ADMINISTRATION OF CEFTIOFUR, PENTOXIFYLLINE AND ALTRENGEST TO TREAT EQUINE PLACENTITIS

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: TERMINATED
• Funding Source: ANIMAL HEALTH
• Reporting Frequency: Annual
• Accession No.: 0222101
• Project No.: FLA-VME-005024
• Project Start Date: Apr 1, 2010
• Project End Date: Mar 31, 2015

Project Director
Macpherson, MA.

Recipient Organization
UNIVERSITY OF FLORIDA
G022 MCCARTY HALL
GAINESVILLE,FL 32611

Performing Department
College of Veterinary Medicine

Non Technical Summary
Foals that are delivered prior to the last week of a normal gestation experience high rates of death. Foal death, in turn, exerts a tremendous financial burden on the equine industry. The single most important cause of abortion and premature delivery in horses is bacterial placentitis (infection of the placenta). Nearly one-third of all premature births or stillborn deliveries in mares are caused by bacteria moving through the cervix and into the placenta. After infection, the placental tissues become inflamed and cause uterine contraction. Premature contractions cause birth of the foal. We believe that the key to preventing premature birth of foals is treating mares with drugs to eliminate bacteria, stop inflammation and quiet the uterus. Recently, our group at the University of Florida showed that infected mares treated with trimethoprim sulfa tablets, pentoxifylline and altrenogest delivered more live foals (83%) when compared to untreated mares (all foals dead). However, over half of the treated mares still had bacteria in their uterus after delivery, suggesting limitations in our choice of antibiotic. Therefore, we propose to test a different antibiotic, ceftiofur, to determine if live foals will be delivered after treatment and bacteria will be cleared from the reproductive tract. Ceftiofur is a strong antibiotic and has excellent effect in killing many different types of bacteria, particularly those that cause placentitis. Furthermore, ceftiofur is administered to horses in the muscle, once daily. Excellent activity against bacteria, tolerability and once daily intramuscular administration are desirable characteristics for an antibiotic used in the field and thus would have applicability to mares with placentitis. In this project, we propose to create infection of the placenta in pregnant mares by putting bacteria directly into the cervix. We will then treat mares with different drugs to see if we can improve foal survival. One group of 6 mares will be treated only with the antibiotic, ceftiofur. The second group of 6 mares will be treated with ceftiofur, pentoxifylline and altrenogest which is similar to our most recent work using trimethoprim sulfa. The final group of 3 mares will not receive any treatment after infection. We expect that the mares administered the combination of ceftiofur, pentoxifylline and altrenogest will deliver healthy, live foals. We expect that mares that are administered only ceftiofur will have a reduction in bacterial infection, but that the treatment will not be effective for stopping preterm labor and foals will be aborted. We expect that the final group of untreated mares will not deliver live foals. Using this model, we will obtain blood and tissue samples to identify ceftiofur in both mares and foals. We will also take uterine samples to see if the antibiotic was effective in clearing bacteria from the uterus after infection. Finally, and most importantly, we will show that ceftiofur, pentoxifylline and altrenogest combined, are good choices for treating mares with placentitis.

Animal Health Component

100%

Research Effort Categories

Basic

(N/A)

Applied

(N/A)

Developmental

(N/A)

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
311	3810	1020	20%
311	3810	1100	20%
311	3810	1180	10%
301	3810	1020	20%
301	3810	1100	20%
301	3810	1180	10%

Knowledge Area
311 - Animal Diseases; 301 - Reproductive Performance of Animals;

Subject Of Investigation
3810 - Horses, ponies, and mules;

Field Of Science
1180 - Pharmacology; 1100 - Bacteriology; 1020 - Physiology;

Keywords

mare

pregnancy

placentitis

infection

ceftiofur

altrenogest

pentoxifylline

abortion

treatment

Goals / Objectives
The loss of foals, as a consequence of premature delivery, has a devastating financial impact on the equine industry. Foals that are delivered prior to the last week of a normal gestation experience high rates of morbidity and mortality. The single most important cause of abortion and premature delivery in horses is bacterial placentitis[1]. Nearly one-third of all premature births, stillborn deliveries and perinatal deaths are caused by bacteria ascending through the vagina and colonizing the placenta [2]. Streptococcus equi subsp zooepidemicus is most commonly implicated in equine placentitis [2,3]. Bacterial infection initiates disease. However, premature delivery is thought to occur because of inflammation and prostaglandin production after placental infection[4-6]. If this hypothesis is correct, the key to overcoming foal loss due to placentitis is to control infection, but also to suppress inflammation and subsequent myometrial activity through control of prostaglandin production. Therefore, our long-range goal is to identify effective treatment strategies that prevent premature delivery of foals from mares with placentitis. The overall objective for this application is to determine if long-term administration of a broad-specturm antimicrobial (ceftiofur), an anti-inflammatory/anticytokine agent (pentoxifylline), and a progestin (altrenogest) to mares with experimentally-induced placentitis will promote delivery of a viable foal. Our central hypothesis is that combined therapy directed at eliminating the sources of bacterial infection, inflammation and myometrial activity will more effectively result in delivery of a viable neonate than administration of antibiotic alone or no treatment. We have formulated this hypothesis based upon results from studies in primates [7-11] and horses [12-16] which indicate that combination therapy is necessary to ameliorate the multifaceted effects of bacterial placental infection. We are particularly well suited to test this hypothesis with our established model of equine placentitis. We propose to test our central hypothesis and, thereby, to attain our overall objective by pursuing the following research objective: 1. Determine whether long term administration of ceftiofur, pentoxifylline and altrenogest improves fetal/neonatal survival in mares with experimentally-induced placentitis when compared to administration of ceftiofur, alone, or no treatment. Our working hypothesis is that combination therapy with an antimicrobial, anti-inflammatory/anti-cytokine agents and progestins is necessary to inhibit fetal infection, reduce inflammatory responses and delay premature parturition, thus improving fetal/neonatal well-being.

Project Methods
We propose to test the efficacy of ceftiofur, alone, as well as in combination with pentoxifylline and altrenogest. Infected, untreated mares will serve as controls. Between gestational Day 280 to 295 each pregnant pony mare will be inoculated with S. zooepidemicus directly into the cervix. Mares will be monitored using physical examination and ultrasonography for visual signs of placentitis beginning the day after inoculation. At the onset of clinical signs, drug treatment will begin for mares in treatment groups. Mares will be randomly assigned to one of three treatment groups. Six mares will administered ceftiofur, alone (Group CEFT); six mares will be administered ceftiofur, pentoxifylline and altrenogest (Group COMBO). Three mares will serve as infected, untreated controls (Group UNTREAT). Mares will be treated until abortion or parturition. Blood samples will be obtained from mares for ceftiofur and progesterone analysis beginning the day after inoculation and continuing for 14 days. Fetal and placental tissue samples will be analyzed for histologic abnormalities and ceftiofur concentrations. Live foals will be monitored for signs of prematurity, septicemia and viability. Serum concentrations of ceftiofur will be obtained from samples immediately following delivery (both mare and foal). Uterine samples will be obtained from mares within 3 hours of delivery to determine efficacy of ceftiofur for eradicating bacteria from the reproductive tract. We expect that the combination of ceftiofur, pentoxifylline and altrenogest will improve foal survival in mares with experimentally-induced placentitis. Further, we expect that mares administered this treatment will have resolution of bacteria within the reproductive tract as demonstrated with immediate post partum uterine culture samples.

Progress 04/01/10 to 03/31/15

Outputs
Target Audience: The target audience for this work is equine veterinarians who participate in reproductive and breeding work. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? This work has provided excellent clinical and scientific training opportunities to the investigators, veterinary residents and veterinary students. How have the results been disseminated to communities of interest? All work relevant to this project has been communicated by presenation at scientific meetings that reach a broad audience to include veterinarians and basic scientists. Further, the work has been published in both abstract and full manuscript form. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? With work done in our laboratory, we have shown the ceftiofur sodium and ceftiofur crystalline free acid (CCFA) do not penetrate the equine placental unit and attain therapeutic concentrations in the fetus. Further, we have shown that combination therapy using CCFA, altrenogest and pentoxifylline does not improve fetal survivability from treated mares with placentitis.

Publications

• Type: Journal Articles Status: Published Year Published: 2012 Citation: Disposition of desfuroylceftiofur acetamide in serum, placental tissue, fetal fluids, and fetal tissues after administration of ceftiofur crystalline free acid (CCFA) to pony mares with placentitis. Macpherson ML, Gigu�re S, Hatzel JN, Pozor M, Benson S, Diaw M, Sanchez LC, Vickroy TW, Tell L, Wetzlich S, Sims J. J Vet Pharmacol Ther. 2013 Feb;36(1):59-67. doi: 10.1111/j.1365-2885.2012.01392.x. Epub 2012 Mar 26

Progress 10/01/11 to 09/30/12

Outputs
OUTPUTS: All data collection for this aspect of the project was completed in 2011. Data were analyzed and published in 2012. Data were also presented at the Annual Meeting for the Society for Theriogenology and the Annual Conference for the American Association of Equine practitioners. Excerpts of these data were presented at the West Coast Equine Reproduction Symposium and the International Society of Italian Equine Veterinarians meeting. All meetings took place in 2012. PARTICIPANTS: Individuals involved in this project are listed in the authorship line. Dr. Jennifer Hatzel and Dr. Momo Diaw were in training programs while this study was conducted. TARGET AUDIENCES: The target audience for this work is the equine veterinarian. The information was disseminated to this population through publication and presentation both nationally and internationally. PROJECT MODIFICATIONS: Not relevant to this project.

Impacts
Data from this project revealed that a commonly used cephalosporin antibiotic does not cross the equine placenta and is not useful for treating mares with placentitis. This led to a subsquent study in 2012 where mares were administered another cephalosporin antibiotic which also did not cross the placenta.

Publications

• Macpherson ML, Giguere S, Hatzel JN,2 Pozor MA, Benson S,4 Diaw M,1 Sanchez LC, Vickroy TW, Tell L, Wetzlich S, Sims J3. Disposition of desfuroylceftiofur acetamide in serum, placental tissue, fetal fluids and fetal tissues after administration of ceftiofur crystalline free acid (CCFA) to pony mares with placentitis. J Vet Therapy and Pharm. 2012 Mar 26. Doi:10.1111/j.1365-2885.2012.01392.x.[Epub ahead of print].
• Hatzel JN,2 Macpherson ML, Giguere S, Pozor M, Benson S, Diaw M,1 Sanchez LC, Tell L, Wetzlich S,4 Simms J,3 Vickroy TW. Administration of ceftiofur crystalline free acid (CCFA) to pony mares with placentitis. Clinical Theriogenology 2012;4(3):431.
• Hatzel JN,2 Macpherson ML, Giguere S, Pozor M, Benson S,4 Diaw M,1 Sanchez LC, Tell L, Wetzlich S,4 Simms J,3 Vickroy TW. Administration of ceftiofur crystalline free acid (CCFA) to pony mares with placentitis. Proc 58th Ann Conv Am Assoc Equine Pract 2012;58:521-522.

Progress 10/01/10 to 09/30/11

Outputs
OUTPUTS: Data from this study has been shared at the following venues: Society for Theriogenology, Milwaukee, WI, Aug 2011; Florida Association of Equine Practitioners, Amelia Island, FL Oct 2011. An abstract for presentation to the American Association of Equine Practitioners was submtted in March 2011 but was denied. Abstracts from the data have been submitted to the Society of Theriogenology (J. Hatzel, et al, 2012) and will be submitted to the AAEP for 2012 (M. Macpherson, et al, 2012) PARTICIPANTS: Margo L. Macpherson, DVM, MS, Diplomate ACT; PI Steeve Giguere, DVM, PhD, Diplomate ACVIM; Co-I Jennifer N. Hatzel, DVM, MS; Graduate student Malgorzata Pozor, DVM, PhD, Diplomate ACT; Co-I Susanne Benson, BS, MS; Biological scientist Mouhamadou Diaw, DVM, Diplomate ACT; resident in reproduction L. Chris Sanchez, DVM, PhD, Diplomate ACVIM; Co-I Thomas W. Vickroy, PhD; Co-I Lisa Tell, DVM, DABVP, DACZM; HPLC Scott Wetzlich, BS; Judd Sims, BS. HPLC Judd Sims, Student worker and recipient of Merial Award This work represents collabortion with University of GA (Giguere) and the University of California Davis (Tell, Wetzlich). Part of the work was supported by Pfizer Animal Health. Assays were performed at the University of California Davis. TARGET AUDIENCES: The target audience for this work is the veterinary community with an empahsis on equine reproduction. The work will be published in a refereed veterinary journal (Journal of Veterinary Pharmacokinetics and Therapeutics) and will hopefully be presented at 1 or 2 veterinary conferences. These data are also very useful to horse owners. PROJECT MODIFICATIONS: In 2012 we will test a sister compound, ceftiofur sodium, to determine if it passes the fetoplacental unit in horses. The drug tested in the original CRIS project was crystalline ceftiofur free acid (CCFA). This drug was not detected in fetal fluids, tissues, foals or placentas suggesting limitations in passage of the drug across the fetoplacental unit.Ceftiofur (and cephalosporins) have excellent application in placental infections for horses. The work from 2012 will confirm the bioavailability of a ceftiofur (sodium) for use in mares with placentitis.

Impacts
Results from this study provided important results for treatment of equine placentitis. A novel, long-acting antibiotic (crystalline ceftiofur free acid) was tested for treatment of this condition in mares with experimentally-induced placentitis. Pharmacokinetic parameters were measured in pregnant mares, fetal and placental tissues. Results showed that the drug is present in the mare's blood stream at expected concentrations after administration. The drug was not detected in fetal fluids, tissues or placentas suggesting that the drug does not penetrate the fetoplacental barrier. Due to the long-acting prepartion of the drug (administration every 96 h) there is a high demand to use it in mares with placentitis. Our data show that the drug would not be acceptable for this use.

Publications

• Macpherson ML, Hatzel JN, Giguere S, et al. Administration of ceftiofur crystalline free acid (CCFA) to pony mares with placentitis. J Vet Pharm Therap, accepted with minor revisions Jan 2012.

Progress 10/01/09 to 09/30/10

Outputs
OUTPUTS: The objective of this project was to examine the ability of ceftiofur, a third generation cephalosporin, to eradicate uterine bacteria in mares with experimentally-induced placentitis as well as ensure delivery of viable foals. Ceftiofur has excellent antimicrobial activity against streptococcal organisms, many Gram negative aerobes, and some anaerobes. Our group hypothesized that long-term treatment with ceftiofur, pentoxifylline, and altrenogest to mares with experimentally-induced placentitis would result in delivery of viable foals as well as eradicate bacterial growth in infected mares and foals. Twelve normal, pregnant pony mares were enrolled in the study between days 280-295 of gestation and assigned to one of three groups: Group CEFT: (n = 3) administered ceftiofur crystalline free acid (long acting ceftiofur, Excede; 6.6 mg/kg, IM, q96h); Group COMBO: (n = 6) administered ceftiofur crystalline free acid (Excede; 6.6 mg/kg, IM, q96h), altrenogest (Regumate; 0.88 mg/kg, PO, q24h), and pentoxifylline (Pentoxifylline, Extended Release; 8.5 mg/kg, PO, q12h); Group UNTREAT: n = 3 mares serving as infected, untreated controls.. Each mare was inoculated with 10*7 CFU S. zooepidemicus, intracervically. At the onset of clinical signs (vulvar discharge, placental thickening, mammary enlargement), drug administration began for mares in treatment groups and was continued until delivery. No viable foals (0/3, 0%) were delivered from mares in Group CEFT. Two viable foals (2/6; 33%) were born to mares in Group COMBO. Two viable foals (2/3, 66%) were delivered from mares in Group UNTREAT. Days from inoculation to delivery were greater in Group TREAT (mean 24.7; range 10-41) than in Group Ceft (mean 15; range 7-25). S. zooepidemicus was the predominant organism recovered from cultures of uterine swabs of the mares that aborted and tissue and blood samples from their fetuses, with E. coli, Enterobacter, and Enterococcus also being found in some samples. Uterine swabs from mares that delivered viable foals and blood samples from their neonates revealed almost no growth upon bacterial culture. Only scant growth of E. coli from a Group COMBO mare and her foal and gram negative bacilli from the other Group COMBO foal were recorded. Placental tissue samples submitted for histopathology revealed necrotic, suppurative lesions in the cervical star region and the umbilical cord near the fetus to be the most common pathological changes in mares that aborted. Analysis of mare serum and placental tissue samples for ceftiofur concentrations are pending but preliminary results suggest poor placental passage of long-acting ceftiofur to fetal circulation or tissues. These data will be submitted in abstract form for the 2011 American Association of Equine Practitioners meeting and in full manuscript to the Journal of Veterinary Pharmacology. PARTICIPANTS: Not relevant to this project. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Not relevant to this project.

Impacts
Preliminary data from this work provided surprising results. Using a model of induced placentitis our laboratory has tested other antimicrobial agents with a much more limited spectrum of bacterial activity. In these studies, the abortion rate has been minimal (10/12 mares delivered live foals). Using a long-acting preparation of ceftiofur in this study resulted in a high abortion rate in treated mares. Further, preliminary date suggest that the drug had poor penetration across fetal membranes and into the fetus. These data are essential to the veterinary community as this drug (ceftiofur CFA) has generated great interest for long-term administration in mares with placentitis. While speculative, it would seem possible that the vehicle for this drug prevented penetration into affected areas this model. The obvious next step would be to administer ceftiofur sodium in a similar model to determine if absorption is better using that preparation. That question will be the target of the next study (2011).

Publications

• No publications reported this period