 | 0 | 100 | 7 | 0 | 100 | 8 | 0 | 100 |
| CNS | 26 | 0 | 100 | 25 | 0 | 100 | 26 | 0 | 100 |
Three 20-mL milk or colostrum samples for microbiological culture were aseptically collected from each cow 5 days before dry off, within 24 hours after calving, and 15 and 30 days after calving. An IMI before treatment was defined as the isolation of ≥ 3 CFUs of a Staphylococcus spp/15 to 20 μL of culture inoculum (milk) in at least 2 of the 3 samples cultured. An IMI was considered cured when a previously identified pathogen was not cultured in any of the samples collected on the subsequent 3 sampling days (within 24 hours after calving and 15 and 30 days after calving).
See Table 1 for remainder of key.
Number of cows from Table 3 that developed a new IMI at calving and 15 and 30 days after calving.
| Pathogen | ||||
|---|---|---|---|---|
| Treatment group | Sample collection day | S aureus | S epidermidis | CNS |
| Tilmicosin-1 | Calving | 2 | 1 | 2 |
| 15 d after calving | 2 | 0 | 0 | |
| 30 d after calving | 0 | 0 | 0 | |
| Tilmicosin-2 | Calving | 1 | 0 | 0 |
| 15 d after calving | 3 | 1 | 0 | |
| 30 d after calving | 1 | 1 | 2 | |
| Ceftiofur | Calving | 2 | 0 | 3 |
| 15 d after calving | 2 | 2 | 0 | |
| 30 d after calving | 1 | 0 | 0 | |
A new IMI was defined as the isolation of a Staphylococcus spp from a gland that had previously cultured negative for that organism.
See Table 3 for remainder of key.
The qualitative CMT test results (data not shown) were consistent with the quantitative SCCs. The quadratic mean ± SD SCC for cows in each treatment group at dry off and 15 and 30 days after calving were summarized (Table 5). The mean SCC did not differ significantly among the 3 treatment groups at dry off or at 15 days after calving. At 30 days after calving, the mean SCC for the cows in the tilmicosin-1 group was significantly lower than that for cows in both the tilmicosin-2 and ceftiofur groups.
Quadratic mean ± SD SCC (cells/mL of milk) for the cows of Table 1 at dry off and 15 and 30 days after calving.
| Sample collection day | |||
|---|---|---|---|
| Treatment group | Dry off | 15 days after calving | 30 days after calving |
| Tilmicosin-1 | 308,679 ± 33,684 | 209,471 ± 34,073 | 175,183 ± 34,292* |
| Tilmicosin-2 | 253,259 ± 39,638 | 170,874 ± 39,638 | 197,759 ± 39,638 |
| Ceftiofur | 335,906 ± 35,944 | 301,484 ± 35,361 | 210,520 ± 34,913 |
Within a column, value differs significantly (P < 0.05) from the corresponding values for the other 2 treatment groups.
See Table 1 for remainder of key.
Discussion
Results of the present study indicated that SC administration of a long-acting experimental formulation of tilmicosin to cows with an IMI caused by Staphylococcus spp at dry off resulted in a 100% cure rate. The efficacy of a parenterally administered antimicrobial to achieve a bacteriologic cure for an IMI is dependent on its ability to penetrate the udder, diffuse into the infected mammary tissue, and remain biologically active.27 This is particularly important in instances when the mammary gland has undergone structural changes such as the development of fibrotic tissue or the formation of abscesses in the infected tissue, both of which are common for IMI caused by Staphylococcus spp10,28
Traditionally, the recommended course of action for most cows with IMIs caused by L-form Staphylococcus spp is culling, or removal from the herd. L-form Staphylococcus spp, particularly S aureus, are able to reside at the intracellular level, which makes them virtually nonresponsive to in vivo treatment.10 Furthermore, the structural changes to the mammary gland commonly associated with IMIs caused by Staphylococcus spp impair the penetration and diffusion of antimicrobials into the infected areas of the gland.10,28 This is especially true for antimicrobials that are administered by the intramammary route. Antimicrobials administered by the intramammary route tend to be only marginally effective at achieving a bacteriologic cure for IMIs caused by Staphylococcus spp.27 Additionally, intramammary administration of an antimicrobial requires the insertion of a cannula into the teat, which has been identified as an important cause of iatrogenically induced IMIs.16,29,30
Treatment of IMIs by parenteral administration of macrolides has been proposed as a method to ensure homogeneous diffusion of clinically relevant concentrations of an antimicrobial into the bovine mammary gland without the difficulties associated with intramammary infusion.31,32 Following parenteral administration to cows, tilmicosin readily diffuses into the mammary tissue and milk18,33 and can also diffuse into the cytoplasm of many cells.18 In 1 study,16 cows administered 10 mg of tilmicosin/kg, SC, once, had milk concentrations of the drug that were considered clinically relevant for the treatment of infections caused by Staphylococcus spp that persisted for 8 to 9 days. Subsequently, on the basis of the results of that study,16 many researchers14,29,34,35 postulated that parenteral administration of tilmicosin to cows will result in concentrations of the drug in milk and mammary gland cells sufficient for the treatment of IMIs caused by Staphylococcus spp. Results of another study26 indicate that the minimum concentration of tilmicosin necessary to inhibit 90% of S aureus isolates is 1.0 μg/mL. In the present study, tilmicosin concentrations > 1.0 μg/mL were achieved in mammary gland secretions for 19 and > 40 days following SC administration of 1 and 2 doses (20 mg/kg, every 20 days) of an experimental preparation of the drug, respectively (Figure 1), and the cure rate for naturally occurring IMIs caused by Staphylococcus spp was 100% for both dosage regimens. In another study,10 the cure rate was only 9.1% for cows that were treated with the commercially available formulation of tilmicosin (5 mg/kg, SC) at dry off and again 4 days later, despite the fact that tilmicosin was detectable in mammary gland secretions for 25.7 days after the last injection. Differences in the IMI cure rates between that study10 and the present study are most likely attributable to differences in the drug formulations used and the higher dose (20 mg/kg vs 5 mg/kg) of tilmicosin that was administered in the present study, which resulted in higher drug concentrations in mammary gland secretions. Also, the cows enrolled in the present study were fairly young; 50% of the cows in both the tilmicosin-1 and tilmicosin-2 groups were in their first or second lactation, and the mean age of those cows was 3.4 and 3.6 years, respectively. The probability that an S aureus IMI will be successfully treated is greater for young cows than for older cows.12 However, half of the cows in both the tilmicosin-1 and tilmicosin-2 groups were in their third or greater lactation, and the cure rate was 100% for all cows, regardless of age.
The pharmacokinetic parameters for tilmicosin in dairy cows following administration of 1 dose (10 mg/kg, SC) of the commercially available formulation of the drug (t1/2β, 33.84 ± 4.60 hours; Cmax, 8.21 ± 2.70 μg/mL; tmax, 24 hours; and MRT, 76.08 ± 7.31 hours)16 were significantly less than the corresponding parameters (t1/2β, 4.7 ± 2.3 days; Cmax, 14.4 ± 2.1 μg/mL; tmax, 3.0 ± 0.5 days; and MRT, 6.8 ± 1.7 days; Table 2) for the cows of the tilmicosin-1 group of the present study following administration of 1 dose (20 mg/kg, SC) of an experimental preparation of the drug. Presumably, this was a reflection of differences in the drug preparations and the fact that the dose of tilmicosin administered in the present study was twice that administered in the other study.16 Tilmicosin is a time-dependent antimicrobial,36 and the pharmacokinetic parameters of the experimental preparation of tilmicosin in milk determined for the present study were consistent with that classification. When the pharmacokinetic parameters for the commercially available formulation of tilmicosin16 were compared with those for the long-acting experimental preparation of tilmicosin used in the present study, the results suggested that the experimental preparation was a better choice than the commercial formulation for dry cow treatment.
Previous studies10,14 have been conducted to investigate the cure rates for IMIs caused by S aureus following intramammary administration of tilmicosin. Infusion of S aureus–infected mammary glands with a sterile solution containing 1,500 mg of tilmicosin at dry off resulted in cure rates of 74.2%10 and 72.5%.14 Thus, it appears that administration of a high dose of tilmicosin is necessary to achieve good efficacy against S aureus IMIs. The total dose of tilmicosin administered SC to the cows of the present study was much higher than 1,500 mg; therefore, it should not be surprising that a 100% cure rate was achieved for IMIs caused by S aureus. However, it is important to note that a tilmicosin preparation designed for intramammary administration is not currently commercially available nor is the experimental preparation of tilmicosin that was injected SC in the present study. Also, tilmicosin is not approved for the treatment of mastitis, and its extralabel use in dairy cows may or may not be legal depending on the country in which the extralabel use is being considered.
In the present study, the mean SCC did not differ significantly among the 3 treatment groups at dry off or 15 days after calving; the mean SCC for the tilmicosin-1 group was significantly lower than that for the ceftiofur and tilmicosin-2 groups at 30 days after calving. Numerically, the mean SCC for the cows in the tilmicosin-1 and tilmicosin-2 groups was consistently lower than that for the cows in the ceftiofur group. A similar finding was reported by investigators of another study10 in which cows were treated at dry off with benzathine cephapirin or tilmicosin by intramammary infusion or tilmicosin by SC administration. However, in that study,10 the mean SCC for cows that received an SC injection of tilmicosin was significantly lower than the mean SCC for cows that received an intramammary infusion of tilmicosin. The investigators of that study10 postulated that direct contact of tilmicosin with the mammary gland epithelium following intramammary infusion caused irritation, which resulted in an increase in the SCC. Given the other environmental factors that affect SCC, further research is required to elucidate the effects of tilmicosin administered by an intramammary route on the SCC of dairy cows.
Qualitative analysis of milk samples for tilmicosin residues revealed that cows in the tilmicosin-2 group had drug residues in their milk for 18 days after calving, which suggested that protocol was not practical for routine use in a commercial field setting. Conversely, none of the cows in the tilmicosin-1 group had tilmicosin residues detected in their milk after calving. The manufacturer of the qualitative assay used in the present study certified that it was capable of detecting tilmicosin; however, the exact limit of detection was unknown, although 40 ppb appeared to be an acceptable value.37
In the present study, cows with Staphylococcus spp–IMIs that were treated with an experimental preparation of tilmicosin (20 mg/kg, SC) at dry off (tilmicosin-1 group) or at dry off and again 20 days later (tilmicosin-2 group) had a 100% cure rate. However, it is important to point out that similar cows treated with a long-acting intramammary preparation of ceftiofur at dry off also had a 100% cure rate, and that product is approved for such use and commercially available throughout the world. Thus, we are not recommending that the experimental preparation of tilmicosin be used instead of or in preference to the approved ceftiofur product. Further research is necessary to elucidate residue elimination patterns of the experimental preparation of tilmicosin following administration of various dry cow treatment protocols to dairy cows before it can be considered for commercial use. Also, its efficacy against other mammary gland pathogens such as Streptococcus uberis10,26 and its effect on bacterial resistance patterns at the herd level warrant further investigation.
Acknowledgments
Supported by Project UNAM/PAPIIT IT200714.
The authors thank Dr. Rocío Angélica Ruiz-Romero for technical assistance.
ABBREVIATIONS
| AUC | Area under the mammary gland secretion concentration–time curve |
| Cmax | Maximum concentration |
| CMT | California mastitis test |
| HPLC | High-performance liquid chromatography |
| IMI | Intramammary infection |
| MRT | Mean residence time |
| SCC | Somatic cell count |
| t1/2β | Elimination half-life |
| tmax | Time to maximum concentration |
Footnotes
Experimental preparation of tilmicosin, manufactured by Casal's Internacional S.A. de C.V. (Guadalajara, Mexico), under Patent MX/E/2014/011982 held by the National Autonomous University of Mexico (UNAM), Instituto Nacional de la Protección Industrial (INPI), Mexico City, Mexico.
Spectramast DC, Zoetis, Florham Park, NJ.
Ekomilk Scan somatic cell counter, Bulteh 2000 Ltd, Stara Zagora, Bulgaria.
Biofoam, DeLaval, Tumba, Sweden.
Valiant Triple Proteccion, ABS Global, Deforest, Wis.
Sigma-Aldrich Corp, Milwaukee, Wis.
JASCO Analytical Instruments, Easton, Md.
Symmetry C18 column and precolumn, Waters Corp, Milford, Mass.
EASYCHROM 2.0, Ampersand International Inc, Beachwood, Ohio.
Acrodisc, Pall Corp, New York, NY.
PKAnalyst, Micromath Scientific Software, St Louis, Mo.
Delvotest, DSM, Heerlen, The Netherlands.
SAS, version 9.1.3, SAS Institute Inc, Cary, NC.
References
1. Halasa T, Østerås O, Hogeveen H, et al. Meta-analysis of dry cow management for dairy cattle. Part 1. Protection against new intramammary infections. J Dairy Sci 2009; 92: 3134–3149.
2. Pezeshki A, Capuco AV, De Spiegeleer B, et al. An integrated view on how the management of the dry period length of lactating cows could affect mammary biology and defence. J Anim Physiol Anim Nutr (Berl) 2010; 94: e7–e30.
3. Cameron M, Keefe GP, Roy JP, et al. Evaluation of selective dry cow treatment following on-farm culture: milk yield and somatic cell count in the subsequent lactation. J Dairy Sci 2015; 98: 2427–2436.
4. Capuco AV, Akers RM, Smith JJ. Mammary growth in Holstein cows during the dry period: quantification of nucleic acids and histology. J Dairy Sci 1997; 80: 477–487.
5. Green MJ, Bradley AJ, Medley GF, et al. Cow, farm, and management factors during the dry period that determine the rate of clinical mastitis after calving. J Dairy Sci 2007; 90: 3764–3776.
6. Scherpenzeel CG, den Uijl IE, van Schaik G, et al. Evaluation of the use of dry cow antibiotics in low somatic cell count cows. J Dairy Sci 2014; 97: 3606–3614.
7. Green MJ, Green LE, Medley GF, et al. Influence of dry period bacterial intramammary infection on clinical mastitis in dairy cows. J Dairy Sci 2002; 85: 2589–2599.
8. Halasa T, Huijps K, Østerås O, et al. Economic effects of bovine mastitis and mastitis management: a review. Vet Q 2007; 29: 18–31.
9. Gundelach Y, Kalscheuer E, Hamann H, et al. Risk factors associated with bacteriological cure, new infection, and incidence of clinical mastitis after dry cow therapy with three different antibiotics. J Vet Sci 2011; 12: 227–233.
10. Nickerson SC, Owens WE, Fox LK, et al. Comparison of tilmicosin and cephapirin as therapeutics for Staphylococcus aureus mastitis at dry-off. J Dairy Sci 1999; 82: 696–703.
11. Dingwell RT, Duffield TF, Leslie KE, et al. The efficacy of intramammary tilmicosin at drying-off, and other risk factors for the prevention of new intramammary infections during the dry period. J Dairy Sci 2002; 85: 3250–3259.
12. Barkema HW, Schukken YH, Zadoks RN. Invited review: the role of cow, pathogen, and treatment regimen in the therapeutic success of bovine Staphylococcus aureus mastitis. J Dairy Sci 2006; 89: 1877–1895.
13. Brouillette E, Grondin G, Shkreta L, et al. In vivo and in vitro demonstration that Staphylococcus aureus is an intracellular pathogen in the presence or absence of fibronectin-binding proteins. Microb Pathog 2003; 35: 159–168.
14. Dingwell RT, Leslie KE, Duffield TF, et al. Efficacy of intramammary tilmicosin and risk factors for cure of Staphylococcus aureus infection in the dry period. J Dairy Sci 2003; 86: 159–168.
15. Experimental preparation of tilmicosin. Patent submission No. 212148. México City: Instituto Mexicano de la Protección Industrial, 2013.
16. Ziv G, Shem-Tov M, Glickman A, et al. Tilmicosin antibacterial activity and pharmacokinetics in cows. J Vet Pharmacol Ther 1995; 18: 340–345.
17. Buret AG. Immuno-modulation and anti-inflammatory benefits of antibiotics: the example of tilmicosin. Can J Vet Res 2010; 74: 1–10.
18. Scorneaux B, Shryock RT. Intracellular accumulation, subcellular distribution, and efflux of tilmicosin in bovine mammary, blood, and lung cells. J Dairy Sci 1999; 82: 1202–1212.
19. Mexican Official Standard NOM-062-ZOO-1999. Technical specifications for the production, care and use of laboratory animals. United Mexican States Department of Agriculture, Livestock, Rural Development, Fisheries and Food. Available at: www.fmvz.unam.mx/fmvz/principal/archivos/062ZOO.PDF. Accessed May 26, 2015.
20. NMC. Procedures for collecting milk samples. Available at: www.nmconline.org/sampling.htm. Accessed Jan 12, 2014.
21. NMC. Using bulk tank milk cultures in a dairy practice. Available at: www.nmconline.org/articles/bulktank.htm. Accessed Jan 12, 2014.
22. Carter GR. Diagnostic procedures in veterinary bacteriology and mycology. 4th ed. Springfield, Ill: Charles C Thomas Publisher Ltd, 1984; 570–577.
23. Parker RM, Patel RK. Determination of tilmicosin in ovine milk using high-performance liquid chromatography. Analyst 1994; 119: 2577–2579.
24. SAS user's guide: statistics (version 9.1.3). Cary, NC: SAS Institute Inc Press, 2012;421–479.
25. Littell CR, Milliken AG, Stroup WW, et al. SAS system for mixed models. Cary, NC: SAS Institute Inc Press, 1996;160–165.
26. Bonnier M, Doré C, Amédéo J, et al. In vitro activity of tylosin and tilmicosin against cocci isolated from bovine mastitis. Revue Méd Vét 2006; 157: 486–489.
27. Gehring R, Smith GW. An overview of factors affecting the disposition of intramammary preparations used to treat bovine mastitis. J Vet Pharmacol Ther 2006; 29: 237–241.
28. Sandgren CH, Waller KP, Emanuelson U. Therapeutic effects of systemic or intramammary antimicrobial treatment of bovine subclinical mastitis during lactation. Vet J 2008; 175: 108–117.
29. Gruet P, Maincent P, Berthelot X, et al. Bovine mastitis and intramammary drug delivery: review and perspectives. Adv Drug Deliv Rev 2001; 50: 245–259.
30. Kalmus P, Simojoki H, Orro T, et al. Efficacy of 5-day parenteral versus intramammary benzylpenicillin for treatment of clinical mastitis caused by gram-positive bacteria susceptible to penicillin in vitro. J Dairy Sci 2014; 97: 2155–2164.
31. Helton-Groce SL, Thomson TD, Readnour RS. A study of tilmicosin in milk following subcutaneous administration to lactating dairy cows. Can Vet J 1993; 34: 619–621.
32. McDougall S, Agnew EK, Cursons R, et al. Parenteral treatment of clinical mastitis with tylosin base or penethamate hydriodide in dairy cattle. J Dairy Sci 2007; 90: 779–789.
33. Avci T, Elmas M. Milk and blood pharmacokinetics of tylosin and tilmicosin following parenteral administrations to cows. Scientific World Journal 2014; 2014: 869096.
34. Owens WE, Nickerson SC, Ray CH. Efficacy of parenterally or intramammarily administered tilmicosin or ceftiofur against Staphylococcus aureus mastitis during lactation. J Dairy Sci 1999; 82: 645–647.
35. Wang W, Song Y, Petrovski K, et al. Development of intramammary delivery systems containing lasalocid for the treatment of bovine mastitis: impact of solubility improvement on safety, efficacy, and milk distribution in dairy cattle. Drug Des Devel Ther 2015; 9: 631–642.
36. Adams HR. Veterinary pharmacology and therapeutics. 8th ed. Ames, Iowa: Iowa State University Press, 2001; 876–882.
37. Sensitivity chart for delvotest SP-NT & delvotest BLF, for antibiotic products commonly used on UK dairy farms. Millwood Products, Bath, England. Available at: www.millwoodproducts.co.uk/public/backend/uploads/imagemanager/documents/Detailed%20SSensitivit%20Chart%20for%20Delvotest%20SP-NT%20&%20BLF.pdf. Accessed Apr 4, 2016.
