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Theriogenology 74 (2010) 402–412

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Treatment efficacy of trimethoprim sulfamethoxazole,pentoxifylline and altrenogest in experimentally

induced equine placentitis

C.S. Baileya,c, M.L. Macphersona,*, M.A. Pozora, M.H.T. Troedssona,d, S. Bensona,S. Giguerea,e, L.C. Sancheza, M.M. LeBlancb, T.W. Vickroya

a University of Florida, Dept of LACS, College of Veterinary Medicine, PO Box 100136, Gainesville FL, USA 32610b Rood and Riddle Equine Hospital, 2150 Georgetown Road, Lexington, KY, USA

c North Carolina State, College of Veterinary Medicine, 4700 Hillsborough St, Raleigh, NC 27606; Troedssond University of Kentucky, Gluck Equine Research Foundation, 108 Gluck Equine Research Center Lexington, KY 40546

e University of Georgia, Dept of LAM, 501 D.W. Brooks Drive, Athens, GA 30602

Received 4 January 2009; received in revised form 11 February 2010; accepted 24 February 2010

bstract

The objective was to determine if long-term treatment with trimethoprim sulfamethoxazole (antimicrobial), pentoxifylline (anti-nflammatory/anti-cytokine) and altrenogest (synthetic progestin), would improve pregnancy outcome in mares with experimentallynduced placentitis. Seventeen normal, pregnant pony mares were enrolled in the study at 280–295 d of pregnancy. Placentitis wasnduced in all mares by intra-cervical inoculation of Streptococcus equi subsp. zooepidemicus (107 CFU). Five mares served as infected,ntreated control animals (Group UNTREAT). Twelve mares (Group TREAT) were infected and given trimethoprim sulfamethoxazole30 mg/kg, PO, q 12h), pentoxifylline (8.5 mg/kg, PO, q 12h) and altrenogest (0.088 mg/kg, PO, q 24h) from the onset of clinical signso delivery of a live foal or abortion. Blood samples were cultured from all foals at delivery and fetal stomach and thoracic contents werebtained for culture from dead fetuses. More mares in Group TREAT delivered viable foals (10/12; 83%; P � 0.05) than mares in GroupNTREAT (0/5; 0%). Ten of 12 foals (83%) in Group TREAT had negative blood cultures at birth. All foals in Group UNTREAT (5/5;00%) had positive cultures from one or more samples (blood, stomach contents, and thoracic fluid). Bacteria were recovered fromterine culture samples in both groups. Streptococcus equi subsp. zooepidemicus was the predominant organism recovered frometal/foal or mare culture samples. The authors inferred that administration of trimethoprim sulfamethoxazole, pentoxifylline andltrenogest may improve the viability of foals from mares with experimentally induced placentitis.ublished by Elsevier Inc.

www.theriojournal.com

aAcadAfm

. Introduction

Placentitis is a common infectious cause of abortion,remature delivery, and neonatal mortality in the horse1–5]. Placentitis generally occurs during late gestation,

* Corresponding author. Tel.: 352 392 2229; fax: 352 392 8289.E-mail address: macphersonm@vetmed.ufl.edu (M.L. Macpher-

lon).

093-691X/$ – see front matter Published by Elsevier Inc.oi:10.1016/j.theriogenology.2010.02.023

nd can be of bacterial, fungal, or viral origin [1,2].scending bacterial infection, through the cervix, oc-

urs most frequently [1–3,6]. The most common caus-tive organism is Streptococcus equi subsp. zooepi-emicus (Strep. equi subsp. zooepidemicus) [1–3,5,7].lthough many mares with placentitis abort acutely,

oals born to mares with chronic placental infectionsay have an accelerated fetal maturation and are more

ikely to survive [6,8]. This provides an incentive for

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403C.S. Bailey et al. / Theriogenology 74 (2010) 402–412

arly and aggressive treatment in mares with symptomsf placentitis.

Most current treatment regimens for equine placen-itis are empirical. Early studies focused on identifyingommon antibiotics in fetal fluids, and from foals, oformal mares after treatment [9,10]. Trimethoprim sul-adiazine and possibly penicillin [9] were identified inhese studies, but gentamicin was not detected [10].

ore recent studies used an in vivo microdialysis tech-ique for continuous drug monitoring in the allantoicuid of pregnant pony mares. In these studies, thereere therapeutic concentrations of penicillin G, genta-icin [11], trimethoprim sulfamethoxazole (TMS), and

entoxifylline (PTX) [12] in allantoic fluid followingreatment. Drugs were detected in allantoic fluid of bothormal and experimentally-infected mares. Further, al-antoic concentrations of all antibiotics were found toeet or exceed published MIC values [13] for at leasth. In addition, tissue concentrations of TMS and PTXere present in aborted fetuses and placentas [14].ares treated with TMS and PTX, after experimentally

nduced placentitis, tended to carry pregnancies longer2–27 d) than infected, untreated, mares (2–14 d).ased on these findings, the authors concluded that therug combination showed promise for treating placen-itis, but that additional therapies might be necessary toddress the multiple facets of this disease process (in-ection, inflammation, and uterine contractility).

Exogenous progestins, most commonly altrenogestRegumate™), are frequently included in treatment oflacentitis. Little definitive information is available re-arding the efficacy of progestin therapy to inhibitremature delivery in late term pregnant mares. Work-rs from Kentucky retrospectively evaluated treatmentecords from mares diagnosed with placentitis over a-y period [15]. Of the 15 cases treated using a com-ination of antibiotics, anti-inflammatory medication,nd altrenogest, 11 mares delivered live-born foals15]. These findings, as well as information from workn other species [15–17], supported a multiprongedpproach to treatment of placentitis. Therefore, the ob-ective of the current study was to determine whetherreatment with trimethoprim sulfamethoxazole (TMS,ral antibiotic), pentoxifylline (PTX, anti-inflamma-ory, and anti-cytokine), and altrenogest (ALT, syn-hetic progestin) would increase the length of preg-ancy and improve neonatal viability in mares withxperimentally induced placentitis. We hypothesizedhat mares treated with TMS, PTX, and ALT wouldaintain pregnancy longer than untreated mares, and

hat the resultant foals would be viable at parturition. c

. Materials and methods

.1. Mares

Seventeen reproductively normal, pregnant ponyares were enrolled in the study at 280–295 d of

regnancy. Baseline inclusion data (systemic parame-ers; combined thickness of the uteroplacental unit [18–1,21], echodensity of fetal fluids, fetal activity, andeart rate within normal limits [22,23] were recordedrior to experimentation. All mares were inoculatedith Strep. equi subsp. zooepidemicus and divided ran-omly into two groups. Five mares served as infected,ntreated control mares (Group UNTREAT). Twelveares were infected and given trimethoprim sulfame-

hoxazole (TMS), altrenogest (ALT), and pentoxifyl-ine (PTX) (Group TREAT). Mares were maintained onasture at the College of Veterinary Medicine, Univer-ity of Florida and were supplemented with hay andoncentrate. This project was approved by the Institu-ional Animal Care and Use Committee of the Univer-ity of Florida.

.2. Bacterial inoculation

Between 280 and 295 d of pregnancy, mares werenoculated, intracervically, with Strep. equi subsp. zoo-pidemicus obtained from a clinical isolate submitted tohe Microbiology Laboratory at the University of Flor-da, College of Veterinary Medicine [24]. A stock so-ution of the bacterial isolate was stored in cryovialsontaining Brucella broth with 10% glycerol and po-ous beads (Cryosaver®, Hardy Diagnostics, Santa

aria, CA, USA) at �80 °C. The bacterial isolate wasensitive to TMS in vitro. Two days before inoculation,n aliquot was thawed and re-plated to confirm a pureulture. On the day of inoculation, a 107 CFU inoculateas made using the MacFarland standards for micro-iology dilutions (McFarland Standard 0.5, Hardy Di-gnostics, Santa Maria, CA, USA). The inoculate of07 CFU of Strep. equi subsp. zooepidemicus was di-uted in 1 mL 0.9% saline immediately prior to inocu-ation.

At the time of inoculation, mares were placed intocks, their tails were wrapped and held laterally, andhe perineum was washed thoroughly with an iodine-ased soap and dried. The inoculum was drawn up inton artificial insemination pipette, alternating with airnd two 0.5 mL saline cushions to prevent loss ofacteria during deposition. Using a gloved arm, the AIipette was passed midway through the cervix usingigital guidance. The inoculum was placed into the

ervical canal approximately 2.5 cm anterior to the

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404 C.S. Bailey et al. / Theriogenology 74 (2010) 402–412

xternal cervical os [24–26]. Placement of the inocu-um into the cervical canal was confirmed with theresence of cervical plug on the operator’s finger.

.3. Mare monitoring

Beginning the day of bacterial inoculation, a com-lete physical exam was performed on each mare in theorning and evening for the duration of the study.

.3.1. Systemic parametersSystemic parameters (temperature, pulse, respira-

ion, digital pulses, gut sounds, mucous membranes,nd general attitude) were recorded until abortion orelivery of a live foal.

.3.2. Vulvar dischargeMares were monitored for presence of vulvar dis-

harge and scored using the following system: 0 � noischarge; 1 � trace amount discharge at vulvar lips;� slight amount discharge; 3 � moderate amount

ischarge; and 4 � significant amount discharge.

.3.3. Mammary gland developmentMammary gland development was scored as fol-

ows: 0 � no development (flat glands); 1 � slightounding of glands; 2 � moderate rounding of glands;� glands developed but teats empty; and 4 � glands

eveloped with teats filled/waxed.

.3.4. Transrectal ultrasoundUsing transrectal ultrasonography (Aloka 900®

ith 5–10MHz linear probe, Aloka Co, Ltd, Tokyo,apan), the combined thickness of the uterus andlacenta (CTUP) was measured for signs of placen-itis (thickening or placental separation) [18,19,21].stablished CTUP measures for normal pregnancy in

ight horse mares were used as standards. Baselineeasures were recorded prior to bacterial inoculation

f each mare. Allantoic and amniotic fluid characterere also monitored during examinations. Fluid wasraded as: 0 � anechoic/black; 1 � hypoechoic/darkray; 2 � echodense/light gray; and 3 � hypere-hoic, non-shadowing/white. Beginning the day afternoculation, transrectal ultrasound examinationsere performed daily on mares, for 7 d, and then

hree times weekly until abortion or delivery.

.3.5. Transabdominal ultrasoundMares were examined using transabdominal ultra-

ound (Aloka 900® with a 2-5MHz curvilinear sectorransducer, Aloka Co, Ltd, Tokyo, Japan) to monitoretal heart rate, fetal activity, fetal fluid character, andlacental separation [22,23]. A minimum of three val-

es were recorded for fetal heart rate and averaged. A m

aseline examination was performed prior to bacterialnoculation. Beginning the day after inoculation, maresere examined using transabdominal ultrasonographyaily for 7 d, and then three times per week untilbortion or delivery of a foal. Fluid character wasraded as for transrectal ultrasound evaluation.

.4. Drug treatments

Drugs were given to mares in Group TREAT, be-inning with the first signs of placentitis (ultrasono-raphic evidence of increased CTUP, placental separa-ion, changes in fluid character, mammary glandevelopment, or vulvar discharge). Trimethoprim sul-amethoxazole (TMS, generic, Vintage Pharmaceuti-als®, Huntsville AL, USA; 30 mg/kg, PO, q 12 h),ltrenogest (ALT � Regumate®, Intervet, Inc, Mills-oro, DE 0.88 mg/kg, PO, q 24 h) and pentoxifyllinePTX � Pentoxifylline, Extended Release, Apotexnc®, Toronto, ON, Canada; 8.5 mg/kg, PO, q 12 h)ere given to mares until abortion or delivery of a live

oal.

.5. Monitoring mares for impending parturition

During the experimental period, mares were moni-ored twice daily for evidence of impending foalingmammary gland enlargement, evidence of mammaryecretions, vulvar softening, laxity of pubic tendons orulva). Once changes consistent with impending foal-ng were noted, mares were monitored frequently byisual observation in the paddock every 2–3 h. Whenvidence of parturition was noted (increased incidencef recumbency, restlessness, inappetence, straining torinate, evidence of fluid from the vulva indicatingupture of the chorioallantois), mares were observedontinuously through parturition until passage of theetal membranes. Mares were allowed to foal normallynless assistance was deemed necessary (dystocia, pre-ature separation of the fetal membranes). Mares and

oals were allowed to bond in the immediate postpar-um period.

.6. Assessment of foals

A physical examination was performed on all foalst delivery. Foals that were able to right themselves,reathe without mechanical assistance, and respond totimuli (nasal and ear stimulation, suckle response)ere deemed viable at birth. Viable foals were ex-ected to require minimal supportive care postpartum,uch as could readily be performed on-site (stimulationo stand for nursing, feeding by nasogastric tube, ad-

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405C.S. Bailey et al. / Theriogenology 74 (2010) 402–412

Within 3 h after birth, jugular blood samples werebtained from live foals for blood culture, completelood count, serum chemistry, and serum cortisol con-entrations. Prior to obtaining blood samples, the foal’seck was clipped and aseptically prepared. A bloodulture bottle (BBL® SEPTI-CHECK™, Becton Dick-nson, Sparks, MD, USA) was inoculated with 8–10L of fresh blood and submitted for culture. Data fromBC, serum chemistry, and serum cortisol concentra-

ions were used to determine the foal’s health status andaturation of the pituitary adrenal axis. Values were

ompared to established laboratory normal values at theniversity of Florida (leukocyte count 5.3–16.8 x 103

ells/�L, neutrophil:lymphocyte ratio �2 [27], cortisoloncentration 60–140 ng/mL[28,29]. Additional bloodamples were taken from viable foals between 8 and4 h after birth and measured for presence of immuno-lobulins (Snap® Equine Immunoglobulin test kit,dexx Pharmaceutical Inc, Greensboro, NC, USA) andortisol concentrations. Blood immunoglobulin con-entrations greater than 800 mg/dL at 12–24 h post-oaling were considered indicative of adequate IgGransfer [30,31].

Foals that were immature or dysmature based onnitial physical examination findings, were unable toreathe without assistance, unresponsive to stimuli, ornable to right themselves at birth, were deemed non-iable. Non-viable foals were subjected to euthanasiasing an overdose of barbiturate (pentobarbital sodiumnd phenytoin in combination—Beuthanasia®, Scher-ng-Plough Animal Health, Kenilworth, NJ, USA).

Blood was obtained, by intracardiac puncture, fromoals that were dead or aborted. Cardiac blood samplesrom dead foals were cultured as described for liveoals. Complete blood count, serum chemistry, andortisol concentrations were not done on blood fromead foals.

.7. Management of viable foals

All viable foals were monitored frequently in therst 24 h post partum. Physical examinations wereerformed on foals, twice daily, for the first 5–7 d ofife. Prophylactic antibiotics were given to all foals for–7 d post-foaling. Choice of antibiotic was based onverall status of the foal after delivery. Those foalsonsidered to be healthy were given ceftiofur sodiumNaxcel® Pfizer Animal Health Inc. New York, NY,SA @ 4 mg/kg, IM, q 12h). Those foals showing

igns of compromise (abnormal blood values, mildaladjustment, significantly abnormal placenta) were

iven Ampicillin (Generic, Webster Veterinary Supply p

nc. Sterling, MA, USA @ 20 mg/kg, IV, q 8 h) andmikacin (Generic, Webster Veterinary Supply Inc.terling, MA, USA @ 25 mg/kg, IV, q 24 h). A naso-astric tube was placed for colostrum administration iniable foals which did not nurse within 3 h after birth,r when the maternal colostrum was poor quality (val-es �23 for sugar measurement and �16 for alcoholeasurement using a wine refractometer: Eclipse™andheld Refractometer, Bellingham & Stanley, Ltd,urnbridge Wells, Kent, UK) [32]. Additionally, foals

hat did not nurse consistently received an indwellingasogastric tube and supplemental feeding with ex-ressed mare’s milk. All foals were treated with aoapy-water enema in the first 24 h of life.

.8. Gross and histologic tissue analysis

Tissues were collected, from fetal membranes andetal tissues of dead or aborted foals, for histopatho-ogic examination. All tissues were collected in themmediate post partum period (within 2 h after expul-ion for fetal membranes or delivery for fetuses/foals).he fetal membranes were evaluated grossly for com-leteness and abnormalities. Samples were procuredrom the pregnant horn, non-pregnant horn, uterineody, umbilicus, and cervical star area. Any other areahat was grossly abnormal was also sampled. A com-lete necropsy was performed on all non-viable foals.amples of lung, liver, kidney, spleen, and adrenalsere collected. Tissues samples were preserved in for-alin prior to histopathologic analysis. Histologic anal-

sis of tissues was performed by a board-certified vet-rinary pathologist.

.9. Uterine culture

A uterine swab for bacterial cultures was obtainedrom all mares within 3 h after foaling. The mare’s tailas wrapped and pulled to the side, and the perineumas aseptically prepared. A double-guarded swab wassed in routine fashion to collect uterine samples. Ev-dence of bacterial growth was documented indepen-ent of severity. An organism was considered dominanthen it represented �50% of the overall bacterialrowth.

.10. Data analysis and statistics

All data were evaluated for normality using a Sha-iro-Wilks test. Dichotomous variables or proportionsere analyzed using a Fisher’s exact test. Dichotomous

esults were reported as number of animals affected/umber of animals in the group and as an affected

ercentage of the group. Time to abortion/delivery was

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406 C.S. Bailey et al. / Theriogenology 74 (2010) 402–412

ompared between groups using a Student’s t-test.hite blood cell counts in neonatal foals were com-

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. Results

.1. Mare monitoring

Placentitis was diagnosed, clinically, based on de-elopment of vulvar discharge in all mares (within 96 hfter inoculation). There were no differences betweenroups for development of vulvar discharge, mammaryland development, or combined thickness of the uterusnd placenta (CTUP) throughout the study. The CTUPas not outside of published reference ranges [19,21]

or any mare at the time of clinical diagnosis. Mares inroup TREAT showed evidence of placental separa-

ion less frequently than mares in Group UNTREATTable 1).

Baseline fetal heart rates were not different betweenroups (TREAT 99.8 � 10.1 bpm; UNTREAT 101.7�5.9 bpm; P � 0.05).

Parturition was either observed or the foal was foundithin 3 h after birth for 16 of 17 mares. One foal was

ound standing in the paddock and was estimated to bereater than 3 h old.

.2. Pregnancy outcome

Mares in Group TREAT carried pregnancies longerP � 0.05) after bacterial inoculation (TREAT 31 �4 d; UNTREAT 8 � 5 d) and delivered more viableoals (TREAT 10/12, 83%; UNTREAT 0/5, 0%; P �.05) than untreated mares. Two foals from GroupREAT were not viable. One fetus was aborted within

able 1ncidence of clinical signs of placentitis after intracervical bacterialnoculation and prior to treatment in pregnant mares.

repartum clinical sign Group

TREAT UNTREAT

ulvar discharge 12/12 (100%) 5/5 (100%)ammary development 0/12 (0%) 0/5 (0%)TUP � 7 mm at diagnosis 0/12 (0%) 0/5 (0%)lacental separation 2/12 (17%)a 4/5 (80%)b

,bWithin a row, groups without a common superscript differed (P �.05).

4 h after fetal death and one fetus was delivered at n

erm with a red-bag delivery. Histologic examination ofung tissue from the fetus revealed non-aerated tissue.

All viable foals stood without assistance within 2 hfter birth. Four viable foals in Group TREAT requiredlacement of nasogastric tubes for one-time feeding,nd two of those foals required indwelling nasogastricubes for 24–72 h. Extensive critical care was not giveno any viable foals. All foals in Group UNTREAT wereeemed unviable. Two fetuses were dead at deliverynd three foals were alive with signs of immaturitysilky hair-coats and floppy ears) and compromise. Oneoal failed to breathe independently (even after intuba-ion and external ventilation), one foal had eyelids thatere still sealed shut and very shallow, weak respira-

ory movements, and one foal had a very slow, fainteartbeat and was unresponsive to therapy.

.3. Peripartum complications

Incidence of peripartum complications was not dif-erent (P � 0.05) between groups (TREAT 4/12, 33%;NTREAT 3/5, 60%). In Group TREAT, three mares

xperienced premature separation of the fetal mem-ranes at parturition and one mare experienced a dys-ocia, which was resolved by manual manipulation. Inroup UNTREAT, two mares experienced a dystocia

nd one mare experienced premature separation of theetal membranes at parturition. All mares expelled fetalembranes within 3 h after foaling.

.4. Gross and histologic tissue analyses

Predominant histopathologic placental and fetal le-ions are shown (Table 2). Differences were not de-ected for incidence of placental lesions between groupsP � 0.05). Mares in Group TREAT tended (P � 0.07)o have a lower incidence of histologic lesions at theervical star than did mares in Group UNTREAT. Twoares in Group TREAT had evidence of funisitis but

id not have inflammatory lesions in the chorionicamples. Placentas from four mares in Group TREATad no histologic inflammatory lesions at parturition.ross lesions at the cervical star were identified in 3 of

he 4 mares that did not have histologic changes at theervical star. All mares in Group UNTREAT had grossnd histologic evidence of disease.

Relevant fetal histological findings are also reportedTable 2). All non-viable fetuses had evidence of bac-eria in the lungs, independent of group. Additionalndings included pulmonary inflammation and passiveongestion of the organs. Statistical comparisons wereot made between groups due to the low number of

on-viable animals in Group TREAT.

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.5. Uterine bacterial culture

Uterine bacterial culture results are reported in Table. Differences were not detected (P � 0.05) between

able 2redominant histological findings of placental and fetal tissuesbtained from mares after experimentally induced placentitis.

nd point Group P value

TREAT UNTREAT

lacental histologic findingslacentitis 8/12 (67%) 5/5 (100%) P � 0.05Lesions at cervical star 6/12 (50%) 5/5 (100%) P � 0.07Funisitis (umbilicus) 5/12 (42%) 4/5 (80%) P � 0.05

etal histologic findingsPulmonary bacteria 2/2 (100%) 5/5 (100%) NAPulmonary inflammation 2/2 (100%) 1/5 (20%) NAPassive congestion (liver,

kidney, spleen, adrenal)2/2 (100%) 4/5 (80%) NA

he incidence of placental pathological lesions was not differentetween treatment groups. Placentitis was detected, histologically,rom all mares in Group UNTREAT but not Group TREAT. Fetalissue lesions are descriptive only. Statistical comparison for inci-ence of fetal pathology was not done, due to insufficient animalumbers in Group TREAT.

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nd points Group

TREAT UNTREAT

terine culturesBacterial growth 8/12 (67%) 5/5 (100%)Predominant Strep. zooepidemicus 7/12 (58%) 5/5 (100%)Enterobacter cloacae 1/12 (8%) 0/5 (0%)

etal/foal bacterial culturesBlood: bacterial growth 1/12 (8%)a 4/5 (80%)b

Blood: Strep. zooepidemicus 0/12 (0%) 2/5 (40%)Blood: other organisms 1/12 (8%) 2/5 (40%)Stomach/thorax: bacterial growth 2/2 (100%) 5/5 (100%)Stomach/thorax: Strep.

zooepidemicus1/2 (50%) 3/5 (60%)

Stomach/thorax: other organisms 1/2 (50%) 2/5 (40%),bWithin a row, groups without a common superscript differed (P �.05). There was no significant difference between groups for thencidence of bacterial infection or recovery of a specific organismrom uterine swabs (P � 0.05). Strep. equi subsp. zooepidemicus washe organism recovered most frequently from uterine samples in bothroups. A uterine swab from one mare in Group TREAT revealedure growth of Enterobacter cloacae. Positive bacterial cultures werebtained from samples of stomach contents and thoracic fluid from allon-viable fetuses (Group TREAT n � 2; Group UNTREAT n � 5).rganisms recovered from foal samples other than Strep. equi subsp.

ooepidemicus included Enterobacter cloacae, Pseudomonas aerugi-

wosa and Actinobacillus lignerisii.

roups for incidence of positive uterine bacterial cul-ure. Four mares from Group TREAT showed norowth from culture of uterine swabs. Strep. equiubsp. zooepidemicus was the organism recovered mostrequently from uterine cultures in both groups. Theterine swab from one mare in Group TREAT revealedure growth of Enterobacter cloacae. Secondary organ-sms which were cultured from uterine swabs includedther �-hemolytic streptococci, Escherichia coli, Pas-eurella multocida, staphylococcal species, and bacilluspecies.

.6. Fetal/foal culture

Bacterial culture results from foals are shown (Table). Foals from Group TREAT had fewer positive bloodultures (P � 0.05) than foals from Group UNTREAT.rganisms other than Strep. equi subsp. zooepidemicus

ecovered from blood included Enterobacter cloacaeGroup TREAT) and Pseudomonas aeruginosa and Ac-inobacillus lignerisii (Group UNTREAT).

Positive bacterial cultures were obtained fromamples of stomach contents and thoracic fluid fromll non-viable fetuses (Group TREAT n �2; GroupNTREAT n �5). Strep. equi subsp. zooepidemicus

nd Enterobacter cloacae were recovered as the pre-ominant organism from one of each fetus in GroupREAT. Three fetuses/foals in Group UNTREATad predominant growth of Strep. equi subsp. zoo-pidemicus, one fetus had predominant growth ofseudomonas aeruginosa, and one fetus had predom-

nant growth of Actinobacillus lignerisii.

.7. Foal serum cortisol and blood analyses

Blood samples for serum cortisol (birth and 24 h),omplete blood count and serum chemistry analysisere obtained from 9 of 10 live foals. Mean serum

ortisol concentrations were 84.3 � 54.7 ng/mL atirth. Seven of 9 foals had cortisol concentrations atirth that were within normal limits [28,29,33,34]. Se-um cortisol concentrations from two foals were low atirth; one foal was clinically compromised (32 ng/mL),hereas the other was normal (52 ng/mL). Mean serum

ortisol concentrations at 24 h were 23.1 � 10.8 ng/mLwithin expected normal values [28]).

White blood cell counts were within establishedaboratory normal values in 5 of 9 foals at birth. Twooals had WBC counts of 5.0–6.9�103 cells/�L andwo had WBC counts which were lower than 5�103

ells/�L. Differential cell-counts were available foreven live foals. The neutrophil to lymphocyte ratio

as �2 in 6 of 7 foals. Serum chemistry values were

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408 C.S. Bailey et al. / Theriogenology 74 (2010) 402–412

ithin laboratory normal limits for all live foals. SerumgG concentrations were � 800 mg/dL in 6 of the 7 liveoals tested (the seventh foal had a serum IgG concen-ration of 507 mg/dL). Serum IgG concentrations werenavailable for three live foals.

. Discussion

In this study, mares with experimentally inducedlacentitis that were given trimethoprim sulfamthox-zole, pentoxifylline, and altrenogest carried pregnan-ies longer and delivered more live foals than un-reated, infected mares. Placentitis was induced usingn established model of intracervical inoculation with07 Strep. equi subsp. zooepidemicus [12,14,24,25].reatment was initiated when clinical signs were iden-

ified. The regimen of trimethoprim sulfamethoxazole,entoxifylline, and altrenogest was selected to treatlacentitis for several reasons. A multimodal approacho treatment was chosen, based on the postulated patho-hysiology that placental infection is initiated by as-ending infection, perpetuated through inflammation,nd terminated with early fetal delivery [35–38]. Inther species, administration of antibiotics, alone, wasffective in eradicating bacterial infection, but notnhibiting amniotic inflammatory mediators (TNF�,L-1�, PGE2 and PGF2�) or uterine contractions16,39,40]. However, administration of ampicillin com-ined with dexamethsone and indomethacin (anti-inflam-atory/immunomodulatory agents) to experimentally in-

ected animals suppressed inflammatory mediators andeduced uterine contractions [17]. Somewhat similar find-ngs were identified in a pony model of ascending placen-itis [14]. Using the same model of induced placentitis ashe present study, infected mares were given an antibiotictrimethoprim sulfamethoxazole) concurrent with an anti-nflammatory agent (pentoxifylline) or no treatment. Ad-inistration of antibiotic or pentoxifylline, alone, was not

ested. Trimethoprim sulfamethoxazole was selected,iven the good oral bioavailability in horses [41], broad-pectrum, bacteriocidal activity against common causativerganisms of placentitis (Strep. equi subsp. zooepidemi-us, Escherichia coli, nocardioform actinomycetes)13,42], and frequent use in clinical practice. Pentoxifyl-ine was selected for its anti-inflammatory/immunomodu-atory properties, including a dose-dependent effect foreducing inflammatory mediators TNF� and IL-1 [43,44].oth drugs were also known to pass fetal membranes andere detectable in allantoic fluid [12] at therapeutic con-

entrations for up to 4 h after administration. When mares

ith experiementally-induced placentitis were given tri- s

ethoprim sulfamethoxazole and pentoxifylline, gestationength tended to be longer in treated mares when com-ared to untreated, infected mares (mean 15 d vs. 4 d; P �.06) [14]. Neonatal survivability was not improved inreated mares. The authors concluded that although treat-ent with trimethoprim sulfamethoxazole and pentoxifyl-

ine tended to prolong gestation in infected mares, that therug combination did not prevent preterm delivery or fetaleath.

In the present study, progestin therapy (altrenogest)as combined with trimthoprim sulfamethoxazole andentoxifylline to determine if adding a proposed toco-ytic agent to the previously studied treatment regimenould not only increase gestation length, but also im-rove neonatal viability [14]. In this study, the majorityf mares (10 of 12; 83%) with placentitis deliverediable foals after treatment with trimethoprim sulfame-hoxazole, pentoxifylline and altrenogest. These dataere in contrast to the work of Graczyk, et al., [14]here only one live foal (1of 6; 16%) was born toares given trimethoprim sulfamethoxazole and pen-

oxifylline without altrenogest. Rationale for the addi-ion of progestin for treatment of induced placentitisas to counteract potential uterine contractility inducedy inflammation. Although not investigated in thistudy, premature uterine contractions have been docu-ented in mares with experimentally induced placen-

itis [45]. Therefore, therapeutic agents that either actirectly to reduce myometrial contractility, or act indi-ectly to counteract the effects of inflammation, are aogical addition to the treatment protocol. In other spe-ies, progesterone decreased myometrial contractionsn vitro and in vivo [46–48]. Physiologically, proges-ins are thought to inhibit formation of myometrial gapunctions, which facilitate uterine contractility [49,50].n vitro studies in the ovine endometrium showed thatrogesterone interfered with the binding of oxytocin tots receptor and inhibited prostaglandin secretion [51]hich could also reduce uterine contractility. In a re-

ent (ex vivo) study [52] using human fetal mononu-lear cells, progesterone rapidly suppressed the fetalnflammatory response. Clinical studies [53–55] havehown a positive treatment effect of progestationalgents when given to women with a previous history ofreterm delivery. Women at high risk for preterm de-ivery (singleton pregnancy, previous history of deliv-ry �37 wk) who were given progesterone, prophylac-ically, had a significant reduction in delivery prior to7 wk gestation when compared to placebo-treated con-rol patients. Results from these multi-center trials were

ignificant enough to prompt a statement from the

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409C.S. Bailey et al. / Theriogenology 74 (2010) 402–412

merican College of Obstetricians and Gynecologistsn support of prophylactic progestin therapy in womenith a known history of preterm labor at less than 37k gestation [56]. As such, the results of this, and other

tudies, support the use of progestins to treat maresith placentitis.However, one must consider other factors in this

tudy that may have lead to the resolution of clinicaligns and higher live foal delivery rate in mares givenreatment, particularly when compared to similarlyreated mares in clinical practice. Early, aggressivereatment may have contributed to successful outcomesn this study. Physical examinations were performed onares twice daily after bacterial inoculation. Treatmentas initiated when clinical signs were first detected

within 4 d after bacterial inoculation in all mares).ucopurulent vulvar discharge was the most important

linical sign of placentitis in this study, although dis-harge production was scant and would have beenissed without vigilant observation. Mammary gland

evelopment was not an indicator of disease in thistudy, nor were ultrasonographic changes in CTUP,etal fluids, or fetal heart rate. These findings contrasthose in a practice setting where precocious mammaryevelopment and milk production are most often therst clinical signs noted in placentitis [41]. A likelyxplanation for differences in clinical signs betweenhis model and naturally occurring placentitis is thathis model used a supraphysiologic dose of bacteria,ntracervically, to induce disease. Naturally occurringlacentitis is probably more insidious in onset, whichould allow more time for clinical signs as such mam-ary development and placental changes to occur. In-

erestingly, only 13 of the 17 inoculated mares in thistudy had histologic evidence of placentitis. Four inoc-lated mares from group TREAT delivered live foals aterm, but did not have histologic evidence of placentitisr positive uterine cultures for Strep. equi subsp. zoo-pidemicus. All four mares developed significant vul-ar discharge, after inoculation, which would be diffi-ult to attribute to vaginitis, alone. Three of the 4 maresad gross evidence of placental lesions at the cervicaltar. Two of the four mares maintained pregnancies forore than 50 d after bacterial inoculation. In contrast,

ll untreated, inoculated mares aborted within 2 wkfter inoculation, a consistent finding in this model14,45]. Presumably, treatment was effective in maresacking histologic evidence of placentitis, and/or wasiven early enough to significantly inhibit bacterialrowth and subsequent inflammation, thus preventing

istologic changes. t

Negative blood cultures from the majority of foals inroup TREAT (10 of 12) provide further evidence for

fficacy of this treatment in a model of ascending placen-itis. In contrast, 4 of 5 foals from Group UNTREAT (allon-survivors) had positive blood cultures. Further, bac-eria were isolated from stomach contents and/or thoracicuid from all five foals in Group UNTREAT. Althoughlood culture results are not definitive for assessment ofeonatal health, additional parameters measured in themmediate postpartum period also indicated that survivingoals were mature and healthy. Serum cortisol samples,btained from live foals, had values consistent with nor-al term foals [28,29,33]. Complete blood count valuesere normal in 7 of the 9 (78%) foals tested at birth, and

ll 10 live foals survived with minimal care in a fieldetting. These data supported the conclusion that the treat-ent protocol in this study can result in the birth a mature,

iable foal after experimental induction of placentitis. It isoteworthy that two fetuses (17%) in Group TREAT didot survive. One fetus had an overwhelming infectionith Strep. equi subsp. zooepidemicus. Samples from the

econd non-viable fetus in Group TREAT showed pre-ominant growth of Enterobacter cloacae, except in thetomach which had mixed growth of Enterobacter cloa-ae and Strep. equi subsp.zooepidemicus. These data mayeflect the limitations of trimethoprim sulfamethoxazoleor treatment, especially of equine streptococcal organ-sms. Although trimethoprim sulfamethoxazole has good,road spectrum antimicrobial activity, efficacy againsttrep. equi subsp.zooepidemicus was limited in a tissuehamber model, and it is reported to have reduced efficacyn a purulent environment [57]. Further, resistance of therug against Streptococcus zooepidemicus has been re-ortedly increasing in recent years [58]. The cause foruperinfection of one fetus with an enterobacter species isess clear. Enterobacter species are inhabitants of the hu-an neonatal gastrointestinal tract [59] and are commonly

dentified in nosocomial infections in human hospitals andeonatal units [60,61]. Enterobacter species are a sourcef bacterial infections in equine neonates [62]. The mostikely explanation for Enterobacter cloacae infection wasntroduction of bacteria at the time inoculation and possi-le secondary opportunistic infection of the immunocom-romised fetus. Purity of the inoculum with Strep. equiubsp. zooepidemicus was confirmed prior to, andmmediately after, the procedure, which suggests thathe enterobacter species were commensal. Some an-mals in Group UNTREAT had a similar pattern ofacterial infection. Whereas all five fetuses fromroup UNTREAT had overwhelming bacterial infec-

ions, Strep. equi subsp. zooepidemicus was the pre-

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410 C.S. Bailey et al. / Theriogenology 74 (2010) 402–412

ominant organism recovered in only 3 of the 5borted fetuses. Again, iatrogenic infection must beonsidered when working in the urogenital tract,articularly after disruption of the cervical plug.

Uterine bacterial culture results from post partumares were an interesting finding of this study. As

xpected, uterine samples obtained from mares inroup UNTREAT contained an overwhelming growthf predominantly Strep. equi subsp. zooepidemicus (5f 5 mares). However, results from uterine cultures inroup TREAT were less predictable. Eight of 12 mares

67%) had positive uterine cultures in the immediateostpartum period in spite of aggressive antibiotic treat-ent. Length of treatment may have influenced pres-

nce of uterine bacteria after delivery. Of the 12 mareshich were treated, one mare was treated for less thanwk (5 d), five were treated for 2–4 wk, and six were

reated for greater than 4 wk before delivery. Maresith negative uterine culture results (no growth) were

ll given treatment for at least 3 wk, and 3 of the 4ares were treated for more than 30 d. The mare

reated for only 5 d aborted with an overwhelmingnfection of Enterobacter cloacae, suggesting contam-nation early in the experimental period. These findingsere clinically relevant in that duration of medical

reatment for mares with placentitis is controversial. Its certainly noteworthy that even after prolonged treat-ent, over half of the mares still had uterine bacteria at

he time of delivery. These data highlighted the need torovide treatment to post partum mares that have hadlacentitis. In this study, mares were given tri-ethoprim sulfamethoxazole and pentoxifylline for

–7 d after delivery and treated with large volumeterine lavage for at least 3 d after delivery. Subsequentterine culture swabs 2 wk after the end of postpartumreatment were negative in 15 of 17 mares (88%).

In summary, mares with experimentally inducedlacentitis, that were given trimethoprim sulfamethox-zole, pentoxifylline and altrenogest, delivered moreive foals than infected, untreated mares. Viable foalsequired minimal supportive care and were mature atirth. Uterine bacteria were not eliminated with theherapy given, thus necessitating additional treatmentor mares in the postpartum period.

cknowledgments

This work was funded by the Grayson-Jockey Clubesearch Foundation, The State of Florida Parimutualagering Trust and Intervet®. The authors are grateful

or the expertise of Dr. Maron Calderwood Mayes who

erformed histological analysis of all tissues. The au-hors also thank Meghan Connor, Lisa Fultz, Ben Moggnd Lien Hiespiel for animal care assistance.

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