Recipient Organization
UNIVERSITY OF ILLINOIS
2001 S. Lincoln Ave.
URBANA,IL 61801
Performing Department
Veterinary Research & Extension
Non Technical Summary
The proposed research project is focused on providing evidence-basis for use or to refute enrofloxacin in late pregnant mares. Enrofloxacin first became available in the veterinary market 30 years ago. Several studies carried out across the world involving most domestic animal species elegantly demonstrated the toxic effects of quinolones to adult horses and newborn foals when given at high doses. However, there is one major unanswered question "Can enrofloxacin be safely administered to pregnant mares?" This question is more relevant now than it was 30 years ago as antimicrobial resistance for common agents represents a serious threat to veterinary medicine. Thus, the ability to use different classes of antimicrobials in pregnant mares will provide additional tools for practicing veterinarians and as a resultproviding a service to the horse industry. We expect that completion of the present research proposal will unequivocally answer this major question. Our central hypothesis, which is based upon strong preliminary data, is that therapeutic doses of enrofloxacin can be safely and efficiently administered to pregnant mares as judged by the lack of lesions in the articular cartilage of long bones of fetuses from enrofloxacin-treated mares. Therefore, two objectives were set forth: (1): Determine plasma (maternal and fetal) and fetal fluids concentrations of enrofloxacin and ciprofloxacin in treated late-term pregnant mares; and (2) Evaluate articular cartilage of long bones in fetuses from mares treated with enrofloxacin.
Animal Health Component
50%
Research Effort Categories
Basic
50%
Applied
50%
Developmental
(N/A)
Goals / Objectives
Our central hypothesis, which is based upon strong preliminary data given below, is that therapeutic doses of enrofloxacin can be safely and therapeutically administered to pregnant mares as judged by lack of lesions in the tendons and articular cartilage of long bones of fetuses from enrofloxacin-treated mares.Hypothesis #1: Enrofloxacin and its active metabolite (ciprofloxacin) cross the placenta and achieve established MICs in maternal plasma, fetal fluids and fetalplasma.Specific Objective #1: Determine plasma (maternal and fetal) and fetal fluids concentrations of enrofloxacin and ciprofloxacin in treated late term pregnant mares.Hypothesis #2: Therapeutic doses (5mg/kg or 10 mg/kg IV, QD) of enrofloxacin administered to late-term pregnant mares do not cause lesions in fetal tendons and articular cartilage.Specific Objective #2: Evaluate articular cartilage of long bones and tendons in fetuses from mares treated with 2 doses of enrofloxacin (5 mg/kg, 10 mg/kg).
Project Methods
Twenty four light breed pregnant mares will be purchased locally. From purchase of the animals to commencement of the study, mares will be maintained in pastures at the Veterinary Medical Research Farm (VMRF) of the College of Veterinary Medicine at the University of Illinois (CVM-UIUC). Immediately prior to the start of the experiment, mares will be transported to the Veterinary Teaching Hospital (VTH) of the CVM-UIUC. The VTH research-designated ward has 35 stalls and 2 stocks USDA approved for research and teaching purposes. Following purchase and quarantine, mares will be used in other activities (e.g. blood sampling, vaccinations, teeth float, and pregnancy diagnosis) associated with the training of professional veterinary students. Costs of animal maintenance until commencement of the present study will be covered with the teaching budget without direct or indirect costs to this project.Before the beginning of the study, a thorough physical and reproductive examination will be performed to evaluate health and pregnancy status. Mares carrying normal pregnancies at ~280 days of gestation will be enrolled in this study, and divided into four groups as follows: 1) control (n=6, no treatment), 2) enrofloxacin (n= 6, therapeutic dose of 5 mg/kg), and 3) enrofloxacin (n=6, therapeutic dose of 10 mg/kg). Enrofloxacin will be administrated intravenously every 24 hours for 10 days for groups 2 and 3. Mares will be examined daily for any abnormalities, especially those pertaining to the musculoskeletal unit, such as lameness, cellulitis, tendonitis, or synovitisas well as for gastrointestinal tract side effects such as colitis. The 280 day gestational age was chosen based on the timing of the spontaneous occurrence of equine placentitis, the most common cause of treatment of pregnant mares with antimicrobials. Blood samples will be collected daily for 11 days in heparinized blood tubes and centrifuged at 600g/x10 minutes. Plasma samples will be harvested and preserved at -80oC until further analyses.Transabdominal ultrasound guided fetal fluid samplingwill be performed at days 1, 5, and 11 after the beginning of the enrofloxacin treatment. Cytologic evaluation of the fetal fluids will be used to confirm the placental compartment origin of each sample. The mare's abdomen will be clipped and surgically prepared. Thereafter, the mare will be sedated (detomidine 0.004 mg/kg/IV and butorphanol 0.004 mg/kg/IV), placed in stocks and fetal fluids will be collected aseptically with transabdominal ultrasound guidance using an echotip spinal needle (18Gx6"; 30 degree short bevel, Chiba-Type spinal needle, with stylet, Echo Block PTC Havel's Cincinnati, OH, USA). One dose of flunixin meglumine (1.1 mg/kg, IV) will be administered to the mare on the fetal fluid sampling days.Twenty four hours after the last dose of enrofloxacin, parturition will be induced with oxytocin (10 units of oxytocin/IV, repeated every 30 minutes until delivery). Fetuses delivered alive will be immediately euthanized with an intracardiac injection (12 mL) of a commercial solution containing sodium pentobarbital (390 mg/ml) and sodium phenytoin (50 mg/ml). Immediately before euthanasia, an aliquot (10 ml) of cardiac fetal blood will be collected, processed and stored in similar manner to maternal plasma. Mares in the control group will be aborted in similar manner.Concentration of enrofloxacin and ciprofloxacin in maternal and fetal plasma, amniotic and allantoic fluids will be determined with the 5500 QTRAP LC/MS/MS system (Sciex, Framingham, MA) in the Metabolomics Laboratory at theRoy J. Carver Biotechnology Center, University of Illinois. Software Analyst 1.6.2 will be used for data acquisition and analysis. The 1200 series HPLC system (Agilent Technologies, Santa Clara, CA) includes a degasser, an auto-sampler, and a binary pump. The LC separation will be performed on an Agilent SB-Aq column (4.6 x 50 mm, 5 µm) with mobile phase A (0.1% formic acid in water) and mobile phase B (0.1% formic acid in acetonitrile). The flow rate will be 0.3 mL/min. The linear gradient will be set as follows: 0-2 minutes, 95%A, 10-13 minutes, 2%A; 13.5-18 minutes, 95%A. The auto-samples will be set at 5 degrees C. The injection volume will be set at 1µL. Mass spectra will be acquired with positive electrospray ionization with the ion spray voltage of 550V. The source temperature will be 450 degrees C. The curtain gas, ion source gas 1, and ion source gas 2 will be 33, 50, and 60 psi, respectively. Multiple reaction monitoring will be used for quantification. Ciprofloxacin m/z 332.1 => m/z 288.1; Enrofloxacin m/z 360.1 => m/z 316.1. Internal standard Ofloxacin will be monitored at m/z 362.1 => m/z 318.1.Following premature induction of parturition, the fetuses will be transported to the necropsy floor at the Veterinary Diagnostic Laboratory. Drs. Roady and Driskell will harvest right and left proximal articular surface humerus, radius, femur, and tibia. These bones will be carefully dissected and the sections from the proximal aspect will be obtained and preserved in formalin until further processing. Specimens will be decalcified, processed and embedded and sectioned for histomorphometric measurements. Two sets of slides will be prepared and stained with hematoxilin eosin (HE) or toluidine blue (TB). Slides stained with HE will be used to assess structural and cellular features, whereas slides stained with TB will be used to evaluate the extracellular matrix (ECM). The intensity and pattern of TB staining can be used to determine whether there is damage in the ECM. Three experienced examiners blinded of the treatment will assess all slides. Evaluation of soft tissue structures will also be performed. At the metacarpal and metatarsal levels, the common digital extensor tendon, superficial flexor tendon, and deep flexor tendon, will be harvested for further evaluation. Tendons will be stained with H&E and toluidine blue, and then histological description of each specimen will be performedusing a light microscope.