Antimicrobial use in species such as sheep and goats is often prescribed without label instructions. The AMDUCA of 1994 eased the scarcity of animal drugs available for use in sheep and goats by permitting veterinarians to use approved animal and human drugs on an extralabel or off-label basis.1 The Act states that, under certain circumstances, veterinarians can use drugs approved for other species, for other diseases and conditions, or at different dosages from those listed on the drug label. One of the practical challenges to implementing the Act's provisions is to determine appropriate dosage and withdrawal times for species not listed on the drug label.
The NRSP-7 is a USDA-funded program that pursues approval of drugs for use in so-called minor species.2 One such NRSP-7 project is extending the label claims for florfenicol in bovine respiratory tract disease to sheep and goats. Florfenicol is a broad-spectrum, primarily bacteriostatic antimicrobial with a range of activity that includes many gram-negative and gram-positive bacteria.3,4 Florfenicol is approved for use in beef cattle (New Animal Drug Application [NADA] 141-063) with respiratory tract disease associated with infection by Mannheimia haemolytica, Pasteurella multocida, and Haemophilus somnus.4 Because the most important respiratory tract disease bacteria isolated from sheep and goats are M haemolytica and P multocida, pursuing minor species approval of florfenicol for sheep and goats was a logical effort. An attractive feature of florfenicol is that it can be administered as a single-dose treatment or administered at 48-hour treatment intervals, protocols that may facilitate treatment of range animals like sheep and goats.
The objective of this study was to determine MIC values of florfenicol for respiratory tract pathogens isolated from sheep and goats and to use results to establish the potential efficacy of florfenicol in sheep and goats for treatment of respiratory disease caused by M haemolytica and P multocida. This can be achieved by contrasting serum time-concentration profiles of florfenicol in sheep and goats with the pathogens' MIC values and by comparing those with serum time-concentration profiles of florfenicol in cattle and cattle pathogen MIC values.
Materials and Methods
Bacterial isolates—Respiratory tract pathogens from ovine and caprine submissions to the Davis and Tulare branches of the California Animal Health and Food Safety Laboratory were evaluated. Forty-one ovine isolates and 36 caprine isolates from 1999 through 2002 were used in the study. Only 1 isolate/premise and submission date was used. Isolates included Mannheimia glucosida (n = 3), M haemolytica (39), Mannheimia varigena (1), P multocida (28), and Pasteurella trehalosii (6). Most (n = 72) isolates were recovered from lung tissue, but 1 nasal cavity isolate, 1 sinus isolate, 1 tracheal isolate, and 2 thoracic cavity isolates were also included. Isolates had previously been frozen and stored by the laboratory and were transferred to tryptic soy agar tubes and sent chilled overnight to the Veterinary Medicine Teaching and Research Center for susceptibility testing.
Antimicrobial susceptibility testing—Upon arrival at the Veterinary Medical Teaching and Resource Center, isolates were restreaked on blood agara and incubated for approximately 12 hours at 37°C. On the following day, a bacterial sampling loop was used to transfer 1 loop's volume of bacterial growth to a vial containing tryptic soy brotha; the vial was kept in an incubator at 37°C until growth in the tube achieved or exceeded 0.5 McFarland turbidity standards (4 to 6 hours). Disk diffusion assay was performed according to CLSI guidelines with the following antimicrobial disksb: florfenicol, 30 mg; amoxicillin-clavulanic acid, 20/10 mg; ceftiofur, 30 mg; tetracycline, 30 mg; and ciprofloxacin, 5 mg.5,6 Zones of inhibition were measured with a calibrated digital measuring device.c The MIC for florfenicol was determined by use of the microbroth dilution technique, according to CLSI guidelines.6 Custom-made 96-well platesd with florfenicol concentrations ranging from 0.12 to 128 mg/mL were obtained. Quality controls for both assays included Streptococcus pneumoniae ATCC 49619, Staphylococcus aureus ATCC 29213, and Escherichia coli ATCC 25922.e
The disk diffusion assay results were stratified by source and bacterium and were summarized by determination of mean, SD, and minimum and maximum zone sizes. The MIC values for florfenicol were similarly stratified, and median, mode, maximum, and minimum values were determined.f Stratified analysis with the nonparametric Kruskal-Wallis testg was used to compare florfenicol MICs among ovine and caprine isolates and between M haemolytica and P multocida.
Results
Results of univariate analysis of MIC values for florfenicol were summarized (Table 1). The MICs for all isolates were low, indicating that the isolates were susceptible to florfenicol. Descriptive statistics for the disk diffusion assay results were tabulated (Table 2). On the basis of CLSI guidelines, most isolates were susceptible to all antimicrobials tested.6 Of the 2 major bacterial species tested, a single M haemolytica isolate had resistance to 1 antimicrobial (tetracycline; zone of inhibition, ≤ 11 mm). Three other isolates (2 isolates of M glucosida and 1 isolate of P trehalosii) were also resistant to tetracycline. All isolates were uniformly susceptible to the other 4 antimicrobials tested.
Values of MIC50 and MIC90 (concentrations at which 50% and 90%, respectively, of isolates tested were inhibited) and mode, minimum, and maximum MIC values (μg/mL) for florfenicol in Mannheimia haemolytica and Pasteurella multocida isolates from sheep and goats with respiratory tract disease.
| Animal | Bacterium | No. | MIC50 | MIC90 | Mode | Min | Max |
|---|---|---|---|---|---|---|---|
| Goat | All isolates | 34 | 0.50 | 1.00 | 0.25 | 0.25 | 2.00 |
| M haemolytica | 25 | 0.50 | 1.00 | 0.50 | 0.25 | 2.00 | |
| P multocida | 7 | 0.25 | 0.50 | 0.25 | 0.25 | 0.50 | |
| Sheep | All isolates | 38 | 0.25 | 1.00 | 0.25 | 0.12 | 1.00 |
| M haemolytica | 13 | 0.50 | 0.50 | 0.50 | 0.25 | 1.00 | |
| P multocida | 19 | 0.25 | 0.50 | 0.25 | 0.25 | 1.00 |
Min = Minimum. Max = Maximum.
Mean, SD, and range of disk diffusion zones (in millimeters) of respiratory tract pathogens for various antimicrobials and stratified by source (ovine or caprine) and bacterium (M haemolytica or P multocida).
| Source | Antimicrobial | Mean | SD | Range |
|---|---|---|---|---|
| All isolates (n = 72) | Florfenicol | 29.7 | 3.7 | 23–40 |
| Amoxicillin-clavulanic acid | 30.9 | 5.8 | 22–58 | |
| Ceftiofur | 36.3 | 5.3 | 24–54 | |
| Tetracycline | 26.2 | 4.7 | 9–35 | |
| Ciprofloxacin | 33.9 | 3.4 | 25–43 | |
| Caprine isolates (34) | Florfenicol | 25.5 | 3.6 | 23–37 |
| Amoxicillin-clavulanic acid | 29.3 | 4.1 | 22–39 | |
| Ceftiofur | 35.1 | 3.5 | 28–42 | |
| Tetracycline | 26.2 | 3.5 | 10–30 | |
| Ciprofloxacin | 33.3 | 2.9 | 28–40 | |
| Ovine isolates (38) | Florfenicol | 30.9 | 3.4 | 26–40 |
| Amoxicillin-clavulanic acid | 32.3 | 6.7 | 23–58 | |
| Ceftiofur | 37.3 | 6.4 | 24–54 | |
| Tetracycline | 26.3 | 5.6 | 9–35 | |
| Ciprofloxacin | 34.4 | 3.8 | 25–43 | |
| Mannheimia haemolytica (38) | Florfenicol | 28.3 | 3.0 | 23–37 |
| Amoxicillin-clavulanic acid | 28.4 | 3.1 | 22–35 | |
| Ceftiofur | 33.7 | 3.0 | 27–41 | |
| Tetracycline | 25.7 | 3.3 | 10–31 | |
| Ciprofloxacin | 33.9 | 2.9 | 28–40 | |
| Pasteurella multocida (26) | Florfenicol | 32.0 | 3.3 | 25–40 |
| Amoxicillin-clavulanic acid | 31.2 | 3.7 | 23–41 | |
| Ceftiofur | 38.1 | 4.6 | 28–49 | |
| Tetracycline | 28.3 | 2.8 | 24–35 | |
| Ciprofloxacin | 34.3 | 3.7 | 27–43 |
Stratified analysis of animal species indicated that ovine isolates had lower (P = 0.01) MIC values for florfenicol than caprine isolates. The MIC values for M haemolytica were slightly higher (P < 0.01) than those for P multocida.
Discussion
This study revealed that pathogens isolated from the respiratory tract of sheep and goats with clinical disease were highly susceptible to florfenicol and other antimicrobials. Results can be used to establish the potential efficacy of florfenicol in sheep and goats for treatment of respiratory disease caused by M haemolytica and P multocida. This can be achieved by contrasting serum time concentration profiles of florfenicol in sheep and goats with the pathogens' MIC values, and by comparing those results with serum time-concentration profiles of florfenicol in cattle and cattle pathogen MIC values.
The MIC values for florfenicol obtained in this study were similar to those obtained from studies3,4 conducted in the United States, Canada, and Europe from 1990 to 1993. Pharmacokinetic studies7–9 have revealed that 3 doses (20 mg/kg) administered SC every 48 hours to sheep yield florfenicol concentrations higher than the target MIC of 0.5 mg/mL for > 108 hours. The drug may therefore be considered a valuable tool for treating sheep and goats with respiratory tract disease. The pathogens tested were similarly sensitive to all antimicrobials tested. Isolates were at least as sensitive to ceftiofur, florfenicol, and tetracycline as those submitted to a diagnostic laboratory in Nebraska from 1995 to 1997.10 The isolates were also all susceptible to ciprofloxacin, but because extralabel use of fluoroquinolones in food animals is prohibited, use of that drug in sheep and goats is prohibited in the United States.
Respiratory tract infections in sheep and goats usually have a multifactorial etiology that includes physical and physiologic stressors and predisposing viral and bacterial infections. Physical factors include weather, animal density, transportation, handling, and ventilation. Underlying infections that predispose small ruminants to infection with M haemolytica and P multocida include parainfluenza 3 virus, adenovirus type 6 and respiratory syncytial virus, Mycoplasma ovipneumonia, and Bordetella pertussis.11 Although results of the present study suggest that precise identification of the causative agent of acute respiratory tract disease may not be necessary before initiating antimicrobial treatment for these pathogens, the clinical success of treatment will be dependent on establishing appropriate dosage regimens that take into account the pharmacokinetic aspects of each drug in addition to the other etiologic and environmental factors.
Bacterial antimicrobial susceptibility is assessed primarily through the use of broth dilution or disk diffusion assays. The relationship between the 2 types of assays has been described,12–14 and both methods yield clinically relevant and reliable information for determining treatment strategy. Some of the bacteria investigated in the present study were difficult to culture and maintain in a viable state for the assays, and several isolates had to be cultured several times before the assay could be performed. In many instances, growth of the pathogens on Mueller-Hinton plates was faint and zone sizes were large. Use of Mueller-Hinton agar plates with sheep blood did not improve bacterial growth or detectability of the inhibitory zones.
Most respiratory tract pathogens from sheep and goats submitted to the diagnostic laboratory in California were susceptible to amoxicillin-clavulanic acid, ciprofloxacin, ceftiofur, florfenicol, and tetracycline. Susceptibility did not appear to vary across host or bacterial species, indicating that bacterial isolation and antimicrobial resistance testing may not be necessary prior to initiating antimicrobial treatment. However, for animals that do not respond favorably to treatment, pathogen identification and antimicrobial susceptibility testing are recommended.
ABBREVIATIONS
| NRSP-7 | National Research Support Project No. 7 |
| MIC | Minimum inhibitory concentration |
| CLSI | Clinical and Laboratory Standards Institute |
| ATCC | American type culture collection |
Hardy Diagnostics, Santa Maria, Calif.
Difco, Becton-Dickinson, Sparks, Md.
Fowler sylvac, Ultra-cal IV, Geneva Gage Inc, Albany, Ore.
TREK Diagnostics Systems Ltd, Cleveland, Ohio.
American Type Culture Collection, Manassas, Va.
Proc Univariate, SAS, version 8.2, SAS Institute Inc, Cary, NC.
Proc Freq, SAS, version 8.2, SAS Institute Inc, Cary, NC.
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