Caseous lymphadenitis has emerged as an important health concern, as it affects all aspects of the small ruminant industry. Efforts to eliminate this disease are arguably most effective by eliminating diseased animals from flocks or herds. Nonetheless, producers in some regions choose to retain affected animals for various reasons such as genetic or sentimental value. The decision to keep affected animals should come with the understanding that CL is not considered a curable disease.
Methods for treatment and control of CL include lancing and flushing of abscesses with potentiated iodine solutions, surgical removal of abscesses, intralesional injection of formalin, and isolation from other animals to prevent disease spread1,2 as well as antimicrobial treatments.3,4 Although the current standard of care for CL lesions is lancing, draining, and flushing abscesses caused by CPT, this creates a potential hazard for spread of the bacterial agent through purulent material to fomites and into the environment during the convalescent period. Although curative in the short term, surgical resection of abscesses does not address recurrence, requires local or general anesthesia, and is a more expensive option than the other reported treatments. Injection of formalin into the lesions is reportedly beneficial1; however, a carcass containing formalin would be considered adulterated and would be unfit for human consumption. The potential for negative public perception related to this practice is also a problem. Antimicrobial protocols have been used for pharmacological treatment of animals with CL with various degrees of efficacy.3,4 Acceptable efficacy of antimicrobials against CPT is hindered because it is a facultative intracellular pathogen that produces thickly encapsulated abscesses.1 Recently, the investigators of the study reported here determined that tulathromycin concentrations sufficient to reduce or eliminate CPT growth can be achieved within surgically implanted tissue chambers (ie, tissue cages) designed to serve as a model for isolated focal abscesses in goats.5 These drug concentrations were detected after a single SC injection or percutaneous injection of the drug directly into the chamber; however, some regional lymph nodes were culture positive for CPT despite having a negative culture result for the CPT-inoculated tissue chamber sample from the same animal.5 Results of that study, together with findings in a randomized clinical trial5 and an observational study6 performed by our research group, led us to conclude that, although not entirely curative, parenteral or intralesional tulathromycin treatment can be an acceptable alternative to lancing, draining, and flushing of CL lesions. The previous study in goats5 established that surgically implanted tissue chambers inoculated with CPT are a suitable model of the abscesses formed by CPT that could be used in future studies to investigate many other facets of this disease.
Previous investigations in our laboratory indicated that CPT is susceptible to oxytetracycline in vitro (unpublished data). Oxytetracycline has moderate lipid solubility, which should allow some penetration into encapsulated lesions and may maintain some bactericidal activity in a purulent environment. Predictability of oxytetracycline activity in purulent material is difficult because although tetracyclines have been shown to bind to divalent cations such as calcium and magnesium, the concentrations of these cations produced by CPT are unknown.7–9 Primary advantages of oxytetracycline administration, compared with tulathromycin treatment, include lower cost, greater availability, and shorter withdrawal time. At present, the cost of oxytetracycline is approximately $0.68/mL in our hospital, whereas the cost of tulathromycin is $5.98/mL. Some forms of injectable oxytetracycline may be available to some animal owners over the counter in the United States, whereas tulathromycin requires a prescription. Finally, results of 1 study10 suggested that an appropriate withdrawal time for a single SC dose of tulathromycin, used in an extralabel manner in sheep, is 36 days. Although the use of oxytetracycline is extralabel in sheep, the 28-day withdrawal period for label use in cattle is sufficient.11
The objectives of the study reported here were to determine concentrations of oxytetracycline within CPT-inoculated and uninoculated tissue chambers following a single IM or intrachamber injection of the drug, to determine the efficacy of these treatments for reduction or elimination of CPT in the inoculated chambers, and to determine whether the organism was present in any regional lymph nodes at the end of the study. We also investigated the MICs of oxytetracycline and other antimicrobials commonly used in sheep and goats for clinically derived isolates of CPT. We hypothesized that measurable concentrations of oxytetracycline would be detectable inside tissue chambers after IM administration, but that these would be less than the MIC for the isolate of CPT used, whereas intrachamber injection of oxytetracycline would reduce or eliminate CPT populations. Finally, we hypothesized that clinically derived isolates of CPT would be susceptible to multiple antimicrobials in vitro.
Materials and Methods
Animals
Ten healthy adult female crossbred sheep obtained from a commercial source (mean weight, 85 kg [range, 82 to 86 kg]) were enrolled in the study. All animals were examined by a veterinarian and found to be clinically normal and free of any visible lesions suggestive of CL. The sheep were housed in a large outdoor paddock prior to surgical implantation of tissue chambers. After the surgery, sheep were housed in individual indoor pens in a biosafety level 2-approved facility. The sheep were fed free-choice Bermuda grass hay and a pelleted ration during the study. All sheep were examined daily for signs of illness and surgical site infections or dehiscence. The study was approved by the Texas A&M University Institutional Animal Care and Use Committee as well as the Institutional Biosafety Committee.
Experimental design
Sterilized tissue chambers were surgically implanted in the paralumbar fossae (1/side) of sheep ≥ 14 days prior to inoculation to allow healing of incision sites. On day −6, 1 randomly selected chamber/sheep was inoculated with CPT in BHIB, and the contralateral chamber, designated as an uninoculated control, was injected with sterile BHIB. A coin flip was used for the randomization. On day 0, immediately prior to administration of oxytetracycline, a fluid sample was collected from each chamber for time 0 bacterial culture and drug concentration determination, and a venous blood sample (5 mL) was collected from each sheep for plasma drug concentration determination. Oxytetracycline (20 mg/kg) was administered by percutaneous injection into the inoculated chamber (n = 4 sheep), percutaneous injection into the uninoculated control chamber (previously injected with sterile BHIB; 1), or IM injection in the cervical region (5). Twelve, 24, 48, 72, 96, and 144 hours after oxytetracycline administration, tissue chamber fluid was collected from all chambers for bacterial culture and drug concentration analysis. Venous blood samples (5 mL) were collected from each sheep at the same times for plasma drug concentration assays. The sheep were humanely euthanized by sodium pentobarbital overdose immediately after collection of the 144-hour posttreatment samples, and the left and right subiliac lymph nodes were collected from each sheep for bacterial culture.
Tissue chambers
Tissue chambers were prepared according to a previously described method.12 Briefly, chambers were constructed of thermoplastica with 4.6-cm internal diameter, 5.2-cm external diameter, and 1.5-cm depth. Thirty-seven 0.6-cm-diameter perforations were distributed over the base and walls of the top portion of the chamber, and the lower portion of the chamber was free of perforations to allow accumulation of fluid in vivo. A silicone rubber membranea covered the top of the chamber and was secured in place by a loop of stainless steel surgical wire around the rim of the cup.12 The assembled chambers were packaged individually and steam sterilized prior to implantation.
Surgical implantation of tissue chambers
The paralumbar fossa was clipped and cleaned, and the region was anesthetized with a proximal paravertebral nerve block by injection of 2% lidocaine solution. The skin was aseptically prepared for surgery with iodinated scrub solution. A vertical skin incision was made in the approximate center of the paralumbar fossa beginning approximately 3 cm ventral to the transverse processes of the lumbar vertebrae and extending approximately 10 cm ventrally. The skin caudal to the incision was undermined to create a pocket for the tissue chamber. The chamber was placed into the pocket just under the skin and secured to the subcutaneous tissue with 4 stay sutures. The chamber was seated so that the area without holes was located ventrally to create a fluid reservoir from which to collect samples. The skin incision was closed with a simple continuous suture pattern and allowed to heal for ≥ 2 weeks prior to CPT inoculation (Figure 1).
Photograph showing the site of a surgically implanted tissue chamber (circled) in the right paralumbar fossa of 1 of 10 sheep used in a study to determine concentrations of oxytetracycline in plasma, CPT-inoculated tissue chambers (used as experimental abscess models), and uninoculated (control) tissue chambers after IM or local (percutaneous intrachamber) administration of the drug (20 mg/kg, once) and to investigate whether CPT growth was reduced or eliminated by these treatments. Each sheep had 1 sterile chamber surgically implanted into each paralumbar fossa; 2 weeks later (day −6), 1 randomly selected chamber/sheep was inoculated by percutaneous injection with CPT (1 × 106 CFUs/mL in BHIB), and the contralateral chamber was injected with sterile BHIB (1 mL/injection). Five sheep received oxytetracycline IM and 5 had a local injection (4 and 1 in CPT-inoculated and control chambers, respectively). Two sheep each had 1 tissue chamber (1 inoculated and 1 control) removed in a second surgery because of dehiscence at the surgical site.
Citation: American Journal of Veterinary Research 80, 6; 10.2460/ajvr.80.6.586
Determination of the MIC of oxytetracycline for the isolate used in inoculations
A CPT isolate derived from a goat with a clinical case of CL was used for inoculation of designated tissue chambers; the same isolate had previously been used in a study5 to evaluate efficacy of tulathromycin against CPT in tissue chambers implanted in goats. The MIC of oxytetracycline for this isolate was determined by the microbroth dilution method of susceptibility testing with a commercially available microbroth systemb according to the manufacturer's recommendations, which are consistent with the methods established by the Clinical Laboratory Standards Institute.13 Antimicrobial susceptibility of the isolate was evaluated 24 and 48 hours after inoculation in the present study by visual inspection of the wells to assess growth of the organism.
Preparation of inoculum and control treatment
Briefly, the isolate was cultured on TSA supplemented with sheep blood (5% concentration) at 37°C in an atmosphere of air with 5% CO2 for 48 hours. Cultures on TSA plates were routinely incubated for 48 hours because of the slow growth characteristics of CPT. After culture for 48 hours, a representative colony was selected and used to inoculate BHIB.c The cultures in BHIB were incubated overnight at 37°C on an orbital shaker at 250 oscillations/min.d The concentration of CPT was determined spectrophotometricallye at an optical density of 600 nm. The bacteria were diluted to a concentration of 1 × 106 CFUs/mL in BHIB for inoculation. Sterile BHIB from the same lot that was used to culture CPT was used for injection into control chambers.
Percutaneous intrachamber injection and sample collection
Prior to injection of the inoculum or sterile BHIB into tissue chambers or sample collection from the chambers, the skin over the chamber was prepared with iodinated scrub solution and rinsed with 91% isopropyl alcohol. A 3-mL syringe with a 1-inch, 18-gauge needle was used to inject 1 mL of the CPT inoculum or sterile BHIB into the designated chambers. The needle was inserted through the skin covering the chamber and was directed ventrally into the region corresponding with the reservoir for fluid. Needle location was verified by the ability to withdraw fluid, which would not be the case if the needle was outside the boundaries of the chamber.
For sampling of chamber fluid, a needle and syringe of the same size were used in the same manner to aspirate 1 mL of fluid from the chambers and transfer the samples directly into anticoagulant-free blood collection tubes. Blood samples (5 mL) were collected directly into heparin-containing blood tubes by jugular venipuncture with a transfer needle.c Blood samples were centrifuged at 4°C for 20 minutes at for collection of plasma. All samples were stored at −80°C for approximately 1 month prior to testing.
Microbial culture of tissue chamber fluid and lymph node samples
Quantitative cultures were performed for chamber fluid samples by the dilution method or the calibrated loop method. For the dilution method, serial 10-fold dilutions of collected fluid were made in PBS and transferred in 100-μL aliquots to TSA plates. For the calibrated loop method, calibrated inoculating loopsf were used to transfer well-defined volumes (1 and 10 μL) of undiluted samples to individual TSA plates. Samples were also directly plated to TSA plates by the swab method to increase the likelihood that low numbers of organisms (< 100 CFUs/mL) would be detected. Plates were incubated at 35 ± 2°C in an atmosphere of air with 5% CO2 for 48 hours; colonies were examined after incubation for 24 and 48 hours and enumerated by counting.
Samples from lymph node tissue were collected by swabs that were used to inoculate TSA plates. The culture conditions were the same as those described for quantitative cultures of chamber fluid. Results from lymph node tissue culture were recorded as positive or negative for CPT.
Determination of oxytetracycline concentrations in plasma and tissue chamber fluid
Acetonitrile,g methanol (liquid chromatography-mass spectrometry grade),g oxytetracycline hydrochloride,h demeclocycline hydrochloride,h and formic acid (liquid chromatography-mass spectrometry grade)i were used to determine oxytetracycline concentrations in sheep plasma and tissue chamber fluid. All chemicals used in the study were of American Chemical Society reagent grade or higher. Demeclocycline hydrochlorideh was used as an internal standard for quantification of oxytetracycline.
Analytes were extracted from chamber fluid and plasma by protein precipitation. Two concentration ranges of calibrators (low level and high level) were prepared. For low-level calibrators, 200 μL of blank matrix was pipetted into a 1.5-mL centrifuge tube. Calibrators of 50, 100, 200, 500, 2,000, 5,000, and 10,000 ng/mL concentration were prepared with 2 μg of the internal standard. For high-level calibrators, 10 μL of blank matrix was pipetted into a 1.5-mL centrifuge tube. Calibrators of 0.1, 0.5, 1.0, 5.0, 20.0, 50.0, and 100.0 mg/mL concentration were prepared with 2 μg of the internal standard. Then 0.5 mL of acetonitrile was added. The tubes were vortexed well and centrifuged for approximately 5 minutes. The clear supernatant was transferred to an autosampler vial for LC-MS-MS analysis. The LC-MS-MS method validation for tissue chamber fluid included 4 concentrations: 200 ng/mL (n = 8), 1,000 ng/mL (9), 2,000 ng/mL (8), and 20,000 ng/mL (9); mean ± SD measured concentrations were 173 ± 11 ng/mL, 1,030 ± 153 ng/mL, 2,106 ± 127 ng/mL, and 18,793 ± 2,026 ng/mL, respectively. The coefficients of variation were 6.2%, 14.8%, 6.0%, and 10.8%, respectively, and accuracy was 86.4%, 103%, 105%, and 94%, respectively. The LC-MS-MS method validation for plasma included concentrations of 200 ng/mL (n = 6) and 2,000 ng/mL (n = 9); mean ± SD measured concentrations were 197 ± 16 ng/mL and 2,168 ± 194 ng/mL, respectively. The coefficients of variation were 8.0% and 8.9%, respectively, and accuracy was 98.3% and 108.4%, respectively.
All LC-MS-MS analyses were conducted by use of a triple quadrupole mass spectrometer with an electrospray ionization source.j The mass spectrometer was calibrated weekly with the calibration standard in accordance with instructions in the instrument manual. The separations were carried out on an express C18 column (internal diameter, 100 × 2.1 mm; particle diameter, 2.7 μm) with a C18 guard column (internal diameter, 5 × 2.1 mm; particle diameter, 2.7 μm)k maintained at 40°C. Mobile phase A contained water with 0.1% formic acid, and mobile phase B contained acetonitrile with 1% formic acid. The following gradient elution was used at a flow rate of 0.5 mL/min: 95% phase A and 5% phase B from 0 to 0.5 minutes, followed by a gradient from 5% to 30% phase B from 0.5 to 1.5 minutes, then a gradient from 30% to 95% phase B from 1.5 to 2 minutes. After holding at 95% phase B from 2 to 3.0 minutes, a return from 95% to 5% phase B took place at 3.0 to 3.1 minutes, followed by holding at 5% phase B from 3.1 to 5.5 minutes. All LC-MS-MS data were collected in positive ion mode by multiple reaction monitoring of the transition m/z 461.2 → m/z 426.1, 283, and 201 for oxytetracycline and m/z 465.1 → m/z 154, 448, and 289 for demeclocycline. The optimized settings for electrospray ionization were as follows: capillary voltage, 4.0 kV; gas temperature, 350°C; gas flow, 10 L/min; and nebulizer pressure, 50 psi. A 0.1- or 10-μL aliquot of extracted analyte was injected for LC-MS-MS analysis.
Pharmacokinetic analysis
Noncompartmental analysis was performed with industry-standard softwarel to estimate pharmacokinetic parameters of interest for oxytetracycline in plasma and tissue chamber fluid. The following parameters were estimated for plasma samples: tmax, Cmax, t1/2λz (calculated as ln2λz, where λz is the terminal slope of the concentration-vs-time curve as estimated by linear regression of time vs log concentration [AUC0-last, calculated by the linear trapezoidal rule]), AUC from time 0 to infinity (calculated by adding the last observed concentration divided by λz to AUC0-last), AUMC from time 0 to the last observed concentration, AUMC from time 0 to infinity, mean residence time estimated from time 0 to the last observed concentration (calculated as AUMC from time 0 to the last observed concentration divided by AUC0-last), and mean residence time estimated from time 0 to infinity (calculated as AUMC from time 0 to infinity divided by AUC from time 0 to infinity). The following parameters were calculated for tissue chamber fluid samples: tmax, Cmax’ and t1/2λz.
Determination of MICs of commonly used antimicrobials for CPT isolates from goats
Fifty-three clinically derived isolates of CPT from goats were obtained from the Texas A&M University Veterinary Medical Teaching Hospital Clinical Microbiology Laboratory repository. Isolates were confirmed as CPT by means of matrix-assisted, laser desorption-ionization time-of-flight mass spectrometry.m Isolates were tested for nitrate reductase activity and synergistic hemolysis with Rhodococcus equi. Isolates that were nitrate-reductase positive (n = 4) were considered likely to be isolates of equine origin and were not investigated further. The remaining 49 isolates, including the isolate used in chambers in the present study, were tested for antimicrobial susceptibility with a commercially available panel of drugs commonly used in food animal medicineb according to the 2013 Clinical Laboratory Standards Institute approved standard.13 Briefly, 10 μL of suspension for each isolate was transferred into 10 mL of cation-adjusted Mueller-Hinton broth for a final bacterial concentration of 1 × 105 CFUs/mL. Each well of the test plate was inoculated with 100 μL of this solution. Each plate included a positive control well that contained no antimicrobial. The plates were sealed and incubated in room air at 35°C. Plates were read by visual inspection after 24 and 48 hours of incubation because of the slow growth habit of the organism. Results were recorded as growth or no growth. Plates were considered invalid if there was no apparent growth in the positive control wells. The assay was tested weekly for quality control purposes with Staphylococcus aureus (American Type Culture Collection No. 29213) and Escherichia coli (American Type Culture Collection No. 25922), and the results obtained for these bacteria were within acceptable ranges.13 The MIC for each isolate was defined as the lowest tested concentration of an antimicrobial to inhibit any visible growth. After the MIC was determined for each drug for each individual isolate, the MIC required to inhibit the growth of 90% of independent isolates (44 of 49 isolates) was determined. Although these isolates were obtained from goats, CPT isolates of sheep or goat origin indiscriminately infect either species.
Results
Two sheep had incisional dehiscence at 1 chamber implantation site each (the CPT-inoculated chamber in a sheep that received oxytetracycline by IM injection and the control chamber in a sheep that had the drug injected percutaneously into the CPT-inoculated chamber) prior to any sampling. These chambers were surgically removed, and incisions were allowed to heal by second intension with daily application of a wound fly control product. Otherwise, all sheep remained healthy for the rest of the study period. On day 0, the remaining 9 tissue chambers inoculated with CPT were culture positive (approx 1 × 105 CFUs/mL to 1 × 109 CFUs/mL), and the remaining 9 control chambers were culture negative.
In the 4 CPT-inoculated chambers that had intrachamber oxytetracycline administration immediately after the time 0 sample collection, no CPT bacterial growth was noted beyond the 48-hour time point (Figure 2). In the sheep that had oxytetracycline injected into the control chamber (n = 1) and the sheep that received oxytetracycline IM (4), the inoculated chambers remained culture positive throughout the study (Figure 3). All control chambers remained culture negative throughout the study. The MIC of oxytetracycline for the CPT isolate used in the study was ≤ 0.5 μg/mL and 1.0 μg/mL after 24 and 48 hours of culture, respectively.
Results of quantitative culture of CPT for samples collected from CPT-inoculated tissue chambers in 4 sheep immediately before and at predetermined time points after percutaneous injection of oxytetracycline into the CPT-inoculated chamber. Each symbol represents 1 sheep. The control chambers in all sheep (n = 9) remained culture negative throughout the study.
Citation: American Journal of Veterinary Research 80, 6; 10.2460/ajvr.80.6.586
Results of quantitative culture of CPT for samples collected from CPT-inoculated tissue chambers in 4 of 5 sheep that received oxytetracycline IM (each represented by a different symbol) and 1 sheep that received the drug by injection into the control chamber (dashed-and-dotted line with no symbol).
Citation: American Journal of Veterinary Research 80, 6; 10.2460/ajvr.80.6.586
Cultures of the regional lymph nodes from both sides of all sheep at the end of the study revealed 1 CPT-positive lymph node in 1 sheep. The implanted tissue chamber inoculated with CPT was ipsilateral to the affected lymph node, and oxytetracycline had been administered by injection into the inoculated chamber. This chamber was culture negative for CPT 48 hours after oxytetracycline administration and throughout the remaining sample collection times.
The time 0 concentration of oxytetracycline in chamber fluid for 1 ewe that received oxytetracycline IM was assumed to be an error (reported concentration, 1.638 μg/mL) and was therefore replaced with 0 μg/mL for the purposes of pharmacokinetic parameter estimates. The mean Cmax of oxytetracycline in plasma after IM administration was more than double that after intrachamber administration (Figure 4). Mean concentrations of oxytetracycline in all tissue chambers of sheep that had intrachamber drug injection were 1 × 102 to 1 × 104 times as high as those in sheep that had IM treatment (Figure 5). Following IM administration, oxytetracycline concentrations over time in tissue chambers mimicked those of plasma concentrations in sheep. Mean t1/2λz of oxytetracycline in plasma after IM or intrachamber administration were similar (57 and 69 hours, respectively), although the drug appeared to be eliminated more rapidly from control chambers than from CPT-inoculated chambers (Tables 1 and 2; Supplementary Tables S1 and S2, available at avmajournals.avma.org/doi/suppl/10.2460/ajvr.80.6.586). The MICs of antimicrobials commonly used in food-producing animals were determined for the 49 clinically derived caprine CPT isolates (Table 3).
Mean and SD plasma concentrations of oxytetracycline for the 10 sheep described in Figure 1 after IM (circles; n = 5) or intrachamber (diamonds; 5) drug administration. The SD is shown in only 1 direction/treatment type to simplify visual interpretation.
Citation: American Journal of Veterinary Research 80, 6; 10.2460/ajvr.80.6.586
Mean and SD oxytetracycline concentrations in CPT-inoculated (n = 9) and control tissue chambers (9) for the 10 sheep described in Figure 1. Oxytetracycline concentrations in CPT-inoculated (crosses) and control (triangles) chambers after intrachamber injection remained substantially higher than those in CPT-inoculated (circles) and control (diamonds) chambers after IM drug injection throughout the study. The SD is shown in only 1 direction to simplify visual interpretation.
Citation: American Journal of Veterinary Research 80, 6; 10.2460/ajvr.80.6.586
Mean ± SD pharmacokinetic parameters for oxytetracycline in plasma as estimated by noncompartmental analysis in a study to determine concentrations of the drug in plasma, CPT-inoculated tissue chambers (used as experimental abscess models), and uninoculated (control) tissue chambers in 10 sheep after IM or local administration and to investigate whether CPT growth was reduced or eliminated by these treatments.
| Oxytetracycline administration | ||
|---|---|---|
| Parameter | IM | IC |
| tmax (h) | 12 ± 0 | 14 ± 5 |
| Cmax (μg/mL) | 4.9 ± 0.9 | 1.3 ± 0.4 |
| λz (h−1) | 0.014 ± 0.006 | 0.011 ± 0.003 |
| t1/2λz (h) | 57 ± 21 | 69 ± 27 |
| AUC0-last (μg·h/mL) | 176.0 ± 38.5 | 80.0 ± 33.1 |
| AUC0–∞(μg·h/mL) | 211.2 ± 62.0 | 104.1 ± 40.2 |
| AUG% extrap (%) | 14 ± 11 | 22 ± 11 |
| AUMC0-last (μg·h2/mL) | 7,807.5 ± 2,714.0 | 4,355.2 ± 1,889.0 |
| AUMC0–∞ (μg·h2/mL) | 16,822.0 ± 12,570.2 | 10,701.5 ± 5,655.5 |
| MRT0-last (h) | 44 ± 8 | 54 ± 3 |
| MRT0–∞ (h) | 73 ± 31 | 100 ± 36 |
Each sheep had 1 sterile chamber surgically implanted into each paralumbar fossa; 2 weeks later (day −6), 1 randomly selected chamber was inoculated by percutaneous injection with CPT (1 × 106 CFUs/mL in BHIB), and the contralateral chamber was injected with sterile BHIB (1 mL/injection). Oxytetracycline was administered at time 0 in a single 20-mg/kg dose IM (n = 5) or percutaneously into CPT-inoculated (4) or control (1) chambers. Concentrations were measured in plasma and in chamber fluid 12, 24, 48, 72, 96, and 144 hours after drug injection.
AUC% extrap = Percentage of the AUC that was extrapolated. AUC0–∞ = AUC from time 0 to infinity. AUMC0–∞ = AUMC from time 0 to infinity. AUMC0-last = AUMC from time 0 to the last measured concentration. IC = Intrachamber. λz = Terminal rate constant. MRT0–∞ = Mean residence time from time 0 to infinity. MRT0-last = Mean residence time from time 0 to the last measured concentration. tmax = Time to maximum concentration.
Mean ± SD pharmacokinetic parameters for oxytetracycline in the CPT-inoculated (n = 9) and control tissue chambers (9) of the same 10 sheep as in Table 1.
| Oxytetracycline administration | ||
|---|---|---|
| Parameter | IM | IC |
| CPT-inoculated chambers | ||
| tmax (h) | 24 ± 0 | 38 ± 32 |
| Cmax (μg/mL) | 2.5 ± 0.8 | 12,600 ± 18,900 |
| λz (h−1) | 0.014 ± 0.003 | 0.0I2 ± 0.005 |
| t1/2λz (h) | 52 ± I2 | 69 ± 33 |
| Control chambers | ||
| tmax (h) | 31 ± 14 | 18 ± 6 |
| Cmax (μg/mL) | 1.5 ± 0.8 | 912.0 ± 1,579.0 |
| λz (h−1) | 0.020 ± 0.006 | 0.010 ± 0.003 |
| t1/2λz (h) | 38 ± 14 | 72 ± 16 |
One chamber of each type (the CPT-inoculated chamber in a sheep that received oxytetracycline IM and the control chamber in a sheep that received the drug by injection in the inoculated chamber) was removed prior to sample collection because of surgical site dehiscence.
See Table 1 for remainder of key.
Results of antimicrobial susceptibility testing for 49 CPT isolates from goats with clinical cases of CL.
| MIC90 (μg/mL) | ||
|---|---|---|
| Antimicrobial | 24 hours | 48 hours |
| Ampicillin | ≤ 0.25 | 0.5 |
| Ceftiofur | 1 | 1 |
| Chlortetracycline | 0.5 | 2 |
| Clindamycin | ≤ 0.25 | ≤ 0.25 |
| Danofloxacin | ≤ 0.12 | ≤ 0.12 |
| Enrofloxacin | ≤ 0.12 | ≤ 0.12 |
| Florfenicol | 2 | 2 |
| Gentamicin | 2 | 2 |
| Neomycin | ≤ 4 | ≤ 4 |
| Oxytetracycline | ≤ 0.5 | 1 |
| Penicillin | 0.25 | 0.25 |
| Spectinomycin | 16 | 16 |
| Sulphadimethoxine | ≤ 256 | ≤ 256 |
| Tiamulin | ≤ 0.5 | ≤ 0.5 |
| Tilmicosin | ≤ 4 | ≤ 4 |
| Trimethoprim-sulfamethoxazole | ≤ 2 | ≤ 2 |
| Tulathromycin | ≤ I | ≤ 1 |
| Tylosin | ≤ 0.5 | ≤ 0.5 |
Fifty-three isolates were obtained from a clinical microbiology laboratory repository and confirmed as CPT by means of matrix-assisted, laser desorption-ionization time-of-flight mass spectrometry. Nitrate-reductase testing revealed that 4 isolates were likely to be of equine origin; these were not investigated further. Each remaining isolate was suspended in cation-adjusted Mueller-Hinton broth (1 × 105 CFUs/mL) and transferred to a commercially available antimicrobial susceptibility test plate that was read after 24 and 48 hours of incubation at 35°C. The antimicrobials tested are commonly used for treatment of food-producing animals; not all the drugs on this list are labeled or approved for administration to sheep in the United States.
MIC90 = MIC at which 90% of the isolates were inhibited.
Discussion
The effectiveness of some tetracyclines has been shown to depend on the AUC-to-MIC ratio, although it has not been established whether oxytetracycline has this characteristic.14–16 For sheep that received oxytetracycline by IM injection in the present study, drug concentrations > 1 μg/mL (MIC for the isolate, 0.5 μg/mL) were achieved in all CPT-inoculated tissue chambers, but all these chambers remained culture positive for CPT throughout the entire study. This suggested that even though the concentrations of oxytetracycline reached concentrations greater than the MIC of the CPT isolate used in the study, they were not maintained at this level long enough to effectively kill CPT. These results supported our hypothesis that there would be detectable concentrations of oxytetracycline within tissue chambers after IM administration owing to the moderate lipid solubility of the drug11 and nullified our hypothesis that oxytetracycline concentrations in the chambers would be less than the MIC of the CPT isolate when the drug was given IM.
Our results indicated that intrachamber administration of a 20-mg/kg dose of oxytetracycline effectively eliminated the CPT isolate ≤ 48 hours after treatment. We have shown the abscess model to be valid9 and are confident the results suggested that in clinical cases of CL, injection of this dose of oxytetracycline into the lesion will effectively eliminate CPT bacterial growth in abscesses in 48 hours. The results supported our hypothesis that injection of oxytetracycline (20 mg/kg) into CPT-inoculated chambers would reduce or eliminate growth of the organism, which was based on our previously conducted but unpublished testing of in vitro susceptibility of CPT to oxytetracycline and the ability of oxytetracycline to maintain bactericidal activity in purulent material.9,14–16
It was not surprising that oxytetracycline concentrations in tissue chambers after intrachamber injection were on the order of 1 × 102 to 1 × 104 times those measured after IM administration. However, the plasma Cmax of oxytetracycline in sheep that received IM treatment was more than twice that of sheep that received intrachamber administration. This fits with the moderately lipid-soluble nature of oxytetracycline leading to moderate penetration into or out of a lesion such as an abscess.
In a previous study,5 we found that tulathromycin administered directly into the CPT-inoculated tissue chambers (n = 6) in goats achieved and maintained concentrations greater than the MIC of that drug for the isolate used throughout the study (360 hours); however, bacterial growth in 3 of those 6 chambers remained throughout the entire study period. In comparison, all CPT-inoculated chambers in which oxytetracycline was injected percutaneously in the present study were culture negative within 48 hours after the treatment and remained so for the duration of the study. We speculate that tulathromycin may not maintain as much bactericidal activity in the presence of purulent material, compared with oxytetracycline. In our study5 of tulathromycin in goats, subcutaneous injection of tulathromycin achieved elimination of CPT growth in some inoculated chambers; however, all the CPT-inoculated chambers maintained growth following IM oxytetracycline administration in the present study. This suggested that the higher lipid solubility of macrolides17 facilitated greater penetration of tulathromycin into the abscess models from the circulation.
Interestingly, the 1 subiliac lymph node that was culture positive for CPT at the end of the present study was from the side of a sheep that contained an inoculated tissue chamber that was injected with oxytetracycline. This chamber had no growth of CPT within 48 hours after drug administration, yet the associated draining lymph node was culture positive at the 144-hour time point. This suggested that the very high concentrations of oxytetracycline inside the chamber did not penetrate beyond the encapsulated lesion to an adequate concentration or for an adequate time to effectively kill the organism in the lymph node. However, because of the unknown CL status of the clinically normal sheep used in the study, the bacteria cultured in the lymph node tissue may not have been the same isolate that was injected into the tissue chamber. In our study9 of tulathromycin in goats with CPT-inoculated tissue chambers, we also found lymph nodes that were culture positive for CPT ipsilateral to inoculated tissue chambers in which percutaneous injection of tulathromycin had eliminated bacterial growth. However, in that study,9 6 of 24 examined subiliac lymph nodes were culture positive at the end of the study, compared with only 1 of 20 in the present investigation. It should be noted, however, that the difference in study periods (360 hours for tulathromycin vs 144 hours for oxytetracycline) could have accounted for this disparity, and the number of animals in both studies was not large enough to determine statistical significance. If the present study had been continued to the 360-hour time point, it is possible that a larger number of CPT-infected regional lymph nodes would have been identified.
On the basis of the MIC data, in general, CPT is susceptible to several of the antimicrobials commonly used in food-producing animals. However, as evidenced in the results of the present study and in previous investigations, the ability of the antimicrobial to effectively penetrate active lesions and eliminate bacteria in vivo is dependent on many factors, some of which are unknown.
Overall, CPT is susceptible to both oxytetracycline and tulathromycin. Although there was not a tulathromycin treatment group in this study and comparisons of efficacy between studies and among species are subjective, it appears that intralesional oxytetracycline treatment may outperform intralesional or SC tulathromycin administration in regard to the apparent elimination of CPT in abscess-type lesions and possibly a lower incidence of lymph node infection. Further, complete elimination of the organism following a single dose of either drug was not achieved, as lymph node tissue from animals in each study tested positive for CPT by means of culture. Our findings also supported those in other studies3,4 where various degrees of success were achieved by treatment of CL in sheep and goats with antimicrobial administration alone. It is also evident that there are many unknown factors influencing whether an antimicrobial will eliminate CPT in vivo. Taken together, results of the present study and our previous study5 in goats supported that CPT is extremely difficult to completely eliminate from small ruminants, and therefore, subsequent recrudescence of CL weeks to months after clinical resolution should be anticipated.
Acknowledgments
All animal experiments were performed at the College of Veterinary Medicine and Biomedical Sciences, Texas A&M University; all pharmacological analyses for oxytetracycline were performed at the Texas A&M Veterinary Medical Diagnostic Laboratory.
No external funding was used for the study. The authors declare there were no conflicts of interest.
The authors thank Austin Driskill for aid in animal care, sample collection, and data entry.
ABBREVIATIONS
| AUC | Area under the plasma concentration-versus-time curve |
| AUC0-last | Area under the plasma concentration-versus-time curve from time 0 to the last measured concentration |
| AUMC | Area under the first moment curve |
| BHIB | Brain-heart infusion broth |
| CL | Caseous lymphadenitis |
| Cmax | Maximum concentration |
| CPT | Corynebacterium pseudotuberculosis |
| LC-MS-MS | Liquid chromatography-tandem mass spectrometry |
| MIC | Minimum inhibitory concentration |
| t1/2λz | Terminal elimination half-life |
| Tmax | Time to maximum concentration |
| TSA | Trypticase soy agar |
Footnotes
Professional Plastics Inc, Fullerton, Calif.
Sensititre Bovine/Porcine MIC Plate, Thermo Fisher Scientific, Pittsburgh, Pa.
Beckton Dickinson and Co, Sparks, Md.
Innova 40, New Brunswick Scientific, Enfield, Conn.
Genesys 10, Thermo Fisher Scientific, Pittsburgh, Pa.
Nunc, Thermo Fisher Scientific, Pittsburgh, Pa.
VWR, Radnor, Pa.
Sigma-Aldrich, St Louis, Mo.
Fisher Scientific, Pittsburgh, Pa.
Agilent 6400, Santa Clara, Calif.
Ascentis Express, St Louis, Mo.
Phoenix WinNonLin, version 8.0.0.3176, Certara, Princeton, NJ.
Bruker Biotyper, Bruker Daltonics Inc, Billerica, Mass.
References
1. Williamson LH. Caseous lymphadenitis in small ruminants. Vet Clin North Am Food Anim Pract 2001;17:359–371.
2. Smith MC, Sherman DM. Subcutaneous swellings. In: Cann C, ed. Goat medicine. Philadelphia: Lea and Febiger, 1994:45–63.
3. Senturk S, Temizel M. Clinical efficacy of rifamycin SV combined with oxytetracycline in the treatment of caseous lymphadenitis in sheep. Vet Rec 2006;159:216–217.
4. Washburn KE, Bissett WT, Fajt VR, et al. Comparison of three treatment regimens for sheep and goats with caseous lymphadenitis. J Am Vet Med Assoc 2009;234:1162–1166.
5. Washburn KE, Fajt VR, Lawhon SD, et al. Caprine abscess model of tulathromycin concentrations in interstitial fluid from tissue chambers inoculated with Corynebacterium pseudotuberculosis following subcutaneous or intrachamber administration. Antimicrob Agents Chemother 2013;57:6295–6304.
6. Washburn KE, Bissett WT, Waldron DF, et al. Serologic and bacteriologic culture prevalence of Corynebacterium pseudotuberculosis infection in goats and sheep and use of Bayesian analysis to determine value of assay results for prediction of future infection. J Am Vet Med Assoc 2013;242:997–1002.
7. Gorden PJ, Ydstie JA, Kleinhenz MD, et al. A study to examine the relationship between metritis severity and depletion of oxytetracycline in plasma and milk after intrauterine infusion. J Dairy Sci 2016;99:8314–8322.
8. Palmer GH, Bywater RJ, Stanton A. Absorption in calves of amoxicillin, ampicillin, and oxytetracycline given in milk replacer, water, or an oral rehydration formulation. Am J Vet Res 1983;44:68–71.
9. Tongaree S, Flanagan DR, Poust RI. The interaction between oxytetracycline and divalent metal ions in aqueous and mixed solvent systems. Pharm Dev Technol 1999;4:581–591.
10. Romanet J, Smith GW, Leavens TL, et al. Pharmacokinetics and tissue elimination of tulathromycin following subcutaneous administration in meat goats. Am J Vet Res 2012;73:1634–1640.
11. Martin-Jimenez T, Craigmill AL, Riviere JE. Extralabel use of oxytetracycline. J Am Vet Med Assoc 1997;211:42–44.
12. Clarke CR, Short CR, Usenik EA, et al. Subcutaneously implanted tissue chambers: a pathophysiological study. Res Vet Sci 1989;47:195–202.
13. Clinical and Laboratory Standards Institute. Performance standards for antimicrobial disk and dilution susceptibility tests for bacteria isolated from animals; approved standard. CLSI document VET01-A4. 4th ed. Wayne, Pa: Clinical and Laboratory Standards Institute, 2013.
14. Agwuh KN, MacGowan A. Pharmacokinetics and pharmacodynamics of the tetracyclines including glycylcyclines. J Antimicrob Chemother 2006;58:256–265.
15. Alfouzan WA, Noel AR, Bowker KE, et al. Pharmacodynamics of minocycline against Acinetobacter baumannii studied in a pharmacokinetic model of infection. Int J Antimicrob Agents 2017;50:715–717.
16. Maaland MG, Papich MG, Turnidge J, et al. Pharmacodynamics of doxycycline and tetracycline against Staphylococcus pseudintermedius: proposal of canine-specific breakpoints for doxycycline. J Clin Microbiol 2013;51:3547–3554.
17. O'Rourke DJ, Baggot JD. Antimicrobial therapy of mastitis. In: Andrews AH, Blowey RW, Boyd H, et al, eds. Bovine medicine: diseases and husbandry of cattle. Oxford, England: Blackwell Science, 2008;391–406.
